G2Cdb::Gene report

Gene id
G00000771
Gene symbol
Vim (MGI)
Species
Mus musculus
Description
vimentin
Orthologue
G00002020 (Homo sapiens)

Databases (11)

Curated Gene
OTTMUSG00000011231 (Vega mouse gene)
Gene
ENSMUSG00000026728 (Ensembl mouse gene)
22352 (Entrez Gene)
1209 (G2Cdb plasticity & disease)
Gene Expression
NM_011701 (Allen Brain Atlas)
g04017 (BGEM)
22352 (Genepaint)
vim (gensat)
Literature
193060 (OMIM)
Marker Symbol
MGI:98932 (MGI)
Protein Sequence
P20152 (UniProt)

Literature (240)

Pubmed - other

  • Neurogenin3 initiates stepwise delamination of differentiating endocrine cells during pancreas development.

    Gouzi M, Kim YH, Katsumoto K, Johansson K and Grapin-Botton A

    During development, pancreatic endocrine cells are specified within the pancreatic epithelium. They subsequently delaminate out of the epithelium and cluster in the mesenchyme to form the islets of Langerhans. Neurogenin3 (Ngn3) is a transcription factor required for the differentiation of all endocrine cells and we investigated its role in their delamination. We observed in the mouse pancreas that most Ngn3-positive cells have lost contact with the lumen of the epithelium, showing that the delamination from the progenitor layer is initiated in endocrine progenitors. Subsequently, in both mouse and chick newly born endocrine cells at the periphery of the epithelium strongly decrease E-cadherin, break-down the basal lamina and cluster into islets of Langerhans. Repression of E-cadherin is sufficient to promote delamination from the epithelium. We further demonstrate that Ngn3 indirectly controls Snail2 protein expression post-transcriptionally to repress E-cadherin. In the chick embryo, Ngn3 independently controls epithelium delamination and differentiation programs.

    Developmental dynamics : an official publication of the American Association of Anatomists 2011;240;3;589-604

  • Cellular expression of the K+-Cl- cotransporter KCC3 in the central nervous system of mouse.

    Shekarabi M, Salin-Cantegrel A, Laganière J, Gaudet R, Dion P and Rouleau GA

    Centre of Excellence in Neuromics, CHUM Research Center and Department of Medicine, University of Montreal, Notre-Dame Hospital, 1560 Sherbrooke East, De-Seve Pavillion, room Y-3616-2, Montréal, QC, H2L 4M1, Canada.

    Potassium/Chloride cotransporters are transmembrane proteins that regulate cell volume and control neuronal activity by transporting K(+) and Cl(-) ions across the plasma membrane. Potassium/Chloride cotransporter 3 (KCC3) mutations are responsible for hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe sensory and motor neuropathy. Two major splice variants, KCC3a and KCC3b, were shown to be expressed in adult mouse tissues. Although KCC3a is mainly expressed in the central nervous system (CNS), its specific cellular expression patterns have not been determined. Here, we used an approach combining in situ hybridization and immunohistochemical techniques to determine the cellular expression of KCC3 in the mouse CNS and showed that KCC3 is mainly expressed in neurons, including a subpopulation of interneurons. Finally, we showed that some non-neuronal cells, such as radial glial-like cells in the spinal cord, also express KCC3.

    Funded by: Canadian Institutes of Health Research: 172248

    Brain research 2011;1374;15-26

  • Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation.

    Li B, Zheng YW, Sano Y and Taniguchi H

    Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

    Unlabelled: Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein.

    Conclusion: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.

    PloS one 2011;6;2;e17092

  • Vimentin binds IRAP and is involved in GLUT4 vesicle trafficking.

    Hirata Y, Hosaka T, Iwata T, Le CT, Jambaldorj B, Teshigawara K, Harada N, Sakaue H, Sakai T, Yoshimoto K and Nakaya Y

    Department of Nutrition and Metabolism, Institute of Health Biosciences, Tokushima University, Tokushima, Japan.

    Insulin-responsive aminopeptidase (IRAP) and GLUT4 are two major cargo proteins of GLUT4 storage vesicles (GSVs) that are translocated from a postendosomal storage compartment to the plasma membrane (PM) in response to insulin. The cytoplasmic region of IRAP is reportedly involved in retention of GSVs. In this study, vimentin was identified using the cytoplasmic domain of IRAP as bait. The validity of this interaction was confirmed by pull-down assays and immunoprecipitation in 3T3-L1 adipocytes. In addition, it was shown that GLUT4 translocation to the PM by insulin was decreased in vimentin-depleted adipocytes, presumably due to dispersing GSVs away from the cytoskeleton. These findings suggest that the IRAP binding protein, vimentin, plays an important role in retention of GSVs.

    Biochemical and biophysical research communications 2011;405;1;96-101

  • Expression of lymphatic endothelium-specific hyaluronan receptor LYVE-1 in the developing mouse kidney.

    Lee HW, Qin YX, Kim YM, Park EY, Hwang JS, Huo GH, Yang CW, Kim WY and Kim J

    Department of Anatomy and MRC for Cell Death Disease Research Center, College of Medicine, The Catholic University of Korea, 505, Banpo-Dong, Seocho-Ku, Seoul, 137-701, Korea.

    Our knowledge of the embryonic development of the lymphatic vessels within the kidney is limited. The aim of this study was to establish the time of appearance and the distribution of intra-renal lymphatic vessels in the developing mouse kidney by using the lymphatic marker, LYVE-1. Kidneys from embryonic day 12 (E12) to E18, from neonates at post-natal day 1 (P1) to P21, and from adults were studied. In the adult mouse kidney, LYVE-1 was expressed mainly in the lymphatic endothelial cells (LECs) and in a subset of endothelial cells in the glomerular capillaries. However, in the developing mouse kidney, LYVE-1 was also expressed transiently in F4/80(+)/CD11b(-) immature macrophages/dendritic cells and in the developing renal vein. LYVE-1(+) lymphatic vessels connected with extra-renal lymphatics were detected in the kidney at E13. F4/80(+)/CD11b(-)/LYVE-1(+) immature macrophages/dendritic cells appeared prior to the appearance of LYVE-1(+) renal lymphatic vessels and were closely intermingled or even formed part of the lymphatic vascular wall. Prox1 was expressed only in the LYVE-1(+) LECs from fetus to adult-hood, but not in LYVE-1(+) endothelial cells of the developing renal vein and macrophages/dendritic cells. Thus, lymphatic vessels of the kidney might originate by extension of extra-renal lymphatics through an active branching process possibly associated with F4/80(+)/CD11b(-)/LYVE-1(+) macrophages/dendritic cells.

    Cell and tissue research 2011;343;2;429-44

  • Reactive glial cells: increased stiffness correlates with increased intermediate filament expression.

    Lu YB, Iandiev I, Hollborn M, Körber N, Ulbricht E, Hirrlinger PG, Pannicke T, Wei EQ, Bringmann A, Wolburg H, Wilhelmsson U, Pekny M, Wiedemann P, Reichenbach A and Käs JA

    Division of Soft Matter Physics, Department of Physics, and Paul Flechsig Institute of Brain Research, Universität Leipzig, Leipzig, Germany.

    Increased stiffness of reactive glial cells may impede neurite growth and contribute to the poor regenerative capabilities of the mammalian central nervous system. We induced reactive gliosis in rodent retina by ischemia-reperfusion and assessed intermediate filament (IF) expression and the viscoelastic properties of dissociated single glial cells in wild-type mice, mice lacking glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)) in which glial cells are consequently devoid of IFs, and normal Long-Evans rats. In response to ischemia-reperfusion, glial cells stiffened significantly in wild-type mice and rats but were unchanged in GFAP(-/-)Vim(-/-) mice. Cell stiffness (elastic modulus) correlated with the density of IFs. These results support the hypothesis that rigid glial scars impair nerve regeneration and that IFs are important determinants of cellular viscoelasticity in reactive glia. Thus, therapeutic suppression of IF up-regulation in reactive glial cells may facilitate neuroregeneration.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2011;25;2;624-31

  • Premutation CGG-repeat expansion of the Fmr1 gene impairs mouse neocortical development.

    Cunningham CL, Martínez Cerdeño V, Navarro Porras E, Prakash AN, Angelastro JM, Willemsen R, Hagerman PJ, Pessah IN, Berman RF and Noctor SC

    Department of Pathology, Institute for Pediatric Regenerative Medicine--Shriners Hospital, School of Medicine, UC Davis, Sacramento, CA 95817, USA.

    Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late adult-onset neurodegenerative disorder caused by a premutation CGG-trinucleotide repeat expansion (55-200 CGG repeats) within the 5'-untranslated region of the FMR1 gene. Although FXTAS generally affects premutation carriers over 50 years of age, cognitive and psychological symptoms can appear in carriers during childhood, suggesting that the FMR1 premutation affects brain function early in life. Recent work with cultured hippocampal neurons from a premutation (Fmr1 CGG knock-in) mouse model revealed impaired development of early postnatal neurons, consistent with the developmental clinical involvement of premutation carriers. In the current work, we show that the presence of premutation CGG-repeat expansions in the mouse Fmr1 gene alters embryonic neocortical development. Specifically, embryonic premutation mice display migration defects in the neocortex and altered expression of neuronal lineage markers. The current data demonstrate that premutation alleles of the Fmr1 gene are associated with defects in developmental programs operating during prenatal stages of brain formation and provide further evidence that the FMR1 premutation has a neurodevelopmental component.

    Funded by: NIA NIH HHS: RL1 AG032119; NINDS NIH HHS: RL1 NS062411, RL1 NS062411-05; PHS HHS: UL1 DEO19583

    Human molecular genetics 2011;20;1;64-79

  • Development of thyroid gland and ultimobranchial body cyst is independent of p63.

    Ozaki T, Nagashima K, Kusakabe T, Kakudo K and Kimura S

    Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

    The ultimobranchial body (UBB) and thyroid primordium are the origins of the thyroid gland that fuse around embryonic day 14.5 of mouse gestation, ultimately giving rise to calcitonin-producing C cells and thyroglobulin-producing follicular cells, respectively. A homeodomain transcription factor NKX2-1 is expressed both in the UBB and the thyroid primordium, and is critical for development of the thyroid gland. In this study, the role of p63 in development of UBB and the thyroid gland was analyzed by histological, immunohistochemical, and electron microscopic analyses using mice with various combinations of Nkx2-1 and p63 wild-type, heterozygous, and null alleles. In the absence of p63, a normal thyroid gland develops, as revealed by expression of thyroglobulin and calcitonin, thus showing that p63 is not required for thyroid development. However, in mice carrying the Nkx2-1-null allele, the UBB remains as a cystic vesicular structure and/or in nested patterns consisting of p63-positive cells surrounding the vesicle and undifferentiated immature cells with occasional cilia lying inside. The cystic UBB was present even in the Nkx2-1;p63 double-null mice. The structure and p63 expression pattern of the UBB cyst strikingly resemble the solid cell nest. These results show that in the absence of NKX2-1, UBB becomes cystic independent of p63, which is likely the origin of SCN.

    Funded by: Intramural NIH HHS: Z99 CA999999, ZIA BC005522-08

    Laboratory investigation; a journal of technical methods and pathology 2011;91;1;138-46

  • Fgf9 signalling stimulates Spred and Sprouty expression in embryonic mouse pancreas mesenchyme.

    Sylvestersen KB, Herrera PL, Serup P and Rescan C

    Hagedorn Research Institute, Department of Developmental Biology, Gentofte, Denmark.

    Epithelial-mesenchymal interactions are critical for normal pancreas development. Fibroblast growth factor (Fgf)-10 is expressed in the pancreatic mesenchyme and its signalling is required for normal growth and regulation of gene expression in the pancreatic epithelium. However, little is known about putative Fgf signalling to the mesenchyme. Here we have examined the embryonic pancreas expression of differentially spliced Fgf receptor isoforms and their targets; the Sprouty (Spry) and Spred family genes which are induced by Fgf signalling. Using qPCR to quantify mRNA levels in microdissected pancreatic epithelium and mesenchyme as well as in FACS isolated Pdx1-GFP(+) and -GFP(-) cell populations we demonstrate that several members of the Spred and Sprouty families are expressed in embryonic mouse pancreas and find Spred1 and -2 as well as Spry2 and -4 to be predominantly expressed in pancreatic mesenchyme. Using embryonic pancreas explant cultures we demonstrate that Spred1/2 and Spry2/4 expression is regulated by Fgf receptor signalling and is increased by treatment with Fgf9, but not by Fgf7 or Fgf10. We extend previous work showing that Fgf9 is expressed in pancreatic mesenchyme, and since Fgf9 is known to activate the mesenchyme-specific "c"-splice forms of Fgf receptors, while Fgf7 and -10 both activate the epithelium-specific "b"-splice forms of Fgf receptors, these results suggest that Fgf signalling is active in the pancreatic mesenchyme, where expression of Spred1/2 and Spry2/4 appear downstream of Fgf9 signalling.

    Gene expression patterns : GEP 2011;11;1-2;105-11

  • Dachshund homologues play a conserved role in islet cell development.

    Kalousova A, Mavropoulos A, Adams BA, Nekrep N, Li Z, Krauss S, Stainier DY and German MS

    Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.

    All metazoans use insulin to control energy metabolism, but they secrete it from different cells: neurons in the central nervous system in invertebrates and endocrine cells in the gut or pancreas in vertebrates. Despite their origins in different germ layers, all of these insulin-producing cells share common functional features and gene expression patterns. In this study, we tested the role in insulin-producing cells of the vertebrate homologues of Dachshund, a transcriptional regulator that marks the earliest committed progenitors of the neural insulin-producing cells in Drosophila. Both zebrafish and mice expressed a single dominant Dachshund homologue in the pancreatic endocrine lineage, and in both species loss of this homologue reduced the numbers of all islet cell types including the insulin-producing β-cells. In mice, Dach1 gene deletion left the pancreatic progenitor cells unaltered, but blocked the perinatal burst of proliferation of differentiated β-cells that normally generates most of the β-cell mass. In β-cells, Dach1 bound to the promoter of the cell cycle inhibitor p27Kip1, which constrains β-cell proliferation. Taken together, these data demonstrate a conserved role for Dachshund homologues in the production of insulin-producing cells.

    Funded by: NIDDK NIH HHS: P30 DK063720, P30 DK063720-02, P30 DK063720-03, P30 DK063720-04, P30 DK063720-05, P30 DK063720-06A1, P30 DK063720-07, R01 DK021344, R01 DK021344-25, R01 DK021344-26, R01 DK021344-27A2, R01 DK021344-28, R01 DK075032, R01DK075032, R01DK21344, U19 DK061245, U19 DK061245-04, U19 DK61245

    Developmental biology 2010;348;2;143-52

  • Nestin is required for the proper self-renewal of neural stem cells.

    Park D, Xiang AP, Mao FF, Zhang L, Di CG, Liu XM, Shao Y, Ma BF, Lee JH, Ha KS, Walton N and Lahn BT

    Department of Human Genetics, University of Chicago, Howard Hughes Medical Institute, Chicago, Illinois, USA. donghyun@uchicago.edu

    The intermediate filament protein, nestin, is a widely employed marker of multipotent neural stem cells (NSCs). Recent in vitro studies have implicated nestin in a number of cellular processes, but there is no data yet on its in vivo function. Here, we report the construction and functional characterization of Nestin knockout mice. We found that these mice show embryonic lethality, with neuroepithelium of the developing neural tube exhibiting significantly fewer NSCs and much higher levels of apoptosis. Consistent with this in vivo observation, NSC cultures derived from knockout embryos show dramatically reduced self-renewal ability that is associated with elevated apoptosis but no overt defects in cell proliferation or differentiation. Unexpectedly, nestin deficiency has no detectable effect on the integrity of the cytoskeleton. Furthermore, the knockout of Vimentin, which abolishes nestin's ability to polymerize into intermediate filaments in NSCs, does not lead to any apoptotic phenotype. These data demonstrate that nestin is important for the proper survival and self-renewal of NSCs, and that this function is surprisingly uncoupled from nestin's structural involvement in the cytoskeleton.

    Funded by: Howard Hughes Medical Institute

    Stem cells (Dayton, Ohio) 2010;28;12;2162-71

  • Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation.

    He S, Pirity MK, Wang WL, Wolf L, Chauhan BK, Cveklova K, Tamm ER, Ashery-Padan R, Metzger D, Nakai A, Chambon P, Zavadil J and Cvekl A

    Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. ales.cvekl@einstein.yu.edu.

    Background: Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation) mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs.

    Results: To examine the roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific αA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (that is, denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in embryonic day 15.5 (E15.5) wild-type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous and Hsf4 homozygous lenses identified multiple genes coregulated by Brg1, Hsf4 and Pax6. DNase IIβ, a key enzyme required for lens fiber cell denucleation, was found to be downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation, for expression of DNase IIβ, for lens fiber cell denucleation and indirectly for retinal development.

    Conclusions: These studies demonstrate a cell-autonomous role for Brg1 in lens fiber cell terminal differentiation and identified DNase IIβ as a potential direct target of SWI/SNF complexes. Brg1 is directly or indirectly involved in processes that degrade lens fiber cell chromatin. The presence of nuclei and other organelles generates scattered light incompatible with the optical requirements for the lens.

    Funded by: NEI NIH HHS: R01 EY014237

    Epigenetics & chromatin 2010;3;1;21

  • Mouse atonal homolog 1 directs intestinal progenitors to secretory cell rather than absorptive cell fate.

    VanDussen KL and Samuelson LC

    Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, MI 48109, USA.

    The Notch-regulated transcription factor mouse atonal homolog 1 (Math1) is required for the development of intestinal secretory cells, as demonstrated by the loss of goblet, endocrine and Paneth cell types in null mice. However, it was unknown whether Math1 is sufficient to induce the program of secretory cell differentiation. To examine the function of Math1 in the differentiation of intestinal epithelial cells, intestinal morphology and epithelial and mesenchymal cell fate were examined by histological staining and marker gene expression in transgenic mice expressing a villin-regulated Math1 transgene. Late prenatal transgenic founders exhibited a gross cellular transformation into a secretory epithelium. The expansion of secretory cells coupled with the almost complete loss of absorptive enterocytes suggested reprogramming of a bipotential progenitor cell. Moreover, Math1 expression inhibited epithelial cell proliferation, as demonstrated by a marked reduction in Ki67 positive cells and blunted villi. Unexpectedly, the transgenic mesenchyme was greatly expanded with increased proliferation. Several mesenchymal cell types were amplified, including smooth muscle and neurons, with maintenance of basic radial patterning. Since transgenic Math1 expression was restricted to the epithelium, these findings suggest that epithelial-mesenchymal signaling is altered by the cellular changes induced by Math1. Thus, Math1 is a key effector directing multipotential precursors to adopt secretory and not absorptive cell fate.

    Funded by: NIDDK NIH HHS: P01 DK062041, P01-DK06241, R01 DK056882, R01 DK078927, R01-DK56882, R01-DK78927; NIGMS NIH HHS: T32 GM008322, T32-GM08322

    Developmental biology 2010;346;2;215-23

  • Keeping the vimentin network under control: cell-matrix adhesion-associated plectin 1f affects cell shape and polarity of fibroblasts.

    Burgstaller G, Gregor M, Winter L and Wiche G

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

    Focal adhesions (FAs) located at the ends of actin/myosin-containing contractile stress fibers form tight connections between fibroblasts and their underlying extracellular matrix. We show here that mature FAs and their derivative fibronectin fibril-aligned fibrillar adhesions (FbAs) serve as docking sites for vimentin intermediate filaments (IFs) in a plectin isoform 1f (P1f)-dependent manner. Time-lapse video microscopy revealed that FA-associated P1f captures mobile vimentin filament precursors, which then serve as seeds for de novo IF network formation via end-to-end fusion with other mobile precursors. As a consequence of IF association, the turnover of FAs is reduced. P1f-mediated IF network formation at FbAs creates a resilient cage-like core structure that encases and positions the nucleus while being stably connected to the exterior of the cell. We show that the formation of this structure affects cell shape with consequences for cell polarization.

    Funded by: Austrian Science Fund FWF: I 413, P 20744

    Molecular biology of the cell 2010;21;19;3362-75

  • Developmental changes of cell adhesion molecule expression in the fetal mouse liver.

    Sugiyama Y, Koike T and Shiojiri N

    Department of Biology, Faculty of Science, Shizuoka University, Shizuoka City, Japan.

    Developmental changes of cell adhesion molecule expression, especially in nonparenchymal cells, have hardly ever been analyzed in the murine liver. The present study was undertaken to immunohistochemically examine the expression of NCAM, ICAM, VCAM, and N-cadherin during mouse liver development and in fetal liver cell cultures. NCAM was transiently expressed in mesenchymal cells of the septum transversum and sinusoidal cells in liver development. In vitro studies demonstrated that desmin-positive stellate cells expressed this cell adhesion molecule. NCAM expression in periportal biliary epithelial cells and connective tissue cells also coincided well with bile duct remodeling processes in the perinatal periods. Expression of ICAM and VCAM was transiently restricted to hepatoblasts, hepatocytes and hemopoietic cells in fetal stages. N-cadherin was expressed not only in hepatoblasts and hepatocytes, but also in nonparenchymal cells such as endothelial cells, stellate cells and connective tissue cells, however the expression was weak. These results suggest that each cell adhesion molecule may play an important role during development in hepatic histogenesis, including hepatoblast/hepatocyte-stellate cell interactions, hemopoiesis, and bile duct morphogenesis.

    Anatomical record (Hoboken, N.J. : 2007) 2010;293;10;1698-710

  • Origin, maturation, and astroglial transformation of secondary radial glial cells in the developing dentate gyrus.

    Brunne B, Zhao S, Derouiche A, Herz J, May P, Frotscher M and Bock HH

    Center of Neurosciences, University of Freiburg, Freiburg, Germany.

    The dentate gyrus is a brain region where neurons are continuously born throughout life. In the adult, the role of its radial glia in neurogenesis has attracted much attention over the past years; however, little is known about the generation and differentiation of glial cells and their relationship to radial glia during the ontogenetic development of this brain structure. Here, we combine immunohistochemical phenotyping using antibodies against glial marker proteins with BrdU birthdating to characterize the development of the secondary radial glial scaffold in the dentate gyrus and its potential to differentiate into astrocytes. We demonstrate that the expression of brain lipid-binding protein, GLAST, and glial fibrillary acidic protein (GFAP) characterizes immature differentiating cells confined to an astrocytic fate in the early postnatal dentate gyrus. On the basis of our studies, we propose a model where immature astrocytes migrate radially through the granule cell layer to adopt their final positions in the molecular layer of the dentate gyrus. Time-lapse imaging of acute hippocampal slices from hGFAP-eGFP transgenic mice provides direct evidence for such a migration mode of differentiating astroglial cells in the developing dentate gyrus.

    Funded by: NHLBI NIH HHS: P01 HL020948, P01 HL020948-210015, P01 HL020948-220015, P01 HL020948-230015, P01 HL020948-240015, P01 HL020948-250015, P01 HL020948-310005, P01 HL020948-320005, P01 HL020948-330005, P01 HL020948-340005, R01 HL063762, R01 HL063762-01, R01 HL063762-02, R01 HL063762-03, R01 HL063762-04, R01 HL063762-05, R01 HL063762-06, R01 HL063762-07, R01 HL063762-08, R01 HL063762-09, R01 HL063762-10, R01 HL063762-10W1, R01 HL063762-11, R01 HL063762-12, R37 HL063762; NINDS NIH HHS: R01 NS043408, R01 NS043408-01, R01 NS043408-02, R01 NS043408-03, R01 NS043408-04, R01 NS043408-05, RC1 NS068697, RC1 NS068697-01, RC1 NS068697-02

    Glia 2010;58;13;1553-69

  • Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline.

    Pulvers JN, Bryk J, Fish JL, Wilsch-Bräuninger M, Arai Y, Schreier D, Naumann R, Helppi J, Habermann B, Vogt J, Nitsch R, Tóth A, Enard W, Pääbo S and Huttner WB

    Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.

    Mutations in ASPM (abnormal spindle-like microcephaly associated) cause primary microcephaly in humans, a disorder characterized by a major reduction in brain size in the apparent absence of nonneurological anomalies. The function of the Aspm protein in neural progenitor cell expansion, as well as its localization to the mitotic spindle and midbody, suggest that it regulates brain development by a cell division-related mechanism. Furthermore, evidence that positive selection affected ASPM during primate evolution has led to suggestions that such a function changed during primate evolution. Here, we report that in Aspm mutant mice, truncated Aspm proteins similar to those causing microcephaly in humans fail to localize to the midbody during M-phase and cause mild microcephaly. A human ASPM transgene rescues this phenotype but, interestingly, does not cause a gain of function. Strikingly, truncated Aspm proteins also cause a massive loss of germ cells, resulting in a severe reduction in testis and ovary size accompanied by reduced fertility. These germline effects, too, are fully rescued by the human ASPM transgene, indicating that ASPM is functionally similar in mice and humans. Our findings broaden the spectrum of phenotypic effects of ASPM mutations and raise the possibility that positive selection of ASPM during primate evolution reflects its function in the germline.

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;38;16595-600

  • Impaired pancreatic development in Hif2-alpha deficient mice.

    Chen H, Houshmand G, Mishra S, Fong GH, Gittes GK and Esni F

    Division of Pediatric General and Thoracic Surgery, Children's Hospital of Pittsburgh, University of Pittsburgh Medical Center, One Children's Drive, 4401 Penn Avenue, Rangos Research Center, Pittsburgh, PA 15244, United States.

    Accumulating data suggest the existence of a link between hypoxia and maintenance of the undifferentiated cell state, but little is known about the cellular signaling mechanisms underlying this process. Recent reports reveal a direct link between components of the hypoxia signaling pathway and Notch pathway in maintaining precursor cells in an undifferentiated state. Here, we report that in the developing mouse pancreas, Hif2-alpha is expressed in pancreatic progenitor cells, but its expression is lost in committed endocrine progenitors as well as in differentiated endocrine and exocrine cells. In an attempt to analyze the function of HIF2-alpha in the developing pancreas, we studied Hif2-alpha(-/-) pancreas. Our analyses revealed that in addition to the decreased size and branching, the Hif2-alpha deficient pancreas also displayed impaired notch signaling and cell differentiation. Finally, we found that HIF2-alpha binds directly to Notch-IC and that the responsible site for this interaction is within the RAM domain of Notch protein. These results suggest that HIF2-alpha is required for normal mouse pancreatic development.

    Biochemical and biophysical research communications 2010;399;3;440-5

  • Intermediate filaments attenuate stimulation-dependent mobility of endosomes/lysosomes in astrocytes.

    Potokar M, Stenovec M, Gabrijel M, Li L, Kreft M, Grilc S, Pekny M and Zorec R

    Celica Biomedical Center, Ljubljana, Slovenia.

    Intermediate filament (IF) proteins upregulation is a hallmark of astrocyte activation and reactive gliosis, but its pathophysiological implications remain incompletely understood. A recently reported association between IFs and directional mobility of peptidergic vesicles allows us to hypothesize that IFs affect vesicle dynamics and exocytosis-mediated astrocyte communication with neighboring cells. Here, we ask whether the trafficking of recycling vesicles (i.e., those fused to and then retrieved from the plasma membrane) and endosomes/lysosomes depends on IFs. Recycling vesicles were labeled by antibodies against vesicle glutamate transporter 1 (VGLUT1) and atrial natriuretic peptide (ANP), respectively, and by lysotracker, which labels endosomes/lysosomes. Quantitative fluorescence microscopy was used to monitor the mobility of labeled vesicles in astrocytes, derived from either wild-type (WT) mice or mice deficient in glial fibrillary acidic protein and vimentin (GFAP(-/-)Vim(-/-)), the latter lacking astrocyte IFs. Stimulation with ionomycin or ATP enhanced the mobility of VGLUT1-positive vesicles and reduced the mobility of ANP-positive vesicles in WT astrocytes. In GFAP(-/-)Vim(-/-) astrocytes, both vesicle types responded to stimulation, but the relative increase in mobility of VGLUT1-positive vesicles was more prominent compared with nonstimulated cells, whereas the stimulation-dependent attenuation of ANP-positive vesicles mobility was reduced compared with nonstimulated cells. The mobility of endosomes/lysosomes decreased following stimulation in WT astrocytes. However, in GFAP(-/-)Vim(-/-) astrocytes, a small increase in the mobility of endosomes/lysosomes was observed. These findings show that astrocyte IFs differentially affect the stimulation-dependent mobility of vesicles. We propose that upregulation of IFs in pathologic states may alter the function of astrocytes by deregulating vesicle trafficking.

    Glia 2010;58;10;1208-19

  • Basal enrichment within neuroepithelia suggests novel function(s) for Celsr1 protein.

    Formstone CJ, Moxon C, Murdoch J, Little P and Mason I

    MRC Centre for Developmental Biology, New Hunts House, Kings College, London SE1 1UL, UK. caroline.formstone@kcl.ac.uk <caroline.formstone@kcl.ac.uk&gt;

    A characteristic of the 7TM-cadherins, Flamingo and Celsr1, is their asymmetric protein distribution and polarized activity at neighboring epithelial cell interfaces along defined axes of planar cell polarity. Here, we describe a novel distribution of Celsr1 protein to the basal surface of neuroepithelial cells within both the early neural tube and a less well-defined group of ventricular zone cells at the midline of the developing spinal cord. Importantly, this basal enrichment is lost in embryos homozygous for a mutant Celsr1 allele. We also demonstrate an intimate association between basal enrichment of Celsr1 protein and dorsal sensory tract morphogenesis, an intriguing spatio-temporal organization of Celsr1 protein along the apico-basal neuroepithelial axis suggestive of multiple Celsr1 protein isoforms and the existence of distinct cell surface Celsr1 protein species with direct signaling potential. Together, these data raise compelling new questions concerning the role of Celsr1 during neural development.

    Funded by: Biotechnology and Biological Sciences Research Council; Medical Research Council; Wellcome Trust

    Molecular and cellular neurosciences 2010;44;3;210-22

  • Sulfatase 1 is an inhibitor of ductal morphogenesis with sexually dimorphic expression in the urogenital sinus.

    Buresh RA, Kuslak SL, Rusch MA, Vezina CM, Selleck SB and Marker PC

    Division of Pharmaceutical Sciences, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, Wisconsin 53705, USA.

    The prostate gland develops from the urogenital sinus in response to circulating androgens. Androgens initiate and stimulate branching morphogenesis in the urogenital sinus via unknown mediators. Heparan sulfate proteoglycans are important extracellular molecules that sequester many growth factors in the extracellular matrix and facilitate signaling by some growth factors as part of ternary complexes that include growth factors, receptors, and heparan sulfate chains. Several enzymes modify the chemical structure of heparan sulfate to further regulate its activity. An examination of these enzymes for sexually dimorphic expression in the urogenital sinus identified Sulfatase 1 (Sulf1) as an enzyme that was down-regulated in the male urogenital sinus coincident with the initiation of prostatic morphogenesis. Down-regulation of Sulf1 was accompanied by an increase in the most highly sulfated forms of heparan sulfate, and a similar increase was observed in female urogenital sinuses treated with testosterone. Inhibiting de novo sulfation of heparan sulfate blocked prostatic morphogenesis, supporting the importance of heparan sulfate modification for prostate development. To functionally test the specific role of Sulf1 during prostate development, Sulf1 was ectopically expressed in the urogenital sinus. It partially inhibited testosterone-stimulated ductal morphogenesis, and it reduced the activation of fibroblast growth factor receptors as well as the ERK1 and ERK2 MAPKs. These data identify sulfatase 1 as an inhibitor of prostatic branching morphogenesis and growth factor signaling that is down-regulated as part of the normal response to androgen action in the male urogenital sinus.

    Funded by: NCI NIH HHS: CA91290, U01 CA091290; NIA NIH HHS: AG024278, R01 AG024278; NIDDK NIH HHS: R01 DK091193; NIGMS NIH HHS: GM054832, R01 GM054832; PHS HHS: CAO0138

    Endocrinology 2010;151;7;3420-31

  • A genomic atlas of mouse hypothalamic development.

    Shimogori T, Lee DA, Miranda-Angulo A, Yang Y, Wang H, Jiang L, Yoshida AC, Kataoka A, Mashiko H, Avetisyan M, Qi L, Qian J and Blackshaw S

    RIKEN-BSI, 2-1 Hirosawa, Wako-shi, Saitama, Japan. tshimogori@brain.riken.jp

    The hypothalamus is a central regulator of many behaviors that are essential for survival, such as temperature regulation, food intake and circadian rhythms. However, the molecular pathways that mediate hypothalamic development are largely unknown. To identify genes expressed in developing mouse hypothalamus, we performed microarray analysis at 12 different developmental time points. We then conducted developmental in situ hybridization for 1,045 genes that were dynamically expressed over the course of hypothalamic neurogenesis. We identified markers that stably labeled each major hypothalamic nucleus over the entire course of neurogenesis and constructed a detailed molecular atlas of the developing hypothalamus. As a proof of concept of the utility of these data, we used these markers to analyze the phenotype of mice in which Sonic Hedgehog (Shh) was selectively deleted from hypothalamic neuroepithelium and found that Shh is essential for anterior hypothalamic patterning. Our results serve as a resource for functional investigations of hypothalamic development, connectivity, physiology and dysfunction.

    Funded by: NINDS NIH HHS: R21 NS067393

    Nature neuroscience 2010;13;6;767-75

  • Lack of cadherins Celsr2 and Celsr3 impairs ependymal ciliogenesis, leading to fatal hydrocephalus.

    Tissir F, Qu Y, Montcouquiol M, Zhou L, Komatsu K, Shi D, Fujimori T, Labeau J, Tyteca D, Courtoy P, Poumay Y, Uemura T and Goffinet AM

    Université catholique de Louvain, Institute of Neuroscience, Developmental Neurobiology, Brussels, Belgium.

    Ependymal cells form the epithelial lining of cerebral ventricles. Their apical surface is covered by cilia that beat in a coordinated fashion to facilitate circulation of the cerebrospinal fluid (CSF). The genetic factors that govern the development and function of ependymal cilia remain poorly understood. We found that the planar cell polarity cadherins Celsr2 and Celsr3 control these processes. In Celsr2-deficient mice, the development and planar organization of ependymal cilia are compromised, leading to defective CSF dynamics and hydrocephalus. In Celsr2 and Celsr3 double mutant ependyma, ciliogenesis is markedly impaired, resulting in lethal hydrocephalus. The membrane distribution of Vangl2 and Fzd3, two key planar cell polarity proteins, was disturbed in Celsr2 mutants, and even more so in Celsr2 and Celsr3 double mutants. Our findings suggest that planar cell polarity signaling is involved in ependymal cilia development and in the pathophysiology of hydrocephalus, with possible implications in other ciliopathies.

    Nature neuroscience 2010;13;6;700-7

  • Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation.

    Lund N, Henrion D, Tiede P, Ziche M, Schunkert H and Ito WD

    Medical Department II, University Hospital Lübeck, Lübeck, Germany.

    The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p(Ser239)-VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.

    Biochemical and biophysical research communications 2010;395;3;401-6

  • Nervous-tissue-specific elimination of microtubule-actin crosslinking factor 1a results in multiple developmental defects in the mouse brain.

    Goryunov D, He CZ, Lin CS, Leung CL and Liem RK

    Department of Pathology and Cell Biology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University College of Physicians and Surgeons, NY, NY 10032, USA.

    The microtubule-actin crosslinking factor 1 (MACF1) is a ubiquitous cytoskeletal linker protein with multiple spliced isoforms expressed in different tissues. The MACF1a isoform contains microtubule and actin-binding regions and is expressed at high levels in the nervous system. Macf1-/- mice are early embryonic lethal and hence the role of MACF1 in the nervous system could not be determined. We have specifically knocked out MACF1a in the developing mouse nervous system using Cre/loxP technology. Mutant mice died within 24-36h after birth of apparent respiratory distress. Their brains displayed a disorganized cerebral cortex with a mixed layer structure, heterotopia in the pyramidal layer of the hippocampus, disorganized thalamocortical and corticofugal fibers, and aplastic anterior and hippocampal commissures. Embryonic neurons showed a defect in traversing the cortical plate. Our data suggest a critical role for MACF1 in neuronal migration that is dependent on its ability to interact with both microfilaments and microtubules.

    Funded by: NINDS NIH HHS: NS40784, NS47711, R01 NS040784, R01 NS040784-04, R01 NS047711, R01 NS047711-04

    Molecular and cellular neurosciences 2010;44;1;1-14

  • Attenuation of reactive gliosis does not affect infarct volume in neonatal hypoxic-ischemic brain injury in mice.

    Järlestedt K, Rousset CI, Faiz M, Wilhelmsson U, Ståhlberg A, Sourkova H, Pekna M, Mallard C, Hagberg H and Pekny M

    Perinatal Center, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

    Background: Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP(-/-)Vim(-/-)) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP(-/-)Vim(-/-) mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown.

    We subjected GFAP(-/-)Vim(-/-) and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP(-/-)Vim(-/-) and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN(+) neurons in the ischemic and contralateral hemisphere was comparable between GFAP(-/-)Vim(-/-) and wild-type mice. In wild-type mice, the number of S100(+) astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0+/-50.1 vs. 85.6+/-34.0, p<0.05). In the GFAP(-/-)Vim(-/-) mice, the number of S100(+) astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP(-/-)Vim(-/-) mice showed an increase in NeuN(+)BrdU(+) (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7+/-7.7; n = 29 versus 2.9+/-3.6; n = 28, respectively, p<0.05), but a comparable number of S100(+)BrdU(+) (surviving newly born) astrocytes.

    Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.

    Funded by: Medical Research Council: G0802853

    PloS one 2010;5;4;e10397

  • Vimentin is a functional partner of hormone sensitive lipase and facilitates lipolysis.

    Shen WJ, Patel S, Eriksson JE and Kraemer FB

    Division of Endocrinology, Stanford University and VA Palo Alto Health Care System, Palo Alto, California 94304, USA.

    Lipolysis involves a number of components including signaling pathways, droplet-associated proteins, and lipases such as hormone-sensitive lipase (HSL). We used surface enhanced laser desorption/ionization time-of-flight mass spectroscopy to identify cellular proteins that might interact with HSL and potentially influence lipolysis. Using recombinant HSL as bait on protein chips, clusters of proteins of 14.7-18.9, 25.8-26.8, 36.1, 44.3-49.1, and 53.7 kDa were identified that interact with HSL, particularly when lysates were examined from beta-agonist treated mouse adipocytes. The ability to detect these interacting proteins was markedly diminished when the adipocytes were treated with insulin. A very similar pattern of proteins was identified when anti-HSL IgG was used as the bait. Following immunocapture, the identification of the prominent 53.7 kDa protein was carried out by tryptic digestion and MS analysis and determined to be vimentin. The interaction of HSL with vimentin, and its hormonal dependence, was confirmed by coimmunoprecipitation. beta-Agonist stimulated lipolysis and the rate of HSL translocation were impaired in vimentin null adipocytes, even though normal amounts of lipases and droplet-associated proteins are expressed. The current studies provide evidence that vimentin participates in lipolysis through direct, hormonally regulated interactions with HSL.

    Funded by: NIA NIH HHS: AG-028908, R01 AG028908-05; NIDDK NIH HHS: R01 DK046942-09

    Journal of proteome research 2010;9;4;1786-94

  • Gene expression profiling of the developing mouse kidney and embryo.

    Shaw L, Johnson PA and Kimber SJ

    Faculty of Life Sciences, The University of Manchester, Core Technology Facility, 46 Grafton St, Manchester, M13 9NT, UK. lisa.shaw@manchester.ac.uk

    The metanephros is formed from the reciprocal inductive interaction of two precursor tissues, the metanephric mesenchyme (MM) and the ureteric bud (UB). The UB induces MM to condense and differentiate forming the glomerulus and renal tubules, whilst the MM induces the UB to differentiate into the collecting tubules of the mature nephron. Uninduced MM is considered the progenitor cell population of the developing metanephros because of its potential to differentiate into more renal cell types than the UB. Previous studies have identified the phenotype of renal precursor cells; however, expression of candidate marker genes have not been analysed in other tissues of the murine embryo. We have assayed up to 19 candidate genes in eight embryonic tissues at five gestation stages of the mouse embryo to identify markers definitively expressed by renal cells during metanephric induction and markers developmentally regulated during kidney maturation. We then analysed their expression in other developing tissues. Results show Dcn, Hoxc9, Mest, Wt1 and Ywhaq were expressed at moderate to high levels during the window of metanephric specification and early differentiation (E10.5-E12.5 dpc), and Hoxc9, Ren1 and Wt1 expression was characteristic of mature renal cells. We demonstrated Cd24a, Cdh11, Mest, Scd2 and Sim2 were regulated during brain development, and Scd2, Cd24a and Sip1 expression was enriched in developing liver. These markers may be useful negative markers of kidney development. Use of a combination of highly expressed and negative markers may aid in the identification and removal of non-renal cells from heterogeneous populations of differentiating stem cells.

    Funded by: Biotechnology and Biological Sciences Research Council

    In vitro cellular & developmental biology. Animal 2010;46;2;155-65

  • Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin.

    Martínez-Estrada OM, Lettice LA, Essafi A, Guadix JA, Slight J, Velecela V, Hall E, Reichmann J, Devenney PS, Hohenstein P, Hosen N, Hill RE, Muñoz-Chapuli R and Hastie ND

    MRC Human Genetics Unit and Institute for Genetics and Molecular Medicine, Edinburgh, UK.

    The epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of the gene encoding Wilms' tumor-1 (Wt1) leads to a reduction in mesenchymal progenitor cells and their derivatives. We show that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during embryonic stem cell differentiation through direct transcriptional regulation of the genes encoding Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages do not form in Wt1-null embryoid bodies, but this effect is rescued by the expression of Snai1, underscoring the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell-based therapies.

    Funded by: Medical Research Council: U.1275.03.013.00001.01

    Nature genetics 2010;42;1;89-93

  • Vimentin and GFAP responses in astrocytes after contusion trauma to the murine brain.

    Ekmark-Lewén S, Lewén A, Israelsson C, Li GL, Farooque M, Olsson Y, Ebendal T and Hillered L

    Department of Neuroscience, Neurosurgery, Uppsala University, Uppsala University Hospital, Uppsala, Sweden.

    Purpose: Astroglial responses after traumatic brain injury are difficult to detect with routine morphological methods. The aims for this study were to compare the temporal and spatial expression pattern of vimentin- and glial fibrillary acidic protein (GFAP) in a weight drop model of mild cerebral contusion injury in the rat. We also wanted to study the vimentin response with immunohistochemistry and vimentin mRNA RT-PCR analysis in severe cortical contusion injury produced by the controlled cortical impact in the mouse.

    Methods: Vimentin and GFAP immunohistochemistry (1 day, 3 days and 7 days) combined with vimentin mRNA RT-PCR analysis (1 h, 4 h, 22 h, 3 days and 7 days) were used after experimental traumatic brain injury in the rat and mouse.

    Results: Increases in post-traumatic vimentin mRNA levels in the cortex and in the hippocampus appeared together with vimentin immunoreactivity in astrocytes in the perimeter of the cortical lesion, in the subcortical white matter and in the hippocampus starting at one day after severe trauma. GFAP immunostaining revealed hypertrophic astrocytes peaking at day 3 in the perifocal cortical region. There was no significant increase in GFAP immunoreactivity in the white matter in the rat. However, in the mouse there was a slight increase in the number of GFAP positive cells in this region, 3 days after trauma. Overall the pattern of vimentin immunoreactivity was very similar in the rat and mouse.

    Conclusions: Vimentin immunoreactivity was more sensitive than the GFAP staining method to demonstrate the distribution and time course of astrocyte reactions after a contusion injury, especially in the white matter distant from the cortical lesion.

    Restorative neurology and neuroscience 2010;28;3;311-21

  • Expression of Pou3f3/Brn-1 and its genomic methylation in developing auditory epithelium.

    Mutai H, Nagashima R, Sugitani Y, Noda T, Fujii M and Matsunaga T

    Laboratory of Auditory Disorders, National Institute of Sensory Organs, National Tokyo Medical Center, Tokyo, Japan.

    In the mammalian cochlea, both the sensory cells-called hair cells (HCs)-and nonsensory cells such as supporting cells (SCs) and mesenchymal cells participate in proper auditory function through the expression of various functional molecules. During development, expression of certain genes is repressed through genomic methylation, one of the major epigenetic regulatory mechanisms. We explored the genomic regions that were differentially methylated in rat auditory epithelium at postnatal day 1 (P1) and P14 using amplification of intermethylated sites (AIMS). An AIMS fragment was mapped to the 3'-flanking region of Pou3f3/Brn-1. Bisulfite-converted PCR and quantitative methylation-specific PCR showed that the methylation frequency of the AIMS region and the adjacent CpG island was increased at P14, when the expression of Pou3f3 and the noncoding RNAs nearby decreased. Expression of de novo DNA methyltransferases 3a and 3b also suggests a role of epigenetic regulation during postnatal inner ear development. Immunohistochemical analysis showed that Pou3f3 was expressed specifically in the SCs and mesenchymal cells in the cochlea and established that Pou3f3 is a new cell-type marker for studying inner ear development. Mice deficient in Pou3f3 or Pou3f2 plus Pou3f3 did not exhibit any abnormality in the embryonic cochlea. Absence of Pou3f3 affected neither the proliferation nor the differentiation activities of HC progenitor cells. Pou3f3 may, however, be important for the maintenance or functional development of the postnatal cochlea. This is the first report to study involvement of an epigenetic regulatory mechanism in the developing mammalian auditory epithelium.

    Developmental neurobiology 2009;69;14;913-30

  • Neuronal expression of vimentin in the Alzheimer's disease brain may be part of a generalized dendritic damage-response mechanism.

    Levin EC, Acharya NK, Sedeyn JC, Venkataraman V, D'Andrea MR, Wang HY and Nagele RG

    University of Medicine and Dentistry of New Jersey/Graduate School of Biomedical Sciences, 2 Medical Center Drive, Stratford, NJ 08084, USA.

    Early pathological features of Alzheimer's disease (AD) include synaptic loss and dendrite retraction, prior to neuronal loss. How neurons respond to this evolving AD pathology remains elusive. In the present study, we used single- and double-label immunohistochemistry to investigate the relationship between neuronal vimentin expression and local brain pathology. Vimentin was localized to neuronal perikarya and dendrites in AD brain, with vimentin-immunopositive neurons prevalent in regions exhibiting intra- and extracellular beta-amyloid(1-42) (Abeta42) deposition. Neuronal co-localization of vimentin and Abeta42 was common in the cerebral cortex, cerebellum and hippocampus. Additionally, neurons in affected brain regions of AD transgenic (Tg2576) mice and in brain tissue subjected to mechanical injury expressed vimentin, while those in comparable regions of control mouse brain did not. Finally, we show that neurons in human fetal brain express vimentin concurrently with periods of rapid neurite extension. Overall, our results suggest that neurons express vimentin as part of an evolutionarily conserved, damage-response mechanism which recapitulates a developmental program used by differentiating neurons to establish dendrites and synaptic connections.

    Brain research 2009;1298;194-207

  • Conditional deletion of beta1-integrin in astroglia causes partial reactive gliosis.

    Robel S, Mori T, Zoubaa S, Schlegel J, Sirko S, Faissner A, Goebbels S, Dimou L and Götz M

    Department of Physiological Genomics, Institute of Physiology, Ludwig-Maximilians University Munich, Munich, Germany.

    Astrocytes play many pivotal roles in the adult brain, including their reaction to injury. A hallmark of astrocytes is the contact of their endfeet with the basement membrane surrounding blood vessels, but still relatively little is known about the signaling mediated at the contact site. Here, we examine the role of beta1-integrin at this interface by its conditional deletion using different Cre lines. Thereby, the protein was reduced only at postnatal stages either in both glia and neurons or specifically only in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and up-regulation of the intermediate filaments GFAP and vimentin as well as pericellular components, such as Tenascin-C and the DSD-1 proteoglycan. In addition, we also observed to a certain degree a non-cell autonomous activation of microglial cells after conditional beta1-integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of beta1-integrin-mediated signaling is not sufficient to elicit proliferation of these cells as observed after brain injury. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood brain barrier disturbances. As these effects did not appear after neuron-specific deletion of beta1-integrin, we conclude that beta1-integrin-mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in beta1-integrin-mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury.

    Glia 2009;57;15;1630-47

  • Lack of aspartoacylase activity disrupts survival and differentiation of neural progenitors and oligodendrocytes in a mouse model of Canavan disease.

    Kumar S, Biancotti JC, Matalon R and de Vellis J

    Intellectual and Developmental Disabilities Research Center, Semel Institute for Neuroscience, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

    Loss of the oligodendrocyte (OL)-specific enzyme aspartoacylase (ASPA) from gene mutation results in the sponginess and loss of white matter (WM) in Canavan disease (CD). This study addresses the fate of OLs during the pathophysiology of CD in an adult ASPA knockout (KO) mouse strain. Massive arrays of neural stem/progenitor cells, immunopositive for PSA-NCAM, nestin, vimentin, and NG2, were observed within the severely affected spongy WM of the KO mouse brain. In these mice, G1-->S cell cycle progression was confirmed by an increase in cdk2-kinase activity, a reduction in mitotic inhibitors p21(Cip1) and p27(Kip1), and an increase in bromodeoxyuridine (BrdU) incorporation. Highly acetylated nuclear histones H2B and H3 were detected in adult KO mouse WM, suggesting the existence of noncompact chromatin as seen during early development. Costaining for BrdU- or Ki67-positive cells with markers for neural progenitors confirmed a continuous generation of OL lineage cells in KO WM. We observed a severe reduction in 21.5- and 18.5-kDa myelin basic protein and PLP/DM20 proteolipid proteins combined with a decrease in myelinated fibers and a perinuclear retention of myelin protein staining, indicating impairment in protein trafficking. Death of OLs, neurons, and astrocytes was identified in every region of the KO brain. Immature OLs constituted the largest population of dying cells, particularly in WM. We also report an early expression of full-length ASPA mRNA in normal mouse brain at embryonic day 12.5, when OL progenitors first appear during development. These findings support involvement of ASPA in CNS development and function.

    Funded by: NICHD NIH HHS: HD-06576, HD04612

    Journal of neuroscience research 2009;87;15;3415-27

  • Sip1 regulates sequential fate decisions by feedback signaling from postmitotic neurons to progenitors.

    Seuntjens E, Nityanandam A, Miquelajauregui A, Debruyn J, Stryjewska A, Goebbels S, Nave KA, Huylebroeck D and Tarabykin V

    Laboratory of Molecular Biology (Celgen), Centre for Human Genetics, KULeuven, Leuven, Belgium.

    The fate of cortical progenitors, which progressively generate neurons and glial cells during development, is determined by temporally and spatially regulated signaling mechanisms. We found that the transcription factor Sip1 (Zfhx1b), which is produced at high levels in postmitotic neocortical neurons, regulates progenitor fate non-cell autonomously. Conditional deletion of Sip1 in young neurons induced premature production of upper-layer neurons at the expense of deep layers, precocious and increased generation of glial precursors, and enhanced postnatal astrocytogenesis. The premature upper-layer generation coincided with overexpression of the neurotrophin-3 (Ntf3) gene and upregulation of fibroblast growth factor 9 (Fgf9) gene expression preceded precocious gliogenesis. Exogenous application of Fgf9 to mouse cortical slices induced excessive generation of glial precursors in the germinal zone. Our data suggest that Sip1 restrains the production of signaling factors in postmitotic neurons that feed back to progenitors to regulate the timing of cell fate switch and the number of neurons and glial cells throughout corticogenesis.

    Nature neuroscience 2009;12;11;1373-80

  • Trps1 functions downstream of Bmp7 in kidney development.

    Gai Z, Zhou G, Itoh S, Morimoto Y, Tanishima H, Hatamura I, Uetani K, Ito M and Muragaki Y

    First Department of Pathology, Wakayama Medical University Medical School, 811-1 Kimiidera, Wakayama 641-0012, Japan.

    During embryonic development, the mesenchyme of the lungs, gut, kidneys, and other tissues expresses Trps1, an atypical member of the GATA-type family of transcription factors. Our previous work suggested the possibility that Trps1 acts downstream of bone morphogenic protein 7 (Bmp7), which is essential for normal renal development. To examine the role of Trps1 during early renal development, we generated Trps1-deficient mice and examined their renal histology. Compared with wild-type mice, Trps1-deficient newborn mice had fewer tubules and glomeruli, an expanded renal interstitium, and numerous uninduced metanephric mesenchymal cells, which resulted in fewer nephrons. In wild-type kidneys, Trps1 expression was present in ureteric buds, cap mesenchyme, and renal vesicles, whereas Trps1 was virtually absent in Bmp7-deficient kidneys. Furthermore, Trps1-deficient kidneys had low levels of Pax2 and Wt1, which are markers of condensed mesenchymal cells, suggesting that a lack of Trps1 affects the differentiation of cap mesenchyme to renal vesicles. In cultured metanephric mesenchymal cells, Bmp7 induced Trps1 and E-cadherin and downregulated vimentin. Knockdown of Trps1 with small interference RNA inhibited this Bmp7-induced mesenchymal-to-epithelial transition. Last, whole-mount in situ hybridization of Wnt9b and Wnt4 demonstrated prolonged branching of ureteric buds and sparse cap mesenchyme in the kidneys of Trps1-deficient mice. Taken together, these findings suggest that normal formation of nephrons requires Trps1, which mediates mesenchymal-to-epithelial transition and ureteric bud branching during early renal development.

    Journal of the American Society of Nephrology : JASN 2009;20;11;2403-11

  • Vimentin intermediate filaments as a template for silica nanotube preparation.

    Gohara R, Liu D, Nakashima K, Takasaki Y and Ando S

    Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Nabeshima, Saga 849-8501, Japan.

    Organic compounds are used as templates to regulate the morphology of inorganic nanostructures. In the present study, we used intermediate filaments (IFs), the major cytoskeleton component of most eukaryotic cells, as a template for hollow silica nanotube preparation. Sol-gel polymerization of tetraethoxysilane proceeded preferentially on the surface of IFs assembled from vimentin protein in vitro, resulting in silica-coated fibres. After removing IFs by calcination, electron microscopy revealed hollow silica nanotubes several micrometers long, with outer diameters of 35-55 nm and an average inner diameter of 10 nm (comparable to that of IFs). Furthermore, the silica nanotubes exhibited a gnarled surface structure with an 18-26 nm repeating pattern (comparable to the 21-nm beading pattern along IFs). Thus, the characteristic morphology of IFs were well replicated into hollow silica nanotubes, suggesting that IFs maybe useful as an organic template.

    Journal of biochemistry 2009;146;5;627-31

  • Vimentin inhibits ATF4-mediated osteocalcin transcription and osteoblast differentiation.

    Lian N, Wang W, Li L, Elefteriou F and Yang X

    Department of Medicine, Vanderbilt University Center for Bone Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

    Activating transcription factor 4 (ATF4) is an osteoblast-enriched transcription factor that regulates osteocalcin transcription and osteoblast terminal differentiation. To identify functional partners of ATF4, we applied ROS17/2.8 osteoblast nuclear extracts and purified recombinant His-ATF4 onto a Ni(+) affinity matrix chromatography column. Vimentin was identified by liquid chromatography-mass spectrometry. Coimmunoprecipitation and pulldown assays revealed that vimentin interacted with ATF4 with its first leucine zipper domain. DNA cotransfection and gel retardation demonstrated that vimentin inhibited the transactivation activity of ATF4 on osteocalcin by preventing it to bind OSE1, the ATF4 binding site on the osteocalcin promoter. Northern hybridization revealed that vimentin was expressed at a high level in immature osteoblasts and a low level in fully differentiated osteoblasts. Down-regulation of vimentin by small interfering RNA induced endogenous osteocalcin transcription in immature osteoblasts. Conversely, ectopic overexpression of vimentin in osteoblasts inhibited osteoblast differentiation as shown by lower alkaline phosphatase activity, delayed mineralization, and decreased expression of osteoblast marker genes such as bone sialoprotein and osteocalcin. Together, our data uncover a novel mechanism whereby a cytoskeletal protein, vimentin, acts as a break on differentiation in immature osteoblasts by interacting with ATF4.

    Funded by: NIDCR NIH HHS: R03 DE017111

    The Journal of biological chemistry 2009;284;44;30518-25

  • Keratins regulate protein biosynthesis through localization of GLUT1 and -3 upstream of AMP kinase and Raptor.

    Vijayaraj P, Kröger C, Reuter U, Windoffer R, Leube RE and Magin TM

    Abteilung für Zellbiochemie, Institut für Biochemie und Molekularbiologie and 2 Bonner Forum Biomedizin, Universität Bonn, 53115 Bonn, Germany.

    Keratin intermediate filament proteins form cytoskeletal scaffolds in epithelia, the disruption of which affects cytoarchitecture, cell growth, survival, and organelle transport. However, owing to redundancy, the global function of keratins has not been defined in full. Using a targeted gene deletion strategy, we generated transgenic mice lacking the entire keratin multiprotein family. In this study, we report that without keratins, embryonic epithelia suffer no cytolysis and maintain apical polarity but display mislocalized desmosomes. All keratin-null embryos die from severe growth retardation at embryonic day 9.5. We find that GLUT1 and -3 are mislocalized from the apical plasma membrane in embryonic epithelia, which subsequently activates the energy sensor adenosine monophosphate kinase (AMPK). Analysis of the mammalian target of rapamycin (mTOR) pathway reveals that AMPK induction activates Raptor, repressing protein biosynthesis through mTORC1's downstream targets S6 kinase and 4E-binding protein 1. Our findings demonstrate a novel keratin function upstream of mTOR signaling via GLUT localization and have implications for pathomechanisms and therapy approaches for keratin disorders and the analysis of other gene families.

    The Journal of cell biology 2009;187;2;175-84

  • Liver X receptors and oxysterols promote ventral midbrain neurogenesis in vivo and in human embryonic stem cells.

    Sacchetti P, Sousa KM, Hall AC, Liste I, Steffensen KR, Theofilopoulos S, Parish CL, Hazenberg C, Richter LA, Hovatta O, Gustafsson JA and Arenas E

    Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Center for Developmental Biology and Regenerative Medicine, Karolinska Institute, Stockholm, Sweden.

    Control over progenitor proliferation and neurogenesis remains a key challenge for stem cell neurobiology and a prerequisite for successful stem cell replacement therapies for neurodegenerative diseases like Parkinson's disease (PD). Here, we examined the function of two nuclear receptors, liver X receptors (Lxralpha and beta) and their ligands, oxysterols, as regulators of cell division, ventral midbrain (VM) neurogenesis, and dopaminergic (DA) neuron development. Deletion of Lxrs reduced cell cycle progression and VM neurogenesis, resulting in decreased DA neurons at birth. Activation of Lxrs with oxysterol ligands increased the number of DA neurons in mouse embryonic stem cells (ESCs) and in wild-type but not Lxralphabeta(-/-) VM progenitor cultures. Likewise, oxysterol treatment of human ESCs (hESCs) during DA differentiation increased neurogenesis and the number of mature DA neurons, while reducing proliferating progenitors. Thus, Lxr ligands may improve current hESC replacement strategies for PD by selectively augmenting the generation of DA neurons.

    Cell stem cell 2009;5;4;409-19

  • Stabilized beta-catenin in lung epithelial cells changes cell fate and leads to tracheal and bronchial polyposis.

    Li C, Li A, Li M, Xing Y, Chen H, Hu L, Tiozzo C, Anderson S, Taketo MM and Minoo P

    Department of Pediatrics, Women's and Children's Hospital, USC Keck School of Medicine, Los Angeles, CA 90033, USA. changgon@usc.edu

    The precise mechanisms by which beta-catenin controls morphogenesis and cell differentiation remain largely unknown. Using embryonic lung development as a model, we deleted exon 3 of beta-catenin via Nkx2.1-cre in the Catnb[+/lox(ex3)] mice and studied its impact on epithelial morphogenesis. Robust selective accumulation of truncated, stabilized beta-catenin was found in Nkx2.1-cre;Catnb[+/lox(ex3)] lungs that were associated with the formation of polyp-like structures in the trachea and main-stem bronchi. Characterization of polyps suggests that accumulated beta-catenin impacts epithelial morphogenesis in at least two ways. "Intracellular" accumulation of beta-catenin blocked differentiation of spatially-appropriate airway epithelial cell types, Clara cells, ciliated cells and basal cells, and activated UCHL1, a marker for pulmonary neuroendocrine cells. There was also evidence for a "paracrine" impact of beta-catenin accumulation, potentially mediated via activation of Bmp4 that inhibited Clara and ciliated, but not basal cell differentiation. Thus, excess beta-catenin can alter cell fate determination by both direct and paracrine mechanisms.

    Funded by: NHLBI NIH HHS: HL075334, HL56590, HL60231, P01 HL060231, R01 HL056590, R01 HL075334, R01 HL075334-05

    Developmental biology 2009;334;1;97-108

  • Dynein is a motor for nuclear rotation while vimentin IFs is a "brake".

    Gerashchenko MV, Chernoivanenko IS, Moldaver MV and Minin AA

    Group of Cell Biology, Institute of Protein Research, Russian Academy of Sciences, Moscow 119334, Russian Federation.

    The positioning of the nucleus is achieved by two interconnected processes, anchoring and migration, both of which are controlled by cytoskeleton structures. Rotation is a special type of nuclear motility in many cell types, but its significance remains unclear. We used a vimentin-null cell line, MFT-16, which shows extensive nuclear rotation to study the phenomenon in detail. By selective disruption of cytoskeletal structures and video-microscopic analysis, nuclear rotation was a microtubule-dependent process that F-actin partially impedes. The dynein-dynactin complex is responsible and inhibiting this motor by expression of a dominant negative mutant of its component P-150 completely stops it. Nuclear rotation is powered by dynein associated with the nuclear envelope along stationary microtubules, centrosomes remaining immobile. We confirmed that vimentin IFs inhibit nuclear rotation, and variant proteins of the mutated wild type gene for vimentin that lacked considerable fragments of the N- and C-terminal domains restored nuclear anchoring. Immunochemical analysis showed that these mutated IFs also bound plectin, arguing for a key role of this cytolinker protein in nuclear anchoring. It is proposed that this versatile machinery guarantees not only rotation and the correct location of a nucleus, but also its orientation in a cell.

    Cell biology international 2009;33;10;1057-64

  • Constitutive activation of Beta-catenin in uterine stroma and smooth muscle leads to the development of mesenchymal tumors in mice.

    Tanwar PS, Lee HJ, Zhang L, Zukerberg LR, Taketo MM, Rueda BR and Teixeira JM

    Vincent Center for Reproductive Biology, Vincent Obstetrics and Gynecology Services, and Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.

    Leiomyomas and other mesenchymally derived tumors are the most common neoplasms of the female reproductive tract. Presently, very little is known about the etiology and progression of these tumors, which are the primary indication for hysterectomies. Dysregulated WNT signaling through beta-catenin is a well-established mechanism for tumorigenesis. We have developed a mouse model that expresses constitutively activated beta-catenin in uterine mesenchyme driven by the expression of Cre recombinase knocked into the Müllerian-inhibiting substance type II receptor promoter locus to investigate its effects on uterine endometrial stroma and myometrium. These mice show myometrial hyperplasia and develop mesenchymal tumors with 100% penetrance that exhibit histological and molecular characteristics of human leiomyomas and endometrial stromal sarcomas. By immunohistochemistry, we also show that both transforming growth factor beta and the mammalian target of rapamycin are induced by constitutive activation of beta-catenin. The prevalence of the tumors was greater in multiparous mice, suggesting that their development may be a hormonally driven process or that changes in uterine morphology during pregnancy and after parturition induce injury and repair mechanisms that stimulate tumorigenesis from stem/progenitor cells, which normally do not express constitutively activated beta-catenin. Additionally, adenomyosis and endometrial gland hyperplasia were occasionally observed in some mice. These results show evidence suggesting that dysregulated, stromal, and myometrial WNT/beta-catenin signaling has pleiotropic effects on uterine function and tumorigenesis.

    Funded by: NICHD NIH HHS: HD052701, R01 HD052701, R01 HD052701-02

    Biology of reproduction 2009;81;3;545-52

  • Transcriptional coactivator with PDZ-binding motif is essential for normal alveolarization in mice.

    Mitani A, Nagase T, Fukuchi K, Aburatani H, Makita R and Kurihara H

    Department of Respiratory Medicine, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.

    Rationale: Transcriptional coactivator with PDZ-binding motif (TAZ) is assumed to act as a coactivator of several transcription factors including smad2/3. In the lung, surfactant protein C (Sftpc) is known to be a downstream target of thyroid transcription factor-1 (TTF-1)-TAZ transcriptional coactivation.

    Objectives: The lung phenotype of Taz-deficient mice was explored.

    Methods: Taz-deficient mice were analyzed pathologically and physiologically. Next, we performed microarray analysis to determine the genes closely related to abnormal lung development. Finally, Taz-heterozygous mice were injected with bleomycin.

    Taz-deficient homozygotes showed abnormal alveolarization during lung development, which caused in adult mice airspace enlargement mimicking emphysema. There was no significant difference in the expression of Sftpc between wild-type and Taz-deficient lungs. Instead, microarray analysis identified some candidate downstream genes related to the pathogenesis, including the connective tissue growth factor (Ctgf) gene, which is required for normal lung development. In vitro studies showed that TAZ up-regulated Ctgf expression not only by reinforcing transforming growth factor-beta/smad signals, but also by interfering in the more proximal Ctgf promoter region (from bp -123 to -76), defined as the TAZ response element. Furthermore, Taz-heterozygous mice were resistant to bleomycin-induced lung fibrosis.

    Conclusions: The results indicate the importance of TAZ in lung alveolarization and its involvement in the pathogenesis of lung fibrosis.

    American journal of respiratory and critical care medicine 2009;180;4;326-38

  • The genesis of cerebellar interneurons and the prevention of neural DNA damage require XRCC1.

    Lee Y, Katyal S, Li Y, El-Khamisy SF, Russell HR, Caldecott KW and McKinnon PJ

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

    Defective responses to DNA single strand breaks underlie various neurodegenerative diseases. However, the exact role of this repair pathway during the development and maintenance of the nervous system is unclear. Using murine neural-specific inactivation of Xrcc1, a factor that is critical for the repair of DNA single strand breaks, we found a profound neuropathology that is characterized by the loss of cerebellar interneurons. This cell loss was linked to p53-dependent cell cycle arrest and occurred as interneuron progenitors commenced differentiation. Loss of Xrcc1 also led to the persistence of DNA strand breaks throughout the nervous system and abnormal hippocampal function. Collectively, these data detail the in vivo link between DNA single strand break repair and neurogenesis and highlight the diverse consequences of specific types of genotoxic stress in the nervous system.

    Funded by: Medical Research Council: G0400959, G0600776; NCI NIH HHS: CA-21765, P30 CA021765, P30 CA21765; NINDS NIH HHS: NS-37956, R01 NS037956, R01 NS037956-09, R56 NS037956; Wellcome Trust: 085284

    Nature neuroscience 2009;12;8;973-80

  • CLRN1 is nonessential in the mouse retina but is required for cochlear hair cell development.

    Geller SF, Guerin KI, Visel M, Pham A, Lee ES, Dror AA, Avraham KB, Hayashi T, Ray CA, Reh TA, Bermingham-McDonogh O, Triffo WJ, Bao S, Isosomppi J, Västinsalo H, Sankila EM and Flannery JG

    Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA. drgeller@gmail.com

    Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5-6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT-PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT-PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.

    PLoS genetics 2009;5;8;e1000607

  • Mammalian Par3 regulates progenitor cell asymmetric division via notch signaling in the developing neocortex.

    Bultje RS, Castaneda-Castellanos DR, Jan LY, Jan YN, Kriegstein AR and Shi SH

    Developmental Biology Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

    Asymmetric cell division of radial glial progenitors produces neurons while allowing self-renewal; however, little is known about the mechanism that generates asymmetry in daughter cell fate specification. Here, we found that mammalian partition defective protein 3 (mPar3), a key cell polarity determinant, exhibits dynamic distribution in radial glial progenitors. While it is enriched at the lateral membrane domain in the ventricular endfeet during interphase, mPar3 becomes dispersed and shows asymmetric localization as cell cycle progresses. Either removal or ectopic expression of mPar3 prevents radial glial progenitors from dividing asymmetrically yet generates different outcomes in daughter cell fate specification. Furthermore, the expression level of mPar3 affects Notch signaling, and manipulations of Notch signaling or Numb expression suppress mPar3 regulation of radial glial cell division and daughter cell fate specification. These results reveal a critical molecular pathway underlying asymmetric cell division of radial glial progenitors in the mammalian neocortex.

    Funded by: NIDA NIH HHS: R01 DA024681, R01 DA024681-01A1, R01 DA024681-02; NIMH NIH HHS: R21 MH083624, R21 MH083624-01, R21 MH083624-02

    Neuron 2009;63;2;189-202

  • The prototype endothelial marker PAL-E is a leukocyte trafficking molecule.

    Keuschnigg J, Henttinen T, Auvinen K, Karikoski M, Salmi M and Jalkanen S

    MediCity Research Laboratory and Department of Medical Microbiology, University of Turku and National Public Health Institute, Turku, Finland.

    Pathologische Anatomie Leiden-endothelium antibody has been used for more than 20 years as a marker for vascular endothelium. Despite its widespread use, the target of this antibody was only recently identified as plasmalemma vesicle-associated protein-1 (PV-1). However, no function has been identified for this molecule. Here we report that activation of human umbilical vein endothelial cells with tumor necrosis factor-alpha resulted in a remarkable redistribution of PV-1 toward the peripheral areas of the cells. Furthermore, in vitro endpoint transmigration experiments showed that transcellularly migrating lymphocytes are surrounded by rings containing PV-1 and caveolin-1. Moreover, PV-1 associates physically with vimentin. In addition, administration of anti-PV-1 antibody during capillary flow assays resulted in a significant inhibition of lymphocyte transmigration through the endothelial cell layer, whereas rolling and adhesion were unaffected. In vivo blockage of PV-1 by an antibody in acute peritonitis and air pouch model resulted in a significant decrease in the number of migrating leukocytes. Here we thus define leukocyte transendothelial migration as the first known function for PV-1.

    Blood 2009;114;2;478-84

  • An appraisal of intermediate filament expression in adult and developing pancreas: vimentin is expressed in alpha cells of rat and mouse embryos.

    Di Bella A, Regoli M, Nicoletti C, Ermini L, Fonzi L and Bertelli E

    Department of Pharmacology Giorgio Segre, Section of Morphology, Via Aldo Moro 2, University of Siena, Siena, Italy.

    Intermediate filaments are frequently used in studies of developmental biology as markers of cell differentiation. To assess whether they can be useful to identify differentiating pancreatic endocrine cells, we examined the pattern of expression of nestin, cytokeratin 20, and vimentin on acetone-fixed cryosections of rat adult and developing pancreas. We also studied vimentin expression in mouse embryonic pancreas at E19. Cytokeratin 20 was found in all pancreatic epithelial cell lineages during the entire development of the rat gland and in the adult animals. Under our experimental conditions, therefore, cytokeratin 20 is not an exclusive marker of rat duct cells. Nestin was detected exclusively in stromal cells either in the adult or developing rat pancreas. Vimentin was observed within cells located in the primitive ducts of rat pancreas starting from E12.5. Their number rapidly increased, reaching its highest level in newborn animals. Vimentin was also spotted in alpha cells starting from E12.5 but disappeared soon after birth, likely identifying immature or recently differentiated alpha cells. In addition, vimentin was observed in duct and alpha cells of mouse developing pancreas showing that its expression in such cells is not an event restricted to the rat. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2009;57;6;577-86

  • The radial glia antibody RC2 recognizes a protein encoded by Nestin.

    Park D, Xiang AP, Zhang L, Mao FF, Walton NM, Choi SS and Lahn BT

    Howard Hughes Medical Institute, Department of Human Genetics, University of Chicago, 929E, 57th, W503B, Chicago, IL 60637, USA. donghyun@uchicago.edu

    The RC2 antibody is widely used to label mouse radial glial cells in the developing central nervous system. While the antibody is known to recognize a 295-kDa intermediate filament proximal protein, the gene encoding the RC2 antigen remains to be identified. Here, we present evidences clearly demonstrating that Nestin encodes the RC2 antigen. First, the RC2 antigen and nestin have the same molecular weight and very similar tissue distribution. Second, genetic manipulations altering nestin expression also exert the same effect on the expression of the RC2 antigen. In particular, Nestin null mutation completely abolishes the RC2 immunoreactivity. Third, the expression of a truncated mouse nestin in Nestin-/- cells produces a small RC2 antigen whose size is the same to that of the truncated nestin. Furthermore, our data suggest that the RC2 antibody recognizes the C-terminal domain of nestin with unidentified posttranslational modification(s).

    Funded by: Howard Hughes Medical Institute

    Biochemical and biophysical research communications 2009;382;3;588-92

  • Leukocyte transmigration is modulated by chemokine-mediated PI3Kgamma-dependent phosphorylation of vimentin.

    Barberis L, Pasquali C, Bertschy-Meier D, Cuccurullo A, Costa C, Ambrogio C, Vilbois F, Chiarle R, Wymann M, Altruda F, Rommel C and Hirsch E

    Department of Genetics, Biology and Biochemistry, Molecular Biotechnology Center, University of Torino, Torino, Italy.

    Phosphoinositide 3-kinase gamma (PI3Kgamma) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine-stimulated WT or PI3Kgamma-null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kgamma signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kgamma or the kinase activity of PI3Kgamma (PI3Kgamma(KD/KD)), phosphorylation of vimentin was reduced. PI3Kgamma-null macrophages displayed impaired chemokine-driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N-terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kgamma-null macrophages with a vimentin mutant mimicking serine phosphorylation of N-terminal residues rescued the transendothelial migration defect. These results define vimentin N-terminal phosphorylation and fiber reorganization as a target of chemokine-dependent PI3Kgamma signaling in leukocytes.

    European journal of immunology 2009;39;4;1136-46

  • Rac is involved in the interkinetic nuclear migration of cortical progenitor cells.

    Minobe S, Sakakibara A, Ohdachi T, Kanda R, Kimura M, Nakatani S, Tadokoro R, Ochiai W, Nishizawa Y, Mizoguchi A, Kawauchi T and Miyata T

    Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, Aichi, Japan.

    The small GTPase Rac regulates neuronal behavior, but whether it also functions in neural progenitor cells has not yet been explored. Here we report that Rac contributes to the regulation of nuclear migration in neocortical progenitor cells. Rac1 is expressed by progenitor cells in a unique spatiotemporal pattern. Cross-sectional immunohistochemical examination revealed intense Rac1 immunoreactivity at the ventricular surface. Similar staining patterns were obtained by immunofluorescence for a Rac-activator, Tiam1, and by reactions to detect the GTP-bound (active) form of Rac. En face inspection of the ventricular surface revealed that apical Rac1 localization was most frequent in M-phase cells, and the endfeet of cells in other cell cycle phases also showed apical Rac1 distribution at lower frequencies. To ask whether progenitor cell behavior prior to and during M phase is Rac-dependent, we monitored individual DiI-labeled progenitor cells live in the presence of a Rac inhibitor, NSC23766. We observed significantly retarded adventricular nuclear migration, as well as cytokinesis failures. Similar inhibitory effects were obtained by forced expression of a dominant-negative Rac1. These results suggest that Rac may play a role in interkinetic nuclear migration in the developing mouse brain.

    Neuroscience research 2009;63;4;294-301

  • Periplakin interactions with lens intermediate and beaded filaments.

    Yoon KH and FitzGerald PG

    Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California, USA.

    Purpose: The lens assembles two systems of intermediated filaments-vimentin intermediate filament (IF) and highly divergent, lens-specific beaded filament (BF)-sequentially as epithelial cells differentiate into fiber cells. The goal of this study was to identify linker proteins that integrate the different lens IF into the biology of the lens fiber cells.

    Methods: Antibodies to periplakin were used in coimmunoprecipitation studies to identify proteins that complex with BF and IF in detergent extracts of mouse lens. GST-periplakin fusion proteins were used to confirm coimmunoprecipitation

    Results: Yeast two-hybrid analysis was used to establish direct linkage between periplakin and BF/IF proteins and to narrow down binding domains. Immunocytochemistry was used to establish spatial and temporal coexpression of periplakin and BF/IF. results. Periplakin is found complexed to BF and IF in the lens. The COOH terminus of periplakin was shown to have a strong affinity for the CP49 rod 2 domain but not its head or rod 1 domains. Low-level affinity was seen between the filensin rod domain and periplakin. Periplakin localization in lens overlapped with BF and IF.

    Conclusions: Despite divergence in primary sequence, predicted secondary structure, and filament structure, CP49 has conserved the capacity to bind a common IF linker protein, periplakin, and shares that binding capacity with the other major lens IF protein, vimentin. This suggests that mutations in periplakin have the potential to emulate the cataract seen in lenses with defective BF proteins.

    Funded by: NCRR NIH HHS: C06 RR-12088-01, C06 RR012088; NEI NIH HHS: EY08747, P30 EY012576, P30 EY12576, R01 EY008747, R01 EY008747-18, R01 EY015560, R01 EY015560-04

    Investigative ophthalmology & visual science 2009;50;3;1283-9

  • Cardiac fibroblasts regulate myocardial proliferation through beta1 integrin signaling.

    Ieda M, Tsuchihashi T, Ivey KN, Ross RS, Hong TT, Shaw RM and Srivastava D

    Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, 94158, USA.

    Growth and expansion of ventricular chambers is essential during heart development and is achieved by proliferation of cardiac progenitors. Adult cardiomyocytes, by contrast, achieve growth through hypertrophy rather than hyperplasia. Although epicardial-derived signals may contribute to the proliferative process in myocytes, the factors and cell types responsible for development of the ventricular myocardial thickness are unclear. Using a coculture system, we found that embryonic cardiac fibroblasts induced proliferation of cardiomyocytes, in contrast to adult cardiac fibroblasts that promoted myocyte hypertrophy. We identified fibronectin, collagen, and heparin-binding EGF-like growth factor as embryonic cardiac fibroblast-specific signals that collaboratively promoted cardiomyocyte proliferation in a paracrine fashion. Myocardial beta1-integrin was required for this proliferative response, and ventricular cardiomyocyte-specific deletion of beta1-integrin in mice resulted in reduced myocardial proliferation and impaired ventricular compaction. These findings reveal a previously unrecognized paracrine function of embryonic cardiac fibroblasts in regulating cardiomyocyte proliferation.

    Funded by: NCRR NIH HHS: C06 RR018928; NHLBI NIH HHS: P01 HL046345, P01 HL089707, P01 HL089707-01A1, R01 HL057181, R01 HL057181-13, R01 HL057181-14, R01 HL080592, R01 HL080592-05, R01 HL080592-06, R01 HL088390, R01 HL094414

    Developmental cell 2009;16;2;233-44

  • Identification of suitable normalizing genes for quantitative real-time RT-PCR analysis of gene expression in fetal mouse gonads.

    Svingen T, Spiller CM, Kashimada K, Harley VR and Koopman P

    Division of Molecular Genetics and Development, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld., Australia.

    In biological research, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) assays are commonly employed to study mRNA abundance in cells and tissues. This type of assay usually relies on assessing transcript abundance relative to constitutively expressed endogenous reference genes. Therefore, it is important that the reference genes themselves are stably expressed in the cells or tissues analyzed, independent of factors such as age, sex, disease or experimental manipulations. Since no gene is expressed at the same level in all cells at all times, suitable reference genes must be identified for the specific cellular system or tissue being investigated. Here, we sought to identify stably expressed endogenous reference genes during embryonic gonad development in the mouse. We measured the transcript abundance of 10 frequently employed normalizing genes, of which 4 were stably expressed in fetal gonads from 11.5 to 14.5 dpc irrespective of sex. Based on our analysis, we suggest that Rn18s, Rps29, Tbp and Sdha are suitable reference genes for qRT-PCR expression studies during early gonad differentiation in the mouse.

    Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation 2009;3;4;194-204

  • Dynamic regulation of Pdx1 enhancers by Foxa1 and Foxa2 is essential for pancreas development.

    Gao N, LeLay J, Vatamaniuk MZ, Rieck S, Friedman JR and Kaestner KH

    Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

    The onset of pancreas development in the foregut endoderm is marked by activation of the homeobox gene Pdx1 (IPF1). Pdx1 is essential for the expansion of the pancreatic primordium and the development of endocrine islets. The control of Pdx1 expression has been only partially elucidated. We demonstrate here that the winged-helix transcription factors Foxa1 and Foxa2 co-occupy multiple regulatory domains in the Pdx1 gene. Compound conditional ablation of both Foxa1 and Foxa2 in the pancreatic primordium results in complete loss of Pdx1 expression and severe pancreatic hypoplasia. Mutant mice exhibit hyperglycemia with severely disrupted acinar and islet development, and die shortly after birth. Assessment of developmental markers in the mutant pancreas revealed a failure in the expansion of the pancreatic anlage, a blockage of exocrine and endocrine cell differentiation, and an arrest at the primitive duct stage. Comparing their relative developmental activity, we find that Foxa2 is the major regulator in promoting pancreas development and cell differentiation. Using chromatin immunoprecipitations (ChIP) and ChIP sequencing (ChIPSeq) of fetal pancreas and islet chromatin, we demonstrate that Foxa1 and Foxa2 predominantly occupy a distal enhancer at -6.4 kb relative to the transcriptional start site in the Pdx1 gene. In addition, occupancy of the well-characterized proximal Pdx1 enhancer by Foxa1 and Foxa2 is developmental stage-dependent. Thus, the regulation of Pdx1 expression by Foxa1 and Foxa2 is a key early event controlling the expansion and differentiation of the pancreatic primordia.

    Funded by: NIDDK NIH HHS: DK055342, P01 DK049210, P01-DK049210, P30 DK050306, P30DK50306, R01 DK055342

    Genes & development 2008;22;24;3435-48

  • Platelet-derived growth factor receptor beta signaling is required for efficient epicardial cell migration and development of two distinct coronary vascular smooth muscle cell populations.

    Mellgren AM, Smith CL, Olsen GS, Eskiocak B, Zhou B, Kazi MN, Ruiz FR, Pu WT and Tallquist MD

    Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA.

    The epicardium plays an essential role in coronary artery formation and myocardial development, but signals controlling the development and differentiation of this tissue are not well understood. To investigate the role of platelet-derived growth factor receptor (PDGFR)beta in development of epicardial-derived vascular smooth muscle cells (VSMCs), we examined PDGFRbeta(-/-) and PDGFRbeta epicardial mutant hearts. We found that PDGFRbeta(-/-) hearts failed to form dominant coronary vessels on the ventral heart surface, had a thinned myocardium, and completely lacked coronary VSMCs (cVSMCs). This constellation of defects was consistent with a primary defect in the epicardium. To verify that these defects were specific to epicardial derivatives, we generated mice with an epicardial deletion of PDGFRbeta that resulted in reduced cVSMCs distal to the aorta. The regional absence of cVSMCs suggested that cVSMCs could arise from 2 sources, epicardial and nonepicardial, and that both were dependent on PDGFRbeta. In the absence of PDGFRbeta signaling, epicardial cells adopted an irregular actin cytoskeleton, leading to aberrant migration of epicardial cells into the myocardium in vivo. In addition, PDGF receptor stimulation promoted epicardial cell migration, and PDGFRbeta-driven phosphoinositide 3'-kinase signaling was critical for this process. Our data demonstrate that PDGFRbeta is required for the formation of 2 distinct cVSMC populations and that loss of PDGFRbeta-PI3K signaling disrupts epicardial cell migration.

    Funded by: NHLBI NIH HHS: R01 HL074257, R01 HL074257-01, R01 HL074257-02, R01 HL074257-03, R01 HL074257-04, R01HL074257

    Circulation research 2008;103;12;1393-401

  • A dominant vimentin mutant upregulates Hsp70 and the activity of the ubiquitin-proteasome system, and causes posterior cataracts in transgenic mice.

    Bornheim R, Müller M, Reuter U, Herrmann H, Büssow H and Magin TM

    Institut für Biochemie and Molekularbiologie, Abteilung für Zellbiochemie und LIMES, Universität Bonn, Nussallee 11, 53115 Bonn, Germany.

    Vimentin is the main intermediate filament (IF) protein of mesenchymal cells and tissues. Unlike other IF-/- mice, vimentin-/- mice provided no evidence of an involvement of vimentin in the development of a specific disease. Therefore, we generated two transgenic mouse lines, one with a (R113C) point mutation in the IF-consensus motif in coil1A and one with the complete deletion of coil 2B of the rod domain. In epidermal keratins and desmin, point mutations in these parts of the alpha-helical rod domain cause keratinopathies and desminopathies, respectively. Here, we demonstrate that substoichiometric amounts of vimentin carrying the R113C point mutation disrupted the endogenous vimentin network in all tissues examined but caused a disease phenotype only in the eye lens, leading to a posterior cataract that was paralleled by the formation of extensive protein aggregates in lens fibre cells. Unexpectedly, central, postmitotic fibres became depleted of aggregates, indicating that they were actively removed. In line with an increase in misfolded proteins, the amounts of Hsp70 and ubiquitylated vimentin were increased, and proteasome activity was raised. We demonstrate here for the first time that the expression of mutated vimentin induces a protein-stress response that contributes to disease pathology in mice, and hypothesise that vimentin mutations cause cataracts in humans.

    Journal of cell science 2008;121;Pt 22;3737-46

  • Plectin deficiency affects precursor formation and dynamics of vimentin networks.

    Spurny R, Gregor M, Castañón MJ and Wiche G

    Department of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter, A-1030 Vienna, Austria.

    Plectin is a typical cytolinker protein that connects intermediate filaments to the other cytoskeletal filament systems and anchors them at membrane-associated junctional sites. One of the most important binding partners of plectin in fibroblasts is the intermediate filament subunit protein vimentin. Previous studies have demonstrated that vimentin networks are highly dynamic structures whose assembly and disassembly is accomplished stepwise via several intermediates. The precursor forms as well as polymerized (filamentous) vimentin are found in the cells in a dynamic equilibrium characterized by the turnover of the subunits within the polymer and the movement of the smaller precursors. To examine whether plectin plays a role in intermediate filament dynamics, we studied vimentin filament formation in plectin-deficient compared to wild-type fibroblasts using GFP-tagged vimentin. Monitoring vimentin and plectin in spreading and dividing cells, we demonstrate that plectin is associated with vimentin from the early stages of assembly and is required for vimentin motility as well as for the stepwise formation of stable filaments. Furthermore, plectin prevents vimentin networks from complete disassembly during mitosis, facilitating the rebuilding of the intermediate filament network in daughter cells.

    Funded by: Austrian Science Fund FWF: P 17862

    Experimental cell research 2008;314;19;3570-80

  • Lgr5 marks cycling, yet long-lived, hair follicle stem cells.

    Jaks V, Barker N, Kasper M, van Es JH, Snippert HJ, Clevers H and Toftgård R

    Karolinska Institutet, Department of Biosciences and Nutrition, Novum, SE-141 57 Huddinge, Sweden.

    In mouse hair follicles, a group of quiescent cells in the bulge is believed to have stem cell activity. Lgr5, a marker of intestinal stem cells, is expressed in actively cycling cells in the bulge and secondary germ of telogen hair follicles and in the lower outer root sheath of anagen hair follicles. Here we show that Lgr5(+) cells comprise an actively proliferating and multipotent stem cell population able to give rise to new hair follicles and maintain all cell lineages of the hair follicle over long periods of time. Lgr5(+) progeny repopulate other stem cell compartments in the hair follicle, supporting the existence of a stem or progenitor cell hierarchy. By marking Lgr5(+) cells during trafficking through the lower outer root sheath, we show that these cells retain stem cell properties and contribute to hair follicle growth during the next anagen. Expression analysis suggests involvement of autocrine Hedgehog signaling in maintaining the Lgr5(+) stem cell population.

    Funded by: NCI NIH HHS: U01CA105491; NIAMS NIH HHS: AR47898

    Nature genetics 2008;40;11;1291-9

  • Role of the aromatic residues in the near-amino terminal motif of vimentin in intermediate filament assembly in vitro.

    Gohara R, Nishikawa S, Takasaki Y and Ando S

    Division of Biopolymer Research, Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Saga 849-8501, Japan.

    Type III and IV intermediate filament (IF) proteins share a conserved sequence motif of -Tyr-Arg-Arg-X-Phe- at the near-amino termini. To characterize significance of the aromatic residues in the motif, we prepared vimentin mutants in which Tyr-10 and Phe-14 are substituted with Asn and Ser (Vim[Y10N], Vim[F14S] and Vim[Y10N, F14S]), and examined assembly properties in vitro by electron microscopy and viscosity measurements. At 2 s after initiation of assembly reaction at pH 7.2 and 150 mM NaCl, all the vimentin mutants formed so-called unit-length filaments (ULFs) that were slightly larger than ULFs of wild-type vimentin. In following filament elongation, Vim[Y10N, F14S] and Vim[Y10N] performed longitudinal annealing of ULFs very rapidly and formed IFs within only 2.5 and 5 min, respectively, while Vim[F14S] and wild-type vimentin gave IFs by 40-60 min. The IFs of Vim[Y10N, F14S] and Vim[Y10N], however, tended to intertwine each other and formed bundles in parts of the specimens. The intertwinements decreased as the salt concentration decreased, and optimal salt concentration for the two mutants to form normal IFs was 50 mM. These results suggest that the aromatic residues, especially Tyr-10, in the motif have a role in controlling intermolecular interactions involved in IF assembly in vitro and suppress undesirable filament intertwinements at physiological ionic strength.

    Journal of biochemistry 2008;144;5;675-84

  • SSAO/VAP-1 protein expression during mouse embryonic development.

    Valente T, Solé M and Unzeta M

    Departament de Bioquimica i Biologia Molecular, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain. tonyvalente@gmail.com

    SSAO/VAP-1 is a multifunctional enzyme depending on in which tissue it is expressed. SSAO/VAP-1 is present in almost all adult mammalian tissues, especially in highly vascularised ones and in adipocytes. SSAO/VAP-1 is an amine oxidase able to metabolise various endogenous or exogenous primary amines. Its catalytic activity can lead to cellular oxidative stress, which has been implicated in several pathologies (atherosclerosis, diabetes, and Alzheimer's disease). The aim of this work is to achieve a study of SSAO/VAP-1 protein expression during mouse embryogenesis. Our results show that SSAO/VAP-1 appears early in the development of the vascular system, adipose tissue, and smooth muscle cells. Moreover, its expression is strong in several epithelia of the sensory organs, as well as in the development of cartilage sites. Altogether, this suggests that SSAO/VAP-1 enzyme could be involved in the differentiation processes that take place during embryonic development, concretely in tissue vascularisation.

    Developmental dynamics : an official publication of the American Association of Anatomists 2008;237;9;2585-93

  • Lis1-Nde1-dependent neuronal fate control determines cerebral cortical size and lamination.

    Pawlisz AS, Mutch C, Wynshaw-Boris A, Chenn A, Walsh CA and Feng Y

    Department of Neurology and Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, 303 E. Superior Street, Chicago, IL 60611, USA.

    Neurons in the cerebral cortex originate predominantly from asymmetrical divisions of polarized radial glial or neuroepithelial cells. Fate control of neural progenitors through regulating cell division asymmetry determines the final cortical neuronal number and organization. Haploinsufficiency of human LIS1 results in type I lissencephaly (smooth brain) with severely reduced surface area and laminar organization of the cerebral cortex. Here we show that LIS1 and its binding protein Nde1 (mNudE) regulate the fate of radial glial progenitors collaboratively. Mice with an allelic series of Lis1 and Nde1 double mutations displayed a striking dose-dependent size reduction and de-lamination of the cerebral cortex. The neocortex of the Lis1-Nde1 double mutant mice showed over 80% reduction in surface area and inverted neuronal layers. Dramatically increased neuronal differentiation at the onset of corticogenesis in the mutant led to overproduction and abnormal development of earliest-born preplate neurons and Cajal-Retzius cells at the expense of progenitors. While both Lis1 and Nde1 are known to regulate the mitotic spindle orientation, only a moderate alteration in mitotic cleavage orientation was detected in the Lis1-Nde1 double deficient progenitors. Instead, a striking change in the morphology of metaphase progenitors with reduced apical attachment to the ventricular surface and weakened lateral contacts to neighboring cells appear to hinder the accurate control of cell division asymmetry and underlie the dramatically increased neuronal differentiation. Our data suggest that maintaining the shape and cell-cell interactions of radial glial neuroepithelial progenitors by the Lis1-Nde1 complex is essential for their self renewal during the early phase of corticogenesis.

    Funded by: Howard Hughes Medical Institute; NIMH NIH HHS: K01MH65338; NINDS NIH HHS: 2R01NS032457, 5R01NS047191

    Human molecular genetics 2008;17;16;2441-55

  • Abnormal reactivity of muller cells after retinal detachment in mice deficient in GFAP and vimentin.

    Verardo MR, Lewis GP, Takeda M, Linberg KA, Byun J, Luna G, Wilhelmsson U, Pekny M, Chen DF and Fisher SK

    Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA. mark.vera@sbcglobal.net

    Purpose: To determine the roles of glial fibrillary acidic protein (GFAP) and vimentin in Müller cell reactivity.

    Methods: Retinal detachments were created in mice deficient for GFAP and vimentin (GFAP(-/-)vim(-/-)) and age-matched wild-type (wt) mice. The reactivity of the retina was studied by immunofluorescence and electron microscopy.

    Results: Müller cell morphology was different and glutamine synthetase immunoreactivity was reduced in the undisturbed GFAP(-/-)vim(-/-) retinas. After retinal detachment, Müller cells formed subretinal glial scars in the wt mice. In contrast, such scars were not observed in GFAP(-/-)vim(-/-) mice. Müller cells, which normally elongate and thicken in response to detachment, appeared compressed, thin, and "spikey" in the GFAP(-/-)vim(-/-) mice. The end foot region of Müller cells in the GFAP(-/-)vim(-/-) mice often sheared away from the rest of the retina during detachment, corroborating earlier results showing decreased resistance of this region in GFAP(-/-)vim(-/-) retinas to mechanical stress. In regions with end foot shearing, ganglion cells showed intense neurite sprouting, as revealed by anti-neurofilament labeling, a response rarely observed in wt mice.

    Conclusions: Müller cells are subtly different in the GFAP(-/-)vim(-/-) mouse retina before detachment. The end foot region of these cells may be structurally reinforced by the presence of the intermediate filament cytoskeleton, and our data suggest a critical role for these proteins in Müller cell reaction to retinal detachment and participation in subretinal gliosis.

    Funded by: NCRR NIH HHS: 1S10RR017753-01; NEI NIH HHS: EY00888, R01 EY000888, R01 EY017641, R01 EY017641-01A2, R37 EY000888

    Investigative ophthalmology & visual science 2008;49;8;3659-65

  • 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

  • Hedgehog signaling is critical for maintenance of the adult coronary vasculature in mice.

    Lavine KJ, Kovacs A and Ornitz DM

    Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Hedgehog (HH) signaling has emerged as a critical pathway involved in the pathogenesis of a variety of tumors. As a result, HH antagonists are currently being evaluated as potential anticancer therapeutics. Conversely, activation of HH signaling in the adult heart may be beneficial, as HH agonists have been shown to increase coronary vessel density and improve coronary function after myocardial infarction. To investigate a potential homeostatic role for HH signaling in the adult heart, we ablated endogenous HH signaling in murine myocardial and perivascular smooth muscle cells. HH signaling was required for proangiogenic gene expression and maintenance of the adult coronary vasculature in mice. In the absence of HH signaling, loss of coronary blood vessels led to tissue hypoxia, cardiomyocyte cell death, heart failure, and subsequent lethality. We further showed that HH signaling specifically controlled the survival of small coronary arteries and capillaries. Together, these data demonstrate that HH signaling is essential for cardiac function at the level of the coronary vasculature and caution against the use of HH antagonists in patients with prior or ongoing heart disease.

    Funded by: NHLBI NIH HHS: HL076664, R01 HL076664

    The Journal of clinical investigation 2008;118;7;2404-14

  • Identification of midbrain floor plate radial glia-like cells as dopaminergic progenitors.

    Bonilla S, Hall AC, Pinto L, Attardo A, Götz M, Huttner WB and Arenas E

    Laboratory of Molecular Neurobiology, MBB, Karolinska Institutet, Scheeles väg 1, A1:2, 171 77 Stockholm, Sweden.

    The floor plate (FP), a signaling center and a structure rich in radial glia-like cells, has been traditionally thought to be devoid of neurons and neuronal progenitors. However, in the midbrain, the FP contains neurons of the dopaminergic (DA) lineage that require contact with radial glia-like cells for their induction. We, therefore, decided to explore the interaction relationship between radial glia and neurons during DA neurogenesis. Taking advantage of a novel FP radial glia-like cell culture system and retroviruses, DA neurons were lineage traced in vitro. In utero BrdU pulse-chases extensively labeled the midbrain FP and traced DA neurons both in vivo and in FP cultures. Moreover, from E9.5 to E13.5 the midbrain FP contained dividing cells only in the most apical part of the neuroepithelium, in cells identified as radial glia-like cells. We, therefore, hypothesized that midbrain FP radial glia-like cells could be DA progenitors and tested our hypothesis in vivo. Lineage tracing of DA progenitors with EGFP in Tis21-EGFP knock-in mice, and genetic fate mapping in GLAST::CreERT2/ZEG mice identified the neuroepithelium of the midbrain FP, and specifically, GLAST+ radial glia-like cells as DA progenitors. Combined, our experiments support the concept that the midbrain FP differs from other FP regions and demonstrate that FP radial glia-like cells in the midbrain are neurogenic and give rise to midbrain DA neurons.

    Glia 2008;56;8;809-20

  • Localization of Nogo and its receptor in the optic pathway of mouse embryos.

    Wang J, Chan CK, Taylor JS and Chan SO

    Department of Anatomy, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Special Administrative Region, China.

    We have investigated the localization of Nogo, an inhibitory protein acting on regenerating axons in the adult central nervous system, in the embryonic mouse retinofugal pathway during the major period of axon growth into the optic chiasm. In the retina, Nogo protein was localized on the neuroepithelial cells at E12 and at later stages (E13-E17) on radial glial cells. Colocalization studies showed expression of Nogo on vimentin-positive glia in the retina and at the optic nerve head but not on most of the TuJ1- and islet-1-immunoreactive neurons. Only a few immature neurons in the ventricular and peripheral regions of the E13 retina were immunoreactive to Nogo. In the ventral diencephalon, Nogo was expressed on radial glia, most strongly on the dense radial glial midline raphe within the chiasm where uncrossed axons turn and in the initial segment of the optic tract. In vitro studies showed that the Nogo receptor (NgR) was expressed on the neurites and growth cones from both the ventral temporal and dorsal nasal quadrant of the retina. In the optic pathway, NgR staining was obvious in the vitreal regions of the retina and on axons in the optic stalk and the optic tract, but not in the chiasm. These expression patterns suggest an interaction of Nogo with its receptor in the mouse retinofugal pathway, which may be involved in guiding axons into the optic pathway and in governing the routing of axons in the optic chiasm.

    Journal of neuroscience research 2008;86;8;1721-33

  • Cited2 is required for fetal lung maturation.

    Xu B, Qu X, Gu S, Doughman YQ, Watanabe M, Dunwoodie SL and Yang YC

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

    Lung maturation at the terminal sac stage of lung development is characterized by a coordinated increase in terminal sac formation and vascular development in conjunction with the differentiation of alveolar type I and type II epithelial cells. The Cited2-Tcfap2a/c complex has been shown to activate transcription of Erbb3 and Pitx2c during mouse development. In this study, we show that E17.5 to E18.5 Cited2-null lungs had significantly reduced terminal sac space due to an altered differentiation of type I and type II alveolar epithelial cells. In addition, E17 Cited2-null lungs exhibited a decrease in the number of apoptotic cells, contributing to the loss in airspace. Consistent with the phenotype, genes associated with alveolar cell differentiation and survival were differentially expressed in Cited2-null fetal lungs compared to those of wild-type littermates. Moreover, expression of Cebpa, a key regulator of airway epithelial maturation, was significantly decreased in Cited2-null fetal lungs. Cited2 and Tcfap2c were present on the Cebpa promoter in E18.5 lungs to activate Cebpa transcription. We propose that the Cited2-Tcfap2c complex controls lung maturation by regulating Cebpa expression. Understanding the function of this complex may provide novel therapeutic strategies for patients with respiratory distress syndromes.

    Funded by: NHLBI NIH HHS: HL075436, R01 HL075436, R21 HL091205, R21 HL091205-01

    Developmental biology 2008;317;1;95-105

  • Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate.

    Joesting MS, Cheever TR, Volzing KG, Yamaguchi TP, Wolf V, Naf D, Rubin JS and Marker PC

    Department of Genetics, Cellular Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA.

    Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1(lacZ) knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of WNT signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical WNT/beta-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the non-canonical WNT/JNK pathway in the prostate.

    Funded by: NIA NIH HHS: AG024278, R01 AG024278-01A2, R01 AG024278-02, R01 AG024278-03; NIDDK NIH HHS: DK069662, R01 DK091193, R21 DK069662-01, R21 DK069662-02

    Developmental biology 2008;317;1;161-73

  • Doublecortin (DCX) and doublecortin-like (DCL) are differentially expressed in the early but not late stages of murine neocortical development.

    Boekhoorn K, Sarabdjitsingh A, Kommerie H, de Punder K, Schouten T, Lucassen PJ and Vreugdenhil E

    Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands.

    During corticogenesis, radial glia-derived neural progenitors divide and migrate along radial fibers to their designated positions within the cortical plate. The microtubule-associated proteins doublecortin (DCX) and doublecortin-like (DCL) are critically involved in neuronal migration and division, and may function in a partially redundant pathway. Since little is known about the important early stages of corticogenesis, when neurogenesis is extensive, we addressed a possible differential role by examining spatiotemporal expression patterns of DCX, DCL, and the radial glia marker vimentin during murine development. We found expression patterns of DCL and DCX to differ remarkably prior to embryonic day (E)13. DCL was already expressed at E9 and largely overlapped with vimentin, whereas DCX expression started modestly from E10/E11 onward. DCL was mainly found in the ventricular zone, often in mitotic cells and in pial-oriented radial fibers. In contrast, DCX was expressed in tangential fibers in the outer cortical regions. After E13, DCX and DCL expression largely overlapped but DCL expression had disappeared from the ventricular zone. Also, DCL levels were attenuated, whereas DCX remained high beyond E17. In conclusion, DCX and DCL are differentially expressed, particularly during early corticogenesis, consistent with their different functional roles. Given its involvement in mitosis, DCL appears to have a unique role in the early neuroepithelium that is different from later developmental stages when DCX is coexpressed.

    The Journal of comparative neurology 2008;507;4;1639-52

  • 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

  • A role for lengsin, a recruited enzyme, in terminal differentiation in the vertebrate lens.

    Wyatt K, Gao C, Tsai JY, Fariss RN, Ray S and Wistow G

    National Eye Institute, National Institutes of Health, Bethesda, MD 20892, USA.

    Lengsin is an eye lens-specific member of the glutamine synthetase (GS) superfamily. Lengsin has no GS activity, suggesting that it has a structural rather than catalytic role in lens. In situ hybridization and immunofluorescence showed that lengsin is expressed in terminally differentiating secondary lens fiber cells. Yeast two-hybrid (Y2H) and recombinant protein experiments showed that full-length lengsin can bind the 2B filament region of vimentin. In affinity chromatography, lengsin also bound the equivalent region of CP49 (BFSP2; phakinin), a related intermediate filament protein specific to the lens. Both the vimentin and CP49 2B fragments bound lengsin in surface plasmon resonance spectroscopy with fast association and slow dissociation kinetics. Lengsin expression correlates with a transition zone in maturing lens fiber cells in which cytoskeleton is reorganized. Lengsin and lens intermediate filament proteins co-localize at the plasma membrane in maturing fiber cells. This suggests that lengsin may act as a component of the cytoskeleton itself or as a chaperone for the reorganization of intermediate filament proteins during terminal differentiation in the lens.

    Funded by: Intramural NIH HHS: Z01 EY000433-04

    The Journal of biological chemistry 2008;283;10;6607-15

  • Six3 inactivation causes progressive caudalization and aberrant patterning of the mammalian diencephalon.

    Lavado A, Lagutin OV and Oliver G

    Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794, USA.

    The homeobox gene Six3 represses Wnt1 transcription. It is also required in the anterior neural plate for the development of the mammalian rostral forebrain. We have now determined that at the 15- to 17-somite stage, the prospective diencephalon is the most-anterior structure in the Six3-null brain, and Wnt1 expression is anteriorly expanded. Consequently, the brain caudalizes, and at the 22- to 24-somite stage, the prospective thalamic territory is the most-anterior structure. At around E11.0, the pretectum replaces this structure. Analysis of Six3;Wnt1 double-null mice revealed that Six3-mediated repression of Wnt1 is necessary for the formation of the rostral diencephalon and that Six3 activity is required for the formation of the telencephalon. These results provide insight into the mechanisms that establish anteroposterior identity in the developing mammalian brain.

    Funded by: NCI NIH HHS: CA-21765; NEI NIH HHS: EY12162

    Development (Cambridge, England) 2008;135;3;441-50

  • Zeb1 links epithelial-mesenchymal transition and cellular senescence.

    Liu Y, El-Naggar S, Darling DS, Higashi Y and Dean DC

    James Graham Brown Cancer Center, Department of Ophthalmology and Visual Sciences, University of Louisville Health Sciences Center, Louisville, KY 40202, USA.

    Overexpression of zinc finger E-box binding homeobox transcription factor 1 (Zeb1) in cancer leads to epithelial-to-mesenchymal transition (EMT) and increased metastasis. As opposed to overexpression, we show that mutation of Zeb1 in mice causes a mesenchymal-epithelial transition in gene expression characterized by ectopic expression of epithelial genes such as E-cadherin and loss of expression of mesenchymal genes such as vimentin. In contrast to rapid proliferation in cancer cells where Zeb1 is overexpressed, this mesenchymal-epithelial transition in mutant mice is associated with diminished proliferation of progenitor cells at sites of developmental defects, including the forming palate, skeleton and CNS. Zeb1 dosage-dependent deregulation of epithelial and mesenchymal genes extends to mouse embryonic fibroblasts (MEFs), and mutant MEFs also display diminished replicative capacity in culture, leading to premature senescence. Replicative senescence in MEFs is classically triggered by products of the Ink4a (Cdkn2a) gene. However, this Ink4a pathway is not activated during senescence of Zeb1 mutant MEFs. Instead, there is ectopic expression of two other cell cycle inhibitory cyclin-dependent kinase inhibitors, p15Ink4b (Cdkn2b) and p21Cdkn1a (Cdkn1a). We demonstrate that this ectopic expression of p15Ink4b extends in vivo to sites of diminished progenitor cell proliferation and developmental defects in Zeb1-null mice.

    Funded by: NCRR NIH HHS: P20 RR018733, RR018733; NEI NIH HHS: EY015636, R21 EY017869, R21 EY017869-01A1, R21 EY017869-02, R24 EY015636

    Development (Cambridge, England) 2008;135;3;579-88

  • Molecular mechanisms of cleft lip formation in CL/Fr mice.

    Nakazawa M, Matsunaga K, Asamura S, Kusuhara H, Isogai N and Muragaki Y

    Department of Plastic and Reconstructive Surgery, Kinki University Hospital, Osaka, Japan.

    CL/Fr mice have a high incidence of cleft lip and the cleft lip is the result of incomplete fusion between the medial and lateral nasal prominences and the maxillary prominence at about day 11.5 of gestation. However, little is known about the molecular mechanisms that are responsible for the incomplete fusion. We made a molecular pathological investigation using 11.5-day CL/Fr embryos. Five embryos were each examined for real-time polymerase chain reaction (PCR) analysis. During the first palatal formation in normal development, an epithelial seam is formed when the medial and lateral nasal prominences first make contact. Some epithelial cells of the epithelial seam then undergo apoptosis, with remaining cells transforming into a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Mesenchymal cells of the medial and lateral nasal prominences then merge across the previous boundary of separation. In CL/Fr mice with cleft lip, neither apoptosis nor EMT occurs in the epithelial cells. Increased expression of claudin 6 mRNA is seen in epithelial cells of epithelial seam in cleft lip compared with that in normal embryos. Slug mRNA expression was also significantly reduced whereas noggin was increased in CL/Fr embryos with cleft lip. We suggest that EMT is prevented in CL/Fr mice with cleft lip by increased expression of claudin 6 and coexistent downregulation of slug in cells of the epithelial seam, and these altered concentrations of transcription factors/repressors prevent fusion of the medial and lateral nasal prominences, leading to clefts of the lip.

    Scandinavian journal of plastic and reconstructive surgery and hand surgery / Nordisk plastikkirurgisk forening [and] Nordisk klubb for handkirurgi 2008;42;5;225-32

  • Patterned neuropathologic events occurring in hyh congenital hydrocephalic mutant mice.

    Páez P, Bátiz LF, Roales-Buján R, Rodríguez-Pérez LM, Rodríguez S, Jiménez AJ, Rodríguez EM and Pérez-Fígares JM

    Departamento de Biología Celular, Genética y Fisiología, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.

    Hyh mutant mice develop long-lasting hydrocephalus and represent a good model for investigating neuropathologic events associated with hydrocephalus. The study of their brains by use of lectin binding, bromodeoxyuridine labeling, immunochemistry, and scanning electron microscopy revealed that certain events related to hydrocephalus followed a well-defined pattern. A program of neuroepithelium/ependyma denudation was initiated at embryonic day 12 and terminated at the end of the second postnatal week. After the third postnatal week the denuded areas remained permanently devoid of ependyma. In contrast, a selective group of ependymal areas resisted denudation throughout the lifespan. Ependymal denudation triggered neighboring astrocytes to proliferate. These astrocytes expressed particular glial markers and formed a superficial cell layer replacing the lost ependyma. The loss of the neuroepithelium/ependyma layer at specific regions of the ventricular walls and at specific stages of brain development would explain the fact that only certain brain structures had abnormal development. Therefore, commissural axons forming the corpus callosum and the hippocampal commissure displayed abnormalities, whereas those forming the anterior and posterior commissures did not; and the brain cortex was not homogenously affected, with the cingular and frontal cortices being the most altered regions. All of these telencephalic alterations developed at stages when hydrocephalus was not yet patent at the lateral ventricles, indicating that abnormal neural development and hydrocephalus are linked at the etiologic level, rather than the former being a consequence of the latter. All evidence collected on hydrocephalic hyh mutant mice indicates that a primary alteration in the neuroepithelium/ependyma cell lineage triggers both hydrocephalus and abnormalities in telencephalic development.

    Journal of neuropathology and experimental neurology 2007;66;12;1082-92

  • Cited2, a coactivator of HNF4alpha, is essential for liver development.

    Qu X, Lam E, Doughman YQ, Chen Y, Chou YT, Lam M, Turakhia M, Dunwoodie SL, Watanabe M, Xu B, Duncan SA and Yang YC

    Department of Pharmacology and Cancer Center, Case Western Reserve University School of Medicine, Cleveland, OH, USA.

    The transcriptional modulator Cited2 is induced by various biological stimuli including hypoxia, cytokines, growth factors, lipopolysaccharide (LPS) and flow shear. In this study, we report that Cited2 is required for mouse fetal liver development. Cited2(-/-) fetal liver displays hypoplasia with higher incidence of cell apoptosis, and exhibits disrupted cell-cell contact, disorganized sinusoidal architecture, as well as impaired lipid metabolism and hepatic gluconeogenesis. Furthermore, we demonstrated the physical and functional interaction of Cited2 with liver-enriched transcription factor HNF4alpha. Chromatin immunoprecipitation (ChIP) assays further confirmed the recruitment of Cited2 onto the HNF4alpha-responsive promoters and the reduced HNF4alpha binding to its target gene promoters in the absence of Cited2. Taken together, this study suggests that fetal liver defects in mice lacking Cited2 result, at least in part, from its defective coactivation function for HNF4alpha.

    Funded by: NHLBI NIH HHS: R01-HL075436-02, R01-HL076919-07

    The EMBO journal 2007;26;21;4445-56

  • 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

  • The intriguing normal acute inflammatory response in mice lacking vimentin.

    Moisan E, Chiasson S and Girard D

    INRS-Institut Armand-Frappier, Université du Québec, Pointe-Claire, PQ, Canada.

    Neutrophils express only two intermediate filament proteins, vimentin and, to a lesser extent, lamin B. Lamin B mutant mice die shortly after birth; however, mice lacking vimentin (vim(-/-)) develop and reproduce normally. Herein, we investigate for the first time the role of vimentin in general inflammation in vivo and in neutrophil functions ex vivo. Using the murine air pouch model, we show that the inflammatory response induced by lipopolysaccharide, interleukin-21 or carageenan is, intriguingly, uncompromised in vim(-/-) mice and that neutrophil functions are not altered ex vivo. Our results suggest that vimentin is dispensable for the establishment of an acute inflammatory response in vivo. In addition, based on several criteria presented in this study, one has to accept the existence of a very complex compensatory mechanism to explain the intriguing normal inflammatory response in absence of vimentin.

    Clinical and experimental immunology 2007;150;1;158-68

  • 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

  • Generation of functional multipotent adult stem cells from GPR125+ germline progenitors.

    Seandel M, James D, Shmelkov SV, Falciatori I, Kim J, Chavala S, Scherr DS, Zhang F, Torres R, Gale NW, Yancopoulos GD, Murphy A, Valenzuela DM, Hobbs RM, Pandolfi PP and Rafii S

    Howard Hughes Medical Institute, Department of Genetic Medicine, Weill Cornell Medical College, New York 10065, USA.

    Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.

    Funded by: Howard Hughes Medical Institute; NEI NIH HHS: K08 EY021171; NHLBI NIH HHS: P01 HL059312, P01 HL059312-060006, P01 HL059312-070006, P01 HL059312-080006, P01 HL059312-090006, P01 HL059312-100006, P01 HL067839, P01 HL067839-010004, P01 HL067839-020004, P01 HL067839-030004, P01 HL067839-040004, P01 HL067839-050004, P50 HL084936, P50 HL084936-010003, P50 HL084936-020003, P50 HL084936-030003, P50 HL084936-040003, R01 HL058707-03, R01 HL058707-04, R01 HL061849, R01 HL061849-02, R01 HL061849-03, R01 HL061849-03S1, R01 HL061849-04, R01 HL061849-05, R01 HL075234, R01 HL075234-01, R01 HL075234-02, R01 HL075234-03, R01 HL075234-04, R01 HL097797, R01 HL097797-01, R01 HL097797-02, R01 HL097797-03, R21 HL083222, R21 HL083222-01, R21 HL083222-02, U01 HL066952, U01 HL066952-010002, U01 HL066952-020002, U01 HL066952-030002, U01 HL066952-040002, U01 HL066952-050002

    Nature 2007;449;7160;346-50

  • Angiopoietin-like protein 1 expression is related to intermuscular connective tissue and cartilage development.

    Lai DM, Tu YK, Hsieh YH, Hsu WM, Lee CC, Cheng WC, Hsieh FJ and Li H

    Division of Neurosurgery, Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan.

    Angptl1, a member of the angiopoietin-related protein family, is known to regulate angiogenesis, but little is known of its potential role in other processes. To identify the expression pattern and possible role of angptl1 during embryogenesis, we used gene targeting to generate angptl1-deficient, nLacZ knockin mice. Staining for beta-galactosidase from embryonic day 9.5 to 6 months of age revealed that angptl1 was initially expressed in the paraxial mesoderm. Expression then shifted to intermuscular connective tissue (fascial plane), joint capsules, and perichondrium (laryngo-trachea, ribs, and long bones), but not the muscles. The vasculature, central and peripheral nervous systems, digestive, respiratory, and other major organ systems did not show any angptl1 expression. This expression pattern suggests that angptl1 is related to development of the connective tissue and cartilage. Lack of phenotype in mutant mice may be due to a functional redundancy from other related factors.

    Developmental dynamics : an official publication of the American Association of Anatomists 2007;236;9;2643-52

  • Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease.

    Mignot C, Delarasse C, Escaich S, Della Gaspera B, Noé E, Colucci-Guyon E, Babinet C, Pekny M, Vicart P, Boespflug-Tanguy O, Dautigny A, Rodriguez D and Pham-Dinh D

    INSERM UMR S 546, Paris, France.

    Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

    Experimental cell research 2007;313;13;2766-79

  • Numb and Numbl are required for maintenance of cadherin-based adhesion and polarity of neural progenitors.

    Rasin MR, Gazula VR, Breunig JJ, Kwan KY, Johnson MB, Liu-Chen S, Li HS, Jan LY, Jan YN, Rakic P and Sestan N

    Department of Neurobiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510, USA.

    The polarity and adhesion of radial glial cells (RGCs), which function as progenitors and migrational guides for neurons, are critical for morphogenesis of the cerebral cortex. These characteristics largely depend on cadherin-based adherens junctions, which anchor apical end-feet of adjacent RGCs to each other at the ventricular surface. Here, we show that mouse numb and numb-like are required for maintaining radial glial adherens junctions. Numb accumulates in the apical end-feet, where it localizes to adherens junction-associated vesicles and interacts with cadherins. Numb and Numbl inactivation in RGCs decreases proper basolateral insertion of cadherins and disrupts adherens junctions and polarity, leading to progenitor dispersion and disorganized cortical lamination. Conversely, overexpression of Numb prolongs RGC polarization, in a cadherin-dependent manner, beyond the normal neurogenic period. Thus, by regulating RGC adhesion and polarity, Numb and Numbl are required for the tissue architecture of neurogenic niches and the cerebral cortex.

    Funded by: Autism Speaks: AS1672; NIA NIH HHS: AG019394; NICHD NIH HHS: HD045481; NINDS NIH HHS: NS047200

    Nature neuroscience 2007;10;7;819-27

  • Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle.

    Spella M, Britz O, Kotantaki P, Lygerou Z, Nishitani H, Ramsay RG, Flordellis C, Guillemot F, Mantamadiotis T and Taraviras S

    Department of Pharmacology, School of Medicine, University of Patras, 26500 Rio, Patras, Greece.

    Nervous system formation integrates control of cellular proliferation and differentiation and is mediated by multipotent neural progenitor cells that become progressively restricted in their developmental potential before they give rise to differentiated neurons and glial cells. Evidence from different experimental systems suggests that Geminin is a candidate molecule linking proliferation and differentiation during nervous system development. We show here that Geminin and its binding partner Cdt1 are expressed abundantly by neural progenitor cells during early mouse neurogenesis. Their expression levels decline at late developmental stages and become undetectable upon differentiation. Geminin and Cdt1 expressing cells also express Sox2 while no overlap is detected with cells expressing markers of a differentiated neuronal phenotype. A fraction of radial glial cells expressing RC2 and Pax6 are also immunoreactive for Geminin and Cdt1. The majority of the Geminin and Cdt1 expressing cell populations appears to be distinct from fate-restricted precursor cells expressing Mash1 or Neurogenin2. Bromo-deoxy-uridine (BrdU) incorporation experiments reveal a cell cycle specific expression in neural progenitor cells, with Geminin being present from S to M phase, while Cdt1 expression characterizes progenitor cells in G1 phase. Furthermore, in vitro differentiation of adult neurosphere cultures shows downregulation of Geminin/Cdt1 in the differentiated state, in line with our data showing that Geminin is present in neural progenitor cells of the CNS during mouse embryogenesis and adulthood and becomes downregulated upon cell fate specification and differentiation. This suggests a role for Geminin in the formation and maintenance of the neural progenitor cells.

    Funded by: Medical Research Council: MC_U117570528

    Neuroscience 2007;147;2;373-87

  • Cellular mechanisms of Müllerian duct formation in the mouse.

    Orvis GD and Behringer RR

    Program in Genes and Development, The University of Texas, Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA.

    Regardless of their sex chromosome karyotype, amniotes develop two pairs of genital ducts, the Wolffian and Müllerian ducts. As the Müllerian duct forms, its growing tip is intimately associated with the Wolffian duct as it elongates to the urogenital sinus. Previous studies have shown that the presence of the Wolffian duct is required for the development and maintenance of the Müllerian duct. The Müllerian duct is known to form by invagination of the coelomic epithelium, but the mechanism for its elongation to the urogenital sinus remains to be defined. Using genetic fate mapping, we demonstrate that the Wolffian duct does not contribute cells to the Müllerian duct. Experimental embryological manipulations and molecular studies show that precursor cells at the caudal tip of the Müllerian duct proliferate to deposit a cord of cells along the length of the urogenital ridge. Furthermore, immunohistochemical analysis reveals that the cells of the developing Müllerian duct are mesoepithelial when deposited, and subsequently differentiate into an epithelial tube and eventually the female reproductive tract. Our studies define cellular and molecular mechanisms for Müllerian duct formation.

    Funded by: NCI NIH HHS: CA 009299; NICHD NIH HHS: HD 30284, R01 HD030284, R01 HD030284-11, R01 HD030284-12, R01 HD030284-13, R01 HD030284-14, R01 HD030284-15

    Developmental biology 2007;306;2;493-504

  • Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin.

    Nakazawa T, Takeda M, Lewis GP, Cho KS, Jiao J, Wilhelmsson U, Fisher SK, Pekny M, Chen DF and Miller JW

    Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

    Purpose: To characterize the reactions of retinal glial cells (astrocytes and Müller cells) to retinal injury in mice that lack glial fibrillary acidic protein (GFAP) and vimentin (GFAP-/-Vim-/-) and to determine the role of glial cells in retinal detachment (RD)-induced photoreceptor degeneration.

    Methods: RD was induced by subretinal injection of sodium hyaluronate in adult wild-type (WT) and GFAP-/-Vim-/- mice. Astroglial reaction and subsequent monocyte recruitment were quantified by measuring extracellular signal-regulated kinase (Erk) and c-fos activation and the level of expression of chemokine monocyte chemoattractant protein (MCP)-1 and by counting monocytes/microglia in the detached retinas. Immunohistochemistry, immunoblotting, real-time quantitative polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used. RD-induced photoreceptor degeneration was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and measurement of outer nuclear layer (ONL) thickness.

    Results: RD-induced reactive gliosis, characterized by GFAP and vimentin upregulation, Erk and c-fos activation, MCP-1 induction, and increased monocyte recruitment in WT mice. Absence of GFAP and vimentin effectively attenuated reactive responses of retinal glial cells and monocyte infiltration. As a result, detached retinas of GFAP-/-Vim-/- mice exhibited significantly reduced numbers of TUNEL-positive photoreceptor cells and increased ONL thickness compared with those of WT mice.

    Conclusions: The absence of GFAP and vimentin attenuates RD-induced reactive gliosis and, subsequently, limits photoreceptor degeneration. Results of this study indicate that reactive retinal glial cells contribute critically to retinal damage induced by RD and provide a new avenue for limiting photoreceptor degeneration associated with RD and other retinal diseases or damage.

    Funded by: NEI NIH HHS: EY003790, EY00888, EY012983, EY014104, P30 EY003790, P30 EY014104, R01 EY000888, R01 EY012983, R01 EY017641, R01 EY017641-01A2, R37 EY000888

    Investigative ophthalmology & visual science 2007;48;6;2760-8

  • Cytoskeleton vimentin disruption of mouse sertoli cells injured by nitrogen mustard in vitro.

    He D, Zhang D, Wei G, Lin T and Li X

    Children's Hospital of Chongqing Medical University, Department of Pediatric Urology, Chongqing, No. 136, Zhongshan 2 RD, Yuzhong District, Chongqing, China 400014. babyuro@163.com

    Reproductive toxicity is one of the potential side effects of anticancer alkylating agents, with potential effects on vimentin intermediate filaments, one of the main components of the Sertoli cytoskeleton. Research suggests (Aumuller et al, 1988; Aumuller et al, 1992) that the highly organized and active Sertoli cytoskeleton is important in spermatogenesis. The aim of the current study was to investigate the effects of alkylating agents on vimentin filament expression in vitro. Sertoli cells, isolated from 20-day-old mice testes, were cultured for 5 days and then incubated with 0, 50, 100, and 200 micromol/L nitrogen mustard (HN2). Morphologic changes in Sertoli cells were observed per 30-minute interval at 12-hour exposure time points to 100 micromol/L HN2. Vimentin expression was investigated by immunocytochemistry at 6 hours and 24 hours posttreatment and reverse transcriptase polymerase chain reaction and Western blot at 12 hours posttreatment with 50, 100, and 200 micromol/L HN2. Exposure to HN2 resulted in a comparatively small Sertoli cell body with diminished cytoplasm. Sertoli cells were shrunk or detached. Cytoskeletal disruption increased with increasing HN2 concentration. The optical density values of vimentin antibody and expression of vimentin mRNA and protein were significantly decreased with increasing concentration of HN2. Significant treatment dose-dependent and time-dependent differences of vimentin mRNA and protein expression levels were also noted. Our data suggest that the change in the biochemical properties of vimentin may indicate that one of the mechanisms of reproductive toxicity resulting from HN2 is disruption of Sertoli cell vimentin filament structure, accompanied by a down-regulation of vimentin expression.

    Journal of andrology 2007;28;3;389-96

  • Synemin is expressed in reactive astrocytes in neurotrauma and interacts differentially with vimentin and GFAP intermediate filament networks.

    Jing R, Wilhelmsson U, Goodwill W, Li L, Pan Y, Pekny M and Skalli O

    Department of Cellular Biology and Anatomy and Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA.

    Immature astrocytes and astrocytoma cells contain synemin and three other intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), vimentin and nestin. Here, we show that, after neurotrauma, reactive astrocytes produce synemin and thus propose synemin as a new marker of reactive astrocytes. Comparison of synemin mRNA and protein levels in brain tissues and astrocyte cultures from wild-type, Vim(-)(/)(-) and Gfap(-)(/)(-)Vim(-)(/)(-) mice showed that in the absence of vimentin, synemin protein was undetectable although synemin mRNA was present at wild-type levels. By contrast, in Gfap(-)(/)(-) astrocytes, synemin protein and mRNA levels, as well as synemin incorporation into vimentin IFs, were unaltered. Biochemical assays with purified proteins suggested that synemin interacts with GFAP IFs like an IF-associated protein rather than like a polymerization partner, whereas the opposite was true for synemin interaction with vimentin. In transfection experiments, synemin did not incorporate into normal, filamentous GFAP networks, but integrated into vimentin and GFAP heteropolymeric networks. Thus, alongside GFAP, vimentin and nestin, reactive astrocytes contain synemin, whose accumulation is suppressed post-transcriptionally in the absence of a polymerization partner. In astrocytes, this partner is vimentin and not GFAP, which implies a functional difference between these two type III IF proteins.

    Funded by: NINDS NIH HHS: NS-35317

    Journal of cell science 2007;120;Pt 7;1267-77

  • Requirement of vimentin filament assembly for beta3-adrenergic receptor activation of ERK MAP kinase and lipolysis.

    Kumar N, Robidoux J, Daniel KW, Guzman G, Floering LM and Collins S

    Program in Endocrine Biology, The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709, USA.

    Catecholamine stimulation of beta-adrenergic receptors (betaAR) in adipocytes activates the cAMP-dependent protein kinase to promote liberation of fatty acids as a fuel source. The adipocyte beta3AR also activates extracellular signal-regulated kinases (ERK)-1 and -2 through direct recruitment and activation of Src kinase. This pathway together with cAMP-dependent protein kinase contributes to maximal beta3AR-stimulated lipolysis. In a search for other molecules that might associate with beta3AR upon agonist stimulation, we identified vimentin using a proteomics approach. Immunoprecipitation of beta3AR from adipocytes in the absence or presence of the beta3AR agonist CL316,243, followed by Western blotting for vimentin confirmed this specific interaction. Since vimentin has also been identified on lipid droplets, the functional consequences of blocking the expression or structural integrity of vimentin intermediate filaments on beta3AR regulation of ERK activation and lipolysis was assessed. Following disruption of intermediate filaments with beta,beta'-iminodipropionitrile, as confirmed by confocal microscopy, beta3AR-stimulated ERK activation was blocked, and lipolysis was reduced by more than 40%. Independently, depletion of vimentin by small hairpin RNA (shRNA) completely inhibited beta3AR-mediated ERK activation and significantly reduced lipolysis. By contrast, disruption of actin-containing microfilaments by cytochalasin D or microtubules by nocodazole had no effect on either lipolysis or ERK activation. These results indicate that vimentin plays an essential role in the signal transduction pathway from beta3AR to the activation ERK and its contribution to lipolysis.

    Funded by: NIDDK NIH HHS: R01-DK53092, R01-DK57698

    The Journal of biological chemistry 2007;282;12;9244-50

  • Oxidation and nitrosylation of cysteines proximal to the intermediate filament (IF)-binding site of plectin: effects on structure and vimentin binding and involvement in IF collapse.

    Spurny R, Abdoulrahman K, Janda L, Rünzler D, Köhler G, Castañón MJ and Wiche G

    Department of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria.

    As an intermediate filament (IF)-based cytolinker protein, plectin plays a key role in the maintenance of cellular cytoarchitecture and serves at the same time as a scaffolding platform for signaling cascades. Consisting of six structural repeats (R1-6) and harboring binding sites for different IF proteins and proteins involved in signaling, the plectin C-terminal domain is of strategic functional importance. Depending on the species, it contains at least 13 cysteines, 4 of which reside in the R5 domain. To investigate the structural and biological functions of R5 cysteines, we used cysteine-to-serine mutagenesis and spectroscopic, biochemical, and functional analyses. Urea-induced unfolding experiments indicated that wild-type R5 in the oxidized, disulfide bond-mediated conformation was more stable than its cysteine-free mutant derivative. The binding affinity of R5 for vimentin was significantly higher, however, when the protein was in the reduced, more relaxed conformation. Of the four R5 cysteines, one (Cys4) was particularly reactive as reflected by its ability to form disulfide bridges with R5 Cys1 and to serve as a target for nitrosylation in vitro. Using immortalized endothelial cell cultures from mice, we show that endogenous plectin is nitrosylated in vivo, and we found that NO donor-induced IF collapse proceeds dramatically faster in plectin-deficient compared with wild-type cells. Our data suggest an antagonistic role of plectin in nitrosylation (oxidative stress)-mediated alterations of IF cytoarchitecture and a possible role of R5 Cys4 as a regulatory switch.

    Funded by: Austrian Science Fund FWF: P 17862

    The Journal of biological chemistry 2007;282;11;8175-87

  • Functions of a jumonji-cyclin D1 pathway in the coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain.

    Takahashi M, Kojima M, Nakajima K, Suzuki-Migishima R and Takeuchi T

    Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan.

    During development of the mouse central nervous system (CNS), most neural progenitor cells proliferate in the ventricular zone (VZ). In many regions of the CNS, neural progenitor cells give rise to postmitotic neurons that initiate neuronal differentiation and migrate out of the VZ to the mantle zone (MZ). Thereafter, they remain in a quiescent state. Here, we found many ectopic mitotic cells and cell clusters expressing neural progenitor or proneural marker genes in the MZ of the hindbrain of jumonji (jmj) mutant embryos. When we examined the expression of cyclin D1, which is repressed by jmj in the repression of cardiac myocyte proliferation, we found many ectopic clusters expressing both cyclin D1 and Musashi 1 in the MZ of mutant embryos. jmj is mainly expressed in the cyclin D1 negative region in the hindbrain, and cyclin D1 expression in the VZ was upregulated in jmj mutant mice. In jmj and cyclin D1 double mutant mice, the ectopic mitosis and formation of the abnormal clusters in the MZ were rescued. These results suggest that a jmj-cyclin D1 pathway is required for the precise coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain.

    Developmental biology 2007;303;2;549-60

  • Embedding of bone samples in methylmethacrylate: a suitable method for tracking LacZ mesenchymal stem cells in skeletal tissues.

    Hannouche D, Raould A, Nizard RS, Sedel L and Petite H

    Laboratoire de Recherches Orthopédiques, CNRS, Faculté de Médecine Lariboisière Saint-Louis, Université Paris 7, 75010 Paris, France. didier.hannouche@lrb.aphp.fr

    Considerable research has been focused on the use of bone marrow-derived mesenchymal stem cells (MSCs) for the repair of non-unions and bone defects. To date, the question of whether transplanted MSCs survive and engraft within newly formed tissue remains unresolved. The development of an easy and reliable method that would allow cell fate monitoring in transplant recipients is a pressing concern for the field of tissue engineering. To demonstrate the presence of transplanted cells in newly formed bone, we established a xenograft nude rat model allowing the detection of murine LacZ MSCs in vivo. MSCs were isolated from transgenic lacZ mice, seeded onto bioabsorbable collagen sponges, and transplanted to repair a calvarial defect in nude rats. As a preliminary step, the histological procedure was adapted to optimize the detection of LacZ cells in bone tissue embedded in methylmethacrylate (MMA). Four fixatives and four fixation times were evaluated. Among all the fixatives tested, 2% formaldehyde/0.2% glutaraldehyde at 4C for 4 days gave the best results for X-gal staining at pH 7.4 on both cell cultures and bone explants. All fixatives were effective for immunodetection of beta-gal. In the chimeric LacZ/nude rat animal model, MSCs were detected in vivo for up to 4 weeks after implantation and contributed to the repair and the neovascularization of the bone defect. LacZ is a suitable phenotypic marker to track MSCs in skeletal tissues embedded in MMA.

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2007;55;3;255-62

  • TGFbeta1 regulation of vimentin gene expression during differentiation of the C2C12 skeletal myogenic cell line requires Smads, AP-1 and Sp1 family members.

    Wu Y, Zhang X, Salmon M, Lin X and Zehner ZE

    Department of Biochemistry and the Massey Cancer Center, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0614, USA.

    Vimentin exhibits a complex pattern of developmental and tissue-specific expression regulated by such growth factors as TGFbeta1, PDGF, FGF, EGF and cytokines. Vimentin is expressed in the more migratory, mesenchymal cell and its expression is often down-regulated to make way for tissue-specific intermediate filaments proteins such as desmin in muscle. Here, we suggest a mechanism to explain how TGFbeta1 contributes to the up-regulation of vimentin expression while blocking myogenesis. TGFbeta1 binds to serine/threonine kinase receptors resulting in the phosphorylation of Smad2 and Smad3, followed by formation of a heteromeric complex with Smad4. The translocation of this complex to the nucleus modulates transcription of selected genes such as vimentin. However, the vimentin gene lacks a consensus TGFbeta1 response element. By transient transfection analysis of vimentin's various promoter elements fused to the CAT reporter gene, we have determined that tandem AP-1 sites surrounded by GC-boxes are required for TGFbeta1 induction. Mutations within this region eliminated the ability of Smad3 to induce reporter gene expression. DNA precipitation and ChIP assays suggest that c-Jun, c-Fos, Smad3 and Sp1/Sp3 interact over this region, but this interaction changes during myogenesis with TGFbeta1 induction.

    Funded by: NHLBI NIH HHS: HL-45422, R01 HL045422; NIDDK NIH HHS: DK073932, R01 DK073932

    Biochimica et biophysica acta 2007;1773;3;427-39

  • Doublecortin-like, a microtubule-associated protein expressed in radial glia, is crucial for neuronal precursor division and radial process stability.

    Vreugdenhil E, Kolk SM, Boekhoorn K, Fitzsimons CP, Schaaf M, Schouten T, Sarabdjitsingh A, Sibug R and Lucassen PJ

    Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden University Medical Center, PO Box 9502, 2300 RA Leiden, The Netherlands. vreugden@lacdr.leidenuniv.nl

    During corticogenesis, progenitors divide within the ventricular zone where they rely on radial process extensions, formed by radial glial cell (RG) scaffolds, along which they migrate to the proper layers of the cerebral cortex. Although the microtubule-associated proteins doublecortin (DCX) and doublecortin-like kinase (DCLK) are critically involved in dynamic rearrangement of the cytoskeletal machinery that allow migration, little is known about their role in early corticogenesis. Here we have functionally characterized a mouse splice-variant of DCLK, doublecortin-like (DCL), exhibiting 73% amino acid sequence identity with DCX over its entire length. Unlike DCX, DCL is expressed from embryonic day 8 onwards throughout the early neuroepithelium. It is localized in mitotic cells, RGs and radial processes. DCL knockdown using siRNA in vitro induces spindle collapse in dividing neuroblastoma cells, whereas overexpression results in elongated and asymmetrical mitotic spindles. In vivo knockdown of the DCLK gene by in utero electroporation significantly reduced cell numbers in the inner proliferative zones and dramatically disrupted most radial processes. Our data emphasize the unique role of the DCLK gene in mitotic spindle integrity during early neurogenesis. In addition, they indicate crucial involvement of DCLK in RG proliferation and their radial process stability, a finding that has thus far not been attributed to DCX or DCLK.

    The European journal of neuroscience 2007;25;3;635-48

  • Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication.

    Ma Z, Kanai M, Kawamura K, Kaibuchi K, Ye K and Fukasawa K

    Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521, USA.

    Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr(199) by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr(199) remains at centrosomes. It has been shown that Thr(199) phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr(199). Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr(199)-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

    Funded by: NCI NIH HHS: CA 95925, R01 CA095925

    Molecular and cellular biology 2006;26;23;9016-34

  • 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

  • Chondromodulin-I maintains cardiac valvular function by preventing angiogenesis.

    Yoshioka M, Yuasa S, Matsumura K, Kimura K, Shiomi T, Kimura N, Shukunami C, Okada Y, Mukai M, Shin H, Yozu R, Sata M, Ogawa S, Hiraki Y and Fukuda K

    Department of Regenerative Medicine and Advanced Cardiac Therapeutics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

    The avascularity of cardiac valves is abrogated in several valvular heart diseases (VHDs). This study investigated the molecular mechanisms underlying valvular avascularity and its correlation with VHD. Chondromodulin-I, an antiangiogenic factor isolated from cartilage, is abundantly expressed in cardiac valves. Gene targeting of chondromodulin-I resulted in enhanced Vegf-A expression, angiogenesis, lipid deposition and calcification in the cardiac valves of aged mice. Echocardiography showed aortic valve thickening, calcification and turbulent flow, indicative of early changes in aortic stenosis. Conditioned medium obtained from cultured valvular interstitial cells strongly inhibited tube formation and mobilization of endothelial cells and induced their apoptosis; these effects were partially inhibited by chondromodulin-I small interfering RNA. In human VHD, including cases associated with infective endocarditis, rheumatic heart disease and atherosclerosis, VEGF-A expression, neovascularization and calcification were observed in areas of chondromodulin-I downregulation. These findings provide evidence that chondromodulin-I has a pivotal role in maintaining valvular normal function by preventing angiogenesis that may lead to VHD.

    Nature medicine 2006;12;10;1151-9

  • Cell cycle deregulation and loss of stem cell phenotype in the subventricular zone of TGF-beta adaptor elf-/- mouse brain.

    Golestaneh N, Tang Y, Katuri V, Jogunoori W, Mishra L and Mishra B

    Laboratory of Developmental Neurobiology, Georgetown University School of Medicine, 3900 Reservoir Road NW, Medical/Dental Building, Room NW 209-213, Washington, DC 20007, USA.

    The mammalian forebrain subependyma contains neural stem cells and other proliferating progenitor cells. Recent studies have shown the importance of TGF-beta family members and their adaptor proteins in the inhibition of proliferation in the nervous system. Previously, we have demonstrated that TGF-beta induces phosphorylation and association of ELF (embryonic liver fodrin) with Smad3 and Smad4 resulting in nuclear translocation. Elf(-/-) mice manifest abnormal neuronal differentiation, with loss of neuroepithelial progenitor cell phenotype in the subventricular zone (SVZ) with dramatic marginal cell hyperplasia and loss of nestin expression. Here, we have analyzed the expression of cell cycle-associated proteins cdk4, mdm2, p21, and pRb family members in the brain of elf(-/-) mice to verify the role of elf in the regulation of neural precursor cells in the mammalian brain. Increased proliferation in SVZ cells of the mutant mice coincided with higher levels of cdk4 and mdm2 expression. A lesser degree of apoptosis was observed in the mutant mice compared to the wild-type control. Elf(-/-) embryos showed elevated levels of hyperphosphorylated forms of pRb, p130 and p107 and decreased level of p21 compared to the wild-type control. These results establish a critical role for elf in the development of a SVZ neuroepithelial stem cell phenotype and regulation of neuroepithelial cell proliferation, suggesting that a mutation in the elf locus renders the cells susceptible to a faster entry into S phase of cell cycle and resistance to senescence and apoptotic stimuli.

    Funded by: NCI NIH HHS: R01 CA106614A; NIDDK NIH HHS: R01 DK56111, R01 DK58637

    Brain research 2006;1108;1;45-53

  • Protein phosphatase 6 subunit with conserved Sit4-associated protein domain targets IkappaBepsilon.

    Stefansson B and Brautigan DL

    Center for Cell Signaling, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

    Protein Ser/Thr phosphatases compose a PPP family that includes type-2 PP2A, PP4, and PP6, each with essential functions. The human PP6 gene rescues sit4(ts) mutants of Saccharomyces cerevisiae, and Sit4 phosphatase function depends on multiple Sit4-associated protein (SAP) subunits. We report here finding a SAPS sequence domain encoded in only a single gene each in Schizosaccharomyces pombe, Caenorhabditis elegans, and Drosophila but in three distinct open reading frames in Xenopus, Mus musculus, and Homo sapiens. The SAPS proteins are more divergent in sequence than PP6. Northern hybridization showed differential distribution of the human SAPS-related mRNA in multiple human tissues, named as PP6R1, PP6R2, and PP6R3. Antibodies were generated, distribution of endogenous PP6, PP6R1, PP6R2, and PP6R3 proteins was examined by immunoblotting, and the abundance of mRNA and protein in various tissues did not match. FLAG-tagged PP6R1 and PP6R2 expressed in HEK293 cells co-precipitated endogenous PP6, but not PP2A or PP4, showing specificity for recognition of phosphatases. The SAPS domain of PP6R1 alone was sufficient for association with PP6, and this predicts that conserved sequence motifs in the SAPS domain accounts for the specificity. FLAG-PP6R1 and FLAG-PP6R2 co-precipitated HA-IkappaBepsilon. Knockdown of PP6 or PP6R1 but not PP6R3 with siRNA significantly enhanced degradation of endogenous IkappaBepsilon in response to tumor necrosis factor-alpha. The results show SAPS domain subunits recruit substrates such as IkappaBepsilon as one way to determine specific functions for PP6.

    Funded by: NCI NIH HHS: CA-77584

    The Journal of biological chemistry 2006;281;32;22624-34

  • Identification of candidate genes at the corticoseptal boundary during development.

    Shen WB, Plachez C, Mongi AS and Richards LJ

    Department of Anatomy and Neurobiology and The Program in Neuroscience, The University of Maryland School of Medicine, Baltimore, MD 21201, USA.

    Cortical midline glia are critical to the formation of the corpus callosum during development. The glial wedge is a population of midline glia that is located at the corticoseptal boundary and expresses repulsive/growth-inhibitory molecules that guide callosal axons as they cross the midline. The glial wedge are the first cells within the cortex to express GFAP and thus may express molecules specific for glial maturation. The corticoseptal boundary is a genetically defined boundary between the cingulate cortex (dorsal telencephalon) and the septum (ventral telencephalon). The correct dorso-ventral position of this boundary is vital to the formation of both the glial wedge and the corpus callosum. Our aim was to identify genes expressed specifically within the glial wedge that might be involved in either glial differentiation, formation of the corticoseptal boundary or development of the corpus callosum. To identify such genes we have performed a differential display PCR screen comparing RNA isolated from the glial wedge with RNA isolated from control tissues such as the neocortex and septum, of embryonic day 17 mouse brains. Using 200 different combinations of primers, we identified and cloned 67 distinct gene fragments. In situ hybridization analysis confirmed the differential expression of many of the genes, and showed that clones G24F3, G39F8 and transcription factor LZIP have specific expression patterns in the telencephalon of embryonic and postnatal brains. An RNase Protection Assay (RPA) revealed that the expression of G39F8, G24F3 and LZIP increase markedly in the telencephalon at E16 and continue to be expressed until at least P0, during the period when the corpus callosum is forming.

    Gene expression patterns : GEP 2006;6;5;471-81

  • Nuclear receptor TLX prevents retinal dystrophy and recruits the corepressor atrophin1.

    Zhang CL, Zou Y, Yu RT, Gage FH and Evans RM

    Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

    During mammalian embryogenesis, precise coordination of progenitor cell proliferation and differentiation is essential for proper organ size and function. The involvement of TLX (NR2E1), an orphan nuclear receptor, has been implicated in ocular development, as Tlx-/- mice exhibit visual impairment. Using genetic and biochemical approaches, we show that TLX modulates retinal progenitor cell proliferation and cell cycle re-entry by directly regulating the expression of Pten and its target cyclin D1. Additionally, TLX finely tunes the progenitor differentiation program by modulating the phospholipase C and mitogen-activated protein kinase (MAPK) pathways and the expression of an array of cell type-specific transcriptional regulators. Consequently, Tlx-/- mice have a dramatic reduction in retina thickness and enhanced generation of S-cones, and develop severe early onset retinal dystrophy. Furthermore, TLX interacts with atrophin1 (Atn1), a corepressor that is involved in human neurodegenerative dentatorubral-pallidoluysian atrophy (DRPLA) and that is essential for development of multiple tissues. Together, these results reveal a molecular strategy by which an orphan nuclear receptor can precisely orchestrate tissue-specific proliferation and differentiation programs to prevent retinal malformation and degeneration.

    Funded by: NICHD NIH HHS: HD27183, R01 HD027183

    Genes & development 2006;20;10;1308-20

  • 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

  • Vimentin function in lymphocyte adhesion and transcellular migration.

    Nieminen M, Henttinen T, Merinen M, Marttila-Ichihara F, Eriksson JE and Jalkanen S

    Turku Centre for Biotechnology, University of Turku and Abo Akademi University, P.O.Box 123, FIN- 20521 Turku, Finland.

    Although the adhesive interactions of leukocytes with endothelial cells are well understood, little is known about the detailed mechanisms underlying the actual migration of leukocytes across the endothelium (diapedesis). Leukocytes have been shown to use both paracellular and transcellular routes for transendothelial migration. Here we show that peripheral blood mononuclear cells (PBMCs; T- and B-lymphocytes) preferentially use the transcellular route. The intermediate filaments of both endothelial cells and lymphocytes formed a highly dynamic anchoring structure at the site of contact between these two cell types. The initiation of this process was markedly reduced in vimentin-deficient (vim(-/-)) PBMCs and endothelial cells. When compared with wild-type PBMCs, vim(-/-) PBMCs showed a markedly reduced capacity to home to mesenteric lymph nodes and spleen. Furthermore, endothelial integrity was compromised in vim(-/-) mice, demonstrating that intermediate filaments also regulate the barrier that governs leukocyte extravasation. Absence of vimentin resulted in highly aberrant expression and distribution of surface molecules critical for homing (ICAM-1 and VCAM-1 on endothelial cells and integrin-beta1 on PBMCs). These data show that intermediate filaments are active in lymphocyte adhesion and transmigration.

    Nature cell biology 2006;8;2;156-62

  • Doublecortin-like kinase functions with doublecortin to mediate fiber tract decussation and neuronal migration.

    Koizumi H, Tanaka T and Gleeson JG

    Neurogenetics Laboratory, Department of Neurosciences, University of California, San Diego, La Jolla, California 93093, USA.

    The potential role of doublecortin (Dcx), encoding a microtubule-associated protein, in brain development has remained controversial. Humans with mutations show profound alterations in cortical lamination, whereas in mouse, RNAi-mediated knockdown but not germline knockout shows abnormal positioning of cortical neurons. Here, we report that the doublecortin-like kinase (Dclk) gene functions in a partially redundant pathway with Dcx in the formation of axonal projections across the midline and migration of cortical neurons. Dosage-dependent genetic effects were observed in both interhemispheric connectivity and migration of cortically and subcortically derived neurons. Surprisingly, RNAi-mediated knockdown of either gene results in similar migration defects. These results indicate the Dcx microtubule-associated protein family is required for proper neuronal migration and axonal wiring.

    Funded by: NINDS NIH HHS: NS41537, NS42749, NS47101, R01 NS041537

    Neuron 2006;49;1;55-66

  • Mutations in vimentin disrupt the cytoskeleton in fibroblasts and delay execution of apoptosis.

    Schietke R, Bröhl D, Wedig T, Mücke N, Herrmann H and Magin TM

    Institut für Physiologische Chemie, Abteilung für Zellbiochemie and LIMES, Universitätsklinikum Bonn, Nussallee 11, D-53115 Bonn, Germany.

    To get new insights into the function of the intermediate filament (IF) protein vimentin in cell physiology, we generated two mutant cDNAs, one with a point mutation in the consensus motif in coil1A (R113C) and one with the complete deletion of coil 2B of the rod domain. In keratins and glia filament protein (GFAP), analogous mutations cause keratinopathies and Alexander disease, respectively. Both mutants prevented filament assembly in vitro and inhibited assembly of wild-type vimentin when present in equal amounts. In stably transfected preadipocytes, these mutants caused the complete disruption of the endogenous vimentin network, demonstrating their dominant-negative behaviour. Cytoplasmic vimentin aggregates colocalised with the chaperones alphaB-crystallin and HSP40. Moreover, vimR113C mutant cells were more resistant against staurosporine-induced apoptosis compared to controls. We hypothesise that mutations in the vimentin gene, like in most classes of IF genes, may contribute to distinct human diseases.

    European journal of cell biology 2006;85;1;1-10

  • PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility.

    Ivaska J, Vuoriluoto K, Huovinen T, Izawa I, Inagaki M and Parker PJ

    VTT Technical Research Centre for Finland, Medical Biotechnology and University of Turku Centre for Biotechnology, Turku, Finland.

    PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.

    The EMBO journal 2005;24;22;3834-45

  • Low-density lipoprotein receptor-related protein (LRP)-2/megalin is transiently expressed in a subpopulation of neural progenitors in the embryonic mouse spinal cord.

    Wicher G, Larsson M, Rask L and Aldskogius H

    Department of Neuroscience, Biomedical Center, Uppsala University, Sweden. Grzegorz.Wicher@neuro.uu.se

    The lipoprotein receptor LRP2/megalin is expressed by absorptive epithelia and involved in receptor-mediated endocytosis of a wide range of ligands. Megalin is expressed in the neuroepithelium during central nervous system (CNS) development. Mice with homozygous deletions of the megalin gene show severe forebrain abnormalities. The possible role of megalin in the developing spinal cord, however, is unknown. Here we examined the spatial and temporal expression pattern of megalin in the embryonic mouse spinal cord using an antibody that specifically recognizes the cytoplasmic part of the megalin molecule. In line with published data, we show expression of megalin in ependymal cells of the central canal from embryonic day (E)11 until birth. In addition, from E11 until E15 a population of cells was found in the dorsal part of the developing spinal cord strongly immunoreactive against megalin. Double labeling showed that most of these cells express vimentin, a marker for immature astrocytes and radial glia, but not brain lipid binding protein (BLBP), a marker for radial glial cells, or glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. These findings indicate that the majority of the megalin-positive cells are astroglial precursors. Megalin immunoreactivity was mainly localized in the nuclei of these cells, suggesting that the cytoplasmic part of the megalin molecule can be cleaved following ligand binding and translocated to the nucleus to act as a transcription factor or regulate other transcription factors. These findings suggest that megalin has a crucial role in the development of astrocytes of the spinal cord.

    The Journal of comparative neurology 2005;492;2;123-31

  • Mouse Polycomb M33 is required for splenic vascular and adrenal gland formation through regulating Ad4BP/SF1 expression.

    Katoh-Fukui Y, Owaki A, Toyama Y, Kusaka M, Shinohara Y, Maekawa M, Toshimori K and Morohashi K

    Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. yfukui@nibb.ac.jp

    Mice with disrupted mammalian PcG (Polycomb group) genes commonly show skeletal transformation of anterior-posterior identities. Disruption of the murine M33 gene, a PcG member, displayed posterior transformation of the vertebral columns and sternal ribs. In addition, failure of T-cell expansion and hypoplasia and sex-reversal of the gonads, have been observed. In the present study, we identified defects in the splenic and adrenal formation of M33-knock-out (KO) mice on a C57BL/6 genetic background. The spleen in these animals was smaller than in the wild-type mice and was spotted red because of nonuniform distribution of blood cells. Histologic examination revealed disorganization of the vascular endothelium and its surrounding structures, and immunohistochemistry demonstrated disturbances in vascular formation and colonization of immature hematopoietic cells. These splenic phenotypes observed in the M33-KO mice were quite similar to those seen in Ad4BP/SF1 (Nr5a1) knock-outs. Moreover, the adrenal glands of M33-KO and Ad4BP/SF1 heterozygous KO mice were smaller than those of the wild-type mice. Western blot, immunohistochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of the M33 knock-outs all indicated significantly low expression of adrenal 4 binding protein/steroidogenic factor-1 (Ad4BP/SF-1), indicating that M33 is an essential upstream regulator of Ad4BP/SF1. In agreement with these observations, chromatin immunoprecipitation assays with adrenocortical Y-1 cells revealed direct binding of the M33-containing PcG to the Ad4BP/SF1 gene locus.

    Blood 2005;106;5;1612-20

  • A dual role of FGF10 in proliferation and coordinated migration of epithelial leading edge cells during mouse eyelid development.

    Tao H, Shimizu M, Kusumoto R, Ono K, Noji S and Ohuchi H

    Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minami-Jyosanjima, Tokushima 770-8506, Japan.

    The development of the eyelid requires coordinated cellular processes of proliferation, cell shape changes, migration and cell death. Mutant mice deficient in the fibroblast growth factor 10 (Fgf10) gene exhibit open-eyelids at birth. To elucidate the roles of FGF10 during eyelid formation, we examined the expression pattern of Fgf10 during eyelid formation and the phenotype of Fgf10-null eyelids in detail. Fgf10 is expressed by mesenchymal cells just beneath the protruding epidermal cells of the nascent eyelid. However, Fgf10-null epithelial cells running though the eyelid groove do not exhibit typical cuboid shape or sufficient proliferation. Furthermore, peridermal clumps are not maintained on the eyelid leading edge, and epithelial extension does not occur. At the cellular level, the accumulation of actin fibers is not observed in the mutant epithelial leading edge. The expression of activin/inhibin betaB (ActbetaB/Inhbb) and transforming growth factor alpha (Tgfa), previously reported to be crucial for eyelid development, is down-regulated in the mutant leading edge, while the onset of sonic hedgehog (Shh) expression is delayed on the mutant eyelid margin. Explant cultures of mouse eyelid primordia shows that the open-eyelid phenotype of the mutant is reduced by exogenous FGF10 protein, and that the expression of ActbetaB and Tgfa is ectopically induced in the thickened eyelid epithelium by the FGF10 protein. These results indicate a dual role of FGF10 in mouse eyelid development, for both proliferation and coordinated migration of eyelid epithelial cells by reorganization of the cytoskeleton, through the regulation of activin, TGFalpha and SHH signaling.

    Development (Cambridge, England) 2005;132;14;3217-30

  • Developmental abnormalities of neuronal structure and function in prenatal mice lacking the prader-willi syndrome gene necdin.

    Pagliardini S, Ren J, Wevrick R and Greer JJ

    Department of Physiology, Centre of Neuroscience, University of Alberta, Edmonton, Alberta, T6G 2S2, Canada.

    Necdin (Ndn) is one of a cluster of genes deleted in the neurodevelopmental disorder Prader-Willi syndrome (PWS). Ndntm2Stw mutant mice die shortly after birth because of abnormal respiratory rhythmogenesis generated by a key medullary nucleus, the pre-Bötzinger complex (preBötC). Here, we address two fundamental issues relevant to its pathogenesis. First, we performed a detailed anatomical study of the developing medulla to determine whether there were defects within the preBötC or synaptic inputs that regulate respiratory rhythmogenesis. Second, in vitro studies determined if the unstable respiratory rhythm in Ndntm2Stw mice could be normalized by neuromodulators. Anatomical defects in Ndntm2Stw mice included defasciculation and irregular projections of axonal tracts, aberrant neuronal migration, and a major defect in the cytoarchitecture of the cuneate/gracile nuclei, including dystrophic axons. Exogenous application of neuromodulators alleviated the long periods of slow respiratory rhythms and apnea, but some instability of rhythmogenesis persisted. We conclude that deficiencies in the neuromodulatory drive necessary for preBötC function contribute to respiratory dysfunction of Ndntm2Stw mice. These abnormalities are part of a more widespread deficit in neuronal migration and the extension, arborization, and fasciculation of axons during early stages of central nervous system development that may account for respiratory, sensory, motor, and behavioral problems associated with PWS.

    The American journal of pathology 2005;167;1;175-91

  • DNER acts as a neuron-specific Notch ligand during Bergmann glial development.

    Eiraku M, Tohgo A, Ono K, Kaneko M, Fujishima K, Hirano T and Kengaku M

    Laboratory for Neural Cell Polarity, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

    Differentiation of CNS glia is regulated by Notch signaling through neuron-glia interaction. Here, we identified Delta/Notch-like EGF-related receptor (DNER), a neuron-specific transmembrane protein, as a previously unknown ligand of Notch during cellular morphogenesis of Bergmann glia in the mouse cerebellum. DNER binds to Notch1 at cell-cell contacts and activates Notch signaling in vitro. In the developing cerebellum, DNER is highly expressed in Purkinje cell dendrites, which are tightly associated with radial fibers of Bergmann glia expressing Notch. DNER specifically binds to Bergmann glia in culture and induces process extension by activating gamma-secretase- and Deltex-dependent Notch signaling. Inhibition of Deltex-dependent, but not RBP-J-dependent, Notch signaling in Bergmann glia suppresses formation and maturation of radial fibers in organotypic slice cultures. Additionally, deficiency of DNER retards the formation of radial fibers and results in abnormal arrangement of Bergmann glia. Thus, DNER mediates neuron-glia interaction and promotes morphological differentiation of Bergmann glia through Deltex-dependent Notch signaling.

    Nature neuroscience 2005;8;7;873-80

  • The forkhead box m1 transcription factor is essential for embryonic development of pulmonary vasculature.

    Kim IM, Ramakrishna S, Gusarova GA, Yoder HM, Costa RH and Kalinichenko VV

    Department of Medicine and Committee on Developmental Biology, The University of Chicago, Chicago, IL 60637, USA.

    Transgenic and gene knock-out studies demonstrated that the mouse Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) is essential for hepatocyte entry into mitosis during liver development, regeneration, and liver cancer. Targeted deletion of Foxm1 gene in mice produces an embryonic lethal phenotype due to severe abnormalities in the development of liver and heart. In this study, we show for the first time that Foxm1(-/-) lungs exhibit severe hypertrophy of arteriolar smooth muscle cells and defects in the formation of peripheral pulmonary capillaries as evidenced by significant reduction in platelet endothelial cell adhesion molecule 1 staining of the distal lung. Consistent with these findings, significant reduction in proliferation of the embryonic Foxm1(-/-) lung mesenchyme was found, yet proliferation levels were normal in the Foxm1-deficient epithelial cells. Severe abnormalities of the lung vasculature in Foxm1(-/-) embryos were associated with diminished expression of the transforming growth factor beta receptor II, a disintegrin and metalloprotease domain 17 (ADAM-17), vascular endothelial growth factor receptors, Polo-like kinase 1, Aurora B kinase, laminin alpha4 (Lama4), and the Forkhead Box f1 transcription factor. Cotransfection studies demonstrated that Foxm1 stimulates transcription of the Lama4 promoter, and this stimulation requires the Foxm1 binding sites located between -1174 and -1145 bp of the mouse Lama4 promoter. In summary, development of mouse lungs depends on the Foxm1 transcription factor, which regulates expression of genes essential for mesenchyme proliferation, extracellular matrix remodeling, and vasculogenesis.

    Funded by: NIDDK NIH HHS: DK 54687-06

    The Journal of biological chemistry 2005;280;23;22278-86

  • The intermediate filament protein vimentin is a new target for epigallocatechin gallate.

    Ermakova S, Choi BY, Choi HS, Kang BS, Bode AM and Dong Z

    Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA.

    Epigallocatechin gallate (EGCG) is the major active polyphenol in green tea. Protein interaction with EGCG is a critical step in the effects of EGCG on the regulation of various key proteins involved in signal transduction. We have identified a novel molecular target of EGCG using affinity chromatography, two-dimensional electrophoresis, and mass spectrometry for protein identification. Spots of interest were identified as the intermediate filament, vimentin. The identification was confirmed by Western blot analysis using an anti-vimentin antibody. Experiments using a pull-down assay with [3H]EGCG demonstrate binding of EGCG to vimentin with a Kd of 3.3 nm. EGCG inhibited phosphorylation of vimentin at serines 50 and 55 and phosphorylation of vimentin by cyclin-dependent kinase 2 and cAMP-dependent protein kinase. EGCG specifically inhibits cell proliferation by binding to vimentin. Because vimentin is important for maintaining cellular functions and is essential in maintaining the structure and mechanical integration of the cellular space, the inhibitory effect of EGCG on vimentin may further explain its anti-tumor-promoting effect.

    Funded by: NCI NIH HHS: CA81064, CA88961

    The Journal of biological chemistry 2005;280;17;16882-90

  • The stomach mesenchymal transcription factor Barx1 specifies gastric epithelial identity through inhibition of transient Wnt signaling.

    Kim BM, Buchner G, Miletich I, Sharpe PT and Shivdasani RA

    Department of Medical Oncology, Dana-Farber Cancer Institute, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.

    Inductive interactions between gut endoderm and the underlying mesenchyme pattern the developing digestive tract into regions with specific morphology and functions. The molecular mechanisms behind these interactions are largely unknown. Expression of the conserved homeobox gene Barx1 is restricted to the stomach mesenchyme during gut organogenesis. Using recombinant tissue cultures, we show that Barx1 loss in the mesenchyme prevents stomach epithelial differentiation of overlying endoderm and induces intestine-specific genes instead. Additionally, Barx1 null mouse embryos show visceral homeosis, with intestinal gene expression within a highly disorganized gastric epithelium. Barx1 directs mesenchymal cell expression of two secreted Wnt antagonists, sFRP1 and sFRP2, and these factors are sufficient replacements for Barx1 function. Canonical Wnt signaling is prominent in the prospective gastric endoderm prior to epithelial differentiation, and its inhibition by Barx1-dependent signaling permits development of stomach-specific epithelium. These results define a transcriptional and signaling pathway of inductive cell interactions in vertebrate organogenesis.

    Funded by: NIDDK NIH HHS: R01-DK61139

    Developmental cell 2005;8;4;611-22

  • Ganglion cells are required for normal progenitor- cell proliferation but not cell-fate determination or patterning in the developing mouse retina.

    Mu X, Fu X, Sun H, Liang S, Maeda H, Frishman LJ and Klein WH

    Department of Biochemistry and Molecular Biology and Graduate program in Genes and Development, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.

    The vertebrate retina develops from an amorphous sheet of dividing retinal progenitor cells (RPCs) through a sequential process that culminates in an exquisitely patterned neural tissue. A current model for retinal development posits that sequential cell-type differentiation is the result of changes in the intrinsic competence state of multipotent RPCs as they advance in time and that the intrinsic changes are influenced by continuous changes in the extracellular environment. Although several studies support the proposition that newly differentiated cells alter the extrinsic state of the developing retina, it is still far from clear what role they play in modifying the extracellular environment and in influencing the properties of RPCs. Here, we specifically ablate retinal ganglion cells (RGCs) as they differentiate, and we determine the impact of RGC absence on retinal development. We find that RGCs are not essential for changing the competence of RPCs, but they are necessary for maintaining sufficient numbers of RPCs by regulating cell proliferation via growth factors. Intrinsic rather than extrinsic factors are likely to play the critical roles in determining retinal cell fate.

    Funded by: NCI NIH HHS: CA16672; NEI NIH HHS: EY006671, EY011930

    Current biology : CB 2005;15;6;525-30

  • Lipid rafts associate with intracellular B cell receptors and exhibit a B cell stage-specific protein composition.

    Mielenz D, Vettermann C, Hampel M, Lang C, Avramidou A, Karas M and Jäck HM

    Division of Molecular Immunology, Department of Internal Medicine III, University of Erlangen, Erlangen, Germany. dmielenz@molmed.uni-erlangen.de

    Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits alpha-1 and beta-2, vimentin, gamma-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;6;3508-17

  • Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve.

    Perlson E, Hanz S, Ben-Yaakov K, Segal-Ruder Y, Seger R and Fainzilber M

    Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel.

    How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.

    Neuron 2005;45;5;715-26

  • Re-establishing the regenerative potential of central nervous system axons in postnatal mice.

    Cho KS, Yang L, Lu B, Feng Ma H, Huang X, Pekny M and Chen DF

    Schepens Eye Research Institute, Program in Neuroscience and Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA.

    At a certain point in development, axons in the mammalian central nervous system lose their ability to regenerate after injury. Using the optic nerve model, we show that this growth failure coincides with two developmental events: the loss of Bcl-2 expression by neurons and the maturation of astrocytes. Before postnatal day 4, when astrocytes are immature, overexpression of Bcl-2 alone supported robust and rapid optic nerve regeneration over long distances, leading to innervation of brain targets by day 4 in mice. As astrocytes matured after postnatal day 4, axonal regeneration was inhibited in mice overexpressing Bcl-2. Concurrent induction of Bcl-2 and attenuation of reactive gliosis reversed the failure of CNS axonal re-elongation in postnatal mice and led to rapid axonal regeneration over long distances and reinnervation of the brain targets by a majority of severed optic nerve fibers up to 2 weeks of age. These results suggest that an early postnatal downregulation of Bcl-2 and post-traumatic reactive gliosis are two important elements of axon regenerative failure in the CNS.

    Funded by: NEI NIH HHS: R01 EY012983

    Journal of cell science 2005;118;Pt 5;863-72

  • Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate progenitor cells, and postmitotic neurons in developing neocortex.

    Englund C, Fink A, Lau C, Pham D, Daza RA, Bulfone A, Kowalczyk T and Hevner RF

    Department of Pathology, University of Washington, Seattle, Washington 98195-7470, USA.

    The developing neocortex contains two types of progenitor cells for glutamatergic, pyramidal-projection neurons. The first type, radial glia, produce neurons and glia, divide at the ventricular surface, and express Pax6, a homeodomain transcription factor. The second type, intermediate progenitor cells, are derived from radial glia, produce only neurons, and divide away from the ventricular surface. Here we show that the transition from radial glia to intermediate progenitor cell is associated with upregulation of Tbr2, a T-domain transcription factor, and downregulation of Pax6. Accordingly, Tbr2 expression in progenitor compartments (the subventricular zone and ventricular zone) rises and falls with cortical plate neurogenesis. The subsequent transition from intermediate progenitor cell to postmitotic neuron is marked by downregulation of Tbr2 and upregulation of Tbr1, another T-domain transcription factor. These findings delineate the transcription factor sequence Pax6 --> Tbr2 --> Tbr1 in the differentiation of radial glia --> intermediate progenitor cell --> postmitotic projection neuron. This transcription factor sequence is modified in preplate neurons, in which Tbr2 is transiently coexpressed with Tbr1, and in the direct differentiation pathway from radial glia --> postmitotic projection neuron, in which Tbr2 is expressed briefly or not at all.

    Funded by: NINDS NIH HHS: K02 NS045018

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;1;247-51

  • Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas.

    Watahiki A, Waki K, Hayatsu N, Shiraki T, Kondo S, Nakamura M, Sasaki D, Arakawa T, Kawai J, Harbers M, Hayashizaki Y and Carninci P

    Genome Science Laboratory, RIKEN, Wako main campus, 2-1 Hirosawa, Wako, Saitama, 351-0198 Japan.

    It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.

    Nature methods 2004;1;3;233-9

  • Increased cell proliferation and neurogenesis in the hippocampal dentate gyrus of old GFAP(-/-)Vim(-/-) mice.

    Larsson A, Wilhelmsson U, Pekna M and Pekny M

    Department of Medical Biochemistry, Sahlgrenska Academy at Göteborg University, Göteborg, Sweden.

    In response to central nervous system (CNS) injury, and more discretely so also during aging, astrocytes become reactive and increase their expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Studies of mice deficient in astrocytic intermediate filaments have provided insights into the function of reactive gliosis. Recently we demonstrated robust integration of retinal transplants (1) and increased posttraumatic synaptic regeneration (2) in GFAP(-/-)Vim(-/-) mice, suggesting that modulation of astrocyte activity affects the permissiveness of the CNS environment for regeneration. Neurogenesis in the adult mammalian CNS is restricted to essentially two regions, the hippocampus and the subventricular zone. Here, we assessed neurogenesis in the hippocampus of 18-month-old GFAP(-/-)Vim(-/-) mice. In the granular layer of the dentate gyrus, cell proliferation/survival was 34% higher and neurogenesis 36% higher in GFAP(-/-)Vim(-/-) mice than in wildtype controls. These findings suggest that the adult hippocampal neurogenesis in healthy old mice can be increased by modulating astrocyte reactivity.

    Neurochemical research 2004;29;11;2069-73

  • Recovery of Na-glucose cotransport activity after renal ischemia is impaired in mice lacking vimentin.

    Runembert I, Couette S, Federici P, Colucci-Guyon E, Babinet C, Briand P, Friedlander G and Terzi F

    INSERM U426, Faculté de Médecine Xavier Bichat, 16 Rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France.

    Vimentin, an intermediate filament protein mainly expressed in mesenchyma-derived cells, is reexpressed in renal tubular epithelial cells under many pathological conditions, characterized by intense cell proliferation. Whether vimentin reexpression is only a marker of cell dedifferentiation or is instrumental in the maintenance of cell structure and/or function is still unknown. Here, we used vimentin knockout mice (Vim(-/-)) and an experimental model of acute renal injury (30-min bilateral renal ischemia) to explore the role of vimentin. Bilateral renal ischemia induced an initial phase of acute tubular necrosis that did not require vimentin and was similar, in terms of morphological and functional changes, in Vim(+/+) and Vim(-/-) mice. However, vimentin was essential to favor Na-glucose cotransporter 1 localization to brush-border membranes and to restore Na-glucose cotransport activity in regenerating tubular cells. We show that the effect of vimentin inactivation is specific and results in persistent glucosuria. We propose that vimentin is part of a structural network that favors carrier localization to plasma membranes to restore transport activity in injured kidneys.

    American journal of physiology. Renal physiology 2004;287;5;F960-8

  • Morphological analysis of glutaraldehyde-fixed vimentin intermediate filaments and assembly-intermediates by atomic force microscopy.

    Ando S, Nakao K, Gohara R, Takasaki Y, Suehiro K and Oishi Y

    Division of Biopolymer Research, Department of Biomolecular Sciences, Saga Medical School, Saga 849-8501, Japan. andohs@post.saga-med.ac.jp

    Atomic force microscopy (AFM) was used to study the morphology of vimentin intermediate filaments (IFs) and their assembly intermediates. At each time after initiation of IF assembly in vitro of recombinant mouse vimentin, the sample was fixed with 0.1% glutaraldehyde and then applied to AFM analysis. When mature vimentin IFs were imaged in air on mica, they appeared to have a width of approximately 28 nm, a height of approximately 4 nm and a length of several micrometers. Taking into account the probe tip's distortion effect, the exact width was evaluated to be approximately 25 nm, suggesting that the filaments flatten on the substrate rather than be cylindrical with a diameter of approximately 10 nm. Vimentin IFs in air clearly demonstrated approximately 21-nm repeating patterns along the filament axis. The three-dimensional profiles of vimentin IFs indicated that the characteristic patterns were presented by repeating segments with a convex surface. The repeating patterns close to 21 nm were also observed by AFM analysis in a physiological solution condition, suggesting that the segments along the filaments are an intrinsic substructure of vimentin IFs. In the course of IF assembly, assembly intermediates were analyzed in air. Many short filaments with a full-width and an apparent length of approximately 78 nm (evaluated length approximately 69 nm) were observed immediately after initiation of the assembly reaction. Interestingly, the short full-width filaments appeared to be composed of the four segments. Further incubation enabled the short full-width filaments to anneal longitudinally into longer filaments with a distinct elongation step of approximately 40 nm, which corresponds to the length of the two segments. To explain these observations, we propose a vimentin IF formation model in which vimentin dimers are supercoiling around the filament axis.

    Biochimica et biophysica acta 2004;1702;1;53-65

  • Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2.

    Kolterud A, Wandzioch E and Carlsson L

    Umeå Centre for Molecular Medicine, Umeå University, 901 87 Umeå, Sweden.

    Liver development is based on reciprocal interactions between ventral foregut endoderm and adjacent mesenchymal tissues. Targeted disruption of the LIM-homeobox gene Lhx2 has revealed that it is important for the expansion of the liver during embryonic development, whereas it appears not to be involved in the induction of hepatic fate. It is not known whether Lhx2 is expressed in the endodermal or mesenchymal portion of the liver, or if the cells normally expressing Lhx2 are absent or present in the liver of Lhx2(-/-) embryos. To address this we have analyzed gene expression from the Lhx2 locus during hepatic development in wild type and Lhx2(-/-) mice. Lhx2 is expressed in cells of the septum transversum mesenchyme adjacent to the liver bud from embryonic day 9. The hepatic cords subsequently migrate into and intermingle with the Lhx2+ cells of the septum transversum mesenchyme. Lhx2 expression is thereafter maintained in a subpopulation of mesenchymal cells in the liver until adult life. In adult liver the Lhx2+ mesenchymal cells co-express desmin, a marker associated with stellate cells. At embryonic day 10.5, cells expressing the mutant Lhx2 allel are present in Lhx2(-/-) livers, and expression of Hlx, hepatocyte growth factor, Hex and Prox1, genes known to be important in liver development, is independent of functional Lhx2 expression. Thus, Lhx2 is specifically expressed in the liver-associated septum transversum mesenchyme that subsequently becomes an integral part of the liver and the formation of these mesenchymal cells does not require functional Lhx2.

    Gene expression patterns : GEP 2004;4;5;521-8

  • Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas.

    Esni F, Ghosh B, Biankin AV, Lin JW, Albert MA, Yu X, MacDonald RJ, Civin CI, Real FX, Pack MA, Ball DW and Leach SD

    Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

    Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.

    Funded by: NCI NIH HHS: CA 70244; NIDDK NIH HHS: DK 61215, DK 62110, T32-DK 077130

    Development (Cambridge, England) 2004;131;17;4213-24

  • Phorbol ester-induced translocation of PKC epsilon to the nucleus in fibroblasts: identification of nuclear PKC epsilon-associating proteins.

    Xu TR and Rumsby MG

    Department of Biology, University of York, York YO10 5DD, UK.

    We show that phorbol ester treatment of NIH 3T3 fibroblasts induces rapid translocation of PKC from a perinuclear site to the nucleus, extending findings in PC12 and NG108-15 cells and in myocytes. We have immunoprecipitated the PKC from nuclei isolated from phorbol ester-treated fibroblasts and identified six proteins which associate with nuclear PKC. These have been characterised as matrin 3, transferrin, Rac GTPase activating protein 1, vimentin, beta-actin and annexin II by MALDI-TOF-MS. We have confirmed that these proteins associate with PKC by gel overlay and/or dot blotting assays. The role of these PKC-associating proteins in the nucleus and their interaction with PKC are considered.

    FEBS letters 2004;570;1-3;20-4

  • Under stress, the absence of intermediate filaments from Müller cells in the retina has structural and functional consequences.

    Lundkvist A, Reichenbach A, Betsholtz C, Carmeliet P, Wolburg H and Pekny M

    Department of Medical Biochemistry, The Sahlgrenska Academy at Göteborg University, Medicinaregatan 9C, SE-41390 Göteborg, Sweden.

    In epithelial and muscle cells, intermediate filaments (IFs) are important for resistance to mechanical stress. The aim of this study was to elucidate whether IFs are also important for providing resistance to mechanical stress in the Müller cells of the retina and whether this has any pathophysiological consequences. We used mice deficient in IF proteins glial fibrillary acidic protein and/or vimentin (GFAP(-/-), Vim(-/-) and GFAP(-/-) Vim(-/-)), and stress on the retina was applied by excision of the eyes immediately post mortem (compared with in situ fixation) or by inducing a neovascular response to oxygen-induced retinopathy (OIR). The structure of unchallenged retinas was normal, but mechanical stress caused local separation of the inner limiting membrane (ILM) and adjacent tissue from the rest of the retina in GFAP(-/-) Vim(-/-) mice and, to a lesser extent, in Vim(-/-) mice. This detachment occurred within the endfeet of Müller cells, structures normally rich in IFs but IF-free in GFAP(-/-) Vim(-/-) mice. Hypoxia-induced neovascularization was comparable in all groups of mice with respect to the retinal surface area occupied by new vessels. However, the vessels traversed the ILM and penetrated the vitreous body less frequently than in wild-type retinas (31-55% in Vim(-/-), 66-79% in GFAP(-/-) Vim(-/-)). We conclude that IFs are important for maintaining the mechanical integrity of Müller-cell endfeet and the inner retinal layers under a mechanical challenge. Furthermore, the absence of IFs in Müller cells leads to an abnormal response of the vascular system to ischemia, specifically decreased ability of newly formed blood vessels to traverse the ILM.

    Journal of cell science 2004;117;Pt 16;3481-8

  • Forced expression of platelet-derived growth factor B in the mouse cerebellar primordium changes cell migration during midline fusion and causes cerebellar ectopia.

    Andrae J, Afink G, Zhang XQ, Wurst W and Nistér M

    Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, SE-751 85 Uppsala, Sweden.

    The platelet-derived growth factor (PDGF) and receptors are expressed in the developing central nervous system and in brain tumors. To investigate the role of PDGF during normal cerebellar development, we created transgenic mice where PDGF-B was introduced into the endogenous Engrailed1 locus (En1). These mice expressed PDGF-B in all types of cells that constitute the developing cerebellum, with localized high expression in the ventral midline of the cerebellar anlage. This affected cell migration in the midline during fusion of the cerebellar anlage and caused misplacement of midline structures. PDGFR-alpha- and laminin alpha1-positive meningeal cells migrated inwards, attracted by the ectopic transgene expression in the ventral neuroepithelium. Other cells followed the meningeal cells and in the adult mouse, cells from all cortical cell layers were found misplaced in the midline. Moreover, the transgene caused an enhancement of capillary vessels. The findings indicate that normal PDGF signaling is important for proper neural tube fusion. It also illustrates that meningeal structures can influence the process.

    Molecular and cellular neurosciences 2004;26;2;308-21

  • Bfsp2 mutation found in mouse 129 strains causes the loss of CP49' and induces vimentin-dependent changes in the lens fibre cell cytoskeleton.

    Sandilands A, Wang X, Hutcheson AM, James J, Prescott AR, Wegener A, Pekny M, Gong X and Quinlan RA

    Department of Molecular and Cellular Pathology, University of Dundee, Dundee DD1 5EH Scotland, UK.

    Here we report the first natural mutation in the mouse Bfsp2 gene. Characterisation of mouse Bfsp2 in the 129X1/SvJ revealed a mutation that deleted the acceptor site of exon 2. This results in exon 1 being erroneously spliced to exon 3 causing a frameshift in the reading frame and the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. RT-PCR studies of lens RNA isolated from 129S1/SvImJ, 129S2/SvPas and 129S4/SvJae strains confirmed the presence of this mutation in these diverse 129 strains and similar mutations were found in both CBA and 101 strains, but not in C3H or C57BL/6J mouse strains. This mutation is predicted to result in a severely truncated protein product called CP49, comprising essentially only exon 1, but polyclonal antibodies to CP49 failed to detect either full length or fragments of CP49 in extracts made from either 129S1/SvImJ or 129S4/SvJae suggesting that these 129 strains lack CP49 protein. Like the knockout of Bfsp2 reported recently, filensin protein levels and its proteolytic processing were altered also in the 129S1/SvImJ and 129S4/SvJae strains compared to C57BL/6J. Electron microscopy of the lens cytoskeleton from 129S2/SvPas revealed similar morphological changes in the cytoskeleton as compared to the CP49 knockout, with beaded and intermediate filaments being apparently replaced by poorly defined filament-like material. Vimentin was a key component of this residual material as shown by immunoelectron microscopy and by the generation of a CP49/vimentin double knockout mouse. This report of a natural mutation in Bfsp2 in the 129 and other mouse strains also has important implications for lens studies that have used the 129X1/SvJ strain in knockout strategies.

    Experimental eye research 2004;78;4;875-89

  • Peroxisomal localization in the developing mouse cerebellum: implications for neuronal abnormalities related to deficiencies in peroxisomes.

    Nagase T, Shimozawa N, Takemoto Y, Suzuki Y, Komori M and Kondo N

    Department of Pediatrics, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. tomoko-n@cc.gifu-u.ac.jp

    In subjects with Zellweger syndrome, the most severe phenotype of peroxisomal biogenesis disorder, brain abnormalities include cortical dysplasia, neuronal heterotopia, and dysmyelination. To clarify the relationship between the lack of peroxisomes and neuronal abnormalities, we investigated peroxisomal localization in the mouse cerebellum, using double immunofluorescent staining for peroxisomal proteins. On immunostaining for peroxisomal matrix protein, while there are few peroxisomes in Purkinje cells, many locate in astroglia, especially soma of Bergmann glia. Clusters of peroxisomes were seen on the inferior side of the Purkinje cell layer in mice on postnatal days 3-5, and with time there was a shift to the superior side. The peroxisomal punctate pattern was seen to be radial and co-localized with Bergmann glial fibers. In cultured cells from the mouse cerebellum, peroxisomes were few in Purkinje cells, whereas many were evident in glial fibrillary acidic protein-positive cells. On the other hand, on immunostaining for peroxisomal membrane protein Pex14p, many particles were seen in Purkinje cells during all developmental stages, which means Purkinje cells possessed empty peroxisomal structures similar to findings of fibroblasts from the Zellweger patients. As peroxisomes in glial cells may control the development of neurons, the neuron-glial interaction and mechanisms of developing central nervous systems deserve ongoing attention.

    Biochimica et biophysica acta 2004;1671;1-3;26-33

  • Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line.

    Shu H, Chen S, Bi Q, Mumby M and Brekken DL

    Protein Chemistry Laboratory, Alliance for Cellular Signaling, University of Texas Southwestern Medical Center, Dallas, TX 75390-9196, USA.

    A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.

    Funded by: NIGMS NIH HHS: U54 GM062114

    Molecular & cellular proteomics : MCP 2004;3;3;279-86

  • GenePaint.org: an atlas of gene expression patterns in the mouse embryo.

    Visel A, Thaller C and Eichele G

    Max Planck Institute of Experimental Endocrinology, Feodor-Lynen-Strasse 7, D-30625 Hannover, Germany.

    High-throughput instruments were recently developed to determine gene expression patterns on tissue sections by RNA in situ hybridization. The resulting images of gene expression patterns, chiefly of E14.5 mouse embryos, are accessible to the public at http://www.genepaint.org. This relational database is searchable for gene identifiers and RNA probe sequences. Moreover, patterns and intensity of expression in approximately 100 different embryonic tissues are annotated and can be searched using a standardized catalog of anatomical structures. A virtual microscope tool, the Zoom Image Server, was implemented in GenePaint.org and permits interactive zooming and panning across approximately 15,000 high-resolution images.

    Nucleic acids research 2004;32;Database issue;D552-6

  • Global expression analysis of gene regulatory pathways during endocrine pancreatic development.

    Gu G, Wells JM, Dombkowski D, Preffer F, Aronow B and Melton DA

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

    To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation.

    Funded by: NIDDK NIH HHS: F32 DK009832, R01 DK065949

    Development (Cambridge, England) 2004;131;1;165-79

  • Nestin expression in pancreatic exocrine cell lineages.

    Delacour A, Nepote V, Trumpp A and Herrera PL

    Department of Morphology, room 5040, University of Geneva Medical School, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland.

    Expression of nestin has been suggested to be a characteristic of pancreatic islet stem cells. To determine whether nestin is indeed expressed in such putative cells during embryonic development, or in the adult pancreas after injury, we performed a cell lineage analysis using two independent lines of transgenic mice encoding Cre recombinase under the control of rat nestin cis-regulatory sequences, each crossed with loxP-bearing R26R mice. F1 animals produced the reporter molecule beta-galactosidase only upon Cre-mediated recombination, thus solely in cells using (or having used) the transgenic nestin promoter. In early pancreatic primordia, beta-galactosidase was observed in mesenchymal and epithelial cells. At later developmental stages or in adults, vast clusters of acinar cells and few ductal cells were labeled, in addition to fibroblasts and vascular cells, but no endocrine cells were tagged by beta-galactosidase. This correlated with the transient expression, observed with an anti-nestin antibody, of endogenous nestin in about 5% of epithelial cells during development (whether in cord-forming arrangements or in nascent acini), and in vascular and mesenchymal structures. After partial pancreatectomy, there was a transient increase of the number of anti-nestin-labeled endothelial cells, but again, no endocrine cells bore beta-galactosidase. Together, these findings show that nestin is expressed in the pancreatic exocrine cell lineage, and suggest that consistent nestin expression is not a major feature of islet endocrine progenitor cells.

    Mechanisms of development 2004;121;1;3-14

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • Cellular and molecular mechanisms of development of the external genitalia.

    Yamada G, Satoh Y, Baskin LS and Cunha GR

    Center for Animal Resources and Development (CARD) and Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Honjo, Kumamoto 860-0811, Japan.

    The limb and external genitalia are appendages of the body wall. Development of these structures differs fundamentally in that masculine development of the external genitalia is androgen dependent, whereas development of the limb is not. Despite this fundamental difference in developmental regulation, epithelial-mesenchymal interactions play key roles in the development of both structures, and similar regulatory molecules are utilized as mediators of morphogenetic cell-cell interactions during development of both the limb and external genitalia. Given the relatively high incidence of hypospadias, a malformation of penile development, it is appropriate and timely to review the morphological, endocrine, and molecular mechanisms of development of the genital tubercle (GT), the precursor of the penis in males and the clitoris in females. Morphological observations comparing development of the GT in humans and mouse emphasize the validity of the mouse as an animal model of GT development and validate the results of experimental studies. Accordingly, the use of mutant mice provides important insights into the roles of specific regulatory molecules in development of the external genitalia. While our current understanding of the morphological and molecular mechanisms of mammalian external genitalia development is still rudimentary, this review summarizes the current state of our knowledge and whenever possible draws from the rich experimental embryology literature on other relevant organs such as the developing limb. Future research on the hormonal and molecular mechanisms of GT development may yield strategies to prevent or reduce the incidence of hypospadias and to elucidate the molecular genetic mechanisms of GT morphogenesis, especially in relation to common organogenetic pathways utilized in other organ systems.

    Funded by: NCI NIH HHS: CA89520, CA91967; NIDDK NIH HHS: DK57246

    Differentiation; research in biological diversity 2003;71;8;445-60

  • Subtractive hybridisation screen identifies sexually dimorphic gene expression in the embryonic mouse gonad.

    McClive PJ, Hurley TM, Sarraj MA, van den Bergen JA and Sinclair AH

    The sex of most mammals is determined by the action of SRY. Its presence initiates testis formation resulting in male differentiation, its absence results in ovary formation and female differentiation. We have used suppression subtraction hybridisation between 12.0-12.5 days postcoitum (dpc) mouse testes and ovaries to identify genes that potentially lie within the Sry pathway. Normalised urogenital ridge libraries comprising 8,352 clones were differentially screened with subtracted probes. A total of 272 candidate cDNAs were tested for qualitative differential expression and localisation by whole mount in situ hybridisation; germ cell-dependent or -independent expression was further resolved using busulfan. Fifty-four genes were identified that showed higher expression in the testis than the ovary. One novel gene may be a candidate for interactions with WT1, based on its localisation to Sertoli cells and map position (16q24.3).

    Genesis (New York, N.Y. : 2000) 2003;37;2;84-90

  • Axonal plasticity and functional recovery after spinal cord injury in mice deficient in both glial fibrillary acidic protein and vimentin genes.

    Menet V, Prieto M, Privat A and Giménez y Ribotta M

    Institut National de la Santé et de la Recherche Médicale U 583, Université de Montpellier II, Place E. Bataillon, F-34095 Montpellier Cedex 05, France.

    The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional disabilities. The inability of neurons to regenerate their axon is appreciably due to an inhospitable environment made of an astrocytic scar. We generated mice knock-out for glial fibrillary acidic protein and vimentin, the major proteins of the astrocyte cytoskeleton, which are upregulated in reactive astrocytes. These animals, after a hemisection of the spinal cord, presented reduced astroglial reactivity associated with increased plastic sprouting of supraspinal axons, including the reconstruction of circuits leading to functional restoration. Therefore, improved anatomical and functional recovery in the absence of both proteins highlights the pivotal role of reactive astrocytes in axonal regenerative failure in adult CNS and could lead to new therapies of spinal cord lesions.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;15;8999-9004

  • Lack of Reelin causes malpositioning of nigral dopaminergic neurons: evidence from comparison of normal and Reln(rl) mutant mice.

    Nishikawa S, Goto S, Yamada K, Hamasaki T and Ushio Y

    Laboratory of Neurobiology, Department of Neurosurgery, Kumamoto University Medical School, Kumamoto 860-8556, 1-1-1 Honjo, Japan.

    The reeler gene (Reln(rl), formerly rl) product Reelin controls neuronal migration and positioning and thereby plays a key role in brain development. Mutation of Reln leads to widespread disruption of laminar cortical regions and ectopia in some brainstem nuclei. In the embryonic striatum of normal mice, a substantial expression of reelin mRNA has been documented; however, the anomalous positioning of neurons in the basal ganglia of reeler mice remains to be studied. We provide first evidence for a potential role of Reelin in the developmental formation of the substantia nigra. In reeler mutant mice lacking Reelin, dopaminergic neurons destined for the substantia nigra fail to migrate laterally and become anomalously clustered just lateral to the ventral tegmental area. Their axons appear to project to striatal patches forming "dopamine islands." Results from the normal mice show that, at the midembryonic stage, Reelin identified with CR-50 is highly concentrated in the ventral mesencephalon, where nigral dopaminergic neurons are in progress to migrate laterally to their eventual position of the adult brain. A combination of CR-50 labeling and anterograde axonal tracing provided evidence that embryonic striatal neurons may supply the ventral portion of the mesencephalon with Reelin through their axonal projections. We hypothesize that Reelin plays a role in the positioning of nigral dopaminergic neurons and that it can act as an environmental cue at a remote site far from its birthplace via a transaxonal delivery system.

    The Journal of comparative neurology 2003;461;2;166-73

  • Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation in the cytokinetic process.

    Goto H, Yasui Y, Kawajiri A, Nigg EA, Terada Y, Tatsuka M, Nagata K and Inagaki M

    Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan.

    Aurora-B is an evolutionally conserved protein kinase that regulates several mitotic events including cytokinesis. We previously demonstrated the possible existence of a protein kinase that phosphorylates at least Ser-72 on vimentin, the most widely expressed intermediate filament protein, in the cleavage furrow-specific manner. Here we showed that vimentin-Ser-72 phosphorylation occurred specifically at the border of the Aurora-B-localized area from anaphase to telophase. Expression of a dominant-negative mutant of Aurora-B led to a reduction of this vimentin-Ser-72 phosphorylation. In vitro analyses revealed that Aurora-B phosphorylates vimentin at approximately 2 mol phosphate/mol of substrate for 30 min and that this phosphorylation dramatically inhibits vimentin filament formation. We further identified eight Aurora-B phosphorylation sites, including Ser-72 on vimentin, and then constructed the mutant vimentin in which these identified sites are changed into Ala. Cells expressing this mutant formed an unusually long bridge-like intermediate filament structure between unseparated daughter cells. We then identified important phosphorylation sites for the bridge phenotype. Our findings indicate that Aurora-B regulates the cleavage furrow-specific vimentin phosphorylation and controls vimentin filament segregation in cytokinetic process.

    The Journal of biological chemistry 2003;278;10;8526-30

  • Localization of ApoER2, VLDLR and Dab1 in radial glia: groundwork for a new model of reelin action during cortical development.

    Luque JM, Morante-Oria J and Fairén A

    Instituto de Neurociencias, Universidad Miguel Hernández, CSIC, Campus de San Juan, E-03550, San Juan de Alicante, Spain. luque@umh.es

    The reelin signaling pathway regulates laminar positioning of radially migrating neurons during cortical development. It has been suggested that reelin secreted by Cajal-Retzius cells in the marginal zone could provide either a stop or an attractant signal for migratory neurons expressing reelin receptors, but the proposed models fail to explain recent experimental findings. Here we provide evidence that the reelin receptor machinery, including the lipoprotein receptors ApoER2 and VLDLR along with the cytoplasmic adaptor protein Dab1, is located in radial glia precursors whose processes span the entire cortical wall from the ventricular zone to the pial surface. Moreover, in reeler mice, defective in reelin, decreased levels of Dab1 in the ventricular zone correspond to an accumulation of the protein in radial end-feet beneath the pia matter. Our results support that neural stem cells receive a functional reelin signal. They are also consistent with a working model of reelin action, according to which reelin signaling on the newborn neuron-inherited radial process regulates perikaryal translocation and positioning.

    Brain research. Developmental brain research 2003;140;2;195-203

  • 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

  • Altered aggregation properties of mutant gamma-crystallins cause inherited cataract.

    Sandilands A, Hutcheson AM, Long HA, Prescott AR, Vrensen G, Löster J, Klopp N, Lutz RB, Graw J, Masaki S, Dobson CM, MacPhee CE and Quinlan RA

    Department of Biochemistry, Medical Science Institutes, University of Dundee, Dundee DD1 5EH, Scotland, UK.

    Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non-neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered gamma-crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of gamma-crystallin mutation, large inclusions containing the altered gamma-crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid-detecting dye, Congo red. In vitro, recombinant mutant gammaB-crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild-type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid-like inclusions. The mutant gamma-crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.

    The EMBO journal 2002;21;22;6005-14

  • Binding of protein kinase B to the plakin family member periplakin.

    van den Heuvel AP, de Vries-Smits AM, van Weeren PC, Dijkers PF, de Bruyn KM, Riedl JA and Burgering BM

    Laboratory of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Stratenum, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

    The serine/threonine kinase protein kinase B (PKB/c-Akt) acts downstream of the lipid kinase phosphoinositide 3-kinase (PI3K) and functions as an essential mediator in many growth-factor-induced cellular responses such as cell cycle regulation, cell survival and transcriptional regulation. PI3K activation generates 3'-phosphorylated phosphatidylinositol lipids (PtdIns3P) and PKB activation requires PtdIns3P-dependent membrane translocation and phosphorylation by upstream kinases. However PKB activation and function is also regulated by interaction with other proteins. Here we show binding of PKB to periplakin, a member of the plakin family of cytolinker proteins. Interaction between PKB and periplakin was mapped to part of the pleckstrin homology (PH) domain of PKB, which is probably not involved in lipid binding, and indeed binding to periplakin did not affect PKB activation. We therefore investigated the possibility that periplakin may act as a scaffold or localization signal for PKB. In cells endogenous periplakin localizes to different cellular compartments, including plasma membrane, intermediate filament structures, the nucleus and mitochondria. Overexpression of the C-terminal part of periplakin, encompassing the PKB binding region, results in predominant intermediate filament localization and little nuclear staining. This also resulted in inhibition of nuclear PKB signalling as indicated by inhibition of PKB-dependent Forkhead transcription factor regulation. These results suggest a possible role for periplakin as a localization signal in PKB-mediated signalling.

    Journal of cell science 2002;115;Pt 20;3957-66

  • 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

  • Sonic hedgehog activates mesenchymal Gli1 expression during prostate ductal bud formation.

    Lamm ML, Catbagan WS, Laciak RJ, Barnett DH, Hebner CM, Gaffield W, Walterhouse D, Iannaccone P and Bushman W

    Department of Urology, Northwestern University Medical School, Chicago, IL 60611, USA.

    Ductal budding in the developing prostate is a testosterone-dependent event that involves signaling between the urogenital sinus epithelium (UGE) and urogenital sinus mesenchyme (UGM). We show here that ductal bud formation is associated with focused expression of Sonic hedgehog (Shh) in the epithelium of nascent prostate buds and in the growing tips of elongating prostate ducts. This pattern of localized Shh expression occurs in response to testosterone stimulation. The gene for the Shh receptor, Ptc1, is expressed in the UGM, as are the members of the Gli gene family of transcriptional regulators (Gli1, Gli2, and Gli3). Expression of Ptc1, Gli1, and Gli2 is localized primarily to mesenchyme surrounding prostate buds, whereas Gli3 is expressed diffusely throughout the UGM. A strong dependence of Gli1 (and Ptc1) expression on Shh signaling is demonstrated by induction of expression in both the intact urogenital sinus and the isolated UGM by exogenous SHH peptide. A similar dependence of Gli2 and Gli3 expression on Shh is not observed. Nonetheless, the chemical inhibitor of Shh signaling, cyclopamine, produced a graded inhibition of Gli gene expression (Gli1>Gli2>Gli3) in urogenital sinus explants that was paralleled by a severe inhibition of ductal budding.

    Funded by: NIDDK NIH HHS: DK 02426, DK 52687; NIEHS NIH HHS: P01 ES10549

    Developmental biology 2002;249;2;349-66

  • Protective role of phosphorylation in turnover of glial fibrillary acidic protein in mice.

    Takemura M, Gomi H, Colucci-Guyon E and Itohara S

    Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Wako 351-0198, Japan.

    Glial fibrillary acidic protein (GFAP), the principal intermediate filament (IF) protein of mature astrocytes in the CNS, plays specific roles in astrocyte functions. GFAP has multiple phosphorylation sites at its N-terminal head domain. To examine the role of phosphorylation at these sites, we generated a series of substitution mutant mice in which phosphorylation sites (Ser/Thr) were replaced by Ala, in different combinations. Gfap(hm3/hm3) mice carrying substitutions at all five phosphorylation sites showed extensive decrease in both filament formation and amounts of GFAP. Gfap(hm1/hm1) and Gfap(hm2/hm2) mice, which carry substitutions at three of five sites and in different combinations, showed differential phenotypes. Although Gfap(hm3/hm3) mice retained GFAP filaments in Bergmann glia in the cerebellum, the (Gfap(hm3/hm3):Vim(-/-)) mice lacked GFAP filaments. Pulse-chase experiments of cultured astrocytes indicated that the Hm3-GFAP encoded by Gfap(hm3) was unstable particularly in the absence of vimentin, another IF protein. These results revealed the role of phosphorylation in turnover of GFAP and a synergistic role of GFAP and vimentin in the dynamics of glial filaments. The data further suggest that each of the phosphorylated sites has a distinct impact on the dynamics of GFAP.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;16;6972-9

  • ELF a beta-spectrin is a neuronal precursor cell marker in developing mammalian brain; structure and organization of the elf/beta-G spectrin gene.

    Tang Y, Katuri V, Iqbal S, Narayan T, Wang Z, Lu RS, Mishra L and Mishra B

    Laboratory of Development Molecular Biology, DVAMC, Washington, District of Columbia, DC 20422, USA.

    Spectrins play a pivotal role in axonal transport, neurite extension, the organization of synaptic vesicles, as well as for protein sorting in the Golgi apparatus and cell membrane. Among spectrins there is great variability in sequence composition, tissue distribution, and function, with two known genes encoding the alpha-chain, and at least five encoding the beta-chain. It remains unclear as to whether novel beta-spectrins such as elf1-4 are distinct genes or beta-G-spectrin isoforms. The role for ELF in the developing nervous system has not been identified to date. In this study we demonstrate the genomic structure of elf-3, as well as the expression of ELF in the developing mouse brain using a peptide specific antibody against its distinctive amino-terminal end. Full genomic structural analyses reveal that elf-3 is composed of 31 exons spanning approximately 67 kb, and confirm that elf and mouse brain beta-G-spectrin share multiple exons, with a complex form of exon/intron usage. In embryonic stages, E9-12, anti-ELF localized to the primary brain vesicular cells that also labeled strongly with anti-nestin but not anti-vimentin. At E12-14, anti-ELF localized to axonal sprouts in the developing neuroblasts of cortex and purkinje cell layer of the cerebellum, as well as in cell bodies in the diencephalon and metencephalon. Double labeling identified significant co-localization of anti-ELF, nestin and dystrophin in sub ventricular zone cells and in stellate-like cells of the developing forebrain. These studies define clearly the expression of ELF, a new isoform of beta-G-spectrin in the developing brain. Based on its expression pattern, ELF may have a role in neural stem cell development and is a marker of axonal sprouting in mid stages of embryonic development.

    Funded by: NIDDK NIH HHS: 1R01 DK 58637, 1R01 DK56111, 1R03 DK53861

    Oncogene 2002;21;34;5255-67

  • A novel type of regulation of the vimentin intermediate filament cytoskeleton by a Golgi protein.

    Gao YS, Vrielink A, MacKenzie R and Sztul E

    Department of Cell Biology, University of Alabama at Birmingham, 35294, USA.

    Whether the highly dynamic structure of the vimentin intermediate filament (IF) cytoskeleton responds to cues from cellular organelles, and what proteins might participate in such events is largely unknown. We have shown previously that the Golgi protein formiminotransferase cyclodeaminase (FTCD) binds to vimentin filaments in vivo and in vitro, and that overexpression of FTCD causes dramatic rearrangements of the vimentin IF cytoskeleton (Gao and Sztul, J. Cell Biol. 152, 877-894, 2001). Using real-time imaging, we now show that FTCD causes bundling of individual thinner vimentin filaments into fibers and that the bundling always originates at the Golgi. FTCD appears to be the molecular "glue" since FTCD cross-links vimentin filaments in vitro. To initiate the analysis of structural determinants required for FTCD function in vimentin dynamics, we used structure-based design to generate individual formiminotransferase (FT) and cyclodeaminase (CD) domains, and to produce an enzymatically inactive FTCD. We show that the intact octameric structure is required for FTCD binding to vimentin filaments and for promoting filament assembly, but that eliminating enzymatic activity does not affect FTCD effects on the vimentin cytoskeleton. Our findings indicate that the Golgi protein FTCD is a potent modulator of the vimentin IF cytoskeleton, and suggest that the Golgi might act as a reservoir for proteins that regulate cytoskeletal dynamics.

    European journal of cell biology 2002;81;7;391-401

  • Expression of genes for alcohol and aldehyde metabolizing enzymes in mouse oocytes and preimplantation embryos.

    Rout UK and Armant DR

    Department of Obstetrics & Gynecology, C.S. Mott Center for Human Growth and Development, Wayne State University School of Medicine, 275 East Hancock, Detroit, MI 48201, USA. aa4306@wayne.edu

    Alcohols and aldehydes are metabolized primarily by alcohol (ADH) and aldehyde (ALDH) dehydrogenase isozymes. Although significant progress has been made towards understanding the involvement of these isozymes in the oxidation of alcohol and aldehydes in the body, it is not known how these compounds are handled during fertilization and preimplantation embryogenesis. In this study, reverse transcription and the polymerase chain reaction (RT-PCR) was used to determine which ADH and ALDH isozymes are expressed at the oocyte, zygote, morula, and blastocyst stages of preimplantation development in the mouse. Transcripts of beta-actin and vimentin, assayed as controls, were detected at all stages, as well as Class III ADH (Adh-2) and Class 3 ALDH (Ahd-4), involved in the detoxification of formaldehyde and aromatic aldehydes, respectively. In contrast, transcripts for the major ethanol oxidizing isozyme, Class I ADH (Adh-1) was not detected during preimplantation development. Cytosolic retinol dehydrogenase (Adh-3) transcripts were marginally detected in oocytes and zygotes. The mRNA for cytosolic retinal dehydrogenase (Ahd-2), microsomal short-chain retinol dehydrogenases (RoDH Type I), and the mitochondrial low-Km acetaldehyde dehydrogenase (Ahd-5) only appeared as maternal transcripts. Microsomal ALDH (Ahd-3), which is induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), was not expressed until the blastocyst stage. ADH and ALDH enzyme systems may guard mouse preimplantation embryos against the toxic effects of industrial pollutants, such as formaldehyde and TCDD, as well as peroxidatic aldehydes generated during lipid peroxidation. The absence of enzymes to convert ethanol to acetaldehyde, coupled with oocyte expression of the acetaldehyde-degrading enzyme, Ahd-5, may be protective for the early embryo.

    Funded by: NIAAA NIH HHS: P50AA07606, R01AA12057

    Reproductive toxicology (Elmsford, N.Y.) 2002;16;3;253-8

  • Thymopoiesis requires Pax9 function in thymic epithelial cells.

    Hetzer-Egger C, Schorpp M, Haas-Assenbaum A, Balling R, Peters H and Boehm T

    Max-Planck-Institut für Immunbiologie, Freiburg, Germany.

    The epithelial thymic anlage develops from the third pharyngeal pouch. Pax9 is expressed in the entire pharyngeal endoderm, and its function is required for normal development of organs derived from pharyngeal pouches. Here, we show that in Pax9 null mice, the thymic anlage develops as an ectopic polyp-like structure in the larynx. It expresses Whn/Foxn1, a marker of thymic epithelium, but fails to perform the normal caudo-ventral movement to the upper mediastinum. The thymic rudiment contains mesenchymal cells, blood vessels and is colonized by T cell progenitors. However, from embryonic day 14.5 onwards, the size of the Pax9 mutant thymus is severely reduced. Whereas expression of TCRbeta chain genes is readily detectable in the mutant thymus, no expression of the TCRgamma chain was detectable. Our results identify a new genetically defined control point of thymopoiesis.

    European journal of immunology 2002;32;4;1175-81

  • Vimentin affects localization and activity of sodium-glucose cotransporter SGLT1 in membrane rafts.

    Runembert I, Queffeulou G, Federici P, Vrtovsnik F, Colucci-Guyon E, Babinet C, Briand P, Trugnan G, Friedlander G and Terzi F

    INSERM U426 and Department Physiology, Faculté de Médecine Xavier Bichat, IFR 02, Université Paris 7, Paris, France.

    It has been reported that vimentin, a cytoskeleton filament that is expressed only in mesenchymal cells after birth, is re-expressed in epithelial cells in vivo under pathological conditions and in vitro in primary culture. Whether vimentin re-expression is only a marker of cellular dedifferentiation or is instrumental in the maintenance of cell structure and/or function is a matter of debate. To address this issue, we used renal proximal tubular cells in primary culture from vimentin-null mice (Vim(-/-)) and from wild-type littermates (Vim(+/+)). The absence of vimentin did not affect cell morphology, proliferation and activity of hydrolases, but dramatically decreased Na-glucose cotransport activity. This phenotype was associated with a specific reduction of SGLT1 protein in the detergent-resistant membrane microdomains (DRM). In Vim(+/+) cells, disruption of these microdomains by methyl-beta-cyclodextrin decreased SGLT1 protein abundance in DRM, a change that was paralleled by a decrease of Na-glucose transport activity. Importantly, we showed that vimentin is located to DRM, but it disappeared after methyl-beta-cyclodextrin treatment. In Vim(-/-) cells, supplementation of cholesterol with cholesterol-methyl-beta-cyclodextrin complexes completely restored Na-glucose transport activity. Interestingly, neither cholesterol content nor cholesterol metabolism changed in Vim(-/-) cells. Our results are consistent with the view that re-expression of vimentin in epithelial cells could be instrumental to maintain the physical state of rafts and, thus, the function of DRM-associated proteins.

    Journal of cell science 2002;115;Pt 4;713-24

  • Phosphorylation and reorganization of vimentin by p21-activated kinase (PAK).

    Goto H, Tanabe K, Manser E, Lim L, Yasui Y and Inagaki M

    Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681, Japan.

    Background: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments.

    Results: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization.

    Conclusion: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation.

    Genes to cells : devoted to molecular & cellular mechanisms 2002;7;2;91-7

  • Multiple influences on the migration of precerebellar neurons in the caudal medulla.

    de Diego I, Kyriakopoulou K, Karagogeos D and Wassef M

    CNRS UMR C8542, Régionalisation Nerveuse, niveau 8, Ecole Normale Supérieure 46, rue d'Ulm 75230 Paris Cedex 05, France.

    Neurons destined to form several precerebellar nuclei are generated in the dorsal neuroepithelium (rhombic lip) of caudal hindbrain. They form two ventrally directed migratory streams, which behave differently. While neurons in the superficial migration migrate in a subpial position and cross the midline to settle into the contralateral hindbrain, neurons in the olivary migration travel deeper in the parenchyma and stop ipsilaterally against the floor plate. In the present study, we compared the behavior of the two neuronal populations in an organotypic culture system that preserves several aspects of their in vivo environment. Both migrations occurred in mouse hindbrain explants dissected at E11.5 even when the floor plate was ablated at the onset of the culture period, indicating that they could rely on dorsoventral cues already distributed in the neural tube. Nevertheless, the local constraints necessary for the superficial migration were more specific than for the olivary migration. Distinct chemoattractive and chemorespulsive signal were found to operate on the migrations. The floor plate exhibited a strong chemoattractive influence on both migrations, which deviated from their normal path in the direction of ectopic floor plate fragments. It was also found to produce a short-range stop signal and to induce inferior olive aggregation. The ventral neural tube was also found to inhibit or slow down the migration of olivary neurons. Interestingly, while ectopic sources of netrin were found to influence both migrations, this effect was locally modulated and affected differentially the successive phases of migration. Consistent with this observation, while neurons in the superficial migration expressed the Dcc-netrin receptor, the migrating olivary neurons did not express Dcc before they reached the midline. Our observations provide a clearer picture of the hierarchy of environmental cues that influence the morphogenesis of these precerebellar nuclei.

    Development (Cambridge, England) 2002;129;2;297-306

  • The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity.

    Pin CL, Rukstalis JM, Johnson C and Konieczny SF

    Department of Paediatrics, Child Health Research Institute, University of Western Ontario, London, Ontario N6C 2V5, Canada.

    The pancreas is a complex organ that consists of separate endocrine and exocrine cell compartments. Although great strides have been made in identifying regulatory factors responsible for endocrine pancreas formation, the molecular regulatory circuits that control exocrine pancreas properties are just beginning to be elucidated. In an effort to identify genes involved in exocrine pancreas function, we have examined Mist1, a basic helix-loop-helix transcription factor expressed in pancreatic acinar cells. Mist1-null (Mist1(KO)) mice exhibit extensive disorganization of exocrine tissue and intracellular enzyme activation. The exocrine disorganization is accompanied by increases in p8, RegI/PSP, and PAP1/RegIII gene expression, mimicking the molecular changes observed in pancreatic injury. By 12 m, Mist1(KO) mice develop lesions that contain cells coexpressing acinar and duct cell markers. Analysis of the factors involved in cholecystokinin (CCK) signaling reveal inappropriate levels of the CCK receptor A and the inositol-1,4,5-trisphosphate receptor 3, suggesting that a functional defect exists in the regulated exocytosis pathway of Mist1(KO) mice. Based on these observations, we propose that Mist1(KO) mice represent a new genetic model for chronic pancreas injury and that the Mist1 protein serves as a key regulator of acinar cell function, stability, and identity.

    Funded by: NIAMS NIH HHS: Z01 AR041115; NIDDK NIH HHS: DK55489, R01 DK055489

    The Journal of cell biology 2001;155;4;519-30

  • Identification of a caspase-9 substrate and detection of its cleavage in programmed cell death during mouse development.

    Nakanishi K, Maruyama M, Shibata T and Morishima N

    Bioarchitect Research Group, RIKEN (Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

    The caspase family of proteases represents the main machinery by which apoptosis occurs. In vitro studies have revealed that upstream caspases are activated in response to apoptotic stimuli, and the active caspases in turn process downstream effector caspases that are involved in the destruction of cellular structure. Caspase-9 is an upstream caspase that can become active in response to cellular damage, including deprivation of growth factors and exposure to oxidative stress in vitro. Little is known, however, about how activation of caspase-9 is temporally and spatially regulated in vivo, e.g. during development. We have identified vimentin as the first example of a caspase-9 substrate that is not a downstream procaspase. Immunohistochemical analysis, using a specific antibody against the vimentin fragments generated by caspase-9, showed that caspase-9 cleaves vimentin in apoptotic cells in the embryonic nervous system and the interdigital regions. This result is consistent with observations that gene knockouts of caspase-9 and its activator, Apaf-1, result in developmental defects in these tissues. Our results show that the specific antibody is useful for in situ detection of caspase-9 activation in programmed cell death.

    The Journal of biological chemistry 2001;276;44;41237-44

  • A programmed ependymal denudation precedes congenital hydrocephalus in the hyh mutant mouse.

    Jiménez AJ, Tomé M, Páez P, Wagner C, Rodríguez S, Fernández-Llebrez P, Rodríguez EM and Pérez-Fígares JM

    Departamento de Biología Celular y Genética, Facultad de Ciencias, Universidad de Málaga, Spain.

    Hydrocephalic hyh mice are born with moderate hydrocephalus and a normal cerebral aqueduct. At about the fifth postnatal day the aqueduct becomes obliterated and severe hydrocephalus develops. The aim of the present investigation was to investigate the mechanism of this hydrocephalus, probably starting during fetal life when the cerebral aqueduct is still patent. By use of immunocytochemistry and scanning electron microscopy, mutant (n = 54) and normal (n = 61) hyh mouse embryos were studied at various developmental stages to trace the earliest microscopic changes occurring in the brains of embryos becoming hydrocephalic. The primary defect begins at an early developmental stage (E-12) and involves cells lining the brain cavities, which detach following a well-defined temporo-spatial pattern. This ependymal denudation mostly involves the ependyma of the basal plate derivatives. There is a relationship between ependymal denudation and ependymal differentiation evaluated by the expression of vimentin and glial fibrillary acidic protein. The ependymal cells had a normal appearance before and after detachment, suggesting that their separation from the ventricular wall might be due to abnormalities in cell adhesion molecules. The process of detachment of the ventral ependyma, clearly visualized under scanning electron microscope, is almost completed before the onset of hydrocephalus. Furthermore, this ependymal denudation does not lead to aqueductal stenosis during prenatal life. Thus, the rather massive ependymal denudation appears to be the trigger of hydrocephalus in this mutant mouse, raising the question about the mechanism responsible for this hydrocephalus. It seems likely that an uncontrolled bulk flow of brain fluid through the extended areas devoid of ependyma may be responsible for the hydrocephalus developed by the hyh mutant embryos. The defect in these embryos also includes loss of the hindbrain floor plate and a delayed in the expression of Reissner fiber glycoproteins by the subcommissural organ.

    Journal of neuropathology and experimental neurology 2001;60;11;1105-19

  • Intermediate filaments regulate astrocyte motility.

    Lepekhin EA, Eliasson C, Berthold CH, Berezin V, Bock E and Pekny M

    Protein Laboratory, Institute of Molecular Pathology, University of Copenhagen, Copenhagen, Denmark.

    Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell-type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.

    Journal of neurochemistry 2001;79;3;617-25

  • Abnormal adherence junctions in the heart and reduced angiogenesis in transgenic mice overexpressing mutant type XIII collagen.

    Sund M, Ylönen R, Tuomisto A, Sormunen R, Tahkola J, Kvist AP, Kontusaari S, Autio-Harmainen H and Pihlajaniemi T

    Collagen Research Unit, Biocenter Oulu, Department of Medical Biochemistry, University of Oulu, PL 5000, 90014 Oulu, Finland.

    Type XIII collagen is a type II transmembrane protein found at sites of cell adhesion. Transgenic mouse lines were generated by microinjection of a DNA construct directing the synthesis of truncated alpha1(XIII) chains. Shortened alpha 1(XIII) chains were synthesized by fibroblasts from mutant mice, and the lack of intracellular accumulation in immunofluorescent staining of tissues suggested that the mutant molecules were expressed on the cell surface. Transgene expression led to fetal lethality in offspring from heterozygous mating with two distinct phenotypes. The early phenotype fetuses were aborted by day 10.5 of development due to a lack of fusion of the chorionic and allantoic membranes. The late phenotype fetuses were aborted by day 13.5 of development and displayed a weak heartbeat, defects of the adherence junctions in the heart with detachment of myofilaments and abnormal staining for the adherence junction component cadherin. Decreased microvessel formation was observed in certain regions of the fetus and the placenta. These results indicate that type XIII collagen has an important role in certain adhesive interactions that are necessary for normal development.

    The EMBO journal 2001;20;18;5153-64

  • A 1.8kb GFAP-promoter fragment is active in specific regions of the embryonic CNS.

    Andrae J, Bongcam-Rudloff E, Hansson I, Lendahl U, Westermark B and Nistér M

    Department of Genetics and Pathology, Rudbeck Laboratory, University Hospital, Uppsala University, SE-751 85 Uppsala, Sweden.

    The intermediate filament glial fibrillary acidic protein (GFAP) constitutes the major cytoskeletal protein in astrocytes (J. Neuroimmunol. 8 (1985) 203) and is traditionally referred to as a specific marker for astrocytes. To identify early glial precursors, we created GFAPpromoter-lacZ transgenic mice, using a 1.8kb 5' fragment of human GFAP. The expression of the transgene was first detected in the neuroepithelium at embryonic day 9.5. It was further found in the ventricular zone of the developing telencephalon, in the cerebellar primordium, trigeminal ganglia, and radial glia. Later, scattered beta-gal+ cells were seen in pons, brain stem and glia limitans. The results indicate that GFAP activity is regulated in a region-specific manner during central nervous system (CNS) development and that the gene is turned on in different cell types independently.

    Mechanisms of development 2001;107;1-2;181-5

  • Role of the intermediate filament protein vimentin in delaying senescence and in the spontaneous immortalization of mouse embryo fibroblasts.

    Tolstonog GV, Shoeman RL, Traub U and Traub P

    Max-Planck-Institut für Zellbiologie, Ladenburg/Heidelberg, Germany.

    Because knockout of the vimentin gene in mice did not produce an immediately obvious, overt, or lethal specific phenotype, the conjecture was made that the mutation affects some subtle cellular functions whose loss manifests itself only when the mutant animals are exposed to stress. In order to substantiate this idea in a tractable in vitro system, primary embryo fibroblasts from wildtype (V(+/+)) and vimentin-knockout (V(-/-)) mice were compared with regard to their growth behavior under the pseudophysiologic conditions of conventional cell culture. Whereas in the course of serial transfer, the V(+/+) fibroblasts progressively reduced their growth potential, passed through a growth minimum around passage 12 (crisis), and, as immortalized cells, resumed faster growth, the V(-/-) fibroblasts also cut down their growth rate but much earlier, and they either did not immortalize or did so at an almost undetectable rate. Cells withdrawing from the cell cycle showed increased concentrations of reactive oxygen species and signs of oxidative damage: enlarged and flattened morphology, large nuclear volume, reinforced stress fiber system as a result of increased contents of actin and associated proteins, prominent extracellular matrix, and perinuclear masses of pathological forms of mitochondria with low membrane potential. The differences in the cell cycle behavior of the V(+/+) and V(-/-) cells in conjunction with the morphologic changes observed in mitotically arrested cells suggests a protective function of vimentin against oxidative cell damage. Because vimentin exhibits affinity for and forms crosslinkage products with recombinogenic nuclear as well as mitochondrial DNA in intact cells, it is credible to postulate that vimentin plays a role in the recombinogenic repair of oxidative damage inflicted on the nuclear and mitochondrial genome throughout the cells' replicative lifespan. Recombinational events mediated by vimentin also appear to take place when the cells pass through the genetically unstable state of crisis to attain immortality. The residual immortalization potential of V(-/-) fibroblasts might be attributable to their capacity to synthesize, in place of vimentin, the tetrameric form of a lacZ fusion protein carrying, in addition to a nuclear localization signal, the N-terminal 59 amino acids of vimentin and thus its DNA-binding site. On the basis of these results and considerations, a major biologic role of vimentin may be to protect animals during development and postnatal life against genetic damage and, because of its contribution to the plasticity of the genome, to allow them to respond to environmental challenges.

    DNA and cell biology 2001;20;9;509-29

  • Inactivation of the glial fibrillary acidic protein gene, but not that of vimentin, improves neuronal survival and neurite growth by modifying adhesion molecule expression.

    Menet V, Giménez y Ribotta M, Chauvet N, Drian MJ, Lannoy J, Colucci-Guyon E and Privat A

    Institut National de la Santé et de la Recherche Médicale U336, Université Montpellier II, F-34095 Montpellier, France. menet@crit.univ-montp2.fr

    Intermediate filaments (IFs) are a major component of the cytoskeleton in astrocytes. Their role is far from being completely understood. Immature astrocytes play a major role in neuronal migration and neuritogenesis, and their IFs are mainly composed of vimentin. In mature differentiated astrocytes, vimentin is replaced by the IF protein glial fibrillary acidic protein (GFAP). In response to injury of the CNS in the adult, astrocytes become reactive, upregulate the expression of GFAP, and reexpress vimentin. These modifications contribute to the formation of a glial scar that is obstructive to axonal regeneration. Nevertheless, astrocytes in vitro are considered to be the ideal substratum for the growth of embryonic CNS axons. In the present study, we have examined the potential role of these two major IF proteins in both neuronal survival and neurite growth. For this purpose, we cocultured wild-type neurons on astrocytes from three types of knock-out (KO) mice for GFAP or/and vimentin in a neuron-astrocyte coculture model. We show that the double KO astrocytes present many features of immaturity and greatly improve survival and neurite growth of cocultured neurons by increasing cell-cell contact and secreting diffusible factors. Moreover, our data suggest that the absence of vimentin is not a key element in the permissivity of the mutant astrocytes. Finally, we show that only the absence of GFAP is associated with an increased expression of some extracellular matrix and adhesion molecules. To conclude, our results suggest that GFAP expression is able to modulate key biochemical properties of astrocytes that are implicated in their permissivity.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;16;6147-58

  • Math5 is required for retinal ganglion cell and optic nerve formation.

    Brown NL, Patel S, Brzezinski J and Glaser T

    Departments of Internal Medicine and Human Genetics, University of Michigan, Ann Arbor, MI 48109-0650, USA. n-brown2@northwestern.edu

    The vertebrate retina contains seven major neuronal and glial cell types in an interconnected network that collects, processes and sends visual signals through the optic nerve to the brain. Retinal neuron differentiation is thought to require both intrinsic and extrinsic factors, yet few intrinsic gene products have been identified that direct this process. Math5 (Atoh7) encodes a basic helix-loop-helix (bHLH) transcription factor that is specifically expressed by mouse retinal progenitors. Math5 is highly homologous to atonal, which is critically required for R8 neuron formation during Drosophila eye development. Like R8 cells in the fly eye, retinal ganglion cells (RGCs) are the first neurons in the vertebrate eye. Here we show that Math5 mutant mice are fully viable, yet lack RGCs and optic nerves. Thus, two evolutionarily diverse eye types require atonal gene family function for the earliest stages of retinal neuron formation. At the same time, the abundance of cone photoreceptors is significantly increased in Math5(-/-) retinae, suggesting a binary change in cell fate from RGCs to cones. A small number of nascent RGCs are detected during embryogenesis, but these fail to develop further, suggesting that committed RGCs may also require Math5 function.

    Funded by: NEI NIH HHS: EY11729, R01 EY014259; NIGMS NIH HHS: GM07544, T32 GM007544

    Development (Cambridge, England) 2001;128;13;2497-508

  • Mouse histamine N-methyltransferase: cDNA cloning, expression, gene cloning and chromosomal localization.

    Wang L, Yan L, McGuire C, Kozak CA, Wang M, Kim UJ, Siciliano M and Weinshilboum RM

    Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School-Mayo Clinic-Mayo Foundation, Rochester, MN 55905 USA.

    Objective: Histamine N-methyltransferase (HNMT) catalyzes the Ntau-methylation of histamine. We set out to clone a mouse liver HNMT cDNA and the mouse HNMT gene as steps toward characterizing molecular genetic mechanisms involved in the regulation of this important histamine-metabolizing enzyme.

    Design: A PCR-based strategy was used to clone both the mouse HNMT cDNA and the gene encoding that cDNA, Hnmt. The cDNA was used both to express recombinant mouse HNMT and to determine the chromosomal localization of Hnmt.

    Results: The mouse liver HNMT cDNA was 1657 bp in length with an 888 bp open reading frame (ORF) that encoded a 296 amino acid protein with a predicted Mr value of approximately 32.5 kDa. The amino acid sequence of the encoded protein was 84% identical to that of human kidney HNMT. Mouse HNMT was expressed in COS-1 cells, and its apparent Km values for histamine and S-adenosyl-L-methionine (Ado-Met), the two cosubstrates for the reaction, were 5.3 and 5.8 microM, respectively. The mouse HNMT gene, Hnmt, spanned approximately 25 kb and had 7 exons. Its structure differed from that of the human gene primarily by the presence of an additional exon at the 5'-terminus. Hnmt mapped to mouse chromosome 2 in an area of conserved synteny to human chromosome 2q, the location of the human gene (2q22) on the basis of fluorescence in situ hybridization.

    Conclusions: Cloning and functional characterization of the mouse HNMT cDNA and gene will now make it possible to study in the mouse molecular genetic mechanisms involved the regulation of this important histamine-metabolizing enzyme.

    Funded by: NCI NIH HHS: R01 CA34936; NIGMS NIH HHS: R01 GM28157, R01 GM35720, UO1 GM61388

    Inflammation research : official journal of the European Histamine Research Society ... [et al.] 2001;50;6;300-8

  • 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

  • Characterization of 'non-N-methyl-D-Aspartate' binding sites for gacyclidine enantiomers in the rat cerebellar and telencephalic structures.

    Hirbec H, Kamenka JM, Privat A and Vignon J

    INSERM U336 DPVSN, Institut de biologie, Montpellier, France.

    Gacyclidine is a non-competitive NMDA receptor antagonist with potent neuroprotective properties. However, we have previously demonstrated that gacyclidine enantiomers [(-) and (+)GK11] interact with other ('non-NMDA') binding sites which may play a role in the lower self-neurotoxicity of this compound relative to the other NMDA receptor antagonists. Evidence for these binding sites has been obtained from autoradiographic and membrane binding experiments. They were found to be expressed at high levels in the molecular layer of the cerebellum, although they can also been seen in the granular layer and in telencephalic regions. The present study was designed to further characterize these gacyclidine 'non-NMDA' binding sites. The pharmacological profiles obtained on cerebellar and telencephalic membrane homogenates showed that they could not be linked directly to the main receptors or uptake complexes of the central nervous system (CNS). However, the comparison of (-) and (+)[(3)H]GK11 binding distribution in different mutant animals bearing specific cellular deficits in the cerebellum has demonstrated that the gacyclidine 'non-NMDA' binding sites are associated with the dendritic trees of Purkinje cells. Interestingly, our study also shows that the radioligand binding to both cerebellar and telencephalic structures could be modulated by endogenous factors which can be removed by a stringent prewashing procedure.

    Journal of neurochemistry 2001;77;1;190-201

  • Loss of alpha3beta1 integrin function results in an altered differentiation program in the mouse submandibular gland.

    Menko AS, Kreidberg JA, Ryan TT, Van Bockstaele E and Kukuruzinska MA

    Department of Anatomy, Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. sue.menko@mail.tju.edu

    Mammalian submandibular gland (SMG) development leads to the establishment of highly organized secretory acinar and nonsecretory ductal epithelial cells. The ability of maturing salivary epithelial cells to attain their differentiated state has been shown to depend, in part, on interactions between extracellular matrix (ECM) proteins and their integrin receptors. In a search for key regulators of salivary cell lineage, we have studied alpha3beta1 integrin, a receptor for the basement membrane protein laminin, by characterizing embryonic day 18 (E18) SMGs isolated from mice carrying a targeted mutation in the alpha3 integrin gene. Transmission electron microscopy studies showed that the mutant SMGs exhibited an aberrant differentiation phenotype with defects in the apical-basal polarity axis and in the basement membrane. Based on immunohistochemistry and Western blot analyses, the alpha3beta1-deficient SMGs had altered expression and/or localization of several ECM and adhesive molecules, including laminin beta1, fibronectin, alpha5 integrin, and E-cadherin. These changes correlated with alterations in the activation state of Ras-extracellular signal-regulated kinase (ERK), as well as the expression and/or localization of Cdc42 and RhoA, two Rho GTPases that regulate the organization of the actin cytoskeleton. We conclude that alpha3beta1 is required for normal salivary cell differentiation and that its absence affects multiple components of adhesive complexes and their associated signalling pathways.

    Funded by: NIDCR NIH HHS: DE10183, DE12831

    Developmental dynamics : an official publication of the American Association of Anatomists 2001;220;4;337-49

  • Parathyroid hormone-related protein maintains mammary epithelial fate and triggers nipple skin differentiation during embryonic breast development.

    Foley J, Dann P, Hong J, Cosgrove J, Dreyer B, Rimm D, Dunbar M, Philbrick W and Wysolmerski J

    Medical Sciences, Indiana University School of Medicine, Bloomington, IN 47405, USA.

    Prior reports have demonstrated that both parathyroid hormone-related protein (PTHrP) and the type I PTH/PTHrP receptor are necessary for the proper development of the embryonic mammary gland in mice. Using a combination of loss-of-function and gain-of-function models, we now report that PTHrP regulates a series of cell fate decisions that are central to the survival and morphogenesis of the mammary epithelium and the formation of the nipple. PTHrP is made in the epithelial cells of the mammary bud and, during embryonic mammary development, it interacts with the surrounding mesenchymal cells to induce the formation of the dense mammary mesenchyme. In response, these mammary-specific mesenchymal cells support the maintenance of mammary epithelial cell fate, trigger epithelial morphogenesis and induce the overlying epidermis to form the nipple. In the absence of PTHrP signaling, the mammary epithelial cells revert to an epidermal fate, no mammary ducts are formed and the nipple does not form. In the presence of diffuse epidermal PTHrP signaling, the ventral dermis is transformed into mammary mesenchyme and the entire ventral epidermis becomes nipple skin. These alterations in cell fate require that PTHrP be expressed during development and they require the presence of the PTH/PTHrP receptor. Finally, PTHrP signaling regulates the epidermal and mesenchymal expression of LEF1 and (&bgr;)-catenin, suggesting that these changes in cell fate involve an interaction between the PTHrP and Wnt signaling pathways.

    Funded by: NIAMS NIH HHS: AR45585; NIDDK NIH HHS: DK45735, DK55501

    Development (Cambridge, England) 2001;128;4;513-25

  • Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.

    Neidhardt L, Gasca S, Wertz K, Obermayr F, Worpenberg S, Lehrach H and Herrmann BG

    Max-Planck-Institut für Immunbiologie, Abt. Entwicklungsbiologie, Stübeweg 51, 79108, Freiburg, Germany.

    We have adapted the whole-mount in situ hybridization technique to perform high-throughput gene expression analysis in mouse embryos. A large-scale screen for genes showing specific expression patterns in the mid-gestation embryo was carried out, and a large number of genes controlling development were isolated. From 35760 clones of a 9.5 d.p.c. cDNA library, a total of 5348 cDNAs, enriched for rare transcripts, were selected and analyzed by whole-mount in situ hybridization. Four hundred and twenty-eight clones revealed specific expression patterns in the 9.5 d.p.c. embryo. Of 361 tag-sequenced clones, 198 (55%) represent 154 known mouse genes. Thirty-nine (25%) of the known genes are involved in transcriptional regulation and 33 (21%) in inter- or intracellular signaling. A large number of these genes have been shown to play an important role in embryogenesis. Furthermore, 24 (16%) of the known genes are implicated in human disorders and three others altered in classical mouse mutations. Similar proportions of regulators of embryonic development and candidates for human disorders or mouse mutations are expected among the 163 new mouse genes isolated. Thus, high-throughput gene expression analysis is suitable for isolating regulators of embryonic development on a large-scale, and in the long term, for determining the molecular anatomy of the mouse embryo. This knowledge will provide a basis for the systematic investigation of pattern formation, tissue differentiation and organogenesis in mammals.

    Mechanisms of development 2000;98;1-2;77-94

  • Teratocarcinomas induced by embryonic stem (ES) cells lacking vimentin: an approach to study the role of vimentin in tumorigenesis.

    Langa F, Kress C, Colucci-Guyon E, Khun H, Vandormael-Pournin S, Huerre M and Babinet C

    Unité de Biologie du Développement, URA C.N.R.S. Institut Pasteur, Paris, France. chbabi@pasteur.fr

    Vimentin is a class III intermediate filament protein widely expressed in the developing embryo and in cells of mesenchymal origin in the adult. Vimentin knock-out mice develop and reproduce without any obvious defect. This is an unexpected finding in view of the high degree of conservation of the vimentin gene among vertebrates. However, it does not exclude the possibility of a role for vimentin in pathological conditions, like tumorigenesis. To address this question directly, we have used a teratocarcinoma model involving the injection of ES cells into syngeneic mice. ES cells lacking vimentin were generated from 129/Sv Vim-/- mice with high efficiency. The absence of vimentin did not affect ES cell morphology, viability or growth rate in vitro. Tumours were induced by subcutaneous injection of either Vim-/- or Vim+/+ ES cells into Vim+/+ and Vim-/- mice, in order to analyse the effect of the absence of vimentin in either the tumorigenic cells or the host mice. No significant differences were found in either tumour incidence, size or vascularization of teratocarcinomas obtained with all possible combinations. Vim-/- ES-derived tumours showed the same cellular composition typical of teratocarcinomas induced by wild-type ES cells together with a very similar apoptotic pattern. Taken together, these results demonstrate that in this model vimentin is not essential for efficient tumour growth and differentiation in vivo.

    Journal of cell science 2000;113 Pt 19;3463-72

  • Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray.

    Tanaka TS, Jaradat SA, Lim MK, Kargul GJ, Wang X, Grahovac MJ, Pantano S, Sano Y, Piao Y, Nagaraja R, Doi H, Wood WH, Becker KG and Ko MS

    Laboratory of Genetics and DNA Array Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6820, USA.

    cDNA microarray technology has been increasingly used to monitor global gene expression patterns in various tissues and cell types. However, applications to mammalian development have been hampered by the lack of appropriate cDNA collections, particularly for early developmental stages. To overcome this problem, a PCR-based cDNA library construction method was used to derive 52,374 expressed sequence tags from pre- and peri-implantation embryos, embryonic day (E) 12.5 female gonad/mesonephros, and newborn ovary. From these cDNA collections, a microarray representing 15,264 unique genes (78% novel and 22% known) was assembled. In initial applications, the divergence of placental and embryonic gene expression profiles was assessed. At stage E12.5 of development, based on triplicate experiments, 720 genes (6.5%) displayed statistically significant differences in expression between placenta and embryo. Among 289 more highly expressed in placenta, 61 placenta-specific genes encoded, for example, a novel prolactin-like protein. The number of genes highly expressed (and frequently specific) for placenta has thereby been increased 5-fold over the total previously reported, illustrating the potential of the microarrays for tissue-specific gene discovery and analysis of mammalian developmental programs.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;16;9127-32

  • Comparative anatomy of the cerebellar cortex in mice lacking vimentin, GFAP, and both vimentin and GFAP.

    Giménez Y Ribotta M, Langa F, Menet V and Privat A

    INSERM U 336, Université Montpellier II, Montpellier Cedex, France.

    In the cerebellum of adult mammals, glial fibrillary acidic protein (GFAP) and vimentin (VIM) are coexpressed in Golgi epithelial cells (GEC), also known as Bergmann glia. In this study we used three transgenic knockout mice (GFAP, VIM and double GFAP and VIM) to analyze the involvement of these proteins in the building of glial filaments and in neuron-glia interactions. The cerebella of VIM, GFAP, and GFAP/VIM mutant mice were processed by the rapid Golgi method and also for electron microscopy. In VIM mutant mice, Bergmann fibers are hypertrophic with thickened appendages. In the electron microscope they appear as large glial profiles devoid of glial filaments, with embedded dendritic thorns and parallel fiber boutons. In addition, signs of degeneration are observed in Purkinje cells. In GFAP mutant mice, GEC exhibit fine, delicate processes, as those seen in wild-type animals, however, a large accumulation of lamellae and granular appendages was observed along their surfaces, which came into contact with each other. The electron microscope exhibited fine and scarce astroglial profiles containing some glial filaments, a stunted glia limitans, and the presence of large extracellular spaces. In double mutant mice, the two phenotypes are expressed but appear attenuated, with a total absence of glial filaments and the general appearance of immaturity for GEC. In conclusion, it appears that the absence of each of the proteins yields a specific phenotype and that the defects are not necessarily additive.

    Glia 2000;31;1;69-83

  • Impaired wound healing in embryonic and adult mice lacking vimentin.

    Eckes B, Colucci-Guyon E, Smola H, Nodder S, Babinet C, Krieg T and Martin P

    Department of Dermatology, University of Cologne, Köln, Germany.

    It is generally assumed that the vimentin intermediate filament network present in most mesenchymally-derived cells is in part responsible for the strength and integrity of these cells, and necessary for any tissue movements that require the generation of significant tractional forces. Surprisingly, we have shown that transgenic KO mice deficient for vimentin are apparently able to undergo embryonic development absolutely normally and go onto develop into adulthood and breed without showing any obvious phenotype. However, fibroblasts derived from these mice are mechanically weak and severely disabled in their capacity to migrate and to contract a 3-D collagen network. To assess whether these functions are necessary for more challenging tissue movements such as those driving in vivo tissue repair processes, we have analysed wound healing ability in wild-type versus vimentin-deficient embryos and adult mice. Wounds in vimentin-deficient adult animals showed delayed migration of fibroblasts into the wound site and subsequently retarded contraction that correlated with a delayed appearance of myofibroblasts at the wound site. Wounds made to vimentin-deficient embryos also failed to heal during the 24 hour culture period it takes for wild-type embryos to fully heal an equivalent wound. By DiI marking the wound mesenchyme and following its fate during the healing process we showed that this impaired healing is almost entirely due to a failure of mesenchymal contraction at the embryonic wound site. These observations reveal an in vivo phenotype for the vimentin-deficient mouse, and challenge the dogma that key morphogenetic events occurring during development require generation of significant tractional forces by mesenchymal cells.

    Journal of cell science 2000;113 ( Pt 13);2455-62

  • Localization of gp130 in the developing and adult mouse cerebellum.

    Ha BK and King JS

    Department of Neuroscience, The Ohio State University, 4068 Graves Hall, 333 W. 10th Avenue, Columbus, OH 43210, USA.

    Interleukin-6 (IL-6) type cytokines show functional redundancy in the immune, hematopoietic, and nervous system, which is believed to result from sharing of the signal transducing receptor gp130. IL-6 type cytokines and their binding receptors have been localized in the adult cerebellum. However, the cellular localization and developmental regulation of gp130 in the cerebellum have not been determined. In the present study the expression pattern of gp130 in the developing and adult mouse cerebellum was investigated. At embryonic day (E)15 and E17, gp130 immunoreactivity is present primarily in fiber bundles that course from the brainstem to the cerebellum. At postnatal day (P)0, gp130 immunoreactivity first appears in the Purkinje cell layer, external granule cell layer, and cerebellar nuclei. As Purkinje cells differentiate, gp130 immunoreactivity progressively extends from the cell body along their developing dendritic arbor. All Purkinje cells show intense gp130 immunoreactivity in their cell bodies by P7. In contrast the gp130 immunoreactivity detected in fiber bundles at E15 and E17 is downregulated postnatally, and cannot be detected after P7. Granule cells show gp130 immunoreactivity at P0 in the external granule cell layer and subsequently in the internal granule cell layer. Astrocytes in the white matter express gp130 at P0, and show intense gp130 immunoreactivity between P7 and P14. As the cerebellum matures gp130 immunoreactivity in the white matter decreases. The present description of the differential spatial and temporal distribution of gp130 provides an initial step in defining specific cellular populations that are potential targets of IL-6 type cytokines during cerebellar ontogeny.

    Funded by: NINDS NIH HHS: NS-08798

    Journal of chemical neuroanatomy 2000;19;3;129-41

  • Cytokeratin, vimentin and E-cadherin immunodetection in the embryonic palate in two strains of mice with different susceptibility to glucocorticoid-induced clefting.

    Montenegro MA, Rojas M, Dominguez S and Vergara A

    Department of Morphology, Biomedical Ciencies Institute, Faculty of Medicine, University of Chile. mmontene@machi.med.uchile.cl

    An immunohistochemical study analyzing the pattern of distribution of some intermediate filament proteins, keratin and vimentin and, one adhesion molecule, cadherin in different stages of developing secondary palate in two strains of mice with different H-2 backgrounds was undertaken to investigate differences between a strain that is susceptible to glucocorticoid-induced cleft palate (A/Sn) and one that is resistant to glucocorticoid-induced cleft palate (C57/BL). The heads of embryos were processed by standard immunohistochemistry with antipancytokeratin (KAE1), antikeratins 18 (K18) and 19 (K19), antivimentin, and anti E-cadherin antibodies. Immunostaining with KAE1 antibody showed differences between the strains. The reaction was stronger in the medial edge epithelia of palatal processes in the A/Sn strain at all stages of palatogenesis. The C57/BL strain showed a weak immunostain to KAE1. Antivimentin antibody stained the mesenchymal cells of palatal processes and K18 and K19 showed no reaction in either strain of mice. Anti E-cadherin antibody was detected in the medial palatal epithelium of both strains of mice and in all stages of palate development. No differences were observed in E-cadherin and vimentin immunostain in palatal epithelium between the strains. The different expression of some cytokeratins in the embryonic palatal epithelium suggests that these intermediate filament proteins may be involved in different susceptibility to glucocorticoid-induced cleft palate in the mouse. The decreased immunoreaction of cytokeratins observed in the resistant strain would facilitate the disappearance of this molecule during the transformation from an epithelial to a mesenchymal phenotype that takes place during the development of the palate. These results may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformation in the embryonic palate.

    Journal of craniofacial genetics and developmental biology 2000;20;3;137-43

  • Altered flow-induced arterial remodeling in vimentin-deficient mice.

    Schiffers PM, Henrion D, Boulanger CM, Colucci-Guyon E, Langa-Vuves F, van Essen H, Fazzi GE, Lévy BI and De Mey JG

    Department of Pharmacology and Toxicology and Cardiovascular Research Institute Maastricht, Universiteit Maastricht, Maastricht, The Netherlands.

    The endothelial cytoskeleton plays a key role in arterial responses to acute changes in shear stress. We evaluated whether the intermediate filament protein vimentin is involved in the structural responses of arteries to chronic changes in blood flow (BF). In wild-type mice (V+/+) and in vimentin-deficient mice (V-/-), the left common carotid artery (LCA) was ligated near its bifurcation, and 4 weeks later, the structures of the occluded and of the contralateral arteries were evaluated and compared with the structures of arteries from sham-operated mice. Body weight and mean carotid artery BF did not differ between the strains, but LCA and right carotid artery (RCA) diameter (737+/-14 microm [LCA] and 723+/-14 microm [RCA] for V-/- versus 808+/-20 microm [LCA] and 796+/-20 microm [RCA] for V+/+) and medial cross-sectional area (CSAm) were significantly smaller in V-/- (21+/-1 and 22+/-2 x 10(3) microm(2) for LCA and RCA, respectively) than in V+/+ (28+/-2 and 28+/-3 x 10(3) microm(2) for LCA and RCA, respectively). In V+/+, LCA ligation eliminated BF in the occluded vessel (before ligation, 0. 35+/-0.02 mL/min) and increased BF from 0.34+/-0.02 to 0.68+/-0.04 mL/min in the RCA. In V-/-, the BF change in the occluded LCA was comparable (from 0.38+/-0.05 mL/min to zero-flow rates), but the BF increase in the RCA was less pronounced (from 0.33+/-0.02 to 0. 50+/-0.05 mL/min). In the occluded LCA of V+/+, arterial diameter was markedly reduced (-162 microm), and CSAm was significantly increased (5 x 10(3) microm(2)), whereas in the high-flow RCA of V+/+, carotid artery diameter and CSAm were not significantly modified. In the occluded LCA of V-/-, arterial diameter was reduced to a lesser extent (-77 microm) and CSAm was increased to a larger extent (10 x 10(3) microm(2)) than in V+/+. In contrast to V+/+, the high-flow RCA of V-/- displayed a significant increase in diameter (52 microm) and a significant increase in CSAm (5 x 10(3) microm(2)). These observations provide the first direct evidence for a role of the cytoskeleton in flow-induced arterial remodeling. Furthermore, they dissociate (1) between acute and chronic arterial responses to altered BF, (2) between alterations of lumen diameter and wall mass during arterial remodeling, and (3) between developmental and imposed flow-induced arterial remodeling.

    Arteriosclerosis, thrombosis, and vascular biology 2000;20;3;611-6

  • Pax-2 expression defines a subset of GABAergic interneurons and their precursors in the developing murine cerebellum.

    Maricich SM and Herrup K

    Alzheimer Research Laboratory, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.

    Pax-2 is a paired box transcription factor expressed in several regions of the developing mammalian central nervous system. First found in the midbrain/hindbrain region, Pax-2 expression is later found in the cerebellum, hindbrain, and spinal cord. We have examined the expression pattern of Pax-2 from embryonic day 12 (E12) through postnatal day 35 (P35) using immunohistochemistry and in situ hybridization. Expression of Pax-2 is found in scattered cells of the cerebellar ventricular zone at E13. Pax-2-expressing cells migrate away from this germinative center to positions in the deep cerebellar nuclei (DCN), internal granule cell layer, molecular layer, and folial white-matter tracts of the cerebellum. Immunocytochemistry of both tissue sections and primary dissociated cultures demonstrates that Pax-2 is expressed by cells of a neuronal lineage, but not by cells of either an astrocytic or oligodendrocytic lineage. Specifically, the presence of Pax-2 identifies the entire population of gamma-aminobutyric acid (GABA)ergic interneurons in the cerebellar cortex (Golgi II, basket and stellate cells) and in the DCN. Bromodeoxyuridase labeling and 4',6-diamino-2-phenylindole (DAPI) staining of cells in M-phase reveals that Pax-2-expressing cells in the folial white-matter tracts of the cerebellum constitute an actively dividing population. We propose that these cells are migratory precursors of the molecular layer interneurons (basket and stellate cells). Our data suggest that the role of Pax-2 in cerebellar development changes after E12, shifting from the specification of an anatomical field to the marking of a specific class of cells. Our findings also suggest a previously uncharacterized relationship among GABAergic interneurons found posterior to the midbrain. Finally, our data support the hypothesis that the basket and stellate cells arise from neuronally restricted, migratory precursors located in the early postnatal cerebellar white matter.

    Funded by: NINDS NIH HHS: NS18381

    Journal of neurobiology 1999;41;2;281-94

  • Renal tubular cells cultured from genetically modified animals.

    Friedlander G, Runembert I, Vrtovsnik F and Terzi F

    INSERM U 426 and Department of Physiology, Faculté de Médecine Xavier-Bichat, Université Denis-Diderot, Paris, France.

    The culture of renal tubular cells from genetically modified animals opens the opportunity of biochemical, cell biology and physiological studies under strictly controlled conditions. Either primary cultures or cell lines can be used. Through two examples of primary cultures of proximal tubular cells obtained from knock-out mice, important information about the function of proteins were obtained. Mice lacking vimentin, an intermediate filament normally reexpressed in tubular cells during regeneration and culture, have a normal tubular function under basal conditions. Proximal cells grown from these animals exhibit a defect in sodium-glucose cotransport activity, most likely related to alterations in the dimer/monomer ratio of the transporter in the apical membranes. These alterations may be important in terms of tubular function during the recovery phase following acute tubular necrosis. The situation is strikingly different with regard to mice lacking HNF-1, a transactivator involved in the transcription of multiple genes. These animals suffer from severe Fanconi syndrome related to decreased expression of proximal transporters including isoforms of sodium-glucose (SGLT2) and sodium-phosphate (NPT1) cotransporters. Whereas transport defects are observed in isolated tubules, they are no longer apparent in cultured proximal cells because the expression of these isoforms is suppressed under culture conditions. These observations illustrate the interest and limits of the in vitro models for studying renal function in transgenic animals.

    Experimental nephrology 1999;7;5-6;407-12

  • Intermediate filament protein partnership in astrocytes.

    Eliasson C, Sahlgren C, Berthold CH, Stakeberg J, Celis JE, Betsholtz C, Eriksson JE and Pekny M

    Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden.

    Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytes in vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.

    The Journal of biological chemistry 1999;274;34;23996-4006

  • Abnormal reaction to central nervous system injury in mice lacking glial fibrillary acidic protein and vimentin.

    Pekny M, Johansson CB, Eliasson C, Stakeberg J, Wallén A, Perlmann T, Lendahl U, Betsholtz C, Berthold CH and Frisén J

    Department of Medical Biochemistry, Gothenburg University, SE-405 30 Gothenburg, Sweden. milos.pekny@medkem.gu.se

    In response to injury of the central nervous system, astrocytes become reactive and express high levels of the intermediate filament (IF) proteins glial fibrillary acidic protein (GFAP), vimentin, and nestin. We have shown that astrocytes in mice deficient for both GFAP and vimentin (GFAP-/-vim-/-) cannot form IFs even when nestin is expressed and are thus devoid of IFs in their reactive state. Here, we have studied the reaction to injury in the central nervous system in GFAP-/-, vimentin-/-, or GFAP-/-vim-/- mice. Glial scar formation appeared normal after spinal cord or brain lesions in GFAP-/- or vimentin-/- mice, but was impaired in GFAP-/-vim-/- mice that developed less dense scars frequently accompanied by bleeding. These results show that GFAP and vimentin are required for proper glial scar formation in the injured central nervous system and that some degree of functional overlap exists between these IF proteins.

    The Journal of cell biology 1999;145;3;503-14

  • Laminin-1 promotes differentiation of fetal mouse pancreatic beta-cells.

    Jiang FX, Cram DS, DeAizpurua HJ and Harrison LC

    Autoimmunity and Transplantation Division, The Walter and Eliza Hall Institute of Medical Research, The Royal Melbourne Hospital, Parkville, Victoria, Australia.

    Extracellular factors that regulate the growth and differentiation of cell lineages in the pancreatic primordia are poorly understood. Identification of these factors for pancreatic islet beta-cells could open new avenues for the treatment of insulin-dependent diabetes. We developed a low cell density serum-free culture system for dissociated pancreatic cells from the 13.5-day mouse fetus and investigated the effects of extracellular matrix proteins on differentiation of islet cells. After 4 days in culture, total cell number decreased by two-thirds, but insulin-positive beta-cell number increased 10-fold. Both of collagens I and IV inhibited cell survival (by >50%), whereas fibronectin had no effect. In the presence of soluble laminin-1, however, the number of beta-cells increased linearly by 60-fold without an increase in the total cell number; glucagon-positive cell number was unchanged, and somatostatin and pancreatic polypeptide-positive cells were not detected. The effect of laminin-1 was completely blocked by a monoclonal rat anti-laminin-1 antibody. In the presence of laminin-1, the thymidine analogue, BrdU, was incorporated into only 2.5% of cells, which were mainly insulin-negative at days 1-3. Laminin-1 appeared, therefore, to induce differentiation of beta-cells from precursor cells in day-13.5 fetal pancreas. Laminin-1 was shown to be expressed in the epithelial basement membrane of the 13.5- to 17.5-day fetal pancreas. These findings provide the first evidence of a role for laminin-1 to promote differentiation of pancreatic beta-cells.

    Diabetes 1999;48;4;722-30

  • Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse.

    Morohashi K, Tsuboi-Asai H, Matsushita S, Suda M, Nakashima M, Sasano H, Hataba Y, Li CL, Fukata J, Irie J, Watanabe T, Nagura H and Li E

    Division of Cell Differentiation, Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki, Japan.

    The spleen has two main functions. The first is to provide a proper microenvironment to lymphoid and myeloid cells, whereas the second involves clearance of abnormal erythrocytes. Ad4BP/SF-1, a product of the mammalian FTZ-F1 gene (mFTZ-F1), was originally identified as a steroidogenic, tissue-specific transcription factor. Immunohistochemical examination of the mammalian spleens confirmed the expression of Ad4BP/SF-1 in endothelial cells of the splenic venous sinuses and pulp vein. In mFtz-F1 gene-disrupted (KO) mice, several structural abnormalities were detected in the spleen, including underdevelopment and nonuniform distribution of erythrocytes. Examination of the spleen of KO fetuses showed failure of development of certain tubular structures during embryogenesis. These structures are normally assembled by Ad4BP/SF-1 immunoreactive cells, and most likely form the vascular system during later stages of development. Other structural abnormalities in the spleen of the KO mice included defects in the tissue distribution of type-IV collagen, laminin, c-kit, and vimentin. These morphologic defects in the vascular system were associated with a decrease in the proportion of hematopoietic cells, although differentiation of these cells was not affected significantly. A high number of abnormal red blood cells containing Howell-Jolly bodies were noted in the KO mice, indicating impaired clearance by the splenic vascular system. We also detected the presence of an mRNA-encoding cholesterol side-chain cleavage P450 in the spleen, resembling the findings in steroidogenic tissues such as the gonads and adrenal cortex. The mRNA transcript was not involved in splenic structural defects as it was detected in the spleens of both normal and KO mice, indicating that the regulatory mechanism of the P450 gene in the spleen is different from that in steroidogenic tissues. Our results indicate that a lack of the mFtz-F1 gene in mice is associated with structural and functional abnormalities of the splenic vascular system.

    Blood 1999;93;5;1586-94

  • Cerebellar defect and impaired motor coordination in mice lacking vimentin.

    Colucci-Guyon E, Giménez Y Ribotta M, Maurice T, Babinet C and Privat A

    Unité de Biologie du Développement, Institut Pasteur, Paris, France.

    Vimentin belongs to the family of intermediate filament (IF) proteins. During the nervous system development in mammals, it is transiently expressed in precursor cells of neuronal and glial lineages, and then it is progressively replaced by other types of IF proteins. Surprisingly, mice knock-out for vimentin develop and reproduce without any apparent defects (Colucci-Guyon et al. Cell 79:679-694, 1994). In adult rodents, Bergmann glia (BG) of the cerebellum continue to express vimentin together with glial fibrillary acidic protein (GFAP). A careful analysis of cerebellar morphology and ultrastructure in mutants showed poorly developed and highly abnormal BG, whereas the migration of granular neurons proceeded normally. Moreover, many Purkinje cells (PC) appeared stunted with a loss of spiny branchlets, and some of them were necrotic. Finally, impaired motor coordination was evidenced by behavioral tests. These observations demonstrate a role for vimentin in contributing to the normal development and morphology of BG and reveal a hitherto unreported functional relationship between BG and PC.

    Glia 1999;25;1;33-43

  • Altered taurine release following hypotonic stress in astrocytes from mice deficient for GFAP and vimentin.

    Ding M, Eliasson C, Betsholtz C, Hamberger A and Pekny M

    Department of Anatomy and Cell Biology, University of Göteborg, PO Box 420, SE-405 30, Göteborg, Sweden.

    Astrocytes maintain their volume in response to changes in osmotic pressure in their environment by an afflux/influx of ions and organic osmoequivalents. The initial swelling of an astrocyte transferred to a hypoosmotic medium is thus reversed within minutes. The mechanisms which trigger this process as well as the sensors for cell volume are largely unknown, however, the cytoskeleton appears to be involved. We have addressed the role of one component of the cytoskeleton, the intermediate filaments, in the maintenance of astrocytic cell volume. Astrocytes from wild type mice were compared with cells from mice deficient for either glial fibrillary acidic protein (GFAP-/-) or vimentin (vimentin-/-) and with astrocytes from mice deficient for both proteins (GFAP-/-vim-/-). Whereas GFAP-/- and vimentin-/- cultured or reactive astrocytes retain intermediate filaments, the GFAP-/-vim-/- astrocytes are completely devoid of these structures. The rate of efflux of the preloaded osmoequivalent 3H-taurine from primary and passaged cultures of astrocytes was monitored. A reduction of NaCl (25 mM) in the perfusion medium led to a 400-900% increase of 3H-taurine afflux in astrocytes from wild type mice. The stimulated efflux was not significantly affected in astrocytes from GFAP-/- or vimentin-/- mice. However, the efflux from astrocytes from GFAP-/-vim-/- mice was 25-46% lower than the wild type levels. The results strengthen the role of the cytoskeleton in astrocyte volume regulation and suggest an involvement of intermediate filaments in the process.

    Brain research. Molecular brain research 1998;62;1;77-81

  • Expression of Kv1.1, a Shaker-like potassium channel, is temporally regulated in embryonic neurons and glia.

    Hallows JL and Tempel BL

    The Virginia Merrill Bloedel Hearing Research Center and the Departments of Pharmacology and Otolaryngology-Head and Neck Surgery, University of Washington School of Medicine, Seattle, Washington 98195, USA.

    Kv1.1, a Shaker-like voltage-gated potassium channel, is strongly expressed in a variety of neurons in adult rodents, in which it appears to be involved in regulating neuronal excitability. Here we show that Kv1.1 is also expressed during embryonic development in the mouse, exhibiting two transient peaks of expression around embryonic day 9.5 (E9.5) and E14.5. Using both in situ hybridization and immunocytochemistry, we have identified several cell types and tissues that express Kv1.1 RNA and protein. At E9.5, Kv1.1 RNA and protein are detected transiently in non-neuronal cells in several regions of the early CNS, including rhombomeres 3 and 5 and ventricular zones in the mesencephalon and diencephalon. At E14.5, several cell types in both the CNS and peripheral nervous system express Kv1.1, including neuronal cells (sensory ganglia and outer aspect of cerebral hemispheres) and glial cells (radial glia, satellite cells, and Schwann cell precursors). These data show that Kv1.1 is expressed transiently in a variety of neuronal and non-neuronal cells during restricted periods of embryonic development. Although the functional roles of Kv1.1 in development are not understood, the cell-specific localization and timing of expression suggest this channel may play a role in several developmental processes, including proliferation, migration, or cell-cell adhesion.

    Funded by: NICHD NIH HHS: N01-HD-6-2915; NIGMS NIH HHS: GM07750; NINDS NIH HHS: NS27206

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;15;5682-91

  • Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts.

    Eckes B, Dogic D, Colucci-Guyon E, Wang N, Maniotis A, Ingber D, Merckling A, Langa F, Aumailley M, Delouvée A, Koteliansky V, Babinet C and Krieg T

    Department of Dermatology, University of Cologne, Federal Republic of Germany.

    Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

    Funded by: NCI NIH HHS: CA-45538; NHLBI NIH HHS: HL-33009

    Journal of cell science 1998;111 ( Pt 13);1897-907

  • Vimentin: the conundrum of the intermediate filament gene family.

    Evans RM

    Department of Pathology, University of Colorado Health Sciences Center, Denver 80262, USA. Robert.Evans@UCHSC.edu

    Intermediate filaments are a major component of the "cytoskeleton" of "higher" eukaryotes. These filaments are composed of a number of different, although structurally related, proteins. Different intermediate filament protein genes are expressed in different tissues. Spontaneous and experimentally produced mutations in the intermediate filament genes indicate that these filaments function to enhance the mechanical stability of epidermal and muscle cells. As a result, the use of transgenic mice with "knockout" or dominant negative mutations in IF genes has become an important approach for investigating the significance of IFs in other cell types. However, a knockout mutation of vimentin (-/-), the intermediate filament protein characteristically expressed in cells of mesenchymal origin, results in very subtle phenotypes that are not obviously related to cell fragility. Although experiments with cultured cells have described a variety of discrete changes in cell properties that are associated with vimentin expression or organization, there is no evidence yet that any of these properties are affected in the vimentin-/- mouse.

    Funded by: NHLBI NIH HHS: HL51850

    BioEssays : news and reviews in molecular, cellular and developmental biology 1998;20;1;79-86

  • Impaired flow-induced dilation in mesenteric resistance arteries from mice lacking vimentin.

    Henrion D, Terzi F, Matrougui K, Duriez M, Boulanger CM, Colucci-Guyon E, Babinet C, Briand P, Friedlander G, Poitevin P and Lévy BI

    Institut National de la Santé et de la Recherche Médicale (INSERM) U 141, IFR 6 (Circulation-Lariboisière), Hôpital Lariboisière, Paris, France. daniel.henrion@inserm.lrb.ap-hop-paris.fr

    The intermediate filament vimentin might play a key role in vascular resistance to mechanical stress. We investigated the responses to pressure (tensile stress) and flow (shear stress) of mesenteric resistance arteries perfused in vitro from vimentin knockout mice. Arteries were isolated from homozygous (Vim-/-, n = 14) or heterozygous vimentin-null mice (Vim+/-, n = 5) and from wild-type littermates (Vim+/+, n = 9). Passive arterial diameter (175+/-15 micron in Vim+/+ at 100 mmHg) and myogenic tone were not affected by the absence of vimentin. Flow-induced (0-150 microl/min) dilation (e. g., 19+/-3 micron dilation at 150 mmHg in Vim+/+) was significantly attenuated in Vim-/- mice (13+/-2 micron dilation, P < 0.01). Acute blockade of nitric oxide synthesis (NG-nitro- L-arginine, 10 microM) significantly decreased flow-induced dilation in both groups, whereas acute blockade of prostaglandin synthesis (indomethacin, 10 microM) had no significant effect. Mean blood pressure, in vivo mesenteric blood flow and diameter, and mesenteric artery media thickness or media to lumen ratio were not affected by the absence of vimentin. Thus, the absence of vimentin decreased selectively the response of resistance arteries to flow, suggesting a role for vimentin in the mechanotransduction of shear stress.

    The Journal of clinical investigation 1997;100;11;2909-14

  • Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle.

    Li Z, Mericskay M, Agbulut O, Butler-Browne G, Carlsson L, Thornell LE, Babinet C and Paulin D

    Station Centrale de Microscopie Electronique, Institut Pasteur, Paris, France.

    A null mutation was introduced into the mouse desmin gene by homologous recombination. The desmin knockout mice (Des -/-) develop normally and are fertile. However, defects were observed after birth in skeletal, smooth, and cardiac muscles (Li, Z., E. Colucci-Guyon, M. Pincon-Raymond, M. Mericskay, S. Pournin, D. Paulin, and C. Babinet. 1996. Dev. Biol. 175:362-366; Milner, D.J., G. Weitzer, D. Tran, A. Bradley, and Y. Capetanaki. 1996. J. Cell Biol. 134:1255- 1270). In the present study we have carried out a detailed analysis of somitogenesis, muscle formation, maturation, degeneration, and regeneration in Des -/- mice. Our results demonstrate that all early stages of muscle differentiation and cell fusion occur normally. However, after birth, modifications were observed essentially in weight-bearing muscles such as the soleus or continually used muscles such as the diaphragm and the heart. In the absence of desmin, mice were weaker and fatigued more easily. The lack of desmin renders these fibers more susceptible to damage during contraction. We observed a process of degeneration of myofibers, accompanied by macrophage infiltration, and followed by a process of regeneration. These cycles of degeneration and regeneration resulted in a relative increase in slow myosin heavy chain (MHC) and decrease in fast MHC. Interestingly, this second wave of myofibrillogenesis during regeneration was often aberrant and showed signs of disorganization. Subsarcolemmal accumulation of mitochondria were also observed in these muscles. The lack of desmin was not compensated by an upregulation of vimentin in these mice either during development or regeneration. Absence of desmin filaments within the sarcomere does not interfere with primary muscle formation or regeneration. However, myofibrillogenesis in regenerating fibers is often abortive, indicating that desmin may be implicated in this repair process. The results presented here show that desmin is essential to maintain the structural integrity of highly solicited skeletal muscle.

    The Journal of cell biology 1997;139;1;129-44

  • Homeobox genes from clusters A and B demonstrate characteristics of temporal colinearity and differential restrictions in spatial expression domains in the branching mouse lung.

    Mollard R and Dziadek M

    Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia. mollard@titus.u-strasbg.fr

    Lung branching morphogenesis is accomplished by reciprocal morphogenetic interactions between the epithelium and its mesenchyme. In order to better understand the molecular mechanisms regulating these interactions in time and space, the expression patterns of Hox genes isolated exclusively from the branching region of the developing lung have been investigated. Reverse transcriptase PCR identified Hoxa-1, Hoxa-3, Hoxa-5, Hoxb-3, Hoxb-4, Hoxb-6, Hoxb-7, and Hoxb-8 transcripts from within this tissue at 11.5 day post coitum (E11.5). Northern blot, in situ hybridization and PCR analyses demonstrated qualitative and quantitative differences in expression patterns for each gene assessed in this region thus providing evidence for Hox gene temporal colinearity. Furthermore, although not within the context of strict anteroposterior definition, Hox genes located within a more 5' region in both clusters were found to have greater spatial expression constrictions when compared to their more 3' counterparts. These Hox genes were also differentially expressed both between and within specific germ cell lineage derivatives. Such patterns of expression suggest that Hox genes play a role in the specification and maturation of lung cell lineage derivatives throughout the pseudoglandular, canalicular and terminal sac phases of lung development.

    The International journal of developmental biology 1997;41;5;655-66

  • Reduction of renal mass is lethal in mice lacking vimentin. Role of endothelin-nitric oxide imbalance.

    Terzi F, Henrion D, Colucci-Guyon E, Federici P, Babinet C, Levy BI, Briand P and Friedlander G

    INSERM U 380, Institut Cochin de Génétique Moléculaire, Paris, France. terzi@cochin.inserm.fr

    Modulation of vascular tone by chemical and mechanical stimuli is a crucial adaptive phenomenon which involves cytoskeleton elements. Disruption, by homologous recombination, of the gene encoding vimentin, a class III intermediate filament protein mainly expressed in vascular cells, was reported to result in apparently normal phenotype under physiological conditions. In this study, we evaluated whether the lack of vimentin affects vascular adaptation to pathological situations, such as reduction of renal mass, a pathological condition which usually results in immediate and sustained vasodilation of the renal vascular bed. Ablation of 3/4 of renal mass was constantly lethal within 72 h in mice lacking vimentin (Vim-/-), whereas no lethality was observed in wild-type littermates. Death in Vim-/- mice resulted from end-stage renal failure. Kidneys from Vim-/- mice synthesized more endothelin, but less nitric oxide (NO), than kidneys from normal animals. In vitro, renal resistance arteries from Vim-/- mice were selectively more sensitive to endothelin, less responsive to NO-dependent vasodilators, and exhibited an impaired flow (shear stress)- induced vasodilation, which is NO dependent, as compared with those from normal littermates. Finally, in vivo administration of bosentan, an endothelin receptor antagonist, totally prevented lethality in Vim-/- mice. These results suggest that vimentin plays a key role in the modulation of vascular tone, possibly via the tuning of endothelin-nitric oxide balance.

    The Journal of clinical investigation 1997;100;6;1520-8

  • Dynamin genes Dnm1 and Dnm2 are located on proximal mouse chromosomes 2 and 9, respectively.

    Klocke R, Augustin A, Ronsiek M, Stief A, van der Putten H and Jockusch H

    Developmental Biology Unit W7, University of Bielefeld, Germany.

    Dynamins, microtubule-binding GTPases, are encoded by at least three genes in mammals. Two distinct gene-specific cDNAs were used to analyze the segregation of dynamin genes Dnm1 and Dnm2 in a mouse interspecies backcross. The nervous system-expressed gene Dnm1 was localized to Chr 2 between the genes for vimentin and nebulin, within a chromosomal region of conserved synteny to human chromosome 9q, consistent with the localization of the human dynamin-1 gene by FISH (see accompanying paper by Newman-Smith et al., 1997, Genomics 41:286-289). The ubiquitously expressed Dnm2 gene was found to be closely linked to the intercellular adhesion molecule-1 gene, Icam1, in a region with homologies to human chromosomes 19p, 8q, and 11q. Potential relations of both loci to disease genes are discussed.

    Genomics 1997;41;2;290-2

  • Transgenic mice carrying chimeric or mutated type III intermediate filament (IF) genes.

    Bloemendal H, Raats JM, Pieper FR, Benedetti EL and Dunia I

    Department of Biochemistry, University of Nijmegen, The Netherlands. H.Bloemendal@bioch.kun.nl

    Mice carrying chimeric, truncated or mutated genes encoding intermediate filament (IF) proteins type III do not show any detectable severe pathology. However, upon (over)expression of the transgene in the eye lens all animals develop lens opacification (cataract). At the cellular level the loss of visual acuity is preceded by interference with the terminal differentiation of lens fibre cells, plasma membrane damage, distorted assembly of the IF cytoskeleton and perturbation of the cytoskeleton-membrane complex. The degree of expression is paralleled by the extent of the damages.

    Cellular and molecular life sciences : CMLS 1997;53;1;1-12

  • Targeted deletion in astrocyte intermediate filament (Gfap) alters neuronal physiology.

    McCall MA, Gregg RG, Behringer RR, Brenner M, Delaney CL, Galbreath EJ, Zhang CL, Pearce RA, Chiu SY and Messing A

    Department of Pathobiological Sciences, University of Wisconsin-Madison, 53706, USA.

    Glial fibrillary acidic protein (GFAP) is a member of the family of intermediate filament structural proteins and is found predominantly in astrocytes of the central nervous system (CNS). To assess the function of GFAP, we created GFAP-null mice using gene targeting in embryonic stem cells. The GFAP-null mice have normal development and fertility, and show no gross alterations in behavior or CNS morphology. Astrocytes are present in the CNS of the mutant mice, but contain a severely reduced number of intermediate filaments. Since astrocyte processes contact synapses and may modulate synaptic function, we examined whether the GFAP-null mice were altered in long-term potentiation in the CA1 region of the hippocampus. The GFAP-null mice displayed enhanced long-term potentiation of both population spike amplitude and excitatory post-synaptic potential slope compared to control mice. These data suggest that GFAP is important for astrocyte-neuronal interactions, and that astrocyte processes play a vital role in modulating synaptic efficacy in the CNS. These mice therefore represent a direct demonstration that a primary defect in astrocytes influences neuronal physiology.

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;13;6361-6

  • Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice.

    Galou M, Colucci-Guyon E, Ensergueix D, Ridet JL, Gimenez y Ribotta M, Privat A, Babinet C and Dupouey P

    Unité de Biochimie des Antigènes, Institut Pasteur, Paris, France.

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed predominantly in astrocytes. The study of its expression in the astrocyte lineage during development and in reactive astrocytes has revealed an intricate relationship with the expression of vimentin, another intermediate filament protein widely expressed in embryonic development. these findings suggested that vimentin could be implicated in the organization of the GFAP network. To address this question, we have examined GFAP expression and network formation in the recently generated vimentin knockout (Vim-) mice. We show that the GFAP network is disrupted in astrocytes that normally coexpress vimentin and GFAP, e.g., those of the corpus callosum or the Bergmann glia of cerebellum. Furthermore, Western blot analysis of GFAP protein content in the cerebellum suggests that posttranslational mechanisms are implicated in the disturbance of GFAP network formation. The role of vimentin in this process was further suggested by transfection of Vim-cultured astrocytes with a vimentin cDNA, which resulted in the normal assembly of the GFAP network. Finally, we examined GFAP expression after stab wound-induced astrogliosis. We demonstrate that in Vim- mice, reactive astrocytes that normally express both GFAP and vimentin do not exhibit GFAP immunoreactivity, whereas those that normally express GFAP only retain GFAP immunoreactivity. Taken together, these results show that in astrocytes, where vimentin is normally expressed with GFAP fails to assemble into a filamentous network in the absence of vimentin. In these cells, therefore, vimentin appears necessary to stabilize GFAP filaments and consequently the network formation.

    The Journal of cell biology 1996;133;4;853-63

  • Fas and Fas ligand in embryos and adult mice: ligand expression in several immune-privileged tissues and coexpression in adult tissues characterized by apoptotic cell turnover.

    French LE, Hahne M, Viard I, Radlgruber G, Zanone R, Becker K, Müller C and Tschopp J

    Department of Dermatology, University of Geneva, Medical School, Switzerland.

    The cell surface receptor Fas (FasR, Apo-1, CD95) and its ligand (FasL) are mediators of apoptosis that have been shown to be implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and one of the two major cytolytic pathways mediated by CD8+ cytolytic T cells. To gain further understanding of the Fas system., we have analyzed Fas and FasL expression during mouse development and in adult tissues. In developing mouse embryos, from 16.5 d onwards, Fas mRNA is detectable in distinct cell types of the developing sinus, thymus, lung, and liver, whereas FasL expression is restricted to submaxillary gland epithelial cells and the developing nervous system. Significant Fas and FasL expression were observed in several nonlymphoid cell types during embryogenesis, and generally Fas and FasL expression were not localized to characteristic sites of programmed cell death. In the adult mouse, RNase protection analysis revealed very wide expression of both Fas and FasL. Several tissues, including the thymus, lung, spleen, small intestine, large intestine, seminal vesicle, prostate, and uterus, clearly coexpress the two genes. Most tissues constitutively coexpressing Fas and FasL in the adult mouse are characterized by apoptotic cell turnover, and many of those expressing FasL are known to be immune privileged. It may be, therefore, that the Fas system is implicated in both the regulation of physiological cell turnover and the protection of particular tissues against potential lymphocyte-mediated damage.

    The Journal of cell biology 1996;133;2;335-43

  • Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation.

    Burmeister M, Novak J, Liang MY, Basu S, Ploder L, Hawes NL, Vidgen D, Hoover F, Goldman D, Kalnins VI, Roderick TH, Taylor BA, Hankin MH and McInnes RR

    Mental Health Research Institute, University of Michigan, Ann Arbor 48109-0720, USA.

    Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

    Funded by: NCI NIH HHS: CA33093; NIGMS NIH HHS: GM18684; NINDS NIH HHS: NS26777; ...

    Nature genetics 1996;12;4;376-84

  • Selective binding of specific mouse genomic DNA fragments by mouse vimentin filaments in vitro.

    Wang X, Tolstonog G, Shoeman RL and Traub P

    Max-Planck-Institut für Zellbiologie, Ladenburg, Germany.

    Mouse vimentin intermediate filaments (IFs) reconstituted in vitro were analyzed for their capacity to select certain DNA sequences from a mixture of about 500-bp-long fragments of total mouse genomic DNA. The fragments preferentially bound by the IFs and enriched by several cycles of affinity binding and polymerase chain reaction (PCR) amplification were cloned and sequenced. In general, they were G-rich and highly repetitive in that they often contained Gn, (GT)n, and (GA)n repeat elements. Other, more complex repeat sequences were identified as well. Apart from the capacity to adopt a Z-DNA and triple helix configuration under superhelical tension, many fragments were potentially able to form cruciform structures and contained consensus binding sites for various transcription factors. All of these sequence elements are known to occur in introns and 5'/3'-flanking regions of genes and to play roles in DNA transcription, recombination and replication. A FASTA search of the EMBL data bank indeed revealed that sequences homologous to the mouse repetitive DNA fragments are commonly associated with gene-regulatory elements. Unexpectedly, vimentin IFs also bound a large number of apparently overlapping, AT-rich DNA fragments that could be aligned into a composite sequence highly homologous to the 234-bp consensus centromere repeat sequence of gamma-satellite DNA. Previous experiments have shown a high affinity of vimentin for G-rich, repetitive telomere DNA sequences, superhelical DNA, and core histones. Taken together, these data support the hypothesis that, after penetration of the double nuclear membrane via an as yet unidentified mechanism, vimentin IFs cooperatively fix repetitive DNA sequence elements in a differentiation-specific manner in the nuclear periphery subjacent to the nuclear lamina and thus participate in the organization of chromatin and in the control of transcription, replication, and recombination processes. This includes aspects of global regulation of gene expression such as the position effects associated with translocation of genes to heterochromatic centromere and telomere regions of the chromosomes.

    DNA and cell biology 1996;15;3;209-25

  • Gene structure and chromosomal localization of the mouse NMDA receptor channel subunits.

    Nagasawa M, Sakimura K, Mori KJ, Bedell MA, Copeland NG, Jenkins NA and Mishina M

    Department of Neuropharmacology, Niigata University, Japan.

    Multiple espilon subunits are major determinants of the diversity of the N-methyl-D-aspartate (NMDA) receptor channel. The four epsilon subunit mRNAs exhibit distinct expression patterns in the brain. In an attempt to elucidate the molecular basis of selective and characteristic expression of the NMDA receptor channel subunits, we have isolated the gene encoding the mouse NMDA receptor epsilon 3 subunit and have determined its structural organization. The epsilon 3 subunit gene spans 17.5 kb and consists of 14 exons. The major transcription start site is 439 bp upstream of the ATG initiation codon as determined by primer extension and S1 nucleas protection analyses. Two polyadenylation sites are 397 (or 398) and 402 bp downstream of the termination codon. The 5'-flanking region of the epsilon 3 subunit gene contains GC-rich segments including consensus sequences for binding of the transcription factors Spl and EGR-1. The murine chromosomal locations of the five NMDA receptor channel subunits, the epsilon 1 (Grin2a), epsilon 2 (Grin2b), epsilon 3 (Grin2c), epsilon 4 (Grin2d) and zeta 1 (Grinl) subunits, were determined using an interspecific backcross mapping panel derived from crosses of [(C57BL/6JxM. spretus) F1xC57BL/6J] mice. Each of these genes mapped to a single chromosome location. The mapping results assigned the five loci to five different mouse autosomes, indicating that they have become well dispersed among mouse chromosomes.

    Funded by: NCI NIH HHS: N01-CO-46001

    Brain research. Molecular brain research 1996;36;1;1-11

  • Functional characterization of the human interleukin-15 receptor alpha chain and close linkage of IL15RA and IL2RA genes.

    Anderson DM, Kumaki S, Ahdieh M, Bertles J, Tometsko M, Loomis A, Giri J, Copeland NG, Gilbert DJ, Jenkins NA et al.

    Department of Molecular Biology, Immunex Corporation, Seattle, Washington 98101, USA.

    Interleukins-2 and -15 (IL-2 and IL-15) are cytokines with overlapping but distinct biological effects. Their receptors share two subunits (the IL-2R beta and -gamma chains) that are essential for signal transduction. The IL-2 receptor requires an additional IL-2-specific alpha subunit for high affinity IL-2 binding. Recently, a murine IL-15-specific alpha subunit was identified, cloned, and shown to be structurally related to IL-2R alpha. However, the murine IL-15R alpha alone bound IL-15 with a 1000-fold higher affinity than that seen with IL-2R alpha and IL-2. We now extend these studies into the human system with the isolation of three differentially spliced human IL-15R alpha variants that are all capable of high affinity binding of IL-15. The cytoplasmic domain of IL-15R alpha, like that of IL-2R alpha, is dispensable for mitogenic signaling, suggesting that the primary role of the alpha chains is to confer high affinity binding. At high concentrations, IL-15, like IL-2, is able to signal through a complex of IL-2R beta and -gamma in the absence of the alpha subunit. Furthermore, the IL15RA and IL2RA genes have a similar intron-exon organization and are closely linked in both human and murine genomes. However, the distribution of expression of the IL-15R alpha is much wider than that of the IL-2R alpha, suggesting a broader range of cellular targets for IL-15.

    Funded by: NCI NIH HHS: CA 01702, CA 21765, CA 23099; ...

    The Journal of biological chemistry 1995;270;50;29862-9

  • Transcriptional regulation of the vimentin-encoding gene in mouse myeloid leukemia M1 cells.

    Nakamura N, Shida M, Hirayoshi K and Nagata K

    Department of Cell Biology, Kyoto University, Japan.

    To investigate the regulatory mechanisms controlling expression of the vimentin-encoding gene (Vim) during mouse myeloid leukemia M1 cell differentiation, mouse Vim was cloned and the transcriptional activity of its 5' promoter region was analysed by chloramphenicol acetyltransferase (CAT) assay. Analyses of various deletion mutants revealed that a 188-bp fragment of the proximal Vim promoter (pVim) was sufficient for effective transcription in M1 cells. This 188-bp sequence is highly conserved between mouse, hamster and human. Further deletion analyses revealed that a minimum promoter element (-44 to +26) is essential for basic promoter function and could respond to cell differentiation. Detailed analyses of mutant and chimeric pVim constructs defined a CCAAT box at -89 to -84 to be an essential positive regulatory element. A G+C-rich element between the CCAAT and TATA boxes was found to act as a strong negative regulatory element in Vim transcription.

    Gene 1995;166;2;281-6

  • Genetic mapping of the interleukin 1 receptor alpha and beta chain genes in the mouse.

    Kozak CA and Leonard WJ

    Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0460, USA.

    Mammalian genome : official journal of the International Mammalian Genome Society 1995;6;11;823-4

  • Cell and molecular analysis of the developing and adult mouse subventricular zone of the cerebral hemispheres.

    Gates MA, Thomas LB, Howard EM, Laywell ED, Sajin B, Faissner A, Götz B, Silver J and Steindler DA

    Department of Anatomy and Neurobiology, University of Tennessee, Memphis 38163, USA.

    The subventricular zone (SVZ) of the lateral ventricle remains mitotically active in the adult mammalian central nervous system (CNS). Recent studies have suggested that this region may contain neuronal precursors (neural stem cells) in adult rodents. A variety of neuronal and glial markers as well as three extracellular matrix (ECM) markers were examined with the hope of understanding factors that may affect the growth and migration of neurons from this region throughout development and in the adult. This study has characterized the subventricular zone of late embryonic, postnatal, and adult mice using several neuronal markers [TuJ1, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), neuron-specific enolase (NSE)], glial markers [RC-2, vimentin, glial fibrillary acidic protein (GFAP), galactocerebroside (Gal-C)], ECM markers [tenascin-C (TN-C), chondroitin sulfate, a chondroitin sulfate proteoglycan termed dermatan sulfate-dependent proteoglycan-1 (DSD-1-PG)], stem-cell marker (nestin), and proliferation-specific marker [bromodeoxyuridine (BrdU)]. TuJ1+ and nestin+ cells (neurons and stem cells, respectively) persist in the region into adulthood, although the numbers of these cells become more sparse as the animal develops, and they appear to be immature compared to the cells in surrounding forebrain structures (e.g., not expressing NSE and having few, if any, processes). Likewise, NADPH-d+ cells are found in and around the SVZ during early postnatal development but become more sparse in the proliferative zone through maturity, and, by adulthood, only a few labeled cells can be found at the border between the SVZ and surrounding forebrain structures (e.g., the striatum), and even smaller numbers of positive cells can be found within the adult SVZ proper. BrdU labeling also seems to decrease significantly after the first postnatal week, but it still persists in the SVZ of adult animals. The disappearance of RC-2+ (radial) glia during postnatal development and the persistence of glial-derived ECM molecules such as tenascin and chondroitin sulfate proteoglycans (as well as other "boundary" molecules) in the adult SVZ may be associated with a persistence of immaturity, cell death, and a lack of cell emigration from the SVZ in the adult.

    Funded by: NINDS NIH HHS: NS25713, NS29225

    The Journal of comparative neurology 1995;361;2;249-66

  • Differential expression of N-CAM, vimentin and MAP1B during initial pathfinding of olfactory receptor neurons in the mouse embryo.

    Aoki K, Osumi-Yamashita N, Ninomiya Y and Eto K

    Department of Developmental Biology, Graduate School of Dentistry, Tokyo Medical and Dental University, Japan.

    Olfactory receptor neurons extend their primary axons from the nasal epithelium to the olfactory bulb primordium via the frontonasal mesenchyme. In the present study, expression of neuronal markers (vimentin and MAP1B) and N-CAM was immunohistochemically investigated in the development of the olfactory system in mouse embryos. Expression of vimentin and MAP1B was first observed at early day 10 of gestation (D10) in the posterosuperior region of the medial nasal epithelium, while N-CAM was initially detected in the mesenchyme adjacent to the vimentin- and MAP1B-positive nasal epithelium. As development proceeded, the localization of neuronal marker-positive cells was mostly included in the N-CAM positive region. In addition, we adopted in situ labelling with vital dye (DiI) to directly determine the localization of the olfactory nerve and N-CAM on the same sections. We demonstrated that most extending axons were located in the N-CAM positive region. These results suggest that the expression of N-CAM plays a crucial role in the initial pathfinding of the olfactory nerve.

    Anatomy and embryology 1995;192;3;211-20

  • Genomic structure and chromosomal localization of the mouse LIM/homeobox gene Lhx3.

    Zhadanov AB, Copeland NG, Gilbert DJ, Jenkins NA and Westphal H

    Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

    We have previously cloned the murine Lhx3 cDNA that encodes a predicted protein containing two tandemly repeated LIM domains and a homeodomain. Early expression of Lhx3 in oral ectoderm that is committed to contribute to the anterior and intermediate lobes of the pituitary and its perseverance in the adult gland strongly suggest an involvement of the gene in mediating and maintaining the differentiation program of this important endocrine system. Additional functions are suggested by the fact that Lhx3 is also expressed bilaterally along the spinal cord and the hindbrain at early stages of mouse development. Here we report the structural organization and chromosomal localization of the Lhx3 gene. The gene is composed of six exons and five introns. Two different exons, Ia and Ib, appear to be alternatively spliced to exon II. The first LIM domain is encoded by exon II and the second by exon III. The homeobox is shared by exons IV and V. We have mapped Lhx3 to the proximal region of mouse chromosome 2 in a region that shares homology with human chromosomes 9q and 10p.

    Funded by: NCI NIH HHS: N01-CO-46000

    Genomics 1995;27;1;27-32

  • Characterization and mapping of three new mammalian ATP-binding transporter genes from an EST database.

    Allikmets R, Gerrard B, Glavac D, Ravnik-Glavac M, Jenkins NA, Gilbert DJ, Copeland NG, Modi W and Dean M

    Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.

    Analysis of the human expressed sequence tag (EST) database identified four clones that contain sequences of previously uncharacterized genes, members of the ATP-binding cassette (ABC) superfamily. Two new ABC genes (EST20237, 31252) are located at Chromosome (Chr) 1q42 and 1q25 respectively in humans, as determined by FISH; at locations distinct from previously mapped genes of this superfamily. Two additional clones, EST 600 and EST 1596, were found to represent different ATP-binding domains of the same gene, ABC2. This gene was localized to 9q34 in humans by FISH and to the proximal region of Chr 2 in mice by linkage analysis. All genes display extensive diversity in sequence and expression pattern. We present several approaches to characterizing EST clones and demonstrate that the analysis of EST clones from different tissues is a powerful approach to identify new members of important gene families. Some drawbacks of using EST databases, including chimerism of cDNA clones, are discussed.

    Funded by: NCI NIH HHS: N0-CO-74101

    Mammalian genome : official journal of the International Mammalian Genome Society 1995;6;2;114-7

  • Nestin mRNA expression correlates with the central nervous system progenitor cell state in many, but not all, regions of developing central nervous system.

    Dahlstrand J, Lardelli M and Lendahl U

    Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.

    Nestin is a recently discovered intermediate filament (IF) gene. Nestin expression has been extensively used as a marker for central nervous system (CNS) progenitor cells in different contexts, based on observations indicating a correlation between nestin expression and this cell type in vivo. To evaluate this correlation in more detail nestin mRNA expression in developing and adult mouse CNS was analysed by in situ hybridization. We find that nestin is expressed from embryonic day (E) 7.75 and that expression is detected in many proliferating CNS regions: at E10.5 nestin is expressed in cells of both the rostral and caudal neural tube, including the radial glial cells; at E15.5 and postnatal day (P) 0 expression is observed largely in the developing cerebellum and in the ventricular and subventricular areas of the developing telencephalon. Furthermore, the transition from a proliferating to a post-mitotic cell state is accompanied by a rapid decrease in nestin mRNA for motor neurons in the ventral spinal cord and for neurons in the marginal layer of developing telencephalon. In contrast to these data we observe two proliferating areas, the olfactory epithelium and the precursor cells of the hippocampal granule neurons, which do not express nestin at detectable levels. Thus, nestin mRNA expression correlates with many, but not all, regions of proliferating CNS progenitor cells. In addition to its temporal and spatial regulation nestin expression also appears to be regulated at the level of subcellular mRNA localization: in columnar neuroepithelial and radial glial cells nestin mRNA is predominantly localized to the pial endfeet.

    Brain research. Developmental brain research 1995;84;1;109-29

  • Normal and pathological expression of GFAP promoter elements in transgenic mice.

    Galou M, Pournin S, Ensergueix D, Ridet JL, Tchélingérian JL, Lossouarn L, Privat A, Babinet C and Dupouey P

    Unité de Biochimie des Antigènes, Institut Pasteur, Paris, France.

    The expression of the glial fibrillary acidic protein (GFAP), a component of astroglial intermediate filaments, is regulated under developmental and pathological conditions. In order to characterize DNA sequences involved in such regulations, we produced transgenic mice bearing 2 kb of the 5' flanking region of the murine GFAP gene linked to the Escherichia coli beta-galactosidase (beta-gal) reporter gene. Seven transgenic lines were obtained. We observed that the regulatory elements present in the transgene GFAP-nls-LacZ direct an expression in the neural and non-neural tissue and target in vivo an unexpected subpopulation of astrocyte. In the developing brain, beta-gal activity and GFAP appeared simultaneously and in the same region, on embryonic day 18 (E18), suggesting that the 2 kb of the promoter contains the regulatory sequences responsible for the perinatal vimentin/GFAP switch. In addition, we demonstrated that the 2 kb sequence of the GFAP promoter used in the transgene possess elements which are activated after a surgical injury, thus permitting to study some aspects of reactive gliosis in these transgenic mice. These transgenic lines provide a useful tool by enabling further studies of astroglial and, probably, neuronal physiologies.

    Glia 1994;12;4;281-93

  • Mice lacking vimentin develop and reproduce without an obvious phenotype.

    Colucci-Guyon E, Portier MM, Dunia I, Paulin D, Pournin S and Babinet C

    Unité de Recherche Associée 361, Centre National de la Recherche Scientifique, Institut Pasteur, France.

    To address the biological role of vimentin in the context of the living organism, we have introduced a null mutation of the vimentin gene into the germ line of mice. Surprisingly, animals homozygous for this mutation developed and reproduced without an obvious phenotype. Immunoblotting, immunofluorescence, and immunogold labeling analysis confirmed the absence of vimentin and of the corresponding filament network. Furthermore, no compensatory expression of another intermediate filament could be demonstrated. While these results leave open the question of the possible role of vimentin in unusual situations or pathological conditions, they show that a conspicuous developmental and cell-specific structure that is an integral part of the cytoskeleton can be eliminated without apparent effect on mouse reproduction and development.

    Cell 1994;79;4;679-94

  • Stage-specific expression of alkaline phosphatase during neural development in the mouse.

    Narisawa S, Hasegawa H, Watanabe K and Millán JL

    La Jolla Cancer Research Foundation, Cancer Research Center, California 92037.

    The expression pattern of tissue nonspecific alkaline phosphatase (TNAP) in the developing neural tube of mouse is reported. Homogeneous AP activity in the neuroepithelium becomes prominent at E8.5. At E9.5, distinctly AP-positive cells appear in the brain and spinal cord area. At stages E10.5 to E12.5, AP positivity is observed between the mesencephalon and the rhombencephalon, along the entire spinal cord and cranial nerves emerging from the myelencephalon. At E13.5, strongly AP positive fibers become prominent in the pons. At E14.5, AP expression in brain tissue is considerably reduced and there is a complete absence of AP activity in the nerve cells and glial cells of adult brain. The choroid plexus remains distinctly positive for AP expression until the adult stage. Northern blot analysis and reverse-transcriptase polymerase chain reaction amplification of RNA indicate that this AP activity results from the expression of the Akp-2 locus. This AP expression pattern is distinct from those reported for the expression of GD3, nestin, Hox 2.3, and Wnt-1 during brain development. We conclude that AP is a useful marker of a subpopulation of neuroectodermal cells present in the neural tube as early as E8.5, at which stages there are no other AP positive intraembryonic cells except PGCs.

    Funded by: NICHD NIH HHS: HD25938

    Developmental dynamics : an official publication of the American Association of Anatomists 1994;201;3;227-35

  • A new in vitro model of murine mesoderm migration: the role of fibronectin and laminin.

    Klinowska TC, Ireland GW and Kimber SJ

    School of Biological Sciences, University of Manchester, UK.

    Examination of the factors involved in primary mesodermal migration in the mouse has been complicated by the lack of a suitable in vitro model. We have developed a new culture system using primitive streak stage embryos denuded of primitive endoderm, which allows easy observation and manipulation of the outgrowing cells. The cells migrating away from these explants were shown by immunocytochemistry to express vimentin and an epitope of the I antigen recognised by the antibody C6, both of which are present on the newly emerged mesoderm and not on the embryonic ectoderm in sections of embryos in utero. Conversely, cytokeratin, stage-specific embryonic antigen 1 (SSEA-1), E-cadherin and desmoplakin are expressed by the embryonic ectoderm but lost during mesoderm formation in vivo. They are absent or expressed very weakly by the migrated cells in vitro. In addition, only explants of the ectoplacental cone (EPC) and visceral endoderm alone, expressed a carbohydrate epitope (recognised by monoclonal antibody BOO6), characteristic of the EPC and primitive endoderm in utero, but absent from mesoderm. Thus we conclude that the cells which outgrow in this system are indeed mesodermal in phenotype. We have confirmed the work of others in demonstrating the presence of fibronectin (FN) and laminin (LN) in the migratory path of the mesoderm, at the ectoderm-visceral endoderm interface. We also report that the beta 1 integrin subunit of the FN and LN receptor is expressed by mesodermal cells at this interface. Using our in vitro model we have examined the role of the extracellular matrix (ECM) in mesodermal migration. Mesodermal cells migrate further and faster on substrates coated with FN or LN, and this increased migration is abolished by appropriate blocking antibodies. We conclude that the ECM, in particular FN and LN, plays an important role in the migration of primary mesodermal cells during gastrulation in the mouse embryo.

    Differentiation; research in biological diversity 1994;57;1;7-19

  • Separation and sequencing of familiar and novel murine proteins using preparative two-dimensional gel electrophoresis.

    Merrick BA, Patterson RM, Witcher LL, He C and Selkirk JK

    Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC.

    Strategies are needed for rapid protein isolation in order to identify disease-related proteins and facilitate the design of oligonucleotides for further molecular inquiry. In our laboratory, C3H10T1/2 murine fibroblasts have been found to express a variety of proteins in various subcellular fractions which are relevant to experimental transformation and carcinogenesis. Preparative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) procedures were developed to identify major cytoplasmic proteins by electroblotting and microsequencing. Isoelectric focusing tube gels were enlarged to 6 mm ID to accommodate larger protein loads at 0.5 to 2 mg protein. Separated proteins were electrotransferred from 6 mm thick slab gels onto 0.22 mu polyvinylidene difluoride membranes. Nearly 100 prominent blotted proteins were stained with Coomassie Brilliant Blue between pI 4.5-7.0 and 18-106 kDa and, of these, 27 prominent and well-resolved proteins were selected for sequencing. Sequences of 14 to 24 amino acid residues in length were obtained from 11 proteins which were identified from computerized databases. Some of these identified proteins had structural or enzymatic functions while others had only recently been discovered, including a newly reported Hsp 70 class member and a novel calcium-binding protein, reticulocalbin. The new heat shock protein has a molecular mass of 75 kDa and has been designated as Grp75, PBP74, CSA or p66mot-1 in mice and humans with purported roles in transformation and antigen processing. Reticulocalbin is an endoplasmic reticular protein which contains six domains of the EF-hand motif associated with high-affinity calcium-binding proteins. It may be involved in protein transport and luminal protein processing. In addition, sequences of 5 to 11 residues in length were also obtained from six other unidentified proteins. Thus, we have found that preparative 2-D PAGE serves as a powerful one-step purification method for protein isolation and characterization from an important in vitro murine model for the study of carcinogenesis.

    Electrophoresis 1994;15;5;735-45

  • Cloning, analysis, and chromosomal localization of myoxin (MYH12), the human homologue to the mouse dilute gene.

    Engle LJ and Kennett RH

    Department of Human Genetics, University of Pennsylvania School of Medicine, Philadelphia 19104.

    The mouse dilute gene encodes a novel type of non-muscle myosin that structurally combines elements from both nonmuscle myosin type I and nonmuscle myosin type II. Phenotypically, mutations in the mouse dilute gene result not only in the lightening of coat color, but also in the onset of severe neurological defects shortly after birth. This may indicate that the mouse dilute gene is important in maintaining the normal neuronal function in the mouse. We report the isolation and sequencing of "myoxin" (MYH12), the human homologue of the mouse dilute gene, and its assignment to human chromosome 15.

    Funded by: NCI NIH HHS: CA-16520; NIDDK NIH HHS: P30-DK19525

    Genomics 1994;19;3;407-16

  • Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development.

    Coleman-Krnacik S and Rosen JM

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

    Mammary gland development is dependent upon local regulatory factors as well as systemic hormones to mediate gland morphogenesis and associated mesenchymal-epithelial interactions. FGF-3 (int-2) has been implicated as an oncogenic growth factor produced locally in mouse mammary tumor virus-induced mammary tumorigenesis. The observation that FGF-3 is not expressed during normal mammary development as well as the high degree of cellular proliferation and angiogenesis that accompany mammary gland growth suggest roles for other FGF family members in this process. In this study, we have examined expression of FGF family members at various stages of mouse mammary growth and tumorigenesis. FGF-1, FGF-2, FGF-4, and FGF-7 were expressed during the ductal stage of mammary development. The majority of FGF-1 gene expression was in the luminal epithelial cells, whereas FGF-2 expression was in the mammary stroma and possibly the myoepithelial cells. The presence of mammary epithelium induced the expression of both FGF-2 and FGF-7 in the stroma. FGF-1 and FGF-2 expression declined during pregnancy and dropped again during lactation, but quantitative analysis showed a much more dramatic decrease in FGF-2 expression. FGF-7 transcripts were also detected during pregnancy and lactation, but an alternate transcript size was observed at these stages. FGF-1, FGF-2, and FGF-7 transcripts were detected in mammary preneoplasias, tumors, and immortal cell lines, but at levels less than those seen during normal mammary growth. These results support the hypothesis that FGF family members play a role in local regulation of mammary development. The differential spatial and temporal pattern of FGF-1, FGF-2 and FGF-7 gene expression indicate that they each have unique functions in the gland.

    Funded by: NCI NIH HHS: CA-09028, CA-16303

    Molecular endocrinology (Baltimore, Md.) 1994;8;2;218-29

  • Isolation and developmental expression of retinoic-acid-induced genes.

    Jonk LJ, de Jonge ME, Vervaart JM, Wissink S and Kruijer W

    Hubrecht Laboratorium, Netherlands Institute for Developmental Biology, Utrecht.

    The vitamin A derivative retinoic acid (RA) is involved in vertebrate anteroposterior axis formation and cellular differentiation and has been shown to modulate the expression of a number of genes implicated in the control of early embryonal development. Under defined culture conditions, all-trans-RA induces differentiation of P19 embryonal carcinoma cells into neural derivatives. In this report, we describe the isolation and partial characterization of 14 RA-regulated genes from P19 cells committed to differentiate along the neural pathway. In addition to the previously recognized genes encoding vimentin, heat stable antigen, neural cadherin, Hox-B2, F52/MacMARCKS, thymosin beta 4b, and the murine homolog of COUP-TF1, we identified 7 novel genes, 5 of which are predominantly expressed in developing neural tissues as shown by in situ hybridization. The results confirm the usefulness of the P19 system in the isolation and study of the regulation of developmentally expressed genes.

    Developmental biology 1994;161;2;604-14

  • Chromosomal localization of the mouse titin gene and its relation to "muscular dystrophy with myositis" and nebulin genes on chromosome 2.

    Müller-Seitz M, Kaupmann K, Labeit S and Jockusch H

    Developmental Biology Unit, University of Bielefeld, Federal Republic of Germany.

    In the mouse, the genes for the structural components of the myofibril titin and nebulin, Ttn and Neb, map to proximal Chr 2, as does the gene for a muscle disease, "muscular dystrophy with myositis," mdm. To facilitate the evaluation of Ttn and Neb as possible candidates for mdm, we have determined their relative map positions, using a Mus spretus/Mus musculus interspecific backcross. The gene order (distances in cM) cenVim-16.9 +/- 4.7-Neb-7.6 +/- 3.0-Ttn, Acra-18.0 +/- 4.9-Pax-6-17.7 +/- 4.9-a ... has been determined. Considering the standard deviations, Neb, Ttn, and Acra could colocalize with mdm. Using Ttn and Neb probes, DNAs from mdm/mdm and mdm/+ mice were tested for restriction fragment variants in comparison to the M. musculus wildtype. No variants have been found with 11 restriction nucleases. Our data corroborate a conserved synteny comprising genes NEB, TTN, CHRNA1 on human Chr 2q.

    Genomics 1993;18;3;559-61

  • Vimentin expression is differentially regulated by IL-2 and IL-4 in murine T cells.

    Hornbeck PV, Garrels JI, Capetanaki Y and Heimer S

    Department of Medicine, University of Maryland School of Medicine, Baltimore 21201.

    IL-2 and IL-4 are T cell growth factors that are produced by different T cell subsets and have distinct roles in lymphocyte biology. Despite their importance in the immune system, little is known about the genes that these lymphokines may specifically control and the interaction of these lymphokines in regulating the expression of their target genes. In this paper, we use the factor-dependent murine T cell line (CT.4R) to investigate the interaction of IL-2 and IL-4 in regulating gene expression. We report that the intermediate filament protein vimentin is differentially regulated by these lymphokines. Cells grown in IL-2 typically express 10- to 20-fold more vimentin and vimentin RNA than those grown in IL-4, but express similar levels of other cytoskeletal proteins including actin and tubulin. Vimentin was specifically induced by IL-2 and apparently suppressed by IL-4 in normal lymph node T cells, suggesting that its differential regulation by these lymphokines is physiologically relevant. We investigated the synergy between IL-2 and IL-4 in regulating the expression of vimentin RNA and compared it to that of two other lymphokine-responsive genes, pancreatic lipase and the IL-2R alpha subunit. Complex regulatory interactions were revealed: IL-4 suppressed the ability of IL-2 to induce vimentin RNA but not IL-2R alpha RNA, whereas IL-2 inhibited the ability of IL-4 to induce lipase RNA. These results indicate that IL-2 and IL-4 can cross-regulate lymphokine-responsive genes and can simultaneously exert both positive and negative regulation of different genes within the same cell.

    Funded by: NIAID NIH HHS: R29 AI30822-01A1

    Journal of immunology (Baltimore, Md. : 1950) 1993;151;8;4013-21

  • Developmentally regulated expression of CGRP in the mouse olfactory pathway.

    Denis-Donini S, Chini B and Vitadello M

    CNR Center of Cytopharmacology, Milano, Italy.

    The pattern of expression of the neuropeptide CGRP and its encoding mRNA has been determined by immunohistochemistry and in situ hybridization in the mouse olfactory pathway during development. Specific CGRP transcripts are first detected at E13 followed by the appearance of the peptide at E15. Both peptide and transcript are present until birth; their expression then appears to be down-regulated since postnatally the peptide is only observed in some olfactory receptor neurons. A monoclonal antibody that specifically recognizes the neurofilament subunit NF-M has been used in order to identify olfactory and trigeminal axons. Our results demonstrate that CGRP is expressed in olfactory neurons and their axons during development, thus supporting further its role as a differentiation factor during olfactory bulb ontogenesis.

    The European journal of neuroscience 1993;5;6;648-56

  • Chromosomal localization of five murine HSP70 gene family members: Hsp70-1, Hsp70-2, Hsp70-3, Hsc70t, and Grp78.

    Hunt CR, Gasser DL, Chaplin DD, Pierce JC and Kozak CA

    Washington University School of Medicine, St. Louis, Missouri 63108.

    The 70,000-D heat shock protein (HSP70) gene family includes both heat-inducible and constitutively expressed genes. We have mapped five murine HSP70 genes to specific sites on three separate chromosomes. Southern blot analysis of Chinese hamster x mouse somatic cell DNAs was used to assign the gene for the 78,000-D glucose-regulated protein (Grp78) to Chromosome (Chr) 2, the male germ cell-specific Hsp70-2 and Hsc70t genes to Chr 12 and Chr 17, respectively, and the heat-inducible Hsp70-3 gene also to Chr 17. Southern blot analysis of DNA from the progeny of two multilocus crosses confirmed the Grp78 location on Chr 2 and suggested the order: centromere-Vim-Abl-Grp78-Hc. Similar analysis also confirmed the initial Hsp70-2 assignment to Chr 12 with the order: Hsp70-2-Aat-Igh. The Hsp70-3 and Hsc70t genes on Chr 17, along with the heat-inducible Hsp70-1 gene, were further localized by Southern blot analysis of genomic clones to the H-2 histocompatibility region with the order: Hsp70-1-Hsp70-3-Hsc70t-Bat-6 (human G7a, valyl-tRNA synthetase).

    Funded by: NCI NIH HHS: R29 CA50503; NIAID NIH HHS: AI15322; NIDCR NIH HHS: DEO9164; ...

    Genomics 1993;16;1;193-8

  • Cloning, analysis, and chromosomal localization of Notch-1, a mouse homolog of Drosophila Notch.

    del Amo FF, Gendron-Maguire M, Swiatek PJ, Jenkins NA, Copeland NG and Gridley T

    Department of Cell and Developmental Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110.

    The Notch gene of Drosophila encodes a large transmembrane protein involved in cell-cell interactions and cell fate decisions in the Drosophila embryo. We report here the isolation of cDNA clones encompassing the full-length coding sequence of Notch-1, a mouse homolog of Drosophila Notch. The predicted amino acid sequence of the Notch-1 protein retains all of the conserved amino acid motifs of Notch and the other vertebrate Notch homologs. The cDNA sequence predicts a 2531-amino-acid protein containing a signal peptide, 36 epidermal growth factor-like repeats, 3 Notch/lin-12 repeats, a transmembrane domain, and 6 cdc10/ankyrin repeats. The Notch-1 gene was localized to the proximal portion of mouse chromosome 2 by mapping with an interspecific backcross panel.

    Funded by: NCI NIH HHS: N01-CO-74101

    Genomics 1993;15;2;259-64

  • A new Pax gene, Pax-9, maps to mouse chromosome 12.

    Wallin J, Mizutani Y, Imai K, Miyashita N, Moriwaki K, Taniguchi M, Koseki H and Balling R

    Department of Developmental Biology, Max Planck Institute of Immunobiology, Freiburg, Germany.

    Members of the Pax gene family have recently been shown to play important roles in mouse embryogenesis. Of eight so far characterized Pax genes, three have been associated with mouse developmental mutants. Here we report the cloning of a new Pax gene, Pax-9. Most of the DNA sequence encoding the highly conserved paired domain has been determined and compared with previously known paired domains. This comparison classifies Pax-9 as a member of the same subgroup as Pax-1/undulated. By analysis of the segregation of a Pax-9 restriction fragment length polymorphism and a large number of simple sequence length polymorphisms in an interspecific C57BL/6 x Mus musculus mollosinus backcross, Pax-9 was mapped close to the D12Nds1 locus on the proximal part of Chromosome (Chr) 12.

    Mammalian genome : official journal of the International Mammalian Genome Society 1993;4;7;354-8

  • Fine genetic mapping of the proximal part of mouse chromosome 2 excludes Pax-8 as a candidate gene for Danforth's short tail (Sd).

    Koseki H, Zachgo J, Mizutani Y, Simon-Chazottes D, Guénet JL, Balling R and Gossler A

    Max-Planck-Institut für Immunobiologie, Freiburg, FRG.

    Danforth's short tail (Sd) is a semidominant mutation of the mouse with effects on the skeleton and the urogenital system. In view of its phenotype and its position in the proximal part of Chromosome (Chr) 2, three genes qualified as possible candidates: Pax-8, a paired box-containing gene; Midkine (Mdk), a retinoic acid-responsive gene; and a new locus (Etl-4) identified by enhancer trapping with a lacZ reporter gene which showed expression in the notochord, the mesonephric mesenchyme, and the apical ectodermal ridge. Three different backcrosses involving all three genes in different combinations were set up and analyzed. From our results we conclude that Sd, Etl-4, Pax-8, and Mdk are independent loci, with Etl-4 being the closest genetic marker (1.1 +/- 1.4 cM) to the Danforth's short tail (Sd) gene.

    Mammalian genome : official journal of the International Mammalian Genome Society 1993;4;6;324-7

  • Homologous chromosomal locations of the four genes for inter-alpha-inhibitor and pre-alpha-inhibitor family in human and mouse: assignment of the ancestral gene for the lipocalin superfamily.

    Salier JP, Simon D, Rouet P, Raguenez G, Muscatelli F, Gebhard W, Guenet JL and Mattei MG

    Institut National de la Santé et de la Recherche Médicale Unit-78, Boisguillaume, France.

    The inter-alpha-inhibitor (I alpha I) and pre-alpha-inhibitor (P alpha I) family is composed of three plasma protease inhibitors, I alpha I, P alpha I, and bikunin, whose chains are encoded by a set of three evolutionarily related heavy (H) chain genes designated H1, H2, and H3 and a fourth gene, the so-called alpha 1-microglobulin/bikunin precursor (AMBP) gene. The latter codes for a precursor that splits into: (i) alpha 1-microglobulin, which belongs to the lipocalin superfamily; and (ii) bikunin, which is made up of two tandemly arranged protease inhibitor domains and belongs to the superfamily of Kunitz-type protease inhibitors. The bikunin chain is found in I alpha I and P alpha I molecules and it is also present as a free molecule in plasma. In human, the AMBP and H2 genes have been mapped to 9q32-q34 and 10p14-p15, respectively, while the H1 and H3 genes are tandemly located at 3p21.1-p21.2. In situ hybridization mappings indicate that the mouse AMBP gene (Intin-4) is located at 4C1----C4, and the H1 (Intin-1) and H3 (Intin-3) genes are colocated at 14A2----C1. In interspecific backcrosses (C57BL/6Pas x Mus spretus) a TaqI restriction variant in (and/or near) the H2 (Intin-2) gene identified a linkage of this gene with other polymorphic loci, which assigns Intin-2 to the centromeric area of chromosome 2. All such assignments are in conserved chromosomal regions between human and mouse. Therefore the genetic events that gave rise to the four I alpha I family genes took place prior to the divergence between human and mouse.(ABSTRACT TRUNCATED AT 250 WORDS)

    Genomics 1992;14;1;83-8

  • Comparative mapping of mouse chromosome 2 and human chromosome 9q: the genes for gelsolin and dopamine beta-hydroxylase map to mouse chromosome 2.

    Pilz A, Moseley H, Peters J and Abbott C

    Department of Genetics and Biometry, University College London, United Kingdom.

    The mapping of human chromosome 9 (HSA9) and mouse chromosome 2 (MMU2) has revealed a conserved syntenic region between the distal end of the long arm of chromosome 9 and proximal mouse chromosome 2. Two genes that map to human chromosome 9q34, gelsolin (GSN) and dopamine beta-hydroxylase (DBH), have not previously been located in the mouse. We have used an interspecific backcross to map each of these genes, by Southern blot analysis, to mouse chromosome 2. Gelsolin (Gsn) is tightly linked to the gene for complement component C5 (Hc), and dopamine beta-hydroxylase (Dbh) is just proximal to the Abelson leukemia virus oncogene (Abl) and alpha-spectrin 2 (Spna-2). The loci for gelsolin and dopamine beta-hydroxylase therefore form part of the conserved synteny between HSA9q and MMU2.

    Genomics 1992;12;4;715-9

  • Isolation and characterization of a library of cDNA clones that are preferentially expressed in the embryonic telencephalon.

    Porteus MH, Brice AE, Bulfone A, Usdin TB, Ciaranello RD and Rubenstein JL

    Nancy Prizker Laboratory of Developmental and Molecular Neurobiology, Department of Psychiatry and Behavioral Sciences, Stanford University, CA 94305.

    In order to isolate genes involved in development of the mammalian telencephalon we employed an efficient cDNA library procedure. By subtracting an adult mouse telencephalic cDNA library from an embryonic day 15 (E15) mouse telencephalic cDNA library we generated two subtracted libraries (ES1 and ES2). We estimate that ES1 contains between 200 and 600 different cDNA clones, which approximates the number of genes that are preferentially expressed in the E15 telencephalon, compared to the adult telencephalon. Northern analysis of 20 different cDNA clones shows that 14 of these are expressed at least 5-fold more in the E15 telencephalon than the adult telencephalon. Limited sequencing of the 14 differentially expressed clones reveals that 10 have no significant identity to sequences in GenBank and EMBL databases, whereas the other 4 have significant sequence identity to vimentin, histone 3.3, topoisomerase I and the B2 repeat element. In situ hybridization using one of the differentially expressed cDNAs, TES-1, demonstrates that it is transiently expressed in the anlage of the basal ganglia. In situ hybridization with another differentially expressed cDNA clone, TES-4, shows that it is specifically expressed in differentiating cells of the neural axis with a distinctive rostral-caudal temporal pattern. These findings, and the methods that we have developed, provide a framework for future investigations of the genetic control of telencephalon development.

    Funded by: NIMH NIH HHS: MH00219, MH10016, MH39437

    Brain research. Molecular brain research 1992;12;1-3;7-22

  • The murine even-skipped-like gene Evx-2 is closely linked to the Hox-4 complex, but is transcribed in the opposite direction.

    Bastian H, Gruss P, Duboule D and Izpisúa-Belmonte JC

    Max-Planck-Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Göttingen, FRG.

    Mammalian genome : official journal of the International Mammalian Genome Society 1992;3;4;241-3

  • Interspecific backcross mice show sex-specific differences in allelic inheritance.

    Siracusa LD, Alvord WG, Bickmore WA, Jenkins NA and Copeland NG

    Mammalian Genetics Laboratory, NCI-Frederick Cancer Research and Development Center, Maryland 21702.

    Transmission distortion is identified as a difference in transmission frequency of two alleles from the normal 1:1 Mendelian segregation in diploid organisms. Transmission distortion can extend over part or all of a chromosome. The recent development of interspecific mouse backcrosses has provided a powerful method for multilocus mapping of entire chromosomes in a single cross, and consequently for identifying distortions in allelic inheritance. We used an interspecific backcross of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice to map molecular loci to mouse chromosome 2 and had previously found that the distal region of the chromosome showed distortions in allelic inheritance. We now report the mapping of five loci (Actc-1, D2Hgu1, His-1, Hox-4.1 and Neb) to chromosome 2, which, in addition to the Abl, Ada, B2m, Bmp-2a, Hc, Emv-15, Fshb, Hck-1, Pax-1, Pck-1, Spna-2 and Vim loci previously mapped in our interspecific backcross, serve as markers to measure allelic inheritance along approximately 75% of mouse chromosome 2. Statistical analyses are used to identify and delimit chromosomal regions showing transmission distortion and to determine whether there are sex-specific differences in allelic inheritance. These studies provide evidence for sex-specific differences in allelic inheritance for chromosome 2 and suggest biological explanations for this form of transmission distortion.

    Funded by: NCI NIH HHS: N01-CO-74101, N01-CO-74103; NIGMS NIH HHS: GM12721-01

    Genetics 1991;128;4;813-21

  • Chromosomal location of the octamer transcription factors, Otf-1, Otf-2, and Otf-3, defines multiple Otf-3-related sequences dispersed in the mouse genome.

    Siracusa LD, Rosner MH, Vigano MA, Gilbert DJ, Staudt LM, Copeland NG and Jenkins NA

    Mammalian Genetics Laboratory, NCI-Frederick Cancer Research and Development Center, Maryland 21702.

    Chromosomal locations have been assigned for the octamer transcription factor, Otf, gene family (previously named the octamer-binding protein, Oct, gene family) using an interspecific backcross of [(C57BL/6J x Mus spretus)F1 x C57BL/6J] mice and the BXH recombinant inbred strains. Molecular probes for Otf-1 and Otf-2 recognized single loci on mouse chromosomes 1 and 7, respectively, whereas probes for Otf-3 recognized a minimum of eight independently segregating loci (designated Otf-3a through Otf-3h). Members of the Otf-3 family mapped to mouse chromosomes 1, 2, 3, 6, 14, 17, and the X chromosome, indicating that the Otf family has become widely dispersed during evolution. Several Otf loci mapped near developmental mutations, raising the possibility that these mutations result from defects in Otf family members.

    Funded by: NCI NIH HHS: N01-CO-74101

    Genomics 1991;10;2;313-26

  • Evidence that Ser-82 is a unique phosphorylation site on vimentin for Ca2(+)-calmodulin-dependent protein kinase II.

    Ando S, Tokui T, Yamauchi T, Sugiura H, Tanabe K and Inagaki M

    Biophysics Unit, Aichi Cancer Center Research Institute, Japan.

    We identified the sites on vimentin that are phosphorylated by Ca2(+)-calmodulin-dependent protein kinase II (CaM-kinase II). Sequential analysis of the purified phosphopeptides demonstrated that the sites are -Thr-Arg-Thr-Tyr-Ser(PO4)38-Leu-Gly-Ser-Ala- and -Val-Arg-Leu-Leu-Gln-Asp-Ser(PO4)82-Val-Asp-, which are located within the amino-terminal head domain of vimentin. For Ser-82 but not Ser-38, the proposed CaM-kinase II recognition amino acid sequence (Arg-X-X-Ser/Thr) was not found. Studies with a series of synthetic peptide analogs corresponding to Ser-82 and its surrounding amino acid sequence indicate that Asp-84 acts as an essential substrate specificity determinant for the Ser-82 phosphorylation by CaM-kinase II. The CaM-kinase II recognition site may be more extensive than heretofore determined.

    Biochemical and biophysical research communications 1991;175;3;955-62

  • The mouse homeobox gene, S8, is expressed during embryogenesis predominantly in mesenchyme.

    Opstelten DJ, Vogels R, Robert B, Kalkhoven E, Zwartkruis F, de Laaf L, Destrée OH, Deschamps J, Lawson KA and Meijlink F

    Hubrecht Laboratorium, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands.

    The murine S8 gene, originally identified by Kongsuwan et al. [EMBO J. 7(1988)2131-2138] encodes a homeodomain which resembles those of the paired family. We studied the expression pattern during mid-gestation embryogenesis of S8 by in situ hybridization. Expression was detected locally in craniofacial mesenchyme, in the limb, the heart and the somites and sclerotomes all along the axis, and was absent from the central and peripheral nervous system, splanchnopleure, and endodermal derivatives. This pattern differs considerably from that of most previously described homeobox containing genes. By genetic analysis, the gene was located on chromosome 2, about 20 cM from the HOX-4 cluster.

    Mechanisms of development 1991;34;1;29-41

  • Mapping HSA10 homologous loci in cattle.

    Threadgill DS and Womack JE

    Department of Veterinary Pathology, Texas A&M University, College Station 77843.

    Loci homologous to those on human chromosome 10 (HSA10) map to five mouse chromosomes, MMU2, MMU7, MMU10, MMU14, and MMU19. In cattle, one unassigned syntenic group (U26) was previously defined by the HSA10/MMU19 isoenzyme marker glutamic-oxaloacetic transaminase 1 (GOT1). To evaluate the syntenic arrangement of other HSA10 loci in cattle, seven genes were physically mapped by segregation analysis in a bovine x hamster hybrid somatic cell panel. The genes mapped include: vimentin (VIM) on HSA10 and MMU2; interleukin 2 receptor (IL2R) on HSA10 and MMU?; ornithine aminotransferase (OAT) on HSA10 and MMU7; hexokinase 1 (HK1) on HSA10 and MMU10; retinol-binding protein 3 (RBP3) on HSA10 and MMU14; plasminogen activator, urokinase type (PLAU) on HSA10 and MMU14; and alpha-2-adrenergic receptor (ADRA2) on HSA10 and MMU19. VIM and IL2R mapped to U11; ADRA2 and OAT mapped to U26; and RBP3, PLAU, and HK1 mapped to U28.

    Cytogenetics and cell genetics 1991;57;2-3;123-6

  • Mouse vimentin: structural relationship to fos, jun, CREB and tpr.

    Capetanaki Y, Kuisk I, Rothblum K and Starnes S

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

    We have isolated and characterized mouse cDNA clones representing the entire coding region of vimentin. RNA blot analysis of different stages during development has revealed differential control in the expression of vimentin mRNA in the different tissues studied. The nucleotide sequence extends 1800 base pairs and contains the 466 amino acid mouse vimentin polypeptide chain, flanked by 90 base pairs 5' and 312 base pairs 3' untranslated region. Conformational analysis of the deduced amino acid sequence was used to localize the three known structural domains: a non-alpha-helical N-terminal head of 81 residues, a rod-like domain of 330 residues arising from three alpha-helices, and a non-alpha-helical C-terminal domain of 55 residues. Amino acid sequence comparisons with other species revealed high sequence conservation of mouse vimentin to hamster (98.7%), human (96%), and chicken (88%) protein. Computer sequence analysis also revealed domains of significant homology between different alpha helical regions of vimentin and the DNA binding-leucine zipper domain of several proto-oncogenes and transcription regulators. Specifically, 50-70% structural similarity was observed between the basic domain of the DNA binding region of the nuclear proto-oncogene products c-fos and its related antigen fra-1, c-jun and the cAMP-responsive DNA binding protein CREB, with part of the N-terminal half region of helix 1b of vimentin. When the leucine zipper domains of all these proteins were compared to vimentin, at least two different regions of similarity in the vimentin molecule were found reaching up to 53% for jun, 60% for fos, and 76% for CREB. Further analysis revealed several domains of significant similarity (50%) between all alpha-helices of the rod domain of vimentin and the N-terminal (approximately 210 residues) activation domain tpr of the oncogenic raf.

    Funded by: NIAMS NIH HHS: AR39617-01

    Oncogene 1990;5;5;645-55

  • Coding sequence and flanking regions of the mouse vimentin gene.

    Hennekes H, Kühn S and Traub P

    Max-Planck-Institut für Zellbiologie, Rosenhof, Ladenburg bei Heidelberg, Federal Republic of Germany.

    Using a polyclonal antibody, a cDNA clone coding for part of mouse vimentin was identified in a lambda gt11 expression library. DNA from this clone was used to screen a genomic library from Ehrlich Ascites Tumor cells for the mouse vimentin gene. A clone was found which contained the whole coding sequence and a large part of the 5'- and 3'-untranslated sequences. It was used to prepare a construct equivalent to a full-length cDNA clone. Extensive homologies to the vimentin sequence from other species were found for the coding and 3'-untranslated sequences and the promoter region.

    Molecular & general genetics : MGG 1990;221;1;33-6

  • Chromosomal localization of the mouse gene coding for vimentin.

    Mattei MG, Lilienbaum A, Lin LZ, Mattei JF and Paulin D

    The chromosomal location of the mouse gene coding for vimentin, one of the intermediate filament subunits, was determined by in situ hybridization using specific H3-labelled DNA probes. There is only one copy of the vimentin gene and it is located on chromosome 2 region A2.

    Genetical research 1989;53;3;183-5

  • Domain- and sequence-specific phosphorylation of vimentin induces disassembly of the filament structure.

    Ando S, Tanabe K, Gonda Y, Sato C and Inagaki M

    Biophysics Unit, Aichi Cancer Center Research Institute, Nagoya, Japan.

    We reported that stoichiometric phosphorylation by either cAMP-dependent protein kinase or protein kinase C induces disassembly of vimentin filaments [Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M., & Sato, C. (1987) Nature 328, 649-652; Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., & Sato, C. (1988) J. Biol. Chem. 263, 5970-5978]. In the present work, we attempted to identify the sites of vimentin phosphorylated by each protein kinase. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-8, Ser-9, Ser-20, Ser-25, Ser-33, and Ser-41 were specifically phosphorylated by protein kinase C, whereas Ser-46 was phosphorylated preferentially by cAMP-dependent protein kinase. Both kinases reacted with Ser-6, Ser-24, Ser-38, Ser-50, and Ser-65. Specific phosphorylation sites for protein kinase C are mostly located close to the amino-terminal side of arginine while those for cAMP-dependent protein kinase are located close to the carboxyl-terminal side of arginine. The phosphorylation sites exclusively occur in the amino-terminal non-alpha-helical head domain, particularly at the beta-turn region. These results provide clues to the molecular mechanisms of phosphorylation-dependent disassembly of vimentin filaments.

    Biochemistry 1989;28;7;2974-9

  • Vimentin cDNA clones covering the complete intermediate-filament protein are found in an EHS tumor cDNA library.

    Wood L, Theriault N and Vogeli G

    Molecular Biology Research, Upjohn Company, Kalamazoo, MI 49001.

    Intermediate filaments are part of the cytoskeleton of most cells. To analyze changes in intermediate filament synthesis, we have isolated two cDNA clones (pV-C25, pV-C877) that cover the complete coding sequence of the murine intermediate filament protein vimentin. The cDNA clones were isolated from a murine Engelbreth-Holm-Swan (EHS) tumor cDNA library by screening under (i) non-stringent conditions with a synthetic oligodeoxynucleotide (oligo), LW-36, which is specific for type-IV collagen, and (ii) stringent conditions with oligo LW75, which was derived from the vimentin clone pV-C25. The cDNA clones contain 38 nucleotides (nt) of the 5'-untranslated region, 1398 nt of the coding region and 7 nt of the 3'-untranslated region. Comparing the mouse sequence with the published sequence for vimentin from hamster, human and chicken, we find shared identities of 99, 97 and 87%, respectively. Since the cDNA clones have been isolated from a basement membrane tumor (EHS) cDNA library, we measured the vimentin mRNA production in EHS tumor cells in culture, and found that this mRNA is half as abundant as mRNA for type-IV mRNA.

    Gene 1989;76;1;171-5

  • Expression of NF-L and NF-M in fibroblasts reveals coassembly of neurofilament and vimentin subunits.

    Monteiro MJ and Cleveland DW

    Johns Hopkins University, School of Medicine, Department of Biological Chemistry, Baltimore, Maryland 21205.

    We have used transient and stable DNA transfection to force synthesis of the mouse NF-L and NF-M genes in nonneuronal cultured animal cells. When the authentic NF-L gene (containing 1.7 kb of sequences 5' to the transcription initiation site) was transfected into L cells, correctly initiated NF-L mRNA was produced from the transfected gene but not the endogenous NF-L genes. Therefore, the normal restriction of NF-L expression to neurons cannot derive exclusively from absence in nonneuronal cells of neuron-specific transcription factors. When the NF-L coding region was linked to the strong promoter from Moloney Murine Sarcoma virus, we obtained high levels of synthesis of NF-L subunits (accumulating to as much as 9% of cell protein in stable cell lines). Although NF-L and NF-M polypeptides are normally expressed exclusively in postmitotic neurons, NF-L or NF-M polypeptides expressed in fibroblasts were efficiently assembled into intermediate filament arrays, thus demonstrating the competence of both NF-L and NF-M to assemble in vivo in the absence of additional neuron-specific factors. As judged by immunofluorescence localization and by the alteration in the solubility of the endogenous vimentin filaments, filaments containing NF-L appeared to be copolymers with vimentin. Neither the alteration in the properties of the vimentin array nor the accumulation of NF-L to a level that made it the second most abundant cellular protein (after actin) had any observable effect on cell viability or growth rate.

    The Journal of cell biology 1989;108;2;579-93

  • Formation of cytoskeletal elements during mouse embryogenesis. Intermediate filaments of the cytokeratin type and desmosomes in preimplantation embryos.

    Jackson BW, Grund C, Schmid E, Bürki K, Franke WW and Illmensee K

    Differentiation; research in biological diversity 1980;17;3;161-79

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
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
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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