G2Cdb::Gene report

Gene id
G00000547
Gene symbol
Mtap2 (MGI)
Species
Mus musculus
Description
microtubule-associated protein 2
Orthologue
G00001796 (Homo sapiens)

Databases (12)

Curated Gene
OTTMUSG00000016448 (Vega mouse gene)
Gene
ENSMUSG00000015222 (Ensembl mouse gene)
17756 (Entrez Gene)
47 (G2Cdb plasticity & disease)
Gene Expression
MGI:97175 (Allen Brain Atlas)
g00614 (BGEM)
EMAGE:1253 (EMAGE)
EMAGE:1238 (EMAGE)
mtap2 (gensat)
Literature
157130 (OMIM)
Marker Symbol
MGI:97175 (MGI)
Protein Sequence
P20357 (UniProt)

Synonyms (2)

  • MAP-2
  • MAP2

Literature (262)

Pubmed - other

  • Multiple phases of expression and regulation of mouse Hoxc8 during early embryogenesis.

    Belting HG, Shashikant CS and Ruddle FH

    Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.

    Hox genes are expressed in dynamic patterns during embryogenesis consistent with their role in axial specifications. To study the distribution of mouse Hoxc8, a homeodomain containing protein, we raised monoclonal antibodies against the least conserved portion of Hoxc8. Using these antibodies, we have examined early and mid-gestation embryos for the distribution of the protein. At the end of gastrulation Hoxc8 is expressed in the caudal portion of the embryo. In the neural tube, an early phase when all cells express Hoxc8 is distinguished from a late phase with predominant expression in differentiating neurons. A comparison of this expression pattern with that of a reporter gene under the control of the early Hoxc8 enhancer demarcates three separate regulatory components: (1) initiation and establishment; (2) maintenance; and (3) downregulation. We propose that Hoxc8 expression during embryogenesis is established in multiple phases. Possible regulatory mechanisms involved in generating such a complex domain of Hox gene expression are discussed.

    Funded by: NIGMS NIH HHS: GM 09966

    The Journal of experimental zoology 

  • Humanized Foxp2 specifically affects cortico-basal ganglia circuits.

    Reimers-Kipping S, Hevers W, Pääbo S and Enard W

    Max Planck Institute for Evolutionary Anthropology, Deutscher Platz 6,D-04103 Leipzig, Germany.

    It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution and influence aspects of speech and language. Recently it was shown that when these substitutions are introduced into the endogenous Foxp2 gene of mice, they increase dendrite length and long-term depression (LTD) in medium spiny neurons of the striatum. Here we investigated if these effects are found in other brain regions. We found that neurons in the cerebral cortex, the thalamus and the striatum have increased dendrite lengths in the humanized mice whereas neurons in the amygdala and the cerebellum do not. In agreement with previous work we found increased LTD in medium spiny neurons, but did not detect alterations of synaptic plasticity in Purkinje cells. We conclude that although Foxp2 is expressed in many brain regions and has multiple roles during mammalian development, the evolutionary changes that occurred in the protein in human ancestors specifically affect brain regions that are connected via cortico-basal ganglia circuits.

    Neuroscience 2011;175;75-84

  • Peripheral contributions to olfactory bulb cell populations (migrations towards the olfactory bulb).

    Blanchart A, Martín-López E, De Carlos JA and López-Mascaraque L

    Instituto Cajal, CSIC, Department of Cellular, Molecular and Developmental Neurobiology, Madrid, Spain.

    The olfactory system represents one of the most suitable models to study interactions between the peripheral and central nervous systems. The developing olfactory epithelium (olfactory placode and pit) gives rise to several cell populations that migrate towards the telencephalic vesicle. One of these cell populations, called the Migratory Mass (MM), accompanies the first emerging olfactory axons from the olfactory placode, but the fate of these cells and their contribution to the Olfactory Bulb (OB) populations has not been properly addressed. To asses this issue we performed ultrasound-guided in utero retroviral injections at embryonic day (E) 11 revealing the MM as an early source of Olfactory Ensheathing Cells in later postnatal stages. Employing a wide number of antibodies to identify the nature of the infected cells we described that those cells generated within the MM at E11 belong to different cell populations both in the mesenchyma, where they envelop olfactory axons and express the most common glial markers, and in the olfactory bulb, where they are restricted to the Olfactory Nerve and Glomerular layers. Thus, the data reveal the existence of a novel progenitor class within the MM, potentially derived from the olfactory placode which gives rise to different neural cell population including some CNS neurons, glia and olfactory ensheathing cells.

    Glia 2011;59;2;278-92

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • Rheb1 is required for mTORC1 and myelination in postnatal brain development.

    Zou J, Zhou L, Du XX, Ji Y, Xu J, Tian J, Jiang W, Zou Y, Yu S, Gan L, Luo M, Yang Q, Cui Y, Yang W, Xia X, Chen M, Zhao X, Shen Y, Chen PY, Worley PF and Xiao B

    The State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, People's Republic of China.

    mTor kinase is involved in cell growth, proliferation, and differentiation. The roles of mTor activators, Rheb1 and Rheb2, have not been established in vivo. Here, we report that Rheb1, but not Rheb2, is critical for embryonic survival and mTORC1 signaling. Embryonic deletion of Rheb1 in neural progenitor cells abolishes mTORC1 signaling in developing brain and increases mTORC2 signaling. Remarkably, embryonic and early postnatal brain development appears grossly normal in these Rheb1f/f,Nes-cre mice with the notable exception of deficits of myelination. Conditional expression of Rheb1 transgene in neural progenitors increases mTORC1 activity and promotes myelination in the brain. In addition the Rheb1 transgene rescues mTORC1 signaling and hypomyelination in the Rheb1f/f,Nes-cre mice. Our study demonstrates that Rheb1 is essential for mTORC1 signaling and myelination in the brain, and suggests that mTORC1 signaling plays a role in selective cellular adaptations, rather than general cellular viability.

    Funded by: NIDA NIH HHS: DA00266-36, P50 DA000266, R37 DA010309, R37 DA010309-16; NIMH NIH HHS: MH068830-05, P50 MH068830, R01 MH053608, R01 MH053608-17

    Developmental cell 2011;20;1;97-108

  • Hyperpolarization-activated cyclic nucleotide-gated channels in olfactory sensory neurons regulate axon extension and glomerular formation.

    Mobley AS, Miller AM, Araneda RC, Maurer LR, Müller F and Greer CA

    Department of Neurosurgery and Neurobiology, Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, Connecticut 06520-8082, USA.

    Mechanisms influencing the development of olfactory bulb glomeruli are poorly understood. While odor receptors (ORs) play an important role in olfactory sensory neuron (OSN) axon targeting/coalescence (Mombaerts et al., 1996; Wang et al., 1998; Feinstein and Mombaerts, 2004), recent work showed that G protein activation alone is sufficient to induce OSN axon coalescence (Imai et al., 2006; Chesler et al., 2007), suggesting an activity-dependent mechanism in glomerular development. Consistent with these data, OSN axon projections and convergence are perturbed in mice deficient for adenylyl cyclase III, which is downstream from the OR and catalyzes the conversion of ATP to cAMP. However, in cyclic nucleotide-gated (CNG) channel knock-out mice OSN axons are only transiently perturbed (Lin et al., 2000), suggesting that the CNG channel may not be the sole target of cAMP. This prompted us to investigate an alternative channel, the hyperpolarization-activated, cyclic nucleotide-gated cation channel (HCN), as a potential developmental target of cAMP in OSNs. Here, we demonstrate that HCN channels are developmentally precocious in OSNs and therefore are plausible candidates for affecting OSN axon development. Inhibition of HCN channels in dissociated OSNs significantly reduced neurite outgrowth. Moreover, in HCN1 knock-out mice the formation of glomeruli was delayed in parallel with perturbations of axon organization in the olfactory nerve. These data support the hypothesis that the outgrowth and coalescence of OSN axons is, at least in part, subject to activity-dependent mechanisms mediated via HCN channels.

    Funded by: NIA NIH HHS: P01 AG028054; NIDCD NIH HHS: F30 DC010324, F32 DC010098, F32 DC010098-01A1, R01 DC000210, R01 DC009817; NIGMS NIH HHS: GM07205, T32 GM007205; NINDS NIH HHS: NS 007224-24, T32 NS007224

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;49;16498-508

  • G1 arrest and differentiation can occur independently of Rb family function.

    Wirt SE, Adler AS, Gebala V, Weimann JM, Schaffer BE, Saddic LA, Viatour P, Vogel H, Chang HY, Meissner A and Sage J

    Department of Pediatrics, Stanford Medical School, Stanford, CA 94305, USA.

    The ability of progenitor cells to exit the cell cycle is essential for proper embryonic development and homeostasis, but the mechanisms governing cell cycle exit are still not fully understood. Here, we tested the requirement for the retinoblastoma (Rb) protein and its family members p107 and p130 in G0/G1 arrest and differentiation in mammalian cells. We found that Rb family triple knockout (TKO) mouse embryos survive until days 9-11 of gestation. Strikingly, some TKO cells, including in epithelial and neural lineages, are able to exit the cell cycle in G0/G1 and differentiate in teratomas and in culture. This ability of TKO cells to arrest in G0/G1 is associated with the repression of key E2F target genes. Thus, G1 arrest is not always dependent on Rb family members, which illustrates the robustness of cell cycle regulatory networks during differentiation and allows for the identification of candidate pathways to inhibit the expansion of cancer cells with mutations in the Rb pathway.

    Funded by: NCI NIH HHS: T32 CA009302

    The Journal of cell biology 2010;191;4;809-25

  • Molecular characterization of the mouse superior lateral parabrachial nucleus through expression of the transcription factor Runx1.

    Zagami CJ and Stifani S

    Centre for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada.

    Background: The ability to precisely identify separate neuronal populations is essential to the understanding of the development and function of different brain structures. This necessity is particularly evident in regions such as the brainstem, where the anatomy is quite complex and little is known about the identity, origin, and function of a number of distinct nuclei due to the lack of specific cellular markers. In this regard, the gene encoding the transcription factor Runx1 has emerged as a specific marker of restricted neuronal populations in the murine central and peripheral nervous systems. The aim of this study was to precisely characterize the expression of Runx1 in the developing and postnatal mouse brainstem.

    Anatomical and immunohistochemical studies were used to characterize mouse Runx1 expression in the brainstem. It is shown here that Runx1 is expressed in a restricted population of neurons located in the dorsolateral rostral hindbrain. These neurons define a structure that is ventromedial to the dorsal nucleus of the lateral lemniscus, dorsocaudal to the medial paralemniscal nucleus and rostral to the cerebellum. Runx1 expression in these cells is first observed at approximately gestational day 12.5, persists into the adult brain, and is lost in knockout mice lacking the transcription factor Atoh1, an important regulator of the development of neuronal lineages of the rhombic lip. Runx1-expressing neurons in the rostral hindbrain produce cholecystokinin and also co-express members of the Groucho/Transducin-like Enhancer of split protein family.

    Conclusion: Based on the anatomical and molecular characteristics of the Runx1-expressing cells in the rostral hindbrain, we propose that Runx1 expression in this region of the mouse brain defines the superior lateral parabrachial nucleus.

    Funded by: Canadian Institutes of Health Research: MOP-42479, MOP-84577

    PloS one 2010;5;11;e13944

  • Increased expression of MAP2 inhibits melanoma cell proliferation, invasion and tumor growth in vitro and in vivo.

    Song Z, He CD, Sun C, Xu Y, Jin X, Zhang Y, Xiao T, Wang Y, Lu P, Jiang Y, Wei H and Chen HD

    Department of Dermatology, No. 1 Hospital of China Medical University, Key Laboratory of Immunodermatology, Ministry of Health (China Medical University), Shenyang, China. szqdalian@yahoo.com

    Malignant melanoma (MM) is characterized by aggressive metastasis and high mortality rate. Microtubule-associated proteins 2 (MAP2) is expressed abundantly in majority of melanocytic nevi and primary melanomas, but absent in metastatic melanomas. To determine whether MAP2 correlates with tumor progression of MM, we investigated the effects of MAP2 inhibition on the biological behaviour of metastatic melanoma in vitro and in vivo. Our results demonstrated that adenovirus-mediated MAP2 induced apoptotic cell death and cell cycle arrest in metastatic human and mouse melanoma cell lines in vitro, and substantially inhibited the growth of melanomas in nude mice in vivo. In addition, intracellular expression of MAP2 was found to induce the morphologic alteration, suppress the migration and invasion and affect the assembly, stabilization and bundling of microtubules in melanoma cells. This is the first study that MAP2 expression significantly inhibits the growth of MM in vivo. Our results suggest that MAP2 may serve as a promising molecular target for therapy and chemoprevention of MM in humans.

    Experimental dermatology 2010;19;11;958-64

  • Enlarged lateral ventricles and aberrant behavior in mice overexpressing PDGF-B in embryonic neural stem cells.

    Niklasson M, Bergström T, Zhang XQ, Gustafsdottir SM, Sjögren M, Edqvist PH, Vennström B, Forsberg M and Forsberg-Nilsson K

    Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Uppsala, Sweden.

    Platelet-derived growth factor (PDGF) is important in central nervous system (CNS) development, and aberrant expression of PDGF and its receptors has been linked to developmental defects and brain tumorigenesis. We previously found that neural stem and progenitor cells in culture produce PDGF and respond to it by autocrine and/or paracrine signaling. We therefore aimed to examine CNS development after PDGF overexpression in neural stem cells in vivo. Transgenic mice were generated with PDGF-B under control of a minimal nestin enhancer element, which is specific for embryonic expression and will not drive adult expression in mice. The resulting mouse showed increased apoptosis in the developing striatum, which suggests a disturbed regulation of progenitor cells. Later in neurodevelopment, in early postnatal life, mice displayed enlarged lateral ventricles. This enlargement remained into adulthood and it was more pronounced in male mice than in transgenic female mice. Nevertheless, there was an overall normal composition of cell types and numbers in the brain and the transgenic mice were viable and fertile. Adult transgenic males, however, showed behavioral aberrations and locomotor dysfunction. Thus, a tightly regulated expression of PDGF during embryogenesis is required for normal brain development and function in mice.

    Experimental cell research 2010;316;17;2779-89

  • Nestin-Cre mice are affected by hypopituitarism, which is not due to significant activity of the transgene in the pituitary gland.

    Galichet C, Lovell-Badge R and Rizzoti K

    Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, London, United Kingdom.

    Nestin-Cre mice express Cre recombinase under control of the rat nestin promoter and central nervous system (CNS) enhancer. While endogenous Nestin is expressed in some other tissues including the pituitary gland, Nestin-Cre mice induce recombination predominantly in the CNS. For this reason, they have been widely used to explore gene function or cell fate in the latter. Pituitary hormonal deficiencies, or hypopituitarism, are associated with a wide range of symptoms and with a significant morbidity. These can have a neural and/or a pituitary origin as the gland's secretions are controlled by the hypothalamus. We report here that Nestin-Cre mice themselves are affected by mild hypopituitarism. Hence, physiological consequences are expected, especially in combination with defects resulting from Cre mediated deletion of any gene under investigation. To further investigate the origin of this phenotype, we re-examined the activity of the transgene. We compared it with expression of Nestin itself in the context of the hypothalamo-pituitary axis, especially in the light of a recent report showing pituitary Nestin-Cre activity, which contrasts with previous data. Our results disagree with those of this recent study and do not support the claim that Nestin positive cells are present in the pituitary anlagen, the Rathke's pouch (RP). Moreover we did not observe any significant activity in the post-natal pituitary, in agreement with the initial report.

    Funded by: Medical Research Council: MC_U117562207, U117512772

    PloS one 2010;5;7;e11443

  • Myocardin-related transcription factors regulate the Cdk5/Pctaire1 kinase cascade to control neurite outgrowth, neuronal migration and brain development.

    Mokalled MH, Johnson A, Kim Y, Oh J and Olson EN

    Department of Molecular Biology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.

    Numerous motile cell functions depend on signaling from the cytoskeleton to the nucleus. Myocardin-related transcription factors (MRTFs) translocate to the nucleus in response to actin polymerization and cooperate with serum response factor (Srf) to regulate the expression of genes encoding actin and other components of the cytoskeleton. Here, we show that MRTF-A (Mkl1) and MRTF-B (Mkl2) redundantly control neuronal migration and neurite outgrowth during mouse brain development. Conditional deletion of the genes encoding these Srf coactivators disrupts the formation of multiple brain structures, reflecting a failure in neuronal actin polymerization and cytoskeletal assembly. These abnormalities were accompanied by dysregulation of the actin-severing protein gelsolin and Pctaire1 (Cdk16) kinase, which cooperates with Cdk5 to initiate a kinase cascade that governs cytoskeletal rearrangements essential for neuron migration and neurite outgrowth. Thus, the MRTF/Srf partnership interlinks two key signaling pathways that control actin treadmilling and neuronal maturation, thereby fulfilling a regulatory loop that couples cytoskeletal dynamics to nuclear gene transcription during brain development.

    Funded by: NHLBI NIH HHS: R01 HL077439, R01 HL093039, R37 HL053351

    Development (Cambridge, England) 2010;137;14;2365-74

  • A transgenic Cre mouse line for the study of cortical and hippocampal development.

    Zhou W, Zhang Y, Li Y, Wei YS, Liu G, Liu DP, Pleasure SJ, Xie W and Zhao C

    Key Laboratory of Developmental Genes and Human Diseases, MOE, Institute of Life Science, Southeast University, Nanjing, Jiangsu, People's Republic of China.

    Wnt signaling regulates cortical and hippocampal development. In a previous study we found that a particular Wnt receptor, Frizzled9 (Fzd9), was selectively expressed in both the developing and adult hippocampus. Taking advantage of the specificity of this promoter, we generated a transgenic cre mouse line using the putative control elements of the Fzd9 gene. In the Fzd9-cre mice, Cre is mainly detected in the developing cortex and hippocampus and is confined to the CA fields and dentate gyrus in adults. Furthermore, by crossing the Fzd9-cre mouse with the ROSA26 reporter line, we examined the activity of Cre and found that it has very high recombination efficiency. Thus, this mouse line will likely prove to be a useful tool for studying cortical and hippocampal development via activation or inactivation of interesting genes.

    Genesis (New York, N.Y. : 2000) 2010;48;5;343-50

  • STRADalpha deficiency results in aberrant mTORC1 signaling during corticogenesis in humans and mice.

    Orlova KA, Parker WE, Heuer GG, Tsai V, Yoon J, Baybis M, Fenning RS, Strauss K and Crino PB

    Department of Neurology, Penn Epilepsy Center, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.

    Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is a rare human autosomal-recessive disorder characterized by abnormal brain development, cognitive disability, and intractable epilepsy. It is caused by homozygous deletions of STE20-related kinase adaptor alpha (STRADA). The underlying pathogenic mechanisms of PMSE and the role of STRADA in cortical development remain unknown. Here, we found that a human PMSE brain exhibits cytomegaly, neuronal heterotopia, and aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) signaling. STRADalpha normally binds and exports the protein kinase LKB1 out of the nucleus, leading to suppression of the mTORC1 pathway. We found that neurons in human PMSE cortex exhibited abnormal nuclear localization of LKB1. To investigate this further, we modeled PMSE in mouse neural progenitor cells (mNPCs) in vitro and in developing mouse cortex in vivo by knocking down STRADalpha expression. STRADalpha-deficient mNPCs were cytomegalic and showed aberrant rapamycin-dependent activation of mTORC1 in association with abnormal nuclear localization of LKB1. Consistent with the observations in human PMSE brain, knockdown of STRADalpha in vivo resulted in cortical malformation, enhanced mTORC1 activation, and abnormal nuclear localization of LKB1. Thus, we suggest that the aberrant nuclear accumulation of LKB1 caused by STRADalpha deficiency contributes to hyperactivation of mTORC1 signaling and disruption of neuronal lamination during corticogenesis, and thereby the neurological features associated with PMSE.

    Funded by: NINDS NIH HHS: NS045877

    The Journal of clinical investigation 2010;120;5;1591-602

  • The apical complex couples cell fate and cell survival to cerebral cortical development.

    Kim S, Lehtinen MK, Sessa A, Zappaterra MW, Cho SH, Gonzalez D, Boggan B, Austin CA, Wijnholds J, Gambello MJ, Malicki J, LaMantia AS, Broccoli V and Walsh CA

    Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center, Division of Genetics, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.

    Cortical development depends upon tightly controlled cell fate and cell survival decisions that generate a functional neuronal population, but the coordination of these two processes is poorly understood. Here we show that conditional removal of a key apical complex protein, Pals1, causes premature withdrawal from the cell cycle, inducing excessive generation of early-born postmitotic neurons followed by surprisingly massive and rapid cell death, leading to the abrogation of virtually the entire cortical structure. Pals1 loss shows exquisite dosage sensitivity, so that heterozygote mutants show an intermediate phenotype on cell fate and cell death. Loss of Pals1 blocks essential cell survival signals, including the mammalian target of rapamycin (mTOR) pathway, while mTORC1 activation partially rescues Pals1 deficiency. These data highlight unexpected roles of the apical complex protein Pals1 in cell survival through interactions with mTOR signaling.

    Funded by: Howard Hughes Medical Institute; NICHD NIH HHS: HD029178, P30 HD018655, P30HD018655, R01 HD029178; NINDS NIH HHS: P01 NS040043, P01 NS40043, R01 NS032457, R01 NS032457-12, R01 NS038097-02, R01NS32457; Telethon: GGP07181

    Neuron 2010;66;1;69-84

  • Dlx5 and Dlx6 regulate the development of parvalbumin-expressing cortical interneurons.

    Wang Y, Dye CA, Sohal V, Long JE, Estrada RC, Roztocil T, Lufkin T, Deisseroth K, Baraban SC and Rubenstein JL

    Department of Psychiatry, Nina Ireland Laboratory of Developmental Neurobiology, University of California, San Francisco, San Francisco, California 94158, USA. Yanling.wang@ucsf.edu

    Dlx5 and Dlx6 homeobox genes are expressed in developing and mature cortical interneurons. Simultaneous deletion of Dlx5 and 6 results in exencephaly of the anterior brain; despite this defect, prenatal basal ganglia differentiation appeared largely intact, while tangential migration of Lhx6(+) and Mafb(+) interneurons to the cortex was reduced and disordered. The migration deficits were associated with reduced CXCR4 expression. Transplantation of mutant immature interneurons into a wild-type brain demonstrated that loss of either Dlx5 or Dlx5&6 preferentially reduced the number of mature parvalbumin(+) interneurons; those parvalbumin(+) interneurons that were present had increased dendritic branching. Dlx5/6(+/-) mice, which appear normal histologically, show spontaneous electrographic seizures and reduced power of gamma oscillations. Thus, Dlx5&6 appeared to be required for development and function of somal innervating (parvalbumin(+)) neocortical interneurons. This contrasts with Dlx1, whose function is required for dendrite innervating (calretinin(+), somatostatin(+), and neuropeptide Y(+)) interneurons (Cobos et al., 2005).

    Funded by: Howard Hughes Medical Institute; NIMH NIH HHS: K05 MH065670, R01 MH049428, R01 MH049428-14, R01 MH049428-15, R37 MH049428, R37 MH049428-16A1, R37 MH049428-17, R37 MH049428-18, R37 MH49428-01; NINDS NIH HHS: 5R01 NS048528, R01 NS048528

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;15;5334-45

  • Neural tube defects and impaired neural progenitor cell proliferation in Gbeta1-deficient mice.

    Okae H and Iwakura Y

    Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

    Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein-coupled receptors (GPCRs), and both Galpha subunits and tightly associated Gbetagamma subunits regulate downstream effector molecules. Compared to Galpha subunits, the physiological roles of individual Gbeta and Ggamma subunits are poorly understood. In this study, we generated mice deficient in the Gbeta1 gene and found that Gbeta1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% Gbeta1(-/-) embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, Gbeta1(-/-) embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist-induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by Galphai and Gbetagamma signaling, were abnormal in Gbeta1(-/-) neural progenitor cells. These data indicate that Gbeta1 is required for normal embryonic neurogenesis.

    Developmental dynamics : an official publication of the American Association of Anatomists 2010;239;4;1089-101

  • Protogenin defines a transition stage during embryonic neurogenesis and prevents precocious neuronal differentiation.

    Wong YH, Lu AC, Wang YC, Cheng HC, Chang C, Chen PH, Yu JY and Fann MJ

    Institute of Neuroscience and Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan.

    Many Ig superfamily members are expressed in the developing nervous system, but the functions of these molecules during neurogenesis are not all clear. Here, we explore the expression and function of one of members of this superfamily, protogenin (PRTG), in the developing nervous system. Expression of PRTG protein is strong in the neural tube of mouse embryos between embryonic days 7.75 and 9.5 but disappears after embryonic day 10.5 when the neural progenitor marker nestin expresses prominently. Perturbation of PRTG activity in P19 embryonal carcinoma cells and in chick embryos, by either RNA interference or a dominant-negative PRTG mutant, increases neuronal differentiation. Using yeast two-hybrid screening and an in situ binding assay, we were able to identify ERdj3 (a stress-inducible endoplasmic reticulum DnaJ homolog) as a putative PRTG ligand. Addition of purified ERdj3 protein into the P19 differentiation assay reduced neurogenesis. This effect was blocked by addition of either a neutralizing antibody against PRTG or purified PRTG ectodomain protein, indicating that the effect of ERdj3 on neurogenesis is mediated through PRTG. Forced expression of ERdj3 in the chick neural tube also impairs neuronal differentiation. Together, these results suggest that expression of PRTG defines a stage between pluripotent epiblasts and committed neural progenitors, and its signaling plays a critical role in suppressing premature neuronal differentiation during early neural development.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;12;4428-39

  • Developmental and age-related changes of peptidylarginine deiminase 2 in the mouse brain.

    Shimada N, Handa S, Uchida Y, Fukuda M, Maruyama N, Asaga H, Choi EK, Lee J and Ishigami A

    Aging Regulation, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.

    Peptidylarginine deiminases (PADs) are a group of posttranslational modification enzymes that citrullinate (deiminate) protein arginine residues in a Ca(2+)-dependent manner. Enzymatic citrullination abolishes positive charges of native protein molecules, inevitably causing significant alterations in their structure and functions. Among the five isoforms of PADs, PAD2 and PAD4 are proved occupants of the central nervous system (CNS), and especially PAD2 is a main PAD enzyme expressed in the CNS. We previously reported that abnormal protein citrullination by PAD2 has been closely associated with the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and prion disease. Protein citrullination in these patients is thought to play a role during the initiation and/or progression of disease. However, the contribution of changes in PAD2 levels, and consequent citrullination, during developmental and aging processes remained unclear. Therefore, we used quantitative real-time RT-PCR, Western blot analysis, and immunohistochemical methods to measure PAD2 expression and localization in the brain during those processes. PAD2 mRNA expression was detected in the brains of mice as early as embryonic day 15, and its expression in cerebral cortex, hippocampus, and cerebellum increased significantly as the animals aged from 3 to 30 months old. No citrullinated proteins were detected during that period. Moreover, we found here, for the first time, that PAD2 localized specifically in the neuronal cells of the cerebral cortex and Purkinje cells of the cerebellum. These findings indicate that, despite PAD2's normally inactive status, it becomes active and citrullinates cellular proteins, but only when the intracellular Ca(2+) balance is upset during neurodegenerative changes.

    Journal of neuroscience research 2010;88;4;798-806

  • ZNF238 is expressed in postmitotic brain cells and inhibits brain tumor growth.

    Tatard VM, Xiang C, Biegel JA and Dahmane N

    The Wistar Institute, Philadelphia, Pennsylvania, USA.

    Brain tumors such as medulloblastoma (MB) and glioblastoma multiforme (GBM) can derive from neural precursors. For instance, many MBs are thought to arise from the uncontrolled proliferation of cerebellar granule neuron precursors (GNP). GNPs normally proliferate in early postnatal stages in mice but then they become postmitotic and differentiate into granule neurons. The proliferation of neural precursors, GNPs, as well as at least subsets of GBM and MB depends on Hedgehog signaling. However, the gene functions that are lost or suppressed in brain tumors and that normally promote the proliferation arrest and differentiation of precursors remain unclear. Here we have identified a member of the BTB-POZ and zinc finger family, ZNF238, as a factor highly expressed in postmitotic GNPs and differentiated neurons. In contrast, proliferating GNPs as well as MB and GBM express low or no ZNF238. Functionally, inhibition of ZNF238 expression in mouse GNPs decreases the expression of the neuronal differentiation markers MAP2 and NeuN and downregulates the expression of the cell cycle arrest protein p27, a regulator of GNP differentiation. Conversely, reinstating ZNF238 expression in MB and GBM cells drastically decreases their proliferation and promotes cell death. It also downregulates cyclin D1 while increasing MAP2 and p27 protein levels. Importantly, ZNF238 antagonizes MB and GBM tumor growth in vivo in xenografts. We propose that the antiproliferative functions of ZNF238 in normal GNPs and possibly other neural precursors counteract brain tumor formation. ZNF238 is thus a novel brain tumor suppressor and its reactivation in tumors could open a novel anticancer strategy.

    Funded by: NCI NIH HHS: CA010815

    Cancer research 2010;70;3;1236-46

  • Gene deletion mutants reveal a role for semaphorin receptors of the plexin-B family in mechanisms underlying corticogenesis.

    Hirschberg A, Deng S, Korostylev A, Paldy E, Costa MR, Worzfeld T, Vodrazka P, Wizenmann A, Götz M, Offermanns S and Kuner R

    Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.

    Semaphorins and their receptors, plexins, are emerging as key regulators of various aspects of neural and nonneural development. Semaphorin 4D (Sema4D) and B-type plexins demonstrate distinct expression patterns over critical time windows during the development of the murine neocortex. Here, analysis of mice genetically lacking plexin-B1 or plexin-B2 revealed the significance of Sema4D-plexin-B signaling in cortical development. Deficiency of plexin-B2 resulted in abnormal cortical layering and defective migration and differentiation of several subtypes of cortical neurons, including Cajal-Retzius cells, GABAergic interneurons, and principal cells in vivo. In contrast, a lack of plexin-B1 did not impact on cortical development in vivo. In various ex vivo assays on embryonic forebrain, Sema4D enhanced the radial and tangential migration of developing neurons in a plexin-B2-dependent manner. These results suggest that Sema4D-plexin-B2 interactions regulate mechanisms underlying cell specification, differentiation, and migration during corticogenesis.

    Molecular and cellular biology 2010;30;3;764-80

  • Sox2 induces neuronal formation in the developing mammalian cochlea.

    Puligilla C, Dabdoub A, Brenowitz SD and Kelley MW

    Section on Developmental Neuroscience and Section on Synaptic Transmission, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892, USA. puligillac@nidcd.nih.gov

    In the cochlea, spiral ganglion neurons play a critical role in hearing as they form the relay between mechanosensory hair cells in the inner ear and cochlear nuclei in the brainstem. The proneural basic helix-loop-helix transcription factors Neurogenin1 (Neurog1) and NeuroD1 have been shown to be essential for the development of otocyst-derived inner ear sensory neurons. Here, we show neural competence of nonsensory epithelial cells in the cochlea, as ectopic expression of either Neurog1 or NeuroD1 results in the formation of neuronal cells. Since the high-mobility-group type transcription factor Sox2, which is also known to play a role in neurogenesis, is expressed in otocyst-derived neural precursor cells and later in the spiral ganglion neurons along with Neurog1 and NeuroD1, we used both gain- and loss-of-function experiments to examine the role of Sox2 in spiral ganglion neuron formation. We demonstrate that overexpression of Sox2 results in the production of neurons, suggesting that Sox2 is sufficient for the induction of neuronal fate in nonsensory epithelial cells. Furthermore, spiral ganglion neurons are absent in cochleae from Sox2(Lcc/Lcc) mice, indicating that Sox2 is also required for neuronal formation in the cochlea. Our results indicate that Sox2, along with Neurog1 and NeuroD1, are sufficient to induce a neuronal fate in nonsensory regions of the cochlea. Finally, we demonstrate that nonsensory cells within the cochlea retain neural competence through at least the early postnatal period.

    Funded by: Intramural NIH HHS: Z99 DC999999

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30;2;714-22

  • Expression of PTPRO in the interneurons of adult mouse olfactory bulb.

    Kotani T, Murata Y, Ohnishi H, Mori M, Kusakari S, Saito Y, Okazawa H, Bixby JL and Matozaki T

    Laboratory of Biosignal Sciences, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan.

    PTPRO is a receptor-type protein tyrosine phosphatase (PTP) with a single catalytic domain in its cytoplasmic region and multiple fibronectin type III-like domains in its extracellular region. In the chick, PTPRO mRNA has been shown to be particularly abundant in embryonic brain, and PTPRO is implicated in axon growth and guidance during embryonic development. However, the temporal and spatial expression of PTPRO protein in the mammalian CNS, particularly in the juvenile and adult mammalian brain, has not been evaluated in any detail. By immunohistofluorescence analysis with a monoclonal antibody to PTPRO, we show that PTPRO is widely expressed throughout the mouse brain from embryonic day 16 to postnatal day 1, while expression is largely confined to the olfactory bulb (OB) and olfactory tubercle in the adult brain. In the OB, PTPRO protein is expressed predominantly in the external plexiform layer, the granule cell layer, and the glomerular layer (GL). In these regions, expression of PTPRO is predominant in interneurons such as gamma-aminobutyric acid (GABA)-ergic or calretinin (CR)-positive granule cells. In addition, PTPRO is expressed in GABAergic, CR-positive, tyrosine hydroxylase-positive, or neurocalcin-positive periglomerular cells in the GL. Costaining of PTPRO with other neuronal markers suggests that PTPRO is likely to be localized to the dendrites or dendritic spines of these olfactory interneurons. Thus, PTPRO might participate in regulation of dendritic morphology or synapse formation of interneurons in the adult mouse OB.

    Funded by: NINDS NIH HHS: NS38920, R01 NS038920

    The Journal of comparative neurology 2010;518;2;119-36

  • The distribution and characterization of endogenous protein arginine N-methyltransferase 8 in mouse CNS.

    Kousaka A, Mori Y, Koyama Y, Taneda T, Miyata S and Tohyama M

    Department of Anatomy and Neuroscience, Graduate School of Medicine, The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Osaka University, 2-2 Yamadaoka, Suita City, Osaka, Japan.

    Protein arginine N-methyltransferase (PRMT) 8 was first discovered from a database search for genes harboring four conserved methyltransferase motifs, which shares more than 80% homology to PRMT1 in amino acid [Lee J, Sayegh J, Daniel J, Clarke S, Bedford MT (2005) PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. J Biol Chem 280:32890-32896]. Interestingly, its tissue distribution is strikingly restricted to mouse CNS. To characterize the function in the CNS neurons, we raised an antiserum against PRMT8 to perform immunohistochemistry (IHC) and Western blot analysis. By IHC, the immunoreactivity of endogenous PRMT8 was broadly distributed in the CNS neurons with markedly intense signals in the cerebellum, hippocampal formation, and cortex, but was not detected in the cerebellar granular layer. In some subset of the neurons, the immunoreactivity was observed in the dendrites and axon bundles. The subcellular localization of the immunoreactivity was dominantly nuclear, arguing against the original report that exogenously expressed PRMT8 localizes to the plasma membrane via the N-terminal myristoylation. A series of the exogenously expressed proteins with different in-frame translation initiation codons was tested for comparison with the endogenous protein in molecular size. The third initiator codon produced the protein that was equivalent in size to the endogenous and showed a similar localizing pattern in PC12 cells. In conclusion, PRMT8 is a neuron-specific nuclear enzyme and the N-terminus does not contain the glycine end for myristoylation target.

    Neuroscience 2009;163;4;1146-57

  • Different timings of Dicer deletion affect neurogenesis and gliogenesis in the developing mouse central nervous system.

    Kawase-Koga Y, Otaegi G and Sun T

    Department of Cell and Developmental Biology, Weill Medical College of Cornell University, New York, New York 10065, USA.

    MicroRNAs, processed by the RNAase III enzyme Dicer, are approximately 22 nucleotide endogenous noncoding small RNAs. The function of Dicer in the mouse central nervous system (CNS) development is not well understood. Here, we show that specifically deleting Dicer expression in the CNS and in the cerebral cortex using two Cre lines results in reduced progenitor numbers, abnormal neuronal differentiation, and thinner cortical wall. Incomplete Dicer deletion during early embryonic stages contributes to normal development of early-born neurons in the cortex and motor neurons in the spinal cord. However, at late embryonic stages when Dicer is completely ablated in the CNS, the migration of late-born neurons in the cortex and oligodendrocyte precursor expansion and differentiation in the spinal cord are greatly affected. Our studies of different timings of Dicer deletion demonstrate the importance of the Dicer-mediated microRNA pathway in regulating distinct phases of neurogenesis and gliogenesis during the CNS development.

    Funded by: NIMH NIH HHS: R01 MH083680, R01 MH083680-01A1, R01MH083680

    Developmental dynamics : an official publication of the American Association of Anatomists 2009;238;11;2800-12

  • Three human ARX mutations cause the lissencephaly-like and mental retardation with epilepsy-like pleiotropic phenotypes in mice.

    Kitamura K, Itou Y, Yanazawa M, Ohsawa M, Suzuki-Migishima R, Umeki Y, Hohjoh H, Yanagawa Y, Shinba T, Itoh M, Nakamura K and Goto Y

    Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan. kitamura@ncnp.gp.jp

    ARX (the aristaless-related homeobox gene) is a transcription factor that participates in the development of GABAergic and cholinergic neurons in the forebrain. Many ARX mutations have been identified in X-linked lissencephaly and mental retardation with epilepsy, and thus ARX is considered to be a causal gene for the two syndromes although the neurobiological functions of each mutation remain unclear. We attempted to elucidate the causal relationships between individual ARX mutations and disease phenotypes by generating a series of mutant mice. We generated three types of mice with knocked-in ARX mutations associated with X-linked lissencephaly (P353R) and mental retardation [P353L and 333ins(GCG)7]. Mice with the P355R mutation (equivalent to the human 353 position) that died after birth were significantly different in Arx transcript/protein amounts, GABAergic and cholinergic neuronal development, brain morphology and lifespan from mice with P355L and 330ins(GCG)7 but considerably similar to Arx-deficient mice with truncated ARX mutation in lissencephaly. Mice with the 330ins(GCG)7 mutation showed severe seizures and impaired learning performance, whereas mice with the P355L mutation exhibited mild seizures and only slightly impaired learning performance. Both types of mutant mice exhibited the mutation-specific lesser presence of GABAergic and cholinergic neurons in the striatum, medial septum and ventral forebrain nuclei when compared with wild-type mice. Present findings that reveal a causal relationship between ARX mutations and the pleiotropic phenotype in mice, suggest that the ARX-related syndrome, including lissencephaly or mental retardation, is caused by only the concerned ARX mutations without the involvement of other genetic factors.

    Human molecular genetics 2009;18;19;3708-24

  • Comparative analysis of uncoupling protein 4 distribution in various tissues under physiological conditions and during development.

    Smorodchenko A, Rupprecht A, Sarilova I, Ninnemann O, Bräuer AU, Franke K, Schumacher S, Techritz S, Nitsch R, Schuelke M and Pohl EE

    Institute of Cell Biology and Neurobiology, Charité - Universitätsmedizin, Berlin, Germany.

    UCP4 is a member of the mitochondrial uncoupling protein subfamily and one of the three UCPs (UCP2, UCP4, UCP5), associated with the nervous system. Its putative functions include thermogenesis, attenuation of reactive oxidative species (ROS), regulation of mitochondrial calcium concentration and involvement in cell differentiation and apoptosis. Here we investigate UCP4's subcellular, cellular and tissue distribution, using an antibody designed specially for this study, and discuss the findings in terms of the protein's possible functions. Western blot and immunohistochemistry data confirmed that UCP4 is expressed predominantly in the central nervous system (CNS), as previously shown at mRNA level. No protein was found in heart, spleen, stomach, intestine, lung, thymus, muscles, adrenal gland, testis and liver. The reports revealing UCP4 mRNA in kidney and white adipose tissue were not confirmed at protein level. The amount of UCP4 varies in the mitochondria of different brain regions, with the highest protein content found in cortex. We show that UCP4 is present in fetal murine brain tissue as early as embryonic days 12-14 (E12-E14), which coincides with the beginning of neuronal differentiation. The UCP4 content in mitochondria decreases as the age of mice increases. UCP4 preferential expression in neurons and its developmental expression pattern under physiological conditions may indicate a specific protein function, e.g. in neuronal cell differentiation.

    Biochimica et biophysica acta 2009;1788;10;2309-19

  • Transient neuronal populations are required to guide callosal axons: a role for semaphorin 3C.

    Niquille M, Garel S, Mann F, Hornung JP, Otsmane B, Chevalley S, Parras C, Guillemot F, Gaspar P, Yanagawa Y and Lebrand C

    Department of Cellular Biology and Morphology, University of Lausanne, Switzerland.

    The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.

    Funded by: Medical Research Council: MC_U117570528

    PLoS biology 2009;7;10;e1000230

  • Targeted inactivation of a developmentally regulated neural plectin isoform (plectin 1c) in mice leads to reduced motor nerve conduction velocity.

    Fuchs P, Zörer M, Reipert S, Rezniczek GA, Propst F, Walko G, Fischer I, Bauer J, Leschnik MW, Lüscher B, Thalhammer JG, Lassmann H and Wiche G

    Department of Biochemistry and Cell Biology, Max F Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.

    Cytolinker proteins stabilize cells mechanically, regulate cytoskeleton dynamics, and provide scaffolds for signaling molecules. For plectin, the prototype of these proteins, an unusual diversity of isoforms has been reported, which show distinct expression patterns, subcellular localizations, and functions. Plectin has been shown to have important functions in skin and muscle, but little is known about its role in neural cells. To address this issue, we generated two knock-out mouse lines, one which was selectively lacking plectin 1c (P1c), the major isoform expressed in neural cells, and another in which plectin was conditionally deleted in neuronal precursor cells. Using isoform-specific antibodies, we found P1c to be expressed late in development and to associate with postsynaptic dendrites of central nervous system neurons, motorneurons of spinal cord, sciatic nerve axons, and Schwann cells. Motor nerve conduction velocity was found significantly reduced in sciatic nerve from P1c-deficient as well as from conditional knock-out mice. This defect was traceable to an increased number of motor nerve fibers with small cross-sectional areas; the thicknesses of axons and of myelin sheaths were unaffected. This is the first report demonstrating an important role of plectin in a major nerve function.

    Funded by: Austrian Science Fund FWF: P 17862, P 20744

    The Journal of biological chemistry 2009;284;39;26502-9

  • The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis.

    Guerrier S, Coutinho-Budd J, Sassa T, Gresset A, Jordan NV, Chen K, Jin WL, Frost A and Polleux F

    Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

    During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in nonneuronal cells filopodia dynamics decrease the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results suggest that F-BAR domains are functionally diverse and highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis.

    Funded by: NICHD NIH HHS: T32 HD046369; NIGMS NIH HHS: TG 5T32GM07205; NINDS NIH HHS: 5F31NS052969-03, F31 NS052969-01A1, F31 NS052969-02, F31 NS052969-03, P30 NS045892-060005, P30 NS045892-070005, P30 NS45892-01, R01 NS067557, R01 NS067557-03

    Cell 2009;138;5;990-1004

  • Target deletion of the cytoskeleton-associated protein palladin does not impair neurite outgrowth in mice.

    Shu RZ, Zhang F, Liu XS, Li CL, Wang L, Tai YL, Wu XL, Yang X, Liao XD, Jin Y, Gu MM, Huang L, Pang XF and Wang ZG

    Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai, China.

    Palladin is an actin cytoskeleton-associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90-92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice.

    PloS one 2009;4;9;e6916

  • Aspects of the neuroendocrine cerebellum: expression of secretogranin II, chromogranin A and chromogranin B in mouse cerebellar unipolar brush cells.

    Nunzi MG and Mugnaini E

    Department of Cell and Molecular Biology, The Feinberg School of Medicine of Northwestern University, Searle 5-474, 320 East Superior Street, Chicago, IL 60611, USA. mg-nunzi@northwestern.edu

    Morphologically distinct neuron classes can be subdivided in sublineages by differential chemical phenotypes that correlate with functional diversity. Here we show by immunocytochemistry that chromogranin A (CgA) chromogranin B (CgB) and secretogranin II (SgII), the principal granins situated in neuronal secretory granules and large dense-core vesicles, are widely but differentially expressed in cells of the mouse cerebellum and terminals of cerebellar afferents. While CgA and CgB were nearly panneuronal, SgII was more restricted in distribution. The cells most intensely immunoreactive for SgII were a class of small, excitatory interneurons enriched in the granular layer of the vestibulocerebellum, the unipolar brush cells (UBCs), although larger neurons likely to be a subset of the Golgi-Lugaro-globular cell population were also distinctly immunopositive; by contrast, Purkinje cells and granule cells were, at best, faintly stained and, stellate, basket cells were unstained. SgII was also present in subsets of mossy fibers, climbing fibers and varicose fibers. Neurons in the cerebellar nuclei and inferior olive were distinctly positive for the three granins. Double-labeling with subset-specific cell class markers indicated that, while both CgA and CgB were present in most UBCs, SgII immunoreactivity was present in the calretinin (CR)-expressing subset, but lacked in metabotropic glutamate receptor 1alpha (mGluR1alpha)-expressing UBCs. Thus, we have identified an additional cell class marker, SgII, which serves to study subtype properties in the UBC population. The abundance of SgII in only one of the two known subsets of UBCs is remarkable, as its expression in other neurons of the cortex was moderate or altogether lacking. The data suggest that the CR-positive UBCs represent a unique neuroendocrine component of the mammalian cerebellar cortex, presumably endowed with transynaptically regulated autocrine or paracrine action/s. Because of the well-known organization of the cerebellar system, several of its neuron classes may represent valuable cellular models to analyze granin functions in situ, in acute slices and in dissociated cell and organotypic slice cultures.

    Funded by: NINDS NIH HHS: NS 09904

    Neuroscience 2009;162;3;673-87

  • Developmental control of CaV1.2 L-type calcium channel splicing by Fox proteins.

    Tang ZZ, Zheng S, Nikolic J and Black DL

    Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, California 90095-1662, USA.

    CaV1.2 voltage-gated calcium channels play critical roles in the control of membrane excitability, gene expression, and muscle contraction. These channels show diverse functional properties generated by alternative splicing at multiple sites within the CaV1.2 pre-mRNA. The molecular mechanisms controlling this splicing are not understood. We find that two exons in the CaV1.2 channel are controlled in part by members of the Fox family of splicing regulators. Exons 9* and 33 confer distinct electrophysiological properties on the channel and show opposite patterns of regulation during cortical development, with exon 9* progressively decreasing its inclusion in the CaV1.2 mRNA over time and exon 33 progressively increasing. Both exons contain Fox protein binding elements within their adjacent introns, and Fox protein expression is induced in cortical neurons in parallel with the changes in CaV1.2 splicing. We show that knocking down expression of Fox proteins in tissue culture cells has opposite effects on exons 9* and 33. The loss of Fox protein increases exon 9* splicing and decreases exon 33, as predicted by the positions of the Fox binding elements and by the pattern of splicing in development. Conversely, overexpression of Fox1 and Fox2 proteins represses exon 9* and enhances exon 33 splicing in the endogenous CaV1.2 mRNA. These effects of Fox proteins on exons 9* and 33 can be recapitulated in transfected minigene reporters. Both the repressive and the enhancing effects of Fox proteins are dependent on the Fox binding elements within and adjacent to the target exons, indicating that the Fox proteins are directly regulating both exons. These results demonstrate that the Fox protein family is playing a key role in tuning the properties of CaV1.2 calcium channels during neuronal development.

    Funded by: Howard Hughes Medical Institute; NIGMS NIH HHS: R01 GM049662, R01 GM049662-16, R01 GM49662

    Molecular and cellular biology 2009;29;17;4757-65

  • Downregulation of functional Reelin receptors in projection neurons implies that primary Reelin action occurs at early/premigratory stages.

    Uchida T, Baba A, Pérez-Martínez FJ, Hibi T, Miyata T, Luque JM, Nakajima K and Hattori M

    Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.

    Reelin signaling is essential for correct development of the mammalian brain. Reelin binds to apolipoprotein E receptor 2 and very low-density lipoprotein receptor and induces phosphorylation of Dab1. However, when and where these reactions occur is essentially unknown, and the primary function(s) of Reelin remain unclear. Here, we used alkaline phosphatase fusion of the receptor-binding region of Reelin to quantitatively investigate the localization of functional Reelin receptors (i.e., those on the plasma membrane as mature forms) in the developing brain. In the wild-type cerebral cortex, they are mainly present in the intermediate and subventricular zones, as well as in radial fibers, but much less in the cell bodies of the cortical plate. Functional Reelin receptors are much more abundant in the Reelin-deficient cortical plate, indicating that Reelin induces their downregulation and that it begins before the neurons migrate out of the intermediate zone. In the wild-type cerebellum, functional Reelin receptors are mainly present in the cerebellar ventricular zone but scarcely expressed by Purkinje cells that have migrated out of it. It is thus strongly suggested that Reelin exerts critical actions on migrating projection neurons at their early/premigratory stages en route to their final destinations, in the developing cerebral cortex and cerebellum.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;34;10653-62

  • The N-Myc-DLL3 cascade is suppressed by the ubiquitin ligase Huwe1 to inhibit proliferation and promote neurogenesis in the developing brain.

    Zhao X, D' Arca D, Lim WK, Brahmachary M, Carro MS, Ludwig T, Cardo CC, Guillemot F, Aldape K, Califano A, Iavarone A and Lasorella A

    Institute for Cancer Genetics, Columbia University Medical Center, New York, NY 10032, USA.

    Self-renewal and proliferation of neural stem cells and the decision to initiate neurogenesis are crucial events directing brain development. Here we show that the ubiquitin ligase Huwe1 operates upstream of the N-Myc-DLL3-Notch pathway to control neural stem cell activity and promote neurogenesis. Conditional inactivation of the Huwe1 gene in the mouse brain caused neonatal lethality associated with disorganization of the laminar patterning of the cortex. These defects stemmed from severe impairment of neurogenesis associated with uncontrolled expansion of the neural stem cell compartment. Loss- and gain-of-function experiments in the mouse cortex demonstrated that Huwe1 restrains proliferation and enables neuronal differentiation by suppressing the N-Myc-DLL3 cascade. Notably, human high-grade gliomas carry focal hemizygous deletions of the X-linked Huwe1 gene in association with amplification of the N-myc locus. Our results indicate that Huwe1 balances proliferation and neurogenesis in the developing brain and that this pathway is subverted in malignant brain tumors.

    Funded by: Medical Research Council: MC_U117570528; NCI NIH HHS: R01 CA085628, R01 CA109755, R01 CA131126, R01 CA131126-01A1, R01CA085628, R01CA109755, R01CA131126, U54 CA121852, U54CA121852

    Developmental cell 2009;17;2;210-21

  • Neuronal 3',3,5-triiodothyronine (T3) uptake and behavioral phenotype of mice deficient in Mct8, the neuronal T3 transporter mutated in Allan-Herndon-Dudley syndrome.

    Wirth EK, Roth S, Blechschmidt C, Hölter SM, Becker L, Racz I, Zimmer A, Klopstock T, Gailus-Durner V, Fuchs H, Wurst W, Naumann T, Bräuer A, de Angelis MH, Köhrle J, Grüters A and Schweizer U

    Neuroscience Research Center, Charité Universitätsmedizin Berlin, Berlin, Germany.

    Thyroid hormone transport into cells requires plasma membrane transport proteins. Mutations in one of these, monocarboxylate transporter 8 (MCT8), have been identified as underlying cause for the Allan-Herndon-Dudley syndrome, an X-linked mental retardation in which the patients also present with abnormally high 3',3,5-triiodothyronine (T(3)) plasma levels. Mice deficient in Mct8 replicate the thyroid hormone abnormalities observed in the human condition. However, no neurological deficits have been described in mice lacking Mct8. Therefore, we subjected Mct8-deficient mice to a comprehensive immunohistochemical, neurological, and behavioral screen. Several behavioral abnormalities were found in the mutants. Interestingly, some of these behavioral changes are compatible with hypothyroidism, whereas others rather indicate hyperthyroidism. We thus hypothesized that neurons exclusively dependent on Mct8 are in a hypothyroid state, whereas neurons expressing other T(3) transporters become hyperthyroid, if they are exposed directly to the high plasma T(3). The majority of T(3) uptake in primary cortical neurons is mediated by Mct8, but pharmacological inhibition suggested functional expression of additional T(3) transporter classes. mRNAs encoding six T(3) transporters, including L-type amino acid transporters (LATs), were coexpressed with Mct8 in isolated neurons. We then demonstrated Lat2 expression in cultured neurons and throughout murine brain development. In contrast, LAT2 is expressed in microglia in the developing human brain during gestation, but not in neurons. We suggest that lack of functional complementation by alternative thyroid hormone transporters in developing human neurons precipitates the devastating neurodevelopmental phenotype in MCT8-deficient patients, whereas Mct8-deficient mouse neurons are functionally complemented by other transporters, for possibly Lat2.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;30;9439-49

  • The transcriptional repressor RP58 is crucial for cell-division patterning and neuronal survival in the developing cortex.

    Okado H, Ohtaka-Maruyama C, Sugitani Y, Fukuda Y, Ishida R, Hirai S, Miwa A, Takahashi A, Aoki K, Mochida K, Suzuki O, Honda T, Nakajima K, Ogawa M, Terashima T, Matsuda J, Kawano H and Kasai M

    Department of Molecular Physiology, Tokyo Metropolitan Institute for Neuroscience, Musashidai, Fuchu, Japan. hokado@tmin.ac.jp

    The neocortex and the hippocampus comprise several specific layers containing distinct neurons that originate from progenitors at specific development times, under the control of an adequate cell-division patterning mechanism. Although many molecules are known to regulate this cell-division patterning process, its details are not well understood. Here, we show that, in the developing cerebral cortex, the RP58 transcription repressor protein was expressed both in postmitotic glutamatergic projection neurons and in their progenitor cells, but not in GABAergic interneurons. Targeted deletion of the RP58 gene led to dysplasia of the neocortex and of the hippocampus, reduction of the number of mature cortical neurons, and defects of laminar organization, which reflect abnormal neuronal migration within the cortical plate. We demonstrate an impairment of the cell-division patterning during the late embryonic stage and an enhancement of apoptosis of the postmitotic neurons in the RP58-deficient cortex. These results suggest that RP58 controls cell division of progenitor cells and regulates the survival of postmitotic cortical neurons.

    Developmental biology 2009;331;2;140-51

  • Maternal diabetes alters transcriptional programs in the developing embryo.

    Pavlinkova G, Salbaum JM and Kappen C

    Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198-5455, USA. gpavlinkova@img.cas.cz

    Background: Maternal diabetes is a well-known risk factor for birth defects, such as heart defects and neural tube defects. The causative molecular mechanisms in the developing embryo are currently unknown, and the pathogenesis of developmental abnormalities during diabetic pregnancy is not well understood. We hypothesized that the developmental defects are due to alterations in critical developmental pathways, possibly as a result of altered gene expression. We here report results from gene expression profiling of exposed embryos from a mouse diabetes model.

    Results: In comparison to normal embryos at mid-gestation, we find significantly altered gene expression levels in diabetes-exposed embryos. Independent validation of altered expression was obtained by quantitative Real Time Polymerase Chain Reaction. Sequence motifs in the promoters of diabetes-affected genes suggest potential binding of transcription factors that are involved in responses to oxidative stress and/or to hypoxia, two conditions known to be associated with diabetic pregnancies. Functional annotation shows that a sixth of the de-regulated genes have known developmental phenotypes in mouse mutants. Over 30% of the genes we have identified encode transcription factors and chromatin modifying proteins or components of signaling pathways that impinge on transcription.

    Conclusion: Exposure to maternal diabetes during pregnancy alters transcriptional profiles in the developing embryo. The enrichment, within the set of de-regulated genes, of those encoding transcriptional regulatory molecules provides support for the hypothesis that maternal diabetes affects specific developmental programs.

    Funded by: NCRR NIH HHS: P20 RR016469, P20 RR018788, P20RR016469, P20RR018788; NICHD NIH HHS: R01 HD034706, R01 HD037804, R01 HD055528, R01 HD055528-02, R01-HD055528, R01-HD34706; NIDDK NIH HHS: P30 DK072476

    BMC genomics 2009;10;274

  • The protocadherin gene Celsr3 is required for interneuron migration in the mouse forebrain.

    Ying G, Wu S, Hou R, Huang W, Capecchi MR and Wu Q

    Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA.

    Interneurons are extremely diverse in the mammalian brain and provide an essential balance for functional neural circuitry. The vast majority of murine cortical interneurons are generated in the subpallium and migrate tangentially over a long distance to acquire their final positions. By using a mouse line with a deletion of the Celsr3 (Flamingo, or Fmi1) gene and a knock-in of the green fluorescent protein reporter, we find that Celsr3, a member of the nonclustered protocadherin (Pcdh) family, is predominantly expressed in the cortical interneurons in adults and in the interneuron germinal zones in embryos. We show that Celsr3 is crucial for interneuron migration in the developing mouse forebrain. Specifically, in Celsr3 knockout mice, calretinin-positive interneurons are reduced in the developing neocortex, accumulated in the corticostriatal boundary, and increased in the striatum. Moreover, the laminar distribution of cortical calbindin-positive cells is altered. Finally, we found that expression patterns of NRG1 (neuregulin-1) and its receptor ErbB4, which are essential for interneuron migration, are changed in Celsr3 mutants. These results demonstrate that the protocadherin Celsr3 gene is essential for both tangential and radial interneuron migrations in a class-specific manner.

    Funded by: Howard Hughes Medical Institute

    Molecular and cellular biology 2009;29;11;3045-61

  • Subcellular dynamics of type II PKA in neurons.

    Zhong H, Sia GM, Sato TR, Gray NW, Mao T, Khuchua Z, Huganir RL and Svoboda K

    Howard Hughes Medical Institute Janelia Farm Research Campus, Ashburn, VA 20147, USA. zhongh@janelia.hhmi.org

    Protein kinase A (PKA) plays multiple roles in neurons. The localization and specificity of PKA are largely controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA in neurons and the roles of specific AKAPs are poorly understood. We imaged the distribution of type II PKA in hippocampal and cortical layer 2/3 pyramidal neurons in vitro and in vivo. PKA was concentrated in dendritic shafts compared to the soma, axons, and dendritic spines. This spatial distribution was imposed by the microtubule-binding protein MAP2, indicating that MAP2 is the dominant AKAP in neurons. Following cAMP elevation, catalytic subunits dissociated from the MAP2-tethered regulatory subunits and rapidly became enriched in nearby spines. The spatial gradient of type II PKA between dendritic shafts and spines was critical for the regulation of synaptic strength and long-term potentiation. Therefore, the localization and activity-dependent translocation of type II PKA are important determinants of PKA function.

    Funded by: Howard Hughes Medical Institute

    Neuron 2009;62;3;363-74

  • Histone deacetylases 1 and 2 control the progression of neural precursors to neurons during brain development.

    Montgomery RL, Hsieh J, Barbosa AC, Richardson JA and Olson EN

    Department of Molecular Biology, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.

    The molecular mechanism by which neural progenitor cells commit to a specified lineage of the central nervous system remains unknown. We show that HDAC1 and HDAC2 redundantly control neuronal development and are required for neuronal specification. Mice lacking HDAC1 or HDAC2 in neuronal precursors show no overt histoarchitectural phenotypes, whereas deletion of both HDAC1 and HDAC2 in developing neurons results in severe hippocampal abnormalities, absence of cerebellar foliation, disorganization of cortical neurons, and lethality by postnatal day 7. These abnormalities in brain formation can be attributed to a failure of neuronal precursors to differentiate into mature neurons and to excessive cell death. These results reveal redundant and essential roles for HDAC1 and HDAC2 in the progression of neuronal precursors to mature neurons in vivo.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;19;7876-81

  • Essential role of Rac1 and Rac3 GTPases in neuronal development.

    Corbetta S, Gualdoni S, Ciceri G, Monari M, Zuccaro E, Tybulewicz VL and de Curtis I

    Cell Adhesion Unit, San Raffaele Scientific Institute and University Vita-Salute San Raffaele, Via Olgettina 58-20132 Milano, Italy.

    Rac GTPases are members of the Rho family regulating the actin cytoskeleton and implicated in neuronal development. Ubiquitous Rac1 and neuron-specific Rac3 GTPases are coexpressed in the developing mammalian brain. We used Cre-mediated conditional deletion of Rac1 in neurons combined with knockout of neuron-specific Rac3 to study the role of these GTPases in neural development. We found that lack of both genes causes motor behavioral defects, epilepsy, and premature death of mice. Deletion of either GTPase does not produce evident phenotypes. Double-knockout mice show specific defects in the development of the hippocampus. Selective impairment of the dorsal hilus of double-knockout animals is associated with alteration in the formation of the hippocampal circuitry. Axonal pathways to and from the dorsal hilus are affected because of the deficit of hilar mossy cells. Moreover, analysis of Rac function in hippocampal cultures shows that spine formation is strongly hampered only in neurons lacking both Rac proteins. These findings show for the first time that both Rac1 and Rac3 are important for the development of the nervous system, wherein they play complementary roles during late stages of neuronal and brain development.

    Funded by: Medical Research Council: MC_U117527252; Telethon: GGP05051

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2009;23;5;1347-57

  • Synaptic NMDA receptor activation stimulates alpha-secretase amyloid precursor protein processing and inhibits amyloid-beta production.

    Hoey SE, Williams RJ and Perkinton MS

    King's College London, Wolfson Centre for Age-Related Diseases, London, United Kingdom.

    Altered amyloid precursor protein (APP) processing leading to increased production and oligomerization of Abeta may contribute to Alzheimer's disease (AD). Understanding how APP processing is regulated under physiological conditions may provide new insights into AD pathogenesis. Recent reports demonstrate that excitatory neural activity regulates APP metabolism and Abeta levels, although understanding of the molecular mechanisms involved is incomplete. We have investigated whether NMDA receptor activity regulates APP metabolism in primary cultured cortical neurons. We report that a pool of APP is localized to the postsynaptic compartment in cortical neurons and observed partial overlap of APP with both NR1 and PSD-95. NMDA receptor stimulation increased nonamyloidogenic alpha-secretase-mediated APP processing, as measured by a 2.5-fold increase in cellular alpha-C-terminal fragment (C83) levels after glutamate or NMDA treatment. This increase was blocked by the NMDA receptor antagonists d-AP5 and MK801 but not by the AMPA receptor antagonist CNQX or the L-type calcium channel blocker nifedipine, was prevented by chelation of extracellular calcium, and was blocked by the alpha-secretase inhibitor TAPI-1. Cotreatment of cortical neurons with bicuculline and 4-AP, which stimulates glutamate release and activates synaptic NMDA receptors, evoked an MK801-sensitive increase in C83 levels. Furthermore, NMDA receptor stimulation caused a twofold increase in the amount of soluble APP detected in the neuronal culture medium. Finally, NMDA receptor activity inhibited both Abeta1-40 release and Gal4-dependent luciferase activity induced by beta-gamma-secretase-mediated cleavage of an APP-Gal4 fusion protein. Altogether, these data suggest that calcium influx through synaptic NMDA receptors promotes nonamyloidogenic alpha-secretase-mediated APP processing.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;14;4442-60

  • Splicing variations in the ligand-binding domain of ApoER2 results in functional differences in the binding properties to Reelin.

    Hibi T, Mizutani M, Baba A and Hattori M

    Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.

    Reelin plays critical roles in brain formation by binding to apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor. Several isoforms and fragments of Reelin are generated by alternative splicing and proteolytic cleavage. In addition, two splice variants of ApoER2 have been recognized, namely, LA1237 and LA12378, that differ in the number of ligand-binding type A (LA) repeats. Here, we quantitatively investigated the affinity between various isoforms/fragments of Reelin and the ApoER2 splice variants. ApoER2-LA1237 bound rather strongly to the Reelin central fragment than to the fragment bearing Reelin repeat 8 (RR8). ApoER2-LA12378 bound comparably to all Reelin fragments without the C-terminal region. These findings suggest that LA8 of ApoER2 and RR8 interfere with the interaction between the Reelin central fragment and ApoER2. Using a monoclonal antibody that only recognizes ApoER2-LA12378, we found that this variant of ApoER2 was expressed in the cerebral cortical wall and in the internal granule cells of the cerebellum during development. Primary-cultured cortical neurons did not express ApoER2-LA12378, and the extent of signal activation by Reelin fragments was well correlated with their affinity for ApoER2-LA1237. Therefore, proteolytic cleavage of Reelin and alternative splicing of ApoER2 may be involved in the fine regulation of Reelin signaling.

    Neuroscience research 2009;63;4;251-8

  • The tumor suppressor Pml regulates cell fate in the developing neocortex.

    Regad T, Bellodi C, Nicotera P and Salomoni P

    Medicial Research Council Toxicology Unit, University of Leicester, Box 138 Lancaster Road, Leicester LE1 7JL, UK.

    The control of cell fate in neural progenitor cells is critical for nervous system development. Nevertheless, the processes involved are only partially known. We found that the expression of the tumor suppressor Pml was restricted to neural progenitor cells (NPCs) in the developing neocortex of the mouse. Notably, in Pml(-/-) cortices, the overall number of proliferating NPCs was increased and transition between the two major progenitor types, radial glial cells and basal progenitors, was impaired. This in turn resulted in reduced differentiation and an overall decrease in the thickness of the cortex wall. In NPCs, Pml regulated the subcellular distribution of the retinoblastoma protein (pRb) and the protein phosphatase 1alpha, triggering pRb dephosphorylation. Together, these findings reveal an unexpected role of Pml in controlling the function of NPCs in the CNS.

    Funded by: Medical Research Council: MC_U132670601

    Nature neuroscience 2009;12;2;132-40

  • AMP-activated protein kinase phosphorylates retinoblastoma protein to control mammalian brain development.

    Dasgupta B and Milbrandt J

    Department of Pathology, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO 63110, USA.

    AMP-activated protein kinase (AMPK) is an evolutionarily conserved metabolic sensor that responds to alterations in cellular energy levels to maintain energy balance. While its role in metabolic homeostasis is well documented, its role in mammalian development is less clear. Here we demonstrate that mutant mice lacking the regulatory AMPK beta1 subunit have profound brain abnormalities. The beta1(-/-) mice show atrophy of the dentate gyrus and cerebellum, and severe loss of neurons, oligodendrocytes, and myelination throughout the central nervous system. These abnormalities stem from reduced AMPK activity, with ensuing cell cycle defects in neural stem and progenitor cells (NPCs). The beta1(-/-) NPC deficits result from hypophosphorylation of the retinoblastoma protein (Rb), which is directly phosphorylated by AMPK at Ser(804). The AMPK-Rb axis is utilized by both growth factors and energy restriction to increase NPC growth. Our results reveal that AMPK integrates growth factor signaling with cell cycle control to regulate brain development.

    Funded by: NIA NIH HHS: AG13730, R01 AG013730, R01 AG013730-11, R01 AG013730-12, R01 AG013730-13; NINDS NIH HHS: NS040745, NS057105, P30 NS057105, R01 NS040745, R01 NS040745-07, R01 NS040745-08, R01 NS040745-09

    Developmental cell 2009;16;2;256-70

  • Reelin stabilizes the actin cytoskeleton of neuronal processes by inducing n-cofilin phosphorylation at serine3.

    Chai X, Förster E, Zhao S, Bock HH and Frotscher M

    Institut für Anatomie und Zellbiologie, Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.

    The extracellular matrix protein Reelin, secreted by Cajal-Retzius cells in the marginal zone of the cortex, controls the radial migration of cortical neurons. Reelin signaling involves the lipoprotein receptors apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VLDLR), the adapter protein Disabled1 (Dab1), and phosphatidylinositol-3-kinase (PI3K). Eventually, Reelin signaling acts on the cytoskeleton; however, these effects on cytoskeletal organization have remained elusive. In Reelin-deficient mutant mice, most cortical neurons are unable to migrate to their destinations, suggesting a role for Reelin signaling in the dynamic cytoskeletal reorganization that is required for neurons to migrate. Here, we show that Reelin signaling leads to serine3 phosphorylation of n-cofilin, an actin-depolymerizing protein that promotes the disassembly of F-actin. Phosphorylation at serine3 renders n-cofilin unable to depolymerize F-actin, thereby stabilizing the cytoskeleton. We provide evidence for ApoER2, Dab1, Src family kinases (SFKs), and PI3K to be involved in n-cofilin serine3 phosphorylation. Phosphorylation of n-cofilin takes place in the leading processes of migrating neurons as they approach the Reelin-containing marginal zone. Immunostaining for phospho-cofilin in dissociated reeler neurons is significantly increased after incubation in Reelin-containing medium compared with control medium. In a stripe choice assay, neuronal processes are stable on Reelin-coated stripes but grow on control stripes by forming lamellipodia. These novel findings suggest that Reelin-induced stabilization of neuronal processes anchors them to the marginal zone which appears to be required for the directional migration process.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2009;29;1;288-99

  • Expression of Disabled 1 suppresses astroglial differentiation in neural stem cells.

    Kwon IS, Cho SK, Kim MJ, Tsai MJ, Mitsuda N, Suh-Kim H and Lee YD

    Department of Anatomy, School of Medicine, Ajou University, Suwon, 443-749, South Korea.

    Disabled 1 (Dab1), a cytoplasmic adaptor protein expressed predominantly in the CNS, transduces a Reelin-initiated signaling that controls neuronal migration and positioning during brain development. To determine the role of Dab1 in neural stem cell (NSC) differentiation, we established a culture of neurospheres derived from the embryonic forebrain of the Dab1(-/-) mice, yotari. Differentiating Dab1(-/-) neurospheres exhibited a higher expression of GFAP, an astrocytic marker, at the expense of neuronal markers. Under Dab1-deficient condition, the expression of NeuroD, a transcription factor for neuronal differentiation, was decreased and the JAK-STAT pathway was evidently increased during differentiation of NSC, suggesting the possible involvement of Dab1 in astrocyte differentiation via JAK-STAT pathway. Notably, expression of neural and glial markers and the level of JAK-STAT signaling molecules were not changed in differentiating NSC by Reelin treatment, indicating that differentiation of NSC is Reelin-independent. Immunohistochemical analyses showed a decrease in the number of neurons and an increase in the number of GFAP-positive cells in developing yotari brains. Our results suggest that Dab1 participates in the differentiation of NSCs into a specific cell lineage, thereby maintaining a balance between neurogenesis and gliogenesis.

    Funded by: NICHD NIH HHS: R37 HD017379, R37 HD017379-16, R37 HD017379-17, R37 HD017379-18, R37 HD017379-19, R37 HD017379-20, R37 HD017379-21

    Molecular and cellular neurosciences 2009;40;1;50-61

  • A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart.

    Kalsotra A, Xiao X, Ward AJ, Castle JC, Johnson JM, Burge CB and Cooper TA

    Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.

    From a large-scale screen using splicing microarrays and RT-PCR, we identified 63 alternative splicing (AS) events that are coordinated in 3 distinct temporal patterns during mouse heart development. More than half of these splicing transitions are evolutionarily conserved between mouse and chicken. Computational analysis of the introns flanking these splicing events identified enriched and conserved motifs including binding sites for CUGBP and ETR-3-like factors (CELF), muscleblind-like (MBNL) and Fox proteins. We show that CELF proteins are down-regulated >10-fold during heart development, and MBNL1 protein is concomitantly up-regulated nearly 4-fold. Using transgenic and knockout mice, we show that reproducing the embryonic expression patterns for CUGBP1 and MBNL1 in adult heart induces the embryonic splicing patterns for more than half of the developmentally regulated AS transitions. These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development.

    Funded by: NHGRI NIH HHS: HG002439, R01 HG002439; NHLBI NIH HHS: R01 HL045565, R01HL45565; NIGMS NIH HHS: R01 GM076493, R01GM076493

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;51;20333-8

  • Wnts acting through canonical and noncanonical signaling pathways exert opposite effects on hippocampal synapse formation.

    Davis EK, Zou Y and Ghosh A

    Division of Biological Sciences, Neurobiology Section, UCSD, La Jolla, CA 92093-0366, USA. edavis@ucsd.edu

    Background: Wnt proteins comprise a large class of signaling molecules that regulate a variety of developmental processes, including synapse formation. Previous studies have shown Wnts to be involved in both the induction and prevention of synapses in a number of different organisms. However, it is not clear whether the influence of Wnts on synapses is a result of Wnts' behavior in different organisms or differences in the activity of different Wnt ligands.

    Results: We used in situ hybridization to show that several Wnt ligands (Wnt3, Wnt5a, Wnt7a, and Wnt7b) and their receptors, Frizzled, are expressed in the developing hippocampus during the period of synapse formation in rodents. We used recombinant Wnt protein or Wnt conditioned media to explore the effects of Wnts on synapses in hippocampal cultures. We found that Wnt7a and Wnt7b activate canonical signaling, whereas Wnt5a activates a noncanonical pathway. The activation of the canonical pathway, either through pathway manipulations or through Wnt stimulation, increases presynaptic inputs. In contrast, exposure to Wnt5a, which activates a noncanonical signaling pathway, decreases the number of presynaptic terminals.

    Conclusion: Our observations suggest that the pro- and antisynaptogenic effects of Wnt proteins are associated with the activation of the canonical and noncanonical Wnt signaling pathways.

    Funded by: NINDS NIH HHS: R01 NS047484

    Neural development 2008;3;32

  • Proteomics analysis identifies phosphorylation-dependent alpha-synuclein protein interactions.

    McFarland MA, Ellis CE, Markey SP and Nussbaum RL

    National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20891, USA.

    Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.

    Funded by: Intramural NIH HHS; NIMH NIH HHS: Z01 MH000279

    Molecular & cellular proteomics : MCP 2008;7;11;2123-37

  • SOX5 postmitotically regulates migration, postmigratory differentiation, and projections of subplate and deep-layer neocortical neurons.

    Kwan KY, Lam MM, Krsnik Z, Kawasawa YI, Lefebvre V and Sestan N

    Department of Neurobiology and Kavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06520, USA.

    Neocortical projection neurons exhibit layer-specific molecular profiles and axonal connections. Here we show that the molecular identities of early-born subplate and deep-layer neurons are not acquired solely during generation or shortly thereafter but undergo progressive postmitotic refinement mediated by SOX5. Fezf2 and Bcl11b, transiently expressed in all subtypes of newly postmigratory early-born neurons, are subsequently downregulated in layer 6 and subplate neurons, thereby establishing their layer 5-enriched postnatal patterns. In Sox5-null mice, this downregulation is disrupted, and layer 6 and subplate neurons maintain an immature differentiation state, abnormally expressing these genes postnatally. Consistent with this disruption, SOX5 binds and represses a conserved enhancer near Fezf2. The Sox5-null neocortex exhibits failed preplate partition and laminar inversion of early-born neurons, loss of layer 5 subcerebral axons, and misrouting of subplate and layer 6 corticothalamic axons to the hypothalamus. Thus, SOX5 postmitotically regulates the migration, postmigratory differentiation, and subcortical projections of subplate and deep-layer neurons.

    Funded by: NINDS NIH HHS: NS054273, R01 NS054273

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;41;16021-6

  • The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone.

    Arnold SJ, Huang GJ, Cheung AF, Era T, Nishikawa S, Bikoff EK, Molnár Z, Robertson EJ and Groszer M

    Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.

    The embryonic subventricular zone (SVZ) is a critical site for generating cortical projection neurons; however, molecular mechanisms regulating neurogenesis specifically in the SVZ are largely unknown. The transcription factor Eomes/Tbr2 is transiently expressed in cortical SVZ progenitor cells. Here we demonstrate that conditional inactivation of Tbr2 during early brain development causes microcephaly and severe behavioral deficits. In Tbr2 mutants the number of SVZ progenitor cells is reduced and the differentiation of upper cortical layer neurons is disturbed. Neurogenesis in the adult dentate gyrus but not the subependymal zone is abolished. These studies establish Tbr2 as a key regulator of neurogenesis in the SVZ.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/F003285/1; Medical Research Council: G0300200; Wellcome Trust: 059312

    Genes & development 2008;22;18;2479-84

  • Expression of liver X receptor beta is essential for formation of superficial cortical layers and migration of later-born neurons.

    Fan X, Kim HJ, Bouton D, Warner M and Gustafsson JA

    Division of Medical Nutrition, Department of Biosciences and Nutrition, Karolinska Institutet, Novum, S-141 86 Stockholm, Sweden.

    Liver X receptor (LXR) beta regulates cholesterol levels in the brain and is essential for maintenance of motor neurons in the spinal cord and dopaminergic neurons in the substantia nigra. Here, we have examined the expression pattern of LXRbeta protein in the cerebral cortex and looked for defects in cortical development in LXRbeta knockout (LXRbeta(-/-)) mice. LXRbeta protein was widely expressed in the mouse brain at later embryonic stages, and the expression pattern in the cerebral cortex was developmentally regulated. In normal postnatal mice, LXRbeta was localized mainly in the upper layers of the cerebral cortex. In LXRbeta(-/-) mice layers II and III were thinner with fewer neurons. Layer I was slightly thicker, whereas layers IV-VI were essentially normal. Consistent with this finding, Brn2 and NeuN expression were decreased in the upper layers in the LXRbeta(-/-) neonatal cortex. The number of S-phase progenitor cells in the cortex between embryonic day (E) 12.5 to E16.5, was similar in WT and LXRbeta(-/-) littermates but BrdU birth dating revealed that late-generated neurons labeled by BrdU injections administered at E14.5 or E16.5, and destined to cortical layers II/III, were disorganized and failed to migrate. The defect in migration appears to be caused by a reduction in the number of vertical processes emanating from the radial glia. These processes are the architectural guides for later-born migrating neurons. Taken together, these findings suggest that LXRbeta expression in the cerebral cortex is involved in cortex lamination and is essential for the migration of late-generated neocortical neurons.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;36;13445-50

  • Mtap2 is a constituent of the protein network that regulates twik-related K+ channel expression and trafficking.

    Sandoz G, Tardy MP, Thümmler S, Feliciangeli S, Lazdunski M and Lesage F

    Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6097, Institut Paul Hamel, 06560 Valbonne Sophia-Antipolis, France.

    Twik-related K+ (TREK) channels produce background currents that regulate cell excitability. In vivo, TREK-1 is involved in neuronal processes including neuroprotection against ischemia, general anesthesia, pain perception, and mood. Recently, we demonstrated that A-kinase anchoring protein AKAP150 binds to a major regulatory domain of TREK-1, promoting drastic changes in channel regulation by polyunsaturated fatty acids, pH, and stretch, and by G-protein-coupled receptors to neurotransmitters and hormones. Here, we show that the microtubule-associated protein Mtap2 is another constituent of native TREK channels in the brain. Mtap2 binding to TREK-1 and TREK-2 does not affect directly channel properties but enhances channel surface expression and current density. This effect relies on Mtap2 binding to microtubules. Mtap2 and AKAP150 interacting sites in TREK-1 are distinct and both proteins can dock simultaneously. Their effects on TREK-1 surface expression and activation are cumulative. In neurons, the three proteins are simultaneously detected in postsynaptic dense bodies. AKAP150 and Mtap2 put TREK channels at the center of a complex protein network that finely tunes channel trafficking, addressing, and regulation.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;34;8545-52

  • Consequence of the loss of Sox2 in the developing brain of the mouse.

    Miyagi S, Masui S, Niwa H, Saito T, Shimazaki T, Okano H, Nishimoto M, Muramatsu M, Iwama A and Okuda A

    Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan.

    The transcription factor Sox2 is expressed at high levels in neural stem and progenitor cells. Here, we inactivated Sox2 specifically in the developing brain by using Cre-loxP system. Although mutant animals did not survive after birth, analysis of late gestation embryos revealed that loss of Sox2 causes enlargement of the lateral ventricles and a decrease in the number of neurosphere-forming cells. However, although their neurogenic potential is attenuated, Sox2-deficient neural stem cells retain their multipotency and self-renewal capacity. We found that expression level of Sox3 is elevated in Sox2 null developing brain, probably mitigating the effects of loss of Sox2.

    FEBS letters 2008;582;18;2811-5

  • Hedgehog signaling is involved in development of the neocortex.

    Komada M, Saitsu H, Kinboshi M, Miura T, Shiota K and Ishibashi M

    Departmant of Anatomy and Developmental Biology, Kyoto University Graduate School of Medicine, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.

    Sonic hedgehog (Shh) function is essential for patterning and cell fate specification, particularly in ventral regions of the central nervous system. It is also a crucial mitogen for cerebellar granule neuron precursors and is important in maintenance of the stem cell niche in the postnatal telencephalon. Although it has been reported that Shh is expressed in the developing dorsal telencephalon, functions of Shh in this region are unclear, and detailed characterization of Shh mRNA transcripts in situ has not been demonstrated. To clarify the roles of Shh signaling in dorsal pallium (neocortex primordium) development, we have knocked out the Shh and Smo genes specifically in the early developing dorsal telencephalon by using Emx1cre mice. The mutants showed a smaller dorsal telencephalon at E18.5, which was caused by cell cycle kinetics defects of the neural progenitor/stem cells. The cell cycle length of the progenitor/stem cells was prolonged, and the number of cycle-exiting cells and neurogenesis were decreased. Birth-date analysis revealed abnormal positioning of neurons in the mutants. The characteristics of the subventricular zone, ventricular zone and subplate cells were also affected. Weak immunoreactivity of Shh was detected in the dorsal telencephalon of wild types. Reduced Shh immunoreactivity in mutant dorsal telencephalons supports the above phenotypes. Our data indicate that Shh signaling plays an important role in development of the neocortex.

    Development (Cambridge, England) 2008;135;16;2717-27

  • Structural requirement of TAG-1 for retinal ganglion cell axons and myelin in the mouse optic nerve.

    Chatzopoulou E, Miguez A, Savvaki M, Levasseur G, Muzerelle A, Muriel MP, Goureau O, Watanabe K, Goutebroze L, Gaspar P, Zalc B, Karagogeos D and Thomas JL

    Institut National de la Santé et de la Recherche Médicale, Unité 711, Université Pierre et Marie Curie, Faculté de Médecine, Hôpital de la Salpêtrière, Institut Fédératif de Recherche 70, F-75013 Paris, France.

    White matter axons organize into fascicles that grow over long distances and traverse very diverse environments. The molecular mechanisms preserving this structure of white matter axonal tracts are not well known. Here, we used the optic nerve as a model and investigated the role of TAG-1, a cell adhesion molecule expressed by retinal axons. TAG-1 was first expressed in the embryonic retinal ganglion cells (RGCs) and later in the postnatal myelin-forming cells in the optic nerve. We describe the consequences of genetic loss of Tag-1 on the developing and adult retinogeniculate tract. Tag-1-null embryos display anomalies in the caliber of RGC axons, associated with an abnormal organization of the astroglial network in the optic nerve. The contralateral projections in the lateral geniculate nucleus are expanded postnatally. In the adult, Tag-1-null mice show a loss of RGC axons, with persistent abnormalities of axonal caliber and additional cytoskeleton and myelination defects. Therefore, TAG-1 is an essential regulator of the structure of RGC axons and their surrounding glial cells in the optic nerve.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;30;7624-36

  • Transcription factor MEF2C influences neural stem/progenitor cell differentiation and maturation in vivo.

    Li H, Radford JC, Ragusa MJ, Shea KL, McKercher SR, Zaremba JD, Soussou W, Nie Z, Kang YJ, Nakanishi N, Okamoto S, Roberts AJ, Schwarz JJ and Lipton SA

    Center for Neuroscience, Aging, and Stem Cell Research, Burnham Institute for Medical Research, La Jolla, CA 92037, USA.

    Emerging evidence suggests that myocyte enhancer factor 2 (MEF2) transcription factors act as effectors of neurogenesis in the brain, with MEF2C the predominant isoform in developing cerebrocortex. Here, we show that conditional knockout of Mef2c in nestin-expressing neural stem/progenitor cells (NSCs) impaired neuronal differentiation in vivo, resulting in aberrant compaction and smaller somal size. NSC proliferation and survival were not affected. Conditional null mice surviving to adulthood manifested more immature electrophysiological network properties and severe behavioral deficits reminiscent of Rett syndrome, an autism-related disorder. Our data support a crucial role for MEF2C in programming early neuronal differentiation and proper distribution within the layers of the neocortex.

    Funded by: NHLBI NIH HHS: R01 HL060853; NICHD NIH HHS: P01 HD029587, P01 HD29587; NINDS NIH HHS: P30 NS057096, R01 NS044326

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;27;9397-402

  • BMP signaling through BMPRIA in astrocytes is essential for proper cerebral angiogenesis and formation of the blood-brain-barrier.

    Araya R, Kudo M, Kawano M, Ishii K, Hashikawa T, Iwasato T, Itohara S, Terasaki T, Oohira A, Mishina Y and Yamada M

    Yamada Research Unit, RIKEN Brain Science Institute, Saitama 351-0198, Japan.

    Bone morphogenetic protein (BMP) signaling is involved in differentiation of neural precursor cells into astrocytes, but its contribution to angiogenesis is not well characterized. This study examines the role of BMP signaling through BMP type IA receptor (BMPRIA) in early neural development using a conditional knockout mouse model, in which Bmpr1a is selectively disrupted in telencephalic neural stem cells. The conditional mutant mice show a significant increase in the number of cerebral blood vessels and the level of vascular endothelial growth factor (VEGF) is significantly upregulated in the mutant astrocytes. The mutant mice also show leakage of immunoglobulin around cerebral microvessels in neonatal mice, suggesting a defect in formation of the blood-brain-barrier. In addition, astrocytic endfeet fail to encircle cortical blood vessels in the mutant mice. These results suggest that BMPRIA signaling in astrocytes regulates the expression of VEGF for proper cerebrovascular angiogenesis and has a role on in the formation of the blood-brain-barrier.

    Funded by: Intramural NIH HHS: Z01 ES071003-10; NIEHS NIH HHS: ES071003-10, Z01 ES071003

    Molecular and cellular neurosciences 2008;38;3;417-30

  • Regulation of neural progenitor cell motility by ceramide and potential implications for mouse brain development.

    Wang G, Krishnamurthy K, Chiang YW, Dasgupta S and Bieberich E

    Program in Developmental Neurobiology, Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia, Augusta, Georgia, USA.

    We provide evidence that the sphingolipid ceramide, in addition to its pro-apoptotic function, regulates neural progenitor (NP) motility in vitro and brain development in vivo. Ceramide (N-palmitoyl d-erythro sphingosine and N-oleoyl d-erythro sphingosine) and the ceramide analog N-oleoyl serinol (S18) stimulate migration of NPs in scratch (wounding) migration assays. Sphingolipid depletion by inhibition of de novo ceramide biosynthesis, or ceramide inactivation using an anti-ceramide antibody, obliterates NP motility, which is restored by ceramide or S18. These results suggest that ceramide is crucial for NP motility. Wounding of the NP monolayer activates neutral sphingomyelinase indicating that ceramide is generated from sphingomyelin. In membrane processes, ceramide is co-distributed with its binding partner atypical protein kinase C zeta/lambda (aPKC), and Cdc42, alpha/beta-tubulin, and beta-catenin, three proteins involved in aPKC-dependent regulation of cell polarity and motility. Sphingolipid depletion by myriocin prevents membrane translocation of aPKC and Cdc42, which is restored by ceramide or S18. These results suggest that ceramide-mediated membrane association of aPKC/Cdc42 is important for NP motility. In vivo, sphingolipid depletion leads to ectopic localization of mitotic or post-mitotic neural cells in the embryonic brain, while S18 restores the normal brain organization. In summary, our study provides novel evidence that ceramide is critical for NP motility and polarity in vitro and in vivo.

    Funded by: NINDS NIH HHS: R01 NS046835, R01NS046835

    Journal of neurochemistry 2008;106;2;718-33

  • Lamination of the cerebral cortex is disturbed in Gli3 mutant mice.

    Friedrichs M, Larralde O, Skutella T and Theil T

    Institute for Animal Developmental and Molecular Biology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany.

    The layered organization of the cerebral cortex develops in an inside-out pattern, a process which is controlled by the secreted protein reelin. Here we report on cortical lamination in the Gli3 hypomorphic mouse mutant Xt(J)/Pdn which lacks the cortical hem, a major source of reelin(+) Cajal Retzius cells in the cerebral cortex. Unlike other previously described mouse mutants with hem defects, cortical lamination is disturbed in Xt(J)/Pdn animals. Surprisingly, these layering defects occur in the presence of reelin(+) cells which are probably derived from an expanded Dbx1(+) progenitor pool in the mutant. However, while these reelin(+) neurons and also Calretinin(+) cells are initially evenly distributed over the cortical surface they form clusters later during development suggesting a novel role for Gli3 in maintaining the proper arrangement of these cells in the marginal zone. Moreover, the radial glial network is disturbed in the regions of these clusters. In addition, the differentiation of subplate cells is affected which serve as a framework for developing a properly laminated cortex.

    Developmental biology 2008;318;1;203-14

  • C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment.

    Voss AK, Britto JM, Dixon MP, Sheikh BN, Collin C, Tan SS and Thomas T

    Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3050, Australia. avoss@wehi.edu.au

    Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.

    Development (Cambridge, England) 2008;135;12;2139-49

  • Expression of TGF-betas in the embryonic nervous system: analysis of interbalance between isoforms.

    Mecha M, Rabadán MA, Peña-Melián A, Valencia M, Mondéjar T and Blanco MJ

    Departamento de Anatomía y Embriología Humana I, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.

    Transforming growth factor-beta (TGF-beta) is a family of growth factors with essential and multiple roles during embryonic development. In mammals, three isoforms (TGF-beta1, TGF-beta2, TGF-beta3) have been described. In the nervous system, the presence of TGF-beta1 has remained undetectable in other structures than meninges and choroids plexus, while TGF-beta2 and TGF-beta3 were considered as the neural members of the family. In the present study, we have analysed the expression pattern of the three isoforms in the neural tube, brain, and spinal cord during development in both mouse and chicken. The data reveal specific patterns for each isoform. This work also shows that both TGF-beta1 and TGF-beta3 are expressed in neural crest cells. In addition, we demonstrate the existence of interbalance between TGF-beta1 and TGF-beta3 with possible functional implications, which, together with the expression of TGF-beta1 in the CNS, represents one of the most important contributions of this work.

    Developmental dynamics : an official publication of the American Association of Anatomists 2008;237;6;1709-17

  • Nuclear pore composition regulates neural stem/progenitor cell differentiation in the mouse embryo.

    Lupu F, Alves A, Anderson K, Doye V and Lacy E

    Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.

    Serving as the primary conduit for communication between the nucleus and the cytoplasm, nuclear pore complexes (NPCs) impact nearly every cellular process. The extent to which NPC composition varies and the functional significance of such variation in mammalian development has not been investigated. Here we report that a null allele of mouse nucleoporin Nup133, a structural subunit of the NPC, disrupts neural differentiation. We find that expression of Nup133 is cell type and developmental stage restricted, with prominent expression in dividing progenitors. Nup133-deficient epiblast and ES cells abnormally maintain features of pluripotency and differentiate inefficiently along the neural lineage. Neural progenitors achieve correct spatial patterning in mutant embryos; however, they are impaired in generating terminally differentiated neurons, as are Nup133 null ES cells. Our results reveal a role for structural nucleoporins in coordinating cell differentiation events in the developing embryo.

    Funded by: NCI NIH HHS: P30 CA008748, P30 CA008748-329039, P30 CA008748-339039, P30 CA008748-349039, P30 CA008748-34S29039, P30 CA008748-359039, P30 CA008748-35S19039, P30 CA008748-369039, P30 CA008748-379039, P30 CA008748-389039; NICHD NIH HHS: 1U01 HD043478, F32 HD042355, F32 HD042355-01, F32 HD042355-02, F32 HD042355-03, HD42355, U01 HD043478, U01 HD043478-01, U01 HD043478-02, U01 HD043478-03, U01 HD043478-04, U01 HD043478-05; NIGMS NIH HHS: R01 GM058726, R01 GM058726-01, R01 GM058726-02, R01 GM058726-03, R01 GM058726-04, R01 GM058726-05, R01 GM058726-06, R01 GM058726-07, R01 GM58726

    Developmental cell 2008;14;6;831-42

  • Regulation of apoptosis and neurite extension by FKBP38 is required for neural tube formation in the mouse.

    Shirane M, Ogawa M, Motoyama J and Nakayama KI

    Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.

    FKBP38 (also known as FKBP8) is a transmembrane chaperone protein that inhibits apoptosis by recruiting the anti-apoptotic proteins Bcl-2 and Bcl-x(L) to mitochondria. We have now generated mice harboring a loss-of-function mutation in Fkbp38. The Fkbp38(-/-) mice die soon after birth manifesting defects in neural tube closure in the thoraco-lumbar-sacral region (spina bifida) as well as skeletal defects including scoliosis, rib deformities, club foot and curled tail. The neuroepithelium is disorganized and that formation of dorsal root ganglia is defective in Fkbp38(-/-) embryos, likely as a result of an increased frequency of apoptosis and aberrant migration of neuronal cells. Furthermore, the extension of nerve fibers in the spinal cord is abnormal in the mutant embryos. To explore the mechanisms underlying these characteristics, we screened for proteins that interact with FKBP38 in the yeast two-hybrid system and thereby identified protrudin, a protein that promotes process formation by regulating membrane trafficking. Protrudin was found to be hyperphosphorylated in the brain of Fkbp38(-/-) mice, suggesting that FKBP38 regulates protrudin-dependent membrane recycling and neurite outgrowth. Together, our findings suggest that FKBP38 is required for neuroectodermal organization during neural tube formation as a result of its anti-apoptotic activity and regulation of neurite extension.

    Genes to cells : devoted to molecular & cellular mechanisms 2008;13;6;635-51

  • The protein dendrite arborization and synapse maturation 1 (Dasm-1) is dispensable for dendrite arborization.

    Mishra A, Knerr B, Paixão S, Kramer ER and Klein R

    Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, 82152 Munich-Martinsried, Germany.

    The development of a highly branched dendritic tree is essential for the establishment of functional neuronal connections. The evolutionarily conserved immunoglobulin superfamily member, the protein dendrite arborization and synapse maturation 1 (Dasm-1) is thought to play a critical role in dendrite formation of dissociated hippocampal neurons. RNA interference-mediated Dasm-1 knockdown was previously shown to impair dendrite, but not axonal, outgrowth and branching (S. H. Shi, D. N. Cox, D. Wang, L. Y. Jan, and Y. N. Jan, Proc. Natl. Acad. Sci. USA 101:13341-13345, 2004). Here, we report the generation and analysis of Dasm-1 null mice. We find that genetic ablation of Dasm-1 does not interfere with hippocampal dendrite growth and branching in vitro and in vivo. Moreover, the absence of Dasm-1 does not affect the modulation of dendritic outgrowth induced by brain-derived neurotrophic factor. Importantly, the previously observed impairment in dendrite growth after Dasm-1 knockdown is also observed when the Dasm-1 knockdown is performed in cultured hippocampal neurons from Dasm-1 null mice. These findings indicate that the dendrite arborization phenotype was caused by off-target effects and that Dasm-1 is dispensable for hippocampal dendrite arborization.

    Molecular and cellular biology 2008;28;8;2782-91

  • Receptor protein tyrosine phosphatases are expressed by cycling retinal progenitor cells and involved in neuronal development of mouse retina.

    Horvat-Bröcker A, Reinhard J, Illes S, Paech T, Zoidl G, Harroch S, Distler C, Knyazev P, Ullrich A and Faissner A

    Department of Cell Morphology and molecular Neurobiology, Faculty of Biology, Ruhr-University-Bochum, Universitaetsstr. 150, 44780 Bochum, Germany.

    Receptor protein tyrosine phosphatases (RPTPs) appear to coordinate many aspects of neural development, including cell proliferation, migration and differentiation. Here we investigated potential roles of RPTPs in the developing mouse retina. Using a degenerate oligonucleotide-based reverse transcription polymerase chain reaction approach, we identified 11 different RPTPs in the retina at embryonic stage 13 (E13). Subsequently, the expression patterns of RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma in the retina from embryonic stages to adult were analyzed in detail using quantitative real-time-PCR, in situ hybridization, immunohistochemistry and Western blotting. At E13, all six RPTPs are expressed in actively cycling retinal progenitor cells and postmitotic newborn retinal neurons. With ongoing maturation, RPTPkappa, RPTPJ, RPTPRR, RPTPsigma, RPTPepsilon and RPTPgamma display a different spatiotemporal regulation of mRNAs and proteins in the pre- and postnatal retina. Finally, in adulthood these six RPTPs localize to distinct cellular compartments of multiple retinal neurons. Additional studies in RPTPgamma(-/-) and RPTPbeta/zeta(-/-) (also known as PTPRZ1, RPTPbeta or RPTPzeta) mice at postnatal stage P1 reveal no apparent differences in retinal laminar organization or in the expression pattern of specific retinal cell-type markers when compared with wild type. However, in RPTPbeta/zeta(-/-) retinas, immunoreactivity of vimentin, a marker of Müller glial cells, is selectively reduced and the morphology of vimentin-immunoreactive radial processes of Müller cells is considerably disturbed. Our results suggest distinct roles of RPTPs in cell proliferation and establishing phenotypes of different retinal cells during retinogenesis as well as later in the maintenance of mature retina.

    Neuroscience 2008;152;3;618-45

  • The spatio-temporal and subcellular expression of the candidate Down syndrome gene Mnb/Dyrk1A in the developing mouse brain suggests distinct sequential roles in neuronal development.

    Hämmerle B, Elizalde C and Tejedor FJ

    Instituto de Neurociencias CSIC-UMH, Universidad Miguel Hernandez-Campus de San Juan, 03550 San Juan, Alicante, Spain.

    It is widely accepted that the neurological alterations in Down syndrome (DS) are principally due to modifications in developmental processes. Accordingly, a large part of the research on DS in recent years has focused on chromosome 21 genes that influence brain development. MNB/DYRK1A is one of the genes on human chromosome 21 that has raised most interest, due to its relationship with the brain functions that are altered in DS. Although a number of interesting experimental mouse models for DS are being developed, we still know little about the expression of Mnb/Dyrk1A during mouse brain development. Here, we report that Mnb/Dyrk1A displays a rather dynamic spatio-temporal expression pattern during mouse central nervous system development. Our data indicate that Mnb/Dyrk1A is specifically expressed in four sequential developmental phases: transient expression in preneurogenic progenitors, cell cycle-regulated expression in neurogenic progenitors, transient expression in recently born neurones, and persistent expression in late differentiating neurones. Our results also suggest that the subcellular localization of MNB/DYRK1A, including its translocation to the nucleus, is finely regulated. Thus, the MNB/DYRK1A protein kinase could be a key element in the molecular machinery that couples sequential events in neuronal development. This rich repertoire of potential functions in the developing central nervous system is suitable to be linked to the neurological alterations in DS through the use of mouse experimental models.

    The European journal of neuroscience 2008;27;5;1061-74

  • Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum.

    Arlotta P, Molyneaux BJ, Jabaudon D, Yoshida Y and Macklis JD

    Massachusetts General Hospital-Harvard Medical School Center for Nervous System Repair, Nayef Al-Rodhan Laboratories, Department of Neurosurgery, Boston, Massachusetts 02114, USA.

    Striatal medium spiny neurons (MSN) are critically involved in motor control, and their degeneration is a principal component of Huntington's disease. We find that the transcription factor Ctip2 (also known as Bcl11b) is central to MSN differentiation and striatal development. Within the striatum, it is expressed by all MSN, although it is excluded from essentially all striatal interneurons. In the absence of Ctip2, MSN do not fully differentiate, as demonstrated by dramatically reduced expression of a large number of MSN markers, including DARPP-32, FOXP1, Chrm4, Reelin, MOR1 (mu-opioid receptor 1), glutamate receptor 1, and Plexin-D1. Furthermore, MSN fail to aggregate into patches, resulting in severely disrupted patch-matrix organization within the striatum. Finally, heterotopic cellular aggregates invade the Ctip2-/- striatum, suggesting a failure by MSN to repel these cells in the absence of Ctip2. This is associated with abnormal dopaminergic innervation of the mutant striatum and dramatic changes in gene expression, including dysregulation of molecules involved in cellular repulsion. Together, these data indicate that Ctip2 is a critical regulator of MSN differentiation, striatal patch development, and the establishment of the cellular architecture of the striatum.

    Funded by: NINDS NIH HHS: NS45523, NS49553

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;3;622-32

  • Neural recognition molecules CHL1 and NB-3 regulate apical dendrite orientation in the neocortex via PTP alpha.

    Ye H, Tan YL, Ponniah S, Takeda Y, Wang SQ, Schachner M, Watanabe K, Pallen CJ and Xiao ZC

    Institute of Molecular and Cell Biology, Singapore, Singapore.

    Apical dendrites of pyramidal neurons in the neocortex have a stereotypic orientation that is important for neuronal function. Neural recognition molecule Close Homolog of L1 (CHL1) has been shown to regulate oriented growth of apical dendrites in the mouse caudal cortex. Here we show that CHL1 directly associates with NB-3, a member of the F3/contactin family of neural recognition molecules, and enhances its cell surface expression. Similar to CHL1, NB-3 exhibits high-caudal to low-rostral expression in the deep layer neurons of the neocortex. NB-3-deficient mice show abnormal apical dendrite projections of deep layer pyramidal neurons in the visual cortex. Both CHL1 and NB-3 interact with protein tyrosine phosphatase alpha (PTPalpha) and regulate its activity. Moreover, deep layer pyramidal neurons of PTPalpha-deficient mice develop misoriented, even inverted, apical dendrites. We propose a signaling complex in which PTPalpha mediates CHL1 and NB-3-regulated apical dendrite projection in the developing caudal cortex.

    The EMBO journal 2008;27;1;188-200

  • Neuronal distribution of spatial in the developing cerebellum and hippocampus and its somatodendritic association with the kinesin motor KIF17.

    Irla M, Saade M, Fernandez C, Chasson L, Victorero G, Dahmane N, Chazal G and Nguyen C

    INSERM-ERM206, laboratoire TAGC, Case 928, Parc Scientifique de Luminy, 13288 Marseille Cedex 9, France.

    We identified the Spatial (Stromal Protein Associated with Thymii and Lymph-node) gene from an adult thymus mouse library of cDNA clones. By RT-PCR, we reported that Spatial was highly expressed in restricted areas of the central nervous system. Here, we characterize the precise cellular localization of Spatial during mouse brain development in the cerebellum, hippocampus and cortex. Five different transcript isoforms have been described for Spatial and among those, only Spatial-epsilon and -beta present a tightly controlled expression. In the cerebellum, Spatial expression is detected in the external precursor granular layer and persists as these cells migrate and differentiate to form the internal granular layer. It is also expressed in differentiating Purkinje cells with a specific somatodendritic distribution. Spatial expression in the hippocampus is spatially and temporally regulated: it is first expressed in the CA3 field, then in CA1 and later in the dentate gyrus. Interestingly, Spatial-beta expression tightly overlaps with the beginning of neuronal differentiation in both structures. Using cultured hippocampal neurons, we show that Spatial also exhibits a somatodendritic distribution and it is concentrated in some synaptic regions. Moreover, the vesicle-like cellular distribution of Spatial protein in dendrites is similar to that described for the kinesin motor protein KIF17. Immunofluorescence analyses show that Spatial colocalizes with KIF17 in dendrites of hippocampal neurons in primary culture. Additionally, coimmunoprecipitation experiments of endogenous proteins from hippocampus confirmed that Spatial and KIF17 physically interact. These findings suggest that Spatial may play a role in neuronal morphogenesis and synaptic plasticity through its interaction with the kinesin motor KIF17 in dendrites.

    Experimental cell research 2007;313;20;4107-19

  • Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains.

    Ngamukote S, Yanagisawa M, Ariga T, Ando S and Yu RK

    Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia, USA.

    Glycosphingolipids (GSLs) and their sialic acid-containing derivatives, gangliosides, are important cellular components and are abundant in the nervous system. They are known to undergo dramatic changes during brain development. However, knowledge on the mechanisms underlying their qualitative and qualitative changes is still fragmentary. In this investigation, we have provided a detailed study on the developmental changes of the expression patterns of GSLs, GM3, GM1, GD3, GD1a, GD2, GD1b, GT1b, GQ1b, A2B5 antigens (c-series gangliosides such as GT3 and GQ1c), Chol-1alpha (GT1aalpha and GQ1balpha), glucosylceramide, galactosylceramide (O1 antigen), sulfatide (O4 antigen), stage-specific embryonic antigen-1 (Lewis x) glycolipids, and human natural killer-1 glycolipid (sulfoglucuronosyl paragloboside) in developing mouse brains [embryonic day 12 (E12) to adult]. In E12-E14 brains, GD3 was a predominant ganglioside. After E16, the concentrations of GD3 and GM3 markedly decreased, and the concentrations of a-series gangliosides, such as GD1a, increased. GT3, glucosylceramide, and stage-specific embryonic antigen-1 were expressed in embryonic brains. Human natural killer-1 glycolipid was expressed transiently in embryonic brains. On the other hand, Chol-1alpha, galactosylceramide, and sulfatide were exclusively found after birth. To provide a better understanding of the metabolic basis for these changes, we analyzed glycogene expression patterns in the developing brains and found that GSL expression is regulated primarily by glycosyltransferases, and not by glycosidases. In parallel studies using primary neural precursor cells in culture as a tool for studying developmental events, dramatic changes in ganglioside and glycosyltransferase gene expression were also detected in neurons induced to differentiate from neural precursor cells, including the expression of GD3, followed by up-regulation of complex a- and b-series gangliosides. These changes in cell culture systems resemble that occurring in brain. We conclude that the dramatic changes in GSL pattern and content can serve as useful markers in neural development and that these changes are regulated primarily at the level of glycosyltransferase gene expression.

    Journal of neurochemistry 2007;103;6;2327-41

  • Very-KIND, a KIND domain containing RasGEF, controls dendrite growth by linking Ras small GTPases and MAP2.

    Huang J, Furuya A and Furuichi T

    Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.

    The regulation of cytoskeletal components in the dendritic shaft core is critical for dendrite elongation and branching. Here, we report that a brain-specific Ras guanine nucleotide exchange factor (RasGEF) carrying two kinase non-catalytic C-lobe domains (KINDs), very-KIND (v-KIND), regulates microtubule-associated protein 2 (MAP2). v-KIND is expressed in developing mouse brain, predominantly in the cerebellar granule cells. v-KIND not only activates Ras small GTPases via the C-terminal RasGEF domain, but also specifically binds to MAP2 via the second KIND domain (KIND2), leading to threonine phosphorylation of MAP2. v-KIND overexpression suppresses dendritic extension and branching of hippocampal neurons and cerebellar granule cells, whereas knockdown of endogenous v-KIND expression promotes dendrite growth. These findings suggest that v-KIND mediates a signaling pathway that links Ras and MAP2 to control dendrite growth.

    The Journal of cell biology 2007;179;3;539-52

  • Targeted deletion of the mitogen-activated protein kinase kinase 4 gene in the nervous system causes severe brain developmental defects and premature death.

    Wang X, Nadarajah B, Robinson AC, McColl BW, Jin JW, Dajas-Bailador F, Boot-Handford RP and Tournier C

    Faculty of Life Sciences, Wellcome Trust Center for Cell-Matrix Research, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom.

    The c-Jun NH2-terminal protein kinase (JNK) is a mitogen-activated protein kinase (MAPK) involved in the regulation of various physiological processes. Its activity is increased upon phosphorylation by the MAPK kinases MKK4 and MKK7. The early embryonic death of mice lacking an mkk4 or mkk7 gene has provided genetic evidence that MKK4 and MKK7 have nonredundant functions in vivo. To elucidate the physiological role of MKK4, we generated a novel mouse model in which the mkk4 gene could be specifically deleted in the brain. At birth, the mutant mice were indistinguishable from their control littermates, but they stopped growing a few days later and died prematurely, displaying severe neurological defects. Decreased JNK activity in the absence of MKK4 correlated with impaired phosphorylation of a subset of physiologically relevant JNK substrates and with altered gene expression. These defects resulted in the misalignment of the Purkinje cells in the cerebellum and delayed radial migration in the cerebral cortex. Together, our data demonstrate for the first time that MKK4 is an essential activator of JNK required for the normal development of the brain.

    Funded by: Biotechnology and Biological Sciences Research Council: BBS/B/0224X; Medical Research Council: G0001285

    Molecular and cellular biology 2007;27;22;7935-46

  • Vitamin K induces osteoblast differentiation through pregnane X receptor-mediated transcriptional control of the Msx2 gene.

    Igarashi M, Yogiashi Y, Mihara M, Takada I, Kitagawa H and Kato S

    The Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

    Vitamin K is a fat-soluble vitamin that serves as a coenzyme for vitamin K-dependent carboxylase. Besides its canonical action, vitamin K binds to the steroid and xenobiotic receptor (SXR)/pregnane X receptor (PXR) and modulates gene transcription. To determine if the osteoprotective action of vitamin K is the result of the PXR/SXR pathway, we screened by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis the PXR/SXR target genes in an osteoblastic cell line (MC3T3-E1) treated with a vitamin K2 (menaquinone 4 [MK4]). Osteoblastic differentiation of MC3T3-E1 cells was induced by MK4. Msx2, an osteoblastogenic transcription factor, was identified as an MK4-induced gene. Functional analysis of the Msx2 gene promoter mapped a vitamin K-responsive element (PXR-responsive element [PXRE]) that was directly bound by a PXR/retinoid X receptor alpha heterodimer. In a chromatin immunoprecipitation analysis, PXR was recruited together with a coactivator, p300, to the PXRE in the Msx2 promoter. MK4-bound PXR cooperated with estrogen-bound estrogen receptor alpha to control transcription at the Msx2 promoter. Knockdown of either PXR or Msx2 attenuated the effect of MK4 on osteoblastic differentiation. Thus, the present study suggests that Msx2 is a target gene for PXR activated by vitamin K and suggests that the osteoprotective action of MK4 in the human mediates, at least in part, a genomic pathway of vitamin K signaling.

    Molecular and cellular biology 2007;27;22;7947-54

  • Neurofibromatosis-1 regulates neuronal and glial cell differentiation from neuroglial progenitors in vivo by both cAMP- and Ras-dependent mechanisms.

    Hegedus B, Dasgupta B, Shin JE, Emnett RJ, Hart-Mahon EK, Elghazi L, Bernal-Mizrachi E and Gutmann DH

    Department of Neurology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    Individuals with neurofibromatosis type 1 (NF1) develop abnormalities of both neuronal and glial cell lineages, suggesting that the NF1 protein neurofibromin is an essential regulator of neuroglial progenitor function. In this regard, Nf1-deficient embryonic telencephalic neurospheres exhibit increased self-renewal and prolonged survival as explants in vivo. Using a newly developed brain lipid binding protein (BLBP)-Cre mouse strain to study the role of neurofibromin in neural progenitor cell function in the intact animal, we now show that neuroglial progenitor Nf1 inactivation results in increased glial lineage proliferation and abnormal neuronal differentiation in vivo. Whereas the glial cell lineage abnormalities are recapitulated by activated Ras or Akt expression in vivo, the neuronal abnormalities were Ras- and Akt independent and reflected impaired cAMP generation in Nf1-deficient cells in vivo and in vitro. Together, these findings demonstrate that neurofibromin is required for normal glial and neuronal development involving separable Ras-dependent and cAMP-dependent mechanisms.

    Funded by: NCI NIH HHS: 1-UO1-CA84314; NIDDK NIH HHS: R03 DK068028, R03 DK068028-01

    Cell stem cell 2007;1;4;443-57

  • Deletion of Shp2 in the brain leads to defective proliferation and differentiation in neural stem cells and early postnatal lethality.

    Ke Y, Zhang EE, Hagihara K, Wu D, Pang Y, Klein R, Curran T, Ranscht B and Feng GS

    Burnham Institute for Medical Research, La Jolla, CA 92037, USA.

    The intracellular signaling controlling neural stem/progenitor cell (NSC) self-renewal and neuronal/glial differentiation is not fully understood. We show here that Shp2, an introcellular tyrosine phosphatase with two SH2 domains, plays a critical role in NSC activities. Conditional deletion of Shp2 in neural progenitor cells mediated by Nestin-Cre resulted in early postnatal lethality, impaired corticogenesis, and reduced proliferation of progenitor cells in the ventricular zone. In vitro analyses suggest that Shp2 mediates basic fibroblast growth factor signals in stimulating self-renewing proliferation of NSCs, partly through control of Bmi-1 expression. Furthermore, Shp2 regulates cell fate decisions, by promoting neurogenesis while suppressing astrogliogenesis, through reciprocal regulation of the Erk and Stat3 signaling pathways. Together, these results identify Shp2 as a critical signaling molecule in coordinated regulation of progenitor cell proliferation and neuronal/astroglial cell differentiation.

    Funded by: NCI NIH HHS: R01 CA078606, R01 CA78606; NIGMS NIH HHS: P20 GM075059, R01 GM053660, R01 GM53660

    Molecular and cellular biology 2007;27;19;6706-17

  • Involvement of the myelin-associated inhibitor Nogo-A in early cortical development and neuronal maturation.

    Mingorance-Le Meur A, Zheng B, Soriano E and del Río JA

    Department of Cell Biology, Cellular and Molecular Basis of Neurodegeneration and Neurorepair, Institute for Research in Biomedicine, University of Barcelona, Barcelona Science Park, Josep Samitier 1-5, 08028 Barcelona, Spain. alemeur@interchange.ubc.ca

    Nogo-A is a myelin-associated protein expressed by neurons and myelinating mature oligodendrocytes in the central nervous system. Although most research has focused on the participation of Nogo-A in the prevention of axonal regeneration and plasticity in the adult, little attention has been paid to the putative functions of Nogo-A during embryonic development. Here we examined the general pattern and cell-specific distribution of Nogo-A in the prenatal mouse telencephalon. In addition, we studied the development of the major axon tracts and radial and tangential migration in Nogo-A/B/C knockout mice. The pattern of Nogo-A showed distinct distribution in radial glia and postmitotic neurons, in which it is particularly enriched in developing axons. Similarly, Nogo-A was enriched at the leading process of tangentially migrating interneurons but not detectable in radial migrating neurons. Although a low level of Nogo-A appears to be on the surface of many cortical neurons, most proteins have intracellular localization. In Nogo-deficient background, neurons displayed early polarization and increased branching in vitro, probably reflecting a cell-intrinsic role of Nogo proteins in branching reduction, and early tangential migration was delayed. On the basis of these observations, we propose that Nogo proteins, particularly Nogo-A, are involved in multiple processes during cortical development.

    Cerebral cortex (New York, N.Y. : 1991) 2007;17;10;2375-86

  • Expression of connexins in embryonic mouse neocortical development.

    Cina C, Bechberger JF, Ozog MA and Naus CC

    Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.

    During embryonic development, young neurons migrate from the ventricular zone to the cortical plate of the cerebral cortex. Disturbances in this neuronal migration have been associated with numerous diseases such as mental retardation, double cortex, Down syndrome, and epilepsy. One possible cause of these neuropathologies is an aberration in normal gap junctional communication. At least 20 connexin (Cx) genes encode gap junction proteins in mice and humans. A proper understanding of the role of specific connexins in the developing brain requires the characterization of their spatial and temporal pattern of expression. In the current study we performed all the experiments on mouse developing cortex at embryonic days (E) 14, 16, and 18, timepoints that are highly active with regard to cortical development. Using reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we found that among the family of gap junction proteins, Cx26, Cx36, Cx37, Cx43, and Cx45 were expressed in the developing cortex of mice, Cx30 and Cx32 were absent, while Cx40 was expressed at a very low level. Our results demonstrate that Cx26 and Cx37 were evenly distributed in the cortical layers of developing brain, while Cx36 and Cx43 were more abundant in the ventricular zone and cortical plate. Cx45 distribution appeared to be more abundant at E18 compared to the other timepoints (E14 and E16). Thus, the present study provides identification and the distribution pattern for Cxs associated with cortical development during normal neuronal migration.

    The Journal of comparative neurology 2007;504;3;298-313

  • Differences in neurogenic potential in floor plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells.

    Ono Y, Nakatani T, Sakamoto Y, Mizuhara E, Minaki Y, Kumai M, Hamaguchi A, Nishimura M, Inoue Y, Hayashi H, Takahashi J and Imai T

    KAN Research Institute Inc., KobeMI R&D Center 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan. y-ono@kan.eisai.co.jp

    Directed differentiation and purification of mesencephalic dopaminergic (mesDA) neurons from stem cells are crucial issues for realizing safe and efficient cell transplantation therapies for Parkinson's disease. Although recent studies have identified the factors that regulate mesDA neuron development, the mechanisms underlying mesDA neuron specification are not fully understood. Recently, it has been suggested that mesencephalic floor plate (FP) cells acquire neural progenitor characteristics to generate mesDA neurons. Here, we directly examined this in a fate mapping experiment using fluorescence-activated cell sorting (FACS) with an FP cell-specific surface marker, and demonstrate that mesencephalic FP cells have neurogenic activity and generate mesDA neurons in vitro. By contrast, sorted caudal FP cells have no neurogenic potential, as previously thought. Analysis of dreher mutant mice carrying a mutation in the Lmx1a locus and transgenic mice ectopically expressing Otx2 in caudal FP cells demonstrated that Otx2 determines anterior identity that confers neurogenic activity to FP cells and specifies a mesDA fate, at least in part through the induction of Lmx1a. We further show that FACS can isolate mesDA progenitors, a suitable transplantation material, from embryonic stem cell-derived neural cells. Our data provide insights into the mechanisms of specification and generation of mesDA neurons, and illustrate a useful cell replacement approach for Parkinson's disease.

    Development (Cambridge, England) 2007;134;17;3213-25

  • Cortical dysplasia and skull defects in mice with a Foxc1 allele reveal the role of meningeal differentiation in regulating cortical development.

    Zarbalis K, Siegenthaler JA, Choe Y, May SR, Peterson AS and Pleasure SJ

    Department of Neurology, University of California, 1550 Fourth Street, San Francisco, CA 94158, USA.

    We report the identification of a hypomorphic mouse allele for Foxc1 (Foxc1(hith)) that survives into adulthood revealing previously unknown roles for Foxc1 in development of the skull and cerebral cortex. This line of mice was recovered in a forward genetic screen using ENU mutagenesis to identify mutants with cortical defects. In the hith allele a missense mutation substitutes a Leu for a conserved Phe at amino acid 107, leading to destabilization of the protein without substantially altering transcriptional activity. Embryonic and postnatal histological analyses indicate that diminished Foxc1 protein expression in all three layers of meningeal cells in Foxc1(hith/hith) mice contributes to the cortical and skull defects in mutant mice and that the prominent phenotypes appear as the meninges differentiate into pia, arachnoid, and dura. Careful analysis of the cortical phenotypes shows that Foxc1(hith/hith) mice display detachment of radial glial endfeet, marginal zone heterotopias, and cortical dyslamination. These abnormalities have some features resembling defects in type 2 (cobblestone) lissencephaly or congenital muscular dystrophies but appear later in corticogenesis because of the delay in breakdown of the basement membrane. Our data reveal that the meninges regulate the development of the skull and cerebral cortex by controlling aspects of the formation of these neighboring structures. Furthermore, we provide evidence that defects in meningeal differentiation can lead to severe cortical dysplasia.

    Funded by: Autism Speaks: AS1625

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;35;14002-7

  • Cajal-Retzius cells and subplate neurons differentially express vesicular glutamate transporters 1 and 2 during development of mouse cortex.

    Ina A, Sugiyama M, Konno J, Yoshida S, Ohmomo H, Nogami H, Shutoh F and Hisano S

    Laboratory of Neuroendocrinology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba 305-3574, Japan.

    In the light of the various neurobiological effects of glutamate in brain development, although some embryonic cells are a probable source of glutamate involved in the development of precursor cells and/or immature neurons, little is known about when and where glutamate plays its crucial roles during corticogenesis. To investigate these roles, we focused on the developmental expression of vesicular glutamate transporter (VGLUT)1 and VGLUT2, which are regarded as the best markers for verifying glutamatergic neuron identity, especially the spatiotemporal distributions of their transcripts and proteins in the developing mouse cortex and hippocampus. In situ hybridization studies revealed that VGLUT1 mRNA is expressed in preplate and marginal zone cells at embryonic day (E)10 and in subplate cells by E13, whereas VGLUT2 mRNA is expressed in preplate and marginal zone cells at E10 and in cells of the subventricular zone by E13. Reverse transcriptase-polymerase chain reaction analysis detected full-length VGLUT1 and VGLUT2 gene transcripts in the embryonic brain. By dual labeling combined with immunostaining for microtubule-associated protein 2 (MAP2) or reelin, we showed that MAP2-positive preplate and marginal zone neurons and subplate neurons express VGLUT1, while reelin-positive preplate and marginal zone cells and MAP2-negative subventricular zone cells express VGLUT2. The present study is the first to provide morphologically reliable evidence showing that Cajal-Retzius cells and subplate neurons are glutamatergic, and that the two cells differentially express VGLUT1 and VGLUT2, respectively, as the specific transport system of glutamate in some events orchestrated by these cells during the cortical development of mice.

    The European journal of neuroscience 2007;26;3;615-23

  • Id sustains Hes1 expression to inhibit precocious neurogenesis by releasing negative autoregulation of Hes1.

    Bai G, Sheng N, Xie Z, Bian W, Yokota Y, Benezra R, Kageyama R, Guillemot F and Jing N

    Laboratory of Molecular Cell Biology, Key Laboratory of Stem Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

    Negative bHLH transcription factor Hes1 can inhibit neural stem cells (NSCs) from precocious neurogenesis through repressing proneural gene expression; therefore, sustenance of Hes1 expression is crucial for NSC pool maintenance. Here we find that Ids, the dominant-negative regulators of proneural proteins, are expressed prior to proneural genes and share an overlapping expression pattern with Hes1 in the early neural tube of chick embryos. Overexpression of Id2 in the chick hindbrain upregulates Hes1 expression and inhibits proneural gene expression and neuronal differentiation. By contrast, Hes1 expression decreases, proneural gene expression expands, and neurogenesis occurs precociously in Id1;Id3 double knockout mice and in Id1-3 RNAi-electroporated chick embryos. Mechanistic studies show that Id proteins interact directly with Hes1 and release the negative feedback autoregulation of Hes1 without interfering with its ability to affect other target genes. These results indicate that Id proteins participate in NSC maintenance through sustaining Hes1 expression in early embryos.

    Funded by: Medical Research Council: MC_U117570528

    Developmental cell 2007;13;2;283-97

  • Neogenin is expressed on neurogenic and gliogenic progenitors in the embryonic and adult central nervous system.

    Fitzgerald DP, Bradford D and Cooper HM

    Queensland Brain Institute, The University of Queensland, Brisbane, Qld, Australia.

    The Netrin/RGMa receptor, Neogenin, has recently been identified on neuronal and gliogenic progenitors, including radial glia in the embryonic mouse cortex and ganglionic eminences, respectively [Fitzgerald, D.P., Cole, S.J., Hammond, A., Seaman, C., Cooper, H.M., 2006a. Characterization of Neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain. Neuroscience 142, 703-716]. Here we have undertaken a detailed analysis of Neogenin expression in the embryonic mouse central nervous system at key developmental time points. We demonstrate that Neogenin protein is present on actively dividing neurogenic precursors during peak phases of neurogenesis (embryonic days 12.5-14.5) in the forebrain, midbrain and hindbrain. Furthermore, we show that Neogenin protein is localized to the cell bodies and glial processes of neurogenic radial glial populations in all these regions. We have also observed Neogenin on gliogenic precursors within the subventricular zones of the forebrain late in development (embryonic day 17.5). Adult neural stem cells found in the subventricular zone of the lateral ventricle of the rodent forebrain are direct descendants of the embryonic striatal radial glial population. Here we show that Neogenin expression is maintained in the neural stem cell population of the adult mouse forebrain. In summary, this study demonstrates that Neogenin expression is a hallmark of many neural precursor populations (neurogenic and gliogenic) in both the embryonic and adult mammalian central nervous system.

    Gene expression patterns : GEP 2007;7;7;784-92

  • Dlx transcription factors promote migration through repression of axon and dendrite growth.

    Cobos I, Borello U and Rubenstein JL

    Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94158, USA. inma.cobos@ucsf.edu

    In the mouse telencephalon, Dlx homeobox transcription factors are essential for the tangential migration of subpallial-derived GABAergic interneurons to neocortex. However, the mechanisms underlying this process are poorly understood. Here, we demonstrate that Dlx1/2 has a central role in restraining neurite growth of subpallial-derived immature interneurons at a stage when they migrate tangentially to cortex. In Dlx1-/-;Dlx2-/- mutants, neurite length is increased and cells fail to migrate. In Dlx1-/-;Dlx2+/- mutants, while the tangential migration of immature interneurons appears normal, they develop dendritic and axonal processes with increased length and decreased branching, and have deficits in their neocortical laminar positions. Thus, Dlx1/2 is required for coordinating programs of neurite maturation and migration. In this regard, we provide genetic evidence that in immature interneurons Dlx1/2 repression of the p21-activated serine/threonine kinase PAK3, a downstream effector of the Rho family of GTPases, is critical in restraining neurite growth and promoting tangential migration.

    Funded by: NIMH NIH HHS: K05 MH065670, R01 MH049428, R01 MH49428

    Neuron 2007;54;6;873-88

  • Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex.

    Ohshima T, Hirasawa M, Tabata H, Mutoh T, Adachi T, Suzuki H, Saruta K, Iwasato T, Itohara S, Hashimoto M, Nakajima K, Ogawa M, Kulkarni AB and Mikoshiba K

    Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. ohshima@brain.riken.go.jp

    The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.

    Development (Cambridge, England) 2007;134;12;2273-82

  • Hand2 is necessary for terminal differentiation of enteric neurons from crest-derived precursors but not for their migration into the gut or for formation of glia.

    D'Autréaux F, Morikawa Y, Cserjesi P and Gershon MD

    Department of Pathology and Cell Biology, Columbia University, P&S, New York, NY 10032, USA.

    Hand genes encode basic helix-loop-helix transcription factors that are expressed in the developing gut, where their function is unknown. We now report that enteric Hand2 expression is limited to crest-derived cells, whereas Hand1 expression is restricted to muscle and interstitial cells of Cajal. Hand2 is developmentally regulated and is intranuclear in precursors but cytoplasmic in neurons. Neurons develop in explants from wild-type but not Hand2(-/-) bowel, although, in both, crest-derived cells are present and glia arise. Similarly, small interfering RNA (siRNA) silencing of Hand2 in enteric crest-derived cells prevents neuronal development. Terminally differentiated enteric neurons do not develop after conditional inactivation of Hand2 in migrating crest-derived cells; nevertheless, conditional Hand2 inactivation does not prevent precursors from expressing early neural markers. We suggest that enteric neuronal development occurs in stages and that Hand2 expression is required for terminal differentiation but not for precursors to enter the neuronal lineage.

    Funded by: NINDS NIH HHS: NS12969, NS15547

    Development (Cambridge, England) 2007;134;12;2237-49

  • Expression and immunolocalization of the plasma membrane monoamine transporter in the brain.

    Dahlin A, Xia L, Kong W, Hevner R and Wang J

    Department of Pharmaceutics, University of Washington, H272J, Health Sciences Building, Seattle, WA 98195, USA.

    High affinity monoamine transporters efficiently terminate neurotransmission through synaptic reuptake of released neurotransmitter. We recently cloned and characterized a novel low-affinity, high capacity plasma membrane monoamine transporter (PMAT) that is strongly expressed in the human brain and efficiently transports 5-HT and dopamine (DA). In efforts to understand the physiological function of PMAT and its relevance in monoaminergic pathways, we cloned the PMAT homolog from the mouse brain, demonstrated its capability for transporting 5-HT and DA, and determined the regional and cellular localization of mouse plasma membrane monoamine transporter (mPMAT) in adult mouse brain by reverse-transcription polymerase chain reaction, non-radioactive in situ hybridization, and immunohistochemical methods. Our results showed that mPMAT mRNA and protein are broadly expressed in the mouse brain and are particularly abundant in forebrain cortex, olfactory tubercle, hippocampus, cerebellum and epithelial cells of the choroid plexus. Dual-immunofluorescence histochemistry with established phenotypic markers microtubule-associated protein (MAP2) and glial fibrillary acidic protein (GFAP) revealed that mPMAT is expressed in neuronal cells but not in astrocytes. mPMAT is co-expressed in many brain regions with the high affinity 5-HT transporter (SERT) and the dopamine transporter (DAT), but is also found in certain sites that receive monoamine innervation but lack significant expression of SERT or DAT. These findings suggest that mPMAT is a widely distributed, neuronally-expressed transporter, which may support the role of 5-HT and DA uptake under certain conditions.

    Funded by: NIGMS NIH HHS: GM066233, R01 GM066233, R01 GM066233-05A1, T32 GM 07750, T32 GM007750

    Neuroscience 2007;146;3;1193-211

  • LKB1 and SAD kinases define a pathway required for the polarization of cortical neurons.

    Barnes AP, Lilley BN, Pan YA, Plummer LJ, Powell AW, Raines AN, Sanes JR and Polleux F

    Neuroscience Center, Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA.

    The polarization of axon and dendrites underlies the ability of neurons to integrate and transmit information in the brain. We show here that the serine/threonine kinase LKB1, previously implicated in the establishment of epithelial polarity and control of cell growth, is required for axon specification during neuronal polarization in the mammalian cerebral cortex. LKB1 polarizing activity requires its association with the pseudokinase Stradalpha and phosphorylation by kinases such as PKA and p90RSK, which transduce neurite outgrowth-promoting cues. Once activated, LKB1 phosphorylates and thereby activates SAD-A and SAD-B kinases, which are also required for neuronal polarization in the cerebral cortex. SAD kinases, in turn, phosphorylate effectors such as microtubule-associated proteins that implement polarization. Thus, we provide evidence in vivo and in vitro for a multikinase pathway that links extracellular signals to the intracellular machinery required for axon specification.

    Funded by: NINDS NIH HHS: P30 NS45892-01

    Cell 2007;129;3;549-63

  • Inactivation of Arx, the murine ortholog of the X-linked lissencephaly with ambiguous genitalia gene, leads to severe disorganization of the ventral telencephalon with impaired neuronal migration and differentiation.

    Colombo E, Collombat P, Colasante G, Bianchi M, Long J, Mansouri A, Rubenstein JL and Broccoli V

    Stem Cell Research Department, San Raffaele Scientific Institute, 20132 Milan, Italy.

    ARX loss-of-function mutations cause X-linked lissencephaly with ambiguous genitalia (XLAG), a severe neurological condition that results in profound brain malformations, including microcephaly, absence of corpus callosum, and impairment of the basal ganglia. Despite such dramatic defects, their nature and origin remain largely unknown. Here, we used Arx mutant mice as a model to characterize the cellular and molecular mechanisms underlying the basal ganglia alterations. In these animals, the early differentiation of this tissue appeared normal, whereas subsequent differentiation was impaired, leading to the periventricular accumulation of immature neurons in both the lateral ganglionic eminence and medial ganglionic eminence (MGE). Both tangential migration toward the cortex and striatum and radial migration to the globus pallidus and striatum were greatly reduced in the mutants, causing a periventricular accumulation of NPY+ or calretinin+ neurons in the MGE. Arx mutant neurons retained their differentiation potential in vitro but exhibited deficits in morphology and migration ability. These findings imply that cell-autonomous defects in migration underlie the neuronal localization defects. Furthermore, Arx mutants lacked a large fraction of cholinergic neurons and displayed a strong impairment of thalamocortical projections, in which major axon fiber tracts failed to traverse the basal ganglia. Altogether, these results highlight the critical functions of Arx in promoting neural migration and regulating basal ganglia differentiation in mice, consistent with the phenotype of XLAG patients.

    Funded by: NIDDK NIH HHS: U19DK072495-01; NIMH NIH HHS: K05 MH065670, R01 MH49428-01; Telethon: GGP04141

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;17;4786-98

  • Cadherin expression in the developing mouse olfactory system.

    Akins MR, Benson DL and Greer CA

    Interdepartmental Neuroscience Graduate Program, Yale University School of Medicine, New Haven, Connecticut 06520-8082, USA.

    Although odor receptors have been implicated in establishing the topography of olfactory sensory neurons (OSNs) in the olfactory bulb (OB), it is likely other molecules are also involved. The cadherins (CDHs) are a large family of cell adhesion molecules that mediate cell:cell interactions elsewhere in the central nervous system. However, their distribution and role in the olfactory system have remained largely unexplored. We previously demonstrated that intracellular binding partners of cadherins, the catenins, have unique spatiotemporal patterns of expression in the developing olfactory system. To further our understanding of cadherin function within the developing olfactory system, we now report on the localization of 11 classical cadherins-CDH1, 2, 3, 4, 5, 6, 8, 10, 11, 13, and 15. We demonstrate the expression of all but CDH5 and CDH15 in neuronal and/or glial cells in primary olfactory structures. CDH1 and CDH2 are expressed by OSNs; CDH2 expression closely parallels that seen for gamma-catenin in OSN axons. CDH3 and CDH11 are expressed by olfactory ensheathing glia, which surround OSN axons in the outer OB. CDH2, CDH4, and CDH6 are expressed within neuropil. CDH2, CDH4, CDH6, CDH8, CDH10, CDH11, and CDH13 are expressed by projection neurons within the main and accessory OBs. We conclude that cadherin proteins in the developing olfactory system are positioned to underlie the formation of the odorant map and local circuits within the OB.

    Funded by: NIDCD NIH HHS: DC00210, DC006291, DC006335, DC006972; NINDS NIH HHS: NS37731, R01 NS037731, R01 NS037731-01A1, R01 NS037731-02, R01 NS037731-02S1, R01 NS037731-03, R01 NS037731-04, R01 NS037731-05A1, R01 NS037731-06, R01 NS037731-07, R01 NS037731-08, R01 NS037731-09A1, R01 NS037731-10

    The Journal of comparative neurology 2007;501;4;483-97

  • Stage-specific association of apolipoprotein A-I and E in developing mouse retina.

    Kurumada S, Onishi A, Imai H, Ishii K, Kobayashi T and Sato SB

    Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

    Purpose: To characterize the intercellular lipid transport systems in differentiating retina.

    Methods: Developing mouse retinas were evaluated for the expression of apolipoproteins (apoE, apoA-I) by Western blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). They were compared with changes in the lipid content and association of retinal proteins, such as postsynaptic density protein 95, glial fibrillary acidic protein, and cellular retinaldehyde-binding protein. Intraretinal distribution of apolipoproteins and their receptors was examined by immunofluorescence and in situ hybridization of prenatal and postnatal retinal sections. In vitro culture of dissociated cells was also examined.

    Results: Although apoE is known to be present in the mature retina, the neonatal retina remarkably expressed apoA-I mRNA and protein. This protein was present until postnatal day (P)3, and its putative receptor, scavenger receptor class B-I, was present until P5 to P7. This state subsequently exhibited a dramatic switchover to an apoE-rich one, in parallel with the stratification. Whereas apoE was synthesized at low levels until P7, apoE mRNA was clearly concentrated in Müller glia cells, which extended long apoE-bound processes to the plexuses and contours of photoreceptor cells. These acceptor cells expressed LDL receptor-related protein 1 as a putative receptor. ApoE genes were not transcribed in ganglion cells, though they were associated with a high level of the protein throughout the development. ApoE protein in ganglion cells initially appeared to be synthesized by astrocytes but later were observed to be supplied from an extraretinal space.

    Conclusions: The present results document several new aspects of apoA-I and apoE in the developing retina. The switchover of the lipoprotein systems runs a parallel course with the differentiation.

    Investigative ophthalmology & visual science 2007;48;4;1815-23

  • Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations.

    Munton RP, Tweedie-Cullen R, Livingstone-Zatchej M, Weinandy F, Waidelich M, Longo D, Gehrig P, Potthast F, Rutishauser D, Gerrits B, Panse C, Schlapbach R and Mansuy IM

    Brain Research Institute, Medical Faculty of the University of Zürich, Switzerland.

    Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.

    Molecular & cellular proteomics : MCP 2007;6;2;283-93

  • Temporal and spatial expression profiles of the Fat3 protein, a giant cadherin molecule, during mouse development.

    Nagae S, Tanoue T and Takeichi M

    Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

    Cadherins constitute a superfamily of cell-cell interaction molecules that participate in morphogenetic processes of animal development. Fat cadherins are the largest members of this superfamily, with 34 extracellular cadherin repeats. Classic Fat, identified in Drosophila, is known to regulate cell proliferation and planar cell polarity. Although 4 subtypes of Fat cadherin, Fat1, Fat2, Fat3, and Fat4/Fat-J, have been identified in vertebrates, their protein localization remains largely unknown. Here we describe the mRNA and protein distributions of Fat3 during mouse development. We found that Fat3 expression was restricted to the nervous system. In the brain, Fat3 was expressed in a variety of regions and axon fascicles. However, its strongest expression was observed in the olfactory bulb and retina. Detailed analysis of Fat3 in the developing olfactory bulb revealed that Fat3 mRNA was mainly expressed by mitral cells and that its proteins were densely localized along the dendrites of these cells as well as in their axons to some extent. Fat3 transcripts in the retina were expressed by amacrine and ganglion cells, and its proteins were concentrated in the inner plexiform layer throughout development. Based on these observations, we suggest that Fat3 plays a role in the interactions between neurites derived from specific subsets of neurons during development.

    Developmental dynamics : an official publication of the American Association of Anatomists 2007;236;2;534-43

  • BAD-LAMP defines a subset of early endocytic organelles in subpopulations of cortical projection neurons.

    David A, Tiveron MC, Defays A, Beclin C, Camosseto V, Gatti E, Cremer H and Pierre P

    Centre d'Immunologie de Marseille-Luminy, Université de la Méditerranée, Case 906, 13288 Marseille cedex 9, France. cremer@ibdm.univ-mrs.fr

    The brain-associated LAMP-like molecule (BAD-LAMP) is a new member of the family of lysosome associated membrane proteins (LAMPs). In contrast to other LAMPs, which show a widespread expression, BAD-LAMP expression in mice is confined to the postnatal brain and therein to neuronal subpopulations in layers II/III and V of the neocortex. Onset of expression strictly parallels cortical synaptogenesis. In cortical neurons, the protein is found in defined clustered vesicles, which accumulate along neurites where it localizes with phosphorylated epitopes of neurofilament H. In primary neurons, BAD-LAMP is endocytosed, but is not found in classical lysosomal/endosomal compartments. Modification of BAD-LAMP by addition of GFP revealed a cryptic lysosomal retention motif, suggesting that the cytoplasmic tail of BAD-LAMP is actively interacting with, or modified by, molecules that promote its sorting away from lysosomes. Analysis of BAD-LAMP endocytosis in transfected HeLa cells provided evidence that the protein recycles to the plasma membrane through a dynamin/AP2-dependent mechanism. Thus, BAD-LAMP is an unconventional LAMP-like molecule and defines a new endocytic compartment in specific subtypes of cortical projection neurons. The striking correlation between the appearance of BAD-LAMP and cortical synatogenesis points towards a physiological role of this vesicular determinant for neuronal function.

    Journal of cell science 2007;120;Pt 2;353-65

  • Expression of Six3 Opposite Strand (Six3OS) during mouse embryonic development.

    Geng X, Lavado A, Lagutin OV, Liu W and Oliver G

    Department of Genetics and Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.

    Recently, sequence analyses have identified a large number of opposite strand transcripts in the vertebrate genome. Although the transcripts appear to be spliced and polyadenylated, many of them are predicted to represent noncoding RNAs. High levels of noncoding transcripts of the Six3 Opposite Strand (Six3OS) were recently identified in the embryonic and postnatal retina of the mouse. In this study, we expanded those initial expression analyses, elucidated in detail the developmental expression profile of mouse Six3OS in the brain and visual system, and compared it with that of Six3. Our results show that Six3OS expression overlaps extensively with that of Six3 and is not altered in Six3-null embryos.

    Funded by: NCI NIH HHS: CA-21765, P30 CA021765; NEI NIH HHS: EY12162, R01 EY012162, R01 EY012162-08; NINDS NIH HHS: NS052386, R01 NS052386

    Gene expression patterns : GEP 2007;7;3;252-7

  • Collapsin response mediator protein 1 mediates reelin signaling in cortical neuronal migration.

    Yamashita N, Uchida Y, Ohshima T, Hirai S, Nakamura F, Taniguchi M, Mikoshiba K, Honnorat J, Kolattukudy P, Thomasset N, Takei K, Takahashi T and Goshima Y

    Department of Molecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan.

    Collapsin response mediator protein 1 (CRMP1) is one of the CRMP family members that mediates signal transduction of axon guidance molecules. Here, we show evidence that CRMP1 is involved in Reelin (Reln) signaling to regulate neuronal migration in the cerebral cortex. In crmp1-/- mice, radial migration of cortical neurons was retarded. This phenotype was not observed in the sema3A-/- and crmp1+/-;sema3A+/- cortices. However, CRMP1 was colocalized with disabled-1 (Dab1), an adaptor protein in Reln signaling. In the Reln(rl/rl) cortex, CRMP1 and Dab1 were expressed at a higher level, yet tyrosine phosphorylated at a lower level. Loss of crmp1 in a dab1 heterozygous background led to the disruption of hippocampal lamination, a Reeler-like phenotype. In addition to axon guidance, CRMP1 regulates neuronal migration by mediating Reln signaling.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;51;13357-62

  • Klf4 and corticosteroids activate an overlapping set of transcriptional targets to accelerate in utero epidermal barrier acquisition.

    Patel S, Xi ZF, Seo EY, McGaughey D and Segre JA

    National Human Genome Research Institute, National Institutes of Health, 49 Convent Drive, Bethesda, MD 20892, USA.

    Premature infants are at an increased risk for infections and dehydration because of incomplete development of the epidermis, which attains its essential function as a barrier only during the last stages of in utero development. When a premature birth is anticipated, antenatal corticosteroids are administered to accelerate lung epithelium differentiation. One pleiotropic, but beneficial, effect of antenatal corticosteroids is acceleration of skin barrier establishment by an unknown mechanism. In mice, the transcription factor Klf4 is both necessary and sufficient, within a developmental field of competence, to establish this skin barrier, as demonstrated by targeted ablation and transgenic expression of Klf4, respectively. Here, we report that Klf4 and corticosteroid treatment coordinately accelerate barrier acquisition in vivo. Transcriptional profiling reveals that the genes regulated by corticosteroids and Klf4 during the critical window of epidermal development significantly overlap. KLF4 activates the proximal promoters of a significant subset of these genes. Dissecting the intersection of the genetic and pharmacological pathways, regulated by KLF4 and corticosteroids, respectively, leads to a mechanistic understanding of the normal process of epidermal development in utero.

    Funded by: Intramural NIH HHS

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;49;18668-73

  • The c-Jun N-terminal kinase activator dual leucine zipper kinase regulates axon growth and neuronal migration in the developing cerebral cortex.

    Hirai S, Cui DF, Miyata T, Ogawa M, Kiyonari H, Suda Y, Aizawa S, Banba Y and Ohno S

    Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama 236-0004, Japan. sh3312@med.yokohama-cu.ac.jp

    Mammalian corticogenesis substantially depends on migration and axon projection of newborn neurons that are coordinated by a yet unidentified molecular mechanism. Dual leucine zipper kinase (DLK) induces activation of c-Jun N-terminal kinase (JNK), a molecule that regulates morphogenesis in various organisms. We show here, using gene targeting in mice, that DLK is indispensable for establishing axon tracts, especially those originating from neocortical pyramidal neurons of the cerebrum. Direct and quantitative analysis of radial migration of pyramidal neurons using slice culture and a time-lapse imaging system revealed that acceleration around the subplate was affected by DLK gene disruption and by administration of a JNK inhibitor. Phosphorylation of JNK substrates, including c-Jun and doublecortin, and of JNK itself at the activation loop were partially affected in brains of DLK-deficient mouse embryos. These data suggest that DLK plays a significant role in the coordinated regulation of radial migration and axon projection by modulating JNK activity.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2006;26;46;11992-2002

  • Neuronal production and precursor proliferation defects in the neocortex of mice with loss of function in the canonical Wnt signaling pathway.

    Zhou CJ, Borello U, Rubenstein JL and Pleasure SJ

    Department of Neurology, UCSF Mission Bay, Box 2722, Rock Hall, 1550 Fourth Street, Room RH-348D, San Francisco, CA 94143-2722, USA.

    To better understand the function of the Wnt pathway in the developing telencephalon, we analyzed neocortical development in low density lipoprotein receptor-related protein (LRP) 6 mutants. LRP6 mutant mice are hypomorphic for the canonical Wnt signaling pathway and have hypoplasia of the developing neocortex. While early telencephalic morphogenesis is largely intact in these mice, probably due to compensation by LRP5, the mutant mice develop a dramatically thinner cortical plate. There is a prominent reduction of neurogenesis leading to a thin cortical plate. Reduced proliferation late in gestation probably also contributes to the hypoplasia. Although there are marked decreases in the numbers of layer 6 and layers 2-4 neurons all laminar identities are generated and there is no evidence of compensatory increases in layer 5 neurons. In addition, LRP6 mutants have partial penetrance of a complex of cortical dysmorphologies resembling those found in patients with developmental forms of epilepsy and mental retardation. These include ventricular and marginal zone heterotopias and cobblestone lissencephaly. This analysis demonstrates that canonical Wnt signaling is required for a diverse array of developmental processes in the neocortex in addition to the previously known roles in regulating precursor proliferation and patterning.

    Funded by: NIMH NIH HHS: K05 MH065670, MH074958, MH66084; NINDS NIH HHS: NS002208, R01 NS34661

    Neuroscience 2006;142;4;1119-31

  • COUP-TFI is required for the formation of commissural projections in the forebrain by regulating axonal growth.

    Armentano M, Filosa A, Andolfi G and Studer M

    TIGEM (Telethon Institute of Genetics and Medicine Disorders Program, Via P. Castellino 111, 80131 Napoli, Italy.

    The transcription factor COUP-TFI (NR2F1), an orphan member of the nuclear receptor superfamily, is an important regulator of neurogenesis, cellular differentiation and cell migration. In the forebrain, COUP-TFI controls the connectivity between thalamus and cortex and neuronal tangential migration in the basal telencephalon. Here, we show that COUP-TFI is required for proper axonal growth and guidance of all major forebrain commissures. Fibres of the corpus callosum, the hippocampal commissure and the anterior commissure project aberrantly and fail to cross the midline in COUP-TFI null mutants. Moreover, hippocampal neurons lacking COUP-TFI have a defect in neurite outgrowth and show an abnormal axonal morphology. To search for downstream effectors, we used microarray analysis and showed that, in the absence of COUP-TFI, expression of various cytoskeleton molecules involved in neuronal morphogenesis is affected. Diminished protein levels of the microtubule-associated protein MAP1B and increased levels of the GTP-binding protein RND2 were confirmed in the developing cortex in vivo and in primary hippocampal neurons in vitro. Therefore, based on morphological studies, gene expression profiling and primary cultured neurons, the present data uncover a previously unappreciated intrinsic role for COUP-TFI in axonal growth in vivo and supply one of the premises for COUP-TFI coordination of neuronal morphogenesis in the developing forebrain.

    Funded by: Telethon: TGM06A04, TGM06S01

    Development (Cambridge, England) 2006;133;21;4151-62

  • Characterization of neogenin-expressing neural progenitor populations and migrating neuroblasts in the embryonic mouse forebrain.

    Fitzgerald DP, Cole SJ, Hammond A, Seaman C and Cooper HM

    Queensland Brain Institute, Neural Migration Laboratory, The University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.

    Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin, a member of the DCC receptor family, has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S, Deitinghoff L, Davis D, Conrad S, Skutella T, Chedotal A, Mueller B, Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors, including neurogenic radial glia, in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast, neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition, we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences, suggesting a role for neogenin in tangential migration. Therefore, neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain.

    Neuroscience 2006;142;3;703-16

  • Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis.

    Ma Y, Li J, Chiu I, Wang Y, Sloane JA, Lü J, Kosaras B, Sidman RL, Volpe JJ and Vartanian T

    Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.

    Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR-NF-kappaB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.

    Funded by: NINDS NIH HHS: P01 NS038475, P01NS038475

    The Journal of cell biology 2006;175;2;209-15

  • TTLL7 is a mammalian beta-tubulin polyglutamylase required for growth of MAP2-positive neurites.

    Ikegami K, Mukai M, Tsuchida J, Heier RL, Macgregor GR and Setou M

    Mitsubishi Kagaku Institute of Life Sciences, Minamiooya, Machida, Tokyo 194-8511, Japan.

    Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform beta-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for beta-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system. Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to beta-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for beta-tubulin and not for alpha-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated beta-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of beta-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (microtubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated beta-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a beta-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells.

    Funded by: NICHD NIH HHS: U01 HD045913-01, U01 HD045913-02, U01 HD045913-03

    The Journal of biological chemistry 2006;281;41;30707-16

  • Cortical migration defects in mice expressing A-RAF from the B-RAF locus.

    Camarero G, Tyrsin OY, Xiang C, Pfeiffer V, Pleiser S, Wiese S, Götz R and Rapp UR

    Institut für Medizinische Strahlenkunde und Zellforschung, Bayerische Julius-Maximilians-Universität, Versbacher-Str. 5, D-97078 Würzburg, Germany.

    We have previously shown that mice lacking the protein kinase B-RAF have defects in both neural and endothelial cell lineages and die around embryonic day 12 (E12). To delineate the function of B-RAF in the brain, B-RAF KIN/KIN mice lacking B-RAF and expressing A-RAF under the control of the B-RAF locus were created. B-RAF KIN/KIN embryos displayed no vascular defects, no endothelial and neuronal apoptosis, or gross developmental abnormalities, and a significant proportion of these animals survived for up to 8 weeks. Cell proliferation in the neocortex was reduced from E14.5 onwards. Newborn cortical neurons were impaired in their migration toward the cortical plate, causing a depletion of Brn-2-expressing pyramidal neurons in layers II, III, and V of the postnatal cortex. Our data reveal that B-RAF is an important mediator of neuronal survival, migration, and dendrite formation and that A-RAF cannot fully compensate for these functions.

    Molecular and cellular biology 2006;26;19;7103-15

  • The Rho-GTPase cdc42 regulates neural progenitor fate at the apical surface.

    Cappello S, Attardo A, Wu X, Iwasato T, Itohara S, Wilsch-Bräuninger M, Eilken HM, Rieger MA, Schroeder TT, Huttner WB, Brakebusch C and Götz M

    GSF, National Research Center for Environment and Health, Institute for Stem Cell Research, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Munich, Germany.

    Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

    Nature neuroscience 2006;9;9;1099-107

  • Embryonic expression of pericentrin suggests universal roles in ciliogenesis.

    Miyoshi K, Onishi K, Asanuma M, Miyazaki I, Diaz-Corrales FJ and Ogawa N

    Department of Brain Science, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. miyoshi@cc.okayama-u.ac.jp

    Pericentrin (Pcnt) is a giant coiled-coil protein known to mediate microtubule organization. It has been recently reported that mitosis-specific centrosomal anchoring of gamma tubulin complexes by Pcnt acts to control mitotic spindle organization, though little is known about the in vivo expression of Pcnt. In this study, we investigated Pcnt expression in mouse embryos. In situ hybridization analysis revealed preferential expression of Pcnt in quiescent G(0) phase cells throughout the embryo with an unexpectedly low expression level in proliferating cells, suggesting that Pcnt might not play an important role in mitotic proliferation. Immunofluorescence analysis confirmed preferential expression of the Pcnt protein in G(0) phase cells. Moreover, Pcnt was shown to be localized to the base of primary cilia in multiple embryonic tissues, in agreement with a recent study demonstrating the involvement of Pcnt in primary cilia formation using cultured mammalian cells.

    Development genes and evolution 2006;216;9;537-42

  • Putative "stemness" gene jam-B is not required for maintenance of stem cell state in embryonic, neural, or hematopoietic stem cells.

    Sakaguchi T, Nishimoto M, Miyagi S, Iwama A, Morita Y, Iwamori N, Nakauchi H, Kiyonari H, Muramatsu M and Okuda A

    Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan.

    Many genes have been identified that are specifically expressed in multiple types of stem cells in their undifferentiated state. It is generally assumed that at least some of these putative "stemness" genes are involved in maintaining properties that are common to all stem cells. We compared gene expression profiles between undifferentiated and differentiated embryonic stem cells (ESCs) using DNA microarrays. We identified several genes with much greater signal in undifferentiated ESCs than in their differentiated derivatives, among them the putative stemness gene encoding junctional adhesion molecule B (Jam-B gene). However, in spite of the specific expression in undifferentiated ESCs, Jam-B mutant ESCs had normal morphology and pluripotency. Furthermore, Jam-B homozygous mutant mice are fertile and have no overt developmental defects. Moreover, we found that neural and hematopoietic stem cells recovered from Jam-B mutant mice are not impaired in their ability to self-renew and differentiate. These results demonstrate that Jam-B is dispensable for normal mouse development and stem cell identity in embryonic, neural, and hematopoietic stem cells.

    Molecular and cellular biology 2006;26;17;6557-70

  • From the Cover: Indispensability of the glutamate transporters GLAST and GLT1 to brain development.

    Matsugami TR, Tanemura K, Mieda M, Nakatomi R, Yamada K, Kondo T, Ogawa M, Obata K, Watanabe M, Hashikawa T and Tanaka K

    Laboratory of Molecular Neuroscience, School of Biomedical Science and Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan.

    Previous in vitro studies have shown that the neurotransmitter glutamate is important in brain development. Paradoxically, loss-of-function mouse models of glutamatergic signaling that are generated by genetic deletion of glutamate receptors or glutamate release show normal brain assembly. We examined the direct consequences on brain development of extracellular glutamate buildup due to the depletion of the glutamate transporters GLAST and GLT1. GLAST/GLT1 double knockout mice show multiple brain defects, including cortical, hippocampal, and olfactory bulb disorganization with perinatal mortality. Here, we report abnormal formation of the neocortex in GLAST/GLT1 mutants. Several essential aspects of neuronal development, such as stem cell proliferation, radial migration, neuronal differentiation, and survival of SP neurons, were impaired. These results provide direct in vivo evidence that GLAST and GLT1 are necessary for brain development through regulation of extracellular glutamate concentration and show that an important mechanism is likely to be maintenance of glutamate-mediated synaptic transmission.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;32;12161-6

  • Characterization of TROY-expressing cells in the developing and postnatal CNS: the possible role in neuronal and glial cell development.

    Hisaoka T, Morikawa Y, Komori T, Sugiyama T, Kitamura T and Senba E

    Department of Anatomy and Neurobiology, Wakayama Medical University, 811-1, Kimiidera, Wakayama 641-8509, Japan.

    A member of the tumor necrosis factor receptor superfamily, TROY, is expressed in the CNS of embryonic and adult mice. In the present study, we characterized TROY-expressing cells in the embryonic and postnatal forebrain. In the early embryonic forebrain, TROY was highly expressed in nestin-positive neuroepithelial cells and radial glial cells, but not in microtubule-associated protein 2-positive postmitotic neurons. During the late embryonic and postnatal development, expression of TROY was observed in radial glial cells and astrocytes, whereas its expression was not detected in neuronal lineage cells. In addition, TROY was exclusively expressed in Musashi-1-positive multipotent/glial progenitors in the postnatal subventricular zone. To investigate the functions of TROY in neural development, we overexpressed TROY in PC12 cells and established stably expressing cell clones. As expected, the signals from overexpressed TROY were constitutively transduced via the activation of the nuclear factor-kappaB and the c-Jun N-terminal kinase pathways in such clones. In addition, upregulation of negative basic helix-loop-helix transcription factors, HES-5 and Id2 proteins, was observed in the TROY-overexpressing clones. Interestingly, the overexpression of TROY in PC12 cells strongly inhibited nerve growth factor-induced neurite outgrowth with reduction of some markers of differentiated neurons, such as neurofilament 150 kDa and neuron-specific beta-tubulin. These findings suggest that the signaling from TROY regulates neuronal differentiation at least in part.

    The European journal of neuroscience 2006;23;12;3149-60

  • Expression and distribution of JNK/SAPK-associated scaffold protein JSAP1 in developing and adult mouse brain.

    Miura E, Fukaya M, Sato T, Sugihara K, Asano M, Yoshioka K and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.

    The c-Jun N-terminal kinase (JNK) is one of the three major mitogen-activated protein kinases (MAPKs) playing key roles in various cellular processes in response to both extracellular and intracellular stimuli. JNK/SAPK-associated protein 1 (JSAP1 also referred to as JIP3) is a JNK-associated scaffold that controls the specificity and efficiency of JNK signaling cascades. Here we studied its expression in mouse brains. JSAP1 mRNA was expressed in developing and adult brains, showing spatial patterns similar to JNK1-3 mRNAs. In embryos, JSAP1 immunolabeling was intense for progenitor cells in the ventricular zone throughout the brain and in the external granular layer of the cerebellum, and for neurons and glial cells differentiating in the mantle zone. In adults, JSAP1 was distributed in various neurons and Bergmann glia, with higher levels in striatal cholinergic interneurons, telencephalic parvalbumin-positive interneurons and cerebellar Purkinje cells. In these neurons, JSAP1 was observed as tiny particulate staining in spines, dendrites, perikarya and axons, where it was often associated with the smooth endoplasmic reticulum (sER) and cell membrane. Immunoblots revealed enriched distribution in the microsomal fraction and cytosolic fraction. Therefore, the characteristic cellular expression and subcellular distribution of JSAP1 might be beneficial for cells to efficiently link external stimuli to the JNK MAPK pathway and other intracellular machineries.

    Journal of neurochemistry 2006;97;5;1431-46

  • Neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells.

    Kimura I, Konishi M, Miyake A, Fujimoto M and Itoh N

    Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Sakyo, Kyoto, Japan.

    Neudesin encodes a secreted signal with neurotrophic activity in neurons. Most neurotrophic factors are involved in neural cell proliferation and/or differentiation. However, the role of neudesin in neural development remains to be elucidated. We examined the expression of neudesin in mouse embryonic cerebral cortex and cultured mouse neural precursor cells and its roles in neural development. Neudesin was expressed in the embryonic cerebral cortex early in development. Its expression was observed mainly in the preplate, where mostly postmitotic neural cells existed. Because neudesin mRNA was expressed in the neural precursor cells before the appearance of neurons, the roles of neudesin in neural development were examined by using the precursor cells. Neudesin significantly promoted neuronal differentiation and overrode the undifferentiated state of the neural precursor cells sustained by fibroblast growth factor 2 (FGF2). In contrast, it inhibited the differentiation of astrocytes. In addition, neudesin transiently promoted neural cell proliferation early in the developmental process. The effect on cell proliferation was distinct from that of FGF2, a self-renewal-promoting factor for neural precursor cells. The differentiation was mediated though activation of the protein kinase A (PKA) and phosphatidylinositol-3 kinase (PI-3K) pathways. In contrast, the proliferation was mediated through the mitogen-activated protein kinase and PKA pathways. The expression profile and activity indicate that neudesin plays unique roles in neural development. The present findings have revealed new potential roles of neudesin in neural cell proliferation and neuronal differentiation.

    Journal of neuroscience research 2006;83;8;1415-24

  • A shared vesicular carrier allows synaptic corelease of GABA and glycine.

    Wojcik SM, Katsurabayashi S, Guillemin I, Friauf E, Rosenmund C, Brose N and Rhee JS

    Department of Molecular Neurobiology, Max-Planck-Institute of Experimental Medicine, D-37075 Göttingen, Germany. wojcik@em.mpg.de

    The type of vesicular transporter expressed by a neuron is thought to determine its neurotransmitter phenotype. We show that inactivation of the vesicular inhibitory amino acid transporter (Viaat, VGAT) leads to embryonic lethality, an abdominal defect known as omphalocele, and a cleft palate. Loss of Viaat causes a drastic reduction of neurotransmitter release in both GABAergic and glycinergic neurons, indicating that glycinergic neurons do not express a separate vesicular glycine transporter. This loss of GABAergic and glycinergic synaptic transmission does not impair the development of inhibitory synapses or the expression of KCC2, the K+ -Cl- cotransporter known to be essential for the establishment of inhibitory neurotransmission. In the absence of Viaat, GABA-synthesizing enzymes are partially lost from presynaptic terminals. Since GABA and glycine compete for vesicular uptake, these data point to a close association of Viaat with GABA-synthesizing enzymes as a key factor in specifying GABAergic neuronal phenotypes.

    Neuron 2006;50;4;575-87

  • Branching and nucleokinesis defects in migrating interneurons derived from doublecortin knockout mice.

    Kappeler C, Saillour Y, Baudoin JP, Tuy FP, Alvarez C, Houbron C, Gaspar P, Hamard G, Chelly J, Métin C and Francis F

    Département de Génétique et Développement, Institut Cochin, F-75014 Paris, France.

    Type I lissencephaly results from mutations in the doublecortin (DCX) and LIS1 genes. We generated Dcx knockout mice to further understand the pathophysiological mechanisms associated with this cortical malformation. Dcx is expressed in migrating interneurons in developing human and mouse brains. Video microscopy analyses of such tangentially migrating neuron populations derived from the medial ganglionic eminence show defects in migratory dynamics. Specifically, the formation and division of growth cones, leading to the production of new branches, are more frequent in knockout cells, although branches are less stable. Dcx-deficient cells thus migrate in a disorganized manner, extending and retracting short branches and making less long-distant movements of the nucleus. Despite these differences, migratory speeds and distances remain similar to wild-type cells. These novel data thus highlight a role for Dcx, a microtubule-associated protein enriched at the leading edge in the branching and nucleokinesis of migrating interneurons.

    Human molecular genetics 2006;15;9;1387-400

  • Comprehensive identification of phosphorylation sites in postsynaptic density preparations.

    Trinidad JC, Specht CG, Thalhammer A, Schoepfer R and Burlingame AL

    Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.

    In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.

    Funded by: NCRR NIH HHS: RR14606; Wellcome Trust

    Molecular & cellular proteomics : MCP 2006;5;5;914-22

  • High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells.

    Fu J, Tay SS, Ling EA and Dheen ST

    Molecular Neurobiology Laboratory, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

    Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs.

    Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2'-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically.

    Results: High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells.

    The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the basis for the defective neural tube patterning observed in embryos of diabetic pregnancies.

    Diabetologia 2006;49;5;1027-38

  • Inactivation of aPKClambda results in the loss of adherens junctions in neuroepithelial cells without affecting neurogenesis in mouse neocortex.

    Imai F, Hirai S, Akimoto K, Koyama H, Miyata T, Ogawa M, Noguchi S, Sasaoka T, Noda T and Ohno S

    Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan.

    In developing mammalian telencephalon, the loss of adherens junctions and cell cycle exit represent crucial steps in the differentiation of neuroepithelial cells into neurons, but the relationship between these cellular events remains obscure. Atypical protein kinase C (aPKC) is known to contribute to junction formation in epithelial cells and to cell fate determination for Drosophila neuroblasts. To elucidate the functions of aPKClambda, one out of two aPKC members, in mouse neocortical neurogenesis, a Nestin-Cre mediated conditional gene targeting system was employed. In conditional aPKClambda knockout mice, neuroepithelial cells of the neocortical region lost aPKClambda protein at embryonic day 15 and demonstrated a loss of adherens junctions, retraction of apical processes and impaired interkinetic nuclear migration that resulted in disordered neuroepithelial tissue architecture. These results are evidence that aPKClambda is indispensable for the maintenance of adherens junctions and may function in the regulation of adherens junction integrity upon differentiation of neuroepithelial cells into neurons. In spite of the loss of adherens junctions in the neuroepithelium of conditional aPKClambda knockout mice, neurons were produced at a normal rate. Therefore, we concluded that, at least in the later stages of neurogenesis, regulation of cell cycle exit is independent of adherens junctions.

    Development (Cambridge, England) 2006;133;9;1735-44

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Neurogenin 2 is required for the development of ventral midbrain dopaminergic neurons.

    Kele J, Simplicio N, Ferri AL, Mira H, Guillemot F, Arenas E and Ang SL

    Laboratory of Molecular Neurobiology, MBB, Karolinska Institutet, Retzius building A1, Stockholm, Sweden.

    Proneural genes are crucial regulators of neurogenesis and subtype specification in many areas of the nervous system; however, their function in dopaminergic neuron development is unknown. We report that proneural genes have an intricate pattern of expression in the ventricular zone of the ventral midbrain, where mesencephalic dopaminergic neurons are generated. Neurogenin 2 (Ngn2) and Mash1 are expressed in the ventral midline, while Ngn1, Ngn2 and Mash1 are co-localized more laterally in the ventricular zone. Ngn2 is also expressed in an intermediate zone immediately adjacent to the ventricular zone at the ventral midline. To examine the function of these genes, we analyzed mutant mice in which one or two of these genes were deleted (Ngn1, Ngn2 and Mash1) or substituted (Mash1 in the Ngn2 locus). Our results demonstrate that Ngn2 is required for the differentiation of Sox2(+) ventricular zone progenitors into Nurr1(+) postmitotic dopaminergic neuron precursors in the intermediate zone, and that it is also likely to be required for their subsequent differentiation into tyrosine hydroxylase-positive dopaminergic neurons in the marginal zone. Although Mash1 normally has no detectable function in dopaminergic neuron development, it could partially rescue the generation of dopaminergic neuron precursors in the absence of Ngn2. These results demonstrate that Ngn2 is uniquely required for the development of midbrain dopaminergic neurons.

    Funded by: Medical Research Council: MC_U117570528, MC_U117570533; Parkinson's UK: G-4068

    Development (Cambridge, England) 2006;133;3;495-505

  • Sex-differences in age-related cognitive decline in C57BL/6J mice associated with increased brain microtubule-associated protein 2 and synaptophysin immunoreactivity.

    Benice TS, Rizk A, Kohama S, Pfankuch T and Raber J

    Department of Behavioral Neuroscience, Oregon Health and Science University, 3181 Southwest Sam Jackson Park Road, Portland, 97239, USA.

    Understanding cognitive aging is becoming more important as the elderly population grows. Here, the effects of age and sex on learning and memory performance were compared in female and male young (3-4 months old) middle-aged (10-12 months old) and old (18-20 months old) wild-type C57BL/6J mice. Old males and females performed worse than young or middle-aged mice in novel location, but not novel object recognition tasks. Old mice, of both sexes, also showed impaired spatial water maze performance during training compared with young or middle-aged mice, however only old females failed to show robust spatial bias during probe trials. While there was no age-difference in passive avoidance performance for males, females showed an age-related decline. There was no difference in cognitive performance between young and middle-age mice of either sex on any task. Cognitive performance was associated with alterations in immunoreactivity of microtubule-associated protein 2-positive dendrites and synaptophysin-positive pre-synaptic terminals in hippocampal CA1, CA3, and dentate, entorhinal cortex, and central nucleus of amygdala. Overall, microtubule-associated protein 2 immunoreactivity was increased in old females compared with both young and middle-age females with no significant difference in males. In contrast, synaptophysin immunoreactivity increased from young to middle-age in females, and from middle-age to old in males; females had higher levels of synaptophysin immunoreactivity than males in middle-age only. Elevated levels of microtubule-associated protein 2 and synaptophysin may constitute a compensatory response to age-related functional decline in mice.

    Funded by: NCRR NIH HHS: RR 00163; NIA NIH HHS: EMF AG-NS-0201, R01 AG20904; NINDS NIH HHS: T32 NS007466-05

    Neuroscience 2006;137;2;413-23

  • Reduced expression of kinase-associated phosphatase in cortical dendrites of MAP2-deficient mice.

    Iriuchijima N, Sato-Harada R, Takano M, Fujio K, Sato T, Goto F and Harada A

    Laboratory of Molecular Traffic, Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371-8512, Japan.

    We previously demonstrated that cAMP-dependent protein kinase was reduced in the dendrites of MAP2-deficient mice. In this study, we compared the expression of various protein phosphatases (PPs) between wild-type and map2(-/-) dendrites. Kinase-associated phosphatase (KAP) was the only PP which showed difference between the two phenotypes: (1) the expression of KAP was reduced in map2(-/-) cortical dendrites, and (2) the amount of KAP bound to microtubules was reduced in map2(-/-) brains. We also demonstrated in cultured neuroblastoma cells that KAP is not only expressed in dividing cells, but also in the neurites of differentiated cells. Our findings propose that KAP, which has been reported to function in cell-cycle control, has an as yet uncovered role in regulating dendritic functions. We also propose MAP2-deficient mice as an ideal system for identifying protein phosphatases essential for dendritic functions.

    Biochemical and biophysical research communications 2005;338;2;1216-21

  • Cystathionine beta-synthase, a key enzyme for homocysteine metabolism, is preferentially expressed in the radial glia/astrocyte lineage of developing mouse CNS.

    Enokido Y, Suzuki E, Iwasawa K, Namekata K, Okazawa H and Kimura H

    Department of Molecular Geneticsy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan. enokido.npat@mri.tmd.ac.jp

    Cystathionine beta-synthase (CBS; EC 4.2.1.22) is a key enzyme in the generation of cysteine from methionine. A deficiency of CBS leads to homocystinuria, an inherited human disease characterized by mental retardation, seizures, psychiatric disturbances, skeletal abnormalities, and vascular disorders; however, the underlying mechanisms remain largely unknown. Here, we show the regional and cellular distribution of CBS in the adult and developing mouse brain. In the adult mouse brain, CBS was expressed ubiquitously, but it is expressed most intensely in the cerebellar molecular layer and hippocampal dentate gyrus. Immunohistochemical analysis revealed that CBS is preferentially expressed in cerebellar Bergmann glia and in astrocytes throughout the brain. At early developmental stages, CBS was expressed in neuroepithelial cells in the ventricular zone, but its expression changed to radial glial cells and then to astrocytes during the late embryonic and neonatal periods. CBS was most highly expressed in juvenile brain, and a striking induction was observed in cultured astrocytes in response to EGF, TGF-alpha, cAMP, and dexamethasone. Moreover, CBS was significantly accumulated in reactive astrocytes in the hippocampus after kainic acid-induced seizures, and cerebellar morphological abnormalities were observed in CBS-deficient mice. Taken together, these results suggest that CBS plays a crucial role in the development and maintenance of the CNS and that radial glia/astrocyte dysfunction might be involved in the complex neuropathological features associated with abnormal homocysteine metabolism.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2005;19;13;1854-6

  • Cyclin-dependent kinase 5 is essential for neuronal cell cycle arrest and differentiation.

    Cicero S and Herrup K

    Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

    Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase with significant homology to cell cycle-related Cdks but is not believed to be active in a typical cell cycle. In Cdk5-deficient embryos and Cdk5 chimeras, migration and survival of postmitotic neurons is compromised in a cell-autonomous manner. In the present study, we show that loss of Cdk5 leads to both failure of neuronal differentiation and loss of cell cycle control. Using specific cytoskeletal proteins as indices of neuronal differentiation, we find that Cdk5-deficient neurons are significantly arrested or delayed in their developmental program both in vivo and in vitro. For example, immunocytochemistry of embryonic day 16 (E16) cortex reveals that the expression of microtubule-associated protein 2c (Map-2c), a marker of mature neurons, is nearly absent in Cdk5(-/-) cells that have migrated to the cortical plate while these same cells continue to express nestin. Similarly, in vitro, Map-2-positive cells are rare in cultures from E16 Cdk5(-/-) embryos. Cell cycle control is also deficient in Cdk5(-/-) cells. In vivo, neurons engaged in cell cycle activities are found in the cortical plate, and, in vitro, class III beta-tubulin-positive cells continue to label with bromodeoxyuridine even after 5 d of incubation. Transfection of a wild-type Cdk5 construct reveals that cell cycle control can be regained in Cdk5(-/-) cells by overexpression of Cdk5. These data indicate that Cdk5 is necessary for both neuronal differentiation and cell cycle inhibition.

    Funded by: NIA NIH HHS: P50-AG08012; NINDS NIH HHS: R01-NS20591

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;42;9658-68

  • Gli3 is required for the specification and differentiation of preplate neurons.

    Theil T

    Institute for Animal Developmental and Molecular Biology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany. ttheil@anatom.uni-tuebingen.de

    During corticogenesis, the cerebral cortex develops a laminated structure which is essential for its function. Early born neurons of the preplate and its derivatives, the marginal zone (MZ) and the subplate (SP), serve as a framework during the cortical lamination process. Here, I report on defects in the generation and specification of these early born cortical neurons in extra-toes (Xt(J)) mice which are defective for the Gli3 zinc finger transcription factor. The Gli3 mutation dramatically disrupts early steps in the cortical lamination process. The MZ, SP and the cortical plate (CP) do not form layers but cortical neurons are arranged in clusters. These defects start to become evident at E12.5 when the cortex forms several protrusions and the ventricular zone becomes undulated. At this stage, cortical progenitor cells start to loose their apical/basal cell polarity correlating with an ectopic expression of Wnt7b in the ventricular zone. In addition, the cellular composition of the preplate is severely altered. Cajal-Retzius cells are reduced in numbers while early born Calretinin(+) neurons are overproduced. These results show that multiple aspects of corticogenesis including the organization of the venticular zone, the apical/basal cell polarity of cortical progenitors and the differentiation of early born cortical neurons are affected in the Gli3 mutant.

    Developmental biology 2005;286;2;559-71

  • Ectopic HOXA5 expression results in abnormal differentiation, migration and p53-independent cell death of superficial dorsal horn neurons.

    Abbott MA, Joksimovic M and Tuggle CK

    Interdepartmental Genetics, Iowa State University, Ames, IA 50011, USA.

    Previously, we reported a line of mice (Hoxa5SV2) that ectopically expresses HOXA5 in the developing cervical and brachial dorsal spinal cord. Animals from this line exhibited a clear loss of cells in the outer lamina of the mature dorsal horn that coincided with an adult phenotype of sensory and motor defects of the forelimb. In this report, we examined the etiology of lost dorsal horn cells. Cells normally fated to populate the outer laminae I-III of the dorsal horn migrated inappropriately, as the percentage of laterally positioned cells in the dorsal horn was significantly reduced in Hoxa5SV2 transgenics. Apoptosis was a major cause of cell loss while proliferation of neurons was not affected in Hoxa5SV2 animals. Although Hoxa5 has been shown in vitro to regulate p53 expression and cause p53-dependent apoptosis, p53 was not required in vivo for the inappropriate apoptosis seen in Hoxa5SV2 mice, or for the normal death of motor neurons. Normal apoptosis is not dependent on Hoxa5, as the level of ventral horn motor neuron apoptosis was not changed in Hoxa5 null animals. As a possible cause of aberrant migration and/or apoptosis of dorsal neurons, misexpression of cell type markers was demonstrated. Further, the expression pattern of laminar markers was altered and sensory fibers aberrantly penetrated the outer lamina of mutants. Our evidence suggests that the loss of dorsal horn neurons in Hoxa5SV2 mutants was due to misexpression of dorsal horn neuronal markers, aberrant migration, and inappropriate apoptosis.

    Funded by: NICHD NIH HHS: R01-HD38463

    Brain research. Developmental brain research 2005;159;2;87-97

  • Phosphorylation of Neurogenin2 specifies the migration properties and the dendritic morphology of pyramidal neurons in the neocortex.

    Hand R, Bortone D, Mattar P, Nguyen L, Heng JI, Guerrier S, Boutt E, Peters E, Barnes AP, Parras C, Schuurmans C, Guillemot F and Polleux F

    Department of Phamacology, Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. Here we demonstrate that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2(Y241F) mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. Our results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity.

    Funded by: NINDS NIH HHS: NS047701-01, P30 NS45892-01

    Neuron 2005;48;1;45-62

  • Neuron-specific relaxation of Igf2r imprinting is associated with neuron-specific histone modifications and lack of its antisense transcript Air.

    Yamasaki Y, Kayashima T, Soejima H, Kinoshita A, Yoshiura K, Matsumoto N, Ohta T, Urano T, Masuzaki H, Ishimaru T, Mukai T, Niikawa N and Kishino T

    Department of Human Genetics, Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan.

    The mouse insulin-like growth factor II receptor (Igf2r) gene and its antisense transcript Air are reciprocally imprinted in most tissues, but in the brain, Igf2r is biallelically expressed despite the imprinted Air expression. To investigate the molecular mechanisms of such brain-specific relaxation of Igf2r imprinting, we analyzed its expression and epigenetic modifications in neurons, glial cells and fibroblasts by the use of primary cortical cell cultures. In glial cells and fibroblasts, Igf2r was maternally expressed and Air was paternally expressed, whereas in the primary cultured neurons, Igf2r was biallelically expressed and Air was not expressed. In the differentially methylated region 2 (DMR2), which includes the Air promoter, allele-specific DNA methylation, differential H3 and H4 acetylation and H3K4 and K9 di-methylation were maintained in each cultured cell type. In DMR1, which includes the Igf2r promoter, maternal-allele-specific DNA hypomethylation, histones H3 and H4 acetylation and H3K4 di-methylation were apparent in glial cells and fibroblasts. However, in neurons, biallelic DNA hypomethylation and biallelic histones H3 and H4 acetylation and H3K4 di-methylation were detected. These data indicate that lack of reciprocal imprinting of Igf2r and Air in the brain results from neuron-specific relaxation of Igf2r imprinting associated with neuron-specific histone modifications in DMR1 and lack of Air expression. Our observation of biallelic Igf2r expression with no Air expression in neurons sheds light on the function of Air as a critical effector in Igf2r silencing and suggests that neuron-specific epigenetic modifications related to the lineage determination of neural stem cells play a critical role in controlling imprinting by antisense transcripts.

    Human molecular genetics 2005;14;17;2511-20

  • Complete loss of Ndel1 results in neuronal migration defects and early embryonic lethality.

    Sasaki S, Mori D, Toyo-oka K, Chen A, Garrett-Beal L, Muramatsu M, Miyagawa S, Hiraiwa N, Yoshiki A, Wynshaw-Boris A and Hirotsune S

    Department of Neuro-Science, Research Center for Genomic Medicine, Saitama Medical School, Japan. shinjih@med.osaka-cu.ac.jp

    Regulation of cytoplasmic dynein and microtubule dynamics is crucial for both mitotic cell division and neuronal migration. NDEL1 was identified as a protein interacting with LIS1, the protein product of a gene mutated in the lissencephaly. To elucidate NDEL1 function in vivo, we generated null and hypomorphic alleles of Ndel1 in mice by targeted gene disruption. Ndel1(-/-) mice were embryonic lethal at the peri-implantation stage like null mutants of Lis1 and cytoplasmic dynein heavy chain. In addition, Ndel1(-/-) blastocysts failed to grow in culture and exhibited a cell proliferation defect in inner cell mass. Although Ndel1(+/-) mice displayed no obvious phenotypes, further reduction of NDEL1 by making null/hypomorph compound heterozygotes (Ndel1(cko/-)) resulted in histological defects consistent with mild neuronal migration defects. Double Lis1(cko/+)-Ndel1(+/-) mice or Lis1(+/-)-Ndel1(+/-) mice displayed more severe neuronal migration defects than Lis1(cko/+)-Ndel1(+/)(+) mice or Lis1(+/-)-Ndel1(+/+) mice, respectively. We demonstrated distinct abnormalities in microtubule organization and similar defects in the distribution of beta-COP-positive vesicles (to assess dynein function) between Ndel1 or Lis1-null MEFs, as well as similar neuronal migration defects in Ndel1- or Lis1-null granule cells. Rescue of these defects in mouse embryonic fibroblasts and granule cells by overexpressing LIS1, NDEL1, or NDE1 suggest that NDEL1, LIS1, and NDE1 act in a common pathway to regulate dynein but each has distinct roles in the regulation of microtubule organization and neuronal migration.

    Funded by: NINDS NIH HHS: NS41030, R01 NS041030

    Molecular and cellular biology 2005;25;17;7812-27

  • P2X(7) receptor-mRNA and -protein in the mouse retina; changes during retinal degeneration in BALBCrds mice.

    Franke H, Klimke K, Brinckmann U, Grosche J, Francke M, Sperlagh B, Reichenbach A, Liebert UG and Illes P

    Rudolf-Boehm-lnstitute of Pharmacology and Toxicology, University of Leipzig, D-04107 Leipzig, Germany.

    A combined real-time PCR/immunohistochemistry study was carried out to investigate whether P2X(7) receptors, known to induce apoptosis and necrosis, may be causally related to the process of retinal degeneration in BALBCrds mice. In the retinae of BALBCrds mice, P2X(7) receptor-mRNA was the highest at an age of 20-40 days, and declined afterwards. At the same time, the P2X(7) receptor-message was constantly low in the retina of control BALBC mice until postnatal day 100. The receptor-mRNA in total brain tissue of both strains of mice was comparable with that of BALBCrds retinae. Double immunofluorescence in combination with laser scanning microscopy was used to study the distribution of P2X(7) receptor-immunoreactivity (IR) on neurons and different glial cell types of the retina. An exclusively neuronal localization of P2X(7)-IR in the ganglion cell layer was found by using either anti-neuronal nuclei or microtubule associated protein-2 as neuronal markers. There was a slight age-dependent decrease in the abundance of neuronal P2X(7)-IR both in BALBCrds or BALBC mice. P2X(7)-IR failed to co-localize with any of the non-neuronal markers used to stain microglial or Müller glial cells. No P2X(7) receptor-IR was found in the retinal ganglion cell layer of P2X(7)(-/-) animals, when compared with the control littermates. Hence, we suggest that, in BALBCrds mice, an early up-regulation of neuronal P2X(7) receptors may cause injury of retinal neurons and thereby functionally contribute to the retinal damage.

    Neurochemistry international 2005;47;4;235-42

  • The Rho-specific GEF Lfc interacts with neurabin and spinophilin to regulate dendritic spine morphology.

    Ryan XP, Alldritt J, Svenningsson P, Allen PB, Wu GY, Nairn AC and Greengard P

    Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, 1230 York Avenue, New York, NY 10021, USA.

    Neurabin and spinophilin are homologous protein phosphatase 1 and actin binding proteins that regulate dendritic spine function. A yeast two-hybrid analysis using the coiled-coil domain of neurabin revealed an interaction with Lfc, a Rho GEF. Lfc was highly expressed in brain, where it interacted with either neurabin or spinophilin. In neurons, Lfc was largely found in the shaft of dendrites in association with microtubules but translocated to spines upon neuronal stimulation. Moreover, expression of Lfc resulted in reduction in spine length and size. Both the translocation and the effect on spine morphology depended on the coiled-coil domain of Lfc. Coexpression of neurabin or spinophilin with Lfc resulted in their clustering together with F-actin, a process that depended on Rho activity. Thus, interaction between Lfc and neurabin/spinophilin selectively regulates Rho-dependent organization of F-actin in spines and is a link between the microtubule and F-actin cytoskeletons in dendrites.

    Funded by: NIDA NIH HHS: DA10044, DA17919, P01 DA010044; NIMH NIH HHS: MH40899

    Neuron 2005;47;1;85-100

  • Constitutively active cytoplasmic c-Jun N-terminal kinase 1 is a dominant regulator of dendritic architecture: role of microtubule-associated protein 2 as an effector.

    Björkblom B, Ostman N, Hongisto V, Komarovski V, Filén JJ, Nyman TA, Kallunki T, Courtney MJ and Coffey ET

    Turku Centre for Biotechnology, Abo Akademi and Turku University, BioCity, FIN-20521 Turku, Finland.

    Normal functioning of the nervous system requires precise regulation of dendritic shape and synaptic connectivity. Here, we report a severe impairment of dendritic structures in the cerebellum and motor cortex of c-Jun N-terminal kinase 1 (JNK1)-deficient mice. Using an unbiased screen for candidate mediators, we identify the dendrite-specific high-molecular-weight microtubule-associated protein 2 (MAP2) as a JNK substrate in the brain. We subsequently show that MAP2 is phosphorylated by JNK in intact cells and that MAP2 proline-rich domain phosphorylation is decreased in JNK1-/- brain. We developed compartment-targeted JNK inhibitors to define whether a functional relationship exists between the physiologically active, cytosolic pool of JNK and dendritic architecture. Using these, we demonstrate that cytosolic, but not nuclear, JNK determines dendritic length and arbor complexity in cultured neurons. Moreover, we confirm that MAP2-dependent process elongation is enhanced after activation of JNK. Using JNK1-/- neurons, we reveal a dominant role for JNK1 over ERK in regulating dendritic arborization, whereas ERK only regulates dendrite shape under conditions in which JNK activity is low (JNK1-/- neurons). These results reveal a novel antagonism between JNK and ERK, potentially providing a mechanism for fine-tuning the dendritic arbor. Together, these data suggest that JNK phosphorylation of MAP2 plays an important role in defining dendritic architecture in the brain.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;27;6350-61

  • Generation and characterization of Rac3 knockout mice.

    Corbetta S, Gualdoni S, Albertinazzi C, Paris S, Croci L, Consalez GG and de Curtis I

    Department of Molecular Biology and Functional Genomics, San Raffaele Scientific Institute, 20132 Milan, Italy.

    Rac proteins are members of the Rho family of GTPases involved in the regulation of actin dynamics. The three highly homologous Rac proteins in mammals are the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3. We show here that Rac3 mRNA is widely and specifically expressed in the developing nervous system, with highest concentration at embryonic day 13 in the dorsal root ganglia and ventral spinal cord. At postnatal day 7 Rac3 appears particularly abundant in populations of projection neurons in several regions of the brain, including the fifth layer of the cortex and the CA1-CA3 region of the hippocampus. We generated mice deleted for the Rac3 gene with the aim of analyzing the function of this GTPase in vivo. Rac3 knockout animals survive embryogenesis and show no obvious developmental defects. Interestingly, specific behavioral differences were detected in the Rac3-deficient animals, since motor coordination and motor learning on the rotarod was superior to that of their wild-type littermates. No obvious histological or immunohistological differences were observed at major sites of Rac3 expression. Our results indicate that, in vivo, Rac3 activity is not strictly required for normal development in utero but may be relevant to later events in the development of a functional nervous system.

    Funded by: Telethon: GGP02190

    Molecular and cellular biology 2005;25;13;5763-76

  • Ephrin signalling controls brain size by regulating apoptosis of neural progenitors.

    Depaepe V, Suarez-Gonzalez N, Dufour A, Passante L, Gorski JA, Jones KR, Ledent C and Vanderhaeghen P

    Institut de Recherches Interdisciplinaires en Biologie Humaine et Moléculaire (IRIBHM), University of Brussels, Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium.

    Mechanisms controlling brain size include the regulation of neural progenitor cell proliferation, differentiation, survival and migration. Here we show that ephrin-A/EphA receptor signalling plays a key role in controlling the size of the mouse cerebral cortex by regulating cortical progenitor cell apoptosis. In vivo gain of EphA receptor function, achieved through ectopic expression of ephrin-A5 in early cortical progenitors expressing EphA7, caused a transient wave of neural progenitor cell apoptosis, resulting in premature depletion of progenitors and a subsequent dramatic decrease in cortical size. In vitro treatment with soluble ephrin-A ligands similarly induced the rapid death of cultured dissociated cortical progenitors in a caspase-3-dependent manner, thereby confirming a direct effect of ephrin/Eph signalling on apoptotic cascades. Conversely, in vivo loss of EphA function, achieved through EphA7 gene disruption, caused a reduction in apoptosis occurring normally in forebrain neural progenitors, resulting in an increase in cortical size and, in extreme cases, exencephalic forebrain overgrowth. Together, these results identify ephrin/Eph signalling as a physiological trigger for apoptosis that can alter brain size and shape by regulating the number of neural progenitors.

    Nature 2005;435;7046;1244-50

  • Mash1 and Math3 are required for development of branchiomotor neurons and maintenance of neural progenitors.

    Ohsawa R, Ohtsuka T and Kageyama R

    Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.

    Basic helix-loop-helix (bHLH) transcription factors are known to play important roles in neuronal determination and differentiation. However, their exact roles in neural development still remain to be determined because of the functional redundancy. Here, we examined the roles of neural bHLH genes Mash1 and Math3 in the development of trigeminal and facial branchiomotor neurons, which derive from rhombomeres 2-4. In Math3-null mutant mice, facial branchiomotor neurons are misspecified, and both trigeminal and facial branchiomotor neurons adopt abnormal migratory pathways. In Mash1;Math3 double-mutant mice, trigeminal and facial branchiomotor neurons are severely reduced in number partly because of increased apoptosis. In addition, neurons with migratory defects are intermingled over the midline from either side of the neural tube. Furthermore, oligodendrocyte progenitors of rhombomere 4 are reduced in number. In the absence of Mash1 and Math3, expression of Notch signaling components is severely downregulated in rhombomere 4 and neural progenitors are not properly maintained, which may lead to intermingling of neurons and a decrease in oligodendrocyte progenitors. These results indicate that Mash1 and Math3 not only promote branchiomotor neuron development but also regulate the subsequent oligodendrocyte development and the cytoarchitecture by maintaining neural progenitors through Notch signaling.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;25;5857-65

  • Frizzled9 protein is regionally expressed in the developing medial cortical wall and the cells derived from this region.

    Zhao C and Pleasure SJ

    Department of Neurology, Graduate Programs in Neuroscience, Developmental Biology and Biomedical Sciences, Room S-268, 513 Parnassus Avenue, University of California, San Francisco, CA 94143, USA. zhaocj@seu.edu.cn

    We used a newly generated antiserum specific for the Wnt receptor Frizzled9 to examine the distribution of this protein in the telencephalon. Previous studies showed expression of the mRNA for Frizzled9 in a medial high to lateral low gradient during cortical development 6. Our studies with the Frizzled9 antiserum supported this localization during mid-gestation mouse embryos. Later in gestation, the expression of Frizzled9 persisted in all cellular derivatives of the medial cortical neuroepithelium. From birth through adulthood, Frizzled9 continues to be expressed in the principal neuronal cells of the hippocampus. Interestingly, Frizzled9 was also expressed by astrocytic cells and dividing neuronal precursors in the adult dentate gyrus. Thus, Frizzled9 expression marks are a regional marker of medially derived cortical derivatives throughout rodent life and will be a useful marker in the study of regulation of cortical arealization.

    Brain research. Developmental brain research 2005;157;1;93-7

  • Differential expression of CRMP1, CRMP2A, CRMP2B, and CRMP5 in axons or dendrites of distinct neurons in the mouse brain.

    Bretin S, Reibel S, Charrier E, Maus-Moatti M, Auvergnon N, Thevenoux A, Glowinski J, Rogemond V, Prémont J, Honnorat J and Gauchy C

    Institut National de la Santé et de la Recherche Médicale U114, Collège de France, 75231 Paris, France. christian.gauchy@college-de-france.fr

    CRMP1, CRMP2, and CRMP5 have been identified as cytosolic proteins relaying semaphorin 3A signalling, one of the molecular cues conducting axon and dendrite growth and guidance. They are highly expressed during brain ontogenesis, but, because of their lower levels in the adult, their distribution in the mature brain is poorly documented. By using specific antibodies, we investigated the cellular distribution of these CRMPs in different adult brain structures and in neural cell cultures with a special focus on the splice variants CRMP2A and CRMP2B. In brain sections of adult mouse, CRMP1, CRMP2B, and CRMP5 were located predominantly in dendrites of specific neuronal populations, such as cortical pyramidal neurons, hippocampal CA1 pyramidal cells, or Purkinje cerebellar cells. On the contrary, CRMP2A was specifically associated with axons of the corpus callosum, bundles of the striatum, and mossy fibers of the hippocampus. In cultures of cortical neurons, CRMP1, CRMP2A, CRMP2B, and CRMP5 were equally distributed throughout cell bodies, axons, or dendrites of neurons, whereas CRMP2A and CRMP5 were completely absent from Purkinje cerebellar cells in 12-day-old animals. By comparison, oligodendrocytes exclusively express CRMP2B and CRMP5 in cell bodies and processes both in situ in the adult brain and in primary cultures. Overall, our results demonstrate specific subcellular localizations of CRMP1, CRMP2A, CRMP2B, and CRMP5 depending on cell types, neuronal compartment, and developmental stage. This study suggests that, beyond their signalling function in axon outgrowth and guidance, CRMPs also play a role in mature neurons both in axons and in dendrites.

    The Journal of comparative neurology 2005;486;1;1-17

  • Baalc, a marker of mesoderm and muscle.

    Satoskar AA, Tanner SM, Weinstein M, Qualman SJ and de la Chapelle A

    Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, 420 West 12th Avenue, TMRF 646, Columbus, OH 43210, USA.

    Transcripts of the Brain and Acute Leukemia, Cytoplasmic (BAALC) gene are expressed in human neuroectodermal tissues and in CD34-positive bone marrow cells. High transcript levels occur in leukemic blasts from some patients with acute myeloid leukemia (AML), where high expression is an independent marker of poor prognosis. To gain insight into the hitherto unknown function of BAALC/Baalc, we studied its protein expression in embryonic and adult mouse tissue by immunohistochemical analysis. Baalc protein was mainly expressed in developing and mature muscle cells (cardiac, skeletal, and smooth) beginning on day E9 (heart). Signal was seen in the pre-muscle mesodermal cells of the dermatomyotome regions, and the derivatives of the lateral plate and intermediate mesoderm such as smooth muscle wall of the esophagus, stomach, the gut tube, bronchi, small blood vessels, and urinary bladder. This pattern continued through the late embryonic stages into adulthood. Baalc appeared to localize in the cytoplasm, adjacent to the cell membrane. This is distinctly observed in adult skeletal muscle cells. Baalc co-localized with known muscle-associated proteins but not with neural crest or neuronal markers. Scattered expression in adult bone marrow hematopoietic cells and weak expression in the brain neuropil also occurred. In conclusion, BAALC/Baalc is a marker of the mesodermal lineage, especially muscle.

    Funded by: NCI NIH HHS: CA09338 T32, CA098933, CA16058

    Gene expression patterns : GEP 2005;5;4;463-73

  • Expression of MUK/DLK/ZPK, an activator of the JNK pathway, in the nervous systems of the developing mouse embryo.

    Hirai S, Kawaguchi A, Suenaga J, Ono M, Cui DF and Ohno S

    Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9, Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. sh3312@med.yokohama-cu.ac.jp

    C-Jun N-terminal kinase (JNK) is implicated in regulating the various cellular events during neural development that include differentiation, apoptosis and migration. MUK/DLK/ZPK is a MAP kinase kinase kinase (MAPKKK) enzyme that activates JNK via MAP kinase kinases (MAPKK) such as MKK7. We show here that the expression of MUK/DLK/ZPK protein in the developing mouse embryo is almost totally specific for the neural tissues, including central, peripheral, and autonomic nervous systems. The only obvious exception is the liver, in which the protein is temporally expressed at around E11. The expression becomes obvious in the neurons of the brain and neural crest tissues at embryonic day 10 (E10) after neuron production is initiated. By E14, MUK/DLK/ZPK proteins are found in various neural tissues including the brain, spinal cord, sensory ganglia (such as trigeminal and dorsal root ganglia), and the sympathetic and visceral nerves. The localization of MUK/DLK/ZPK protein in neural cells almost consistently overlapped that of betaIII-tubulin, a neuron specific tubulin isoform, and both proteins were more concentrated in axons than in cell bodies and dendrites. The intensely overlapping localization of betaIII-tubulin and MUK/DLK/ZPK indicated that this protein kinase is tightly associated with the microtubules of neurons.

    Gene expression patterns : GEP 2005;5;4;517-23

  • FGFR3 regulates brain size by controlling progenitor cell proliferation and apoptosis during embryonic development.

    Inglis-Broadgate SL, Thomson RE, Pellicano F, Tartaglia MA, Pontikis CC, Cooper JD and Iwata T

    Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine, University of Glasgow, Beatson Laboratories for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK.

    Mice with the K644E kinase domain mutation in fibroblast growth factor receptor 3 (Fgfr3) (EIIa;Fgfr3(+/K644E)) exhibited a marked enlargement of the brain. The brain size was increased as early as E11.5, not secondary to the possible effect of Fgfr3 activity in the skeleton. Furthermore, the mutant brains showed a dramatic increase in cortical thickness, a phenotype opposite to that in FGF2 knockout mice. Despite this increased thickness, cortical layer formation was largely unaffected and no cortical folding was observed during embryonic days 11.5-18.5 (E11.5-E18.5). Measurement of cortical thickness revealed an increase of 38.1% in the EIIa;Fgfr3(+/K644E) mice at E14.5 and the advanced appearance of the cortical plate was frequently observed at this stage. Unbiased stereological analysis revealed that the volume of the ventricular zone (VZ) was increased by more than two fold in the EIIa;Fgfr3(+/K644E) mutants at E14.5. A relatively mild increase in progenitor cell proliferation and a profound decrease in developmental apoptosis during E11.5-E14.5 most likely accounts for the dramatic increase in total telecephalic cell number. Taken together, our data suggest a novel function of Fgfr3 in controlling the development of the cortex, by regulating proliferation and apoptosis of cortical progenitors.

    Funded by: Biotechnology and Biological Sciences Research Council: BBS/B/08736; NINDS NIH HHS: NS41930, NS44310

    Developmental biology 2005;279;1;73-85

  • Proteomic analysis of in vivo phosphorylated synaptic proteins.

    Collins MO, Yu L, Coba MP, Husi H, Campuzano I, Blackstock WP, Choudhary JS and Grant SG

    Division of Neuroscience, University of Edinburgh, Edinburgh EH8 9JZ, UK.

    In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.

    The Journal of biological chemistry 2005;280;7;5972-82

  • Novel function of neuronal PAS domain protein 1 in erythropoietin expression in neuronal cells.

    Ohsawa S, Hamada S, Kakinuma Y, Yagi T and Miura M

    Department of Genetics, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.

    The basic helix loop helix-PAS (bHLH-PAS) transcription factors have diverse roles in physiologic responses to the environment and in early development. One bHLH-PAS protein, neuronal PAS domain protein 1 (NPAS1), is reported to be expressed only in the central nervous system beginning at the late embryonic stage, but its function is unknown. Using an anti-NPAS1 antibody, we have shown that NPAS1 expression in cerebral cortex was observed first around embryonic Day 16.5 (E16.5) and was then dispersed throughout the region as cortical development progressed. From the similarity with hypoxia-inducible factor-1alpha (HIF-1alpha), we investigated whether NPAS1 regulates transcription of erythropoietin (EPO), which is the target of HIF-1alpha and is expressed in the brain during the early developmental stage. In the present study, we have shown that NPAS1 binds to the enhancer region of Epo in vivo. A luciferase reporter assay revealed a repressive effect of NPAS1 on hypoxia-responsive element-regulated gene expression. These results raise the possibility that NPAS1 plays a role in late central nervous system development by modulating EPO expression in response to cellular oxygen level.

    Journal of neuroscience research 2005;79;4;451-8

  • Neuronal pentraxin with chromo domain (NPCD) is a novel class of protein expressed in multiple neuronal domains.

    Chen B and Bixby JL

    Department of Molecular & Cellular Pharmacology, Neurological Surgery, and Neuroscience Program, The Miami Project to Cure Paralysis, University of Miami School of Medicine, Lois Pope LIFE Center, Miami, Florida 33136, USA.

    The receptor tyrosine phosphatase PTPRO is involved in axon guidance, but its intracellular signaling mechanisms are unknown. Signals generated through PTPRO must involve interaction of the intracellular domain with substrates and/or signaling proteins. By screening for proteins interacting with PTPRO's intracellular domain, we have identified a new class of cytoplasmic protein. This novel protein, NPCD (Neuronal Pentraxin with Chromo Domain), has multiple cytoplasmic isoforms generated by alternative splicing that are selectively expressed in neurons. These cytoplasmic NPCD isoforms are composed of a neuronal pentraxin domain (formerly thought exclusively extracellular) linked to a chromo domain (formerly thought exclusively nuclear); this protein motif organization is unprecedented. NPCD isoforms are expressed in numerous regions of the central nervous system, where they are present in distinct subcellular arrangements in different brain regions. NPCD isoforms are mainly associated with the inner side of the plasma membrane in brain neurons and rat PC12 cells in vitro; they are present in cell bodies, processes, and growth cones. The biochemical complexity and neuronal expression pattern of NPCD, together with its interaction with PTPRO, suggests involvement in multiple neuronal processes.

    Funded by: NINDS NIH HHS: NS 38920

    The Journal of comparative neurology 2005;481;4;391-402

  • Role of presenilin-1 in cortical lamination and survival of Cajal-Retzius neurons.

    Wines-Samuelson M, Handler M and Shen J

    Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

    Presenilin-1 (PS1), the major causative gene of familial Alzheimer disease, regulates neuronal differentiation and Notch signaling during early neural development. To investigate the role of PS1 in neuronal migration and cortical lamination of the postnatal brain, we circumvented the perinatal lethality of PS1-null mice by generating a conditional knockout (cKO) mouse in which PS1 inactivation is restricted to neural progenitor cells (NPCs) and NPC-derived neurons and glia. BrdU birthdating analysis revealed that many late-born neurons fail to migrate beyond the early-born neurons to arrive at their appropriate positions in the superficial layer, while the migration of the early-born neurons is largely normal. The migration defect of late-born neurons coincides with the progressive reduction of radial glia in PS1 cKO mice. In contrast to the premature loss of Cajal-Retzius (CR) neurons in PS1-null mice, generation and survival of CR neurons are unaffected in PS1 cKO mice. Furthermore, the number of proliferating meningeal cells, which have been shown to be important for the survival of CR neurons, is increased in PS1-null mice but not in PS1 cKO mice. These findings show a cell-autonomous role for PS1 in cortical lamination and radial glial development, and a non-cell-autonomous role for PS1 in CR neuron survival.

    Funded by: NINDS NIH HHS: NS42818

    Developmental biology 2005;277;2;332-46

  • Biochemical characterization of the mammalian Cux2 protein.

    Gingras H, Cases O, Krasilnikova M, Bérubé G and Nepveu A

    Molecular Oncology Group, McGill University Health Center, Canada; Department of Biochemistry, McGill University, Canada.

    The CCAAT displacement protein (CDP) and cux (Cut homeobox) genes were originally identified as the human and mouse orthologs of Drosophila melanogaster cut. More recently, vertebrates were found to possess a second cut orthologs that was generated by gene duplication: Cux2. We report the initial biochemical characterization of the Cux2 protein in tissue culture and in vitro. We generated four polyclonal antibodies that were able to recognize the human and mouse Cux2 protein but displayed little or no cross-reactivity towards CDP1 and Cux1. The expression of the Cux2 protein was convincingly detected in only one among 19 neuronal cell lines: the SH-SY5Y human neuroblastoma cell line. CDP/Cux proteins contain four DNA binding domains, three Cut repeat (CR1, CR2 and CR3) and one Cut homeodomain (HD). Purified fusion proteins containing either CR1CR2, CR2CR3HD or CR3HD exhibited similar DNA binding specificities as the corresponding domains of Cux1, but their DNA binding kinetics were much more rapid. Similarly, the full-length Cux2 protein made rapid but transient interactions with DNA. We did not observe an N-terminally processed Cux2 isoform equivalent to the Cux1 p110 isoform. Whereas Cux1 can function as a repressor or activator in a promoter-specific manner, Cux2 functioned exclusively as a transcriptional repressor in NIH3T3 cells. Overall, our results suggest that the Cux1 and Cux2 proteins carry distinct biochemical functions. Cux2 is able, like Cux1, to perform the CCAAT-displacement activity. However, Cux2 is unlikely to execute transcriptional regulatory functions that require stable interaction with DNA.

    Gene 2005;344;273-85

  • The axon guidance defect of the telencephalic commissures of the JSAP1-deficient brain was partially rescued by the transgenic expression of JIP1.

    Ha HY, Cho IH, Lee KW, Lee KW, Song JY, Kim KS, Yu YM, Lee JK, Song JS, Yang SD, Shin HS and Han PL

    Department of Neuroscience, Neuroscience Research Center and Medical Research Institute, Ewha Womans University School of Medicine, Seoul 110-783, Korea.

    The JNK interacting protein, JSAP1, has been identified as a scaffold protein for mitogen-activated protein kinase (MAPK) signaling pathways and as a linker protein for the cargo transport along the axons. To investigate the physiological function of JSAP1 in vivo, we generated mice lacking JSAP1. The JSAP1 null mutation produced various developmental deficits in the brain, including an axon guidance defect of the corpus callosum, in which phospho-FAK and phospho-JNK were distributed at reduced levels. The axon guidance defect of the corpus callosum in the jsap1-/- brain was correlated with the misplacement of glial sling cells, which reverted to their normal position after the transgenic expression of JNK interacting protein 1(JIP1). The transgenic JIP1 partially rescued the axon guidance defect of the corpus callosum and the anterior commissure of the jsap1-/- brain. The JSAP1 null mutation impaired the normal distribution of the Ca+2 regulating protein, calretinin, but not the synaptic vesicle marker, SNAP-25, along the axons of the thalamocortical tract. These results suggest that JSAP1 is required for the axon guidance of the telencephalic commissures and the distribution of cellular protein(s) along axons in vivo, and that the signaling network organized commonly by JIP1 and JSAP1 regulates the axon guidance in the developing brain.

    Developmental biology 2005;277;1;184-99

  • Selective ablation of alphav integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death.

    McCarty JH, Lacy-Hulbert A, Charest A, Bronson RT, Crowley D, Housman D, Savill J, Roes J and Hynes RO

    Center for Cancer Research, Massachusetts Institute of Technology, 40 Ames Street, E17-227, Cambridge, MA 02139, USA.

    Mouse embryos genetically null for all alphav integrins develop intracerebral hemorrhage owing to defective interactions between blood vessels and brain parenchymal cells. Here, we have used conditional knockout technology to address whether the cerebral hemorrhage is due to primary defects in vascular or neural cell types. We show that ablating alphav expression in the vascular endothelium has no detectable effect on cerebral blood vessel development, whereas deletion of alphav expression in central nervous system glial cells leads to embryonic and neonatal cerebral hemorrhage. Conditional deletion of alphav integrin in both central nervous system glia and neurons also leads to cerebral hemorrhage, but additionally to severe neurological defects. Approximately 30% of these mutants develop seizures and die by 4 weeks of age. The remaining mutants survive for several months, but develop axonal deterioration in the spinal cord and cerebellum, leading to ataxia and loss of hindlimb coordination. Collectively, these data provide evidence that alphav integrins on embryonic central nervous system neural cells, particularly glia, are necessary for proper cerebral blood vessel development, and also reveal a novel function for alphav integrins expressed on axons in the postnatal central nervous system.

    Funded by: NHLBI NIH HHS: P01 HL66105-03

    Development (Cambridge, England) 2005;132;1;165-76

  • Analysis of neuronal subpopulations in mice over-expressing suppressor of cytokine signaling-2.

    Ransome MI and Turnley AM

    Neural Regeneration Laboratory, Centre for Neuroscience, University of Melbourne, Melbourne, Victoria 3010, Australia.

    Developing an understanding of factors that regulate development of the nervous system is important if we hope to be able to repair the nervous system after injury or disease. Suppressor of cytokine signaling-2 (SOCS2) is an intracellular regulator of cytokine signaling that blocks the inhibitory effects of growth hormone on neuronal differentiation and promotes neurogenesis. Here we examine the effect of SOCS2 over-expression on brain development by assessing density and soma size of different neuronal populations in the somatosensory cortex and striatum of SOCS2 transgenic mice compared with wildtype C57BL/6 mice. There were no significant differences in brain weight, cortical thickness or striatal area between mice of either genotype. Analysis of NeuN positive neuronal cell density showed a modest but significant 9% increase across layers 2-6 of SOCS2 transgenic cortex, while cortical interneuron subpopulations were variably affected. In the cortex, parvalbumin and somatostatin expressing neuron densities were unaffected, while calretinin and calbindin positive neuronal densities increased by 48% and 45% respectively. There was no apparent difference in glial fibrillary acidic protein positive astrocyte numbers in layers 1 or 6b of cortex. Furthermore, soma sizes of calretinin and calbindin positive cortical neurons were significantly smaller than wildtype, although there was no difference in size of Cresyl Violet-stained layer 5 projection neurons nor of parvalbumin or somatostatin positive cortical neurons. Additionally, synaptic density and dendritic branching were found to be increased in SOCS2 transgenic cortex. These effects on calretinin and calbindin positive cortical neurons and cortical neuronal circuitry were not observed in the striatum of SOCS2-Tg brains. However, striatal cholinergic interneurons were significantly smaller in SOCS2-Tg brains. At embryonic day 14.5, proliferation and apoptosis in the developing telencephalon were similar in each genotype. Therefore, over-expression of SOCS2 variably affects different cortical regions and neuronal populations, with the predominant effect appearing to be on interneurons and neuronal connectivity in the cortex.

    Neuroscience 2005;132;3;673-87

  • Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment.

    Schurov IL, Handford EJ, Brandon NJ and Whiting PJ

    Merck Sharp & Dohme, The Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR, UK. irina_schurov@merck.com

    Disrupted in Schizophrenia 1 (DISC1) was identified as a potential susceptibility gene for schizophrenia due to its disruption by a balanced t(1;11) (q42;q14) translocation, which has been shown to cosegregate with major psychiatric disease in a large Scottish family. We have recently presented evidence that DISC1 exists in a neurodevelopmentally regulated protein complex with Nudel. In this study, we report the protein expression profile of DISC1 in the adult and developing mouse brain utilizing immunohistochemistry and quantitative Western blot. In the adult mouse brain, DISC1 is expressed in neurons within various brain areas including the olfactory bulb, cortex, hippocampus, hypothalamus, cerebellum and brain stem. During development, DISC1 protein is detected at all stages, from E10 to 6 months old, with two significant peaks of protein expression of a DISC1 isoform at E13.5 and P35. Interestingly, these time points correspond to critical stages during mouse development, the active neurogenesis period in the developing brain and the period of puberty. Together, these results suggest that DISC1 may play a critical role in brain development, consistent with the neurodevelopmental hypothesis of the etiology of schizophrenia.

    Molecular psychiatry 2004;9;12;1100-10

  • Neural progenitor cells do not differentiate prematurely in presenilin-1 null mutant mice.

    Wen PH, De Gasperi R, Gama Sosa MA and Elder GA

    Department of Psychiatry, Mount Sinai School of Medicine, New York, NY 10029, USA.

    Mice with a null mutation of the presenilin-1 (PS1-/-) gene die during late intrauterine life or shortly after birth and exhibit defects in cortical development. A previous report suggested that neurons differentiate prematurely in PS1-/- brain [M. Handler, X. Yang, J. Shen, Presenilin-1 regulates neuronal differentiation during neurogenesis, Development 127 (2000) 2593-2606]. Here we reexamined the issue of whether premature neuronal differentiation occurs in PS1-/- brain using fresh cell suspensions from embryonic E11.5 and E13.5 telencephalon where individual cell phenotypes can be easily determined with cell type specific markers. Immunostaining with seven neuronal specific markers (MAP2, beta-III tubulin, GABA, reelin, GluR2/3, calbindin, and calretinin) failed to reveal any evidence of premature neuronal differentiation in PS1-/- telencephalon. We also determined the fraction of cells expressing the neural progenitor marker nestin and found no evidence for premature depletion of neural progenitor cells in PS1-/- telencephalon. Moreover, based on MAP2 staining of tissue sections from E12.5 embryos the topography of newly generated neurons also appeared to be undisturbed in the telencephalon of PS1-/- embryos. These studies thus argue that premature neuronal differentiation is unlikely to be a core pathophysiological feature underlying the aberrant cortical development that occurs in PS1-/- brain.

    Funded by: NIA NIH HHS: 1R01AG020139

    Neuroscience letters 2004;371;2-3;249-54

  • Genetic influences on ovulation of primary oocytes in LT/Sv strain mice.

    Everett CA, Auchincloss CA, Kaufman MH, Abbott CM and West JD

    Medical Genetics, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

    A high proportion of LT/Sv strain oocytes arrest in meiotic metaphase I (MI) and are ovulated as diploid primary oocytes rather than haploid secondary oocytes. (Mus musculus castaneus x LT/SvKau)F1 x LT/SvKau backcross females were analysed for the proportion of oocytes that arrested in MI and typed by PCR for a panel of microsatellite DNA sequences (simple sequence repeat polymorphisms) that differed between strain LT/SvKau and M. m. castaneus. This provided a whole genome scan of 86 genetic markers distributed over all 19 autosomes and the X chromosome, and revealed genetic linkage of the MI arrest phenotype to markers on chromosomes 1 and 9. Identification of these two chromosomal regions should facilitate the identification of genes involved in mammalian oocyte maturation and the control of meiosis.

    Reproduction (Cambridge, England) 2004;128;5;565-71

  • Id4 regulates neural progenitor proliferation and differentiation in vivo.

    Yun K, Mantani A, Garel S, Rubenstein J and Israel MA

    Department of Pediatrics and Genetics and the Norris Cotton Cancer Center, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, 1 Medical Center Drive, Lebanon, NH 03756, USA.

    The mechanisms that determine whether a precursor cell re-enters the cell cycle or exits and differentiates are crucial in determining the types and numbers of cells that constitute a particular organ. Here, we report that Id4 is required for normal brain size, and regulates lateral expansion of the proliferative zone in the developing cortex and hippocampus. In its absence, proliferation of stem cells in the ventricular zone (VZ) is compromised. In early cortical progenitors, Id4 is required for the normal G1-S transition. By contrast, at later ages, ectopically positioned proliferating cells are found in the mantle zone of the Id4-/- cortex. These observations, together with evidence for the premature differentiation of early cortical stem cells, indicate that Id4 has a unique and complex function in regulating neural stem cell proliferation and differentiation.

    Funded by: NCI NIH HHS: CA84280

    Development (Cambridge, England) 2004;131;21;5441-8

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex.

    Demyanenko GP, Schachner M, Anton E, Schmid R, Feng G, Sanes J and Maness PF

    Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.

    We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.

    Funded by: NIMH NIH HHS: MH064056; NINDS NIH HHS: NS26620, P30NS045892

    Neuron 2004;44;3;423-37

  • Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members.

    Herms J, Anliker B, Heber S, Ring S, Fuhrmann M, Kretzschmar H, Sisodia S and Müller U

    Zentrum für Neuropathologie und Prionforschung, Universität München, München, Germany.

    The Alzheimer's disease beta-amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor-like proteins, termed APLP1 and APLP2. We previously documented that APLP2-/-APLP1-/- and APLP2-/-APP-/- mice die postnatally, while APLP1-/-APP-/- mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double-mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.

    The EMBO journal 2004;23;20;4106-15

  • TrkB regulates neocortex formation through the Shc/PLCgamma-mediated control of neuronal migration.

    Medina DL, Sciarretta C, Calella AM, Von Bohlen Und Halbach O, Unsicker K and Minichiello L

    European Molecular Biology Laboratory, Monterotondo, Italy.

    The generation of complex neuronal structures, such as the neocortex, requires accurate positioning of neurons and glia within the structure, followed by differentiation, formation of neuronal connections, and myelination. To understand the importance of TrkB signaling during these events, we have used conditional and knockin mutagenesis of the TrkB neurotrophin receptor, and we now show that this tyrosine kinase receptor, through docking sites for the Shc/FRS2 adaptors and phospholipase Cgamma (PLCgamma), coordinates these events in the cerebral cortex by (1) controlling cortical stratification through the timing of neuronal migration during cortex formation, and (2) regulating both neuronal and oligodendrocyte differentiation. These results provide genetic evidence that TrkB regulates important functions throughout the formation of the cerebral cortex via recruitment of the Shc/FRS2 adaptors and PLCgamma.

    The EMBO journal 2004;23;19;3803-14

  • The Tlx gene regulates the timing of neurogenesis in the cortex.

    Roy K, Kuznicki K, Wu Q, Sun Z, Bock D, Schutz G, Vranich N and Monaghan AP

    Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    The tailless (tlx) gene is a forebrain-restricted transcription factor. Tlx mutant animals exhibit a reduction in the size of the cerebral hemispheres and associated structures (Monaghan et al., 1997). Superficial cortical layers are specifically reduced, whereas deep layers are relatively unaltered (Land and Monaghan, 2003). To determine whether the adult laminar phenotype has a developmental etiology and whether it is associated with a change in proliferation/differentiation decisions, we examined the cell cycle and neurogenesis in the embryonic cortex. We found that there is a temporal and regional requirement for the Tlx protein in progenitor cells (PCs). Neurons prematurely differentiate at all rostrocaudal levels up to mid-neurogenesis in mutant animals. Heterozygote animals have an intermediate phenotype indicating there is a threshold requirement for Tlx in early cortical neurogenesis. Our studies indicate that PCs in the ventricular zone are sensitive to loss of Tlx in caudal regions only; however, PCs in the subventricular zone are altered at all rostrocaudal levels in tlx-deficient animals. Furthermore, we found that the cell cycle is shorter from embryonic day 9.5 in tlx-/- embryos. At mid-neurogenesis, the PC population becomes depleted, and late PCs have a longer cell cycle in tlx-deficient animals. Consequently, later generated structures, such as upper cortical layers, the dentate gyrus, and the olfactory bulbs, are severely reduced. These studies indicate that tlx is an essential intrinsic regulator in the decision to proliferate or differentiate in the developing forebrain.

    Funded by: NIMH NIH HHS: 5R01MH060774, R01 MH060774, R01 MH060774-01, R01 MH060774-02, R01 MH060774-03, R01 MH060774-04

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;38;8333-45

  • A screen for downstream effectors of Neurogenin2 in the embryonic neocortex.

    Mattar P, Britz O, Johannes C, Nieto M, Ma L, Rebeyka A, Klenin N, Polleux F, Guillemot F and Schuurmans C

    University of Calgary, Calgary, Alberta, Canada T2N 4N1.

    Neurogenin (Ngn) 1 and Ngn2 encode basic-helix-loop-helix transcription factors expressed in the developing neocortex. Like other proneural genes, Ngns participate in the specification of neural fates and neuronal identities, but downstream effectors remain poorly defined. We set out to identify Ngn2 effectors in the cortex using a subtractive hybridization screen and identified several regionally expressed genes that were misregulated in Ngn2 and Ngn1;Ngn2 mutants. Included were genes down-regulated in germinal zone progenitors (e.g., Nlgn1, Unc5H4, and Dcc) and in postmitotic neurons in the cortical plate (e.g., Bhlhb5 and NFIB) and subplate (e.g., Mef2c, srGAP3, and protocadherin 9). Further analysis revealed that Ngn2 mutant subplate neurons were misspecified and that thalamocortical afferents (TCAs) that normally target this layer instead inappropriately projected towards the germinal zone. Strikingly, EphA5 and Sema3c, which encode repulsive guidance cues, were down-regulated in the Ngn2 and Ngn1;Ngn2 mutant germinal zones, providing a possible molecular basis for axonal targeting defects. Thus, we identified several new components of the differentiation cascade(s) activated downstream of Ngn1 and Ngn2 and provided novel insights into a new developmental process controlled by these proneural genes. Further analysis of the genes isolated in our screen should provide a fertile basis for understanding the molecular mechanisms underlying corticogenesis.

    Developmental biology 2004;273;2;373-89

  • Expression levels of cytoskeletal proteins indicate pathological aging of S100B transgenic mice: an immunohistochemical study of MAP-2, drebrin and GAP-43.

    Shapiro LA and Whitaker-Azmitia PM

    Program in Biopsychology, Department of Psychology, State University of New York, Stony Brook, NY 11794-2500, USA.

    S100B is a calcium-binding protein found within astroglial cells. When released, S100B has extracellular neurotrophic effects involving the neuronal cytoskeleton. The gene for S100B is located on chromosome 21 and levels of the protein are elevated in Down Syndrome (DS) and Alzheimer's Disease (AD). Thus, overexpression of S100B may be related to the cytoskeletal abnormalities seen in these disorders. Transgenic mice overexpressing human S100B were examined for cytoskeletal changes as young (70 days) and aged (200 days) adults, using immunochemical staining of the dendritic associated protein, MAP-2, the growth-associated protein-43 (GAP-43) and the dendritic spine marker, drebrin. As young adults, the S100B transgenic mice exhibited significantly greater MAP-2-immunoreactivity in the hippocampus, however as older adults, the animals exhibited less staining. In both the CD1 control animals and the S100B animals, the immunoreactivity of drebrin increased with age, however there were no significant between group differences. Finally, the older S100B animals showed more GAP-43 staining than the control animals, suggesting that synaptic remodeling could take place, possibly in response to the loss of MAP-2-ir dendrites. Overall, the data suggest that S100B overexpression leads to changes in cytoskeletal markers. The longitudinal effects of S100B overexpression are discussed with relevance to aging and pathology.

    Funded by: NINDS NIH HHS: R01 NS 042555

    Brain research 2004;1019;1-2;39-46

  • Genomic analysis of mouse retinal development.

    Blackshaw S, Harpavat S, Trimarchi J, Cai L, Huang H, Kuo WP, Weber G, Lee K, Fraioli RE, Cho SH, Yung R, Asch E, Ohno-Machado L, Wong WH and Cepko CL

    Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA.

    The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE). The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Müller glia and mitotic progenitor cells were found to be highly similar, suggesting that Müller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length ("noncoding RNAs") were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.

    Funded by: NCI NIH HHS: P20 CA096470, P20 CA96470; NEI NIH HHS: EY08064, R01 EY008064

    PLoS biology 2004;2;9;E247

  • Lmx1b controls the differentiation and migration of the superficial dorsal horn neurons of the spinal cord.

    Ding YQ, Yin J, Kania A, Zhao ZQ, Johnson RL and Chen ZF

    Departments of Anesthesiology, Psychiatry, Molecular Biology and Pharmacology, Washington University School of Medicine Pain Center, St. Louis, MO 63110, USA.

    The differentiation and migration of superficial dorsal horn neurons and subsequent ingrowth of cutaneous afferents are crucial events in the formation of somatosensory circuitry in the dorsal spinal cord. We report that the differentiation and migration of the superficial dorsal horn neurons are regulated by the LIM homeobox gene Lmx1b, and its downstream targets Rnx and Drg11, two transcription factors implicated in the development of dorsal horn circuitry. An analysis of Lmx1b mutants shows that Lmx1b normally acts to maintain the expression of the Ebf genes and to repress the Zic genes. Lmx1b mutants also exhibit the disruption of the cutaneous afferent ingrowth, suggesting that the dorsal horn cells might provide important cues guiding sensory axons into the dorsal spinal cord. Our results thus indicate that Lmx1b has a pivotal role in genetic cascades that control the assembly of circuitry in the superficial dorsal horn.

    Funded by: NINDS NIH HHS: NS43968-01

    Development (Cambridge, England) 2004;131;15;3693-703

  • Foxg1 suppresses early cortical cell fate.

    Hanashima C, Li SC, Shen L, Lai E and Fishell G

    Developmental Genetics Program and the Department of Cell Biology, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, 540 First Avenue, New York, NY 10016, USA.

    During mammalian cerebral corticogenesis, progenitor cells become progressively restricted in the types of neurons they can produce. The molecular mechanism that determines earlier versus later born neuron fate is unknown. We demonstrate here that the generation of the earliest born neurons, the Cajal-Retzius cells, is suppressed by the telencephalic transcription factor Foxg1. In Foxg1 null mutants, we observed an excess of Cajal-Retzius neuron production in the cortex. By conditionally inactivating Foxg1 in cortical progenitors that normally produce deep-layer cortical neurons, we demonstrate that Foxg1 is constitutively required to suppress Cajal-Retzius cell fate. Hence, the competence to generate the earliest born neurons during later cortical development is actively suppressed but not lost.

    Funded by: NEI NIH HHS: EY11124; NICHD NIH HHS: HD29584; NINDS NIH HHS: NS32993, NS39007

    Science (New York, N.Y.) 2004;303;5654;56-9

  • Foxp1 gene expression in projection neurons of the mouse striatum.

    Tamura S, Morikawa Y, Iwanishi H, Hisaoka T and Senba E

    Department of Anatomy and Neurobiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-8509, Japan.

    The developmental processes of maturation in the CNS are the result of specific events including mitogenesis, differentiation, and cell death which occur in a precise spatial and temporal manner. It has been reported that many transcription factors, including forkhead transcription factors, play a key role in these processes. First, we examined the expression pattern of the forkhead transcription factor Foxp1 in the adult CNS. Foxp1 was highly expressed in the striatum and moderately in the cerebral cortex, CA1/2 subfields of the hippocampus, and several thalamic nuclei. In situ hybridization combined with immunohistochemistry in the striatum of adult mice revealed that Foxp1 mRNA was detected in a subset of projection neurons, not in interneurons. In addition, the expression of Foxp1 mRNA was observed in the developing basal ganglia with the exception of the globus pallidus. Thus, Foxp1 mRNA was expressed in a subset of striatal projection neurons, probably the matrix neurons. The expression pattern of Foxp1 mRNA suggests that Foxp1 may play a role in the development and formation of a circuit in the basal ganglia, which is involving the matrix neurons.

    Neuroscience 2004;124;2;261-7

  • An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis.

    Calegari F and Huttner WB

    Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307, Dresden, Germany.

    The G1 phase of the cell cycle of neuroepithelial cells, the progenitors of all neurons of the mammalian central nervous system, has been known to lengthen concomitantly with the onset and progression of neurogenesis. We have investigated whether lengthening of the G1 phase of the neuroepithelial cell cycle is a cause, rather than a consequence, of neurogenesis. As an experimental system, we used whole mouse embryo culture, which was found to exactly reproduce the temporal and spatial gradients of the onset of neurogenesis occurring in utero. Olomoucine, a cell-permeable, highly specific inhibitor of cyclin-dependent kinases and G1 progression, was found to significantly lengthen, but not arrest, the cell cycle of neuroepithelial cells when used at 80 microM. This olomoucine treatment induced, in the telencephalic neuroepithelium of embryonic day 9.5 to 10.5 mouse embryos developing in whole embryo culture to embryonic day 10.5, (i) the premature up-regulation of TIS21, a marker identifying neuroepithelial cells that have switched from proliferative to neuron-generating divisions, and (ii) the premature generation of neurons. Our data indicate that lengthening G1 can alone be sufficient to induce neuroepithelial cell differentiation. We propose a model that links the effects of cell fate determinants and asymmetric cell division to the length of the cell cycle.

    Journal of cell science 2003;116;Pt 24;4947-55

  • Expression of the Prader-Willi gene Necdin during mouse nervous system development correlates with neuronal differentiation and p75NTR expression.

    Andrieu D, Watrin F, Niinobe M, Yoshikawa K, Muscatelli F and Fernandez PA

    Neurogenèse et Morphogenèse dans le Développement et chez l'Adulte, CNRS UMR 6156, Institut de Biologie du Développement de Marseille (IBDM), Case 907, Campus de Luminy, 13288 Cedex 9, Marseille, France.

    The expression pattern of Necdin, a gene involved in the etiology of Prader-Willi syndrome and a member of the MAGE family of genes, is described during mouse nervous system development. Using RNA in situ hybridization, immunohistochemical staining, and colocalization with neuronal differentiation markers, we found that Necdin RNA and protein are expressed within post-mitotic neurons at all stages studied. From E10 to E12, Necdin is detected in all developing neurons, in both central and peripheral nervous system, with the highest expression levels in the diencephalon and the hindbrain. After E13, Necdin is expressed in specific structures of the nervous system, in particular the hypothalamus, the thalamus, and the pons, suggesting a specific developmental role therein. In addition, Necdin expression is also detected in non-neural tissues, such as the somites, the developing limb buds, the first branchial arches, the tong, and the axial muscles. Recently, Necdin and other MAGE proteins were found to interact in vitro with the intracellular domain of the p75NTR neurotrophin receptor, but this interaction has not been validated in vivo. We report here that the spatial and temporal expression of p75NTR is included in Necdin expression domain. These results are in agreement with Necdin proposed role on cell cycle arrest, inhibition of apoptosis and facilitation of neuronal differentiation in vitro, and with hypothalamic cellular deficiencies reported in mice with abrogation of the Necdin gene. Furthermore, they are also consistent with the putative role of Necdin in signaling events promoted by p75NTR during mouse nervous system development.

    Gene expression patterns : GEP 2003;3;6;761-5

  • Multimodal tangential migration of neocortical GABAergic neurons independent of GPI-anchored proteins.

    Tanaka D, Nakaya Y, Yanagawa Y, Obata K and Murakami F

    Graduate School of Frontier Biosciences, Osaka University, Machikaneyama 1-3, Toyonaka, Osaka 560-8531, Japan.

    Neuronal migration is crucial for the construction of neuronal architecture such as layers and nuclei. Most inhibitory interneurons in the neocortex derive from the basal forebrain and migrate tangentially; however, little is known about the mode of migration of these neurons in the cortex. We used glutamate decarboxylase (Gad)67-green fluorescent protein (GFP) knock-in embryonic mice with expression of GFP in gamma-aminobutyric acid (GABA)-ergic neurons and performed time-lapse analysis. In coronal slices, many GFP-positive neurons in the lower intermediate zone (IZ) and subventricular zone (SVZ) showed robust tangential migration from lateral to medial cortex, while others showed radial and non-radial migration mostly towards the pial surface. In flat-mount preparations, GFP-positive neurons of the marginal zone (MZ) showed multidirectional tangential migration. Some of these neurons descended toward the cortical plate (CP). Intracortical migration of these neurons was largely unaffected by a treatment that cleaves glycosylphosphatidylinositol (GPI) anchors. These findings suggest that tangential migration of cortical interneurons from lateral to medial cortex predominantly occurs in the IZ/SVZ and raise the possibility that a part of the pial surface-directed neurons in the IZ/SVZ reach the MZ, whereby they spread into the whole area of the cortex. At least a part of these neurons may descend toward the CP. Our results also suggest that intracortical migration of GABAergic neurons occurs independent of GPI-anchored proteins.

    Development (Cambridge, England) 2003;130;23;5803-13

  • Differential distribution of Rac1 and Rac3 GTPases in the developing mouse brain: implications for a role of Rac3 in Purkinje cell differentiation.

    Bolis A, Corbetta S, Cioce A and de Curtis I

    Cell Adhesion Unit, Department of Molecular Biology and Functional Genomics, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.

    Rac3 is one of the three known Rac GTPases in vertebrates. Rac3 shows high sequence homology to Rac1, and its transcript is specifically expressed in the developing nervous system, where its localization and function are unknown. By using Rac3-specific antibodies, we show that the endogenous Rac3 protein is differentially expressed during mouse brain development, with a peak of expression at times of neuronal maturation and synaptogenesis. Comparison with Rac1 shows clear-cut differences in the overall distribution of the two GTPases in the developing brain, and in their subcellular distribution in regions of the brain where both proteins are expressed. At P7, Rac3 staining is particularly marked in the deep cerebellar nuclei and in the pons, where it shows a discontinuous distribution around the neuronal cell bodies, in contrast with the diffuse staining of Rac1. Rac3 does not evidently co-localize with pre- and post-synaptic markers, nor with GFAP-positive astrocytes, but it clearly co-localizes with actin filaments, and with the terminal portions of calbindin-positive Purkinje cell axons in the deep cerebellar nuclei. Our data implicate Rac3 in neuronal differentiation, and support a specific role of this GTPase in actin-mediated remodelling of Purkinje cell neuritic terminals at time of synaptogenesis.

    Funded by: Telethon: GGP02190

    The European journal of neuroscience 2003;18;9;2417-24

  • Loss of K-Cl co-transporter KCC3 causes deafness, neurodegeneration and reduced seizure threshold.

    Boettger T, Rust MB, Maier H, Seidenbecher T, Schweizer M, Keating DJ, Faulhaber J, Ehmke H, Pfeffer C, Scheel O, Lemcke B, Horst J, Leuwer R, Pape HC, Völkl H, Hübner CA and Jentsch TJ

    Zentrum für Molekulare Neurobiologie, ZMNH, Universität Hamburg, Falkenried 94, D-20251 Hamburg, Germany.

    K-Cl co-transporters are encoded by four homologous genes and may have roles in transepithelial transport and in the regulation of cell volume and cytoplasmic chloride. KCC3, an isoform mutated in the human Anderman syndrome, is expressed in brain, epithelia and other tissues. To investigate the physiological functions of KCC3, we disrupted its gene in mice. This severely impaired cell volume regulation as assessed in renal tubules and neurons, and moderately raised intraneuronal Cl(-) concentration. Kcc3(-/-) mice showed severe motor abnormalities correlating with a progressive neurodegeneration in the peripheral and CNS. Although no spontaneous seizures were observed, Kcc3(-/-) mice displayed reduced seizure threshold and spike-wave complexes on electrocorticograms. These resembled EEG abnormalities in patients with Anderman syndrome. Kcc3(-/-) mice also displayed arterial hypertension and a slowly progressive deafness. KCC3 was expressed in many, but not all cells of the inner ear K(+) recycling pathway. These cells slowly degenerated, as did sensory hair cells. The present mouse model has revealed important cellular and systemic functions of KCC3 and is highly relevant for Anderman syndrome.

    The EMBO journal 2003;22;20;5422-34

  • Transient loss of microtubule-associated protein 2 immunoreactivity after moderate brain injury in mice.

    Huh JW, Raghupathi R, Laurer HL, Helfaer MA and Saatman KE

    Department of Anesthesia and Critical Care, The Children's Hospital of Philadelphia, Pennsylvania, USA.

    Microtubule-associated protein 2 (MAP2) is important for microtubule stability and neural plasticity and appears to be among the most vulnerable of the cytoskeletal proteins under conditions of neuronal injury. To evaluate the acute effects of moderate severity traumatic brain injury on MAP2, anesthetized, adult male C57BL/6 mice were subjected to controlled cortical impact brain injury. At 5 min, 15 min, 90 min, 4 h, and 24 h following brain injury (n = 4 injured and n = 1 sham-injured per time point), mice were sacrificed and immunohistochemistry was performed on coronal brain sections. Profound decreases in MAP2 immunolabeling were observed in the ipsilateral cortex and hippocampal dentate hilus at 5 min postinjury and in the ipsilateral hippocampal CA3 area by 4 h postinjury. Decreases in MAP2 labeling occurred prior to notable neuronal cell loss. Interestingly, cortical MAP2 immunoreactivity returned by 90 min postinjury, but the recovery was short-lived within the core in comparison to the periphery of the impact site. Partial restoration of MAP2 immunoreactivity was also observed in the ipsilateral CA3 and dentate hilus by 24 h postinjury. Our data corroborate that MAP2 is an early and sensitive marker for neuronal damage following traumatic brain injury. Acute MAP2 loss, however, may not necessarily presage neuronal death, even following moderate severity traumatic brain injury. Rather, to the best of our knowledge, our data are the first to suggest an intrinsic ability of the traumatized brain for MAP2 recovery after injury of moderate severity.

    Funded by: NINDS NIH HHS: P50-NS08803, R01-NS41561

    Journal of neurotrauma 2003;20;10;975-84

  • Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation.

    Petrone A, Battaglia F, Wang C, Dusa A, Su J, Zagzag D, Bianchi R, Casaccia-Bonnefil P, Arancio O and Sap J

    Department of Pharmacology, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA.

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha (RPTPalpha) regulates SRC family kinases, potassium channels and NMDA receptors. Here, we report that absence of RPTPalpha compromises correct positioning of pyramidal neurons during development of mouse hippocampus. Thus, RPTPalpha is a novel member of the functional class of genes that control radial neuronal migration. The migratory abnormality likely results from a radial glial dysfunction rather than from a neuron-autonomous defect. In spite of this aberrant development, basic synaptic transmission from the Schaffer collateral pathway to CA1 pyramidal neurons remains intact in Ptpra(-/-) mice. However, these synapses are unable to undergo long-term potentiation. Mice lacking RPTPalpha also underperform in the radial-arm water-maze test. These studies identify RPTPalpha as a key mediator of neuronal migration and synaptic plasticity.

    Funded by: NCI NIH HHS: CA87005, R01 CA087005; NINDS NIH HHS: NS40045, R01 NS040045

    The EMBO journal 2003;22;16;4121-31

  • Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain.

    Cheng A, Wang S, Cai J, Rao MS and Mattson MP

    Laboratory of Neurosciences, National Institute on Aging Gerontology Research Center, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.

    Nitric oxide (NO) is believed to act as an intercellular signal that regulates synaptic plasticity in mature neurons. We now report that NO also regulates the proliferation and differentiation of mouse brain neural progenitor cells (NPCs). Treatment of dissociated mouse cortical neuroepithelial cluster cell cultures with the NO synthase inhibitor L-NAME or the NO scavenger hemoglobin increased cell proliferation and decreased differentiation of the NPCs into neurons, whereas the NO donor sodium nitroprusside inhibited NPC proliferation and increased neuronal differentiation. Brain-derived neurotrophic factor (BDNF) reduced NPC proliferation and increased the expression of neuronal NO synthase (nNOS) in differentiating neurons. The stimulatory effect of BDNF on neuronal differentation of NPC was blocked by L-NAME and hemoglobin, suggesting that NO produced by the latter cells inhibited proliferation and induced neuronal differentiation of neighboring NPCs. A similar role for NO in regulating the switch of neural stem cells from proliferation to differentiation in the adult brain is suggested by data showing that NO synthase inhibition enhances NPC proliferation and inhibits neuronal differentiation in the subventricular zone of adult mice. These findings identify NO as a paracrine messenger stimulated by neurotrophin signaling in newly generated neurons to control the proliferation and differentiation of NPC, a novel mechanism for the regulation of developmental and adult neurogenesis.

    Developmental biology 2003;258;2;319-33

  • Neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of Ube3a.

    Yamasaki K, Joh K, Ohta T, Masuzaki H, Ishimaru T, Mukai T, Niikawa N, Ogawa M, Wagstaff J and Kishino T

    Department of Human Genetics, School of Medicine, Nagasaki University, 1-12-4 Sakamoto-machi, Nagasaki, Japan.

    The human UBE3A gene shows brain-specific partial imprinting, and lack of a maternally inherited allele causes Angelman syndrome (AS), which is characterized by neurobehavioral anomalies. In several AS model mice, imprinted Ube3a expression is detected predominantly in the hippocampus, cerebellar Purkinje cells and the olfactory bulb. Therefore, imprinting of mouse Ube3a is thought to be region-specific with different levels of silencing of the paternal Ube3a allele in different brain regions. To determine cell types of imprinted Ube3a expression, we analyzed its imprinting status in embryonic brain cells by using primary cortical cell cultures. RT-PCR and immunofluorescence were performed to determine the allelic expression of the gene. The Ube3a gene encodes two RNA transcripts in the brain, sense and antisense. The sense transcript was expressed maternally in neurons but biallelically in glial cells in the embryonic brain, whereas the antisense transcript was expressed only in neurons and only from the paternal allele. Our data present evidence of brain cell type-specific imprinting, i.e. neuron-specific imprinting of Ube3a in primary brain cell cultures. Reciprocal imprinting of sense and antisense transcripts present only in neurons suggests that the neuron-specific imprinting mechanism is related to the lineage determination of neural stem cells.

    Human molecular genetics 2003;12;8;837-47

  • Neuronal lineage-specific induction of phospholipase Cepsilon expression in the developing mouse brain.

    Wu D, Tadano M, Edamatsu H, Masago-Toda M, Yamawaki-Kataoka Y, Terashima T, Mizoguchi A, Minami Y, Satoh T and Kataoka T

    Division of Molecular Biology, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

    Phospholipase C is a key enzyme of intracellular signal transduction in the central nervous system. We and others recently discovered a novel class of phospholipase C, phospholipase Cepsilon, which is regulated by Ras and Rap small GTPases. As a first step toward analysis of its function, we have examined the spatial and temporal expression patterns of phospholipase Cepsilon during mouse development by in situ hybridization and immunohistochemistry. Around embryonic day 10.5, abundant expression of phospholipase Cepsilon is observed specifically in the outermost layer of the neural tube. On embryonic day 12 and later, it is observed mainly in the marginal zone of developing brain and spinal cord as well as in other regions undergoing neuronal differentiation, such as the retina and olfactory epithelium. The phospholipase Cepsilon-expressing cells almost invariably express microtubule-associated protein 2, but hardly express nestin or glial fibrillary acidic protein, indicating that the expression of phospholipase Cepsilon is induced specifically in cells committed to the neuronal lineage. The expression of phospholipase Cepsilon persists in the terminally differentiated neurons and exhibits no regional specificity. Further, an in vitro culture system of neuroepithelial stem cells is employed to show that abundant expression of phospholipase Cepsilon occurs in parallel with the loss of nestin expression as well as with the induction of microtubule-associated protein 2 expression and neuronal morphology. Also, glial fibrillary acidic protein-positive glial lineage cells do not exhibit the high phospholipase Cepsilon expression. These results suggest that the induction of phospholipase Cepsilon expression may be a specific event associated with the commitment of the neural precursor cells to the neuronal lineage.

    The European journal of neuroscience 2003;17;8;1571-80

  • Inactivation of the nuclear receptor coactivator RAP250 in mice results in placental vascular dysfunction.

    Antonson P, Schuster GU, Wang L, Rozell B, Holter E, Flodby P, Treuter E, Holmgren L and Gustafsson JA

    Department of Biosciences at Novum, Karolinska Institutet, Novum, S-14157 Huddinge, Sweden. per.antonson@cbt.si.se

    Coactivators constitute a diverse group of proteins that are essential for optimal transcriptional activity of nuclear receptors. In the past few years many coactivators have been identified but it is still unclear whether these proteins interact indiscriminately with all nuclear receptors and whether there is some redundancy in their functions. We have previously cloned and characterized RAP250 (ASC-2/PRIP/TRBP/NRC), an LXXLL-containing coactivator for nuclear receptors. In order to study its biological role, Rap250 null mice were generated by gene targeting. Here we show that genetic disruption of Rap250 results in embryonic lethality at embryonic day (E) 13.5. Histological examination of placentas revealed a dramatically reduced spongiotrophoblast layer, a collapse of blood vessels in the region bordering the spongiotrophoblast, and labyrinthine layers in placentas from Rap250(-/-) embryos. These findings suggest that the lethality of Rap250(-/-) embryos is the result of obstructed placental blood circulation. Moreover, the transcriptional activity of PPAR gamma is reduced in fibroblasts derived from Rap250(-/-) embryos, suggesting that RAP250 is an essential coactivator for this nuclear receptor in the placenta. Our results demonstrate that RAP250 is necessary for placental development and thus essential for embryonic development.

    Molecular and cellular biology 2003;23;4;1260-8

  • Neutral amino acid transporter ASCT1 is preferentially expressed in L-Ser-synthetic/storing glial cells in the mouse brain with transient expression in developing capillaries.

    Sakai K, Shimizu H, Koike T, Furuya S and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.

    Nonessential amino acid L-Ser plays an essential role in neuronal survival and differentiation, through preferential expression of the L-Ser biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH), in particular in glial cells but not in neurons. To seek the molecular candidates responsible for glia-borne L-Ser transport, we performed histochemical analyses on amino acid transporter ASCT1, which prefers small neutral amino acids, such as Ala, Ser, Cys, and Thr, and mediates their obligatory exchange. At early developmental stages, neuroepithelial cells constituting the ventricular zone expressed ASCT1 mRNA and protein ubiquitously. Thereafter, ASCT1 expression was gradually downregulated in neuronal populations during the late embryonic and neonatal periods, whereas its high expression was transmitted to radial glial cells and then to astrocytes. High levels of ASCT1 were also detected in the olfactory ensheathing glia. The preferential glial expression of ASCT1 was consistent with that of 3PGDH, and their extensive colocalization was demonstrated at the cellular level. Moreover, high cellular contents of L-Ser were revealed in these glial cells by using a specific antibody to L-Ser. These results strongly suggest that a large amount of L-Ser is synthesized and stored in these glial cells and is released through ASCT1 in exchange for other extracellular substrates. In addition, we observed prominent expression of ASCT1 in capillary endothelial cells of embryonic and neonatal brains. Therefore, ASCT1 appears to be regulated to meet metabolic demands by differentiating and mature neurons through the transport of glia- and blood-borne small neutral amino acids.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;2;550-60

  • BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

    Stryke D, Kawamoto M, Huang CC, Johns SJ, King LA, Harper CA, Meng EC, Lee RE, Yee A, L'Italien L, Chuang PT, Young SG, Skarnes WC, Babbitt PC and Ferrin TE

    Department of Pharmaceutical Chemistry, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.

    The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

    Funded by: NCRR NIH HHS: P41 RR001081, P41 RR01081; NHLBI NIH HHS: U01 HL066621, U01 HL66621

    Nucleic acids research 2003;31;1;278-81

  • Neurogenin3 participates in gliogenesis in the developing vertebrate spinal cord.

    Lee J, Wu Y, Qi Y, Xue H, Liu Y, Scheel D, German M, Qiu M, Guillemot F, Rao M and Gradwohl G

    IGBMC, Universite Louis Pasteur, 1 rue Laurent Fries, BP10142, 67404 Illkirch Cedex, CU de Strasbourg, France.

    To study the role of basic helix-loop-helix (bHLH) transcription factors in gliogenesis, we examined whether bHLH transcription factors were expressed in glial precursor cells and participated in regulating oligodendrocyte and astrocyte development. As assessed by reverse transcription-polymerase chain reaction (RT-PCR), Neurogenin3 (Ngn3) was transiently expressed in bipotential glial cells fated to become either oligodendrocytes or astrocytes. Mice lacking Ngn3 displayed a loss of Nkx2.2 expression, a transcription factor required for proper oligodendrogliogenesis. Furthermore, a reduction in the expression of myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP), markers for mature oligodendrocytes and astrocytes, was observed in the Ngn3 null mice. Overexpression of Ngn3 was sufficient to drive expression from the PLP promoter in transient cotransfection assays. Overall, the data suggest that Ngn3 may regulate glial differentiation at a developmental stage prior to the segregation of the oligodendrocyte and astrocyte lineage.

    Developmental biology 2003;253;1;84-98

  • Connective tissue growth factor: a novel marker of layer VII neurons in the rat cerebral cortex.

    Heuer H, Christ S, Friedrichsen S, Brauer D, Winckler M, Bauer K and Raivich G

    Max-Planck Institute for Experimental Endocrinology, Feodor-Lynen-Str. 7, D-30625 Hannover, Germany.

    Connective tissue growth factor (CTGF) belongs to a family of secreted, extracellular matrix-associated proteins that are involved in the regulation of cellular functions such as adhesion, migration, mitogenesis, differentiation and survival. Recent studies have also suggested the up-regulation of CTGF in response to trauma, scar formation and excitotoxicity in the CNS. To further elucidate the localization and regulation of this molecule in the rat brain we performed in situ hybridization experiments and found a very strong and selective expression of CTGF messenger ribonucleic acid (mRNA) on the band of layer VII neurons throughout the adult cerebral cortex. Similarly strong neuronal expression was also present in the dorsal endopiriform nucleus, extending rostrally from the ventrocaudal cortical layer VII, and in the deep layers of the olfactory glomeruli and the accessory olfactory nucleus. Double in situ hybridization confirmed selective CTGF mRNA expression on a subpopulation (approximately 35%) of microtubule-associated protein 2 mRNA-positive neurons in the cortical layer VII and the dorsal endopiriform nucleus. The nucleus of lateral olfactory tract showed moderate signal intensity; other parts of the forebrain, mesencephalon and brain stem only revealed a very weak level of CTGF mRNA expression. Non-neuronal expression was rare, considerably weaker than on cortical layer VII neurons, and normally associated with blood vessels. Developmental analysis of CTGF mRNA expression in embryonic and postnatal mouse also showed a moderately late onset at embryonic day 16-18, and confirmed the presence of CTGF mRNA in cortical layer VII in a second rodent species. Interestingly, injury experiments using direct cerebral trauma or injection of excitotoxic kainic acid into rat brain failed to up-regulate CTGF mRNA after injury and during the ensuing period of neuronal cell death, gliosis and neural scar tissue formation. Altogether, the current data suggest a constitutive role of CTGF, particularly in the adult cerebral cortex. In view of the strong ascending projections of subplate neurons into cortical layer 1, this molecule may be involved in the modulation of synaptic input to apical dendrites of pyramidal neurons.

    Neuroscience 2003;119;1;43-52

  • Deletion of the N-terminus of murine map2 by gene targeting disrupts hippocampal ca1 neuron architecture and alters contextual memory.

    Khuchua Z, Wozniak DF, Bardgett ME, Yue Z, McDonald M, Boero J, Hartman RE, Sims H and Strauss AW

    Vanderbilt University Medical Center, Department of Pediatrics, B3307 MCN, 1161 21 Avenue South, Nashville, TN 37232, USA. zaza.khuchua@vanderbilt.edu

    Microtubule-associated protein-2 (MAP2) is a brain specific A-kinase anchoring protein that targets the cyclic AMP-dependent protein kinase holoenzyme (PKA) to microtubules. Phosphorylation of MAP2 by different protein kinases is crucial for neuronal growth. The N-terminus of MAP2 contains the binding site for regulatory subunit II of cAMP-dependent protein kinase (PKA-RIIbeta). Using homologous recombination, we created a mutant line of mice (delta1-158) that express truncated MAP2 lacking the N-terminal peptide and the PKA binding site. Deletion of the PKA binding site from the MAP2 gene resulted in decreased efficiency of MAP2 phosphorylation. Biochemical and immunohistochemical studies demonstrate major changes in the morphology of hippocampal neurons in delta1-158 mice. Behavioral tests indicate that delta1-158 mice were impaired (exhibited less conditioned freezing) relative to Wild-Type (WT) controls during a test of contextual, but not during auditory cue, fear conditioning when tested at 8 weeks or 8 months of age. The delta1-158 mice displayed a heightened sensitivity to shock at 8 weeks, but not at 8 months of age. We conclude that PKA binding to MAP2 and MAP2 phosphorylation is essential for the selective development of contextual memory.

    Funded by: NHLBI NIH HHS: HL52350; NIA NIH HHS: AG11355

    Neuroscience 2003;119;1;101-11

  • Gamma protocadherins are required for survival of spinal interneurons.

    Wang X, Weiner JA, Levi S, Craig AM, Bradley A and Sanes JR

    Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

    The murine genome contains approximately 70 protocadherin (Pcdh) genes. Many are expressed in the nervous system, suggesting that Pcdhs may specify neuronal connectivity. Here, we analyze the 22 contiguous genes of the Pcdh-gamma cluster. Individual neurons express subsets of Pcdh-gamma genes. Pcdh-gamma proteins are present in most neurons and associated with, but not confined to, synapses. Early steps in neuronal migration, axon outgrowth, and synapse formation proceed in mutant mice lacking all 22 Pcdh-gamma genes. At late embryonic stages, however, dramatic neurodegeneration leads to neonatal death. In mutant spinal cord, many interneurons are lost, but sensory and motor neurons are relatively spared. In cultures from mutant spinal cord, neurons differentiate and form synapses but then die. Thus, Pcdh-gamma genes are dispensable for at least some aspects of connectivity but required for survival of specific neuronal types.

    Neuron 2002;36;5;843-54

  • Pitx2 distinguishes subtypes of terminally differentiated neurons in the developing mouse neuroepithelium.

    Martin DM, Skidmore JM, Fox SE, Gage PJ and Camper SA

    Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, 48109, USA. donnamm@umich.edu

    Pitx2, a homeodomain transcription factor, is essential for normal development of pituitary, eyes, heart, and teeth. In the developing mouse brain, Pitx2 (Rieg, Ptx2, Otlx2, Brx1) mRNA is expressed in discrete regions of the diencephalon, mesencephalon, and rhombencephalon. While prior reports have provided an overview of the temporal and regional specificity of Pitx2 mRNA expression in the brain, the precise cell types that express PITX2 are not known. In this study, we analyzed Pitx2 mRNA and PITX2 protein expression in individual cells of the developing e10.5-e14.5 mouse CNS using multiple markers of cellular proliferation and differentiation. We identified Pitx2 expression in nestin-positive neural progenitors and in postmitotic, developing neurons. In the diencephalon, PITX2 is expressed in neurons of the zona limitans intrathalamica and mammillary region and in gamma-aminobutyric acid (GABA)-producing neurons of the zona incerta. In the mesencephalon, PITX2-labeled nuclei also appear in differentiated neurons, some of which are GABAergic and destined to occupy superior colliculus. Our results suggest that PITX2 expression in postmitotic neurons may contribute to development of GABAergic and other differentiated neuronal phenotypes.

    Funded by: NEI NIH HHS: P30 EY007003, R01 EY014126; NICHD NIH HHS: K08 HD040288-01, KO8 HD 40288, R01 HD34283

    Developmental biology 2002;252;1;84-99

  • Modulation of the notch signaling by Mash1 and Dlx1/2 regulates sequential specification and differentiation of progenitor cell types in the subcortical telencephalon.

    Yun K, Fischman S, Johnson J, Hrabe de Angelis M, Weinmaster G and Rubenstein JL

    Nina Ireland Laboratory of Developmental Neurobiology, Department of Psychiatry, LPPI, University of California, San Francisco, 401 Parnassus, Box 0984, 94143-0984, USA.

    Notch signaling has a central role in cell fate specification and differentiation. We provide evidence that the Mash1 (bHLH) and Dlx1 and Dlx2 (homeobox) transcription factors have complementary roles in regulating Notch signaling, which in turn mediates the temporal control of subcortical telencephalic neurogenesis in mice. We defined progressively more mature subcortical progenitors (P1, P2 and P3) through their combinatorial expression of MASH1 and DLX2, as well as the expression of proliferative and postmitotic cell markers at E10.5-E11.5. In the absence of Mash1, Notch signaling is greatly reduced and 'early' VZ progenitors (P1 and P2) precociously acquire SVZ progenitor (P3) properties. Comparing the molecular phenotypes of the delta-like 1 and Mash1 mutants, suggests that Mash1 regulates early neurogenesis through Notch-and Delta-dependent and -independent mechanisms. While Mash1 is required for early neurogenesis (E10.5), Dlx1 and Dlx2 are required to downregulate Notch signaling during specification and differentiation steps of 'late' progenitors (P3). We suggest that alternate cell fate choices in the developing telencephalon are controlled by coordinated functions of bHLH and homeobox transcription factors through their differential affects on Notch signaling.

    Funded by: NIMH NIH HHS: K02 MH01046-01, R01 MH49428-01, R01 MH51561-01A1

    Development (Cambridge, England) 2002;129;21;5029-40

  • Frizzled-3 is required for the development of major fiber tracts in the rostral CNS.

    Wang Y, Thekdi N, Smallwood PM, Macke JP and Nathans J

    Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Many ligand/receptor families are known to contribute to axonal growth and targeting. Thus far, there have been no reports implicating Wnts and Frizzleds in this process, despite their large numbers and widespread expression within the CNS. In this study, we show that targeted deletion of the mouse fz3 gene leads to severe defects in several major axon tracts within the forebrain. In particular, fz3(-/-) mice show a complete loss of the thalamocortical, corticothalamic, and nigrostriatal tracts and of the anterior commissure, and they show a variable loss of the corpus callosum. Peripheral nerve fibers and major axon tracts in the more caudal regions of the CNS are mostly or completely unaffected. Cell proliferation in the ventricular zone and cell migration to the developing cortex proceed normally until at least embryonic day 14. Extensive cell death in the fz3(-/-) striatum occurs late in gestation, perhaps secondary to the nearly complete absence of long-range connections. In contrast, there is little cell death, as assayed by terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling, in the cortex. These data provide the first link between Frizzled signaling and axonal development.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;19;8563-73

  • MAPK-upstream protein kinase (MUK) regulates the radial migration of immature neurons in telencephalon of mouse embryo.

    Hirai S, Kawaguchi A, Hirasawa R, Baba M, Ohnishi T and Ohno S

    Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan. sh3312@med.yokohama-cu.ac.jp

    The radial migration of differentiating neurons provides an essential step in the generation of laminated neocortex, although its molecular mechanism is not fully understood. We show that the protein levels of a JNK activator kinase, MUK/DLK/ZPK, and JNK activity increase potently and temporally in newly generated neurons in developing mouse telencephalon during radial migration. The ectopic expression of MUK/DLK/ZPK in neural precursor cells in utero impairs radial migration, whereas it allows these cells to leave the ventricular zone and differentiate into neural cells. The MUK/DLK/ZPK protein is associated with dotted structures that are frequently located along microtubules and with Golgi apparatus in cultured embryonic cortical cells. In COS-1 cells, MUK/DLK/ZPK overexpression impairs the radial organization of microtubules without massive depolymerization. These results suggest that MUK/DLK/ZPK and JNK regulate radial cell migration via microtubule-based events.

    Development (Cambridge, England) 2002;129;19;4483-95

  • MAP2 is required for dendrite elongation, PKA anchoring in dendrites, and proper PKA signal transduction.

    Harada A, Teng J, Takei Y, Oguchi K and Hirokawa N

    Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Japan.

    Microtubule-associated protein 2 (MAP2) is a major component of cross-bridges between microtubules in dendrites, and is known to stabilize microtubules. MAP2 also has a binding domain for the regulatory subunit II of cAMP-dependent protein kinase (PKA). We found that there is reduction in microtubule density in dendrites and a reduction of dendritic length in MAP2-deficient mice. Moreover, there is a significant reduction of various subunits of PKA in dendrites and total amounts of various PKA subunits in hippocampal tissue and cultured neurons. In MAP2-deficient cultured neurons, the induction rate of phosphorylated CREB after forskolin stimulation was much lower than in wild-type neurons. Therefore, MAP2 is an anchoring protein of PKA in dendrites, whose loss leads to reduced amount of dendritic and total PKA and reduced activation of CREB.

    The Journal of cell biology 2002;158;3;541-9

  • Ectopic expression of Gcm1 induces congenital spinal cord abnormalities.

    Nait-Oumesmar B, Stecca B, Fatterpekar G, Naidich T, Corbin J and Lazzarini RA

    Department of Molecular, Cellular and Developmental Biology and. Department of Radiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

    Brief ectopic expression of Gcm1 in mouse embryonic tail bud profoundly affects the development of the nervous system. All mice from 5 independently derived transgenic lines exhibited either one or both of two types of congenital spinal cord pathologies: failure of the neural tube to close (spina bifida) and multiple neural tubes (diastematomyelia). Because the transgene is expressed only in a restricted caudal region and only for a brief interval (E8.5 to E13.5), there was no evidence of embryonic lethality. The dysraphisms develop during the period and within the zone of transgene expression. We present evidence that these dysraphisms result from an inhibition of neuropore closure and a stimulation of secondary neurulation. After transgene expression ceases, the spinal dysraphisms are progressively resolved and the neonatal animals, while showing signs of scarring and tissue resorption, have a closed vertebral column. The multiple spinal cords remain but are enclosed in a single spinal column as in the human diastematomyelia. The animals live a normal life time, are fertile and do not exhibit any obvious weakness or motor disabilities.

    Funded by: NINDS NIH HHS: R01 NS39836

    Development (Cambridge, England) 2002;129;16;3957-64

  • Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex.

    Shinozaki K, Miyagi T, Yoshida M, Miyata T, Ogawa M, Aizawa S and Suda Y

    Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan.

    Emx1 and Emx2, mouse orthologs of the Drosophila head gap gene, ems, are expressed during corticogenesis. Emx2 null mutants exhibit mild defects in cortical lamination. Segregation of differentiating neurons from proliferative cells is normal for the most part, however, reelin-positive Cajal-Retzius cells are lost by the late embryonic period. Additionally, late-born cortical plate neurons display abnormal position. These types of lamination defects are subtle in the Emx1 mutant cortex. In the present study we show that Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall was diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone was nearly normal; moreover, no apparent increase in cell death or tetraploid cell number was observed. However, tangential migration of cells from the ganglionic eminence into the neocortex was greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon did not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerged normally, but the laminar structure subsequently formed was completely abnormal. Furthermore, both corticofugal and corticopetal fibers were predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons were found throughout E11.5-E18.5. Thus, this investigation suggests that laminar organization in the cortex or the production of Cajal-Retzius cells and subplate neurons is interrelated to the tangential movement of cells from the ganglionic eminence under the control of Emx1 and Emx2.

    Development (Cambridge, England) 2002;129;14;3479-92

  • Early onset of NMDA receptor GluR epsilon 1 (NR2A) expression and its abundant postsynaptic localization in developing motoneurons of the mouse hypoglossal nucleus.

    Oshima S, Fukaya M, Masabumi N, Shirakawa T, Oguchi H and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.

    Oro-facial sensorimotor function conducted by the brainstem is vital to newborn mammals, and N-methyl-D-aspartate (NMDA) receptors play an important role in the regulation. Here we examined the expression of NMDA receptor subunits in the mouse hypoglossal nucleus from embryonic day 13 (E13) through postnatal day 21 (P21). Compared with other brainstem regions, early onset of GluRepsilon1 (NR2A) mRNA expression was conspicuous to the embryonic hypoglossal nucleus. The expression peaked at P1-P7, when other brainstem regions just started to express it. At P1, GluRepsilon1 subunit was localized to asymmetrical synapses on motoneuron dendrites, particularly, on the postsynaptic junction membrane. In developing motoneurons, expressions of GluRepsilon2 (NR2B), GluRepsilon4 (NR2D), and GluRzeta1 (NR1) mRNAs were accompanied. Until P21, however, all of these subunits were down-regulated with particular reduction for GluRepsilon2 and GluRepsilon4 mRNAs. Similar patterns of temporal expressions were observed in motoneurons of other brainstem motor nuclei. Taking that high levels of GluRepsilon1, GluRepsilon2, and GluRzeta1 subunits are also found in the adult hippocampus and cerebral cortex, it can be assumed that NMDA receptors in developing motoneurons are highly potent and potentially involved in structural and functional development of the brainstem motor system.

    Neuroscience research 2002;43;3;239-50

  • BET, a novel neuronal transmembrane protein with multiple EGF-like motifs.

    Nishizumi H, Komiyama T, Miyabayashi T, Sakano S and Sakano H

    Department of Biophysics, Central Technology Laboratory, Asahi Kasei Corporation, 2-1 Samejima, Fuji, Shizuoka 416-8501, Japan.

    Using the signal sequence trap method, we have identified a cDNA clone coding for a type I transmembrane protein, BET, with 10 epidermal growth factor (EGF) motifs in the extracellular domain. In situ hybridization revealed that the bet mRNA is specifically expressed in the mitral/tufted cells in the olfactory bulb, Purkinje cells in the cerebellum, and pyramidal cells in the hippocampus. Using polyclonal antibodies, we have demonstrated that the BET protein is highly glycosylated and is localized in patches in the dendrites and in the somata of neurons. Since the predicted structure of BET shares many similarities with the Notch ligands, this novel protein may play crucial roles in establishing the neuronal networks in the olfactory system, cerebellum, and hippocampus. BET is the first transmembrane protein containing only multiple EGF-like repeats specifically expressed in the brain.

    Neuroreport 2002;13;6;909-15

  • Abeta-degrading endopeptidase, neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology.

    Fukami S, Watanabe K, Iwata N, Haraoka J, Lu B, Gerard NP, Gerard C, Fraser P, Westaway D, St George-Hyslop P and Saido TC

    Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan.

    Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.

    Neuroscience research 2002;43;1;39-56

  • Participation of structural microtubule-associated proteins (MAPs) in the development of neuronal polarity.

    González-Billault C, Engelke M, Jiménez-Mateos EM, Wandosell F, Cáceres A and Avila J

    Centro de Biología Molecular Severo Ochoa, CSIC, Universidad Autónoma de Madrid, Campus Cantoblanco, Madrid, Spain.

    Several lines of evidence have indicated that changes in the structure of neuronal cytoskeleton provide the support for the dramatic morphological changes that occur during neuronal differentiation. It has been proposed that microtubule-associated proteins can contribute to the development of this phenomenon by controlling the dynamic properties of microtubules. In this report we have characterized the effect of the combined suppression of MAP1B and tau, and MAP1B and MAP2 on neuronal polarization in cultured hippocampal cells grown on a laminin-containing substrate. We have taken advantage of the use of a mouse line deficient in MAP1B expression obtained by the gene trapping approach. In addition to this engineered mice line we used the antisense oligonucleotide approach to induce the suppression of tau or MAP2, in wild type and MAP1B-deficient neurons. Together these results show a synergistic role for MAP1B/MAP2 and MAP1B/TAU.

    Journal of neuroscience research 2002;67;6;713-9

  • Transcriptional regulation of cortical neuron migration by POU domain factors.

    McEvilly RJ, de Diaz MO, Schonemann MD, Hooshmand F and Rosenfeld MG

    Howard Hughes Medical Institute, Department and School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92037-0648, USA.

    The identification of pathways mediated by the kinase Cdk5 and the ligand reelin has provided a conceptual framework for exploring the molecular mechanisms underlying proper lamination of the developing mammalian cerebral cortex. In this report, we identify a component of the regulation of Cdk5-mediated cortical lamination by genetic analysis of the roles of the class III POU domain transcription factors, Brn-1 and Brn-2, expressed during the development of the forebrain and coexpressed in most layer II-V cortical neurons. Brn-1 and Brn-2 appear to critically control the initiation of radial migration, redundantly regulating the cell-autonomous expression of the p35 and p39 regulatory subunits of Cdk5 in migrating cortical neurons, with Brn-1(-/-)/Brn-2(-/-) mice exhibiting cortical inversion.

    Science (New York, N.Y.) 2002;295;5559;1528-32

  • Rescue of ataxia and preplate splitting by ectopic expression of Reelin in reeler mice.

    Magdaleno S, Keshvara L and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA.

    The gene mutated in reeler (reelin) encodes a protein secreted by neurons in the developing brain that controls laminar positioning of migrating cells in the CNS by an unknown mechanism. To investigate Reelin function, we used the nestin promoter to express Reelin ectopically in the ventricular zone and other brain regions in transgenic mice. In the presence of the endogenous protein, ectopic Reelin did not alter cell migration in the neocortex or the cerebellum. However, in the reeler background, ectopic Reelin induced tyrosine phosphorylation of Dab-1 in the ventricular zone and rescued some, but not all, of the neuroanatomic and behavioral abnormalities characteristic of reeler. These results indicate that Reelin does not function simply as a positional signal. Rather, it appears to participate in multiple events critical for neuronal migration and cell positioning.

    Funded by: NINDS NIH HHS: R01 NS36558

    Neuron 2002;33;4;573-86

  • Nitric oxide affects the phosphorylation state of microtubule-associated protein 2 (MAP-2) and neurofilament: an immunocytochemical study in the brain of rats and neuronal nitric oxide synthase (nNOS)-knockouts.

    Rothe F, Possel H and Wolf G

    Institute of Medical Neurobiology, University of Magdeburg, D-39120 Magdeburg, Germany. fritz.rothe@medizin.uni-magdeburg.de

    Alterations in function and specific cellular location of cytoskeletal elements are characterized by changes in their phosphorylation state. On this background we studied immunocytochemically the distribution pattern of neurofilament (NF) in its phosphorylated (P-NF) and nonphosphorylated (NP-NF) form and of microtubule-associated protein-2 (MAP-2) in the rat and mouse brain. Neurons that are strongly positive for neuronal nitric oxide synthase (nNOS)-immunoreactivity (IR) showed, interestingly, neither P-NF- nor MAP-2-IR. In contrast, nNOS-negative neuronal cell elements exhibited an intense IR and specific location for both antigens throughout the brain. As a model we chose the dorsolateral tegmental nucleus (LDT) of knockout (nNOS(-/-)) mice in which the main splice isoform nNOSalpha is lacking, but a low nNOS-activity persists, apparently due to the splice isoforms nNOSbeta and gamma. The principal neurons of such nNOS(-/-)-mice, which are equivalent to the nNOS-containing neurons in the LDT of wild-type mice exhibited a decreased nitrotyrosine-IR and an increased phosphotyrosine-IR if compared to those of wild-type mice. The same neurons failed to show NF-IR and MAP-2-IR, though. When the residual nNOS activity in nNOS(-/-)-mice was inhibited by treatment with N-omega-nitro-L-arginine methyl ester (L-NAME) the principal neurons displayed a moderate MAP-2 and NF-staining. NO and NO-derived oxygen species are suggested to modulate the balance between the activities of kinases and phosphatases, thus changing phosphorylation levels for NF, MAP-2, and, possibly, other proteins in neurons and adjacent cell elements.

    Nitric oxide : biology and chemistry 2002;6;1;9-17

  • Characterization of mouse homolog of brain acyl-CoA hydrolase: molecular cloning and neuronal localization.

    Kuramochi Y, Takagi-Sakuma M, Kitahara M, Emori R, Asaba Y, Sakaguchi R, Watanabe T, Kuroda J, Hiratsuka K, Nagae Y, Suga T and Yamada J

    Department of Clinical Biochemistry, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, 192-0392, Tokyo, Japan.

    Acyl-CoA hydrolase could provide a mechanism via its potency to modulate cellular concentrations of acyl-CoAs for the regulation of various cellular events including fatty acid metabolism and gene expression. However, only limited evidence of this is available. To better understand the physiological role of this enzyme, we characterized a mouse brain acyl-CoA hydrolase, mBACH. The cloned cDNA for mBACH encoded a 338-amino-acid polypeptide with >95% identity to the human and rat homologs, indicating that the BACH gene is highly conserved among species. This was supported by the similarity in genomic organization of the BACH gene between humans and mice. Bacterially expressed mBACH was highly active against long-chain acyl-CoAs with a relatively broad specificity for chain length. While palmitoyl-CoA hydrolase activity was widely distributed in mouse tissues, it was marked in the brain, consistent with mBACH being almost exclusively distributed in this tissue, where >80% of the enzyme activity was explained by mBACH present in the cytosol. Immunohistochemistry demonstrated a neuronal localization of mBACH in both the central and peripheral nervous systems. In neurons, mBACH was distributed throughout the cell body and neurites. Although four isoforms except mBACH itself, that may be generated by the alternative use of exons of a single mBACH gene, were cloned, their mRNA levels in the brain were estimated to be negligible. However, a 50-kDa polypeptide besides the major one of 43-kDa seemed to be translated from the mBACH mRNA with differential in-frame ATG triplets used as the initiation codon. These findings will contribute to the functional analysis of the BACH gene using mice including genetic studies.

    Brain research. Molecular brain research 2002;98;1-2;81-92

  • Functional peculiarities of MAP2 in DBA/2J inbred mice as a component of genetic predisposition to seizures.

    Chulanova TA, Echikov SN, Sadovnikov VB and Shchipakina TG

    Group of the Mechanisms of Synaptic Transmission, Institute of Theoretical and Experimental Biophysics, Pushchino. neurochem@mail.ru

    We compared the content of high molecular microtubule-associated protein-2 and its phosphorylation by cAMP- and Ca(2+)/calmodulin-dependent protein kinases in the brain of DBA/2J and C57Bl/6 inbred mice. The revealed differences in protein content and phosphorylation can be attributed to the mechanisms mediating audiogenic seizures in DBA/2J mice and to differential sensitivity of these inbred lines to seizure-inducing factors.

    Bulletin of experimental biology and medicine 2001;132;5;1058-61

  • Rna-binding protein Musashi2: developmentally regulated expression in neural precursor cells and subpopulations of neurons in mammalian CNS.

    Sakakibara S, Nakamura Y, Satoh H and Okano H

    Division of Neuroanatomy, Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.

    Musashi1 (Msi1) is a mammalian neural RNA-binding protein highly enriched in neural precursor cells that are capable of generating both neurons and glia during embryonic and postnatal CNS development. Here, we identified Musashi2 (Msi2), a novel mammalian RNA-binding protein that exhibits high sequence similarity to Msi1. The Msi2 transcript appeared to be distributed ubiquitously in a wide variety of tissues, consistent with the mRNA distribution of its Xenopus homolog, xrp1. However, the present study revealed cell type-specific and developmentally regulated expression of Msi2 in the mammalian CNS. Interestingly, Msi2 was expressed prominently in precursor cells in the ventricular zone and subventricular zone with the same pattern as Msi1 throughout CNS development. In the postnatal and adult CNS, this concurrent expression of Msi2 and Msi1 was seen in cells of the astrocyte lineage, including ependymal cells, a possible source for postnatal CNS stem cells. During neurogenesis, the expression of both Msi2 and Msi1 was lost in most postmitotic neurons, whereas Msi2 expression persisted in a subset of neuronal lineage cells, such as parvalbumin-containing GABA neurons in the neocortex and neurons in several nuclei of the basal ganglia. Msi2 may have a unique role that is required for the generation and/or maintenance of specific neuronal lineages. Furthermore, in vitro studies showed that Msi2 and Msi1 have similar RNA-binding specificity. These two RNA-binding proteins may exert common functions in neural precursor cells by regulating gene expression at the post-transcriptional level.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;20;8091-107

  • 3-Phosphoglycerate dehydrogenase, a key enzyme for l-serine biosynthesis, is preferentially expressed in the radial glia/astrocyte lineage and olfactory ensheathing glia in the mouse brain.

    Yamasaki M, Yamada K, Furuya S, Mitoma J, Hirabayashi Y and Watanabe M

    Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-8638, Japan.

    l-Serine is synthesized from glycolytic intermediate 3-phosphoglycerate and is an indispensable precursor for the synthesis of proteins, membrane lipids, nucleotides, and neuroactive amino acids d-serine and glycine. We have recently shown that l-serine and its interconvertible glycine act as Bergmann glia-derived trophic factors for cerebellar Purkinje cells. To investigate whether such a metabolic neuron-glial relationship is fundamental to the developing and adult brain, we examined by in situ hybridization and immunohistochemistry the cellular expression of 3-phosphoglycerate dehydrogenase (3PGDH), the initial step enzyme for de novo l-serine biosynthesis in animal cells. At early stages when the neural wall consists exclusively of the ventricular zone, neuroepithelial stem cells expressed 3PGDH strongly and homogeneously. Thereafter, 3PGDH expression was downregulated and eventually disappeared in neuronal populations, whereas its high expression was transmitted to the radial glia and later to astrocytes in the gray and white matters. In addition, 3PGDH was highly expressed throughout development in the olfactory ensheathing glia, a specialized supporting cell that thoroughly ensheathes olfactory nerves. These results establish a fundamental link of the radial glia/astrocyte lineage and olfactory ensheathing glia to l-serine biosynthesis in the brain. We discuss this finding in the context of the hypothesis that 3PGDH expression in these glia cells contributes to energy metabolism in differentiating and differentiated neurons and other glia cells, which are known to be vulnerable to energy loss.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;19;7691-704

  • Synergistic effects of MAP2 and MAP1B knockout in neuronal migration, dendritic outgrowth, and microtubule organization.

    Teng J, Takei Y, Harada A, Nakata T, Chen J and Hirokawa N

    Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

    MAP1B and MAP2 are major members of neuronal microtubule-associated proteins (MAPs). To gain insights into the function of MAP2 in vivo, we generated MAP2-deficient (map2(-/-)) mice. They developed without any apparent abnormalities, which indicates that MAP2 is dispensable in mouse survival. Because previous reports suggest a functional redundancy among MAPs, we next generated mice lacking both MAP2 and MAP1B to test their possible synergistic functions in vivo. Map2(-/-)map1b(-/-) mice died in their perinatal period. They showed not only fiber tract malformations but also disrupted cortical patterning caused by retarded neuronal migration. In spite of this, their cortical layer maintained an "inside-out" pattern. Detailed observation of primary cultures of hippocampal neurons from map2(-/-)map1b(-/-) mice revealed inhibited microtubule bundling and neurite elongation. In these neurons, synergistic effects caused by the loss of MAP2 and MAP1B were more apparent in dendrites than in axons. The spacing of microtubules was reduced significantly in map2(-/-)map1b(-/-) mice in vitro and in vivo. These results suggest that MAP2 and MAP1B have overlapping functions in neuronal migration and neurite outgrowth by organizing microtubules in developing neurons both for axonal and dendritic morphogenesis but more dominantly for dendritic morphogenesis.

    The Journal of cell biology 2001;155;1;65-76

  • Extensive neuronal localization and neurotrophic function of fibroblast growth factor 8 in the nervous system.

    Tanaka A, Kamiakito T, Hakamata Y, Fujii A, Kuriki K and Fukayama M

    Department of Pathology, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Kawachi, 329-0498, Tochigi, Japan. atanaka@jichi.ac.jp

    Fibroblast growth factor (FGF) 8 has been well established to play a critical role in the early development of the central nervous system (CNS). We report here extensive neuronal localization and neurotrophic function of FGF8 in the nervous system. In sections of mouse embryos at E10.5, FGF8 was immunohistochemically found in neurons at the marginal zones of the CNS and in the dorsal root ganglia (DRG). Neuronal localization of FGF8 was marked at later embryonic stages and in adults, involving most of the central and peripheral neurons, including intermuscular enteric neurons, DRGs, and paraaortic sympathetic ganglia. Functionally, FGF8 promoted neurite outgrowth in human neuroblastoma SK-N-MC cells as well as in rat pheochromocytoma PC12 cells, suggesting that FGF8 acts as a neurotrophic factor. FGF8 also supported neuronal survival and differentiation in cultured human neural progenitor cells. In a cell growth assay, treatment with 50 ng/ml FGF8 on human cultured neuroblastoma SK-N-MC and IMR32 cells attenuated the growth of both. In accordance with these in vitro findings, the immunohistochemical analysis on human neurological diseases showed that FGF8 expression is evident in differentiating histological types of neuroblastoma and ganglioneuroblastoma, and that the levels of FGF8 immunoreactivity in the substantia nigra from Parkinson's disease are significantly lower than those in age-matched controls. Taken together, the present findings strongly suggest that FGF8 acts as a more generalized neurotrophic factor than previously reported.

    Brain research 2001;912;2;105-15

  • Platelet-derived growth factor receptor-alpha in ventricular zone cells and in developing neurons.

    Andrae J, Hansson I, Afink GB and Nistér M

    Department of Genetics and Pathology, Uppsala University, Uppsala, SE-751 85, Sweden.

    Cells in the early neuroepithelium differentiate and give rise to all cells in the central nervous system (CNS). The ways from a multipotent CNS stem cell to specialized neurons and glia are not fully understood. Using immunohistochemistry we found that neuroepithelial cells express the platelet-derived growth factor receptor-alpha (PDGFR-alpha) in the neural plate at embryonic day 8.5 and onwards in the neural tube. The protein was polarized to ventricular endfeet. Furthermore, PDGFR-alpha expression was localized to cells undergoing early neuronal development. We also found PDGFR-alpha expression in developing granule cells in the postnatal cerebellum, in Purkinje cells in the adult cerebellum and on processes of developing dorsal root ganglion cells. Previous reports mainly describe PDGFR-alpha expression in oligodendrocyte precursors and glial cells. We believe, in line with a few previous reports, that the PDGFR-alpha in addition marks a pool of undifferentiated cells, which are able to differentiate into neurons.

    Molecular and cellular neurosciences 2001;17;6;1001-13

  • Disruption of KCC2 reveals an essential role of K-Cl cotransport already in early synaptic inhibition.

    Hübner CA, Stein V, Hermans-Borgmeyer I, Meyer T, Ballanyi K and Jentsch TJ

    Zentrum für molekulare Neurobiologie Hamburg, ZMNH, Universität Hamburg, Martinistr. 52, D-20246, Hamburg, Germany.

    Synaptic inhibition by GABA(A) and glycine receptors, which are ligand-gated anion channels, depends on the electrochemical potential for chloride. Several potassium-chloride cotransporters can lower the intracellular chloride concentration [Cl(-)](i), including the neuronal isoform KCC2. We show that KCC2 knockout mice died immediately after birth due to severe motor deficits that also abolished respiration. Sciatic nerve recordings revealed abnormal spontaneous electrical activity and altered spinal cord responses to peripheral electrical stimuli. In the spinal cord of wild-type animals, the KCC2 protein was found at inhibitory synapses. Patch-clamp measurements of embryonic day 18.5 spinal cord motoneurons demonstrated an excitatory GABA and glycine action in the absence, but not in the presence, of KCC2, revealing a crucial role of KCC2 for synaptic inhibition.

    Neuron 2001;30;2;515-24

  • Combinatorial expression patterns of LIM-homeodomain and other regulatory genes parcellate developing thalamus.

    Nakagawa Y and O'Leary DD

    Molecular Neurobiology Laboratory, The Salk Institute, La Jolla, California 92037, USA.

    The anatomical and functional organization of dorsal thalamus (dTh) and ventral thalamus (vTh), two major regions of the diencephalon, is characterized by their parcellation into distinct cell groups, or nuclei, that can be histologically defined in postnatal animals. However, because of the complexity of dTh and vTh and difficulties in histologically defining nuclei at early developmental stages, our understanding of the mechanisms that control the parcellation of dTh and vTh and the differentiation of nuclei is limited. We have defined a set of regulatory genes, which include five LIM-homeodomain transcription factors (Isl1, Lhx1, Lhx2, Lhx5, and Lhx9) and three other genes (Gbx2, Ngn2, and Pax6), that are differentially expressed in dTh and vTh of early postnatal mice in distinct but overlapping patterns that mark nuclei or subsets of nuclei. These genes exhibit differential expression patterns in dTh and vTh as early as embryonic day 10.5, when neurogenesis begins; the expression of most of them is detected as progenitor cells exit the cell cycle. Soon thereafter, their expression patterns are very similar to those that we observe postnatally, indicating that unique combinations of these genes mark specific cell groups from the time they are generated to their later differentiation into nuclei. Our findings suggest that these genes act in a combinatorial manner to control the specification of nuclei-specific properties of thalamic cells and the differentiation of nuclei within dTh and vTh. These genes may also influence the pathfinding and targeting of thalamocortical axons through both cell-autonomous and non-autonomous mechanisms.

    Funded by: NINDS NIH HHS: R01 NS31558

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;8;2711-25

  • p190 RhoGAP is the principal Src substrate in brain and regulates axon outgrowth, guidance and fasciculation.

    Brouns MR, Matheson SF and Settleman J

    MGH Cancer Center, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA.

    The Src tyrosine kinases have been implicated in several aspects of neural development and nervous system function; however, their relevant substrates in brain and their mechanism of action in neurons remain to be established clearly. Here we identify the potent Rho regulatory protein, p190 RhoGAP (GTPase-activating protein), as the principal Src substrate detected in the developing and mature nervous system. We also find that mice lacking functional p190 RhoGAP exhibit defects in axon guidance and fasciculation. p190 RhoGAP is co-enriched with F-actin in the distal tips of axons, and overexpressing p190 RhoGAP in neuroblastoma cells promotes extensive neurite outgrowth, indicating that p190 RhoGAP may be an important regulator of Rho-mediated actin reorganization in neuronal growth cones. p190 RhoGAP transduces signals downstream of cell-surface adhesion molecules, and we find that p190-RhoGAP-mediated neurite outgrowth is promoted by the extracellular matrix protein laminin. Together with the fact that mice lacking neural adhesion molecules or Src kinases also exhibit defects in axon outgrowth, guidance and fasciculation, our results suggest that p190 RhoGAP mediates a Src-dependent adhesion signal for neuritogenesis to the actin cytoskeleton through the Rho GTPase.

    Nature cell biology 2001;3;4;361-7

  • Tbr1 regulates differentiation of the preplate and layer 6.

    Hevner RF, Shi L, Justice N, Hsueh Y, Sheng M, Smiga S, Bulfone A, Goffinet AM, Campagnoni AT and Rubenstein JL

    Nina Ireland Laboratory of, Developmental Neurobiology, Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA.

    During corticogenesis, early-born neurons of the preplate and layer 6 are important for guiding subsequent neuronal migrations and axonal projections. Tbr1 is a putative transcription factor that is highly expressed in glutamatergic early-born cortical neurons. In Tbr1-deficient mice, these early-born neurons had molecular and functional defects. Cajal-Retzius cells expressed decreased levels of Reelin, resulting in a reeler-like cortical migration disorder. Impaired subplate differentiation was associated with ectopic projection of thalamocortical fibers into the basal telencephalon. Layer 6 defects contributed to errors in the thalamocortical, corticothalamic, and callosal projections. These results show that Tbr1 is a common genetic determinant for the differentiation of early-born glutamatergic neocortical neurons and provide insights into the functions of these neurons as regulators of cortical development.

    Funded by: NIMH NIH HHS: K02 MH01046; NINDS NIH HHS: NS01973, R01 NS34661; Telethon: F.2

    Neuron 2001;29;2;353-66

  • Cell migration from the ganglionic eminences is required for the development of hippocampal GABAergic interneurons.

    Pleasure SJ, Anderson S, Hevner R, Bagri A, Marin O, Lowenstein DH and Rubenstein JL

    Neurodevelopmental Disorders Laboratory, Department of Neurology, University of California, San Francisco, CA 94143, USA.

    GABAergic interneurons have major roles in hippocampal function and dysfunction. Here we provide evidence that, in mice, virtually all of these cells originate from progenitors in the basal telencephalon. Immature interneurons tangentially migrate from the basal telencephalon through the neocortex to take up their final positions in the hippocampus. Disrupting differentiation in the embryonic basal telencephalon (lateral and medial ganglionic eminences) through loss of Dlx1/2 homeobox function blocks the migration of virtually all GABAergic interneurons to the hippocampus. On the other hand, disrupting specification of the medial ganglionic eminence through loss of Nkx2.1 homeobox function depletes the hippocampus of a distinct subset of hippocampal interneurons. Loss of hippocampal interneurons does not appear to have major effects on the early development of hippocampal projection neurons nor on the pathfinding of afferrent tracts.

    Funded by: NIDA NIH HHS: R01DA12462; NIMH NIH HHS: R01 MH49428-01, R01 MH51561-01A1

    Neuron 2000;28;3;727-40

  • Mammalian achaete-scute and atonal homologs regulate neuronal versus glial fate determination in the central nervous system.

    Tomita K, Moriyoshi K, Nakanishi S, Guillemot F and Kageyama R

    Institute for Virus Research, Kyoto University, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, Japan.

    Whereas vertebrate achaete-scute complex (as-c) and atonal (ato) homologs are required for neurogenesis, their neuronal determination activities in the central nervous system (CNS) are not yet supported by loss-of-function studies, probably because of genetic redundancy. Here, to address this problem, we generated mice double mutant for the as-c homolog Mash1 and the ato homolog Math3. Whereas in Mash1 or Math3 single mutants neurogenesis is only weakly affected, in the double mutants tectal neurons, two longitudinal columns of hindbrain neurons and retinal bipolar cells were missing and, instead, those cells that normally differentiate into neurons adopted the glial fate. These results indicated that Mash1 and Math3 direct neuronal versus glial fate determination in the CNS and raised the possibility that downregulation of these bHLH genes is one of the mechanisms to initiate gliogenesis.

    The EMBO journal 2000;19;20;5460-72

  • Perinatal lethality of microtubule-associated protein 1B-deficient mice expressing alternative isoforms of the protein at low levels.

    González-Billault C, Demandt E, Wandosell F, Torres M, Bonaldo P, Stoykova A, Chowdhury K, Gruss P, Avila J and Sánchez MP

    Centro de Biología Molecular, Universidad Autónoma de Madrid, Spain.

    Microtubule-associated protein 1B (MAP1B) has been implicated in axogenesis in cultured cells. To gain insight into the functions that MAP1B plays in vivo, we analyzed a strain of Map1B mutant mice generated by a gene trapping approach. Homozygous mice die on the first day after birth, probably due to a severe abnormal development of the nervous system. They present alterations in the structure of several brain regions. The normal Map1B gene yields different protein isoforms from alternatively spliced transcripts. The smaller isoforms were present in wild type, hetero-, and homozygous mice, but their expression was higher in the mutants than in the wild-type. Moreover, trace amounts of MAP1B protein were also observed in Map1B homozygous mutants, indicating an alternative splicing around the gene trap insertion. Thus, the Map1B gene trapped mutation reported in this work did not generated a null mutant, but a mouse with a drastic deficiency in MAP1B expression. Analyses of these mice indicate the presence of several neural defects and suggest the participation of MAP1B in neuronal migration.

    Molecular and cellular neurosciences 2000;16;4;408-21

  • Combinatorial roles of the nuclear receptor corepressor in transcription and development.

    Jepsen K, Hermanson O, Onami TM, Gleiberman AS, Lunyak V, McEvilly RJ, Kurokawa R, Kumar V, Liu F, Seto E, Hedrick SM, Mandel G, Glass CK, Rose DW and Rosenfeld MG

    Howard Hughes Medical Institute, Department of Biology, University of California, San Diego, La Jolla 92093, USA.

    Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicated that N-CoR was a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase were also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems.

    Funded by: NIDDK NIH HHS: 1 RO1 DK54802-O1A1

    Cell 2000;102;6;753-63

  • Abnormal polarization and axon outgrowth in retinal ganglion cells lacking the POU-domain transcription factor Brn-3b.

    Wang SW, Gan L, Martin SE and Klein WH

    Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030, USA.

    The POU domain transcription factor Brn-3b (also called Brn-3.2) is essential for the normal development of retinal ganglion cells (RGCs) in the mouse. Without Brn-3b, RGCs commit to their fate and migrate to the ganglion cell layer, but most cells die during fetal development. An earlier report (L. Gan et al., 1999, Dev. Biol. 210, 469-480) suggested that cell death was caused by abnormal axon formation. Here, we use retinal explants from wild-type and mutant embryos to show that brn-3b-deficient RGCs are not properly polarized and tend to form dendrites rather than axons. Compared with wild-type explants, neurites of RGCs from brn-3b-deficient retinal explants grew slower, were shorter, and did not fasciculate properly. Mutant neurites had more microtubules than wild-type controls, and the arrangement of microtubules and neurofilaments was characteristic of dendrites rather than axons. Neurites from individual mutant RGCs displayed abnormal polarity and had dendrite-like branches extending outward from their main axis. Most mutant RGCs exhibited abnormal migratory behavior, and their neurites labeled intensely with the dendrite marker MAP-2. A small number of mutant RGCs were not migratory, and their neurites were longer and labeled positively for the axon marker tau-1, suggesting that some RGCs were not as severely affected by the absence of Brn-3b as others. Although tau-1 was not observed in most mutant neurites, it did accumulate in mutant cell bodies, implying that the absence of Brn-3b caused a defect in axon transport. Thus, Brn-3b appears to control the activity of genes that function in establishing RGC polarity, and without Brn-3b, RGCs cannot extend normal axons.

    Funded by: NCI NIH HHS: CA16672; NEI NIH HHS: EY10608, EY11930

    Molecular and cellular neurosciences 2000;16;2;141-56

  • Developmental history of the subplate and developing white matter in the murine neocortex. Neuronal organization and relationship with the main afferent systems at embryonic and perinatal stages.

    Del Río JA, Martínez A, Auladell C and Soriano E

    Department of Cell Biology, Faculty of Biology and Neuroscience Research Center, (C.E.R.N.), University of Barcelona, E-08028 Barcelona, Spain. jario@porthos.bio.ub.es

    The neuronal diversity of the subplate and developing white matter in the mouse was studied using a variety of neuronal markers. The subplate was first visible in lateral cortical areas at E13, coinciding with the emergence of the cortical plate. During prenatal development, this layer was formed by morphologically heterogeneous neurons, subsets of which were immunoreactive for GABA- and calcium-binding proteins. From E18 onwards, a few subplate cells also contained neuropeptides. Colocalization experiments demonstrated that the percentages of neurons immunoreactive for each antigen were similar to those described in adult neocortex. By E15, subplate cells had received synaptic contacts. Moreover, a second early-neuronal population was conspicuous from E13 in the lower intermediate zone: the intermediate-subventricular population. Unlike subplate cells, these neurons were morphologically uniform, smaller and horizontally oriented. Nevertheless, a few of these cells also appeared within the ventricular zone, with a perpendicular/ oblique orientation. Most of these cells were GABA-positive and showed calbindin immunoreactivity. At the electron microscopic level, no synaptic contacts were found in these neurons. Tracing studies using DiI showed that subplate neurons were the first to send axons outside the neocortex towards the ganglionic eminence at E13. At E14, subplate axons and ingrowing thalamic fibers met in the striate primordium. Subplate cells retained their projection to the thalamus during prenatal development. Thalamocortical axons reached the subplate at E15, and 1 day later began to invade the upper cortical layers. Early callosal axons, in contrast, did not run through the subplate to reach the contralateral hemisphere, nor did subplate cells send out callosal fibers. Callosal axons ran just above the subventricular zone, intermingled with the intermediate-subventricular neuronal population. We conclude that the subplate neuronal population has a chemical heterogeneity reminiscent of that of the adult cortex and is crucial to the establishment of thalamocortical relationships, whereas the intermediate-subventricular neurons constituted a particular GABAergic population, which includes resident cells and tangentially migrating postmitotic neurons spatially related to the development of callosal connections.

    Cerebral cortex (New York, N.Y. : 1991) 2000;10;8;784-801

  • Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptor-containing vesicle transport.

    Setou M, Nakagawa T, Seog DH and Hirokawa N

    Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan.

    Experiments with vesicles containing N-methyl-D-aspartate (NMDA) receptor 2B (NR2B subunit) show that they are transported along microtubules by KIF17, a neuron-specific molecular motor in neuronal dendrites. Selective transport is accomplished by direct interaction of the KIF17 tail with a PDZ domain of mLin-10 (Mint1/X11), which is a constituent of a large protein complex including mLin-2 (CASK), mLin-7 (MALS/Velis), and the NR2B subunit. This interaction, specific for a neurotransmitter receptor critically important for plasticity in the postsynaptic terminal, may be a regulatory point for synaptic plasticity and neuronal morphogenesis.

    Science (New York, N.Y.) 2000;288;5472;1796-802

  • Mouse numb is an essential gene involved in cortical neurogenesis.

    Zhong W, Jiang MM, Schonemann MD, Meneses JJ, Pedersen RA, Jan LY and Jan YN

    Howard Hughes Medical Institute and Departments of Physiology and Biochemistry, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California, San Francisco, CA 94143, USA.

    During neurogenesis of the mammalian neocortex, neural progenitor cells divide to generate daughter cells that either become neurons or remain as progenitor cells. The mouse numb (m-numb) gene encodes a membrane-associated protein that is asymmetrically localized to the apical cell membrane of dividing cortical progenitor cells and may be segregated to only the apical daughter cell that has been suggested to remain as a progenitor cell. To examine m-numb function during neural development, we generated a loss-of-function mutant allele of m-numb. Mice homozygous for this mutation exhibit severe defects in cranial neural tube closure and precocious neuron production in the forebrain and die around embryonic day 11.5 (E11. 5). These findings suggest that m-numb is an essential gene that plays a role in promoting progenitor cell fate during cortical neurogenesis.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;12;6844-9

  • Presenilin-1 regulates neuronal differentiation during neurogenesis.

    Handler M, Yang X and Shen J

    Center for Neurologic Diseases, Department of Neurology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

    Mutations in Presenilin-1 (PS1) are a major cause of familial Alzheimer's disease. Our previous studies showed that PS1 is required for murine neural development. Here we report that lack of PS1 leads to premature differentiation of neural progenitor cells, indicating a role for PS1 in a cell fate decision between postmitotic neurons and neural progenitor cells. Neural proliferation and apoptotic cell death during neurogenesis are unaltered in PS1(-/-) mice, suggesting that the reduction in the neural progenitor cells observed in the PS1(-/-) brain is due to premature differentiation of progenitor cells, rather than to increased apoptotic cell death or decreased cell proliferation. In addition, the premature neuronal differentiation in the PS1(-/-) brain is associated with aberrant neuronal migration and disorganization of the laminar architecture of the developing cerebral hemisphere. In the ventricular zone of PS1(-/-) mice, expression of the Notch1 downstream effector gene Hes5 is reduced and expression of the Notch1 ligand Dll1 is elevated, whereas expression of Notch1 is unchanged. The level of Dll1 transcripts is also increased in the presomitic mesoderm of PS1(-/-) embryos, while the level of Notch1 transcripts is unchanged, in contrast to a previous report (Wong et al., 1997, Nature 387, 288-292). These results provide direct evidence that PS1 controls neuronal differentiation in association with the downregulation of Notch signalling during neurogenesis.

    Development (Cambridge, England) 2000;127;12;2593-606

  • Involvement of the TRAP220 component of the TRAP/SMCC coactivator complex in embryonic development and thyroid hormone action.

    Ito M, Yuan CX, Okano HJ, Darnell RB and Roeder RG

    Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021, USA.

    The TRAP220 component of the TRAP/SMCC complex, a mammalian homologof the yeast Mediator that shows diverse coactivation functions, interacts directly with nuclear receptors. Ablation of the murine Trap220 gene revealed that null mutants die during an early gestational stage with heart failure and exhibit impaired neuronal development with extensive apoptosis. Primary embryonic fibroblasts derived from null mutants show an impaired cell cycle regulation and a prominent decrease of thyroid hormone receptor function that is restored by ectopic TRAP220 but no defect in activation by Gal4-RARalpha/RXRalpha, p53, or VP16. Moreover, haploinsufficient animals show growth retardation, pituitary hypothyroidism, and widely impaired transcription in certain organs. These results indicate that TRAP220 is essential for a wide range of physiological processes but also that it has gene- and activator-selective functions.

    Molecular cell 2000;5;4;683-93

  • Demarcation of early mammalian cortical development by differential expression of fringe genes.

    Ishii Y, Nakamura S and Osumi N

    Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, 4-1-1 Ogawa-higashi, Kodaira, Tokyo, Japan.

    Fringe has originally been found in Drosophila as a gene encoding a putative secreted protein which regulates the sensitivity of Notch signaling pathway to different ligands. We show that three members of murine fringe gene family, Lunatic fringe (L-fng), Manic fringe (M-fng) and Radical fringe (R-fng), show related patterns of expression in the developing cerebral wall. L-fng is expressed in immature cells in the ventricular zone. M-fng is upregulated transiently in maturing neurons when they leave the ventricular zone (VZ). R-fng is upregulated in more mature neurons when they enter the preplate and cortical plate. These patterns suggest that the transition from immature to mature neurons involves sequential changes in the member of fringe family genes expressed. More detailed expression analyses of fringe genes and immunohistochemistry for neuron-specific class III beta-tubulin suggest a mode of neurogenesis which might underlie the histogenesis of the cerebral cortex. A proliferative population situated outside of the VZ is defined as M-fng-positive/BrdU-positive cells, which constitutes about 10-20% of the total S-phase cells in the cerebral wall of embryonic day 10.5-12.5. We found that M-fng is expressed in mitotic figures outside the VZ and some of them react with the antibody against class III beta-tubulin. These observations suggest that a significant number of proliferative cells exist outside the VZ, which supply neurons during early cortical development.

    Brain research. Developmental brain research 2000;119;2;307-20

  • The lack of Emx2 causes impairment of Reelin signaling and defects of neuronal migration in the developing cerebral cortex.

    Mallamaci A, Mercurio S, Muzio L, Cecchi C, Pardini CL, Gruss P and Boncinelli E

    Department of Biological and Technological Research (DIBIT), Istituto Scientifico H. San Raffaele, 20132 Milano, Italy. mallamaci.antonio@hsr.it

    Neocorticogenesis in mice homozygous for an Emx2 null allele is the topic of this article. The development of both main components of neocortex, primordial plexiform layer derivatives and cortical plate, was analyzed, paying special attention to radial migration of neurons forming the cortical plate. The products of the Reelin gene, normally playing a key role in orchestrating radial migration of these neurons, display normal distribution at the beginning of the cortical neuronogenesis but are absent in the neocortical marginal zone of the mutant mice at the time when the cortical plate is laid down. As a consequence, the development of radial glia is impaired, and neurons making up the cortical plate display abnormal migration patterns. In addition, restricted defects along the rostrocaudal and the mediolateral axes are present in the subplate, suggesting an Emx2-specific role in priming the proper development of this layer.

    Funded by: Telethon: E.0450

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;3;1109-18

  • Spatial, temporal and subcellular localization of islet-brain 1 (IB1), a homologue of JIP-1, in mouse brain.

    Pellet JB, Haefliger JA, Staple JK, Widmann C, Welker E, Hirling H, Bonny C, Nicod P, Catsicas S, Waeber G and Riederer BM

    Institut de Biologie Cellulaire et de Morphologie, IBCM, Rue du Bugnon 9, 1005 Lausanne, Switzerland.

    Islet-brain 1 (IB1) was recently identified as a DNA-binding protein of the GLUT2 gene promoter. The mouse IB1 is the rat and human homologue of the Jun-interacting protein 1 (JIP-1) which has been recognized as a key player in the regulation of c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. JIP-1 is involved in the control of apoptosis and may play a role in brain development and aging. Here, IB1 was studied in adult and developing mouse brain tissue by in situ hybridization, Northern and Western blot analysis at cellular and subcellular levels, as well as by immunocytochemistry in brain sections and cell cultures. IB1 expression was localized in the synaptic regions of the olfactory bulb, retina, cerebral and cerebellar cortex and hippocampus in the adult mouse brain. IB1 was also detected in a restricted number of axons, as in the mossy fibres from dentate gyrus in the hippocampus, and was found in soma, dendrites and axons of cerebellar Purkinje cells. After birth, IB1 expression peaks at postnatal day 15. IB1 was located in axonal and dendritic growth cones in primary telencephalon cells. By biochemical and subcellular fractionation of neuronal cells, IB1 was detected both in the cytosolic and membrane fractions. Taken together with previous data, the restricted neuronal expression of IB1 in developing and adult brain and its prominent localization in synapses suggest that the protein may be critical for cell signalling in developing and mature nerve terminals.

    The European journal of neuroscience 2000;12;2;621-32

  • Epistatic and independent functions of caspase-3 and Bcl-X(L) in developmental programmed cell death.

    Roth KA, Kuan C, Haydar TF, D'Sa-Eipper C, Shindler KS, Zheng TS, Kuida K, Flavell RA and Rakic P

    Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110, USA. kroth@pathology.wustl.edu

    The number of neurons in the mammalian brain is determined by a balance between cell proliferation and programmed cell death. Recent studies indicated that Bcl-X(L) prevents, whereas Caspase-3 mediates, cell death in the developing nervous system, but whether Bcl-X(L) directly blocks the apoptotic function of Caspase-3 in vivo is not known. To examine this question, we generated bcl-x/caspase-3 double mutants and found that caspase-3 deficiency abrogated the increased apoptosis of postmitotic neurons but not the increased hematopoietic cell death and embryonic lethality caused by the bcl-x mutation. In contrast, caspase-3, but not bcl-x, deficiency changed the normal incidence of neuronal progenitor cell apoptosis, consistent with the lack of expression of Bcl-X(L) in the proliferative population of the embryonic cortex. Thus, although Caspase-3 is epistatically downstream to Bcl-X(L) in postmitotic neurons, it independently regulates apoptosis of neuronal founder cells. Taken together, these results establish a role of programmed cell death in regulating the size of progenitor population in the central nervous system, a function that is distinct from the classic role of cell death in matching postmitotic neuronal population with postsynaptic targets.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;1;466-71

  • The bHLH gene hes1 as a repressor of the neuronal commitment of CNS stem cells.

    Nakamura Y, Sakakibara Si, Miyata T, Ogawa M, Shimazaki T, Weiss S, Kageyama R and Okano H

    Department of Neuroanatomy, Biomedical Research Center, Osaka University, Suita, Osaka 565-0871, Japan.

    Hes1 is one of the basic helix-loop-helix transcription factors that regulate mammalian CNS development, and its loss- and gain-of-function phenotypes indicate that it negatively regulates neuronal differentiation. Here we report that Hes1(-/-) mice expressed both early (TuJ1 and Hu) and late (MAP2 and Neurofilament) neuronal markers prematurely, and that there were approximately twice the normal number of neurons in the Hes1(-/-) brain during early neural development. However, immunochemical analyses of sections and dissociated cells using neural progenitor markers, including nestin, failed to detect any changes in Hes1(-/-) progenitor population. Therefore, further characterization of neural progenitor cells that discriminated between multipotent and monopotent cells was performed using two culture methods, low-density culture, and a neurosphere assay. We demonstrate that the self-renewal activity of multipotent progenitor cells was reduced in the Hes1(-/-) brain, and that their subsequent commitment to the neuronal lineage was accelerated. The Hes1(-/-) neuronal progenitor cells were functionally abnormal, in that they divided, on average, only once, and then generated two neurons, (instead of one progenitor cell and one neuron), whereas wild-type progenitor cells divided more. In addition, some Hes1(-/-) progenitors followed an apoptotic fate. The overproduction of neurons in the early Hes1(-/-) brains may reflect this premature and immediate generation of neurons as well as a net increase in the number of neuronal progenitor cells. Taken together, we conclude that Hes1 is important for maintaining the self-renewing ability of progenitors and for repressing the commitment of multipotent progenitor cells to a neuronal fate, which is critical for the correct number of neurons to be produced and for the establishment of normal neuronal function.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;1;283-93

  • Ebf1 controls early cell differentiation in the embryonic striatum.

    Garel S, Marín F, Grosschedl R and Charnay P

    Unité 368 de l'Institut National de la Santé et de la Recherche Médicale, Ecole Normale Supérieure, 75230 Paris Cedex 05, France.

    Ebf1/Olf-1 belongs to a small multigene family encoding closely related helix-loop-helix transcription factors, which have been proposed to play a role in neuronal differentiation. Here we show that Ebf1 controls cell differentiation in the murine embryonic striatum, where it is the only gene of the family to be expressed. Ebf1 targeted disruption affects postmitotic cells that leave the subventricular zone (SVZ) en route to the mantle: they appear to be unable to downregulate genes normally restricted to the SVZ or to activate some mantle-specific genes. These downstream genes encode a variety of regulatory proteins including transcription factors and proteins involved in retinoid signalling as well as adhesion/guidance molecules. These early defects in the SVZ/mantle transition are followed by an increase in cell death, a dramatic reduction in size of the postnatal striatum and defects in navigation and fasciculation of thalamocortical fibres travelling through the striatum. Our data therefore show that Ebf1 plays an essential role in the acquisition of mantle cell molecular identity in the developing striatum and provide information on the genetic hierarchies that govern neuronal differentiation in the ventral telencephalon.

    Development (Cambridge, England) 1999;126;23;5285-94

  • Dual role of brain factor-1 in regulating growth and patterning of the cerebral hemispheres.

    Dou CL, Li S and Lai E

    Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. c-dou@ski.mskcc.org

    Brain factor-1 (BF-1) is a winged-helix (WH) transcription factor with a restricted pattern of expression in the neural tube. In the embryo, BF-1 is localized to the progenitor cells of the most rostral neural tube, the telencephalic neuroepithelium. Expression of BF-1 persists in the adult brain in the structures derived from the telencephalon, including the cerebral cortex, the hippocampus, the olfactory bulbs and the basal ganglia. Targeted disruption of the BF-1 gene in mice results in hypoplasia of the cerebral hemispheres. Proliferation of the telencephalic neuroepithelium is decreased and neuronal differentiation occurs prematurely. The forebrain of the BF-1 (-/-) mutant also displays dorsal-ventral patterning defects. Development of the ventral (basal) region of the telencephalon is more severely affected than the dorsal region. These anomalies are associated with the ectopic expression of BMP4 in the dorsal telencephalic neuroepithelium and the loss of shh in the ventral telencephalon. These results raise the possibility that BF-1 may modulate both progenitor cell proliferation and regional patterning by regulating the expression or activity of inductive signals which act on the telencephalic neuroepithelium.

    Cerebral cortex (New York, N.Y. : 1991) 1999;9;6;543-50

  • Identification and genetic mapping of differentially expressed genes in mice differing at the If1 interferon regulatory locus.

    Kozak CA, Su Y, Raj NB and Pitha PM

    Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Building 4, Room 329, 4 Center Drive MSC 0460, Bethesda, Maryland 20982-0460, USA.

    A subtractive cDNA library was used to identify differentially expressed genes in mouse strains that differ at If1, a locus that regulates response to interferon induction by Newcastle Disease Virus infection. Among the isolated clones, sequence analysis identified the ribosomal proteins L37a and S8 as well as cDNAs for thymosine beta4, the QM transcriptional factor, and a novel genetic sequence. Analysis of two multilocus mouse crosses showed that the thymosine beta4 gene, Ptmb4, is present as a single-copy gene that maps to distal Chr X. The L37a, S8, and QM clones are all members of large multilocus families. These five clones were used to determine the map locations for 37 loci, of which 31 had not previously been described. The novel genetic sequence, D3Ppr1, mapped to distal Chr 3 near the position of the If1 locus, suggesting it may be a candidate for this regulatory gene.

    Funded by: NIAID NIH HHS: AI19737

    Mammalian genome : official journal of the International Mammalian Genome Society 1999;10;9;853-7

  • Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum.

    Sussel L, Marin O, Kimura S and Rubenstein JL

    Center for Neurobiology and Psychiatry, Department of Psychiatry and University of California at San Francisco, CA 94143-0984, USA.

    The telencephalon is organized into distinct longitudinal domains: the cerebral cortex and the basal ganglia. The basal ganglia primarily consists of a dorsal region (striatum) and a ventral region (pallidum). Within the telencephalon, the anlage of the pallidum expresses the Nkx2.1 homeobox gene. A mouse deficient in Nkx2.1 function does not form pallidal structures, lacks basal forebrain TrkA-positive neurons (probable cholinergic neurons) and has reduced numbers of cortical cells expressing GABA, DLX2 and calbindin that migrate from the pallidum through the striatum and into the cortex. We present evidence that these phenotypes result from a ventral-to-dorsal transformation of the pallidal primordium into a striatal-like anlage.

    Funded by: NIMH NIH HHS: K02 MH01046-01

    Development (Cambridge, England) 1999;126;15;3359-70

  • Abnormalities in neuronal process extension, hippocampal development, and the ventricular system of L1 knockout mice.

    Demyanenko GP, Tsai AY and Maness PF

    Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7260, USA.

    In humans, mutations in the L1 cell adhesion molecule are associated with a neurological syndrome termed CRASH, which includes corpus callosum agenesis, mental retardation, adducted thumbs, spasticity, and hydrocephalus. A mouse model with a null mutation in the L1 gene (Cohen et al., 1997) was analyzed for brain abnormalities by Nissl and Golgi staining and immunocytochemistry. In the motor, somatosensory, and visual cortex, many pyramidal neurons in layer V exhibited undulating apical dendrites that did not reach layer I. The hippocampus of L1 mutant mice was smaller than normal, with fewer pyramidal and granule cells. The corpus callosum of L1-minus mice was reduced in size because of the failure of many callosal axons to cross the midline. Enlarged ventricles and septal abnormalities were also features of the mutant mouse brain. Immunoperoxidase staining showed that L1 was abundant in developing neurons at embryonic day 18 (E18) in wild-type cerebral cortex, hippocampus, and corpus callosum and then declined to low levels with maturation. In the E18 cortex, L1 colocalized with microtubule-associated protein 2, a marker of dendrites and somata. These new findings suggest new roles for L1 in the mechanism of cortical dendrite differentiation, as well as in guidance of callosal axons and regulation of hippocampal development. The phenotype of the L1 mutant mouse indicates that it is a potentially valuable model for the human CRASH syndrome.

    Funded by: NICHD NIH HHS: HD35170; NINDS NIH HHS: NS26620

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;12;4907-20

  • Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.

    Trommsdorff M, Gotthardt M, Hiesberger T, Shelton J, Stockinger W, Nimpf J, Hammer RE, Richardson JA and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235-9046, USA.

    Layering of neurons in the cerebral cortex and cerebellum requires Reelin, an extracellular matrix protein, and mammalian Disabled (mDab1), a cytosolic protein that activates tyrosine kinases. Here, we report the requirement for two other proteins, cell surface receptors termed very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2). Both receptors can bind mDab1 on their cytoplasmic tails and are expressed in cortical and cerebellar layers adjacent to layers that express Reelin. mDab1 expression is upregulated in knockout mice that lack both VLDLR and ApoER2. Inversion of cortical layers and absence of cerebellar foliation in these animals precisely mimic the phenotype of mice lacking Reelin or mDab1. These findings suggest that VLDLR and ApoER2 participate in transmitting the extracellular Reelin signal to intracellular signaling processes initiated by mDab1.

    Funded by: NHLBI NIH HHS: HL20948, R37 HL063762; NIA NIH HHS: AG12300

    Cell 1999;97;6;689-701

  • Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5.

    Zhao Y, Sheng HZ, Amini R, Grinberg A, Lee E, Huang S, Taira M and Westphal H

    Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA.

    The mammalian hippocampus contains the neural circuitry that is crucial for cognitive functions such as learning and memory. The development of such circuitry is dependent on the generation and correct placement of the appropriate number and types of neurons. Mice lacking function of the LIM homeobox gene Lhx5 showed a defect in hippocampus development. Hippocampal neural precursor cells were specified and proliferated, but many of them failed to either exit the cell cycle or to differentiate and migrate properly. Lhx5 is therefore essential for the regulation of precursor cell proliferation and the control of neuronal differentiation and migration during hippocampal development.

    Science (New York, N.Y.) 1999;284;5417;1155-8

  • The cytoskeleton of the myenteric neurons during murine embryonic life.

    Faussone-Pellegrini MS, Matini P and DeFelici M

    Department of Human Anatomy and Histology, University of Florence, Italy. s_faussone@cesit1.unifi.it

    The organization of the cytoskeleton has been studied during mouse differentiation in cells of the myenteric neuronal lineage. The entire gut was examined starting from day 12.5 of embryonic life (E12.5) until birth (P0). Immunocytochemistry was performed to evaluate the expression of five of the most represented neurofilaments proteins (the low, NF-L, medium, NF-M, and heavy, NF-H, molecular weight subunits, alpha-internexin and peripherin) and of two of the microtubule-associated proteins (MAPI and MAP2a+2b). In parallel, the appearance in the differentiating myenteric neurons of filamentous and microtubular structures and their intracytoplasmatic distribution were observed under the electron microscope. A differential immunohistochemical expression of the structural proteins was found. Immature cells expressed alpha-internexin, peripherin, NF-M and MAP1 by day E12.5; alpha-internexin expression was strong in these cells, but gradually decreased with age and was practically absent in adulthood. Conversely, the expression of the other three proteins increased with cell differentiation and was still present in adulthood. NF-L and NF-H expression appeared later, by day E16.5, and was weak for the entire pre- and postnatal life. MAP2a+2b was never expressed. Under the electron microscope, at day E12.5 the cytoskeleton was already organized in filamentous and microtubular structures. At this age neurofilaments were few and mainly located in the cell processes, and microtubules were numerous and mainly assembled in the neuritic growth cones, together with synaptic vesicles. With ageing, neurofilaments and microtubules were ubiquitous in the neuron. Data obtained demonstrate that cytoskeletal proteins gradually accumulate in the cells of the neuronal lineage in parallel with the organization of the cytoskeletal structures, which in turn mediate important neural events by the earliest stages of murine embryonic life, including growth of nerve processes and initiation of axonal transport.

    Anatomy and embryology 1999;199;5;459-69

  • Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation.

    Ohtsuka T, Ishibashi M, Gradwohl G, Nakanishi S, Guillemot F and Kageyama R

    Institute for Virus Research, Kyoto University, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, USA.

    While the transmembrane protein Notch plays an important role in various aspects of development, and diseases including tumors and neurological disorders, the intracellular pathway of mammalian Notch remains very elusive. To understand the intracellular pathway of mammalian Notch, the role of the bHLH genes Hes1 and Hes5 (mammalian hairy and Enhancer-of-split homologues) was examined by retrovirally misexpressing the constitutively active form of Notch (caNotch) in neural precursor cells prepared from wild-type, Hes1-null, Hes5-null and Hes1-Hes5 double-null mouse embryos. We found that caNotch, which induced the endogenous Hes1 and Hes5 expression, inhibited neuronal differentiation in the wild-type, Hes1-null and Hes5-null background, but not in the Hes1-Hes5 double-null background. These results demonstrate that Hes1 and Hes5 are essential Notch effectors in regulation of mammalian neuronal differentiation.

    The EMBO journal 1999;18;8;2196-207

  • Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice.

    Kobayashi T, Minowa O, Kuno J, Mitani H, Hino O and Noda T

    Department of Experimental Pathology, Cancer Institute, Tokyo, Japan.

    Germ-line mutations of the human TSC2 tumor suppressor gene cause tuberous sclerosis (TSC), a disease characterized by the development of hamartomas in various organs. In the Eker rat, however, a germ-line Tsc2 mutation gives rise to renal cell carcinomas with a complete penetrance. The molecular mechanism for this phenotypic difference between man and rat is currently unknown, and the physiological function of the TSC2/Tsc2 product (tuberin) is not fully understood. To investigate these unsolved problems, we have generated a Tsc2 mutant mouse. Tsc2 heterozygous mutant (Tsc2+/-) mice developed renal carcinomas with a complete penetrance, as seen in the Eker rat, but not the angiomyolipomas characteristic of human TSC, confirming the existence of a species-specific mechanism of tumorigenesis caused by tuberin deficiency. Unexpectedly, approximately 80% of Tsc2+/- mice also developed hepatic hemangiomas that are not observed in either TSC or the Eker rat. Tsc2 homozygous (Tsc2-/-) mutants died around embryonic day 10.5, indicating an essential function for tuberin in mouse embryonic development. Some Tsc2-/- embryos exhibited an unclosed neural tube and/or thickened myocardium. The latter is associated with increased cell density that may be a reflection of loss of a growth-suppressive function of tuberin. The mouse strain described here should provide a valuable experimental model to analyze the function of tuberin and its association with tumorigenesis.

    Cancer research 1999;59;6;1206-11

  • Essential roles for the Abl and Arg tyrosine kinases in neurulation.

    Koleske AJ, Gifford AM, Scott ML, Nee M, Bronson RT, Miczek KA and Baltimore D

    Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA. anthony.koleske@yale.edu

    The Abl and Arg tyrosine kinases play fundamental roles in the development and function of the central nervous system. Arg is most abundant in adult mouse brain, especially in synapse-rich regions. arg(-/-) mice develop normally but exhibit multiple behavioral abnormalities, suggesting that arg(-/-) brains suffer from defects in neuronal function. Embryos deficient in both Abl and Arg suffer from defects in neurulation and die before 11 days postcoitum (dpc). Although they divide normally, abl(-/-)arg(-/-) neuroepithelial cells display gross alterations in their actin cytoskeleton. We find that Abl and Arg colocalize with each other and with actin microfilaments at the apical surface of the developing neuroepithelium. Thus, Abl and Arg play essential roles in neurulation and can regulate the structure of the actin cytoskeleton.

    Funded by: NCI NIH HHS: CA51462; NIAAA NIH HHS: AA5122; NIDA NIH HHS: DA02632

    Neuron 1998;21;6;1259-72

  • Requirement for early-generated neurons recognized by monoclonal antibody lot1 in the formation of lateral olfactory tract.

    Sato Y, Hirata T, Ogawa M and Fujisawa H

    Division of Biological Science, Nagoya University Graduate School of Science, Chikusa-ku, Nagoya 464-8602, Japan.

    During development, mitral cells, the main output neurons of the olfactory bulb, project axons into a very narrow part of the telencephalon and form an axonal bundle called the lateral olfactory tract (LOT). The present study shows that before the first mitral cell axons elongate, the LOT position is already marked with a subset of early-generated neurons that are recognized by monoclonal antibody lot1 (lot cells). Mitral cell axons choose the lot cell position for their growth pathway and maintain a close contact with the cells until LOT formation is completed. Ablation of lot cells prevented LOT formation in organotypic culture. These results suggest that lot cells are "guidepost cells" for mitral cell axons.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;19;7800-10

  • Disabled-1 acts downstream of Reelin in a signaling pathway that controls laminar organization in the mammalian brain.

    Rice DS, Sheldon M, D'Arcangelo G, Nakajima K, Goldowitz D and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.

    Mutation of either reelin (Reln) or disabled-1 (Dab1) results in widespread abnormalities in laminar structures throughout the brain and ataxia in reeler and scrambler mice. Both exhibit the same neuroanatomical defects, including cerebellar hypoplasia with Purkinje cell ectopia and disruption of neuronal layers in the cerebral cortex and hippocampus. Despite these phenotypic similarities, Reln and Dab1 have distinct molecular properties. Reln is a large extracellular protein secreted by Cajal-Retzius cells in the forebrain and by granule neurons in the cerebellum. In contrast, Dab1 is a cytoplasmic protein which has properties of an adapter protein that functions in phosphorylation-dependent intracellular signal transduction. Here, we show that Dab1 participates in the same developmental process as Reln. In scrambler mice, neuronal precursors are unable to invade the preplate of the cerebral cortex and consequently, they do not align within the cortical plate. During development, cells expressing Dab1 are located next to those secreting Reln at critical stages of formation of the cerebral cortex, cerebellum and hippocampus, before the first abnormalities in cell position become apparent in either reeler or scrambler. In reeler, the major populations of displaced neurons contain elevated levels of Dab1 protein, although they express normal levels of Dab1 mRNA. This suggests that Dab1 accumulates in the absence of a Reln-evoked signal. Taken together, these results indicate that Dab1 functions downstream of Reln in a signaling pathway that controls cell positioning in the developing brain.

    Funded by: NCI NIH HHS: P30 CA21765, T32 CA09346; NINDS NIH HHS: R01 NS36558

    Development (Cambridge, England) 1998;125;18;3719-29

  • BDNF regulates reelin expression and Cajal-Retzius cell development in the cerebral cortex.

    Ringstedt T, Linnarsson S, Wagner J, Lendahl U, Kokaia Z, Arenas E, Ernfors P and Ibáñez CF

    Department of Neuroscience, Karolinska Institute, Stockholm, Sweden. thomas.ringstedt@mbb.ki.se

    Cajal-Retzius (CR) cells of the cerebral cortex express receptors for the neurotrophin brain-derived neurotrophic factor (BDNF) and downregulate expression of the extracellular matrix protein Reelin during early postnatal development, coincident with the onset of cortical BDNF expression. During this period, mice lacking BDNF have elevated levels of Reelin in CR cells. Acute BDNF stimulation of cortical neuron cultures and overexpression of BDNF in the developing brain of transgenic mice prior to the onset of endogenous production causes a profound, dose-dependent reduction of Reelin expression in CR cells. In addition, overexpression of BDNF produces gaps and heterotopias in the marginal zone and disorganization and aggregation of cortical CR cells and induces several other malformations, including aberrant cortical lamination, similar to the phenotype of reeler mutant mice, which lack Reelin. These results demonstrate a role for BDNF on cortical CR cells and identify Reelin as a direct effector of this neurotrophin during brain development.

    Neuron 1998;21;2;305-15

  • Genetic mapping of the mouse ferritin light chain gene and 11 pseudogenes on 11 mouse chromosomes.

    Filie JD, Buckler CE and Kozak CA

    Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460, USA.

    We typed the progeny of two sets of genetic crosses to determine the map locations for loci containing sequences related to the ferritin light chain (Ft11) gene. Twelve loci were positioned on 11 different chromosomes. One of these genes mapped to a position on Chr 7 predicted to contain the expressed gene on the basis of the previously determined position of the human homolog on 19q13.3-q13.4.

    Mammalian genome : official journal of the International Mammalian Genome Society 1998;9;2;111-3

  • Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene.

    Zhang XQ, Afink GB, Svensson K, Jacobs JJ, Günther T, Forsberg-Nilsson K, van Zoelen EJ, Westermark B and Nistér M

    Department of Pathology, University of Uppsala, University Hospital, Sweden.

    The platelet-derived growth factor alpha-receptor (PDGFR-alpha) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5'-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR-alpha mRNA, while they failed to specify reporter expression in PDGFR-alpha expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.

    Mechanisms of development 1998;70;1-2;167-80

  • Vinculin knockout results in heart and brain defects during embryonic development.

    Xu W, Baribault H and Adamson ED

    The Burnham Institute, La Jolla, CA 92037, USA.

    The vinculin gene codes for a cytoskeletal protein, found in focal adhesion plaques and in cell-cell adherens junctions. Vinculin was inactivated by homologous recombination using a targeting vector in embryonic stem (ES) cells. The heterozygous ES cells were introduced into mice by established procedures to produce heterozygous animals that were normal and fertile. No homozygous vinculin-/- embryos were born and analyses during the gestational period showed that the vinculin null embryos were small and abnormal from day E8 but some survived until E10. The most prominent defect was lack of midline fusion of the rostral neural tube, producing a cranial bilobular appearance and attenuation of cranial and spinal nerve development. Heart development was curtailed at E9.5, with severely reduced and akinetic myocardial and endocardial structures. Mutant embryos were 30-40% smaller, somites and limbs were retarded and ectodermal tissues were sparse and fragile. Fibroblasts (MEF) isolated from mutant embryos were shown to have reduced adhesion to fibronectin, vitronectin, laminin and collagen compared to wild-type levels. In addition, migration rates over these substrata were two-fold higher and the level of focal adhesion kinase (FAK) activity was three-fold higher. We conclude that vinculin is necessary for normal embryonic development, probably because of its role in the regulation of cell adhesion and locomotion, cell behaviors essential for normal embryonic morphogenesis, although specific roles in neural and cardiac development cannot be ruled out.

    Funded by: NCI NIH HHS: CA 28427; NIAMS NIH HHS: AR41816

    Development (Cambridge, England) 1998;125;2;327-37

  • Disruption of hippocampal development in vivo by CR-50 mAb against reelin.

    Nakajima K, Mikoshiba K, Miyata T, Kudo C and Ogawa M

    Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Tsukuba, Ibaraki 305, Japan. nakjima@rtc.riken.go.jp

    We previously generated a monoclonal alloantibody, CR-50, by immunizing reeler mutant mice with homogenates of normal embryonic brains. This antibody recently was shown to recognize a Reelin protein, which is coded by the recently identified candidate gene for the reeler mutation. However, it is still unclear whether Reelin, especially the CR-50 epitope region, is indeed responsible for the reeler phenotype in vivo. Here we show that Reelin is localized on Cajal-Retzius neurons in the hippocampus and that intraventricular injection of CR-50 at the embryonic stage disrupts the organized development of the hippocampus in vivo, converting it to a reeler pattern. Labeling experiments with 5-bromodeoxyuridine demonstrated that the labeled cells in the stratum pyramidale of the CR-50-treated mice were distributed in a pattern similar to that of reeler. Thus, Cajal-Retzius neurons play a crucial function in hippocampus development, and the CR-50 epitope on Reelin plays a central role in this function.

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;15;8196-201

  • Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons.

    Anderson SA, Qiu M, Bulfone A, Eisenstat DD, Meneses J, Pedersen R and Rubenstein JL

    Center for Neurobiology and Psychiatry, Department of Psychiatry, University of California at San Francisco, 94143, USA.

    The striatum has a central role in many neurobiological processes, yet little is known about the molecular control of its development. Inroads to this subject have been made, due to the discovery of transcription factors, such as the Dlx genes, whose expression patterns suggest that they have a role in striatal development. We report that mice lacking both Dlx-1 and Dlx-2 have a time-dependent block in striatal differentiation. In these mutants, early born neurons migrate into a striatum-like region, which is enriched for markers of the striosome (patch) compartment. However, later born neurons accumulate within the proliferative zone. Several lines of evidence suggest that mutations in Dlx-1 and Dlx-2 produce abnormalities in the development of the striatal subventricular zone and in the differentiation of striatal matrix neurons.

    Funded by: NICHD NIH HHS: NICDPO-1 HD26732; NIMH NIH HHS: K02 MH01046-01, R01 MH49428-01; Telethon: TGM06S01

    Neuron 1997;19;1;27-37

  • Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord.

    Ozaki S and Snider WD

    Department of Neurology, Washington University School of Medicine, St Louis, Missouri 63110, USA.

    The formation of laminar-specific projections is a key event in the development of appropriate neuronal connections in many regions of the central nervous system. In order to provide a framework for defining functions of molecules related to spinal laminar targeting of dorsal root ganglion neurons in mice, we have characterized the initial trajectories of sensory axons in relation to the maturation of their target laminae in the spinal cord. We show that morphological and biochemical differentiation of distinct clusters of neurons in the dorsal region of the spinal cord precedes initial collateral branching from sensory axons. Between embryonic day (E) 12.5 and E13.5, sensory axons develop swelling ("nodes") along their entire intraspinal extent and elaborate interstitial collateral branches from these nodes. Collaterals from the different classes of sensory axons then penetrate the gray matter of the spinal cord sequentially. Each class of sensory axons projects directly to its target lamina, never branching into inappropriate laminae en route. Some cutaneous afferents traverse the entire width of the spinal cord to reach superficial laminae on the contralateral side, strictly avoiding both the ventral spinal cord and inappropriate laminae of the deep dorsal horn. The pathways taken by developing sensory afferents are compatible with the idea that cells in inappropriate laminae exert inhibitory influences on sensory axons which regulate their laminar specificity.

    Funded by: NINDS NIH HHS: R01 NS31768; PHS HHS: O1 50757

    The Journal of comparative neurology 1997;380;2;215-29

  • Distribution of a reeler gene-related antigen in the developing cerebellum: an immunohistochemical study with an allogeneic antibody CR-50 on normal and reeler mice.

    Miyata T, Nakajima K, Aruga J, Takahashi S, Ikenaka K, Mikoshiba K and Ogawa M

    Molecular Neurobiology Laboratory, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.

    We have immunohistochemically investigated the expression of a reeler gene-related antigen in the mouse cerebellum by using a monoclonal antibody, CR-50. This antibody probes a distinct allelic antigen present in normal but not in reeler mutant mice, and this antigen is localized in the brain regions in which morphological abnormalities occur in reeler mice (Ogawa et al., Neuron 14: 899-912, 1995). The developing normal cerebellum showed transient immunoreactivity to CR-50 in a limited set of neurons and in the extracellular space near the pial surface. An early population of CR-50-labeled cells emerged on embryonic day (E) 13 along the dorsal cerebellar surface, comprising the nuclear transitory zone (NTZ). Bromodeoxyuridine labeling revealed the time of origin of these cells to be at E11-12. From E14 to E18, some CR-50-labeled cells were stacked in the inner border of the external granular layer (EGL), whereas others were scattered in deep areas, such as the cerebellar nuclei and the surrounding intermediate zone or white matter. In the first postnatal week, these subcortical structures became immunonegative. However, CR-50 antigen was continuously produced until the second postnatal week by another population of cells occupying i) the premigratory zone (PMZ), the inner half of the EGL, and ii) the internal granular layer (IGL). These later CR-50-positive cells were smaller than the earlier type and showed the morphology typical of granule neurons. Both types of CR-50-labeled cells were positive for a DNA-binding protein, zic. By treating living cerebellar slices with CR-50, the extracellular antigen was localized as a puncutate staining pattern in the NTZ, PMZ, and molecular layer (ML), but not in the subcortical regions and IGL. Purkinje cells were negative for CR-50 and aligned as a monolayer adjacent to the PMZ, though their dendritic trees were closely associated with the extracellular CR-50-antigen in the PMZ and ML. Staining of dissociated cells suggested that the extracellular antigen is initially present throughout the surfaces of the CR-50/anti-zic double positive neurons, and is then rearranged to concentrate on their processes contacting with Purkinje cells. The spatiotemporal expressions of the CR-50 antigen in the cerebellum are consistent with the possibility that this antigen is involved in cell-cell interactions related to the histogenetic assembly of Purkinje cells.

    The Journal of comparative neurology 1996;372;2;215-28

  • Genetic mapping of the human and mouse phospholipase C genes.

    Lyu MS, Park DJ, Rhee SG and Kozak CA

    Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

    To determine chromosome positions for 10 mouse phospholipase C (PLC) genes, we typed the progeny of two sets of genetic crosses for inheritance of restriction enzyme polymorphisms of each PLC. Four mouse chromosomes, Chr 1, 11, 12, and 19, contained single PLC genes. Four PLC loci, Plcb1, Plcb2, Plcb4, and Plcg1, mapped to three sites on distal mouse Chr 2. Two PLC genes, Plcd1 and Plcg2, mapped to distinct sites on Chr 8. We mapped the human homologs of eight of these genes to six chromosomes by analysis of human x rodent somatic cell hybrids. The map locations of seven of these genes were consistent with previously defined regions of conserved synteny; Plcd1 defines a new region of homology between human Chr 3 and mouse Chr 8.

    Mammalian genome : official journal of the International Mammalian Genome Society 1996;7;7;501-4

  • Dispersion of chromogranin/secretogranin secretory protein family loci in mammalian genomes.

    Mahata SK, Kozak CA, Szpirer J, Szpirer C, Modi WS, Gerdes HH, Huttner WB and O'Connor DT

    Department of Medicine, University of California, San Diego, California, 92161, USA.

    Chromogranin A, chromogranin B, and secretogranin II, members of the chromogranin/secretogranin secretory protein family, are overexpressed in some human hereditary maladies and may have arisen, in part, from common ancestor genes. To understand better the mammalian chromosomal dispersion of this gene family and to facilitate studies of these genes in human illnesses and their animal models, we positioned the locus of each member in the rat, mouse, and human genomes. Our results indicate that each locus lies in a region of locally syntenic chromosomal homology across the three species.

    Genomics 1996;33;1;135-9

  • The chromosomal distribution of mouse odorant receptor genes.

    Sullivan SL, Adamson MC, Ressler KJ, Kozak CA and Buck LB

    Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA.

    Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

    Funded by: NIDCD NIH HHS: 1-R01-DC01662-01, 5-F32-DC00188-01, P01 DC000347-10

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;2;884-8

  • Report and abstracts of the Fourth International Workshop on Human Chromosome 2 Mapping 1996.

    Spur NK, Bashir R, Bushby K, Cox A, Cox S, Hilde Brandt F, Hill N, Kao FT, Krols L, Marzella R, Miller N, Nothwang HG, Rocchi M, Sarfarazi M, Stratakis CA, Wallgren-Petterson C and Naylor S

    Cytogenetics and cell genetics 1996;73;4;255-73

  • Winged helix transcription factor BF-1 is essential for the development of the cerebral hemispheres.

    Xuan S, Baptista CA, Balas G, Tao W, Soares VC and Lai E

    Cell Biology and Genetics Program, Cornell University Graduate School of Medical Sciences, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

    We generated mice with a null mutation of the forebrain-restricted transcription factor BF-1 to examine its function in brain development. Heterozygous animals have an apparently normal phenotype. Homozygous null BF-1 mutants die at birth and have a dramatic reduction in the size of the cerebral hemispheres. The development of the ventral telencephalon is more severely affected than that of the dorsal telencephalon. Telencephalic neuroepithelial cells are specified in the BF-1 mutant, but their proliferation is reduced. Dorsal telencephalic neuroepithelial cells also differentiate prematurely, leading to early depletion of the progenitor population. These results suggest that BF-1 controls the morphogenesis of the telencephalon by regulating the rate of neuroepithelial cell proliferation and the timing of neuronal differentiation.

    Funded by: NICHD NIH HHS: R01HD29584

    Neuron 1995;14;6;1141-52

  • Distinct adhesive behaviors of neurons and neural precursor cells during regional differentiation in the mammalian forebrain.

    Whitesides JG and LaMantia AS

    Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Prior to the emergence of the major functional subdivisions of the mammalian forebrain--the neocortex, hippocampus, olfactory bulb, basal ganglia, and basal forebrain--the lateral aspect of the telencephalic vesicle is distinguished by early neuronal differentiation assessed by MAP2 and GAP43 expression and increased expression of the Ca(2+)-independent/immunoglobulin superfamily cell adhesion molecules (CAMs) NCAM, L1, and TAG-1. In contrast, the ventral and medial aspects of the vesicle show little early neuronal differentiation and intermediate or undetectable levels of CAM expression. We asked whether cells from these three regions acquire distinct adhesive and recognition properties that reflect their position, state of neuronal differentiation, and level of CAM expression. In a dissociation/reaggregation assay, cells from the lateral telencephalic vesicle form the largest reaggregates while ventral reaggregates are of intermediate size and medial reaggregates are the smallest. This differential adhesion has a Ca(2+)-independent component, and cells in reaggregates from each region maintain expression of CAMs and other neuronal markers consistent with their region of origin. Furthermore, cells from the lateral telencephalon can specifically sort out from medial cells. Little adhesivity is observed prior to early neuronal differentiation and the expression of Ca(2+)-independent CAMs, when the forebrain is still a prosencephalic vesicle, nor does it follow the pattern of detectable CAM expression once forebrain rudiments are formed. Thus, cells in the early developing forebrain acquire distinct adhesive and recognition properties that reflect the concurrent emergence of regional differences in neuronal differentiation and CAM expression. These differences are transient and can only be detected in the telencephalic vesicle before and during the morphogenesis of rudiments of major forebrain subdivisions.

    Funded by: NICHD NIH HHS: HD29178

    Developmental biology 1995;169;1;229-41

  • Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice.

    Motoyama N, Wang F, Roth KA, Sawa H, Nakayama K, Nakayama K, Negishi I, Senju S, Zhang Q, Fujii S et al.

    Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110.

    bcl-x is a member of the bcl-2 gene family, which may regulate programmed cell death. Mice were generated that lacked Bcl-x. The Bcl-x-deficient mice died around embryonic day 13. Extensive apoptotic cell death was evident in postmitotic immature neurons of the developing brain, spinal cord, and dorsal root ganglia. Hematopoietic cells in the liver were also apoptotic. Analyses of bcl-x double-knockout chimeric mice showed that the maturation of Bcl-x-deficient lymphocytes was diminished. The life-span of immature lymphocytes, but not mature lymphocytes, was shortened. Thus, Bcl-x functions to support the viability of immature cells during the development of the nervous and hematopoietic systems.

    Science (New York, N.Y.) 1995;267;5203;1506-10

  • DLX-2, MASH-1, and MAP-2 expression and bromodeoxyuridine incorporation define molecularly distinct cell populations in the embryonic mouse forebrain.

    Porteus MH, Bulfone A, Liu JK, Puelles L, Lo LC and Rubenstein JL

    Nina Ireland Laboratory of Developmental Biology, Department of Psychiatry, University of California, San Francisco 94143-0984.

    Recently, the Dlx family of homeobox genes have been identified as candidates for regulating patterning and differentiation of the forebrain. We have made a polyclonal antiserum to the protein product of the Dlx-2 gene. Using this antiserum, we have characterized the spatial and temporal pattern of DLX-2 protein expression during murine development and in the adult mouse brain. These studies demonstrate that, like the mRNA from the Dlx-2 gene, DLX-2 protein is expressed in mouse embryonic forebrain, limbs, tail, genital tubercle, and branchial arches. Within the embryonic forebrain, DLX-2 protein is expressed within specific transverse and longitudinal domains. Analysis of expression within the wall of the forebrain shows that DLX-2 is expressed in proliferative regions including the ventricular and subventricular zones. DLX-2 is expressed in the same cells as MASH-1, a marker of relatively undifferentiated cells, but in a reciprocal fashion to MAP-2, a marker of terminal neuronal differentiation. A number of DLX-2-expressing cells, but not all, can be labeled with bromodeoxyuridine (BrdU). Using the patterns of DLX-2, MASH-1, MAP-2 expression, and bromodeoxyuridine incorporation, we identify four molecularly distinct populations of cells that may correspond to different stages of neuronal differentiation in the mouse basal forebrain, in which DLX-2 is expressed at the transition from proliferation to terminal differentiation.

    Funded by: NIMH NIH HHS: KO2 MH01046-01, R01 MH49428-01

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1994;14;11 Pt 1;6370-83

  • The murine dilute suppressor gene encodes a cell autonomous suppressor.

    Moore KJ, Swing DA, Copeland NG and Jenkins NA

    Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Maryland 21702.

    The murine dilute suppressor gene (dsu) suppresses the coat-color phenotype of three pigment mutations, dilute (d), ashen (ash) and leaden (ln), that each produce adendritic melanocytes. Suppression is due to the ability of dsu to partially restore (ash and ln), or almost completely restore (d), normal melanocyte morphology. While the ash and ln gene products have yet to be identified, the d gene encodes a novel myosin heavy chain (myosin 12), which is speculated to be necessary for the elaboration, maintenance, and/or function of melanocyte cell processes. To begin to discriminate between different models of dsu action, we have produced aggregation chimeras between mice homozygous for dsu and mice homozygous for d to determine if dsu acts cell autonomously or cell nonautonomously. In addition, we have further refined the map location of dsu in order to examine a number of possible dsu candidate genes mapping in the region and to provide a genetic basis for the positional cloning of dsu.

    Funded by: NCI NIH HHS: N01-CO-74101

    Genetics 1994;138;2;491-7

  • Embryonic expression pattern of amyloid protein precursor suggests a role in differentiation of specific subsets of neurons.

    Salbaum JM and Ruddle FH

    Department of Biology, Yale University, New Haven, Connecticut 06520.

    Immunohistochemical analysis revealed the temporal and spatial expression pattern of the amyloid protein precursor (APP) during the development of the mouse embryo. APP was first detected at day 9.5 of gestation in motor neurons of the hind brain and the spinal cord. APP proteins were also evident in cells of the floor plate, and in neurons of the cranial, dorsal root, and sympathetic ganglia shortly after their formation. Except for floor plate cells, APP expression was restricted to differentiated neurons. Comparison with the expression of microtubule-associated protein 2 (MAP-2), a marker for neurodifferentiation, showed that APP is expressed on a subset of differentiated neurons. APP can also serve as an early marker for the developing nuclei of the hind brain. The onset of APP expression in neurons appeared to be correlated with axonal outgrowth, whereas later expression of APP may be associated with functional specialization in the developing nervous system.

    Funded by: NHLBI NIH HHS: 1-RO1-HL49560

    The Journal of experimental zoology 1994;269;2;116-27

  • Murine chromosomal location of the mu and kappa opioid receptor genes.

    Kozak CA, Filie J, Adamson MC, Chen Y and Yu L

    Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.

    Opioid receptors are the membrane proteins that mediate the pain-relieving effect of opioid drugs, such as morphine and fentanyl as well as endogenous opioid peptides enkephalins and endorphins. Using cDNAs for the mu and the kappa opioid receptors, we mapped the chromosomal locations of their genes in mouse. Multilocus cross analysis located the mu receptor gene Oprm on Chr 10 and the kappa receptor gene Oprk1 on Chr 1. Both genes are near centromere, with no markers more centromeric. These data indicate that the two opioid receptors are different gene products, ruling out the possibility that they may be differential splicing products from the same gene.

    Funded by: NINDS NIH HHS: NS01557, NS28190

    Genomics 1994;21;3;659-61

  • Embryonic neurons of the developing optic chiasm express L1 and CD44, cell surface molecules with opposing effects on retinal axon growth.

    Sretavan DW, Feng L, Puré E and Reichardt LF

    Department of Physiology, University of California San Francisco.

    The first retinal ganglion cell axons arriving at the embryonic mouse ventral diencephalon encounter an inverted V-shaped neuronal array defining the midline and posterior boundaries of the future optic chiasm. These neurons express L1, an immunoglobulin superfamily molecule known to promote retinal axon outgrowth, and CD44, a cell surface molecule that we find inhibits embryonic retinal axon growth in vitro. Incoming retinal axons do not penetrate this L1/CD44 neuron array, but turn to establish the characteristic X-shaped optic chiasm along the anterior border of this array. These results suggest that L1/CD44 neurons may serve as an anatomical template for retinal axon pathways at the embryonic mouse ventral diencephalon.

    Funded by: NINDS NIH HHS: P01 NS016033, P01 NS016033-17A10014

    Neuron 1994;12;5;957-75

  • Organization of the embryonic and early postnatal murine hippocampus. I. Immunocytochemical characterization of neuronal populations in the subplate and marginal zone.

    Soriano E, Del Río JA, Martínez A and Supèr H

    Unit of Cell Biology, Faculty of Biology, University of Barcelona, Spain.

    Immunocytochemical techniques were used to characterize the neuronal populations in the hippocampal subplate and marginal zone from embryonic day 13 (E13) to postnatal day 5 (P5). Sections were processed for the visualization of microtubule-associated protein 2 (MAP2) and other antigens such as neurotransmitters, neuropeptides, calcium-binding proteins and a synaptic antigen (Mab SMI81). At E13-E14, only the ventricular zone and the primitive plexiform layer were recognized. Some cells in the later stratum displayed MAP2-, gamma-aminobutyric acid (GABA)- and calretinin immunoreactivities. From E15 onwards, the hippocampal and dentate plates became visible. Neurons in the plexiform layers were immunoreactive at E15-E16, whereas the hippocampal and dentate plates showed immunostaining two or three days later. Between E15 and E19 the following populations were distinguished in the plexiform layers: the subventricular zone displayed small neurons that reacted with MAP2 and GABA antibodies; the subplate (prospective stratum oriens) was poorly populated by MAP2- and GABA-positive cells; the inner marginal zone (future stratum radiatum) was heavily populated by multipolar GABAergic cells; the outer marginal zone (stratum lacunosum-moleculare) displayed horizontal neurons that showed glutamate- and calretinin immunoreactivities, their morphology being reminiscent of neocortical Cajal-Retzius cells. Thus, each plexiform layer was populated by a characteristic neuronal population whose distribution did not overlap. Similar segregated neuronal populations were also found in the developing dentate gyrus. At perinatal stages, small numbers of neurons in the plexiform layers began to express calbindin D-28K and neuropeptides. During early postnatal stages, neurons in the subplate and inner marginal zones were transformed into resident cells of the stratum oriens and radiatum, respectively. In contrast, calretinin-positive neurons in the stratum lacunosum-moleculare disappeared at postnatal stages. At E15-E19, SMI81-immunoreactive fibers were observed in the developing white matter, subplate and outer marginal zone, which suggests that these layers are sites of early synaptogenesis. At P0-P5, SMI81 immunoreactivity became homogeneously distributed within the hippocampal layers. The present results show that neurons in the hippocampal subplate and marginal zones have a more precocious morphological and neurochemical differentiation than the neurons residing in the principal cell layers. It is suggested that these early maturing neurons may have a role in the targeting of hippocampal afferents, as subplate cells do in the developing neocortex.

    The Journal of comparative neurology 1994;342;4;571-95

  • An isoform of microtubule-associated protein 2 (MAP2) containing four repeats of the tubulin-binding motif.

    Doll T, Meichsner M, Riederer BM, Honegger P and Matus A

    Friedrich Miescher Institute, Basel, Switzerland.

    Microtubule-associated protein 2 (MAP2) exists in both high- and low-molecular mass isoforms, each of which has a tubulin-binding domain consisting of 3 imperfect tandem repeats of 31 amino acids containing a more highly conserved 18 amino acid 'core' sequence. We describe here a novel form of low molecular mass MAP2 (MAP2c) that contains an additional 4th repeat of this tubulin-binding motif. Like the 3 previously known repeat sequences, this 4th copy is highly conserved between MAP2 and the two other known members of the same gene family, tau and MAP4. In each of these three genes the additional 4th repeat is inserted between the 1st and 2nd repeats of the 3-repeat form of the molecule. Experiments with brain cell cultures, in which the relative proportions of neurons and glia had been manipulated by drug treatment, showed that 4-repeat MAP2c is associated with glial cells whereas 3-repeat MAP2c is expressed in neurons. Whereas 3-repeat MAP2c is expressed early in development and then declines, the level of 4-repeat MAP2c increases later in development, corresponding to the relatively late differentiation of glial cells compared to neurons. When transfected into non-neuronal cells, the 4-repeat version of MAP2c behaved indistinguishably from the 3-repeat form in stabilising and rearranging cellular microtubules. The presence of an additional 4th repeat of the tubulin-binding motif in all three members of the MAP2 gene family suggests that this variant arose prior to their differentiation from an ancestral gene.

    Journal of cell science 1993;106 ( Pt 2);633-9

  • Regulation of tubulin, Tau and microtubule associated protein 2 expression during mouse brain development.

    Charrière-Bertrand C and Nunez J

    INSERM U 282, Hôpital Henri Mondor, Crétail, France.

    The level of three microtubule proteins, tubulin, Tau and MAP2 and of their encoding mRNA was studied in the mouse brain at an early developmental stage (3 days postnatal) and in adulthood. The level of the mRNA encoding both tubulin and Tau decreased by 85% between these two stages whereas the encoded proteins decreased only by 50% during the same period. Thus, the level of these proteins seems to be regulated both negatively and positively by transcriptional and post translational mechanisms. In vitro transcription assays, performed with nuclei isolated at different postnatal stages, showed that the tubulin and Tau transcripts are produced with some variations during mouse brain development. However these fluctuations are much less important than the drops of the steady state levels of tubulin and Tau mRNA seen in vivo. Thus, the decrease in transcripts levels does not seem to result from reduced transcriptional activities, and can be ascribed to changes in mRNA stability occurring during brain development, i.e. to a post transcriptional mechanism. The situation is even more complex for MAP2: its encoding mRNA level remains constant during development whereas the in vitro transcription activity decreases markedly during the same period. Finally, MAP2 protein level increases during development although its encoding mRNA level remains constant suggesting that this protein is stabilized by a post translational mechanism.

    Neurochemistry international 1992;21;4;535-41

  • Expression of necdin, an embryonal carcinoma-derived nuclear protein, in developing mouse brain.

    Aizawa T, Maruyama K, Kondo H and Yoshikawa K

    Department of Molecular Biology, Tokyo Institute of Psychiatry, Japan.

    Necdin is a polypeptide sequence encoded by neural differentiation-specific mRNA derived from embryonal carcinoma cells. We have examined the expression of necdin and its mRNA in cultured cells and mouse brain by Northern blot analysis and immunohistochemistry. Among various established cell lines including neuroblastoma and glioma cells, only differentiated embryonal carcinoma cells (P19 and F9) expressed necdin mRNA. Necdin immunoreactivity was localized in the nuclei of differentiated neurons derived from P19 cells. Necdin mRNA was detected throughout brain regions of adult mouse; the relative abundances in the hypothalamus and midbrain were the highest, whereas those in the olfactory bulb and cerebellum were the lowest. In developing mouse brain, necdin mRNA was expressed during early periods of neuronal generation and differentiation, and the peak levels were attained during postnatal days 1-4. Necdin immunoreactivity was not detected in the neural stem cells on embryonic day 10, but was concentrated in the nuclei of brain cells, mostly neurons, at advanced stages of differentiation. The majority of differentiated neurons in the brain had necdin-immunoreactive nuclei on postnatal day 33. Thus, necdin may represent a valuable molecular marker for differentiated neurons both in vitro and in vivo.

    Brain research. Developmental brain research 1992;68;2;265-74

  • Assignment of the microtubule-associated protein 2 gene to mouse chromosome 1.

    Lafuse WP, Brown D and Zwilling BS

    Department of Medical Microbiology and Immunology, Ohio State University, Columbus 43210.

    Funded by: NIAID NIH HHS: R01 AI22249

    Mammalian genome : official journal of the International Mammalian Genome Society 1992;3;1;48-51

  • Complete sequence of a cDNA encoding mouse MAP2.

    Wang D, Lewis SA and Cowan NJ

    Department of Biochemistry, NYU Medical Center 10016.

    Nucleic acids research 1988;16;23;11369-70

  • Dendritic and axonal distribution of the microtubule-associated proteins MAP2 and tau in the cerebellum of the nervous mutant mouse.

    Brion JP, Guilleminot J and Nunez J

    Laboratoire d'Anatomie Pathologique, Université Libre de Bruxelles, Belgium.

    The fate of the different types of axons and dendrites in the nervous mutant mouse has been studied with antibodies raised against the two major microtubule-associated proteins, MAP2 and tau. These proteins are specific markers of dendrites and axons, respectively. (1) Immunoblot analysis of cerebellar extracts showed that MAP2 concentration is markedly reduced (by approximately 90%) in the adult mutant. A 60% decrease was already noticed at day 20 postnatal, i.e., when all the Purkinje cells are present in their normal location and in apparent normal number. (2) Immunohistochemical studies performed at an adult stage with anti MAP2 antibodies showed marked alterations in the shape of the dendrites of the rare surviving Purkinje cells present in the lateral sections of the cerebellum of the mutant. In the vermis, where 50% of the cells survive in adulthood, the MAP2 antibody revealed both clusters of cells with a normal density and an intricated and extensive pattern of dendritic arborization and isolated cells showing either an apparently normal or an altered dendritic tree. (3) At day 20 postnatal the same antibody revealed, in the lateral sections severe abnormalities of the dendrites of the Purkinje cells which were different from those seen in adulthood in the vermis. Thus, although few or any Purkinje cells are dead at this stage, a large proportion of them have already profound dendritic alterations. In contrast, in the vermis the Purkinje cells and their dendritic tree are undistinguishable at this stage from those of the unaffected normal mice. (4) Immunoblot and immunohistochemical studies performed with the anti Tau antibody suggested that the majority of the axonal fibers of the cerebellum were present both at day 20 postnatal and at later adult stages. This suggests that, although deprived of their postsynaptic targets these axons can survive for a long time after Purkinje cell death. However, an anti-neurofilament monoclonal antibody which stains specifically the axons of the basket cells, revealed an altered morphology of the basket cell nest in the regions devoid of Purkinje cells. (5) In conclusion the alterations in the morphology of dendrites seem to represent an early event of Purkinje cell degeneration and to be correlated with a marked decrease in expression of MAP2. It remains unclear, however, whether such changes in expression of MAP2 represent a primary effect of the mutation or if it is only a precocious result of Purkinje cell degeneration.

    Brain research. Developmental brain research 1988;44;2;221-32

  • Microtubule-associated protein MAP2 shares a microtubule binding motif with tau protein.

    Lewis SA, Wang DH and Cowan NJ

    Department of Biochemistry, New York University Medical Center, NY 10016.

    The microtubule-associated protein MAP2 is a prominent large-sized component of purified brain microtubules that, like the 36- to 38-kilodalton tau proteins, bears antigenic determinants found in association with the neurofibrillary tangles of Alzheimer's disease. The complete sequence of mouse brain MAP2 was determined from a series of overlapping cloned complementary DNAs. The sequence of the carboxyl-terminal 185 amino acids is very similar (67 percent) to a corresponding region of tau protein, and includes a series of three imperfect repeats, each 18 amino acids long and separated by 13 or 14 amino acids. A subcloned fragment spanning the first two of the 18-amino acid repeats was expressed as a polypeptide by translation in vitro. This polypeptide copurified with microtubules through two successive cycles of polymerization and depolymerization, whereas a control polypeptide derived from the amino-terminal region of MAP2 completely failed to copurify. These data imply that the carboxyl-terminal domain containing the 18-amino acid repeats constitutes the microtubule binding site in MAP2. The occurrence of these repeats in tau protein suggests that these may be a general feature of microtubule binding proteins.

    Science (New York, N.Y.) 1988;242;4880;936-9

  • Brain-specific expression of MAP2 detected using a cloned cDNA probe.

    Lewis SA, Villasante A, Sherline P and Cowan NJ

    We describe the isolation of a set of overlapping cDNAs encoding mouse microtubule associated protein 2 (MAP2), using an anti-MAP antiserum to screen a mouse brain cDNA expression library cloned in bacteriophage lambda gt11. The authenticity of these clones was established by the following criteria: (a) three non-identical clones each expressing a MAP2 immunoreactive fusion protein were independently isolated from the expression library; each of these clones cross-hybridized at the nucleic acid level; (b) anti-MAP antiserum was affinity purified using nitrocellulose-bound fusion protein; these antibodies detected only MAP2 in an immunoblot experiment of whole brain microtubule protein; (c) a series of cDNA "walking" experiments was done so as to obtain a non-overlapping cloned fragment corresponding to a different part of the same mRNA molecule. Upon subcloning this non-overlapping fragment into plasmid expression vectors, a fusion protein was synthesized that was immunoreactive with an anti-MAP2 specific antiserum. Thus, a single contiguous cloned mRNA molecule encodes at least two MAP2-specific epitopes; (d) the cloned cDNA probes detect an mRNA species in mouse brain that is of a size (approximately 9 kb) consistent with the coding capacity required by a 250,000-D protein. The MAP2-specific cloned cDNA probes were used in RNA blot transfer experiments to assay for the presence of MAP2 mRNA in a variety of mouse tissues. Though brain contained abundant quantities of MAP2 mRNA, no corresponding sequences were detectable in RNA prepared from liver, kidney, spleen, stomach, or thymus. We conclude that the expression of MAP2 is brain-specific. Use of the MAP2 specific cDNA probes in genomic Southern blot transfer experiments showed the presence of a single gene encoding MAP2 in mouse. The microheterogeneity of MAP2 is therefore ascribable either to alternative splicing within a single gene, or to posttranslational modification(s), or both. Under conditions of low stringency, the mouse MAP2 cDNA probe cross-hybridizes with genomic sequences from rat, human, and (weakly) chicken, but not with sequences in frog, Drosophila, or sea urchin DNA. Thus, there is significant interspecies divergence of MAP2 sequences. The implications of the above observations are discussed in relationship to the potential biological function of MAP2.

    The Journal of cell biology 1986;102;6;2098-105

Gene lists (9)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000005 G2C Mus musculus Mouse mGluR5 Mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000007 G2C Mus musculus Mouse NRC Mouse NRC adapted from Collins et al (2006) 186
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
L00000021 G2C Mus musculus Pocklington M3 Cluster 3 (mouse) from Pocklington et al (2006) 30
L00000060 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus (ortho) 748
L00000062 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus 984
L00000070 G2C Mus musculus BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list (ortho) 1461
L00000072 G2C Mus musculus BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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