G2Cdb::Gene report

Gene id
G00000606
Gene symbol
Des (MGI)
Species
Mus musculus
Description
desmin
Orthologue
G00001855 (Homo sapiens)

Databases (9)

Curated Gene
OTTMUSG00000019416 (Vega mouse gene)
Gene
ENSMUSG00000026208 (Ensembl mouse gene)
13346 (Entrez Gene)
1006 (G2Cdb plasticity & disease)
Gene Expression
NM_010043 (Allen Brain Atlas)
13346 (Genepaint)
Literature
125660 (OMIM)
Marker Symbol
MGI:94885 (MGI)
Protein Sequence
P31001 (UniProt)

Literature (246)

Pubmed - other

  • Physiology, structure, and susceptibility to injury of skeletal muscle in mice lacking keratin 19-based and desmin-based intermediate filaments.

    Lovering RM, O'Neill A, Muriel JM, Prosser BL, Strong J and Bloch RJ

    Department of Physiology, University of Maryland, Baltimore, 21201, USA. rlovering@som.umaryland.edu

    Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. Our previous results show that the tibialis anterior (TA) muscles of mice lacking keratin 19 (K19) lose costameres, accumulate mitochondria under the sarcolemma, and generate lower specific tension than controls. Here we compare the physiology and morphology of TA muscles of mice lacking K19 with muscles lacking desmin or both proteins [double knockout (DKO)]. K19-/- mice and DKO mice showed a threefold increase in the levels of creatine kinase (CK) in the serum. The absence of desmin caused a larger change in specific tension (-40%) than the absence of K19 (-19%) and played the predominant role in contractile function (-40%) and decreased tolerance to exercise in the DKO muscle. By contrast, the absence of both proteins was required to obtain a significantly greater loss of contractile torque after injury (-48%) compared with wild type (-39%), as well as near-complete disruption of costameres. The DKO muscle also showed a significantly greater misalignment of myofibrils than either mutant alone. In contrast, large subsarcolemmal gaps and extensive accumulation of mitochondria were only seen in K19-null TA muscles, and the absence of both K19 and desmin yielded milder phenotypes. Our results suggest that keratin filaments containing K19- and desmin-based intermediate filaments can play independent, complementary, or antagonistic roles in the physiology and morphology of fast-twitch skeletal muscle.

    Funded by: NIAMS NIH HHS: 1R01-AR-059179, K01-AR-053235, R01-AR-55928, T32-AR-07592

    American journal of physiology. Cell physiology 2011;300;4;C803-13

  • Septum transversum-derived mesothelium gives rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver.

    Asahina K, Zhou B, Pu WT and Tsukamoto H

    Southern California Research Center for ALPD and Cirrhosis and Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA. asahina@usc.edu

    Unlabelled: The septum transversum mesenchyme (STM) signals to induce hepatogenesis from the foregut endoderm. Hepatic stellate cells (HSCs) are sinusoidal pericytes assumed to originate from the STM and participate in mesenchymal-epithelial interaction in embryonic and adult livers. However, the developmental origin of HSCs remains elusive due to the lack of markers for STM and HSCs. We previously identified submesothelial cells (SubMCs) beneath mesothelial cells (MCs) as a potential precursor for HSCs in developing livers. In the present study, we reveal that both STM in embryonic day (E) 9.5 and MC/SubMCs in E12.5 share the expression of activated leukocyte cell adhesion molecule (Alcam), desmin, and Wilms tumor 1 homolog (Wt1). A cell lineage analysis using MesP1(Cre) /Rosa26lacZ(flox) mice identifies the mesodermal origin of the STM, HSCs, and perivascular mesenchymal cells (PMCs). A conditional cell lineage analysis using the Wt1(CreERT2) mice demonstrates that Wt1(+) STM gives rise to MCs, SubMCs, HSCs, and PMCs during liver development. Furthermore, we find that Wt1(+) MC/SubMCs migrate inward from the liver surface to generate HSCs and PMCs including portal fibroblasts, smooth muscle cells, and fibroblasts around the central veins. On the other hand, the Wt1(+) STM and MC/SubMCs do not contribute to sinusoidal endothelial cells, Kupffer cells, and hepatoblasts.

    Conclusion: our results demonstrate that HSCs and PMCs are derived from MC/SubMCs, which are traced back to mesodermal STM during liver development.

    Funded by: NIAAA NIH HHS: P50 AA011999, P50 AA011999-12, P50AA011999, R24 AA012885, R24 AA012885-10, R24AA12885

    Hepatology (Baltimore, Md.) 2011;53;3;983-95

  • DOT1L regulates dystrophin expression and is critical for cardiac function.

    Nguyen AT, Xiao B, Neppl RL, Kallin EM, Li J, Chen T, Wang DZ, Xiao X and Zhang Y

    Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, NC 27599, USA.

    Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin-glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.

    Funded by: Howard Hughes Medical Institute; NCI NIH HHS: CA119133, R01 CA119133

    Genes & development 2011;25;3;263-74

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

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

    Telethon Institute of Genetics and Medicine, Naples, Italy.

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

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

    PLoS biology 2011;9;1;e1000582

  • β8 integrin and band 4.1B cooperatively regulate morphogenesis of the embryonic heart.

    Jung Y, Kissil JL and McCarty JH

    Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

    Morphogenesis of the heart is regulated by various cues, including growth factors and extracellular matrix (ECM) proteins. The mechanisms by which cardiac cells properly integrate these cues to regulate growth, differentiation, and migration remain poorly understood. Here we have used genetic strategies in mice to identify αvβ8 integrin and its cytoskeletal adaptor protein, Band 4.1B, as essential regulators of cardiac morphogenesis. We demonstrate that approximately 60% of mouse embryos genetically null for β8 integrin and Band 4.1B display cardiovascular phenotypes and die by E11.5. This premature death is due, in part, to defective development of the cardiac outflow tract (OFT), with reduced expression of smooth muscle α-actin (SMAα-actin) in OFT cells derived from the cardiac neural crest. These data are the first to identify cell adhesion and signaling pathways regulated by αvβ8 integrin and Band 4.1B as essential for normal formation and function of the heart during embryogenesis.

    Funded by: NCI NIH HHS: CA-16672

    Developmental dynamics : an official publication of the American Association of Anatomists 2011;240;1;271-7

  • Myotubularin controls desmin intermediate filament architecture and mitochondrial dynamics in human and mouse skeletal muscle.

    Hnia K, Tronchère H, Tomczak KK, Amoasii L, Schultz P, Beggs AH, Payrastre B, Mandel JL and Laporte J

    Department of Neurobiology and Genetics, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

    Muscle contraction relies on a highly organized intracellular network of membrane organelles and cytoskeleton proteins. Among the latter are the intermediate filaments (IFs), a large family of proteins mutated in more than 30 human diseases. For example, mutations in the DES gene, which encodes the IF desmin, lead to desmin-related myopathy and cardiomyopathy. Here, we demonstrate that myotubularin (MTM1), which is mutated in individuals with X-linked centronuclear myopathy (XLCNM; also known as myotubular myopathy), is a desmin-binding protein and provide evidence for direct regulation of desmin by MTM1 in vitro and in vivo. XLCNM-causing mutations in MTM1 disrupted the MTM1-desmin complex, resulting in abnormal IF assembly and architecture in muscle cells and both mouse and human skeletal muscles. Adeno-associated virus-mediated ectopic expression of WT MTM1 in Mtm1-KO muscle reestablished normal desmin expression and localization. In addition, decreased MTM1 expression and XLCNM-causing mutations induced abnormal mitochondrial positioning, shape, dynamics, and function. We therefore conclude that MTM1 is a major regulator of both the desmin cytoskeleton and mitochondria homeostasis, specifically in skeletal muscle. Defects in IF stabilization and mitochondrial dynamics appear as common physiopathological features of centronuclear myopathies and desmin-related myopathies.

    Funded by: NINDS NIH HHS: P50 NS040828

    The Journal of clinical investigation 2011;121;1;70-85

  • Pericytes are required for blood-brain barrier integrity during embryogenesis.

    Daneman R, Zhou L, Kebede AA and Barres BA

    UCSF Department of Anatomy, 513 Parnassus Avenue, HSW1301, San Francisco, California 94143-0452, USA. Richard.daneman@ucsf.edu

    Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.

    Funded by: NINDS NIH HHS: R01 NS045621, R01 NS045621-04, R01-NS045621

    Nature 2010;468;7323;562-6

  • Endothelin receptor type A expression defines a distinct cardiac subdomain within the heart field and is later implicated in chamber myocardium formation.

    Asai R, Kurihara Y, Fujisawa K, Sato T, Kawamura Y, Kokubo H, Tonami K, Nishiyama K, Uchijima Y, Miyagawa-Tomita S and Kurihara H

    Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

    The avian and mammalian heart originates from two distinct embryonic regions: an early differentiating first heart field and a dorsomedially located second heart field. It remains largely unknown when and how these subdivisions of the heart field divide into regions with different fates. Here, we identify in the mouse a subpopulation of the first (crescent-forming) field marked by endothelin receptor type A (Ednra) gene expression, which contributes to chamber myocardium through a unique type of cell behavior. Ednra-lacZ/EGFP-expressing cells arise in the ventrocaudal inflow region of the early linear heart tube, converge to the midline, move anteriorly along the outer curvature and give rise to chamber myocardium mainly of the left ventricle and both atria. This movement was confirmed by fluorescent dye-labeling and transplantation experiments. The Ednra-lacZ/EGFP-expressing subpopulation is characterized by the presence of Tbx5-expressing cells. Ednra-null embryonic hearts often demonstrate hypoplasia of the ventricular wall, low mitotic activity and decreased Tbx5 expression with reciprocal expansion of Tbx2 expression. Conversely, endothelin 1 stimulates ERK phosphorylation and Tbx5 expression in the early embryonic heart. These results indicate that early Ednra expression defines a subdomain of the first heart field contributing to chamber formation, in which endothelin 1/Ednra signaling is involved. The present finding provides an insight into how subpopulations within the crescent-forming (first) heart field contribute to the coordination of heart morphogenesis through spatiotemporally defined cell movements.

    Development (Cambridge, England) 2010;137;22;3823-33

  • 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

  • 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

  • Focal Mullerian duct retention in male mice with constitutively activated beta-catenin expression in the Mullerian duct mesenchyme.

    Tanwar PS, Zhang L, Tanaka Y, Taketo MM, Donahoe PK and Teixeira JM

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

    Müllerian-inhibiting substance (MIS), which is produced by fetal Sertoli cells shortly after commitment of the bipotential gonads to testicular differentiation, causes Müllerian duct (MD) regression. In the fetal female gonads, MIS is not expressed and the MDs will differentiate into the internal female reproductive tract. We have investigated whether dysregulated β-catenin activity affects MD regression by expressing a constitutively activated nuclear form of β-catenin in the MD mesenchyme. We show that constitutively activated (CA) β-catenin causes focal retention of MD tissue in the epididymides and vasa deferentia. In adult mutant mice, the retained MD tissues express α-smooth muscle actin and desmin, which are markers for uterine differentiation. MD retention inhibited the folding complexity of the developing epididymides and usually led to obstructive azoospermia by spermatoceles. The MDs of urogenital ridges from mutant female embryos showed less regression with added MIS in organ culture compared with control MDs when analyzed by whole mount in situ hybridization for Wnt7a as a marker for the MD epithelium. CA β-catenin did not appear to affect expression of either MIS in the embryonic testes or its type II receptor (AMHR2) in the MD mesenchyme nor did it inhibit pSmad1/5/8 nuclear accumulation, suggesting that dysregulated β-catenin must inhibit MD regression independently of MIS signaling. These studies suggest that dysregulated Wnt/β-catenin signaling in the MD mesenchyme might also be a contributing factor in persistent Müllerian duct syndrome, a form of male pseudohermaphroditism, and development of spermatoceles.

    Funded by: NICHD NIH HHS: HD057201, R01 HD052701

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;37;16142-7

  • Rapid remodeling of airway vascular architecture at birth.

    Ni A, Lashnits E, Yao LC, Baluk P and McDonald DM

    Cardiovascular Research Institute, Comprehensive Cancer Center, Department of Anatomy, University of California, San Francisco, California, USA.

    Recent advances have documented the development of lung vasculature before and after birth, but less is known of the growth and maturation of airway vasculature. We sought to determine whether airway vasculature changes during the perinatal period and when the typical adult pattern develops. On embryonic day 16.5 mouse tracheas had a primitive vascular plexus unlike the adult airway vasculature, but instead resembling the yolk sac vasculature. Soon after birth (P0), the primitive vascular plexus underwent abrupt and extensive remodeling. Blood vessels overlying tracheal cartilage rings regressed from P1 to P3 but regrew from P4 to P7 to form the hierarchical, segmented, ladder-like adult pattern. Hypoxia and HIF-1α were present in tracheal epithelium over vessels that survived but not where they regressed. These findings reveal the plasticity of airway vasculature after birth and show that these vessels can be used to elucidate factors that promote postnatal vascular remodeling and maturation.

    Funded by: NCI NIH HHS: CA82923; NHLBI NIH HHS: HL24136, HL59157, HL96511, P01 HL024136-30, P01 HL024136-31A1, P01 HL024136-31A18647, R01 HL059157, R01 HL059157-13

    Developmental dynamics : an official publication of the American Association of Anatomists 2010;239;9;2354-66

  • Foxp1 coordinates cardiomyocyte proliferation through both cell-autonomous and nonautonomous mechanisms.

    Zhang Y, Li S, Yuan L, Tian Y, Weidenfeld J, Yang J, Liu F, Chokas AL and Morrisey EE

    Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

    Cardiomyocyte proliferation is high in early development and decreases progressively with gestation, resulting in the lack of a robust cardiomyocyte proliferative response in the adult heart after injury. Little is understood about how both cell-autonomous and nonautonomous signals are integrated to regulate the balance of cardiomyocyte proliferation during development. In this study, we show that a single transcription factor, Foxp1, can control the balance of cardiomyocyte proliferation during development by targeting different pathways in the endocardium and myocardium. Endocardial loss of Foxp1 results in decreased Fgf3/Fgf16/Fgf17/Fgf20 expression in the heart, leading to reduced cardiomyocyte proliferation. This loss of myocardial proliferation can be rescued by exogenous Fgf20, and is mediated, in part, by Foxp1 repression of Sox17. In contrast, myocardial-specific loss of Foxp1 results in increased cardiomyocyte proliferation and decreased differentiation, leading to increased myocardial mass and neonatal demise. We show that Nkx2.5 is a direct target of Foxp1 repression, and Nkx2.5 expression is increased in Foxp1-deficient myocardium. Moreover, transgenic overexpression of Nkx2.5 leads to increased cardiomyocyte proliferation and increased ventricular mass, similar to the myocardial-specific loss of Foxp1. These data show that Foxp1 coordinates the balance of cardiomyocyte proliferation and differentiation through cell lineage-specific regulation of Fgf ligand and Nkx2.5 expression.

    Funded by: NHLBI NIH HHS: HL071589, HL100405, R01 HL071589, U01 HL100405

    Genes & development 2010;24;16;1746-57

  • EphB-ephrin-B2 interactions are required for thymus migration during organogenesis.

    Foster KE, Gordon J, Cardenas K, Veiga-Fernandes H, Makinen T, Grigorieva E, Wilkinson DG, Blackburn CC, Richie E, Manley NR, Adams RH, Kioussis D and Coles MC

    Molecular Immunology, National Institute of Medical Research, London NW7 1AA, United Kingdom.

    Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.

    Funded by: Medical Research Council: G0601156, MC_U117532048

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;30;13414-9

  • Jaw muscularization requires Dlx expression by cranial neural crest cells.

    Heude E, Bouhali K, Kurihara Y, Kurihara H, Couly G, Janvier P and Levi G

    Evolution des Régulations Endocriniennes, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7221, Muséum National d'Histoire Naturelle, 75005 Paris, France.

    The origin of active predation in vertebrates is associated with the rise of three major, uniquely derived developmental characteristics of the head: (i) migratory cranial neural crest cells (CNCCs) giving rise to most skeletal skull elements; (ii) expression of Dlx genes by CNCCs in the Hox-free first pharyngeal arch (PA1); and (iii) muscularization of PA1 derivatives. Here we show that these three innovations are tightly linked. Expression of Dlx genes by CNCCs is not only necessary for head skeletogenesis, but also for the determination, differentiation, and patterning of cephalic myogenic mesoderm leading to masticatory muscle formation. In particular, inactivation of Dlx5 and Dlx6 in the mouse results in loss of jaw muscles. As Dlx5/6 are not expressed by the myogenic mesoderm, our findings imply an instructive role for Dlx5/6-positive CNCCs in muscle formation. The defect in muscularization does not result from the loss of mandibular identity observed in Dlx5/6(-/-) mice because masticatory muscles are still present in EdnRA(-/-) mutants, which display a similar jaw transformation. The genesis of jaws and their muscularization should therefore be seen as an integrated Dlx-dependent developmental process at the origin of the vertebrate head. The role of Dlx genes in defining gnathostome jaw identity could, therefore, be secondary to a more primitive function in the genesis of the oral skeletomuscular system.

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;25;11441-6

  • Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage.

    Finzsch M, Schreiner S, Kichko T, Reeh P, Tamm ER, Bösl MR, Meijer D and Wegner M

    Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

    Mutations in the transcription factor SOX10 cause neurocristopathies, including Waardenburg-Hirschsprung syndrome and peripheral neuropathies in humans. This is partly attributed to a requirement for Sox10 in early neural crest for survival, maintenance of pluripotency, and specification to several cell lineages, including peripheral glia. As a consequence, peripheral glia are absent in Sox10-deficient mice. Intriguingly, Sox10 continues to be expressed in these cells after specification. To analyze glial functions after specification, we specifically deleted Sox10 in immature Schwann cells by conditional mutagenesis. Mutant mice died from peripheral neuropathy before the seventh postnatal week. Nerve alterations included a thinned perineurial sheath, increased lipid and collagen deposition, and a dramatically altered cellular composition. Nerve conduction was also grossly aberrant, and neither myelinating nor nonmyelinating Schwann cells formed. Instead, axons of different sizes remained unsorted in large bundles. Schwann cells failed to develop beyond the immature stage and were unable to maintain identity. Thus, our study identifies a novel cause for peripheral neuropathies in patients with SOX10 mutations.

    The Journal of cell biology 2010;189;4;701-12

  • von Hippel-Lindau protein regulates transition from the fetal to the adult circulatory system in retina.

    Kurihara T, Kubota Y, Ozawa Y, Takubo K, Noda K, Simon MC, Johnson RS, Suematsu M, Tsubota K, Ishida S, Goda N, Suda T and Okano H

    Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

    In early neonates, the fetal circulatory system undergoes dramatic transition to the adult circulatory system. Normally, embryonic connecting vessels, such as the ductus arteriosus and the foramen ovale, close and regress. In the neonatal retina, hyaloid vessels maintaining blood flow in the embryonic retina regress, and retinal vessels take over to form the adult-type circulatory system. This process is regulated by a programmed cell death switch mediated by macrophages via Wnt and angiopoietin 2 pathways. In this study, we seek other mechanisms that regulate this process, and focus on the dramatic change in oxygen environment at the point of birth. The von Hippel-Lindau tumor suppressor protein (pVHL) is a substrate recognition component of an E3-ubiquitin ligase that rapidly destabilizes hypoxia-inducible factor alphas (HIF-alphas) under normoxic, but not hypoxic, conditions. To examine the role of oxygen-sensing mechanisms in retinal circulatory system transition, we generated retina-specific conditional-knockout mice for VHL (Vhl(alpha)(-CreKO) mice). These mice exhibit arrested transition from the fetal to the adult circulatory system, persistence of hyaloid vessels and poorly formed retinal vessels. These defects are suppressed by intraocular injection of FLT1-Fc protein [a vascular endothelial growth factor (VEGF) receptor-1 (FLT1)/Fc chimeric protein that can bind VEGF and inhibit its activity], or by inactivating the HIF-1alpha gene. Our results suggest that not only macrophages but also tissue oxygen-sensing mechanisms regulate the transition from the fetal to the adult circulatory system in the retina.

    Funded by: Howard Hughes Medical Institute

    Development (Cambridge, England) 2010;137;9;1563-71

  • Six1 and Six4 gene expression is necessary to activate the fast-type muscle gene program in the mouse primary myotome.

    Niro C, Demignon J, Vincent S, Liu Y, Giordani J, Sgarioto N, Favier M, Guillet-Deniau I, Blais A and Maire P

    Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.

    While the signaling pathways and transcription factors active in adult slow- and fast-type muscles begin to be characterized, genesis of muscle fiber-type diversity during mammalian development remains unexplained. We provide evidence showing that Six homeoproteins are required to activate the fast-type muscle program in the mouse primary myotome. Affymetrix transcriptomal analysis of Six1(-/-)Six4(-/-) E10.5 somites revealed the specific down-regulation of many genes of the fast-type muscle program. This data was confirmed by in situ hybridization performed on Six1(-/-)Six4(-/-) embryos. The first mouse myocytes express both fast-type and slow-type muscle genes. In these fibers, Six1 and Six4 expression is required to specifically activate fast-type muscle genes. Chromatin immunoprecipitation experiments confirm the binding of Six1 and Six4 on the regulatory regions of these muscle genes, and transfection experiments show the ability of these homeoproteins to activate specifically identified fast-type muscle genes. This in vivo wide transcriptomal analysis of the function of the master myogenic determinants, Six, identifies them as novel markers for the differential activation of a specific muscle program during mammalian somitic myogenesis.

    Developmental biology 2010;338;2;168-82

  • Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice.

    Chandana EP, Maeda Y, Ueda A, Kiyonari H, Oshima N, Yamamoto M, Kondo S, Oh J, Takahashi R, Yoshida Y, Kawashima S, Alexander DB, Kitayama H, Takahashi C, Tabata Y, Matsuzaki T and Noda M

    Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8501, Japan.

    Background: Developmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear.

    Results: We found that Reck is abundantly expressed in the cells associated with blood vessels undergoing angiogenesis or remodelling in the uteri of pregnant female mice. Some of the Reck-positive vessels show morphological features consistent with non-sprouting angiogenesis. Treatment with a vector expressing a small hairpin RNA against Reck severely disrupts the formation of blood vessels with a compact, round lumen. Similar defects were found in the vasculature of Reck-deficient or Reck conditional knockout embryos.

    Conclusions: Our findings implicate Reck in vascular remodeling, possibly through non-sprouting angiogenesis, in both maternal and embyonic tissues.

    BMC developmental biology 2010;10;84

  • Neogenin regulates skeletal myofiber size and focal adhesion kinase and extracellular signal-regulated kinase activities in vivo and in vitro.

    Bae GU, Yang YJ, Jiang G, Hong M, Lee HJ, Tessier-Lavigne M, Kang JS and Krauss RS

    Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029, USA

    A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.

    Funded by: NIAMS NIH HHS: AR46207, R01 AR046207

    Molecular biology of the cell 2009;20;23;4920-31

  • Pax3:Foxc2 reciprocal repression in the somite modulates muscular versus vascular cell fate choice in multipotent progenitors.

    Lagha M, Brunelli S, Messina G, Cumano A, Kume T, Relaix F and Buckingham ME

    CNRS URA 2578, Département de Biologie du Développement, Institut Pasteur, 75015 Paris, France.

    Maintenance of multipotency and how cells exit this state to adopt a specific fate are central questions in stem cell biology. During vertebrate development, multipotent cells of the dorsal somite, the dermomyotome, give rise to different lineages such as vascular smooth and skeletal muscle, regulated by the transcription factors Foxc2 and Pax3, respectively. Here we show reciprocal inhibition between Pax3 and Foxc2 in the mouse embryo. Using both genetic approaches and manipulation of external signals in somite explants, we demonstrate that the Pax3:Foxc2 ratio modulates myogenic versus vascular cell fates. This provides insight into how cell fate choices are orchestrated by these lineage genes in the dermomyotome.

    Developmental cell 2009;17;6;892-9

  • Numb promotes an increase in skeletal muscle progenitor cells in the embryonic somite.

    Jory A, Le Roux I, Gayraud-Morel B, Rocheteau P, Cohen-Tannoudji M, Cumano A and Tajbakhsh S

    Stem Cells & Development, Department of Developmental Biology Pasteur Institute, Centre National de la Recherche Scientifique (CNRS) URA 2578, 75724 Paris Cedex 15, France.

    Multiple cell types arise from cells in the dermomyotome of the somite that express Pax3 and Pax7, and myogenesis is regulated by Notch signaling. The asymmetric cell fate determinant Numb is thought to promote differentiation of skeletal muscle and other lineages by negatively regulating Notch signaling. We used transgenesis to overexpress Numb spatiotemporally in Pax3(+)/Pax7(+) somitic stem and progenitor cells in mouse embryos using a spatiotemporally regulated enhancer element from the Myf5 locus that can target muscle progenitor cells prior to cell commitment. Molecular analyses as well as examination of dermal and skeletal muscle cell fates in vivo show that although Numb is thought to be associated with muscle differentiation, unexpectedly the common stem/progenitor pool size for these lineages is increased in Numb-transgenic embryos. Prospective isolation of the relevant transgenic cells and analysis by quantitative reverse-transcription polymerase chain reaction demonstrated that, in this context, canonical Notch targets are not significantly downregulated. These findings were corroborated using a Notch reporter mouse during the formation of somites and prior to lineage segregation. Thus, we propose that Numb can regulate the self-renewal of dermal and muscle progenitors during a lineage progression.

    Stem cells (Dayton, Ohio) 2009;27;11;2769-80

  • Talin 1 and 2 are required for myoblast fusion, sarcomere assembly and the maintenance of myotendinous junctions.

    Conti FJ, Monkley SJ, Wood MR, Critchley DR and Müller U

    The Scripps Research Institute, Department of Cell Biology and Institute of Childhood and Neglected Diseases, La Jolla, CA 92037, USA.

    Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1 regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we show that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and sarcomere assembly, resembling defects in muscle lacking beta1 integrins. Talin 1/2-deficient myoblasts express functionally active beta1 integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial for skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.

    Funded by: NIMH NIH HHS: MH078833; NINDS NIH HHS: NS046456

    Development (Cambridge, England) 2009;136;21;3597-606

  • Integrin-linked kinase controls vascular wall formation by negatively regulating Rho/ROCK-mediated vascular smooth muscle cell contraction.

    Kogata N, Tribe RM, Fässler R, Way M and Adams RH

    Vascular Development Laboratory, UK London Research Institute, London WC2A 3PX, United Kingdom.

    Vascular smooth muscle cells (VSMCs) form contractile layers around larger blood vessels in a process that is essential for the formation of a fully functional vasculature. Here, we show that integrin-linked kinase (ILK) is required for the formation of a unitary layer of aligned VSMCs around arterioles and the regulation of blood vessel constriction in mice. In the absence of ILK, activated Rho/ROCK signaling induces the elevated phosphorylation of myosin light chain leading to abnormally enhanced VSMC contraction in vitro and in vivo. Our findings identify ILK as a key component regulating vascular wall formation by negatively modulating VSMC contractility.

    Funded by: Cancer Research UK

    Genes & development 2009;23;19;2278-83

  • The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium.

    Remme CA, Verkerk AO, Hoogaars WM, Aanhaanen WT, Scicluna BP, Annink C, van den Hoff MJ, Wilde AA, van Veen TA, Veldkamp MW, de Bakker JM, Christoffels VM and Bezzina CR

    Heart Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. c.a.remme@amc.uva.nl

    Cardiac sodium channels are responsible for conduction in the normal and diseased heart. We aimed to investigate regional and transmural distribution of sodium channel expression and function in the myocardium. Sodium channel Scn5a mRNA and Na(v)1.5 protein distribution was investigated in adult and embryonic mouse heart through immunohistochemistry and in situ hybridization. Functional sodium channel availability in subepicardial and subendocardial myocytes was assessed using patch-clamp technique. Adult and embryonic (ED14.5) mouse heart sections showed low expression of Na(v)1.5 in the HCN4-positive sinoatrial and atrioventricular nodes. In contrast, high expression levels of Na(v)1.5 were observed in the HCN4-positive and Cx43-negative AV or His bundle, bundle branches and Purkinje fibers. In both ventricles, a transmural gradient was observed, with a low Na(v)1.5 labeling intensity in the subepicardium as compared to the subendocardium. Similar Scn5a mRNA expression patterns were observed on in situ hybridization of embryonic and adult tissue. Maximal action potential upstroke velocity was significantly lower in subepicardial myocytes (mean +/- SEM 309 +/- 32 V/s; n = 14) compared to subendocardial myocytes (394 +/- 32 V/s; n = 11; P < 0.05), indicating decreased sodium channel availability in subepicardium compared to subendocardium. Scn5a and Na(v)1.5 show heterogeneous distribution patterns within the cardiac conduction system and across the ventricular wall. This differential distribution of the cardiac sodium channel may have profound consequences for conduction disease phenotypes and arrhythmogenesis in the setting of sodium channel disease.

    Basic research in cardiology 2009;104;5;511-22

  • Expression pattern and role of Galectin1 during early mouse myogenesis.

    Shoji H, Deltour L, Nakamura T, Tajbakhsh S and Poirier F

    Department of Developmental Biology, Institut Jacques Monod, CNRS 7592, Paris-Diderot University, Paris, France.

    Galectin1, the prototype member of a family of carbohydrate binding proteins, is involved in muscle stem cell behavior and in tissue regeneration after muscle injury in adult mice. Here, we addressed the question of when this gene is first acting in the muscle lineage. We found that Galectin1 is an early marker of myogenesis as the transcripts and protein are initially confined to the somites, starting from day 9.0 of embryogenesis. We next investigated its relationship with the muscle determination factors, Myf5 and Myod. By comparing the spatio-temporal distribution of Galectin1 transcripts in control and Myf5 null mutant embryos, we were able to establish that it acts downstream of Myf5. However, early myogenesis does not seem affected in Galectin1 null mutant embryos indicating that, unlike in the adult, Galectin1 does not play a role in muscle fate acquisition during development.

    Development, growth & differentiation 2009;51;7;607-15

  • MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice.

    Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, Chen JF, Deng Z, Gunn B, Shumate J, Willis MS, Selzman CH and Wang DZ

    Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, North Carolina, USA.

    MicroRNAs (miRNAs) are a class of small noncoding RNAs that have gained status as important regulators of gene expression. Here, we investigated the function and molecular mechanisms of the miR-208 family of miRNAs in adult mouse heart physiology. We found that miR-208a, which is encoded within an intron of alpha-cardiac muscle myosin heavy chain gene (Myh6), was actually a member of a miRNA family that also included miR-208b, which was determined to be encoded within an intron of beta-cardiac muscle myosin heavy chain gene (Myh7). These miRNAs were differentially expressed in the mouse heart, paralleling the expression of their host genes. Transgenic overexpression of miR-208a in the heart was sufficient to induce hypertrophic growth in mice, which resulted in pronounced repression of the miR-208 regulatory targets thyroid hormone-associated protein 1 and myostatin, 2 negative regulators of muscle growth and hypertrophy. Studies of the miR-208a Tg mice indicated that miR-208a expression was sufficient to induce arrhythmias. Furthermore, analysis of mice lacking miR-208a indicated that miR-208a was required for proper cardiac conduction and expression of the cardiac transcription factors homeodomain-only protein and GATA4 and the gap junction protein connexin 40. Together, our studies uncover what we believe are novel miRNA-dependent mechanisms that modulate cardiac hypertrophy and electrical conduction.

    The Journal of clinical investigation 2009;119;9;2772-86

  • Paracrine Hedgehog signaling in stomach and intestine: new roles for hedgehog in gastrointestinal patterning.

    Kolterud A, Grosse AS, Zacharias WJ, Walton KD, Kretovich KE, Madison BB, Waghray M, Ferris JE, Hu C, Merchant JL, Dlugosz AA, Kottmann AH and Gumucio DL

    Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-2200, USA.

    Hedgehog signaling is critical in gastrointestinal patterning. Mice deficient in Hedgehog signaling exhibit abnormalities that mirror deformities seen in the human VACTERL (vertebral, anal, cardiac, tracheal, esophageal, renal, limb) association. However, the direction of Hedgehog signal flow is controversial and the cellular targets of Hedgehog signaling change with time during development. We profiled cellular Hedgehog response patterns from embryonic day 10.5 (E10.5) to adult in murine antrum, pyloric region, small intestine, and colon.

    Methods: Hedgehog signaling was profiled using Hedgehog pathway reporter mice and in situ hybridization. Cellular targets were identified by immunostaining. Ihh-overexpressing transgenic animals were generated and analyzed.

    Results: Hedgehog signaling is strictly paracrine from antrum to colon throughout embryonic and adult life. Novel findings include the following: mesothelial cells of the serosa transduce Hedgehog signals in fetal life; the hindgut epithelium expresses Ptch but not Gli1 at E10.5; the 2 layers of the muscularis externa respond differently to Hedgehog signals; organogenesis of the pyloric sphincter is associated with robust Hedgehog signaling; dramatically different Hedgehog responses characterize stomach and intestine at E16; and after birth, the muscularis mucosa and villus smooth muscle consist primarily of Hedgehog-responsive cells and Hh levels actively modulate villus core smooth muscle.

    Conclusions: These studies reveal a previously unrecognized association of paracrine Hedgehog signaling with several gastrointestinal patterning events involving the serosa, pylorus, and villus smooth muscle. The results may have implications for several human anomalies and could potentially expand the spectrum of the human VACTERL association.

    Funded by: NHLBI NIH HHS: T32 HL007622, T32-HL07622; NICHD NIH HHS: T32 HD007505, T32-HD007505; NIDDK NIH HHS: P01 DK062041, P01 DK62041, R01 DK065850, R01 DK065850-03, R01 DK065850-04; NIGMS NIH HHS: T32 GM007315

    Gastroenterology 2009;137;2;618-28

  • The small G-proteins Rac1 and Cdc42 are essential for myoblast fusion in the mouse.

    Vasyutina E, Martarelli B, Brakebusch C, Wende H and Birchmeier C

    Max-Delbrück-Center for Molecular Medicine, Department of Neuroscience, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.

    Rac1 and Cdc42 are small G-proteins that regulate actin dynamics and affect plasma membrane protrusion and vesicle traffic. We used conditional mutagenesis in mice to demonstrate that Rac1 and Cdc42 are essential for myoblast fusion in vivo and in vitro. The deficit in fusion of Rac1 or Cdc42 mutant myoblasts correlates with a deficit in the recruitment of actin fibers and vinculin to myoblast contact sites. Comparison of the changes observed in mutant myogenic cells indicates that Rac1 and Cdc42 function in a nonredundant and not completely overlapping manner during the fusion process. Our genetic analysis demonstrates thus that the function of Rac in myoblast fusion is evolutionarily conserved from insects to mammals and that Cdc42, a molecule hitherto not implicated in myoblast fusion, is essential for the fusion of murine myoblasts.

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;22;8935-40

  • Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates.

    Sambasivan R, Gayraud-Morel B, Dumas G, Cimper C, Paisant S, Kelly RG, Kelly R and Tajbakhsh S

    Institut Pasteur, Stem Cells & Development, CNRS URA 2578, Université de la Méditerranée, Campus de Luminy, Marseille, France.

    Genetic regulatory networks governing skeletal myogenesis in the body are well understood, yet their hierarchical relationships in the head remain unresolved. We show that either Myf5 or Mrf4 is necessary for initiating extraocular myogenesis. Whereas Mrf4 is dispensable for pharyngeal muscle progenitor fate, Tbx1 and Myf5 act synergistically for governing myogenesis in this location. As in the body, Myod acts epistatically to the initiating cascades in the head. Thus, complementary pathways, governed by Pax3 for body, and Tbx1 for pharyngeal muscles, but absent for extraocular muscles, activate the core myogenic network. These diverse muscle progenitors maintain their respective embryonic regulatory signatures in the adult. However, these signatures are not sufficient to ensure the specific muscle phenotypes, since the expected differentiated phenotype is not manifested when satellite cells are engrafted heterotopically. These findings identify novel genetic networks that may provide insights into myopathies which often affect only subsets of muscles.

    Developmental cell 2009;16;6;810-21

  • Unraveling the genetic landscape of bladder development in mice.

    Price KL, Woolf AS and Long DA

    Nephro-Urology Unit, University College London Institute of Child Health, London, United Kingdom.

    Purpose: To better understand the pathobiology of human congenital bladder abnormalities and disorders associated with dedifferentiation, such as bladder cancer, we must first unravel the biology of normal bladder development. Therefore, we performed microarray analysis focusing on determining the gene expression profile at the initiation of bladder development.

    RNA was extracted from embryonic day 13 and 18 mouse bladders (anatomically equivalent to 7 and 13 weeks of human gestation) and gene expression was evaluated using microarrays. Alterations in select genes of biological interest were confirmed using real-time quantitative polymerase chain reaction and localization was determined by immunohistochemistry.

    Results: The genetic profile in the initiating mouse bladder at embryonic day 13 was dominated by transcription factors, retinoic acid signaling genes, Eph/ephrin bidirectional signaling molecules and genes associated with regulating cell cycle and differentiation. Later in development at embryonic day 18 genes associated with smooth muscle, innervation and epithelial differentiation were up-regulated. In addition, we examined the functional role of midkine, which was highly expressed at embryonic day 13, using organ culture and to our knowledge we provide the first evidence that this growth factor up-regulates molecules associated with bladder smooth muscle differentiation.

    Conclusions: These data provide novel insights into molecules that orchestrate bladder development and highlight genes that may be involved in diseases associated with abnormal differentiation.

    The Journal of urology 2009;181;5;2366-74

  • Biochemical and mechanical dysfunction in a mouse model of desmin-related myopathy.

    Maloyan A, Osinska H, Lammerding J, Lee RT, Cingolani OH, Kass DA, Lorenz JN and Robbins J

    Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Ohio 45229-3039, USA.

    An R120G mutation in alphaB-crystallin (CryAB(R120G)) causes desmin-related myopathy (DRM). In mice with cardiomyocyte-specific expression of the mutation, CryAB(R120G)-mediated DRM is characterized by CryAB and desmin accumulations within cardiac muscle, mitochondrial deficiencies, activation of apoptosis, and heart failure (HF). Excessive production of reactive oxygen species (ROS) is often a hallmark of HF and treatment with antioxidants can sometimes prevent the progression of HF in terms of contractile dysfunction and cardiomyocyte survival. It is unknown whether blockade of ROS is beneficial for protein misfolding diseases such as DRM. We addressed this question by blocking the activity of xanthine oxidase (XO), a superoxide-generating enzyme that is upregulated in our model of DRM. The XO inhibitor oxypurinol was administered to CryAB(R120G) mice for a period of 1 or 3 months. Mitochondrial function was dramatically improved in treated animals in terms of complex I activity and conservation of mitochondrial membrane potential. Oxypurinol also largely restored normal mitochondrial morphology. Surprisingly, however, cardiac contractile function and cardiac compliance were unimproved, indicating that the contractile deficit might be independent of mitochondrial dysfunction and the initiation of apoptosis. Using magnetic bead microrheology at the single cardiomyocyte level, we demonstrated that sarcomeric disarray and accumulation of the physical aggregates resulted in significant changes in the cytoskeletal mechanical properties in the CryAB(R120G) cardiomyocytes. Our findings indicate that oxypurinol treatment largely prevented mitochondrial deficiency in DRM but that contractility was not improved because of mechanical deficits in passive cytoskeletal stiffness.

    Funded by: NHLBI NIH HHS: 01HL087862, P01HL059408, P01HL69799, P50HL074728, P50HL077101, R01 HL082792-04, R01 HL082792-05; NINDS NIH HHS: R01 NS059348-04

    Circulation research 2009;104;8;1021-8

  • Prox1 maintains muscle structure and growth in the developing heart.

    Risebro CA, Searles RG, Melville AA, Ehler E, Jina N, Shah S, Pallas J, Hubank M, Dillard M, Harvey NL, Schwartz RJ, Chien KR, Oliver G and Riley PR

    Molecular Medicine Unit, UCL Institute of Child Health, London WC1N 1EH, UK.

    Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the murine homeobox transcription factor Prox1 results in the disruption of expression and localisation of sarcomeric proteins, gross myofibril disarray and growth-retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of the genes encoding the structural proteins alpha-actinin, N-RAP and zyxin, which collectively function to maintain an actin-alpha-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and the manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study reveals an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling foetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.

    Funded by: British Heart Foundation; Medical Research Council: G0400153; NHLBI NIH HHS: R01 HL073402, R01 HL073402-06, R01-HL073402

    Development (Cambridge, England) 2009;136;3;495-505

  • Severe myopathy mutations modify the nanomechanics of desmin intermediate filaments.

    Kreplak L and Bär H

    Department of Physics and Atmospheric Science, Dalhousie University, Halifax, Nova Scotia, Canada B3H 3J5. kreplak@dal.ca

    Mutations in the intermediate filament (IF) protein desmin cause severe forms of myofibrillar myopathy characterized by partial aggregation of the extrasarcomeric desmin cytoskeleton and structural disorganization of myofibrils. In contrast to prior expectations, we showed that some of the known disease-causing mutations, such as DesA360P, DesQ389P and DesD399Y, are assembly-competent and do allow formation of bona fide IFs in vitro and in vivo. We also previously demonstrated that atomic force microscopy can be employed to measure the tensile properties of single desmin IFs. Using the same approach on filaments formed by the aforementioned mutant desmins, we now observed two different nanomechanical behaviors: DesA360P exhibited tensile properties similar to that of wild-type desmin IFs, whereas DesQ389P and DesD399Y exhibited local variations in their tensile properties along the filament length. Based on these findings, we hypothesize that DesQ389P and DesD399Y may cause muscle disease by altering the specific biophysical properties of the desmin filaments, thereby compromising both its mechanosensing and mechanotransduction ability.

    Journal of molecular biology 2009;385;4;1043-51

  • Disruption of striated preferentially expressed gene locus leads to dilated cardiomyopathy in mice.

    Liu X, Ramjiganesh T, Chen YH, Chung SW, Hall SR, Schissel SL, Padera RF, Liao R, Ackerman KG, Kajstura J, Leri A, Anversa P, Yet SF, Layne MD and Perrella MA

    Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02115, USA.

    Background: The striated preferentially expressed gene (Speg) generates 4 different isoforms through alternative promoter use and tissue-specific splicing. Depending on the cell type, Speg isoforms may serve as markers of striated or smooth muscle differentiation.

    To elucidate function of Speg gene isoforms, we disrupted the Speg gene locus in mice by replacing common exons 8, 9, and 10 with a lacZ gene. beta-Galactosidase activity was detected in cardiomyocytes of the developing heart starting at day 11.5 days post coitum (dpc). beta-Galactosidase activity in other cell types, including vascular smooth muscle cells, did not begin until 18.5 dpc. In the developing heart, protein expression of only Spegalpha and Spegbeta isoforms was present in cardiomyocytes. Homozygous Speg mutant hearts began to enlarge by 16.5 dpc, and by 18.5 dpc, they demonstrated dilation of right and left atria and ventricles. These cardiac abnormalities in the absence of Speg were associated with a cellular hypertrophic response, myofibril degeneration, and a marked decrease in cardiac function. Moreover, Speg mutant mice exhibited significant neonatal mortality, with increased death occurring by 2 days after birth.

    Conclusions: These findings demonstrate that mutation of the Speg locus leads to cardiac dysfunction and a phenotype consistent with a dilated cardiomyopathy.

    Funded by: NHLBI NIH HHS: HL60788, HL65639, K08 HL076286, R01 HL060788, R01 HL060788-09, R01 HL060788-10, R01 HL060788-11, R01 HL065639, R01 HL065639-03, R01 HL065639-04, R01 HL065639-05, R01 HL102897; NIGMS NIH HHS: GM53249, R01 GM053249, R01 GM053249-10, R01 GM053249-11, R01 GM053249-12

    Circulation 2009;119;2;261-8

  • Reassessment of Isl1 and Nkx2-5 cardiac fate maps using a Gata4-based reporter of Cre activity.

    Ma Q, Zhou B and Pu WT

    Department of Cardiology, Children's Hospital Boston, 300 Longwood Ave, Boston, MA 02115, USA.

    Isl1 and Nkx2-5-expressing cardiovascular progenitors play pivotal roles in cardiogenesis. Previously reported Cre-based fate-mapping studies showed that Isl1 progenitors contribute predominantly to the derivatives of the second heart field, and Nkx2-5 progenitors contributed mainly to the cardiomyocyte lineage. However, partial recombination of Cre reporter genes can complicate interpretation of Cre fate-mapping experiments. We found that a Gata4-based Cre-activated reporter was recombined by Isl1(Cre) and Nkx2-5(Cre) in a substantially broader domain than previously reported using standard Cre-activated reporters. The expanded Isl1 and Nkx2-5 cardiac fate maps were remarkably similar, and included extensive contributions to cardiomyocyte, endocardial, and smooth muscle lineages in all four cardiac chambers. These data indicate that Isl1 is expressed in progenitors of both primary and secondary heart fields, and that Nkx2-5 is expressed in progenitors of cardiac endothelium and smooth muscle, in addition to cardiomyocytes. These results have important implications for our understanding of cardiac lineage diversification in vivo, and for the interpretation of Cre-based fate maps.

    Funded by: NHLBI NIH HHS: P50 HL074734, P50 HL074734-01, SCCOR 1 PO1 HL074734

    Developmental biology 2008;323;1;98-104

  • Use of dual section mRNA in situ hybridisation/immunohistochemistry to clarify gene expression patterns during the early stages of nephron development in the embryo and in the mature nephron of the adult mouse kidney.

    Georgas K, Rumballe B, Wilkinson L, Chiu HS, Lesieur E, Gilbert T and Little MH

    Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD, 4072, Australia.

    The kidney is the most complex organ within the urogenital system. The adult mouse kidney contains in excess of 8,000 mature nephrons, each of which can be subdivided into a renal corpuscle and 14 distinct tubular segments. The histological complexity of this organ can make the clarification of the site of gene expression by in situ hybridisation difficult. We have defined a panel of seven antibodies capable of identifying the six stages of early nephron development, the tubular nephron segments and the components of the renal corpuscle within the embryonic and adult mouse kidney. We have analysed in detail the protein expression of Wt1, Calb1 Aqp1, Aqp2 and Umod using these antibodies. We have then coupled immunohistochemistry with RNA in situ hybridisation in order to precisely identify the expression pattern of different genes, including Wnt4, Umod and Spp1. This technique will be invaluable for examining at high resolution, the structure of both the developing and mature nephron where standard in situ hybridisation and histological techniques are insufficient. The use of this technique will enhance the expression analyses of genes which may be involved in nephron formation and the function of the mature nephron in the mouse.

    Funded by: NIDDK NIH HHS: DK070136-02

    Histochemistry and cell biology 2008;130;5;927-42

  • Nkx2-5- and Isl1-expressing cardiac progenitors contribute to proepicardium.

    Zhou B, von Gise A, Ma Q, Rivera-Feliciano J and Pu WT

    Harvard Stem Cell Institute and Department of Cardiology, Children's Hospital Boston, 300 Longwood Avenue, Boston, MA 02115, USA.

    Correct delineation of the hierarchy of cardiac progenitors is a key step to understanding heart development, and will pave the way for future use of cardiac progenitors in the treatment of heart disease. Multipotent Nkx2-5 and Isl1 cardiac progenitors contribute to cardiomyocyte, smooth muscle, and endothelial lineages, which constitute the major lineages of the heart. Recently, progenitors located within the proepicardium and epicardium were reported to differentiate into cardiomyocytes, as well as smooth muscle and endothelial cells. However, the relationship of these proepicardial progenitors to the previously described Nkx2-5 and Isl1 cardiac progenitors is incompletely understood. To address this question, we performed in vivo Cre-loxP-based lineage tracing. Both Nkx2-5- and Isl1-expressing progenitors contributed to the proepicardium and expressed Wt1 and Tbx18, markers of proepicardial progenitor cells. Interestingly, Nkx2-5 knockout resulted in abnormal proepicardial development and decreased expression of Wt1, suggesting a functional role for Nkx2-5 in proepicardium formation. Taken together, these results suggest that Nkx2-5 and/or Isl1 cardiac progenitors contribute to proepicardium during heart development.

    Funded by: NHLBI NIH HHS: R01 HL094683, R21 HL089417, R21 HL089417-01

    Biochemical and biophysical research communications 2008;375;3;450-3

  • The atypical Rac activator Dock180 (Dock1) regulates myoblast fusion in vivo.

    Laurin M, Fradet N, Blangy A, Hall A, Vuori K and Côté JF

    Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada H2W 1R7.

    Dock1 (also known as Dock180) is a prototypical member of a new family of atypical Rho GTPase activators. Genetic studies in Drosophila and Caenorhabditis elegans have demonstrated that Dock1 orthologues in these organisms have a crucial role in activating Rac GTPase signaling. We generated mutant alleles of the closely related Dock1 and Dock5 genes to study their function in mammals. We report that while Dock5 is dispensable for normal mouse embryogenesis, Dock1 has an essential role in embryonic development. A dramatic reduction of all skeletal muscle tissues is observed in Dock1-null embryos. Mechanistically, this embryonic defect is attributed to a strong deficiency in myoblast fusion, which is detectable both in vitro and in vivo. Furthermore, we have uncovered a contribution of Dock5 toward myofiber development. These studies identify Dock1 and Dock5 as critical regulators of the fusion step during primary myogenesis in mammals and demonstrate that a specific component of the myoblast fusion machinery identified in Drosophila plays an evolutionarily conserved role in higher vertebrates.

    Funded by: NCI NIH HHS: CA102583, P01 CA102583

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;40;15446-51

  • A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization.

    Mavroidis M, Panagopoulou P, Kostavasili I, Weisleder N and Capetanaki Y

    Cell Biology Division, Center of Basic Research, Biomedical Research Foundation, Academy of Athens, Athens, Greece.

    A missense mutation (Ile 451 to Met) at the tail domain of the muscle-specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. The Ile451Met mutation is located inside a conserved motif in the desmin tail domain, believed to have a potential role in the lateral packing of type III intermediate filaments. Nevertheless, the role of the type III intermediate filament tail domain remains elusive. To further study the role of this domain in the function of cardiomyocytes and in the development of cardiomyopathy, we generated transgenic mice expressing the mutant desmin(I451M) in the cardiac tissue. Analysis of hearts from transgenic animals revealed that mutant desmin loses its Z-disc localization but it can still associate with the intercalated discs, which, however, have an altered architecture, resembling other examples of dilated cardiomyopathy. This is the first report demonstrating a critical role of the desmin head and tail domains in the formation of the IF scaffold around Z discs. It is further suggested that in cardiomyocytes, an interplay between desmin tail and head domains is taking place, which potentially protects the amino terminus of desmin from specific proteases. The fact that the association with intercalated discs seems unchanged suggests that this association must take place through the desmin tail, in contrast to the head domain that is most possibly involved in the Z-disc binding.

    Funded by: NHLBI NIH HHS: HL-98

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;9;3318-27

  • Fgf9 signaling regulates small intestinal elongation and mesenchymal development.

    Geske MJ, Zhang X, Patel KK, Ornitz DM and Stappenbeck TS

    Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.

    Short bowel syndrome is an acquired condition in which the length of the small intestine is insufficient to perform its normal absorptive function. Current therapies are limited as the developmental mechanisms that normally regulate elongation of the small intestine are poorly understood. Here, we identify Fgf9 as an important epithelial-to-mesenchymal signal required for proper small intestinal morphogenesis. Mouse embryos that lack either Fgf9 or the mesenchymal receptors for Fgf9 contained a disproportionately shortened small intestine, decreased mesenchymal proliferation, premature differentiation of fibroblasts into myofibroblasts and significantly elevated Tgfbeta signaling. These findings suggest that Fgf9 normally functions to repress Tgfbeta signaling in these cells. In vivo, a small subset of mesenchymal cells expressed phospho-Erk and the secreted Tgfbeta inhibitors Fst and Fstl1 in an Fgf9-dependent fashion. The p-Erk/Fst/Fstl1-expressing cells were most consistent with intestinal mesenchymal stem cells (iMSCs). We found that isolated iMSCs expressed p-Erk, Fst and Fstl1, and could repress the differentiation of intestinal myofibroblasts in co-culture. These data suggest a model in which epithelial-derived Fgf9 stimulates iMSCs that in turn regulate underlying mesenchymal fibroblast proliferation and differentiation at least in part through inhibition of Tgfbeta signaling in the mesenchyme. Taken together, the interaction of FGF and TGFbeta signaling pathways in the intestinal mesenchyme could represent novel targets for future short bowel syndrome therapies.

    Funded by: NIDDK NIH HHS: DK071619, R01 DK071619, R01 DK071619-01A2, T32 DK007130, T32 DK007130-33, T32 DK007130-34, T32DK0713034

    Development (Cambridge, England) 2008;135;17;2959-68

  • Serum response factor is required for sprouting angiogenesis and vascular integrity.

    Franco CA, Mericskay M, Parlakian A, Gary-Bobo G, Gao-Li J, Paulin D, Gustafsson E and Li Z

    UPMC Univ Paris 06, UMR 7079, Physiology and Physiopathology, 75005 Paris, France.

    Serum response factor (SRF) is a transcription factor that controls the expression of cytoskeletal proteins and immediate early genes in different cell types. Here, we found that SRF expression is restricted to endothelial cells (ECs) of small vessels such as capillaries in the mouse embryo. EC-specific Srf deletion led to aneurysms and hemorrhages from 11.5 days of mouse development (E11.5) and lethality at E14.5. Mutant embryos presented a reduced capillary density and defects in EC migration, with fewer numbers of filopodia in tip cells and ECs showing defects in actin polymerization and intercellular junctions. We show that SRF is essential for the expression of VE-cadherin and beta-actin in ECs both in vivo and in vitro. Moreover, knockdown of SRF in ECs impaired VEGF- and FGF-induced in vitro angiogenesis. Taken together, our results demonstrate that SRF plays an important role in sprouting angiogenesis and small vessel integrity in the mouse embryo.

    Developmental cell 2008;15;3;448-61

  • Expression and localization of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in murine and human placentas.

    Satoh H, Kiyota E, Terasaki Y, Sawamura T, Takagi K, Mizuta H and Takeya M

    Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.

    Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is one of the scavenger receptors that recognizes oxidized low-density lipoprotein as a major ligand. The placenta is a major source of prooxidant during pregnancy, and the level of placental oxidative stress increases rapidly at the end of the first trimester and tapers off later in gestation. In our study, we evaluated placental expression of LOX-1 during different gestational stages in mice and humans. We used immunohistochemistry and ISH to identify LOX-1-expressing cells in murine and human placentas. In both species, higher expression of LOX-1 mRNA during early to midgestational stages compared with late gestation-corresponding to the increased oxidative stress in early pregnancy-was shown by real-time RT-PCR. In murine placenta, we showed that LOX-1-expressing cells were fibroblast-like stromal cells in metrial glands and decidua basalis and that they were glycogen trophoblast cells in the junctional and labyrinth zones. In the human, LOX-1 expression was detected in villous cytotrophoblasts in both first trimester and term placentas. These localization patterns of LOX-1 in murine and human placentas suggest the possible involvement of LOX-1 in high oxidative stress conditions of pregnancy.

    The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 2008;56;8;773-84

  • Role of desmin in active force transmission and maintenance of structure during growth of urinary bladder.

    Scott RS, Li Z, Paulin D, Uvelius B, Small JV and Arner A

    Dept. of Physiology and Pharmacology, Karolinska Institutet, v Eulers v 8, SE 171 77 Stockholm, Sweden.

    Role of the intermediate filament protein desmin in hypertrophy of smooth muscle was examined in desmin-deficient mice (Des(-/-)). A partial obstruction of the urethra was created, and after 9-19 days bladder weight increased approximately threefold in both Des(-/-) and wild type (Des(+/+)) animals. Bladder growth was associated with the synthesis of actin and myosin. In the hypertrophic Des(+/+) bladder, the relative content of desmin increased. In Des(-/-)mice, desmin was absent. No alterations in the amount of vimentin were observed. Although Des(-/-) obstructed bladders were capable of growth, they had structural changes with a partial disruption of the wall. Des(-/-)bladders had slightly lower passive stress and significantly lower active stress compared with Des(+/+). Des(-/-)preparations had lower shortening velocity. During hypertrophy, these structural and mechanical alterations in the Des(-/-)urinary bladder became more pronounced. In conclusion, desmin in the bladder smooth muscle is not needed for growth but has a role in active force transmission and maintenance of wall structure.

    American journal of physiology. Cell physiology 2008;295;2;C324-31

  • Leukemia inhibitory factor regulates microvessel density by modulating oxygen-dependent VEGF expression in mice.

    Kubota Y, Hirashima M, Kishi K, Stewart CL and Suda T

    Department of Cell Differentiation, Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo, Japan. ykubo33@sc.itc.keio.ac.jp

    To meet tissue requirements for oxygen, capillaries must be properly distributed without excess or shortage. In this process, tissue oxygen concentration is well known to determine capillary density via the hypoxia-induced cascade, in which HIFs and VEGF play key roles. However, some additional mechanisms modulating this cascade are suggested to be involved in precise capillary network formation. Here, we showed that leukemia inhibitory factor (LIF) was predominantly expressed in developing endothelium, while its receptor was expressed in surrounding cells such as retinal astrocytes. The retinas of Lif(-/-) mice displayed increased microvessel density accompanied by sustained tip cell activity, due to increased VEGF expression by astrocytes in the vascularized area. Lif(-/-) mice resisted hyperoxygen insult in the oxygen-induced retinopathy model, whereas they paradoxically had increased numbers of neovascular tufts. In an in vitro study, LIF inhibited hypoxia-induced VEGF expression and proliferation in cultured astrocytes. Lif(-/-) mice also exhibited similarly increased microvessel density and upregulated VEGF in various tissues outside the retina. Together, these findings suggest that tissues and advancing vasculature communicate to ensure adequate vascularization using LIF as well as oxygen, which suggests a new strategy for antiangiogenic therapy in human diseases such as diabetic retinopathy and cancer.

    The Journal of clinical investigation 2008;118;7;2393-403

  • Desmin mediates TNF-alpha-induced aggregate formation and intercalated disk reorganization in heart failure.

    Panagopoulou P, Davos CH, Milner DJ, Varela E, Cameron J, Mann DL and Capetanaki Y

    Cell Biology Division, Center of Basic Research, and 2Cardiovascular Research Division, Center of Clinical Research, Biomedical Research Foundation, Academy of Athens, Athens 11527, Greece.

    We explored the involvement of the muscle-specific intermediate filament protein desmin in the model of tumor necrosis factor alpha (TNF-alpha)-induced cardiomyopathy. We demonstrate that in mice overexpressing TNF-alpha in the heart (alpha-myosin heavy chain promoter-driven secretable TNF-alpha [MHCsTNF]), desmin is modified, loses its intercalated disk (ID) localization, and forms aggregates that colocalize with heat shock protein 25 and ubiquitin. Additionally, other ID proteins such as desmoplakin and beta-catenin show similar localization changes in a desmin-dependent fashion. To address underlying mechanisms, we examined whether desmin is a substrate for caspase-6 in vivo as well as the implications of desmin cleavage in MHCsTNF mice. We generated transgenic mice with cardiac-restricted expression of a desmin mutant (D263E) and proved that it is resistant to caspase cleavage in the MHCsTNF myocardium. The aggregates are diminished in these mice, and D263E desmin, desmoplakin, and beta-catenin largely retain their proper ID localization. Importantly, D263E desmin expression attenuated cardiomyocyte apoptosis, prevented left ventricular wall thinning, and improved the function of MHCsTNF hearts.

    Funded by: NHLBI NIH HHS: P50 HL-O6H, R01 HL58081, R01 HL73017

    The Journal of cell biology 2008;181;5;761-75

  • Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms.

    Konieczny P, Fuchs P, Reipert S, Kunz WS, Zeöld A, Fischer I, Paulin D, Schröder R and Wiche G

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

    Dysfunction of plectin, a 500-kD cytolinker protein, leads to skin blistering and muscular dystrophy. Using conditional gene targeting in mice, we show that plectin deficiency results in progressive degenerative alterations in striated muscle, including aggregation and partial loss of intermediate filament (IF) networks, detachment of the contractile apparatus from the sarcolemma, profound changes in myofiber costameric cytoarchitecture, and decreased mitochondrial number and function. Analysis of newly generated plectin isoform-specific knockout mouse models revealed that IF aggregates accumulate in distinct cytoplasmic compartments, depending on which isoform is missing. Our data show that two major plectin isoforms expressed in muscle, plectin 1d and 1f, integrate fibers by specifically targeting and linking desmin IFs to Z-disks and costameres, whereas plectin 1b establishes a linkage to mitochondria. Furthermore, disruption of Z-disk and costamere linkages leads to the pathological condition of epidermolysis bullosa with muscular dystrophy. Our findings establish plectin as the major organizer of desmin IFs in myofibers and provide new insights into plectin- and desmin-related muscular dystrophies.

    Funded by: Austrian Science Fund FWF: P 17862

    The Journal of cell biology 2008;181;4;667-81

  • Interaction of surfactant protein A with the intermediate filaments desmin and vimentin.

    Garcia-Verdugo I, Synguelakis M, Degrouard J, Franco CA, Valot B, Zivy M, Chaby R and Tanfin Z

    Institut de Biochimie et Biophysique Moléculaire et Cellulaire, UMR-8619 du CNRS, Université de Paris-Sud, 91400 Orsay, France. ignaciogarciaverdugo@yahoo.com

    Surfactant protein A (SP-A), a member of the collectin family that modulates innate immunity, has recently been involved in the physiology of reproduction. Consistent with the activation of ERK-1/2 and COX-2 induced by SP-A in myometrial cells, we reported previously the presence of two major proteins recognized by SP-A in these cells. Here we identify by mass spectrometry one of these SP-A targets as the intermediate filament (IF) desmin. In myometrial preparations derived from desmin-deficient mice, the absence of binding of SP-A to any 50 kDa protein confirmed the identity of this SP-A-binding site as desmin. Our data based on partial chymotrypsin digestion of pure desmin suggested that SP-A recognizes especially its rod domain, which is known to play an important role during the assembly of desmin into filaments. In line with that, electron microscopy experiments showed that SP-A inhibits in vitro the polymerization of desmin filaments. SP-A also recognized in vitro polymerized filaments in a calcium-dependent manner at a physiological ionic strength but not the C1q receptor gC1qR. Furthermore, Texas Red-labeled SP-A colocalized with desmin filaments in myometrial cells. Interestingly, vimentin, the IF characteristic of leukocytes, is one of the major proteins recognized by SP-A in protein extracts of U937 cells after PMA-induced differentiation of this monocytic cell line. Interaction of SP-A with vimentin was further confirmed using recombinant vimentin in solid-phase binding assays. The ability of SP-A to interact with desmin and vimentin, and to prevent polymerization of desmin monomers, shed light on unexpected and wider biological roles of this collectin.

    Biochemistry 2008;47;18;5127-38

  • Tissue distribution and subcellular localization of the cardiac sodium channel during mouse heart development.

    Domínguez JN, de la Rosa A, Navarro F, Franco D and Aránega AE

    Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Paraje de las Lagunillas, s/n, Jaén 23071, Spain.

    Aims: The aim of this study was to analyse the mRNA expression levels and protein distribution of the cardiac sodium channel Scn5a/Nav1.5 during mouse cardiogenesis.

    Scn5a mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E17.5 as well as postnatal and adult hearts. In addition, Scn5a protein (Nav1.5) distribution was analysed by immunohistochemistry and confocal microscopy. Scn5a mRNA levels displayed a peak at stage E11.5, decreased during the subsequent stages and then steadily increased from E17.5 onwards, and throughout the postnatal to the adult stages. Immunohistochemistry experiments revealed comparable distribution of Nav1.5 between the different cardiac chambers at early embryonic stages. During the foetal stages, Nav1.5 showed an enhanced expression in the trabeculated myocardium and in the bundle branches. At the subcellular level, Nav1.5 and Scn1b double-immunostaining analysis is consistent with the presence of both sodium channel subunits in the T-tubule system and the intercalated discs.

    Conclusion: Our results demonstrate that the cardiac sodium channel, Nav1.5, shows a dynamic expression pattern during mouse heart development, indicating that it could play an important role in the acquisition of a mature pattern of conduction and contraction during cardiogenesis.

    Cardiovascular research 2008;78;1;45-52

  • Bmp in podocytes is essential for normal glomerular capillary formation.

    Ueda H, Miyazaki Y, Matsusaka T, Utsunomiya Y, Kawamura T, Hosoya T and Ichikawa I

    Division of Kidney and Hypertension, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8, Nishishinbashi, Minato-ku, Tokyo, Japan. hi-ro@jikei.ac.jp

    Bone morphogenetic protein (BMP) 4 exerts multiple biological effects on kidney and ureter development. To examine the role of BMP4 in glomerular morphogenesis, we generated transgenic mice with altered BMP4 function in podocytes by conferring tissue-specificity with the nephrin (Nphs1) promoter. At birth, Tg(Nphs1-Nog) mice, which had loss of BMP4 function in podocytes, were found to have glomerular microaneurysms, collapsed glomerular capillary tufts, enlarged Bowman's capsules, and fewer normal proximal tubules. Conversely, Tg(Nphs1-Bmp4) mice, which had increased BMP4 function in podocytes, demonstrated defects in glomerular capillary formation, but podocytes were not appreciably affected. The Tg(Nphs1-Nog) and Tg(Nphs1-Bmp4) mice shared morphological characteristics with the previously reported podocyte-specific Vegf-A over-expressing and knockout mice, respectively. Consistent with the morphological similarity, in situ hybridization revealed an intense signal for podocyte expression of Vegf in Tg(Nphs1-Nog) mice, whereas the signal was markedly suppressed in Tg(Nphs1-Bmp4) mice. However, in vitro studies with metanephroi failed to demonstrate a direct interaction between BMP4 or Noggin and VEGF in podocytes. Instead, immunostaining showed that phosphorylated Smads, the mediators of BMP signaling, are present in endothelial and/or mesangial cells, but not in podocytes, within the developing glomeruli. Therefore, this study suggests that podocyte-derived BMP plays an important role in glomerular capillary formation, perhaps by acting on non-podocyte glomerular cells in a paracrine fashion.

    Journal of the American Society of Nephrology : JASN 2008;19;4;685-94

  • Contribution of neural crest-derived cells in the embryonic and adult thymus.

    Foster K, Sheridan J, Veiga-Fernandes H, Roderick K, Pachnis V, Adams R, Blackburn C, Kioussis D and Coles M

    Molecular Immunology, National Institute for Medical Research, Edinburgh, UK.

    Neural crest (NC)-derived mesenchyme has previously been shown to play an important role in the development of fetal thymus. Using Wnt1-Cre and Sox10-Cre mice crossed to Rosa26(eYfp) reporter mice, we have revealed NC-derived mesenchymal cells in the adult murine thymus. We report that NC-derived cells infiltrate the thymus before day 13.5 of embryonic development (E13.5) and differentiate into cells with characteristics of smooth muscle cells associated with large vessels, and pericytes associated with capillaries. In the adult organ at 3 mo of age, these NC-derived perivascular cells continue to be associated with the vasculature, providing structural support to the blood vessels and possibly regulating endothelial cell function.

    Funded by: Cancer Research UK; Medical Research Council

    Journal of immunology (Baltimore, Md. : 1950) 2008;180;5;3183-9

  • Targeted deletion of Dicer in the heart leads to dilated cardiomyopathy and heart failure.

    Chen JF, Murchison EP, Tang R, Callis TE, Tatsuguchi M, Deng Z, Rojas M, Hammond SM, Schneider MD, Selzman CH, Meissner G, Patterson C, Hannon GJ and Wang DZ

    Carolina Cardiovascular Biology Center, Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599, USA.

    Cardiovascular disease is the leading cause of human morbidity and mortality. Dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy associated with heart failure. Here, we report that cardiac-specific knockout of Dicer, a gene encoding a RNase III endonuclease essential for microRNA (miRNA) processing, leads to rapidly progressive DCM, heart failure, and postnatal lethality. Dicer mutant mice show misexpression of cardiac contractile proteins and profound sarcomere disarray. Functional analyses indicate significantly reduced heart rates and decreased fractional shortening of Dicer mutant hearts. Consistent with the role of Dicer in animal hearts, Dicer expression was decreased in end-stage human DCM and failing hearts and, most importantly, a significant increase of Dicer expression was observed in those hearts after left ventricle assist devices were inserted to improve cardiac function. Together, our studies demonstrate essential roles for Dicer in cardiac contraction and indicate that miRNAs play critical roles in normal cardiac function and under pathological conditions.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;6;2111-6

  • Abnormal heart development and lung remodeling in mice lacking the hypoxia-inducible factor-related basic helix-loop-helix PAS protein NEPAS.

    Yamashita T, Ohneda O, Nagano M, Iemitsu M, Makino Y, Tanaka H, Miyauchi T, Goto K, Ohneda K, Fujii-Kuriyama Y, Poellinger L and Yamamoto M

    Department of Regenerative Medicine, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8575, Japan.

    Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3alpha gene by alternative splicing, replacing the first exon of HIF-3alpha with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3alpha. NEPAS suppressed reporter gene expression driven by HIF-1alpha and HIF-2alpha. By generating mice with a targeted disruption of the NEPAS/HIF-3alpha locus, we found that homozygous mutant mice (NEPAS/HIF-3alpha(-)(/)(-)) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin 1 and platelet-derived growth factor beta was increased in the lung endothelial cells of NEPAS/HIF-3alpha-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1alpha and HIF-2alpha are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.

    Molecular and cellular biology 2008;28;4;1285-97

  • Human muscular fetal cells: a potential cell source for muscular therapies.

    Hirt-Burri N, de Buys Roessingh AS, Scaletta C, Gerber S, Pioletti DP, Applegate LA and Hohlfeld J

    Pediatric Surgery Laboratory, University Hospital Lausanne, CHUV, CI/02/60, Lausanne, Switzerland. Nathalie.Burri@chuv.ch

    Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.

    Pediatric surgery international 2008;24;1;37-47

  • Mice expressing L345P mutant desmin exhibit morphological and functional changes of skeletal and cardiac mitochondria.

    Kostareva A, Sjöberg G, Bruton J, Zhang SJ, Balogh J, Gudkova A, Hedberg B, Edström L, Westerblad H and Sejersen T

    Department of Woman and Child Health and Center for Molecular Medicine, Karolinska Institute, L8:02, Stockholm 17176, Sweden.

    Desmin mutations underlie inherited myopathies/cardiomyopathies with varying severity and involvement of the skeletal and cardiac muscles. We developed a transgenic mouse model expressing low level of the L345P desmin mutation (DESMUT mice) in order to uncover changes in skeletal and cardiac muscles caused by this mutation. The most striking ultrastructural changes in muscle from DESMUT mice were mitochondrial swelling and vacuolization. The mitochondrial Ca(2+) level was significantly increased in skeletal and cardiac myocytes from DESMUT mice compared to wild type cells during and after contractions. In isolated DESMUT soleus muscles, contractile function and recovery from fatigue were impaired. A SHIRPA screening test for neuromuscular performance demonstrated decreased motor function in DESMUT compared to WT mice. Echocardiographic changes in DESMUT mice included left ventricular wall hypertrophy and a decreased left ventricular chamber dimension. The results imply that low levels of L345P desmin acts, at least partially, by a dominant negative effect on mitochondria.

    Journal of muscle research and cell motility 2008;29;1;25-36

  • Wt1 and retinoic acid signaling are essential for stellate cell development and liver morphogenesis.

    Ijpenberg A, Pérez-Pomares JM, Guadix JA, Carmona R, Portillo-Sánchez V, Macías D, Hohenstein P, Miles CM, Hastie ND and Muñoz-Chápuli R

    MRC Human Genetics Unit, Western General Hospital, Edinburgh, UK.

    Previous studies of knock-out mouse embryos have shown that the Wilms' tumor suppressor gene (Wt1) is indispensable for the development of kidneys, gonads, heart, adrenals and spleen. Using OPT (Optical Projection Tomography) we have found a new role for Wt1 in mouse liver development. In the absence of Wt1, the liver is reduced in size, and shows lobing abnormalities. In normal embryos, coelomic cells expressing Wt1, GATA-4, RALDH2 and RXRalpha delaminate from the surface of the liver, intermingle with the hepatoblasts and incorporate to the sinusoidal walls. Some of these cells express desmin, suggesting a contribution to the stellate cell population. Other cells, keeping high levels of RXRalpha immunoreactivity, are negative for stellate or smooth muscle cell markers. However, coelomic cells lining the liver of Wt1-null embryos show decreased or absent RALDH2 expression, the population of cells expressing high levels of RXRalpha is much reduced and the proliferation of hepatoblasts and RXRalpha-positive cells is significantly decreased. On the other hand, the expression of smooth muscle cell specific alpha-actin increases throughout the liver, suggesting an accelerated and probably anomalous differentiation of stellate cell progenitors. We describe a similar retardation of liver growth in RXRalpha-null mice as well as in chick embryos after inhibition of retinoic acid synthesis. We propose that Wt1 expression in cells delaminating from the coelomic epithelium is essential for the expansion of the progenitor population of liver stellate cells and for liver morphogenesis. Mechanistically, at least part of this effect is mediated via the retinoic acid signaling pathway.

    Funded by: Medical Research Council: MC_U127527180; NICHD NIH HHS: N01-HD-2-3144

    Developmental biology 2007;312;1;157-70

  • Regulation of skeletal muscle sarcomere integrity and postnatal muscle function by Mef2c.

    Potthoff MJ, Arnold MA, McAnally J, Richardson JA, Bassel-Duby R and Olson EN

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

    Myocyte enhancer factor 2 (MEF2) transcription factors cooperate with the MyoD family of basic helix-loop-helix (bHLH) transcription factors to drive skeletal muscle development during embryogenesis, but little is known about the potential functions of MEF2 factors in postnatal skeletal muscle. Here we show that skeletal muscle-specific deletion of Mef2c in mice results in disorganized myofibers and perinatal lethality. In contrast, neither Mef2a nor Mef2d is required for normal skeletal muscle development in vivo. Skeletal muscle deficient in Mef2c differentiates and forms normal myofibers during embryogenesis, but myofibers rapidly deteriorate after birth due to disorganized sarcomeres and a loss of integrity of the M line. Microarray analysis of Mef2c null muscles identified several muscle structural genes that depend on MEF2C, including those encoding the M-line-specific proteins myomesin and M protein. We show that MEF2C directly regulates myomesin gene transcription and that loss of Mef2c in skeletal muscle results in improper sarcomere organization. These results reveal a key role for Mef2c in maintenance of sarcomere integrity and postnatal maturation of skeletal muscle.

    Molecular and cellular biology 2007;27;23;8143-51

  • Proper perinuclear localization of the TRIM-like protein myospryn requires its binding partner desmin.

    Kouloumenta A, Mavroidis M and Capetanaki Y

    Cell Biology Division, Center of Basic Research, Biomedical Research Foundation Academy of Athens, Athens 11527, Greece.

    Desmin, the muscle-specific intermediate filament protein, surrounds the Z disks and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles, and the nucleus. In an attempt to explore the molecular mechanisms of these associations, we performed a yeast two-hybrid screening of a cardiac cDNA library. We showed that the desmin amino-terminal domain (N-(1-103)) binds to a 413-kDa TRIM-like protein, myospryn, originally identified as the muscle-specific partner of dysbindin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1). Binding of desmin with myospryn was confirmed with glutathione S-transferase pulldown assays and coimmunoprecipitation experiments. Western blot analysis revealed that the complex immunoprecipitated by desmin antibodies, in addition to myospryn, contained the BLOC-1 components dysbindin and pallidin. Deletion analysis revealed that only the (N-(1-103)) fragment of desmin binds to myospryn carboxyl terminus and that this association takes place through the 24-amino acid-long carboxyl-terminal end of the SPRY domain of myospryn. Using an antibody against the COOH terminus of myospryn, we demonstrated that myospryn colocalizes with desmin at the periphery of the nucleus, in close proximity to the endoplasmic reticulum, of mouse neonatal cardiomyocytes. In adult heart muscle, the two proteins colocalize, predominantly at intercalated disks and costameres. We also showed that myospryn colocalizes with lysosomes. Using desmin null hearts, we determined that desmin is required for both the proper perinuclear localization of myospryn, as well as the proper positioning of lysosomes, thus suggesting a potential role of desmin intermediate filaments in lysosomes and lysosome-related organelle biogenesis and/or positioning.

    The Journal of biological chemistry 2007;282;48;35211-21

  • Dmrt2 and Pax3 double-knockout mice show severe defects in embryonic myogenesis.

    Seo KW

    Department of Biochemistry and Molecular Biology, MD Anderson Cancer Center, Houston, TX, USA. kwangwons@yahoo.com

    Myogenesis is one of the critical developmental processes in mammals. Several transcription factors from the dermomyotome affect embryonic myogenesis. Among these, Dmrt2 and Pax3 were tested for genetic and functional interactions during embryonic myogenesis by evaluating myogenin and desmin expression patterns in Dmrt2-Pax3 mutant mouse embryos. In doubly homozygous mutant embryos, myogenin expression was reduced, and the expression pattern was altered dramatically. In Pax3-knockout mouse embryos, the pattern of Dmrt2 expression was altered, suggesting that Pax3 is important in maintaining the epaxial dermomyotome. Even though Pax3 and Dmrt2 are expressed in similar tissue- and developmental-stage-specific manners during dermomyotomal development, they appear to have independent roles in mammalian myogenesis. The processes characteristic of embryonic myogenesis are similar to those occurring during muscle regeneration in adults. Therefore, these results may provide insight into the pathogenesis of innate muscular dystrophy and may lead to the development of drugs to promote muscle repair after injury.

    Comparative medicine 2007;57;5;460-8

  • Desmin stimulates differentiation of cardiomyocytes and up-regulation of brachyury and nkx2.5.

    Hofner M, Höllrigl A, Puz S, Stary M and Weitzer G

    Max F. Perutz Laboratories, Department of Medical Biochemistry, Medical University of Vienna, Dr. Bohrgasse 9, A1030 Vienna, Austria.

    Desmin contributes to structural integrity and function of the myocardium but its function seems to be redundant in early cardiomyogenesis in the desmin null mouse model. To test the hypothesis that desmin also plays a supportive role in cardiomyogenic commitment and early differentiation of cardiomyocytes we investigated cardiomyogenesis in embryoid bodies expressing different desmin alleles. Constitutive expression of desmin and increased synthesis during mesoderm formation led to the up-regulation of brachyury and nkx2.5 genes, accelerated early cardiomyogenesis and resulted in the development of large, proliferating, highly interconnected, and synchronously beating cardiomyocyte clusters, whereas desmin null cardiomyocytes featured an opposite phenotype. In contrast, constitutive expression of amino-terminally truncated desmin(Delta1-48) interfered with the beginning of cardiomyogenesis, caused down-regulation of mesodermal and myocardial transcription factors, and hampered myofibrillogenesis and survival of cardiomyocytes. These results provide first evidence that a type III intermediate filament protein takes part in regulating the differentiation of mesoderm to cardiomyocytes at the very beginning of cardiomyogenesis.

    Funded by: Austrian Science Fund FWF: P 18659-B12

    Differentiation; research in biological diversity 2007;75;7;605-15

  • ROCK2 and its alternatively spliced isoform ROCK2m positively control the maturation of the myogenic program.

    Pelosi M, Marampon F, Zani BM, Prudente S, Perlas E, Caputo V, Cianetti L, Berno V, Narumiya S, Kang SW, Musarò A and Rosenthal N

    EMBL Mouse Biology Unit, Campus Buzzati-Traverso, via Ramarini 32, 00016 Monterotondo-Scalo, Roma, Italy. mpelosi@embl-monterotondo.it

    Signal transduction cascades involving Rho-associated kinases (ROCK), the serine/threonine kinases downstream effectors of Rho, have been implicated in the regulation of diverse cellular functions including cytoskeletal organization, cell size control, modulation of gene expression, differentiation, and transformation. Here we show that ROCK2, the predominant ROCK isoform in skeletal muscle, is progressively up-regulated during mouse myoblast differentiation and is highly expressed in the dermomyotome and muscle precursor cells of mouse embryos. We identify a novel and evolutionarily conserved ROCK2 splicing variant, ROCK2m, that is preferentially expressed in skeletal muscle and strongly up-regulated during in vivo and in vitro differentiation processes. The specific knockdown of ROCK2 or ROCK2m expression in C2C12 myogenic cells caused a significant and selective impairment of the expression of desmin and of the myogenic regulatory factors Mrf4 and MyoD. We demonstrate that in myogenic cells, ROCK2 and ROCK2m are positive regulators of the p42 and p44 mitogen-activated protein kinase-p90 ribosomal S6 kinase-eucaryotic elongation factor 2 intracellular signaling pathways and, thereby, positively regulate the hypertrophic effect elicited by insulin-like growth factor 1 and insulin, linking the multifactorial functions of ROCK to an important control of the myogenic maturation.

    Molecular and cellular biology 2007;27;17;6163-76

  • Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption.

    Huang L, Min JN, Masters S, Mivechi NF and Moskophidis D

    Center for Molecular Chaperones/Radiobiology and Cancer Virology, Medical College of Georgia, 1410 Laney Walker Blvd, CN3143, Augusta, GA 30912, USA.

    The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.

    Funded by: NCI NIH HHS: CA062130, CA121951, CA132640

    Genesis (New York, N.Y. : 2000) 2007;45;8;487-501

  • Crim1KST264/KST264 mice implicate Crim1 in the regulation of vascular endothelial growth factor-A activity during glomerular vascular development.

    Wilkinson L, Gilbert T, Kinna G, Ruta LA, Pennisi D, Kett M and Little MH

    Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia 4072.

    Crim1, a transmembrane cysteine-rich repeat-containing protein that is related to chordin, plays a role in the tethering of growth factors at the cell surface. Crim1 is expressed in the developing kidney; in parietal cells, podocytes, and mesangial cells of the glomerulus; and in pericytes that surround the arterial vasculature. A gene-trap mouse line with an insertion in the Crim1 gene (Crim1(KST264/KST264)) displayed perinatal lethality with defects in multiple organ systems. This study further analyzed the defects that are present within the kidneys of these mice. Crim1(KST264/KST264) mice displayed abnormal glomerular development, illustrated by enlarged capillary loops, podocyte effacement, and mesangiolysis. When outbred, homozygotes that reached birth displayed podocyte and glomerular endothelial cell defects and marked albuminuria. The podocytic co-expression of Crim1 with vascular endothelial growth factor-A (VEGF-A) suggested a role for Crim1 in the regulation of VEGF-A action. Crim1 and VEGF-A were shown to interact directly, providing evidence that cysteine-rich repeat-containing proteins can bind to non-TGF-beta superfamily ligands. Crim1(KST264/KST264) mice display a mislocalization of VEGF-A within the developing glomerulus, as assessed by immunogold electron microscopy and increased activation of VEGF receptor 2 (Flk1) in the glomerular endothelial cells, suggesting that Crim1 regulates the delivery of VEGF-A by the podocytes to the endothelial cells. This is the first in vivo demonstration of regulation of VEGF-A delivery and supports the hypothesis that Crim1 functions to regulate the release of growth factors from the cell of synthesis.

    Journal of the American Society of Nephrology : JASN 2007;18;6;1697-708

  • Identification of skeletal muscle mutations in tail snips from neonatal mice using immunohistochemistry.

    Lovering RM, O'Neill A, Roche JA and Bloch RJ

    Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA. rlovering@som.umaryland.edu

    BioTechniques 2007;42;6;702, 704

  • Exercise reverses preamyloid oligomer and prolongs survival in alphaB-crystallin-based desmin-related cardiomyopathy.

    Maloyan A, Gulick J, Glabe CG, Kayed R and Robbins J

    Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.

    The R120G mutation in the small heat shock-like protein alphaB-crystallin (CryAB(R120G)) causes desmin-related myopathy (DRM), which is characterized by the formation of desmin- and CryAB-containing aggregates within muscle fibers. Mice with cardiac-specific overexpression of CryAB(R120G) develop cardiomyopathy at 3 months and die at 6-7 months from heart failure (HF). Previous studies showed that overexpression of CryAB(R120G) results in accumulation of preamyloid oligomer (PAO). PAO is considered to be the cytotoxic entity in many of the protein misfolding-based neurodegenerative diseases. On the basis of data from mouse models of neurodegenerative diseases showing that exercise or environmental enrichment reduces the amyloid oligomer level and improves cognitive ability, we hypothesized that CryAB(R120G)-induced DRM would also respond favorably to prolonged voluntary exercise, reducing HF symptoms and rescuing the mice from premature death. Six months of voluntary exercise in CryAB(R120G) animals resulted in 100% survival at a time when all unexercised mice had died. After 22 weeks of exercise, PAO levels were decreased by 47% compared with the unexercised CryAB(R120G) control mice (P = 0.00001). Although CryAB(R120G) expression led to decreased levels of the metallomembrane endopeptidase neprilysin, normal levels were maintained in the exercised CryAB(R120G) mice, and in vitro loss-of-function and gain-of-function experiments using adenovirus-infected cardiomyocytes confirmed the importance of neprilysin in ameliorating PAO accumulation. The data demonstrate that voluntary exercise slows the progression to HF in the CryAB(R120G) DRM model and that PAO accumulation is mediated, at least in part, by decreased neprilysin activity.

    Funded by: NHLBI NIH HHS: HL52318, HL56370, HL60546, P50 HL052318, R01 HL056370, R01 HL69799

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;14;5995-6000

  • Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box M1 transcription factor.

    Ramakrishna S, Kim IM, Petrovic V, Malin D, Wang IC, Kalin TV, Meliton L, Zhao YY, Ackerson T, Qin Y, Malik AB, Costa RH and Kalinichenko VV

    Department of Medicine, University of Chicago, Chicago, Illinois.

    The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 -/- mouse line to demonstrate that Foxm1-deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in Foxm1 -/- hearts were associated with a reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21(Cip1) protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in Tie2-Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the -9,259/-9,288-bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxm1 regulates expression of genes essential for the proliferation of cardiomyocytes during heart development.

    Funded by: NHLBI NIH HHS: HL 84151-01

    Developmental dynamics : an official publication of the American Association of Anatomists 2007;236;4;1000-13

  • RBP-J (Rbpsuh) is essential to maintain muscle progenitor cells and to generate satellite cells.

    Vasyutina E, Lenhard DC, Wende H, Erdmann B, Epstein JA and Birchmeier C

    Max Delbrück Center for Molecular Medicine, Robert Rössle Strasse 10, 13125 Berlin, Germany.

    In the developing muscle, a pool of myogenic progenitor cells is formed and maintained. These resident progenitors provide a source of cells for muscle growth in development and generate satellite cells in the perinatal period. By the use of conditional mutagenesis in mice, we demonstrate here that the major mediator of Notch signaling, the transcription factor RBP-J, is essential to maintain this pool of progenitor cells in an undifferentiated state. In the absence of RBP-J, these cells undergo uncontrolled myogenic differentiation, leading to a depletion of the progenitor pool. This results in a lack of muscle growth in development and severe muscle hypotrophy. In addition, satellite cells are not formed late in fetal development in conditional RBP-J mutant mice. We conclude that RBP-J is required in the developing muscle to set aside proliferating progenitors and satellite cells.

    Funded by: NHLBI NIH HHS: P01 HL 075215, P01 HL075215

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;11;4443-8

  • Fetal muscle-derived cells can repair dystrophic muscles in mdx mice.

    Auda-Boucher G, Rouaud T, Lafoux A, Levitsky D, Huchet-Cadiou C, Feron M, Guevel L, Talon S, Fontaine-Pérus J and Gardahaut MF

    CNRS UMR 6204, Faculté des Sciences et des Techniques, 44322 Nantes Cedex 3, France.

    We have previously reported that CD34(+) cells purified from mouse fetal muscles can differentiate into skeletal muscle in vitro and in vivo when injected into muscle tissue of dystrophic mdx mice. In this study, we investigate the ability of such donor cells to restore dystrophin expression, and to improve the functional muscle capacity of the extensor digitorum longus muscle (EDL) of mdx mice. For this purpose green fluorescent-positive fetal GFP(+)/CD34(+) cells or desmin(+)/(-)LacZ/CD34(+) cells were transplanted into irradiated or non-irradiated mdx EDL muscle. Donor fetal muscle-derived cells predominantly fused with existing fibers. Indeed more than 50% of the myofibers of the host EDL contained donor nuclei delivering dystrophin along 80-90% of the length of their sarcolemma. The presence of significant amounts of dystrophin (about 60-70% of that found in a control wild-type mouse muscle) was confirmed by Western blot analyses. Dystrophin expression also outcompeted that of utrophin, as revealed by a spatial shift in the distribution of utrophin. At 1 month post-transplant, the recipient muscle appeared to have greater resistance to fatigue than control mdx EDL muscle during repeated maximal contractions.

    Experimental cell research 2007;313;5;997-1007

  • Molecular pathway for the localized formation of the sinoatrial node.

    Mommersteeg MT, Hoogaars WM, Prall OW, de Gier-de Vries C, Wiese C, Clout DE, Papaioannou VE, Brown NA, Harvey RP, Moorman AF and Christoffels VM

    Center for Heart Failure Research, Academic Medical Centre, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands.

    The sinoatrial node, which resides at the junction of the right atrium and the superior caval vein, contains specialized myocardial cells that initiate the heart beat. Despite this fundamental role in heart function, the embryonic origin and mechanisms of localized formation of the sinoatrial node have not been defined. Here we show that subsequent to the formation of the Nkx2-5-positive heart tube, cells bordering the inflow tract of the heart tube give rise to the Nkx2-5-negative myocardial cells of the sinoatrial node and the sinus horns. Using genetic models, we show that as the myocardium of the heart tube matures, Nkx2-5 suppresses pacemaker channel gene Hcn4 and T-box transcription factor gene Tbx3, thereby enforcing a progressive confinement of their expression to the forming Nkx2-5-negative sinoatrial node and sinus horns. Thus, Nkx2-5 is essential for establishing a gene expression border between the atrium and sinoatrial node. Tbx3 was found to suppress chamber differentiation, providing an additional mechanism by which the Tbx3-positive sinoatrial node is shielded from differentiating into atrial myocardium. Pitx2c-deficient fetuses form sinoatrial nodes with indistinguishable molecular signatures at both the right and left sinuatrial junction, indicating that Pitx2c functions within the left/right pathway to suppress a default program for sinuatrial node formation on the left. Our molecular pathway provides a mechanism for how pacemaker activity becomes progressively relegated to the most recently added components of the venous pole of the heart and, ultimately, to the junction of the right atrium and superior caval vein.

    Funded by: NICHD NIH HHS: HD047858, R01 HD33082

    Circulation research 2007;100;3;354-62

  • Association between the muscle-specific proteins desmin and caveolin-3 in muscle cells.

    Mermelstein CS, Martins ER, Portilho DM and Costa ML

    Departamento de Histologia e Embriologia, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. mermelstein@ufrj.br

    The muscle-specific intermediate filament protein desmin is expressed in mononucleated myoblasts and in differentiated myotubes. Desmin has been shown to associate with the sarcolemma in specific structures, such as neuromuscular junctions and the dystrophin-associated protein complex. Since these are specialized membrane regions, the study of a possible association between desmin and liquid-ordered membrane microdomains is of particular interest. We have carried out an analysis of the association between desmin and the muscle-specific protein caveolin-3, a major component of caveolar microdomains. Our results demonstrate that (1) desmin precisely co-localizes with caveolin-3 in myoblasts and multinucleated myotubes, (2) caveolin-3 is up-regulated during in vitro chick muscle development, (3) desmin is detectable in caveolae-enriched membrane fractions prepared from skeletal muscle, and (4) caveolin-3 co-immunoprecipitates with desmin. We have thus shown, for the first time, an association between the intermediate filament protein desmin and caveolin-3 in myogenic cells.

    Cell and tissue research 2007;327;2;343-51

  • Premature myogenic differentiation and depletion of progenitor cells cause severe muscle hypotrophy in Delta1 mutants.

    Schuster-Gossler K, Cordes R and Gossler A

    Institute for Molecular Biology, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, Hannover, D-30625 Lower Saxony, Germany.

    In vertebrates, skeletal myogenesis is initiated by the generation of myoblasts followed by their differentiation to myocytes and the formation of myofibers. The determination of myoblasts and their differentiation are controlled by muscle regulatory factors that are activated at specific stages during myogenesis. During late embryonic and fetal stages a distinct population of resident proliferating progenitor cells is the major source of myogenic cells. How the differentiation of myoblasts and progenitor cells is regulated is not clear. We show that in mouse embryos the Notch ligand Delta1 (Dll1) controls both differentiation of early myoblasts and maintenance of myogenic progenitor cells. Early dermomyotome-derived myoblasts are determined normally in Dll1 mutant embryos, but their differentiation is accelerated, leading to a transient excess of myotomal muscle fibers. Similarly, migratory hypaxial myogenic cells colonize the limb buds and activate muscle regulatory factor expression normally, but muscle differentiation progresses more rapidly. Resident progenitor cells defined by Pax3/Pax7 expression are formed initially, but they are progressively lost and virtually absent at embryonic day 14.5. Muscle growth declines beginning around embryonic day 12, leading to subsequent severe muscle hypotrophy in hypomorphic Dll1 fetuses. We suggest that premature and excessive differentiation leads to depletion of progenitor cells and cessation of muscle growth, and we conclude that Dll1 provides essential signals that are required to prevent uncontrolled differentiation early and ensure sustained muscle differentiation during development.

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;2;537-42

  • PDGF signaling specificity is mediated through multiple immediate early genes.

    Schmahl J, Raymond CS and Soriano P

    Program in Developmental Biology and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109, USA.

    Growth factor signaling leads to the induction or repression of immediate early genes, but how these genes act collectively as effectors of downstream processes remains unresolved. We have used gene trap-coupled microarray analysis to identify and mutate multiple platelet-derived growth factor (PDGF) intermediate early genes in mice. Mutations in these genes lead to a high frequency of phenotypes that affect the same cell types and processes as those controlled by the PDGF pathway. We conclude that these genes form a network that controls specific processes downstream of PDGF signaling.

    Funded by: NCI NIH HHS: CA09657; NICHD NIH HHS: HD24875, R01 HD024875, R37 HD025326; NIGMS NIH HHS: GM071158

    Nature genetics 2007;39;1;52-60

  • Blood vessels and desmin control the positioning of nuclei in skeletal muscle fibers.

    Ralston E, Lu Z, Biscocho N, Soumaka E, Mavroidis M, Prats C, Lømo T, Capetanaki Y and Ploug T

    Office of Science and Technology, National Institute of Arthritis, Musculoskeletal and Skin Diseases/NIH, Bethesda, MD 20892, USA. ralstone@mail.nih.gov

    Skeletal muscle fibers contain hundreds to thousands of nuclei which lie immediately under the plasmalemma and are spaced out along the fiber, except for a small cluster of specialized nuclei at the neuromuscular junction. How the nuclei attain their positions along the fiber is not understood. Here we show that the nuclei are preferentially localized near blood vessels (BV), particularly in slow-twitch, oxidative fibers. Thus, in rat soleus muscle fibers, 81% of the nuclei appear next to BV. Lack of desmin markedly perturbs the distribution of nuclei along the fibers but does not prevent their close association with BV. Consistent with a role for desmin in the spacing of nuclei, we show that denervation affects the organization of desmin filaments as well as the distribution of nuclei. During chronic stimulation of denervated muscles, new BV form, along which muscle nuclei align themselves. We conclude that the positioning of nuclei along muscle fibers is plastic and that BV and desmin intermediate filaments each play a distinct role in the control of this positioning.

    Funded by: NIAMS NIH HHS: AR 39617

    Journal of cellular physiology 2006;209;3;874-82

  • Fibroblast growth factor 10 is required for survival and proliferation but not differentiation of intestinal epithelial progenitor cells during murine colon development.

    Sala FG, Curtis JL, Veltmaat JM, Del Moral PM, Le LT, Fairbanks TJ, Warburton D, Ford H, Wang K, Burns RC and Bellusci S

    UMR144-CNRS/Institut Curie, 75248 Paris cedex 05, France.

    Epithelial-mesenchymal interactions that govern the development of the colon from the primitive gastrointestinal tract are still unclear. In this study, we determine the temporal-spatial expression pattern of Fibroblast growth factor 10 (Fgf10), a key developmental gene, in the colon at different developmental stages. We found that Fgf10 is expressed in the mesenchyme of the distal colon, while its main receptor Fgfr2-IIIb is expressed throughout the entire intestinal epithelium. We demonstrate that Fgf10 inactivation leads to decreased proliferation and increased cell apoptosis in the colonic epithelium at E10.5, therefore resulting in distal colonic atresia. Using newly described Fgf10 hypomorphic mice, we show that high levels of FGF10 are dispensable for the differentiation of the colonic epithelium. Our work unravels for the first time the pivotal role of FGF10 in the survival and proliferation of the colonic epithelium, biological activities which are essential for colonic crypt formation.

    Developmental biology 2006;299;2;373-85

  • Transgenic mice expressing PAX3-FKHR have multiple defects in muscle development, including ectopic skeletal myogenesis in the developing neural tube.

    Finckenstein FG, Davicioni E, Osborn KG, Cavenee WK, Arden KC and Anderson MJ

    Childrens Hospital Los Angeles Research Institute, Los Angeles, CA 90027, USA.

    The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of alveolar RMS by influencing the commitment and differentiation of the myogenic cell lineage.

    Transgenic research 2006;15;5;595-614

  • Impact of disease mutations on the desmin filament assembly process.

    Bär H, Mücke N, Ringler P, Müller SA, Kreplak L, Katus HA, Aebi U and Herrmann H

    Department of Cardiology, University of Heidelberg, D-69120 Heidelberg, Germany.

    It has been documented that mutations in the human desmin gene lead to a severe type of myofibrillar myopathy, termed more specifically desminopathy, which affects cardiac and skeletal as well as smooth muscle. We showed recently that 14 recombinant versions of these disease-causing desmin variants, all involving single amino acid substitutions in the alpha-helical rod domain, interfere with in vitro filament formation at distinct stages of the assembly process. We now provide mechanistic details of how these mutations affect the filament assembly process by employing analytical ultracentrifugation, time-lapse electron microscopy of negatively stained and glycerol-sprayed/low-angle rotary metal-shadowed samples, quantitative scanning transmission electron microscopy, and viscometric studies. In particular, the soluble assembly intermediates of two of the mutated proteins exhibit unusually high s-values, compatible with octamers and other higher-order complexes. Moreover, several of the six filament-forming mutant variants deviated considerably from wild-type desmin with respect to their filament diameters and mass-per-length values. In the heteropolymeric situation with wild-type desmin, four of the mutant variants caused a pronounced "hyper-assembly", when assayed by viscometry. This indicates that the various mutations may cause abortion of filament formation by the mutant protein at distinct stages, and that some of them interfere severely with the assembly of wild-type desmin. Taken together, our findings provide novel insights into the basic intermediate filament assembly mechanisms and offer clues as to how amino acid changes within the desmin rod domain may interfere with the normal structural organization of the muscle cytoskeleton, eventually leading to desminopathy.

    Journal of molecular biology 2006;360;5;1031-42

  • Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development.

    Orii KE, Lee Y, Kondo N and McKinnon PJ

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

    The repair of DNA double-strand breaks (DSBs) occurs via nonhomologous end-joining (NHEJ) or homologous recombination (HR). These mechanistically distinct pathways are critical for maintenance of genomic integrity and organismal survival. Although inactivation of either pathway leads to embryonic lethality, here we show selective requirements for each DNA DSB repair pathway at different stages of mammalian nervous system development. DNA damage-induced apoptosis resulting from inactivation of HR (Xrcc2 deficiency) only occurred in proliferating neural precursor cells, whereas disruption of NHEJ (DNA ligase IV deficiency) mainly affected differentiating cells at later developmental stages. Therefore, these data suggest that NHEJ is dispensable for a substantial portion of early development because DSB repair during this period utilizes HR. Moreover, DNA damage-induced apoptosis required the ataxia telangiectasia mutated (Atm) kinase after disruption of NHEJ, but not HR, during neurogenesis. However, embryonic lethality arising from disruption of either repair pathway was rescued by loss of p53 and resulted in specific tumor types reflective of the particular DSB repair pathway inactivated. Thus, these data reveal distinct tissue- and cell-type requirements for each DNA DSB repair pathway during neural development and provide insights for understanding the contributions of DNA DSB responses to disease.

    Funded by: NCI NIH HHS: CA 21765, P30 CA 21765, P30 CA021765; NINDS NIH HHS: NS 37956, R01 NS037956, R56 NS037956

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;26;10017-22

  • Desmin modulates lung elastic recoil and airway responsiveness.

    Shardonofsky FR, Capetanaki Y and Boriek AM

    University of Texas Southwestern Medical Center, Department of Pediatrics, 5323 Harry Hines Blvd., Dallas, TX 75390-9063, USA. felix.shardonofsky@utsouthwestern.edu

    Desmin is a structural protein that is expressed in smooth muscle cells of both airways and alveolar ducts. Therefore, desmin could be well situated to participate in passive and contractile force transmission in the lung. We hypothesized that desmin modulates lung compliance, lung recoil pressure, and airway contractile response. To test this hypothesis, respiratory system complex impedance (Zin,rs) at different positive end-expiratory pressure (PEEP) levels and quasi-static pressure-volume data were obtained in desmin-null and wild-type mice at baseline and during methacholine administration. Airways and lung tissue properties were partitioned by fitting Zin,rs to a constant-phase model. Relative to controls, desmin-null mice showed 1) lower values for lung stiffness and recoil pressure at baseline and induced airway constriction, 2) greater negative PEEP dependence of H and airway resistance under baseline conditions and cholinergic stimulation, and 3) airway hyporesponsiveness. These results demonstrate that desmin is a load-bearing protein that stiffens the airways and consequently the lung and modulates airway contractile response.

    Funded by: NHLBI NIH HHS: HL 02839, HL 63134

    American journal of physiology. Lung cellular and molecular physiology 2006;290;5;L890-6

  • Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo.

    Bajanca F, Luz M, Raymond K, Martins GG, Sonnenberg A, Tajbakhsh S, Buckingham M and Thorsteinsdóttir S

    Department of Animal Biology and Centre for Environmental Biology, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal.

    We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogenin-negative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5(nlacZ/nlacZ) (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the alpha6beta1 integrin, a laminin receptor, suggesting that integrin alpha6beta1-laminin interactions are required for myotomal laminin matrix assembly. Blocking alpha6beta1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5(nlacZ/nlacZ) embryos. Furthermore, inhibition of alpha6beta1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that alpha6beta1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining alpha6beta1 expression on myogenic precursor cells, and that alpha6beta1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by alpha6beta1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.

    Development (Cambridge, England) 2006;133;9;1635-44

  • Large-scale identification of genes implicated in kidney glomerulus development and function.

    Takemoto M, He L, Norlin J, Patrakka J, Xiao Z, Petrova T, Bondjers C, Asp J, Wallgard E, Sun Y, Samuelsson T, Mostad P, Lundin S, Miura N, Sado Y, Alitalo K, Quaggin SE, Tryggvason K and Betsholtz C

    Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large-scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non-glomerular kidney tissue, FACS-sorted podocytes and brain capillaries, which identified over 300 glomerular cell-enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus-enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.

    The EMBO journal 2006;25;5;1160-74

  • Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1.

    Ohkawa Y, Marfella CG and Imbalzano AN

    Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

    Myogenin is required not for the initiation of myogenesis but instead for skeletal muscle formation through poorly understood mechanisms. We demonstrate in cultured cells and, for the first time, in embryonic tissue, that myogenic late genes that specify the skeletal muscle phenotype are bound by MyoD prior to the initiation of gene expression. At the onset of muscle specification, a transition from MyoD to myogenin occurred at late gene loci, concomitant with loss of HDAC2, the appearance of both the Mef2D regulator and the Brg1 chromatin-remodeling enzyme, and the opening of chromatin structure. We further demonstrated that ectopic expression of myogenin and Mef2D, in the absence of MyoD, was sufficient to induce muscle differentiation in a manner entirely dependent on Brg1. These results indicate that myogenin specifies the muscle phenotype by cooperating with Mef2D to recruit an ATP-dependent chromatin-remodeling enzyme that alters chromatin structure at regulatory sequences to promote terminal differentiation.

    Funded by: NIGMS NIH HHS: GM56244, R01 GM056244

    The EMBO journal 2006;25;3;490-501

  • Targeted disruption of the DM domain containing transcription factor Dmrt2 reveals an essential role in somite patterning.

    Seo KW, Wang Y, Kokubo H, Kettlewell JR, Zarkower DA and Johnson RL

    Department of Biochemistry and Molecular Biology, University of Texas, MD Anderson Cancer Center, Houston, 77030, USA.

    Dmrt2 is expressed in the dermomyotome of developing vertebrate somites. To determine the role of Dmrt2 during mouse embryonic development, we generated a null mutation of Dmrt2 via homologous recombination in embryonic stem cells. Dmrt2 heterozygous mice derived from these cells are phenotypically normal. However, Dmrt2 homozygotes die soon after birth. The cause of death is likely due to abnormal rib and sternal development, leading to an inability to breathe. Loss of Dmrt2 leads to embryonic somite patterning defects, first evidenced at embryonic day (E) 10.5 and more pronounced by E11.5. Notably, both the dermomyotome and myotome fail to adopt a normal epithelial morphology in the absence of Dmrt2. Accompanying these morphological defects are alterations in the expression patterns of dermomyotomal and myotomal transcription factors including Pax3, Paraxis, Myf5, myogenin, Mrf4 and MyoD. Despite these defects, embryos harvested from E13.5 onwards exhibited relatively normal muscle pattern and mass, suggesting that early myotomal defects are corrected by a Dmrt2-independent mechanism. Taken together, our results define an essential function for Dmrt2 in somite development and provide evidence that DM domain genes have been co-opted into other critical developmental pathways distinct from that of sex determination or differentiation.

    Developmental biology 2006;290;1;200-10

  • Impairment of the ubiquitin-proteasome system in desminopathy mouse hearts.

    Liu J, Chen Q, Huang W, Horak KM, Zheng H, Mestril R and Wang X

    Cardiovascular Research Institute, South Dakota Health Research Foundation, University of South Dakota School of Medicine and Sioux Valley Hospitals and Health System, Sioux Falls, South Dakota 57105, USA.

    Protein misfolding and aberrant aggregation are associated with many severe disorders, such as neural degenerative diseases, desmin-related myopathy (DRM), and congestive heart failure. Intrasarcoplasmic amyloidosis and increased ubiquitinated proteins are observed in human failing hearts. The pathogenic roles of these derangements in the heart remain unknown. The ubiquitin-proteasome system (UPS) plays a central role in intracellular proteolysis and regulates critical cellular processes. In cultured cells, aberrant aggregation by a mutant (MT) or misfolded protein impairs the UPS. However, this has not been demonstrated in intact animals, and it is unclear how the UPS is impaired. Cross-breeding UPS reporter mice with a transgenic mouse model of DRM featured by aberrant protein aggregation in cardiomyocytes, we found that overexpression of MT-desmin but not normal desmin protein impairs UPS proteolytic function in the heart. The primary defect does not appear to be in the ubiquitination or the proteolytic activity of the 20S proteasome, because ubiquitinated proteins and the peptidase activities of 20S proteasomes were significantly increased rather than decreased in the DRM heart. Therefore, the defect resides apparently in the entry of ubiquitinated proteins into the 20S proteasome. Consistent with this notion, key components (Rpt3 and Rpt5) of 19S proteasomes were markedly decreased, while major components of 20S proteasomes were increased. Additional experiments with HEK cells suggest that proteasomal malfunction observed in MT-desmin hearts is not secondary to cardiac malfunction or to disruption of desmin filaments. Thus, UPS impairment may represent an important pathogenic mechanism underlying cardiac disorders with abnormal protein aggregation.

    Funded by: NHLBI NIH HHS: R01 HL072166, R01-HL-72166

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2006;20;2;362-4

  • Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly.

    Foo SS, Turner CJ, Adams S, Compagni A, Aubyn D, Kogata N, Lindblom P, Shani M, Zicha D and Adams RH

    Vascular Development Laboratory, Cancer Research UK London Research Institute, London WC2A 3PX, UK.

    New blood vessels are initially formed through the assembly or sprouting of endothelial cells, but the recruitment of supporting pericytes and vascular smooth muscle cells (mural cells) ensures the formation of a mature and stable vascular network. Defective mural-cell coverage is associated with the poorly organized and leaky vasculature seen in tumors or other human diseases. Here we report that mural cells require ephrin-B2, a ligand for Eph receptor tyrosine kinases, for normal association with small-diameter blood vessels (microvessels). Tissue-specific mutant mice display perinatal lethality; vascular defects in skin, lung, gastrointestinal tract, and kidney glomeruli; and abnormal migration of smooth muscle cells to lymphatic capillaries. Cultured ephrin-B2-deficient smooth muscle cells are defective in spreading, focal-adhesion formation, and polarized migration and show increased motility. Our results indicate that the role of ephrin-B2 and EphB receptors in these processes involves Crk-p130(CAS) signaling and suggest that ephrin-B2 has some cell-cell-contact-independent functions.

    Cell 2006;124;1;161-73

  • Mice deficient in the chemokine receptor CXCR4 exhibit impaired limb innervation and myogenesis.

    Odemis V, Lamp E, Pezeshki G, Moepps B, Schilling K, Gierschik P, Littman DR and Engele J

    Institute of Anatomy, University of Leipzig, Medical Faculty, Liebigstr. 13, 04103 Leipzig, Germany.

    The chemokine CXCL12/SDF-1 and its receptor CXCR4 regulate the development and the function of the hematopoietic system and control morphogenesis of distinct brain areas. Here, we demonstrate that inactivation of CXCR4 results in a massive loss of spinal cord motoneurons and dorsal root ganglion neurons and, subsequently, in a reduced innervation of the developing mouse fore- and hindlimbs. However, only the death of sensory neurons seems to be a direct consequence of receptor inactivation as suggested by the observations that DRG neurons, but not motoneurons, of wild-type animals express CXCR4 and respond to CXCL12 with an increase in cell survival. In contrast, the increased death of motoneurons in CXCR4-deficient animals seems to result from impaired limb myogenesis and a subsequent loss of muscle-derived neurotrophic support. In summary, our findings unravel a previously unrecognized complex role of CXCL12/CXCR4 in the control of limb neuromuscular development.

    Molecular and cellular neurosciences 2005;30;4;494-505

  • Severe muscle disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages.

    Bär H, Mücke N, Kostareva A, Sjöberg G, Aebi U and Herrmann H

    Department of Molecular Genetics, German Cancer Research Center, D-69120 Heidelberg, Germany.

    Desmin is the major intermediate filament (IF) protein of muscle. Recently, mutations of the desmin gene have been reported to cause familial or sporadic forms of human skeletal, as well as cardiac, myopathy, termed desmin-related myopathy (DRM). The impact of any of these mutations on filament assembly and integration into the cytoskeletal network of myocytes is currently not understood, despite the fact that all cause the same histopathological defect, i.e., desmin aggregation. To gain more insight into the molecular basis of this process, we investigated how mutations within the alpha-helical rod domain of desmin affect both the assembly of the recombinant protein in vitro as well as the filament-forming capacity in cDNA-transfected cells. Whereas 6 of 14 mutants assemble into seemingly normal IFs in the test tube, the other mutants interfere with the assembly process at distinct stages, i.e., tetramer formation, unit-length filament (ULF) formation, filament elongation, and IF maturation. Correspondingly, the mutants with in vitro assembly defects yield dot-like aggregates in transfected cells, whereas the mutants that form IFs constitute a seemingly normal IF cytoskeleton in the cellular context. At present, it is entirely unclear why the latter mutant proteins also lead to aggregate formation in myocytes. Hence, these findings may be a starting point to dissect the contribution of the individual subdomains for desmin pathology and, eventually, the development of therapeutic interventions.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;42;15099-104

  • Evaluation of candidate markers for the peritubular myoid cell lineage in the developing mouse testis.

    Jeanes A, Wilhelm D, Wilson MJ, Bowles J, McClive PJ, Sinclair AH and Koopman P

    Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

    Despite the importance of peritubular myoid (PM) cells in the histogenesis of the fetal testis, understanding the origin and function of these cells has been hampered by the lack of suitable markers. The current study was aimed at identifying molecular markers for PM cells during the early stages of testis development in the mouse embryo. Expression of candidate marker genes was tested by section in situ hybridisation, in some instances followed by immunofluorescent detection of protein products. Collagen type-I, inhibinbetaA, caldesmon 1 and tropomyosin 1 were found to be expressed by early-stage PM cells. These markers were also expressed in subsets of interstitial cells, most likely reflecting their common embryological provenance from migrating mesonephric cells. Although not strictly specific for PM cells, these markers are likely to be useful in studying the biology of early PM cells in the fetal testis.

    Reproduction (Cambridge, England) 2005;130;4;509-16

  • Stroma-derived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts.

    Amatangelo MD, Bassi DE, Klein-Szanto AJ and Cukierman E

    Basic Science/Tumor Cell Biology, Fox Chase Cancer Center, Philadelphia, PA 19111-2497, USA

    Stromagenesis is a host reaction of connective tissue that, when induced in cancer, produces a progressive and permissive mesenchymal microenvironment, thereby supporting tumor progression. The stromal microenvironment is complex and comprises several cell types, including fibroblasts, the primary producers of the noncellular scaffolds known as extracellular matrices. The events that support tumor progression during stromagenesis are for the most part unknown due to the lack of suitable, physiologically relevant, experimental model systems. In this report, we introduce a novel in vivo-like three-dimensional system derived from tumor-associated fibroblasts at diverse stages of tumor development that mimic the stromagenic features of fibroblasts and their matrices observed in vivo. Harvested primary stromal fibroblasts, obtained from different stages of tumor development, did not retain in vivo stromagenic characteristics when cultured on traditional two-dimensional substrates. However, they were capable of effectively maintaining the tumor-associated stromal characteristics within three-dimensional cultures. In this study, we demonstrate that in vivo-like three-dimensional matrices appear to have the necessary topographical and molecular information sufficient to induce desmoplastic stroma differentiation of normal fibroblasts.

    The American journal of pathology 2005;167;2;475-88

  • Dosage-dependent requirement for mouse Vezf1 in vascular system development.

    Kuhnert F, Campagnolo L, Xiong JW, Lemons D, Fitch MJ, Zou Z, Kiosses WB, Gardner H and Stuhlmann H

    Department of Cell Biology, Division of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

    Vezf1 is an early development gene that encodes a zinc finger transcription factor. In the developing embryo, Vezf1 is expressed in the yolk sac mesoderm and the endothelium of the developing vasculature and, in addition, in mesodermal and neuronal tissues. Targeted inactivation of Vezf1 in mice reveals that it acts in a closely regulated, dose-dependent fashion on the development of the blood vascular and lymphatic system. Homozygous mutant embryos display vascular remodeling defects and loss of vascular integrity leading to localized hemorrhaging. Ultrastructural analysis shows defective endothelial cell adhesion and tight junction formation in the mutant vessels. Moreover, in heterozygous embryos, haploinsufficiency is observed that is characterized by lymphatic hypervascularization associated with hemorrhaging and edema in the jugular region; a phenotype reminiscent of the human congenital lymphatic malformation syndrome cystic hygroma.

    Funded by: NHLBI NIH HHS: R01 HL065738, R01 HL65738; NICHD NIH HHS: R29 HD31534; NIDDK NIH HHS: T32 DK007757, T32 DK07757

    Developmental biology 2005;283;1;140-56

  • Genetic inactivation of the transcription factor TIF-IA leads to nucleolar disruption, cell cycle arrest, and p53-mediated apoptosis.

    Yuan X, Zhou Y, Casanova E, Chai M, Kiss E, Gröne HJ, Schütz G and Grummt I

    Division of Molecular Biology of the Cell II, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.

    Growth-dependent regulation of rRNA synthesis is mediated by TIF-IA, a basal transcription initiation factor for RNA polymerase I. We inactivated the murine TIF-IA gene by homologous recombination in mice and embryonic fibroblasts (MEFs). TIF-IA-/- embryos die before or at embryonic day 9.5 (E9.5), displaying retardation of growth and development. In MEFs, Cre-mediated depletion of TIF-IA leads to disruption of nucleoli, cell cycle arrest, upregulation of p53, and induction of apoptosis. Elevated levels of p53 after TIF-IA depletion are due to increased binding of ribosomal proteins, such as L11, to MDM2 and decreased interaction of MDM2 with p53 and p19(ARF). RNAi-induced loss of p53 overcomes proliferation arrest and apoptosis in response to TIF-IA ablation. The striking correlation between perturbation of nucleolar function, elevated levels of p53, and induction of cell suicide supports the view that the nucleolus is a stress sensor that regulates p53 activity.

    Molecular cell 2005;19;1;77-87

  • Serine protease HtrA1 is developmentally regulated in trophoblast and uterine decidual cells during placental formation in the mouse.

    Nie G, Li Y and Salamonsen LA

    Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia. guiying.nie@phimr.monash.edu.au

    Development of a hemochorial placenta involves trophoblast proliferation, differentiation, and invasion into the uterus to promote blood flow to the embryo. Trophoblast invasion is tightly controlled by expression of specific proteases in the trophoblast and highly coordinated activities in the uterus. One uterine event essential for placentation is the developmentally regulated formation and regression of the decidua. In mice, decidual regression takes place in a temporal- and spatial-specific manner that is coordinated with placental development. In this study, we identified that the serine protease HtrA1 (high temperature requirement factor A1) was specifically expressed in differentiated trophoblast cells, especially the giant cells, during the early stages of placental development. A high level of HtrA1 expression was also detected in decidua capsularis specifically at the decidual-trophoblast interface where active involution occurs. Thus, we have identified a previously unknown role for HtrA1 as a protease potentially important for trophoblast differentiation/invasion and uterine decidual regression during placental development.

    Developmental dynamics : an official publication of the American Association of Anatomists 2005;233;3;1102-9

  • A Pax3/Pax7-dependent population of skeletal muscle progenitor cells.

    Relaix F, Rocancourt D, Mansouri A and Buckingham M

    C.N.R.S. URA 2578, Department of Developmental Biology, Pasteur Institute, 75724 Paris Cedex 15, France. frelaix@pasteur.fr

    During vertebrate development, successive phases of embryonic and fetal myogenesis lead to the formation and growth of skeletal muscles. Although the origin and molecular regulation of the earliest embryonic muscle cells is well understood, less is known about later stages of myogenesis. We have identified a new cell population that expresses the transcription factors Pax3 and Pax7 (paired box proteins 3 and 7) but no skeletal-muscle-specific markers. These cells are maintained as a proliferating population in embryonic and fetal muscles of the trunk and limbs throughout development. Using a stable green fluorescent protein (GFP) reporter targeted to Pax3, we demonstrate that they constitute resident muscle progenitor cells that subsequently become myogenic and form skeletal muscle. Late in fetal development, these cells adopt a satellite cell position characteristic of progenitor cells in postnatal muscle. In the absence of both Pax3 and Pax7, further muscle development is arrested and only the early embryonic muscle of the myotome forms. Cells failing to express Pax3 or Pax7 die or assume a non-myogenic fate. We conclude that this resident Pax3/Pax7-dependent progenitor cell population constitutes a source of myogenic cells of prime importance for skeletal muscle formation, a finding also of potential value in the context of cell therapy for muscle disease.

    Nature 2005;435;7044;948-53

  • Pax3/Pax7 mark a novel population of primitive myogenic cells during development.

    Kassar-Duchossoy L, Giacone E, Gayraud-Morel B, Jory A, Gomès D and Tajbakhsh S

    Department of Developmental Biology, Pasteur Institute, Centre Nationale de la Recherche Scientifique Unité de Recherche Associeé 2578, 75724 Paris, Cedex 15, France.

    Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.

    Genes & development 2005;19;12;1426-31

  • Morphogenesis of the right ventricle requires myocardial expression of Gata4.

    Zeisberg EM, Ma Q, Juraszek AL, Moses K, Schwartz RJ, Izumo S and Pu WT

    Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, USA.

    Mutations in developmental regulatory genes have been found to be responsible for some cases of congenital heart defects. One such regulatory gene is Gata4, a zinc finger transcription factor. In order to circumvent the early embryonic lethality of Gata4-null embryos and to investigate the role of myocardial Gata4 expression in cardiac development, we used Cre/loxP technology to conditionally delete Gata4 in the myocardium of mice at an early and a late time point in cardiac morphogenesis. Early deletion of Gata4 by Nkx2-5Cre resulted in hearts with striking myocardial thinning, absence of mesenchymal cells within the endocardial cushions, and selective hypoplasia of the RV. RV hypoplasia was associated with downregulation of Hand2, a transcription factor previously shown to regulate formation of the RV. Cardiomyocyte proliferation was reduced, with a greater degree of reduction in the RV than in the LV. Late deletion of Gata4 by Cre recombinase driven by the alpha myosin heavy chain promoter did not selectively affect RV development or generation of endocardial cushion mesenchyme but did result in marked myocardial thinning with decreased cardiomyocyte proliferation, as well as double-outlet RV. Our results demonstrate a general role of myocardial Gata4 in regulating cardiomyocyte proliferation and a specific, stage-dependent role in regulating the morphogenesis of the RV and the atrioventricular canal.

    Funded by: NHLBI NIH HHS: 1PO1 HL074734, K08 HL004387, K08 HL004387-04, P50 HL074734

    The Journal of clinical investigation 2005;115;6;1522-31

  • Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2-5 null mutants.

    Dupays L, Jarry-Guichard T, Mazurais D, Calmels T, Izumo S, Gros D and Théveniau-Ruissy M

    Laboratoire de Génétique et Physiologie du Développement (UMR CNRS 6545), Institut de Biologie du Développement de Marseille, Université de la Méditerranée, 13288 Marseille cedex 9, France.

    In humans, mutations of the gene encoding the transcription factor Nkx2-5 result in the heart in electrical conduction defects and morphological abnormalities. In this organ Nkx2-5 is expressed in both the myocardium and the endocardium. Connexins (Cxs) are gap junction channel proteins that have been shown to be involved in both heart development and cardiac electrical conduction, suggesting a possible correlation between expression of Cxs and Nkx2-5. To evaluate this correlation, the expression of Cxs has been investigated in the cardiovascular system of wild-type and Nkx2-5-/- 9.2 days post-conception (dpc) mouse embryos. The disruption of the Nkx2-5 gene results in the loss of Cx43 in the heart, due in part to the poor development of the ventricular trabecular network, as well as specific downregulation of Cx45 gene expression. In addition, the nuclear translocation of NFATc1 in the endocardial endothelial cells is inhibited in the Nkx2-5-/- embryos. These results indicate for the first time that Nkx2-5 is involved in the transcriptional regulation of the Cx45 gene expression. In the mutant embryos the aorta is collapsed, and the vascular endothelial Cxs, Cx40 and Cx37, are no longer expressed in its posterior region. Poor development of the trabeculae and downregulation of Cx45 may contribute both to failure of the myocardial function and to hemodynamic insufficiency. The latter, in turn, may result in the dysregulation of Cx40 and -37 expressions along the whole length of the aorta. Direct or indirect effects of Nkx2-5 inactivation on the Cx45 gene expression could explain the absence of the endocardial cushions in the heart of Nkx2-5-/- embryos.

    Journal of molecular and cellular cardiology 2005;38;5;787-98

  • Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo.

    Grifone R, Demignon J, Houbron C, Souil E, Niro C, Seller MJ, Hamard G and Maire P

    Département Génétique, Développement et Pathologie Moléculaire, Institut Cochin--INSERM 567, CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques 75014 Paris, France.

    In mammals, Six5, Six4 and Six1 genes are co-expressed during mouse myogenesis. Six4 and Six5 single knockout (KO) mice have no developmental defects, while Six1 KO mice die at birth and show multiple organ developmental defects. We have generated Six1Six4 double KO mice and show an aggravation of the phenotype previously reported for the single Six1 KO. Six1Six4 double KO mice are characterized by severe craniofacial and rib defects, and general muscle hypoplasia. At the limb bud level, Six1 and Six4 homeogenes control early steps of myogenic cell delamination and migration from the somite through the control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by apoptosis. At the interlimb level, epaxial Met expression is abolished, while it is preserved in Pax3-deficient embryos. Within the myotome, absence of Six1 and Six4 impairs the expression of the myogenic regulatory factors myogenin and Myod1, and Mrf4 expression becomes undetectable. Myf5 expression is correctly initiated but becomes restricted to the caudal region of each somite. Early syndetomal expression of scleraxis is reduced in the Six1Six4 embryo, while the myotomal expression of Fgfr4 and Fgf8 but not Fgf4 and Fgf6 is maintained. These results highlight the different roles played by Six proteins during skeletal myogenesis.

    Development (Cambridge, England) 2005;132;9;2235-49

  • Baalc, a marker of mesoderm and muscle.

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

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

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

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

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

  • Temporal and spatial expression pattern of beta1 sodium channel subunit during heart development.

    Domínguez JN, Navarro F, Franco D, Thompson RP and Aránega AE

    Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Paraje de las Lagunillas, s/n, 23071 Jaén, Spain.

    Objectives: The aim of this study is to analyze Scn1b mRNA expression levels and protein distribution of Scn1b, a putative modulator of the pore-forming Na(+) channel subunit in the heart, during mouse cardiac development.

    Methods: Scn1b mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E18.5 as well as in postnatal and adult heart. Scn1b protein distribution and subcellular localization during cardiogenesis were analyzed by immunohistochemistry and confocal microscopy.

    Results: Scn1b mRNA showed a dynamic expression pattern, peaking at stage E12.5 and decreasing at E15.5. Scn1b mRNA increased at later embryonic and neonatal stages, being maximal in the adult heart. Immunohistochemistry experiments revealed comparable distribution of Scn1b protein between the different cardiac chambers at early embryonic stages. With further development, Scn1b protein showed an enhanced expression in the trabeculated myocardium and the bundle branches. At the subcellular level in later embryonic and postnatal mouse cardiomyocytes, Scn1b was present in T-tubules as identified by immunostaining of alpha-actinin, and in the intercalated disks as identified by immunostaining of connexin 43.

    Conclusion: These results demonstrate that Scn1b is expressed during mouse heart development, suggesting it can play an important role in the action potential configuration of the cardiomyocytes during heart morphogenesis.

    Cardiovascular research 2005;65;4;842-50

  • Alterations in the heart mitochondrial proteome in a desmin null heart failure model.

    Fountoulakis M, Soumaka E, Rapti K, Mavroidis M, Tsangaris G, Maris A, Weisleder N and Capetanaki Y

    Center of Basic Research, Foundation for Biomedical Research, Academy of Athens, Soranou Efessiou 4, 11527 Athens, Greece; F. Hoffmann-La Roche Ltd., Center for Medical Genomics, Basel, Switzerland.

    Desmin, the major muscle-specific intermediate filament (IF) protein, is essential for mitochondrial behavior and function and maintenance of healthy muscle. Mice null for desmin develop dilated cardiomyopathy characterized by extensive cardiomyocyte death, fibrosis, calcification and eventual heart failure. We sought to investigate the heart mitochondrial proteome of wild type and desmin null mice in order to understand the cardiac and skeletal myopathy phenotype of desmin deficiency. The proteins were analyzed by 2-D electrophoresis, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Three hundred and eighty different gene products were identified, about 50% of which were enzyme subunits. Cytoskeletal and muscle-specific proteins, calcium-binding proteins, proteins with various other functions and about 70 unknown, hypothetical or poorly described gene products, were also identified. We have observed differences in most metabolic pathways, in apoptosis, calcium homeostasis, calcification and fibrosis and in different signaling pathways linked or not to mitochondrial function. The most significant changes were observed in ketone body and acetate metabolism, NADH shuttle proteins, amino-acid metabolism proteins and respiratory enzymes. Several of these changes are consistent with the known phenotype of desmin deficiency.

    Funded by: NIAMS NIH HHS: AR 39617

    Journal of molecular and cellular cardiology 2005;38;3;461-74

  • Epithelial hedgehog signals pattern the intestinal crypt-villus axis.

    Madison BB, Braunstein K, Kuizon E, Portman K, Qiao XT and Gumucio DL

    Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0616, USA.

    Morphological development of the small intestinal mucosa involves the stepwise remodeling of a smooth-surfaced endodermal tube to form finger-like luminal projections (villi) and flask-shaped invaginations (crypts). These remodeling processes are orchestrated by instructive signals that pass bidirectionally between the epithelium and underlying mesenchyme. Sonic (Shh) and Indian (Ihh) hedgehog are expressed in the epithelium throughout these morphogenic events, and mice lacking either factor exhibit intestinal abnormalities. To examine the combined role of Shh and Ihh in intestinal morphogenesis, we generated transgenic mice expressing the pan-hedgehog inhibitor, Hhip (hedgehog interacting protein) in the epithelium. We demonstrate that hedgehog (Hh) signaling in the neonatal intestine is paracrine, from epithelium to Ptch1-expressing subepithelial myofibroblasts (ISEMFs) and smooth muscle cells (SMCs). Strong inhibition of this signal compromises epithelial remodeling and villus formation. Surprisingly, modest attenuation of Hh also perturbs villus patterning. Desmin-positive smooth muscle progenitors are expanded, and ISEMFs are mislocalized. This mesenchymal change secondarily affects the epithelium: Tcf4/beta-catenin target gene activity is enhanced, proliferation is increased, and ectopic precrypt structures form on villus tips. Thus, through a combined Hh signal to underlying ISEMFs, the epithelium patterns the crypt-villus axis, ensuring the proper size and location of the emerging precrypt compartment.

    Funded by: NHLBI NIH HHS: T32-HL07505; NIDDK NIH HHS: P01 DK062041

    Development (Cambridge, England) 2005;132;2;279-89

  • Dysmorphogenesis of kidney cortical peritubular capillaries in angiopoietin-2-deficient mice.

    Pitera JE, Woolf AS, Gale NW, Yancopoulos GD and Yuan HT

    Institute of Child Health, University College London, London, UK.

    Angiopoietin-2 (Ang-2) modulates Tie-2 receptor activation. In mouse kidney maturation, Ang-2 is expressed in arteries, with lower levels in tubules, whereas Tie-2 is expressed by endothelia. We hypothesized that Ang-2 deficiency disrupts kidney vessel patterning. The normal renal cortical peritubular space contains fenestrated capillaries, which have few pericytes; they receive water and solutes which proximal tubules reclaim from the glomerular filtrate. In wild-type neonates, alpha smooth muscle actin (alpha SMA), platelet-derived growth factor receptor beta (PDGFR beta), and desmin-expressing cells were not prominent in this compartment. In Ang-2 null mutants, alpha SMA, desmin, and PDGFR beta prominently immunolocalized in cortical peritubular locations. Some alpha SMA-positive cells were closely associated with CD31- and Tie-2-positive peritubular capillary endothelia, and some of the alpha SMA-positive cells expressed PDGFR beta, desmin, and neural/glial cell 2 (NG2), consistent with a pericyte-like identity. Immunoblotting suggested an increase of total and tyrosine-phosphorylated Tie-2 proteins in null mutant versus wild-type kidneys, and electron microscopy confirmed disorganized capillaries and adjacent cells in cortical peritubular spaces in mutant neonate kidneys. Hence, Ang-2 deficiency causes dysmorphogenesis of cortical peritubular capillaries, with adjacent cells expressing pericyte-like markers; we speculate the latter effect is caused by disturbed paracrine signaling between endothelial and surrounding mesenchymal precursor cells.

    The American journal of pathology 2004;165;6;1895-906

  • Positive regulation of myogenic bHLH factors and skeletal muscle development by the cell surface receptor CDO.

    Cole F, Zhang W, Geyra A, Kang JS and Krauss RS

    Brookdale Department of Molecular, Cell, and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.

    Skeletal myogenesis is controlled by bHLH transcription factors of the MyoD family that, along with MEF-2 factors, comprise a positive feedback network that maintains the myogenic transcriptional program. Cell-cell contact between muscle precursors promotes myogenesis, but little is known of the underlying mechanisms. CDO, an Ig superfamily member, is a component of a cell surface receptor complex found at sites of cell-cell contact that positively regulates myogenesis in vitro. We report here that mice lacking CDO display delayed skeletal muscle development. Additionally, satellite cells from these mice differentiate defectively in vitro. CDO functions to activate myogenic bHLH factors via enhanced heterodimer formation, most likely by inducing hyperphosphorylation of E proteins. The Cdo gene is, in turn, a target of MyoD. The promyogenic effect of cell-cell contact is therefore linked to the activity of myogenic bHLH factors. Furthermore, the myogenic positive feedback network extends from the cell surface to the nucleus.

    Funded by: NCI NIH HHS: CA59474, CA78207; NIAMS NIH HHS: AR42607

    Developmental cell 2004;7;6;843-54

  • Progress of cell proliferation in striated muscle tissues during development of the mouse tongue.

    Nagata J and Yamane A

    Department of Pharmacology, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan.

    The developmental stages of and places for the proliferation of tongue muscle cells have not yet been determined. To determine the stages of and places for proliferation between embryonic day (E) 9 and birth, we analyzed the expression of cyclin D1 mRNA and the immunolocalization for proliferating cell nuclear antigen (PCNA). The ratio of PCNA-positive nuclei to total nuclei (PCNA-labeling index) was obtained in the anterior, middle, and posterior regions. Cyclin D1 mRNA was highly expressed between E11 and E13, but decreased thereafter until birth. The distribution of PCNA-positive cell nuclei was consistent with that of myogenic cells in the occipital somites at E9. The PCNA-labeling index was highest at E11, then decreased until birth without a significant difference among the 3 regions. These findings suggest that some tongue muscle progenitor cells begin proliferation in the occipital somites at E9, and that the proliferation in the whole tongue region occurred most actively between E11 and E13, then decreased until birth without regional differences.

    Journal of dental research 2004;83;12;926-9

  • Lhx2-/- mice develop liver fibrosis.

    Wandzioch E, Kolterud A, Jacobsson M, Friedman SL and Carlsson L

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

    Liver fibrosis is a wound-healing response to chronic injury of any type and is characterized by a progressive increase in deposition of extracellular matrix (ECM) proteins, the major source of which are activated hepatic stellate cells (HSCs). Because the LIM homeobox gene Lhx2 is expressed in HSCs and liver development in Lhx2(-/-) mice is disrupted, we analyzed liver development in Lhx2(-/-) embryos in detail. Lhx2(-/-) embryos contain numerous activated HSCs and display a progressively increased deposition of the ECM proteins associated with liver fibrosis, suggesting that Lhx2 inhibits HSC activation. Transfection of Lhx2 cDNA into a human HSC line down-regulates expression of genes characteristic of activated HSCs. Moreover, the Lhx2(-/-) liver display a disrupted cellular organization and an altered gene expression pattern of the intrahepatic endodermal cells, and the increased deposition of ECM proteins precedes these abnormalities. Collectively these results show that Lhx2 negatively regulates HSC activation, and its inactivation in developing HSCs appears therefore to mimic the signals that are triggered by the wound-healing response to chronic liver injury. This study establishes a spontaneous and reproducible animal model for hepatic fibrosis and reveals that Lhx2 expression in HSCs is important for proper cellular organization and differentiation of the liver.

    Funded by: NIDDK NIH HHS: DK56621

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;47;16549-54

  • GATA4 is a dosage-sensitive regulator of cardiac morphogenesis.

    Pu WT, Ishiwata T, Juraszek AL, Ma Q and Izumo S

    Department of Cardiology, Children's Hospital Boston, Boston, MA 02115, USA. wpu@enders.tch.harvard.edu

    Normal heart development is orchestrated by a set of highly conserved transcription factors that includes GATA4, Nkx2-5, and Tbx5. Heterozygous mutation of each of these genes causes congenital heart disease in humans. In mouse models, haploinsufficiency for Nkx2-5 or Tbx5 resulted in an increased incidence of structural heart disease, confirming that normal heart development is sensitive to small changes in expression levels of Nkx2-5 and Tbx5. However, mice haploinsufficient for GATA4 have not been reported to have cardiac abnormalities. We generated two new GATA4 alleles, GATA4(H) and GATA4(flox). GATA4(flox/flox) embryos expressed 50% less GATA4 protein in the heart and survived normally. In contrast, GATA4(H/H) embryos expressed 70% less GATA4 protein in the heart and died between days 13.5 and 16.5 of gestation. These embryos had common atrioventricular canal (CAVC), double outlet right ventricle (DORV), hypoplastic ventricular myocardium, and normal coronary vasculature. Myocardial hypoplasia was associated with diminished cardiomyocyte proliferation. Hemodynamic measurements demonstrated that these embryos had normal systolic function, severe diastolic dysfunction, and atrioventricular regurgitation. Surprisingly, expression levels of the putative GATA4 target genes ANF, BNP, MEF2C, Nkx2-5, cyclin D2, and BMP4 were unchanged in mutant hearts, suggesting that GATA4 is not a dose-limiting regulator of the expression of these genes during later stages of embryonic cardiac development. These data demonstrate that multiple aspects of embryonic cardiac morphogenesis and function are exquisitely sensitive to small changes in GATA4 expression levels.

    Funded by: NHLBI NIH HHS: K08 HL004387-04, P50 HL61036

    Developmental biology 2004;275;1;235-44

  • Rescue of cardiac defects in id knockout embryos by injection of embryonic stem cells.

    Fraidenraich D, Stillwell E, Romero E, Wilkes D, Manova K, Basson CT and Benezra R

    Cancer Biology and Genetics Program, Department of Medicine, Weill Medical College of Cornell University, New York, NY 10021, USA.

    We report that Id knockout mouse embryos display multiple cardiac defects, but mid-gestation lethality is rescued by the injection of 15 wild-type embryonic stem (ES) cells into mutant blastocysts. Myocardial markers altered in Id mutant cells are restored to normal throughout the chimeric myocardium. Intraperitoneal injection of ES cells into female mice before conception also partially rescues the cardiac phenotype with no incorporation of ES cells. Insulin-like growth factor 1, a long-range secreted factor, in combination with WNT5a, a locally secreted factor, likely account for complete reversion of the cardiac phenotype. Thus, ES cells have the potential to reverse congenital defects through Id-dependent local and long-range effects in a mammalian embryo.

    Funded by: NCI NIH HHS: R01 CA107429-01, R01CA107429; NHLBI NIH HHS: K01 HL076568-01, KO1HL076568

    Science (New York, N.Y.) 2004;306;5694;247-52

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Conversion of ES cells to columnar epithelia by hensin and to squamous epithelia by laminin.

    Takito J and Al-Awqati Q

    Dept. of Medicine, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA.

    Single-layered epithelia are the first differentiated cell types to develop in the embryo, with columnar and squamous types appearing immediately after blastocyst implantation. Here, we show that mouse embryonic stem cells seeded on hensin or laminin, but not fibronectin or collagen type IV, formed hemispheric epithelial structures whose outermost layer terminally differentiated to an epithelium that resembled the visceral endoderm. Hensin induced columnar epithelia, whereas laminin formed squamous epithelia. At the egg cylinder stage, the distal visceral endoderm is columnar, and these cells begin to migrate anteriorly to create the anterior visceral endoderm, which assumes a squamous shape. Hensin expression coincided with the dynamic appearance and disappearance of columnar cells at the egg cylinder stage of the embryo. These expression patterns, and the fact that hensin null embryos (and those already reported for laminin) die at the onset of egg cylinder formation, support the view that hensin and laminin are required for terminal differentiation of columnar and squamous epithelial phenotypes during early embryogenesis.

    Funded by: NCRR NIH HHS: RR 10506; NIDDK NIH HHS: DK 20999, R01 DK020999-25

    The Journal of cell biology 2004;166;7;1093-102

  • Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice.

    Kassar-Duchossoy L, Gayraud-Morel B, Gomès D, Rocancourt D, Buckingham M, Shinin V and Tajbakhsh S

    Stem Cells and Development, Department of Developmental Biology, CNRS URA 2578, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.

    In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells. Two determination factors of the basic helix-loop-helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts. In the absence of these factors, progenitor cells remain multipotent and can change their fate. Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes. Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the new Myf5:Myod double-null mice only when Mrf4 expression is not compromised. This finding contradicts the widely held view that myogenic identity is conferred solely by Myf5 and Myod, and identifies Mrf4 as a determination gene. We revise the epistatic relationship of the MRFs, in which both Myf5 and Mrf4 act upstream of Myod to direct embryonic multipotent cells into the myogenic lineage.

    Nature 2004;431;7007;466-71

  • Lineage and morphogenetic analysis of the cardiac valves.

    de Lange FJ, Moorman AF, Anderson RH, Männer J, Soufan AT, de Gier-de Vries C, Schneider MD, Webb S, van den Hoff MJ and Christoffels VM

    Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

    We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.

    Circulation research 2004;95;6;645-54

  • 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

  • Immunolocalization of extracellular matrix components and integrins during mouse liver development.

    Shiojiri N and Sugiyama Y

    Department of Biology, Faculty of Science, Shizuoka University, Oya 836, Shizuoka 422-8529, Japan. sbnshio@ipc.shizuoka.ac.jp

    Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent connective tissue, the mechanism of which is still unclear. This study was undertaken to analyze the immunolocalization of extracellular matrix components and their cellular receptors during mouse liver development, with special attention given to biliary differentiation and vascular development. In young fetal mouse liver, primitive structures of sinusoids were developed between hepatic cords associated with hematopoietic cells demonstrated by immunohistochemistry of basal laminar components, the alpha6 integrin subunit, and PECAM-1. Portal veins and hepatic veins showed different staining intensities of alpha2, alpha3, and alpha6 integrin subunits from early stages of development. Anti-beta4 integrin subunit antibodies reacted with portal veins, but not with hepatic veins after perinatal stages. Their different phenotypes may be related to the preferential differentiation of periportal bile ducts. In intrahepatic bile duct development, periportal hepatoblasts adjacent to the connective tissue were immunostained for each basal laminar component on the basal side at almost the same time; alpha3, alpha5, alpha6, and beta4 integrin subunits were immunohistochemically detectable later than the basal laminar components. These staining patterns of intrahepatic bile duct cells clearly differed from those of extrahepatic bile duct cells from the beginning of their development, suggesting that these ducts are of different origins. In conclusion, the vascular structures, including sinusoids, portal veins, and hepatic veins, develop from early stages of liver development, and the extracellular matrix components may play important roles in biliary differentiation and vascular development. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

    Hepatology (Baltimore, Md.) 2004;40;2;346-55

  • Null mutation of calpain 3 (p94) in mice causes abnormal sarcomere formation in vivo and in vitro.

    Kramerova I, Kudryashova E, Tidball JG and Spencer MJ

    Department of Pediatrics and Mattel Children's Hospital, David Geffen School of Medicine at University of California, Los Angeles 90095-1606, USA.

    The giant protein titin serves a primary role as a scaffold for sarcomere assembly; however, proteins that mediate this remodeling have not been identified. One potential mediator of this process is the protease calpain 3 (C3), the protein mutated in limb girdle muscular dystrophy type 2A. To test the hypothesis that C3 mediates remodeling during myofibrillogenesis, C3 knockout (C3KO) mice were generated. The C3KO mice were atrophic containing small foci of muscular necrosis. Myogenic cells fused normally in vitro, but lacked well-organized sarcomeres, as visualized by electron microscopy (EM). Titin distribution was normal in longitudinal sections from the C3KO mice; however, EM of muscle fibers showed misaligned A-bands. In vitro studies revealed that C3 can bind and cleave titin and that some mutations that are pathogenic in human muscular dystrophy result in reduced affinity of C3 for titin. These studies suggest a role for C3 in myofibrillogenesis and sarcomere remodeling.

    Funded by: NIAMS NIH HHS: AR047855, AR47721, AR48177

    Human molecular genetics 2004;13;13;1373-88

  • Mutations in two matrix metalloproteinase genes, MMP-2 and MT1-MMP, are synthetic lethal in mice.

    Oh J, Takahashi R, Adachi E, Kondo S, Kuratomi S, Noma A, Alexander DB, Motoda H, Okada A, Seiki M, Itoh T, Itohara S, Takahashi C and Noda M

    Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.

    The matrix metalloproteinase (MMP) family (approximately 25 members in mammals) has been implicated in extracellular matrix remodeling associated with embryonic development, cancer formation and progression, and various other physiological and pathological events. Inactivating mutations in individual matrix metalloproteinase genes in mice described so far, however, are nonlethal at least up to the first few weeks after birth, suggesting functional redundancy among MMP family members. Here, we report that mice lacking two MMPs, MMP-2 (nonmembrane type) and MT1-MMP (membrane type), die immediately after birth with respiratory failure, abnormal blood vessels, and immature muscle fibers reminiscent of central core disease. In the absence of MMP-2 and MT1-MMP, myoblast fusion in vitro is also significantly retarded. These findings suggest functional overlap in mice between the two MMPs with distinct molecular natures.

    Oncogene 2004;23;29;5041-8

  • A conserved enhancer element that drives FGF4 gene expression in the embryonic myotomes is synergistically activated by GATA and bHLH proteins.

    Iwahori A, Fraidenraich D and Basilico C

    Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA.

    FGF4 is the earliest member of the fibroblast growth factor (FGF) family expressed during embryogenesis where it plays essential roles in post-implantation development and limb growth and patterning. The expression of the Fgf4 gene in specific developmental stages, including the ICM of the blastocyst, the myotomes, and the limb bud AER, is regulated by distinct enhancer elements (Hom) in the 3' UTR. We previously identified the Hom3a region as the major DNA element responsible for Fgf4 expression in the myotomes and AER, and showed that a conserved E-box is a target for the myogenic bHLH transcription factors MYF5 and MYOD. To further define the cis- and trans-acting elements that determine Hom3a activity, we conducted a mutational analysis of the ability of the Hom3a region to drive lacZ expression in the myotomes of transgenic mice. We identified a minimal enhancer of 226nt that contains four elements, including the E-box, necessary to drive gene expression in the myotomes. One of these elements is a binding site for the GATA family of transcription factors, and we show here that GATA 1-4 and 6 can synergize with MYF5 or MYOD to activate transcription of a reporter plasmid driven by a portion of the Hom3a enhancer including the GATA site and the E-box. In line with this finding, we could show a direct interaction between MYF5/MYOD and GATA-3 or GATA-4, mediated by the N-terminal and bHLH domains of MYF5/MYOD and the C-terminal zing finger domain of GATA-3/4. To further study the role of the Hom3a enhancer in directing Fgf4 expression and the function of FGF4 in limb and muscle development, we generated mutant mice in which the Fgf4 Hom3a region had been deleted (Delta3a). In situ hybridization analysis of sections from Delta3a/ Delta3a embryos at E11.5 showed a drastically reduced expression of Fgf4 mRNA in the myotomes and AER. However, these mice developed normally and show no limb or muscle defects, and the same was true of heterozygous mice in which one Fgf4 allele carried the Hom3a deletion and the other was a null allele (Delta3a/Fgf4(-)). Together, these results show that Hom3a is the major DNA enhancer element directing Fgf4 expression in myotomes and limb bud AER, and that its activity in the myotomes results at least in part from the synergistic action of GATA and bHLH myogenic factors that bind to evolutionary conserved sequences in the Hom3a enhancer. However, expression of Fgf4 in the myotomes or AER of murine embryos does not appear to be essential for muscle or limb development.

    Funded by: NCI NIH HHS: CA078925

    Developmental biology 2004;270;2;525-37

  • Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality.

    Parlakian A, Tuil D, Hamard G, Tavernier G, Hentzen D, Concordet JP, Paulin D, Li Z and Daegelen D

    Laboratoire de Biologie Moléculaire de la Différenciation, Université Paris 7, 75005 Paris, France.

    Serum response factor (SRF) is at the confluence of multiple signaling pathways controlling the transcription of immediate-early response genes and muscle-specific genes. There are active SRF target sequences in more than 50 genes expressed in the three muscle lineages including normal and diseased hearts. However, the role of SRF in heart formation has not been addressed in vivo thus far due to the early requirement of SRF for mesoderm formation. We have generated a conditional mutant of SRF by using Cre-LoxP strategy that will be extremely useful to study the role of SRF in embryonic and postnatal cardiac functions, as well as in other tissues. This report shows that heart-specific deletion of SRF in the embryo by using a new beta MHC-Cre transgenic mouse line results in lethal cardiac defects between embryonic day 10.5 (E10.5) and E13.5, as evidenced by abnormally thin myocardium, dilated cardiac chambers, poor trabeculation, and a disorganized interventricular septum. At E9.5, we found a marked reduction in the expression of essential regulators of heart development, including Nkx2.5, GATA4, myocardin, and the SRF target gene c-fos prior to overt maldevelopment. We conclude that SRF is crucial for cardiac differentiation and maturation, acting as a global regulator of multiple developmental genes.

    Molecular and cellular biology 2004;24;12;5281-9

  • Divergent functions of murine Pax3 and Pax7 in limb muscle development.

    Relaix F, Rocancourt D, Mansouri A and Buckingham M

    Centre National de la Recherche Scientifique (CNRS) URA 2578, Department of Developmental Biology, Pasteur Institute, 75724 Paris Cedex 15, France.

    Pax genes encode evolutionarily conserved transcription factors that play critical roles in development. Pax3 and Pax7 constitute one of the four Pax subfamilies. Despite partially overlapping expression domains, mouse mutations for Pax3 and Pax7 have very different consequences. To investigate the mechanism of these contrasting phenotypes, we replaced Pax3 by Pax7 by using gene targeting in the mouse. Pax7 can substitute for Pax3 function in dorsal neural tube, neural crest cell, and somite development, but not in the formation of muscles involving long-range migration of muscle progenitor cells. In limbs in which Pax3 is replaced by Pax7, the severity of the muscle phenotype increases as the number of Pax7 replacement alleles is reduced, with the forelimb more affected than the hindlimb. We show that this hypomorphic activity of Pax7 is due to defects in delamination, migration, and proliferation of muscle precursor cells with inefficient activation of c-met in the hypaxial domain of the somite. Despite this, overall muscle patterning is retained. We conclude that functions already prefigured by the single Pax3/7 gene present before vertebrate radiation are fulfilled by Pax7 as well as Pax3, whereas the role of Pax3 in appendicular muscle formation has diverged, reflecting the more recent origin of this mode of myogenesis.

    Genes & development 2004;18;9;1088-105

  • Glomulin is predominantly expressed in vascular smooth muscle cells in the embryonic and adult mouse.

    McIntyre BA, Brouillard P, Aerts V, Gutierrez-Roelens I and Vikkula M

    Laboratory of Human Molecular Genetics, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, Avenue Hippocrate 74 (+5), BP 75.39, 1200 Brussels, Belgium.

    Mutations in the glomulin gene result in dominantly inherited vascular lesions of the skin known as glomuvenous malformations (GVMs). These lesions are histologically distinguished by their distended vein-like channels containing characteristic 'glomus cells', which appear to be incompletely or improperly differentiated vascular smooth muscle cells (VSMCs). The function of glomulin is currently unknown. We studied glomulin expression during murine development (E9.5 days post-coitum until adulthood) by non-radioactive in situ hybridization. Glomulin was first detected at E10.5 dpc in cardiac outflow tracts. Later, it showed strong expression in VSMCs as well as a limited expression in the perichondrium. At E11.5-14.5 dpc glomulin RNA was most abundant in the walls of the large vessels. At E16.5 dpc expression was also detectable in smaller arteries and veins. The high expression of glomulin in murine vasculature suggests an important role for glomulin in blood vessel development and/or maintenance, which is supported by the vascular phenotype seen in GVM patients with mutations in this gene.

    Gene expression patterns : GEP 2004;4;3;351-8

  • Regional expression of L-type calcium channel subunits during cardiac development.

    Acosta L, Haase H, Morano I, Moorman AF and Franco D

    Department of Experimental Biology, University of Jaén, Jaén, Spain.

    The contraction of cardiomyocytes is initiated by the entrance of extracellular calcium through specific calcium channels. Within the myocardium, L-type calcium channels are most abundant. In the heart, the main pore-forming subunit is the alpha1C, although there is a larger heterogeneity on auxiliary beta subunits. We have analyzed the distribution pattern of different alpha1C and beta subunits during cardiac development by immunohistochemistry. We observed homogeneous expression of alpha1C and beta subunits within the early tubular heart, whereas regional differences are observed during the late embryogenesis. beta2 and beta4 show differential expression within the embryonic myocardium. alpha1CD1 displays only a transient enhanced expression in the ventricular conduction system. In adult heart, the expression of the different calcium channel subunits analyzed is homogeneous along the entire myocardium except for alpha1CD1 that is practically undetectable. These findings suggest that beta subunits might play a major role in conferring calcium handling heterogeneity within the developing embryonic myocardium, while alpha1C subunits might contribute just transiently.

    Developmental dynamics : an official publication of the American Association of Anatomists 2004;230;1;131-6

  • Structural and functional roles of desmin in mouse skeletal muscle during passive deformation.

    Shah SB, Davis J, Weisleder N, Kostavassili I, McCulloch AD, Ralston E, Capetanaki Y and Lieber RL

    Departments of Bioengineering and Orthopaedics, Biomedical Sciences Graduate Group, University of California and Veterans Administration Medical Centers, San Diego, California 92161, USA.

    Mechanical interactions between desmin and Z-disks, costameres, and nuclei were measured during passive deformation of single muscle cells. Image processing and continuum kinematics were used to quantify the structural connectivity among these structures. Analysis of both wild-type and desmin-null fibers revealed that the costamere protein talin colocalized with the Z-disk protein alpha-actinin, even at very high strains and stresses. These data indicate that desmin is not essential for mechanical coupling of the costamere complex and the sarcomere lattice. Within the sarcomere lattice, significant differences in myofibrillar connectivity were revealed between passively deformed wild-type and desmin-null fibers. Connectivity in wild-type fibers was significantly greater compared to desmin-null fibers, demonstrating a significant functional connection between myofibrils that requires desmin. Passive mechanical analysis revealed that desmin may be partially responsible for regulating fiber volume, and consequently, fiber mechanical properties. Kinematic analysis of alpha-actinin strain fields revealed that knockout fibers transmitted less shear strain compared to wild-type fibers and experienced a slight increase in fiber volume. Finally, linkage of desmin intermediate filaments to muscle nuclei was strongly suggested based on extensive loss of nuclei positioning in the absence of desmin during passive fiber loading.

    Funded by: NHLBI NIH HHS: T32 HL007089, T32HL07089; NIAMS NIH HHS: AR40050, R01 AR040050

    Biophysical journal 2004;86;5;2993-3008

  • Cytoskeletal disruption and small heat shock protein translocation immediately after lengthening contractions.

    Koh TJ and Escobedo J

    School of Kinesiology, University of Illinois at Chicago, Chicago, Illinois 60608, USA. tjkoh@uic.edu

    The purposes of this study were to determine whether, immediately after lengthening contractions, 1) levels of specific force-transmitting cytoskeletal elements are reduced in skeletal muscle cells and 2) cytosolic small heat shock proteins (HSPs) translocate to structures prone to disruption. Western blot analysis demonstrated decreased concentrations of z-disk proteins alpha-actinin and plectin and membrane scaffolding proteins dystrophin and beta-spectrin in muscle exposed to lengthening contractions compared with contralateral control muscle. Lengthening contractions also resulted in immediate translocation of constitutively expressed HSP25 and alphaB-crystallin from the soluble to the insoluble fraction of muscle homogenates, and cryosections showed translocation from a diffuse, cytosolic localization to striations that corresponded to z-disks. Lengthening contraction-induced translocation of HSP25 and alphaB-crystallin was associated with phosphorylation of these small HSPs, which may trigger their protective activity. In summary, these findings demonstrate loss of z-disk and membrane scaffolding proteins immediately after lengthening contractions, and concomitant translocation of HSP25 and alphaB-crystallin to the z-disk, which may help to stabilize or repair cytoskeletal elements at this site.

    American journal of physiology. Cell physiology 2004;286;3;C713-22

  • Elimination by necrosis, not apoptosis, of embryonic extraocular muscles in the muscular dysgenesis mutant of the mouse.

    Heimann P, Kuschel T and Jockusch H

    Developmental Biology and Molecular Pathology, W7, Bielefeld University, 33501 Bielefeld, Germany.

    Muscular dysgenesis (mdg) in the mouse is a loss-of-function mutation of the skeletal muscle isoform of the voltage-sensor Ca2+ channel of skeletal muscle (DHP receptor alpha1 subunit, Cchl1a3, Chr1), which is essential for excitation-contraction coupling. Affected individuals (genotype mdg/mdg, phenotype MDG) are unable to breathe and die perinatally. We introduce here extraocular muscles in the study of MDG myopathy and show that, despite their developmental origin from head placodes, they are affected like trunk and limb muscles. MDG myotubes in situ are eliminated by necrosis, not apoptosis.

    Cell and tissue research 2004;315;2;243-7

  • Bcl-2 overexpression corrects mitochondrial defects and ameliorates inherited desmin null cardiomyopathy.

    Weisleder N, Taffet GE and Capetanaki Y

    Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

    One of the hallmarks of cardiomyopathy and heart failure is pronounced and progressive cardiomyocyte death. Understanding the mechanisms involved in cardiomyocyte cell death is a topic of great interest for treatment of cardiac disease. Mice null for desmin, the muscle-specific member of the intermediate filament gene family, develop cardiomyopathy characterized by extensive cardiomyocyte death, fibrosis, calcification, and eventual heart failure. The earliest ultrastructural defects are observed in mitochondria. In the present study, we have demonstrated that these mitochondrial abnormalities are the primary cause of the observed cardiomyopathy and that these defects can be ameliorated by overexpression of bcl-2 in desmin null heart. Overexpression of bcl-2 in the desmin null heart results in correction of mitochondrial defects, reduced occurrence of fibrotic lesions in the myocardium, prevention of cardiac hypertrophy, restoration of cardiomyocyte ultrastructure, and significant improvement of cardiac function. Furthermore, we have found that loss of desmin also diminishes the capacity of mitochondria to resist exposure to calcium, a defect that can be partially restored by bcl-2 overexpression. These results point to a unique function for desmin in protection of mitochondria from calcium exposure that can be partially rescued by overexpression of bcl-2. We show that bcl-2 cardiac overexpression has provided significant improvement of an inherited form of cardiomyopathy, revealing the potential for bcl-2, and perhaps other genes in the family, as therapeutic agents for heart disease of many types, including inherited forms.

    Funded by: NIA NIH HHS: AG17899, R01 AG017899; NIAMS NIH HHS: R01 AR039617

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;3;769-74

  • Heart and head defects in mice lacking pairs of connexins.

    Simon AM, McWhorter AR, Dones JA, Jackson CL and Chen H

    Department of Physiology, University of Arizona, Tucson, AZ 85724, USA. amsimon@u.arizona.edu

    Gene ablation studies in mice have revealed roles for gap junction proteins (connexins) in heart development. Of the 20 connexins in vertebrates, four are expressed in developing heart: connexin37 (Cx37), connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). Although each cardiac connexin has a different pattern of expression, some heart cells coexpress multiple connexins during cardiac morphogenesis. Since different connexins could have overlapping functions, some developmental phenotypes may only become evident when more than one connexin is ablated. In this study, we interbred Cx40(-/-) and Cx43(-/-) mice to generate mice lacking both Cx40 and Cx43. Cx40(-/-)Cx43(-/-) mice die around embryonic day 12.5 (E12.5), much earlier than either Cx40(-/-) or Cx43(-/-) mice, and they exhibit malformed hearts with ventricles that are abnormally rotated, suggesting a looping defect. Some Cx40(-/-)Cx43(-/-) animals also develop head defects characteristic of exencephaly. In addition, we examined mice lacking both Cx40 and Cx37 and found a high incidence of atrial and ventricular septal defects at birth. These results provide further evidence for the importance of gap junctions in embryonic development. Moreover, ablating different pairs of cardiac connexins results in distinct heart defects, suggesting both common and unique functions for Cx40, Cx43, and Cx37 during cardiac morphogenesis.

    Funded by: NHLBI NIH HHS: HL64232

    Developmental biology 2004;265;2;369-83

  • Persistent expression of the ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the developing and adult heart.

    Martin CM, Meeson AP, Robertson SM, Hawke TJ, Richardson JA, Bates S, Goetsch SC, Gallardo TD and Garry DJ

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8573, USA.

    Stem cells are important in the maintenance and repair of adult tissues. A population of cells, termed side population (SP) cells, has stem cell characteristics as they have been shown to contribute to diverse lineages. In this study, we confirm that Abcg2 is a determinant of the SP cell phenotype. Therefore, we examined Abcg2 expression during murine embryogenesis and observed robust expression in the blood islands of the E8.5 yolk sac and in developing tissues including the heart. During the latter stages of embryogenesis, Abcg2 identifies a rare cell population in the developing organs. We further establish that the adult heart contains an Abcg2 expressing SP cell population and these progenitor cells are capable of proliferation and differentiation. We define the molecular signature of cardiac SP cells and compare it to embryonic stem cells and adult cardiomyocytes using emerging technologies. We propose that the cardiac SP cell population functions as a progenitor cell population for the development, maintenance, and repair of the heart.

    Funded by: NIAMS NIH HHS: AR47850

    Developmental biology 2004;265;1;262-75

  • Cardiomyocyte-specific desmin rescue of desmin null cardiomyopathy excludes vascular involvement.

    Weisleder N, Soumaka E, Abbasi S, Taegtmeyer H and Capetanaki Y

    Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza Texas Medical Center, Houston, TX 77030, USA.

    Mice deficient in desmin, the muscle-specific member of the intermediate filament gene family, display defects in all muscle types and particularly in the myocardium. Desmin null hearts develop cardiomyocyte hypertrophy and dilated cardiomyopathy (DCM) characterized by extensive myocyte cell death, calcific fibrosis and multiple ultrastructural defects. Several lines of evidence suggest impaired vascular function in desmin null animals. To determine whether altered capillary function or an intrinsic cardiomyocyte defect is responsible for desmin null DCM, transgenic mice were generated to rescue desmin expression specifically to cardiomyocytes. Desmin rescue mice display a wild-type cardiac phenotype with no fibrosis or calcification in the myocardium and normalization of coronary flow. Cardiomyocyte ultrastructure is also restored to normal. Markers of hypertrophy upregulated in desmin null hearts return to wild-type levels in desmin rescue mice. Working hearts were perfused to assess coronary flow and cardiac power. Restoration of a wild-type cardiac phenotype in a desmin null background by expression of desmin specifically within cardiomyocyte indicates that defects in the desmin null heart are due to an intrinsic cardiomyocytes defect rather than compromised coronary circulation.

    Funded by: NHLBI NIH HHS: R01-HL43133; NIAMS NIH HHS: AR39617

    Journal of molecular and cellular cardiology 2004;36;1;121-8

  • 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

  • Loss of desmin leads to impaired voluntary wheel running and treadmill exercise performance.

    Haubold KW, Allen DL, Capetanaki Y and Leinwand LA

    Dept. of Molecular, Cellular, and Developmental Biology, Univ. of Colorado, Campus Box 347, Boulder, CO 80309-0347, USA.

    We examined voluntary wheel running and forced treadmill running exercise performance of wild-type mice and mice null for the desmin gene. When given access to a cage wheel, desmin null mice spent less time running and ran less far than wild-type mice. Wild-type mice showed a significant training effect with prolonged voluntary wheel running, as evidenced by an increase in mean running speed across the 3-wk exercise period, whereas desmin null mice did not. Desmin null mice also performed less well in acute treadmill stress and endurance tests compared with wild-type mice. We also evaluated serum creatine kinase (CK) activity in wild-type and desmin null mice in response to running. Voluntary running did not result in elevated CK activity in either wild-type or desmin null mice, whereas downhill treadmill running caused significant increases in serum CK activity in both wild-type and desmin null mice. However, the increase in serum CK was significantly less in desmin null mice than in wild-type mice. These results suggest that the lack of desmin adversely affects the ability of mice to engage in both chronic and acute bouts of endurance running exercise but that this decrement in performance is not associated with an increase in serum CK activity.

    Funded by: NHLBI NIH HHS: HL-56510

    Journal of applied physiology (Bethesda, Md. : 1985) 2003;95;4;1617-22

  • A significant reduction of the diaphragm in mdx:MyoD-/-(9th) embryos suggests a role for MyoD in the diaphragm development.

    Inanlou MR, Dhillon GS, Belliveau AC, Reid GA, Ying C, Rudnicki MA and Kablar B

    Department of Anatomy and Neurobiology, Dalhousie University, Faculty of Medicine, 5850 College Street, B3H 1X5, Halifax, NS, Canada.

    To further investigate the role of MyoD during skeletal myogenesis, we backcrossed mdx mutant mice (lacking dystrophin) with MyoD knock-out mice to obtain viable mice with MyoD allele on a pure mdx background. However, after nine generations of backcrossing, it was not possible to obtain a viable mdx:MyoD-/- phenotype (designated as: mdx:MyoD-/-(9th)). The compound-mutant embryos were examined just before birth. Essentially normal Myf5-dependent and most of the MyoD-dependent musculature was observed. By contrast, the skeletal muscle compartment of the diaphragm was significantly reduced. The mesenchymal compartment of the diaphragm was intact and no herniations were observed. Other examined organs (e.g., liver, kidney, brain, etc.) showed no histological abnormalities. Pulmonary hypoplasia was determined as the cause of neonatal death. Therefore, using a different approach, our new data supplement our previous findings and suggest an essential role for MyoD in development of skeletal muscle of the diaphragm. The failure of mdx:MyoD-/-(9th) diaphragm to develop normally is not caused by a reduced number of satellite cells, but from the inability of stem cells to progress through the myogenic program. Our data also suggest that functions of MyoD and Myf5 (and the respective muscle precursor cell sub-populations) are not entirely redundant by term, as previously suggested, since Myf5 is not capable of fully substituting for MyoD in the diaphragm development.

    Developmental biology 2003;261;2;324-36

  • Laminin alpha5 chain is required for intestinal smooth muscle development.

    Bolcato-Bellemin AL, Lefebvre O, Arnold C, Sorokin L, Miner JH, Kedinger M and Simon-Assmann P

    INSERM U381, Ontogenèse et Pathologie du Système Digestif, Avenue Molière, Strasbourg, France.

    Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.

    Developmental biology 2003;260;2;376-90

  • Myf5 and MyoD activation define independent myogenic compartments during embryonic development.

    Kablar B, Krastel K, Tajbakhsh S and Rudnicki MA

    Department of Anatomy and Neurobiology, Dalhousie University, 5859 University Avenue, B3H 4H7, Halifax, NS, Canada. bkablar@dal.ca

    Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, we examined the sites of epaxial, hypaxial, and cephalic myogenesis at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins revealed that myogenic precursor cells were not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5(nlacZ)-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appeared to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/-2.5lacZ-expression (-20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments.

    Developmental biology 2003;258;2;307-18

  • A genetic model for a central (septum transversum) congenital diaphragmatic hernia in mice lacking Slit3.

    Yuan W, Rao Y, Babiuk RP, Greer JJ, Wu JY and Ornitz DM

    Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    Congenital diaphragmatic hernia (CDH) is a significant cause of pediatric mortality in humans with a heterogeneous and poorly understood etiology. Here we show that mice lacking Slit3 developed a central (septum transversum) CDH. Slit3 encodes a member of the Slit family of guidance molecules and is expressed predominantly in the mesothelium of the diaphragm during embryonic development. In Slit3 null mice, the central tendon region of the diaphragm fails to separate from liver tissue because of abnormalities in morphogenesis. The CDH progresses through continuous growth of the liver into the thoracic cavity. This study establishes the first genetic model for CDH and identifies a previously unsuspected role for Slit3 in regulating the development of the diaphragm.

    Funded by: NCI NIH HHS: R01 CA114197, R01 CA114197-01A2; NEI NIH HHS: R01 EY014576, R01 EY014576-03; NIDCD NIH HHS: DC04289; NIGMS NIH HHS: R01 GM070967, R01 GM070967-02

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;9;5217-22

  • Caspase proteolysis of desmin produces a dominant-negative inhibitor of intermediate filaments and promotes apoptosis.

    Chen F, Chang R, Trivedi M, Capetanaki Y and Cryns VL

    Division of Endocrinology, Metabolism, and Molecular Medicine, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.

    Caspase cleavage of key cytoskeletal proteins, including several intermediate filament proteins, triggers the dramatic disassembly of the cytoskeleton that characterizes apoptosis. Here we describe the muscle-specific intermediate filament protein desmin as a novel caspase substrate. Desmin is cleaved selectively at a conserved Asp residue in its L1-L2 linker domain (VEMD downward arrow M(264)) by caspase-6 in vitro and in myogenic cells undergoing apoptosis. We demonstrate that caspase cleavage of desmin at Asp(263) has important functional consequences, including the production of an amino-terminal cleavage product, N-desmin, which is unable to assemble into intermediate filaments, instead forming large intracellular aggregates. Moreover, N-desmin functions as a dominant-negative inhibitor of filament assembly, both for desmin and the structurally related intermediate filament protein vimentin. We also show that stable expression of a caspase cleavage-resistant desmin D263E mutant partially protects cells from tumor necrosis factor-alpha-induced apoptosis. Taken together, these results indicate that caspase proteolysis of desmin at Asp(263) produces a dominant-negative inhibitor of intermediate filaments and actively participates in the execution of apoptosis. In addition, these findings provide further evidence that the intermediate filament cytoskeleton has been targeted systematically for degradation during apoptosis.

    Funded by: NINDS NIH HHS: NS31957

    The Journal of biological chemistry 2003;278;9;6848-53

  • Foxf1 +/- mice exhibit defective stellate cell activation and abnormal liver regeneration following CCl4 injury.

    Kalinichenko VV, Bhattacharyya D, Zhou Y, Gusarova GA, Kim W, Shin B and Costa RH

    Department of Molecular Genetics, College of Medicine, University of Illinois at Chicago, 60607, USA.

    Previous studies have shown that haploinsufficiency of the splanchnic and septum transversum mesoderm Forkhead Box (Fox) f1 transcriptional factor caused defects in lung and gallbladder development and that Foxf1 heterozygous (+/-) mice exhibited defective lung repair in response to injury. In this study, we show that Foxf1 is expressed in hepatic stellate cells in developing and adult liver, suggesting that a subset of stellate cells originates from septum transversum mesenchyme during mouse embryonic development. Because liver regeneration requires a transient differentiation of stellate cells into myofibroblasts, which secrete type I collagen into the extracellular matrix, we examined Foxf1 +/- liver repair following carbon tetrachloride injury, a known model for stellate cell activation. We found that regenerating Foxf1 +/- liver exhibited defective stellate cell activation following CCl(4) liver injury, which was associated with diminished induction of type I collagen, alpha-smooth muscle actin, and Notch-2 protein and resulted in severe hepatic apoptosis despite normal cellular proliferation rates. Furthermore, regenerating Foxf1 +/- livers exhibited decreased levels of interferon-inducible protein 10 (IP-10), delayed induction of monocyte chemoattractant protein 1 (MCP-1) levels, and aberrantly elevated expression of transforming growth factor beta1. In conclusion, Foxf1 +/- mice exhibited abnormal liver repair, diminished activation of hepatic stellate cells, and increased pericentral hepatic apoptosis following CCl(4) injury.

    Funded by: NHLBI NIH HHS: R01 HL62446-04; NIDDK NIH HHS: R01 DK54687-04

    Hepatology (Baltimore, Md.) 2003;37;1;107-17

  • Lower active force generation and improved fatigue resistance in skeletal muscle from desmin deficient mice.

    Balogh J, Li Z, Paulin D and Arner A

    Department of Physiological Sciences, Lund University, BMC F11, Tornavägen 10, SE-221 84, Lund, Sweden.

    The mechanical effects of the intermediate filament protein desmin was examined in desmin deficient mice (Des-/-) and their wild type control (Des+/+). Active force generation was determined in intact soleus muscles and in skinned single fibres from soleus and psoas. A decreased force generation of skinned muscle fibres from Des-/- mice and a tendency towards decreased active force in intact soleus muscle were detected. Concentrations of the contractile protein actin and myosin were not altered in Des-/- muscles. Ca(2+)-sensitivity of skinned single fibres in Des-/- muscles was unchanged compared to Des+/+. Using a protocol with repeated short tetani an increased fatigue resistance was found in the intact soleus muscles from Des-/- mice. In conclusion, desmin intermediate filaments are required for optimal generation or transmission of active force in skeletal muscle. Although other studies have shown that the desmin intermediate filaments appear to influence Ca(2+)-handling, the Ca(2+)-sensitivity of the contractile filaments is not altered in skeletal muscle of Des-/- mice. Previous studies have reported a switch towards slower myosin isoforms in slow skeletal muscle of Des-/- mice. The increased fatigue resistance show that this change is reflected in the physiological function of the muscle.

    Journal of muscle research and cell motility 2003;24;7;453-9

  • CYR61 (CCN1) is essential for placental development and vascular integrity.

    Mo FE, Muntean AG, Chen CC, Stolz DB, Watkins SC and Lau LF

    Department of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607-7170, USA.

    CYR61 (CCN1) is a member of the CCN family of secreted matricellular proteins that includes connective tissue growth factor (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). First identified as the product of a growth factor-inducible immediate-early gene, CYR61 is an extracellular matrix-associated angiogenic inducer that functions as a ligand of integrin receptors to promote cell adhesion, migration, and proliferation. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. To understand the functions of CYR61 during development, we have disrupted the Cyr61 gene in mice. We show here that Cyr61-null mice suffer embryonic death: approximately 30% succumbed to a failure in chorioallantoic fusion, and the reminder perished due to placental vascular insufficiency and compromised vessel integrity. These findings establish CYR61 as a novel and essential regulator of vascular development. CYR61 deficiency results in a specific defect in vessel bifurcation (nonsprouting angiogenesis) at the chorioallantoic junction, leading to an undervascularization of the placenta without affecting differentiation of the labyrinthine syncytiotrophoblasts. This unique phenotype is correlated with impaired Vegf-C expression in the allantoic mesoderm, suggesting that CYR61-regulated expression of Vegf-C plays a role in vessel bifurcation. The genetic and molecular basis of vessel bifurcation is presently unknown, and these findings provide new insight into this aspect of angiogenesis.

    Funded by: NCI NIH HHS: CA46565, CA76541, R01 CA046565, R01 CA076541, R29 CA076541

    Molecular and cellular biology 2002;22;24;8709-20

  • Excessive microvascular adaptation to changes in blood flow in mice lacking gene encoding for desmin.

    Loufrani L, Li Z, Lévy BI, Paulin D and Henrion D

    Institut National de la Santé et de la Recherche Médicale U 541, IFR Circulation-Paris-Nord, Paris, France.

    Objective: Desmin, an intermediate filament, has a key role in the integrity of myocytes, and its absence induces cardiomyopathies. Mice lacking desmin (Des-/- group) exhibit microvascular dysfunction leading to smooth muscle hyporeactivity. We investigated the effect of the absence of desmin in mice (Des-/- mice versus Des+/+ mice) on the adaptation of mesenteric arteries to changes in blood flow.

    With the use of selective ligations of second-order mesenteric arteries, blood flow was either diminished (low flow [LF]) or elevated (high flow [HF]); respective LF to HF values were 136+/-18 to 206+/-29 microL/min for Des+/+ mice and 119+/-14 to 189+/-24 microL/min for Des-/-mice in daughter arteries. Two weeks after ligation, arteries were mounted in an arteriograph, allowing the measurement of diameter under controlled conditions of pressure and flow. In HF arteries, diameter changes in response to increases in pressure were higher in Des-/- mice than in Des+/+ mice. Conversely, in LF arteries, diameter was lower in Des-/- mice. Flow-dependent dilation was higher in HF arteries and lower in LF arteries than in control arteries. This adaptation was lower in Des-/- mice than in Des+/+ mice (11.6+/-3.1% versus 25.5+/-4.8% dilation, respectively). Endothelial NO synthase expression increased in HF arteries in both strains.

    Conclusions: These findings provide a demonstration of the role of the intermediate filament desmin in microvascular remodeling. This dysfunction might take place in desmin-related myopathies.

    Arteriosclerosis, thrombosis, and vascular biology 2002;22;10;1579-84

  • Passive stiffness changes in soleus muscles from desmin knockout mice are not due to titin modifications.

    Anderson J, Joumaa V, Stevens L, Neagoe C, Li Z, Mounier Y, Linke WA and Goubel F

    Unité de Biomécanique et Génie Biomédical, CNRS-UMR 6600, Université de Technologie de Compiègne, BP 20529, 60205 Compiègne cedex, France.

    Passive stiffness was found to be increased in mouse soleus muscles lacking desmin. Because titin is considered to be the major source of muscle elasticity, the stiffening might be explainable by titin adaptation. To test this, passive mechanical properties of single skinned fibres of soleus muscles from desmin knockout and control mice were analysed by using various extension tests. Titin expression was studied by SDS-gel electrophoresis. Absence of desmin did not modify either electrophoretic mobility of the titin band (3700 kDa) or optical density-unit ratios between bands for titin and nebulin (congruent with 0.3) and bands for titin and myosin heavy chain (congruent with 0.08). Elastic properties of fibres were not altered in the absence of desmin since passive tensions were similar under quasi-static (56-66 kN m(-2)) and dynamic (100-118 kN m(-2)) conditions whatever the kind of fibre. Thus, titin is unlikely to be responsible for the large increase in passive stiffness observed in whole soleus muscles when desmin is lacking.

    Pflugers Archiv : European journal of physiology 2002;444;6;771-6

  • Sarcolemmal organization in skeletal muscle lacking desmin: evidence for cytokeratins associated with the membrane skeleton at costameres.

    O'Neill A, Williams MW, Resneck WG, Milner DJ, Capetanaki Y and Bloch RJ

    Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

    The sarcolemma of fast-twitch muscle is organized into "costameres," structures that are oriented transversely, over the Z and M lines of nearby myofibrils, and longitudinally, to form a rectilinear lattice. Here we examine the role of desmin, the major intermediate filament protein of muscle in organizing costameres. In control mouse muscle, desmin is enriched at the sarcolemmal domains that lie over nearby Z lines and that also contain beta-spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin -/- quadriceps, by contrast, most costameric structures are stable. Alternatively, Z line domains may be lost, whereas domains oriented longitudinally or lying over M lines are retained. Experiments with pan-specific antibodies to intermediate filament proteins and to cytokeratins suggest that control and desmin -/- muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin -/- muscle and redistribute with beta-spectrin at the sarcolemma when costameres are lost. Our results suggest that desmin associates with and selectively stabilizes the Z line domains of costameres, but that cytokeratins associate with all three domains of costameres, even in the absence of desmin.

    Funded by: NHLBI NIH HHS: HL64304; NIAMS NIH HHS: AR39617; NINDS NIH HHS: NS17282

    Molecular biology of the cell 2002;13;7;2347-59

  • beta8 integrins are required for vascular morphogenesis in mouse embryos.

    Zhu J, Motejlek K, Wang D, Zang K, Schmidt A and Reichardt LF

    Howard Hughes Medical Institute and Department of Physiology, University of California, San Francisco, San Francisco, CA 94143, USA.

    In order to assess the in vivo function of integrins containing the beta8 subunit, we have generated integrin beta8-deficient mice. Ablation of beta8 results in embryonic or perinatal lethality with profound defects in vascular development. Sixty-five percent of integrin beta8-deficient embryos die at midgestation, with evidence of insufficient vascularization of the placenta and yolk sac. The remaining 35% die shortly after birth with extensive intracerebral hemorrhage. Examination of brain tissue from integrin beta8-deficient embryos reveals abnormal vascular morphogenesis resulting in distended and leaky capillary vessels, as well as aberrant brain capillary patterning. In addition, endothelial cell hyperplasia is found in these mutant brains. Expression studies show that integrin beta8 transcripts are localized in endodermal cells surrounding endothelium in the yolk sac and in periventricular cells of the neuroepithelium in the brain. We propose that integrin beta8 is required for vascular morphogenesis by providing proper cues for capillary growth in both yolk sac and embryonic brain. This study thus identifies a molecule crucial for vascular patterning in embryonic yolk sac and brain.

    Funded by: NINDS NIH HHS: NS19090, R01 NS019090-20

    Development (Cambridge, England) 2002;129;12;2891-903

  • Mechanical function of intermediate filaments in arteries of different size examined using desmin deficient mice.

    Wede OK, Löfgren M, Li Z, Paulin D and Arner A

    Department of Physiological Sciences, Lund University, Lund, Sweden.

    Protein composition and mechanical function of intermediate filaments were examined in arteries of different sizes using desmin deficient mice (Des-/-) and their wild-type controls (Des+/+). Using SDS-PAGE gels and Western blots we found a gradient in desmin expression in the arterial tree; the desmin content increased from the elastic artery aorta, via the muscular mesenteric artery to the resistance-sized mesenteric microarteries approximately 150 microm in diameter in Des+/+ mice. Mechanical experiments were performed on the aorta, the mesenteric artery and resistance-sized arteries using wire myographs. For aorta and mesenteric artery, no differences in passive or active circumference- stress relations were found between Des-/- and Des+/+ mice. In microarteries, both passive and active stress were lower in the Des-/- group. In conclusion, large elastic and muscular arteries contain a relatively low amount of desmin, and the desmin intermediate filaments do not seem to play a major role in the mechanical properties of these larger arterial vessels. In the microarteries, where expression of desmin is high, desmin plays a role in supporting both passive and active tension.

    The Journal of physiology 2002;540;Pt 3;941-9

  • Ectopic expression of desmin in the epidermis of transgenic mice permits development of a normal epidermis.

    Kirfel J, Peters B, Grund C, Reifenberg K and Magin TM

    Institute of Physiological Chemistry and Bonner Forum Biomedizin, University of Bonn, Nussallee 11, 53115 Bonn, Germany.

    Cell architecture is largely based on the interaction of cytoskeletal proteins, which include intermediate filaments (IF), microfilaments, microtubules, as well as their type-specific membrane-attachment structures and associated proteins. In order to further our understanding of IF proteins and to address the fundamental issue whether different IF perform unique functions in different tissues, we expressed a desmin transgene in the basal epidermis of mice. Ectopic expression of desmin led to the formation of an additional, keratin-independent IF cytoskeleton and did not interfere with the keratin-desmosome interaction. We show that ectopic expression of a type III IF protein in basal keratinocytes did not interfere with the normal epidermal architecture and the program of terminal differentiation. This demonstrated that keratinocytes suffered no obvious detrimental effects from extra desmin filaments in their cytoplasm. In addition, we asked whether stable expression of desmin could rescue K5 null mice, which served as a model for severe EBS. Transgenic mice ectopically expressing desmin in the basal layer were mated with K5 heterozygous deficient animals to generate desmin rescue mice and analysed. In summary, our study support the notion that the different IF like desmin or keratins composing a IF network in vivo are central to cytoskeletal architecture and design in cells.

    Differentiation; research in biological diversity 2002;70;1;56-68

  • Extensive induction of important mediators of fibrosis and dystrophic calcification in desmin-deficient cardiomyopathy.

    Mavroidis M and Capetanaki Y

    Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA.

    Mice lacking the intermediate filament protein desmin demonstrate abnormal mitochondria behavior, disruption of muscle architecture, and myocardial degeneration with extensive calcium deposits and fibrosis. These abnormalities are associated with cardiomyocyte hypertrophy, cardiac chamber dilation and eventually with heart failure. In an effort to elucidate the molecular mechanisms leading to the observed pathogenesis, we have analyzed gene expression changes in cardiac tissue using differential display polymerase chain reaction and cDNA atlas array methods. The most substantial changes were found in genes coding the small extracellular matrix proteins osteopontin and decorin that are dramatically induced in the desmin-null myocardium. We further analyzed their expression pattern both at the RNA and protein levels and we compared their spatial expression with the onset of calcification. Extensive osteopontin localization is observed by immunohistochemistry in the desmin-null myocardium in areas with massive myocyte death, as well as in hypercellular regions with variable degrees of calcification and fibrosis. Osteopontin is consistently co-localized with calcified deposits, which progressively are transformed to psammoma bodies surrounded by decorin, especially in the right ventricle. These data together with the observed up-regulation of transforming growth factor-beta1 and angiotensin-converting enzyme, could explain the extensive fibrosis and dystrophic calcification observed in the heart of desmin-null mice, potentially crucial events leading to heart failure.

    Funded by: NIAMS NIH HHS: R01 AR039617

    The American journal of pathology 2002;160;3;943-52

  • Hepatocyte growth factor is essential for migration of myogenic cells and promotes their proliferation during the early periods of tongue morphogenesis in mouse embryos.

    Amano O, Yamane A, Shimada M, Koshimizu U, Nakamura T and Iseki S

    Department of Histology and Embryology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan. oamano@dent.meikai.ac.jp

    Temporal and spatial occurrence of hepatocyte growth factor (HGF) and its cognate receptor c-Met in the mouse mandibular development was investigated by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction. HGF was first recognized in the mesenchymal cells of the first branchial arch at the 10th day of gestation (E10), before tongue formation, whereas HGF receptor (c-Met) -positive myogenic cells first appeared at E11 in the center of mandibles. By E12, HGF turned to be colocalized with c-Met in the differentiating tongue myoblasts. Between E14 and E16, HGF disappeared, whereas c-Met remained, in the tongue myoblasts. The levels of HGF mRNA in the developing tongue decreased in accordance with the increase of desmin mRNA levels from E11 to E17. These in vivo results strongly suggest that the HGF/c-Met system takes part in the earlier stages of tongue development. To elucidate this hypothesis, the antisense oligodeoxyribonucleotide (A-ODN) for mouse HGF mRNA was added to the organ culture system of mandible with serumless, defined medium. Mandibular arches from E10 mouse embryos were cultured at 37 degrees C for 10 days in the absence or presence of A-ODN, control (sense) oligonucleotide (C-ODN), or A-ODN plus recombinant HGF. In the control mandibular explants cultured without HGF or ODN, the anterior two-third of the tongue derived from the first branchial arch was formed. It contained abundant desmin-positive myoblasts and was equivalent to the tongue of E14-E15. In contrast, in the presence of A-ODN in the medium, neither the swelling nor myogenic cells were found in the tongue-forming region of explants, and myogenic cells accumulated behind the tongue-forming region. Such dysplasia of tongue was never induced in the presence of C-ODN or A-ODN plus recombinant HGF in the medium. The effect of A-ODN appeared to be developmental stage-specific, because tongue dysplasia occurred when A-ODN was present during the earlier 4 days but not during the later 4 days of the culture. Furthermore, recombinant HGF added to the culture without ODNs during the earlier 4 days caused elevation in the number of mitotic myoblasts. These results suggest that HGF regulates both the migration and proliferation of myogenic cells during the earlier stages of tongue development.

    Developmental dynamics : an official publication of the American Association of Anatomists 2002;223;2;169-79

  • Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex.

    Poon E, Howman EV, Newey SE and Davies KE

    Department of Human Anatomy, University of Oxford, South Parks Road, Oxford OX13QX, United Kingdom.

    We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with alpha-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies.

    The Journal of biological chemistry 2002;277;5;3433-9

  • Hearts from mice lacking desmin have a myopathy with impaired active force generation and unaltered wall compliance.

    Balogh J, Merisckay M, Li Z, Paulin D and Arner A

    Department of Physiological Sciences, Lund University, BMC F11, Tornavägen 10, SE-221 84 Lund, Sweden.

    Objective: Desmin intermediate filaments are key structures in the cytoskeleton of cardiac muscle. Since they are associated with Z-discs and intercalated discs, they may have a role in sarcomere alignment or force transmission. We have explored the mechanical function of the desmin filaments in the cardiac wall by comparing desmin-deficient (Des-/-) and wild-type (Des+/+) mice.

    Methods: The Langendorff technique was used to examine the contractility of the whole heart. Rate of force generation, Ca(2+)-sensitivity and force per cross-sectional area were measured in skinned ventricle muscle preparations.

    Results: Des-/- mice have a cardiomyopathy with increased heart weight. Diastolic pressure was increased at all filling volumes in the Des-/- group. Since passive wall stress (i.e. force per area) was unchanged, the alteration in diastolic pressure is a consequence of the thicker ventricle wall. Developed pressure, rate of pressure increase and developed wall stress were significantly reduced, suggesting that active force generation of the contractile apparatus is reduced in Des-/-. Concentrations of actin and myosin in the ventricle were unaltered. Measurements in skinned muscle preparations showed a lower active force development with unaltered Ca(2+)-sensitivity and rate of tension development.

    Conclusion: It is suggested that the intermediate filaments have a role in active force generation of cardiac muscle, possibly by supporting sarcomere alignment or force transmission. The desmin filaments do not contribute the passive elasticity of the ventricle wall. Des-/- mice provide a model for genetic cardiomyopathy where the main factor contributing to altered cardiac performance is a decrease in active force generation, possibly in combination with a loss of functional contractile units.

    Cardiovascular research 2002;53;2;439-50

  • Evidence for increased myofibrillar mobility in desmin-null mouse skeletal muscle.

    Shah SB, Su FC, Jordan K, Milner DJ, Fridén J, Capetanaki Y and Lieber RL

    Department of Orthopaedics, Veterans Affairs Medical Center and University of California at San Diego, San Diego, CA 92093, USA.

    Quantitative electron microscopy was used to characterize the longitudinal mobility of myofibrils during muscle extension to investigate the functional roles of skeletal muscle intermediate filaments. Extensor digitorum longus fifth toe muscles from wild-type (+/+) and desmin-null (des -/-) animals were passively stretched to varying lengths, and the horizontal displacement of adjacent Z-disks in neighboring myofibrils (Deltax(myo)) and average sarcomere length (SL) were calculated. At short SL (<2.20 microm), wild-type and desmin-null Deltax(myo) were not significantly different, although there was a trend towards greater Z-disk misalignment in muscles from knockout animals (Deltax(myo) 0.34+/-0.04 microm versus 0.22+/-0.09 microm; P>0.2; means +/- S.E.M.). However, at higher SL (>2.90 microm), muscles from knockout animals displayed a dramatically increased Deltax(myo) relative to wild-type muscles (0.49+/-0.10 microm versus 0.25+/-0.07 microm; P<0.05). The results, which establish a maximum extension of the desmin network surrounding the Z-disk, provide what we believe to be the first quantitative estimation of the functional limits of the desmin intermediate filament system in the presence of an intact myofibrillar lattice. The existence of a limit on the extension of desmin suggests a mechanism for the recruitment of desmin into a network of force transmission, whether as a longitudinal load bearer or as a component in a radial force-transmission system.

    Funded by: PHS HHS: 40050

    The Journal of experimental biology 2002;205;Pt 3;321-5

  • Selective microvascular dysfunction in mice lacking the gene encoding for desmin.

    Loufrani L, Matrougui K, Li Z, Levy BI, Lacolley P, Paulin D and Henrion D

    Institut National de la Santé et de la Recherche Médicale (INSERM) U 541, IFR-Circulation-Paris-Nord, Paris VII University, Paris, France.

    The intermediate filament desmin has a key role in the integrity and contractility of skeletal and cardiac myocytes. Its absence or aggregation leads to cardiomyopathies. In arteries desmin is distributed heterogeneously; vascular disorders might also occur in its absence. We studied endothelial and muscular functions in arteries from mice lacking desmin (des-/-), compared with control (des+/+). Carotid and mesenteric resistance arteries were mounted in vitro in arteriographs. Desmin was located exclusively in smooth muscle cells. In arteries from des-/- mice, pressure-induced (myogenic) tone was unchanged, but agonist-induced tone decreased in resistance arteries (no change in large arteries). Flow (shear stress)- and acetylcholine-induced, endothelium-dependent dilation, as well as endothelium-independent dilation, were also decreased in resistance arteries. To our knowledge, this is the first study of vascular contractile and dilatory functions in arteries lacking desmin. Although vascular reactivity was normal in large arteries, it decreased strongly in small resistance arteries. Thus, desmin is required in vascular smooth muscle cells and in resistance arteries, for efficient control of vascular tone and consequently for an optimal blood flow supply. This microvascular defect found in the absence of desmin might play a major role in myopathies seen in desmin-related diseases.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2002;16;1;117-9

  • Smooth muscle differentiation and cell turnover in mouse detrusor development.

    Smeulders N, Woolf AS and Wilcox DT

    Nephro-Urology Unit, Institute of Child Health, University College London, London, United Kingdom.

    Purpose: We systematically analyzed detrusor muscle differentiation in normal mice with a focus on cell turnover (proliferation and apoptosis) as well as on expression of the muscle specific proteins alpha-smooth muscle actin and desmin.

    The stages examined were embryonic days 14 and 18, and postnatal day 1 and week 6, representing a period spanning organ inception to postnatal maturity. Alpha-smooth muscle actin, desmin and proliferating cell nuclear antigen were assessed by immunohistochemical testing of whole bladders and Western blot analysis of dissected detrusor layers. Apoptosis was detected in tissue sections by end-labeling.

    Results: Alpha-smooth muscle actin was expressed by the detrusor layer throughout maturation with levels significantly increasing from embryonic days 14 to 18 and cytoplasmic staining gaining in uniformity postnatally. Desmin expression in the detrusor was insignificant at embryonic day 14 but increased progressively thereafter. Proliferating cell nuclear antigen expression in the detrusor was highest at organ inception and fell stepwise at each developmental stage with low levels postnatally. Apoptosis in the detrusor was only detected at embryonic day 14.

    Conclusions: These results demonstrate that morphological growth of the mouse detrusor muscle is accompanied by complex serial changes in the expression of muscle specific proteins and in cell turnover. Strikingly, detrusor muscle cell differentiation and proliferation are inversely related. These detailed studies may serve as a comparison for future experiments involving aberrant mouse bladder development.

    The Journal of urology 2002;167;1;385-90

  • Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

    Zhang JQ, Elzey B, Williams G, Lu S, Law DJ and Horowits R

    Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

    N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.

    Biochemistry 2001;40;49;14898-906

  • 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

  • Divergent expression of delayed rectifier K(+) channel subunits during mouse heart development.

    Franco D, Demolombe S, Kupershmidt S, Dumaine R, Dominguez JN, Roden D, Antzelevitch C, Escande D and Moorman AF

    Experimental Molecular Cardiology Group, AMC, University of Amsterdam, Amsterdam, The Netherlands. dfranco@ujaen.es

    The repolarization phase of the cardiac action potential is dependent on transmembrane K(+) currents. The slow (I(Ks)) and fast (I(Kr)) components of the delayed-rectifier cardiac K(+) current are generated by pore-forming alpha subunits KCNQ1 and KCNH2, respectively, in association with regulatory beta-subunit KCNE1, KCNE2 and perphaps KCNE3. In the present study we have investigated the distribution of transcripts encoding these five potassium channel-forming subunits during mouse heart development as well as the protein distribution of KCNQ1 and KCNH2. KCNQ1 and KCNH2 mRNAs (and protein) are first expressed at embryonic day (E) 9.5, showing comparable levels of expression within the atrial and ventricular myocardium during the embryonic and fetal stages. In contrast, the beta-subunits display a more dynamic pattern of expression during development. KCNE1 expression is first observed at E9.5 throughout the entire myocardium and progressively is confined to the ventricular myocardium. With further development (E16.5), KCNE1 expression is mainly confined to the compact ventricular myocardium. KCNE2 is first expressed at E9.5 and it is restricted already to the atrial myocardium. KCNE3 is first expressed at E8.5 throughout the myocardium and with further development, it becomes restricted to the atrial myocardium. The fact that alpha subunits are homogeneously distributed within the myocardium, whereas the beta subunits display a regionalized expression profile during cardiac development, suggest that differences in the slow and fast component of the delayed-rectifier cardiac K(+) currents between the atrial and the ventricular cardiomyocytes are mainly determined by differential beta-subunit distribution.

    Cardiovascular research 2001;52;1;65-75

  • 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

  • Effects of desmin gene knockout on mice heart mitochondria.

    Lindén M, Li Z, Paulin D, Gotow T and Leterrier JF

    Groupe de Biologie des Interactions Cellulaires, UMR CNRS 6558, Poitiers, France. Monica@dbb.su.se

    In heart tissue from mice lacking the intermediate filament (IF) desmin, mitochondria show an abnormal shape and distribution (Thornell et al., 1997). In the present study we have isolated heart mitochondria from desmin null (D-/-) and control (D+/+) mice, and analyzed their composition by SDS-PAGE, immunoblotting, and enzyme measurements. We found both in vitro and in situ that the conventional kinesin, the microtubule-associated plus-end directed motor, was frequently associated with D+/+ heart mitochondria, but not with D-/- heart mitochondria, suggesting that the positioning of mitochondria in heart is a dynamic event involving the IF desmin, the molecular motor kinesin, and, most likely, the microtubules (MT) network. Furthermore, an increased capacity in energy production was found, as indicated by a threefold higher creatine kinase activity in heart mitochondria from D-/- compared to D+/+ mice. We also observed a significantly lower amount of cytochrome c in heart mitochondria from D-/- mice, and a relocalization of Bcl-2, which may indicate an apoptotic condition in the cell leading to the earlier reported pathological events, such as cardiomyocytes degeneration and calcinosis of the heart (Thornell et al., 1997).

    Journal of bioenergetics and biomembranes 2001;33;4;333-41

  • Passive stiffness is increased in soleus muscle of desmin knockout mouse.

    Anderson J, Li Z and Goubel F

    Unité de Biomécanique et Génie Biomédical, Unité Mixte de Recherche 6600, Centre National de la Recherche Scientifique et Université de Technologie, BP 20529, F-60205 Compiègne, France.

    The effects of an absence of desmin on passive stiffness of skeletal muscle were analyzed using soleus muscles from desmin knockout mice. Stiffness was evaluated by analyzing the passive tension induced by a ramp-and-hold extension test. This test showed that passive resistance to stretch was largely increased for muscles lacking desmin. This study could facilitate interpretation of changes in muscle mechanics observed in humans affected by desmin-related myopathies.

    Muscle & nerve 2001;24;8;1090-2

  • Mechanical properties and structure of carotid arteries in mice lacking desmin.

    Lacolley P, Challande P, Boumaza S, Cohuet G, Laurent S, Boutouyrie P, Grimaud JA, Paulin D, Lamazière JM and Li Z

    Institut National de la Santé et de la Recherche Médicale, INSERM U337, 15 Rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France. lacolley@ccr.jussieu.fr

    Objective: Our aim was to determine in desmin homozygous mutant mice the viscoelastic properties, the mechanical strength and the structure of the carotid artery.

    Methods: To assess the viscoelastic properties of large arteries, we have performed an in vivo analysis of the diameter-, and distensibility-pressure curves of the common carotid artery (CCA) in homozygous (Des -/-), heterozygous (Des +/-) and wild-type (Des +/+) mice. To evaluate the mechanical strength, we have measured the in vitro intraluminal pressure producing the rupture of the carotid artery wall. The structure analysis of the arterial wall was based on histology and electronic microscopy.

    Results: A lower distensibility and an increase of arterial wall viscosity were observed in Des -/- compared with Des +/+. Arterial thickness of Des -/- was similar to those of Des +/+, without changes in elastin and collagen contents. Electron microscopy revealed that the perimeter of cellular fingerlike-projections was smaller in Des -/-, indicating that the cells have lost part of their connections to the extracellular matrix. The rupture pressure was significantly lower in Des -/- (1500+/-200 mmHg) compared with Des +/+ (2100+/-80 mmHg) indicating a lower mechanical strength of the vascular wall. No significant difference was found between Des +/- and Des +/+.

    Conclusion: The desmin is essential to maintain proper viscoelastic properties, structure and mechanical strength of the vascular wall.

    Cardiovascular research 2001;51;1;178-87

  • Signals transduced by Ca(2+)/calcineurin and NFATc3/c4 pattern the developing vasculature.

    Graef IA, Chen F, Chen L, Kuo A and Crabtree GR

    Department of Developmental Biology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305, USA.

    Vascular development requires an orderly exchange of signals between growing vessels and their supporting tissues, but little is known of the intracellular signaling pathways underlying this communication. We find that mice with disruptions of both NFATc4 and the related NFATc3 genes die around E11 with generalized defects in vessel assembly as well as excessive and disorganized growth of vessels into the neural tube and somites. Since calcineurin is thought to control nuclear localization of NFATc proteins, we introduced a mutation into the calcineurin B gene that prevents phosphatase activation by Ca(2+) signals. These CnB mutant mice exhibit vascular developmental abnormalities similar to the NFATc3/c4 null mice. We show that calcineurin function is transiently required between E7.5 and E8.5. Hence, early calcineurin/NFAT signaling initiates the later cross-talk between vessels and surrounding tissues that pattern the vasculature.

    Cell 2001;105;7;863-75

  • Differential expression of the actin-binding proteins, alpha-actinin-2 and -3, in different species: implications for the evolution of functional redundancy.

    Mills M, Yang N, Weinberger R, Vander Woude DL, Beggs AH, Easteal S and North K

    Neurogenetics Research Unit, Children's Hospital at Westmead, Westmead, Sydney, NSW 2145, Australia.

    The alpha-actinins are a multigene family of four actin-binding proteins related to dystrophin. The two skeletal muscle isoforms of alpha-actinin (ACTN2 and ACTN3) are major structural components of the Z-line involved in anchoring the actin-containing thin filaments. In humans, ACTN2 is expressed in all muscle fibres, while ACTN3 expression is restricted to a subset of type 2 fibres. We have recently demonstrated that alpha-actinin-3 is absent in approximately 18% of individuals in a range of human populations, and that homozygosity for a premature stop codon (577X) accounts for most cases of true alpha-actinin-3 deficiency. Absence of alpha-actinin-3 is not associated with an obvious disease phenotype, raising the possibility that ACTN3 is functionally redundant in humans, and that alpha-actinin-2 is able to compensate for alpha-actinin-3 deficiency. We now present data concerning the expression of ACTN3 in other species. Genotyping of non-human primates indicates that the 577X null mutation has likely arisen in humans. The mouse genome contains four orthologues which all map to evolutionarily conserved syntenic regions for the four human genes. Murine Actn2 and Actn3 are differentially expressed, spatially and temporally, during embryonic development and, in contrast to humans, alpha-actinin-2 expression does not completely overlap alpha-actinin-3 in postnatal skeletal muscle, suggesting independent function. Furthermore, sequence comparison of human, mouse and chicken alpha-actinin genes demonstrates that ACTN3 has been conserved over a long period of evolutionary time, implying a constraint on evolutionary rate imposed by continued function of the gene. These observations provide a real framework in which to test theoretical models of genetic redundancy as they apply to human populations. In addition we highlight the need for caution in making conclusions about gene function from the phenotypic consequences of loss-of-function mutations in animal knockout models.

    Funded by: NIAMS NIH HHS: K02 AR02026, R01 AR44345

    Human molecular genetics 2001;10;13;1335-46

  • Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy.

    Pashmforoush M, Pomiès P, Peterson KL, Kubalak S, Ross J, Hefti A, Aebi U, Beckerle MC and Chien KR

    UCSD-Salk Program in Molecular Medicine and the UCSD Institute of Molecular Medicine, University of California at San Diego, La Jolla, California, USA.

    Although cytoskeletal mutations are known causes of genetically based forms of dilated cardiomyopathy, the pathways that link these defects with cardiomyopathy are unclear. Here we report that the alpha-actinin-associated LIM protein (ALP; Alp in mice) has an essential role in the embryonic development of the right ventricular (RV) chamber during its exposure to high biomechanical workloads in utero. Disruption of the gene encoding Alp (Alp) is associated with RV chamber dilation and dysfunction, directly implicating alpha-actinin-associated proteins in the onset of cardiomyopathy. In vitro assays showed that Alp directly enhances the capacity of alpha-actinin to cross-link actin filaments, indicating that the loss of Alp function contributes to destabilization of actin anchorage sites in cardiac muscle. Alp also colocalizes at the intercalated disc with alpha-actinin and gamma-catenin, the latter being a known disease gene for human RV dysplasia. Taken together, these studies point to a novel developmental pathway for RV dilated cardiomyopathy via instability of alpha-actinin complexes.

    Nature medicine 2001;7;5;591-7

  • Sarcomere number regulation maintained after immobilization in desmin-null mouse skeletal muscle.

    Shah SB, Peters D, Jordan KA, Milner DJ, Fridén J, Capetanaki Y and Lieber RL

    Department of Orthopaedics, Biomedical Sciences Graduate Group, Veterans Affairs and University of California Medical Centers, San Diego, CA 92161, USA.

    The serial sarcomere number of skeletal muscle changes in response to chronic length perturbation. The role of the intermediate filament desmin in regulating these changes was investigated by comparing the architectural adaptations of the tibialis anterior, extensor digitorum longus (EDL) and soleus from wild-type mice with those of homozygous desmin knockout mice after hindlimb immobilization. After 28 days, serial sarcomere number increased significantly in the lengthened wild-type tibialis anterior (by approximately 9%) and EDL (by approximately 17%). Surprisingly, muscles from desmin knockout mice also experienced significant serial remodeling, with the serial sarcomere number of the tibialis anterior increasing by approximately 10% and that of the EDL by approximately 27%. A consistent result was observed in the shortened soleus: a significant decrease in sarcomere number was observed in the muscles from both wild-type (approximately 26%) and knockout (approximately 12%) mice. Thus, although desmin is not essential for sarcomerogenesis or sarcomere subtraction in mouse hindlimb muscles, the results do suggest subtle differences in the nature of sarcomere number adaptation. We speculate that desmin may play a role in regulating the optimal arrangement of sarcomeres within the muscle or in sensing the magnitude of the immobilization effect itself.

    Funded by: NIAMS NIH HHS: AR40050, AR45358

    The Journal of experimental biology 2001;204;Pt 10;1703-10

  • Expression of heparin/heparan sulfate interacting protein/ribosomal protein l29 during the estrous cycle and early pregnancy in the mouse.

    Julian J, Das SK, Dey SK, Baraniak D, Ta VT and Carson DD

    Department of Biological Sciences, University of Delaware, Newark 19707, USA.

    Using a variety of approaches, we have examined the expression of the heparin/heparan sulfate (Hp/HS) interacting protein/ribosomal protein L29 (HIP/RPL29) in mouse uteri during the estrous cycle and early pregnancy. HIP/RPL29 selectively binds heparin and HS and may promote HS-dependent embryo adhesion. HIP/RPL29 was prominently expressed in both luminal and glandular epithelia under almost all conditions, including the phase of embryo attachment. In contrast, differences were noted in HIP/RPL29 expression in the stromal compartment both during the estrous cycle and during early pregnancy. Most notably, HIP/RPL29 accumulated in decidua, where it displayed a pattern complementary to that of pericellular deposition of the HS proteoglycan, perlecan. HIP/RPL29 protein was detected in implanted embryos at both initial and later stages of implantation; however, embryonic HIP/RPL29 mRNA accumulation was more pronounced at later stages (Day 7.5 postcoitum). In situ hybridization revealed similar spatial changes for HIP/RPL29 mRNA during these different physiological states. Whereas differences in the spatial pattern of HIP/RPL29 protein and mRNA expression were demonstrable, little change was detected in the level of HIP/RPL29 mRNA or protein in total endometrial extracts. Mouse blastocysts attached, but did not outgrow, on surfaces coated with recombinant murine HIP/RPL29. Surprisingly, soluble glycosaminoglycans including heparin, low molecular weight heparin, or chondroitin sulfate were not able to inhibit embryo attachment to HIP/RPL29-coated surfaces. These latter observations indicate that embryonic cell surface components other than HS proteoglycans can promote binding to HIP/RPL29 expressed by uterine cells.

    Funded by: NICHD NIH HHS: HD 25235, HD 29963, HD 29968; NIEHS NIH HHS: ES 07814

    Biology of reproduction 2001;64;4;1165-75

  • Sonic hedgehog is a survival factor for hypaxial muscles during mouse development.

    Krüger M, Mennerich D, Fees S, Schäfer R, Mundlos S and Braun T

    Institute of Physiological Chemistry, University of Halle-Wittenberg, Hollystr. 1, Germany.

    Sonic hedgehog (Shh) has been proposed to function as an inductive and trophic signal that controls development of epaxial musculature in vertebrate embryos. In contrast, development of hypaxial muscles was assumed to occur independently of Shh. We here show that formation of limb muscles was severely affected in two different mouse strains with inactivating mutations of the Shh gene. The limb muscle defect became apparent relatively late and initial stages of hypaxial muscle development were unaffected or only slightly delayed. Micromass cultures and cultures of tissue fragments derived from limbs under different conditions with or without the overlaying ectoderm indicated that Shh is required for the maintenance of the expression of myogenic regulatory factors (MRFs) and, consecutively, for the formation of differentiated limb muscle myotubes. We propose that Shh acts as a survival and proliferation factor for myogenic precursor cells during hypaxial muscle development. Detection of a reduced but significant level of Myf5 expression in the epaxial compartment of somites of Shh homozygous mutant embryos at E9.5 indicated that Shh might be dispensable for the initiation of myogenesis both in hypaxial and epaxial muscles. Our data suggest that Shh acts similarly in both somitic compartments as a survival and proliferation factor and not as a primary inducer of myogenesis.

    Development (Cambridge, England) 2001;128;5;743-52

  • Molecular characterization of the ventricular conduction system in the developing mouse heart: topographical correlation in normal and congenitally malformed hearts.

    Franco D and Icardo JM

    Department of Experimental Biology, Faculty of Experimental and Health Sciences, University of Jaén, Paraje Las Lagunillas s/n, 23071, Jaén, Spain. dfranco@ujaen.es

    Objectives: Within the adult heart, it is convention to distinguish the conduction system and working (atrial and ventricular) myocardium. The adult conduction system (CS) comprises the sinoatrial (SAN), and atrioventricular (AVN) nodes, the atrioventricular bundle (AVB), the bundle branches and the peripheral Purkinje fibers, each of which display distinct functional properties and distinct profile of gene expression. Characterization of the mouse cardiac conduction system during development is rudimentary at present, even though genetically-modified mice are an increasing source of information regarding cardiac function and embryonic heart development.

    Methods: We have performed a detailed study of the pattern of expression of myosin heavy chain (MHC), myosin light chain (MLC), troponin I (TnI) isoforms, connexin 43 (Cx43), desmin and alpha-smooth muscle actin (alpha-SMA), in the ventricular conduction system of normal and congenitally malformed mouse hearts (iv background) from embryonic day 14.5 to 19.5.

    Results: The AVN is characterized by co-expression of MHC and MLC isoforms and no detectable expression of Cx43, desmin or alpha-SMA. The AVB expresses betaMHC and MLC2v, but no alphaMHC, MLC2a, Cx43, desmin or alpha-SMA. The right and left bundle branches display enhanced expression of desmin and alpha-SMA but no Cx43. The normal expression profile is maintained in congenitally malformed hearts such as double-outlet right ventricle and common atrioventricular canal. Three-dimensional reconstruction of the conduction system shows normal arrangement of the bundle branches in congenitally malformed hearts, but abnormal location and/or extension of the AVN.

    Conclusions: Molecular characterization allows to follow the development of the CS in both, normal and malformed mouse hearts. Normal phenotypic expression of the CS is independent of heart situs but shows minor modifications in the presence of heart malformations. It is concluded that the AVN derives from the atrioventricular canal myocardium, the bundle of His from the ventricular myocardium, and the bundle branches from the ventricular trabeculations. Our results do not provide evidence to support an extra-cardiac origin of the ventricular CS.

    Cardiovascular research 2001;49;2;417-29

  • Defects in development of the kidney, heart and eye vasculature in mice homozygous for a hypomorphic Notch2 mutation.

    McCright B, Gao X, Shen L, Lozier J, Lan Y, Maguire M, Herzlinger D, Weinmaster G, Jiang R and Gridley T

    The Jackson Laboratory, Bar Harbor, ME 04609, USA.

    The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the in vivo role of the Notch2 gene, we constructed a targeted mutation, Notch2(del1). Unexpectedly, we found that alternative splicing of the Notch2(del1) mutant allele leads to the production of two different in-frame transcripts that delete either one or two EGF repeats of the Notch2 protein, suggesting that this allele is a hypomorphic Notch2 mutation. Mice homozygous for the Notch2(del1) mutation died perinatally from defects in glomerular development in the kidney. Notch2(del1)/Notch2(del1 )mutant kidneys were hypoplastic and mutant glomeruli lacked a normal capillary tuft. The Notch ligand encoded by the Jag1 gene was expressed in developing glomeruli in cells adjacent to Notch2-expressing cells. We show that mice heterozygous for both the Notch2(del1) and Jag1(dDSL) mutations exhibit a glomerular defect similar to, but less severe than, that of Notch2(del1)/Notch2(del1 )homozygotes. The co-localization and genetic interaction of Jag1 and Notch2 imply that this ligand and receptor physically interact, forming part of the signal transduction pathway required for glomerular differentiation and patterning. Notch2(del1)/Notch2(del1 )homozygotes also display myocardial hypoplasia, edema and hyperplasia of cells associated with the hyaloid vasculature of the eye. These data identify novel developmental roles for Notch2 in kidney, heart and eye development.

    Funded by: NCI NIH HHS: CA34196; NINDS NIH HHS: NS36437

    Development (Cambridge, England) 2001;128;4;491-502

  • Desmin integrates the three-dimensional mechanical properties of muscles.

    Boriek AM, Capetanaki Y, Hwang W, Officer T, Badshah M, Rodarte J and Tidball JG

    Department of Medicine, Baylor College of Medicine, Houston, Texas 77030, USA. boriek@bcm.tmc.edu

    Striated muscle is a linear motor whose properties have been defined in terms of uniaxial structures. The question addressed here is what contribution is made to the properties of this motor by extramyofilament cytoskeletal structures that are not aligned in parallel with the myofilaments. This question arose from observations that transverse loads increase muscle force production in diaphragm but not in the hindlimb muscle, thereby indicating the presence of structures that couple longitudinal and transverse properties of diaphragmatic muscle. Furthermore, we find that the diaphragms of null mutants for the cytoskeletal protein desmin show 1) significant reductions in coupling between the longitudinal and transverse properties, indicating for the first time a role for a specific protein in integrating the three-dimensional mechanical properties of muscle, 2) significant reductions in the stiffness and viscoelasticity of muscle, and 3) significant increases in tetanic force production. Thus desmin serves a complex mechanical function in diaphragm muscle by contributing both to passive stiffness and viscoelasticity and to modulation of active force production in a three-dimensional structural network. Our finding changes the paradigm of force transmission among cells by placing our understanding of the function of the cytoskeleton in the context of the structural and mechanical complexity of muscles.

    Funded by: NHLBI NIH HHS: HL-63134; NIAMS NIH HHS: AR-39617, AR-40343

    American journal of physiology. Cell physiology 2001;280;1;C46-52

  • Pro- and anti-apoptotic members of the Bcl-2 family in skeletal muscle: a distinct role for Bcl-2 in later stages of myogenesis.

    Dominov JA, Houlihan-Kawamoto CA, Swap CJ and Miller JB

    Myogenesis Research Laboratory, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.

    Apoptotic myonuclei appear during myogenesis and in diseased muscles. To investigate cell death regulation in skeletal muscle, we examined how members of the Bcl-2 family of apoptosis regulators are expressed and function in the C2C12 muscle cell line and in primary muscle cells at different stages of development. Both anti-apoptotic (Bcl-W, Bcl-X(L)) and pro-apoptotic (Bad, Bak, Bax) members of the Bcl-2 family were expressed in developing skeletal muscle in vivo. Each was also expressed in embryonic (E11-12), fetal (E15-16), and neonatal muscle stem cells, myoblasts, and myotubes in vitro. In contrast, Bcl-2 expression was limited to a small group of mononucleate, desmin-positive, myogenin-negative muscle cells that were seen in fetal and neonatal, but not embryonic, muscle cell cultures. The cell surface protein Sca-1, which is associated with muscle and blood stem cells, was found on approximately 1/2 of these Bcl-2-positive cells. Loss of Bcl-2 did not affect expression of other family members, because neonatal muscles of wild-type and Bcl-2-null mice had similar amounts of Bcl-X(L), Bcl-W, Bad, Bak, and Bax mRNAs. Loss of Bcl-2 did have functional consequences; however, because neonatal muscles of Bcl-2-null mice had only approximately 2/3 as many fast muscle fibers as muscles in wild-type mice. Thus, Bcl-2 function is required for particular stages of fetal and postnatal myogenesis.

    Developmental dynamics : an official publication of the American Association of Anatomists 2001;220;1;18-26

  • Delayed embryonic development of mouse masseter muscle correlates with delayed MyoD family expression.

    Yamane A, Ohnuki Y and Saeki Y

    Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama, Japan. GAH03667@nifty.ne.jp

    While the masseter muscle is known to have several unique developmental characteristics as compared with other skeletal muscles, little is known about its myogenesis. Thus, we examined the expression of myogenic marker and of myoD family gene mRNA from embryonic day (E) 11 to birth. The obtained results were compared with our earlier results of the mouse tongue muscle, which is also involved in oral functions. The mRNA quantities were determined by means of the reverse-transcription and competitive-polymerase chain-reaction techniques. The expression of myogenic marker mRNA indicated that differentiation and maturation in the masseter began at E13 as in the tongue, and were not yet completed at birth, although they were completed in the tongue. The expression of myoD, myogenin, and myf5 mRNA peaked later in the masseter (E17) than in the tongue (E13). The expression of MRF4 mRNA began later in the masseter (E15) than in the tongue (E13). These results suggest that the delayed expression of the myoD family genes in the masseter correlates with delayed differentiation and maturation, probably due to the later functional requirements of the masseter than of the tongue.

    Journal of dental research 2000;79;12;1933-6

  • Generation of tension by skinned fibers and intact skeletal muscles from desmin-deficient mice.

    Wieneke S, Stehle R, Li Z and Jockusch H

    Developmental Biology Unit, University of Bielefeld, Bielefeld, D-33501, Germany.

    We have investigated the physiological role of desmin in skeletal muscle by measuring isometric tension generated in skinned fibres and intact skeletal muscles from desmin knock-out (DES-KO) mice. About 80% of skinned single extensor digitorum longus (EDL) fibres from adult DES-KO mice generated tensions close to that of wild-type (WT) controls. Weights and maximum tensions of intact EDL but not of soleus (SOL) muscles were lowered in DES-KO mice. Repeated contractions with stretch did not affect subsequent isometric tension in EDL muscles of DES-KO mice. Tension during high frequency fatigue (HFF) declined faster and this deficiency was compensated in DES-KO EDL muscles by 5 mM caffeine which had no influence on HFF in WT EDL. Furthermore, caffeine evoked twitch potentiation was higher in DES-KO than in WT muscles. We conclude that desmin is not essential for acute tensile strength but rather for optimal activation of intact myofibres during E-C coupling.

    Biochemical and biophysical research communications 2000;278;2;419-25

  • Immunohistochemical analysis of development of desmin-positive hepatic stellate cells in mouse liver.

    Nitou M, Ishikawa K and Shiojiri N

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

    Development of desmin-positive hepatic stellate cells was studied in mice using double immunofluorescent techniques and in vitro cultures with special attention given to their cell lineages. Several studies recently reported on the presence of cells that are immunologically reactive with both antidesmin and anticytokeratin antibodies in young fetal rat livers, and suggested the possibility that these cells give rise to hepatocytes and hepatic stellate cells. At early stages of mouse liver development, stellate cells with desmin-positive filaments were scattered in the liver parenchyma. However, the stellate cells definitely differed from hepatoblasts and hepatocytes in terms of their morphology and expression of desmin and hepatoblast and hepatocyte-specific E-cadherin in the liver. Fetal hepatoblasts and hepatocytes did not react with antidesmin antibodies, nor did desmin-positive stellate cells express E-cadherin in vivo and in vitro. Thus it is likely that desmin-positive stellate cells and hepatoblasts belong to different cell lineages. In the fetal liver, the desmin-positive stellate cells surrounded blood vessels, and extended their processes to haematopoietic cells and megakaryocytes. Many, but not all, hepatoblasts and hepatocytes were observed to be associated with the stellate cells. At fetal stages, cellular processes positive for desmin in the stellate cells were also thick compared with those in the adult liver, in which desmin-positive stellate cells lay in Disse's space and were closely associated with all hepatocytes. These developmental changes in the geography of desmin-positive cells in the liver parenchyma and their morphology may be associated with their maturation and interactions with other cell types.

    Journal of anatomy 2000;197 Pt 4;635-46

  • 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

  • An overlapping CArG/octamer element is required for regulation of desmin gene transcription in arterial smooth muscle cells.

    Mericskay M, Parlakian A, Porteu A, Dandré F, Bonnet J, Paulin D and Li Z

    Laboratoire de Biologie Moléculaire de la Différenciation, Université Denis Diderot Paris 7, 2, place Jussieu, Paris, 75005, France.

    The desmin gene encodes an intermediate filament protein that is present in skeletal, cardiac, and smooth muscle cells. This study shows that the 4-kb upstream region of the murine desmin promoter directs expression of a lacZ reporter gene throughout the heart from E7.5 and in skeletal muscle and vascular smooth muscle cells from E9. 5. The distal fragment (-4005/-2495) is active in arterial smooth muscle cells but not in venous smooth muscle cells or in the heart in vivo. It contains a CArG/octamer overlapping element (designated CArG4) that can bind the serum response factor (SRF) and an Oct-like factor. The desmin distal fragment can replace a SM22alpha regulatory region (-445/-126) that contains two CArG boxes, to cis-activate a minimal (-125/+65) SM22alpha promoter fragment in arterial smooth muscle cells of transgenic embryos. lacZ expression was abolished when mutations were introduced into the desmin CArG4 element that abolished the binding of SRF and/or Oct-like factor. These data suggest that a new type of combined CArG/octamer element plays a prominent role in the regulation of the desmin gene in arterial smooth muscle cells, and SRF and Oct-like factor could cooperate to drive specific expression in these cells.

    Developmental biology 2000;226;2;192-208

  • Desmin knockout muscles generate lower stress and are less vulnerable to injury compared with wild-type muscles.

    Sam M, Shah S, Fridén J, Milner DJ, Capetanaki Y and Lieber RL

    Departments of Orthopaedics, Bioengineering, and Applied Mechanics and Engineering Sciences, Biomedical Sciences Graduate Group, University of California, San Diego and Veterans Administration Medical Centers, San Diego, California 92161, USA.

    The functional role of the skeletal muscle intermediate filament system was investigated by measuring the magnitude of muscle force loss after cyclic eccentric contraction (EC) in normal and desmin null mouse extensor digitorum longus muscles. Isometric stress generated was significantly greater in wild-type (313 +/- 8 kPa) compared with knockout muscles (276 +/- 13 kPa) before EC (P < 0.05), but 1 h after 10 ECs, both muscle types generated identical levels of stress ( approximately 250 kPa), suggesting less injury to the knockout. Differences in injury susceptibility were not explained by the different absolute stress levels imposed on wild-type versus knockout muscles (determined by testing older muscles) or by differences in fiber length or mechanical energy absorbed. Morphometric analysis of longitudinal electron micrographs indicated that Z disks from knockout muscles were more staggered (0.36 +/- 0. 03 microm) compared with wild-type muscles (0.22 +/- 0.03 microm), which may indicate that the knockout cytoskeleton is more compliant. These data demonstrate that lack of the intermediate filament system decreases isometric stress production and that the desmin knockout muscle is less vulnerable to mechanical injury.

    Funded by: NIAMS NIH HHS: AR40050

    American journal of physiology. Cell physiology 2000;279;4;C1116-22

  • Thoracic skeletal defects in myogenin- and MRF4-deficient mice correlate with early defects in myotome and intercostal musculature.

    Vivian JL, Olson EN and Klein WH

    Department of Biochemistry and Molecular Biology and Graduate Program in Genes & Development, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77050, USA.

    Myogenin and MRF4 are skeletal muscle-specific bHLH transcription factors critical for muscle development. In addition to a variety of skeletal muscle defects, embryos homozygous for mutations in myogenin or MRF4 display phenotypes in the thoracic skeleton, including rib fusions and sternal defects. These skeletal defects are likely to be secondary because myogenin and MRF4 are not expressed in the rib cartilage or sternum. In this study, the requirement for myogenin and MRF4 in thoracic skeletal development was further examined. When a hypomorphic allele of myogenin and an MRF4-null mutation were placed together, the severity of the thoracic skeletal defects was greatly increased and included extensive rib cartilage fusion and fused sternebrae. Additionally, new rib defects were observed in myogenin/MRF4 compound mutants, including a failure of the rib cartilage to contact the sternum. These results suggested that myogenin and MRF4 share overlapping functions in thoracic skeletal formation. Spatial expression patterns of skeletal muscle-specific markers in myogenin- and MRF4-mutant embryos revealed early skeletal muscle defects not previously reported. MRF4-/- mice displayed abnormal intercostal muscle morphology, including bifurcation and fusion of adjacent intercostals. myogenin/MRF4-mutant combinations displayed ventral myotome defects, including a failure to express normal levels of myf5. The results suggested that the early muscle defects observed in myogenin and MRF4 mutants may cause subsequent thoracic skeletal defects, and that myogenin and MRF4 have overlapping functions in ventral myotome differentiation and intercostal muscle morphogenesis.

    Developmental biology 2000;224;1;29-41

  • [The expression of connexin 43 and desmin in viral myocarditis].

    Zhu Y, Mao Z, Lou D and Zhang H

    Department of Pathology, Medical School, Zhejiang University, Hangzhou 310031, China.

    Objective: To study the cytobiological basis of constriction dysfunction and arrhythmia in cardiac muscle cells with viral myocarditis.

    Methods: The expression of connexin 43 and desmin in cardiac muscle cells of mice with experimental viral myocarditis was determined by immunohistochemistry.

    Results: In normal mice, connexin 43 and desmin are located in the intercalated disks of cardiac muscle, and the latter also revealed a positive immunoreactivity in the cross striations of sarcomeres. In viral myocarditis, the expression of both became much weakened, can even become negative.

    Conclusion: In myocarditis, expression of connexin 43 and desmin in the involved cardiac muscle cells was inhibited, resulting in dysfunction of gap junctional communication and arrhythmia.

    Zhonghua bing li xue za zhi = Chinese journal of pathology 2000;29;4;288-90

  • Myf-5 revisited: loss of early myotome formation does not lead to a rib phenotype in homozygous Myf-5 mutant mice.

    Kaul A, Köster M, Neuhaus H and Braun T

    Institute of Physiological Chemistry, University of Halle-Wittenberg, Germany.

    Cell 2000;102;1;17-9

  • Essential role of p38alpha MAP kinase in placental but not embryonic cardiovascular development.

    Adams RH, Porras A, Alonso G, Jones M, Vintersten K, Panelli S, Valladares A, Perez L, Klein R and Nebreda AR

    European Molecular Biology Laboratory, Heidelberg, Germany.

    p38alpha MAP kinase is activated in response to many cellular stresses and also regulates the differentiation and/or survival of various cell types in vitro, including skeletal muscle cells and cardiomyocytes. Here we show that targeted inactivation of the mouse p38alpha gene results in embryonic lethality at midgestation correlating with a massive reduction of the myocardium and malformation of blood vessels in the head region. However, this defect appears to be secondary to insufficient oxygen and nutrient transfer across the placenta. When the placental defect was rescued, p38alpha(-/-) embryos developed to term and were normal in appearance. Our results indicate that p38alpha is required for placental organogenesis but is not essential for other aspects of mammalian embryonic development.

    Molecular cell 2000;6;1;109-16

  • Characterization of a zebrafish (Danio rerio) desmin cDNA: an early molecular marker of myogenesis.

    Loh SH, Chan WT, Gong Z, Lim TM and Chua KL

    Department of Biological Sciences, National University of Singapore.

    Desmin is a muscle-specific protein and a constitutive subunit of the intermediate filaments (IF) in skeletal, cardiac and smooth muscles. It is an early marker of skeletal muscle myogenesis. We have characterized a clone of desmin cDNA from an embryonic zebrafish (Danio rerio) cDNA library. The full-length cDNA comprised 1798 nucleotides, encoding a protein of 473 amino acids. The predicted amino acid sequence of the zebrafish desmin shares a high degree of similarity to other vertebrate desmins, but also contains a sequence at the carboxyl terminal of the tail domain that is unique to the zebrafish. It carries many features which are distinctive of IF subunit proteins. These include the T/SSYRRXF/Y motif in the head domain, and the intermediate filament signature consensus, [I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E], located in the carboxyl terminus of the central helical rod. Unlike other 3' UTR sequences, the 3' UTR of the zebrafish cDNA sequence has two CAYUG elements flanking a single polyadenylation site. The temporal and spatial expression patterns of desmin mRNA during early zebrafish development were studied. The onset of desmin expression occurred at the 1-3 somite stage (11 hpf). It increased throughout somitogenesis, with maximum expression at the Prim-6 stage (25 hpf), and decreasing expression towards the protruding-mouth stage (72 hpf). Desmin mRNA was initially localised exclusively to the somites, but was subsequently also detected in other musculature in the developing heart and fins. The onset of expression and the spatial localization of desmin mRNA in the zebrafish coincides with that reported for MyoD and myogenin.

    Differentiation; research in biological diversity 2000;65;5;247-54

  • Transdifferentiation of esophageal smooth to skeletal muscle is myogenic bHLH factor-dependent.

    Kablar B, Tajbakhsh S and Rudnicki MA

    Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.

    Previously, coexpression of smooth and skeletal differentiation markers, but not myogenic regulatory factors (MRFs), was observed from E16.5 mouse fetuses in a small percentage of diaphragm level esophageal muscle cells, suggesting that MRFs are not involved in the process of initiation of developmentally programmed transdifferentiation in the esophagus. To investigate smooth-to-skeletal esophageal muscle transition, we analyzed Myf5nlacZ knock-in mice, MyoD-lacZ and myogenin-lacZ transgenic embryos with a panel of the antibodies reactive with myogenic regulatory factors (MRFs) and smooth and skeletal muscle markers. We observed that lacZ-expressing myogenic precursors were not detected in the esophagus before E15.5, arguing against the hypothesis that muscle precursor cells populate the esophagus at an earlier stage of development. Rather, the expression of the MRFs initiated in smooth muscle cells in the upper esophagus of E15.5 mouse embryos and was immediately followed by the expression of skeletal muscle markers. Moreover, transdifferentiation was markedly delayed or absent only in the absence of Myf5, suggesting that appropriate initiation and progression of smooth-to-skeletal muscle transdifferentiation is Myf5-dependent. Accordingly, the esophagus of Myf5(-/-):MyoD(-/-)embryos completely failed to undergo skeletal myogenesis and consisted entirely of smooth muscle. Lastly, extensive proliferation of muscularis precursor cells, without programmed cell death, occurred concomitantly with esophageal smooth-to-skeletal muscle transdifferentiation. Taken together, these results indicate that transdifferentiation is the fate of all smooth muscle cells in the upper esophagus and is normally initiated by Myf5.

    Development (Cambridge, England) 2000;127;8;1627-39

  • Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4.

    Valdez MR, Richardson JA, Klein WH and Olson EN

    Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard, Dallas, Texas, 75235-9148, USA.

    The basic helix-loop-helix (bHLH) transcription factors-MyoD, Myf5, myogenin, and MRF4-can each activate the skeletal muscle-differentiation program in transfection assays. However, their functions during embryogenesis, as revealed by gene-knockout studies in mice, are distinct. MyoD and Myf5 have redundant functions in myoblast specification, whereas myogenin and either MyoD or MRF4 are required for differentiation. Paradoxically, myoblasts from myogenin mutant or MyoD/MRF4 double-mutant neonates differentiate normally in vitro, despite their inability to differentiate in vivo, suggesting that the functions of the myogenic bHLH factors are influenced by the cellular environment and that the specific myogenic defects observed in mutant mice do not necessarily reflect essential functions of these factors. Understanding the individual roles of these factors is further complicated by their ability to cross-regulate one another's expression. To investigate the functions of Myf5 in the absence of contributions from other myogenic bHLH factors, we generated triple-mutant mice lacking myogenin, MyoD, and MRF4. These mice appear to contain a normal number of myoblasts, but in contrast to myogenin or MyoD/MRF4 mutants, differentiated muscle fibers fail to form in vivo and myoblasts from neonates of this triple-mutant genotype are unable to differentiate in vitro. These results suggest that physiological levels of Myf5 are insufficient to activate the myogenic program in the absence of other myogenic factors and suggest that specialized functions have evolved for the myogenic bHLH factors to switch on the complete program of muscle gene expression.

    Developmental biology 2000;219;2;287-98

  • Defective glomerulogenesis in the absence of laminin alpha5 demonstrates a developmental role for the kidney glomerular basement membrane.

    Miner JH and Li C

    Renal Division, Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri, 63110, USA.

    Laminins are major components of all basement membranes. They are a diverse group of alpha/beta/gamma heterotrimers formed from five alpha, three beta, and three gamma chains. Laminin alpha5 is a widely expressed chain found in many embryonic and adult basement membranes. During embryogenesis, alpha5 has a role in disparate developmental processes, including neural tube closure, digit septation, and placentation. Here, we analyzed kidney development in Lama5 mutant embryos and found a striking defect in glomerulogenesis associated with an abnormal glomerular basement membrane (GBM). This correlates with failure of the developmental switch in laminin alpha chain deposition in which alpha5 replaces alpha1 in the GBM at the capillary loop stage of glomerulogenesis. In the absence of a normal GBM, glomerular epithelial cells were in disarray, and endothelial and mesangial cells were extruded from within the constricting glomerulus, leading to a complete absence of vascularized glomeruli. In addition, a minority of Lama5 mutant mice lacked one or both kidneys, indicating that laminin alpha5 is also important in earlier kidney development. Our results demonstrate a dual role for laminin alpha5 in kidney development, illustrate a novel defect in glomerulogenesis, and indicate a heretofore unappreciated developmental role for the GBM in influencing the behavior of epithelial and endothelial cells.

    Funded by: NIDDK NIH HHS: P50 DK45181

    Developmental biology 2000;217;2;278-89

  • Expression of myogenic regulatory factors during the development of mouse tongue striated muscle.

    Yamane A, Mayo M, Shuler C, Crowe D, Ohnuki Y, Dalrymple K and Saeki Y

    Department of Pharmacology, School of Dental Medicine, Tsurumi University, Yokohama, Japan. gah03667@nifty.ne.jp

    While the role of myogenic regulatory factors (MRFs) in skeletal myogenesis has been well evaluated in limb and trunk muscles, very little is known about their role in tongue myogenesis. Here the expression of MRF mRNA in mouse tongue muscle was examined during development from embryonic day (E)11 to birth and compared them with that in hind-limb muscle. Desmin, muscle creatine kinase and troponin C mRNAs were used as markers for myoblast determination, myotubule formation and myofibre maturation, respectively. The mRNA quantities were determined by competitive reverse transcriptase-polymerase chain reaction. The expression profile of desmin mRNA indicated that myoblast determination occurred before E11 in both the tongue and hind-limb muscles; the profile of muscle creatine kinase and troponin C mRNAs indicated that myotubule formation and myofibre maturation began between E11 and 13 in both tongue and hind-limb muscles, but ended 2 days earlier in the tongue than in the hind limb. Expression of myoD and myogenin mRNAs began at E11, increased, and showed peak values earlier in the tongue muscle (E13) than in the hind-limb muscle (E15). Expression of MRF4 mRNA appeared earlier in the tongue (E13) than in the hind-limb muscle (E15) and increased in both muscles after that. These results suggest that myotubule formation and myofibre maturation in the tongue muscle progress faster than in the hind-limb muscle, a result of earlier expression of myoD, myogenin, and MRF4 in response to earlier functional demands such as suckling immediately after birth.

    Archives of oral biology 2000;45;1;71-8

  • The absence of desmin leads to cardiomyocyte hypertrophy and cardiac dilation with compromised systolic function.

    Milner DJ, Taffet GE, Wang X, Pham T, Tamura T, Hartley C, Gerdes AM and Capetanaki Y

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

    Desmin is the muscle-specific member of the intermediate filament family of cytoskeletal proteins, expressed both in striated and smooth muscle tissues. In mature striated muscle fibers, the desmin filament lattice surrounds the Z-discs, interconnects them to each other and links the entire contractile apparatus to the sarcolemmal cytoskeleton, cytoplasmic organelles and the nucleus. There have been increasing reports of human cardiomyopathies associated with abnormal accumulation and aggregation of desmin filaments. Recently identified desmin mutations in humans suffering from skeletal muscle myopathy and cardiomyopathy suggest that these diseases might arise as a consequence of impaired function of desmin filaments. Previous generation of desmin null mice in our laboratory demonstrated that the absence of desmin results in myocyte ultrastructural defects and myocyte cell death leading to fibrosis and calcification of the myocardium. However, the effects that these defects have on cardiac function were not addressed. To further our understanding of desmin function in vivo, and in order to address the direct involvement of desmin in cardiomyopathy, we investigated the effect of the absence of desmin on myocardial mass, myocyte size and shape, changes in gene expression and cardiac systolic and diastolic function in mice. Morphometric characterization of isolated cardiomyocytes demonstrated a 24% increase in cell volume in the desmin null mice, solely due to an increase in transverse section area, suggesting for the first time that mice lacking the intermediate filament protein desmin develop concentric cardiomyocyte hypertrophy. This type of hypertrophy was accompanied by induction of embryonic gene expression and later by ventricular dilatation, and compromised systolic function. These results demonstrate that desmin is essential for normal cardiac function, and they suggest that the absence of an intact desmin filament system, rather than accumulation of the protein, may be responsible for the pathology seen in some of the desmin associated cardiomyopathies.

    Funded by: NIA NIH HHS: AG13251; NIAMS NIH HHS: AR 39617-09

    Journal of molecular and cellular cardiology 1999;31;11;2063-76

  • Early development of the myotome in the mouse.

    Venters SJ, Thorsteinsdóttir S and Duxson MJ

    Department of Anatomy and Structural Biology, University of Otago, Dunedin, New Zealand. vensa566@student.otago.ac.nz

    The structure and development of the myotome has been extensively studied in birds and amphibians but few studies have systematically addressed its development in mammals. We have used a transgenic mouse carrying an nLacZ marker coupled to a myosin light chain 3F promoter to describe the structure of the developing mammalian myotome. Through studies of transgene expression pattern, coupled with immunohistochemistry for the muscle structural proteins desmin and slow myosin heavy chain we describe a gradient of maturity for the cells within the developing myotome. Our results show that the earliest myocytes of the mammalian myotome span the rostrocaudal extent of the somite and have single large nuclei which localise centrally within the myotome. Throughout the period of study the myotome is more mature ventrally than dorsally and cells comprising the medial aspect of the myotome are younger than those lying laterally. Immunohistochemistry for the earliest expressed muscle regulatory factor (myf-5) is used to define areas of the myotome contributing new myogenic cells. In the early myotome small, round, myf-5-expressing cells are found extensively within the dorsomedial aspect of the dermamyotome and also within the entire rostral and caudal dermamyotomal lips. They subsequently appear within the central zone of the myotome, adjacent to the medially curled rostral and caudal dermamyotomal lips, and there begin to elongate symmetrically. As the myotome enlarges, myf-5 expression is always restricted to the most medial aspect of the myotome, adjacent to the least mature myocytes, marking the site of addition of new myogenic cells. Together, these results allow development of a model of mammalian myotome formation where growth occurs medially by addition of new cells from both rostral and caudal dermamyotome lips, while more mature myocytes are displaced laterally. Furthermore, early myotomal myocytes differentiate in the absence of MyoD expression, unlike later myotomal myocytes. This, along with their distinct morphology, suggests these cells may form a separate lineage of pioneer myogenic cells.

    Developmental dynamics : an official publication of the American Association of Anatomists 1999;216;3;219-32

  • In vivo modeling of myosin binding protein C familial hypertrophic cardiomyopathy.

    Yang Q, Sanbe A, Osinska H, Hewett TE, Klevitsky R and Robbins J

    Department of Pediatrics, Division of Molecular Cardiovascular Biology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229-3039, USA.

    Myosin binding protein C (MyBP-C) is an integral part of the striated muscle sarcomere. As is the case for other sarcomeric genes in human populations, multiple mutations within the gene have been linked to familial hypertrophic cardiomyopathy. Although some MyBP-C lesions are the result of missense mutations, most show truncated polypeptides lacking either the myosin or myosin and titin binding sites. Previously, we generated transgenic (TG) mice with cardiac-specific expression of a MyBP-C mutant lacking the myosin and titin binding domains. Surprisingly, the mutant protein was stable and made up a majority of the MyBP-C species, with concomitant reductions in endogenous MyBP-C such that overall MyBP-C stoichiometry was conserved. In the present study, we created a second series of TG mice that express, in the heart, a mutant MyBP-C lacking only the myosin binding site. In contrast to the previous data for the MyBP-C lacking both titin and myosin binding sites, only very modest levels of protein were found, consistent with data obtained from human biopsies in which mutated MyBP-C could not be detected. Despite normal levels of wild-type MyBP-C, there were significant changes in the structure and ultrastructure of the heart. Fiber mechanics showed decreased unloading shortening velocity, maximum shortening velocity, and relative maximal power output.

    Funded by: NHLBI NIH HHS: HL41496, HL56370, HL56620; ...

    Circulation research 1999;85;9;841-7

  • Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knock-out mice.

    Carlsson L, Li Z, Paulin D and Thornell LE

    Department of Integrative Medical Biology, Section for Anatomy, Umeå University, Umeå, SE-901 87, Sweden.

    Desmin, the main component of intermediate filaments (IFs) in mature skeletal muscle, forms an interlinking scaffold around myofibrils with connections to the sarcolemma and the nuclear membrane. Desmin is enriched in neuromuscular and myotendinous junctions. Mice lacking the desmin gene develop normally and reproduce. However, postnatally they develop a cardiomyopathy and a dystrophy in highly used muscles. We have investigated whether and how neuromuscular and myotendinous junctions are affected and whether nestin compensates for the lack of desmin in the knock-out (K/O) mice. We show that neither neuromuscular nor myotendinous junctions were markedly affected in the desmin K/O mice. In neuromuscular junctions nestin was present between the postjunctional folds and the subneural nuclei and between the nucleus and the myofibrillar cytoskeleton. In myotendinous junctions nestin was present between myofibrils at the Z-disc level and in longitudinal strands close to and at the junction. Nestin expression at these specialized sites, as well as during myogenesis and myofibrillogenesis, is independent of the presence of desmin. In desmin K/O mice nestin was also found in regenerating myofibers. The presence of nestin at neuromuscular and myotendinous junctions might provide enough strength for preservation and organization of the junctional areas, although desmin is lacking.

    Experimental cell research 1999;251;1;213-23

  • MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

    Saga Y, Miyagawa-Tomita S, Takagi A, Kitajima S, Miyazaki Ji and Inoue T

    Cellular and Molecular Toxicology Division, National Institute of Health Sciences, Setagaya-ku, Tokyo 158, Japan. saga@nihs.go.jp

    The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.

    Development (Cambridge, England) 1999;126;15;3437-47

  • Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.

    Carmeliet P, Lampugnani MG, Moons L, Breviario F, Compernolle V, Bono F, Balconi G, Spagnuolo R, Oosthuyse B, Dewerchin M, Zanetti A, Angellilo A, Mattot V, Nuyens D, Lutgens E, Clotman F, de Ruiter MC, Gittenberger-de Groot A, Poelmann R, Lupu F, Herbert JM, Collen D and Dejana E

    Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium. peter.carmelilet@med.kuleuven.ac.be

    Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.

    Cell 1999;98;2;147-57

  • Downregulation of connexin 45 gene products during mouse heart development.

    Alcoléa S, Théveniau-Ruissy M, Jarry-Guichard T, Marics I, Tzouanacou E, Chauvin JP, Briand JP, Moorman AF, Lamers WH and Gros DB

    Laboratoire de Génétique et Physiologie du Développement, Institut de Biologie du Développement de Marseille, CNRS/INSERM/AP Marseille/Université de la Méditerranée, Campus de Luminy, Marseille.

    The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.

    Circulation research 1999;84;12;1365-79

  • Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.

    Hellström M, Kalén M, Lindahl P, Abramsson A and Betsholtz C

    Department of Medical Biochemistry, Göteborg University, Box 440, SE 405 30 Göteborg, Sweden. christer.betsholtz@medkem.gu.se

    Development of a vascular system involves the assembly of two principal cell types - endothelial cells and vascular smooth muscle cells/pericytes (vSMC/PC) - into many different types of blood vessels. Most, if not all, vessels begin as endothelial tubes that subsequently acquire a vSMC/PC coating. We have previously shown that PDGF-B is critically involved in the recruitment of pericytes to brain capillaries and to the kidney glomerular capillary tuft. Here, we used desmin and alpha-smooth muscle actin (ASMA) as markers to analyze vSMC/PC development in PDGF-B-/- and PDGFR-beta-/- embryos. Both mutants showed a site-specific reduction of desmin-positive pericytes and ASMA-positive vSMC. We found that endothelial expression of PDGF-B was restricted to immature capillary endothelial cells and to the endothelium of growing arteries. BrdU labeling showed that PDGFR-beta-positive vSMC/PC progenitors normally proliferate at sites of endothelial PDGF-B expression. In PDGF-B-/- embryos, limb arterial vSMC showed a reduced BrdU-labeling index. This suggests a role of PDGF-B in vSMC/PC cell proliferation during vascular growth. Two modes of vSMC recruitment to newly formed vessels have previously been suggested: (1) de novo formation of vSMC by induction of undifferentiated perivascular mesenchymal cells, and (2) co-migration of vSMC from a preexisting pool of vSMC. Our data support both modes of vSMC/PC development and lead to a model in which PDGFR-beta-positive vSMC/PC progenitors initially form around certain vessels by PDGF-B-independent induction. Subsequent angiogenic sprouting and vessel enlargement involves PDGF-B-dependent vSMC/PC progenitor co-migration and proliferation, and/or PDGF-B-independent new induction of vSMC/PC, depending on tissue context.

    Development (Cambridge, England) 1999;126;14;3047-55

  • Functions of c-Jun in liver and heart development.

    Eferl R, Sibilia M, Hilberg F, Fuchsbichler A, Kufferath I, Guertl B, Zenz R, Wagner EF and Zatloukal K

    Department of Pathology, University of Graz, A-8036 Graz, Austria.

    Mice lacking the AP-1 transcription factor c-Jun die around embryonic day E13.0 but little is known about the cell types affected as well as the cause of embryonic lethality. Here we show that a fraction of mutant E13.0 fetal livers exhibits extensive apoptosis of both hematopoietic cells and hepatoblasts, whereas the expression of 15 mRNAs, including those of albumin, keratin 18, hepatocyte nuclear factor 1, beta-globin, and erythropoietin, some of which are putative AP-1 target genes, is not affected. Apoptosis of hematopoietic cells in mutant livers is most likely not due to a cell-autonomous defect, since c-jun-/- fetal liver cells are able to reconstitute all hematopoietic compartments of lethally irradiated recipient mice. A developmental analysis of chimeras showed contribution of c-jun-/- ES cell derivatives to fetal, but not to adult livers, suggesting a role of c-Jun in hepatocyte turnover. This is in agreement with the reduced mitotic and increased apoptotic rates found in primary liver cell cultures derived from c-jun-/- fetuses. Furthermore, a novel function for c-Jun was found in heart development. The heart outflow tract of c-jun-/- fetuses show malformations that resemble the human disease of a truncus arteriosus persistens. Therefore, the lethality of c-jun mutant fetuses is most likely due to pleiotropic defects reflecting the diversity of functions of c-Jun in development, such as a role in neural crest cell function, in the maintenance of hepatic hematopoiesis and in the regulation of apoptosis.

    The Journal of cell biology 1999;145;5;1049-61

  • YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis.

    Moore AW, McInnes L, Kreidberg J, Hastie ND and Schedl A

    MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK.

    The Wilms' Tumour gene WT1 has important functions during development. Knock-out mice were shown to have defects in the urogenital system and to die at embryonic day E13.5, probably due to heart failure. Using a lacZ reporter gene inserted into a YAC construct, we demonstrate that WT1 is expressed in the early proepicardium, the epicardium and the subepicardial mesenchymal cells (SEMC). Lack of WT1 leads to severe defects in the epicardial layer and a concomitant absence of SEMCs, which explains the pericardial bleeding and subsequent embryonic death observed in Wt1 null embryos. We further show that a human-derived WT1 YAC construct is able to completely rescue heart defects, but only partially rescues defects in the urogenital system. Analysis of the observed hypoplastic kidneys demonstrate a continuous requirement for WT1 during nephrogenesis, in particular, in the formation of mature glomeruli. Finally, we show that the development of adrenal glands is also severely affected in partially rescued embryos. These data demonstrate a variety of new functions for WT1 and suggest a general requirement for this protein in the formation of organs derived from the intermediate mesoderm.

    Funded by: Wellcome Trust

    Development (Cambridge, England) 1999;126;9;1845-57

  • Catenary cultures of embryonic gastrointestinal tract support organ morphogenesis, motility, neural crest cell migration, and cell differentiation.

    Hearn CJ, Young HM, Ciampoli D, Lomax AE and Newgreen D

    The Murdoch Institute, Parkville, Victoria, Australia. hearnc@cryptic.rch.unimelb.edu.au

    The embryonic gastrointestinal tract develops from a simple tube into a coiled, flexed, and regionalized structure. The changes in gut morphology coincide with the differentiation of multiple cell types in concentric layers, and include colonization by migratory neuron precursors, and the development of gastrointestinal motility. We describe a reliable method for growing embryonic mouse intestine in vitro by the attachment of segments of intestinal tract by their cut ends, with the intervening region suspended in the culture medium. These are termed "catenary cultures." E11-E11.5 mouse midgut, hindgut, or mid- plus hindgut segments were grown in catenary culture for up to 10 days and their growth, morphology, cell differentiation, ability to support neural precursor migration, and contractile activity were assessed. The increase in size of the cultured explants was not large, but morphogenesis proceeded, best exemplified by elongation of the caecum. Cell differentiation also proceeded. In the mucosa, goblet cells differentiated. Muscle layers, characterized by desmin expression, and kit-positive interstitial cells of Cajal differentiated in the correct positions. Where segments initially included neural precursors in a small sub-region, these migrated and proliferated to form uniform neuronal networks throughout the entire explant, and the cells expressed the neuron markers nitric oxide synthase and neuron specific enolase. Gut motility was attained 5-6 days into the culture period, and both contractile- and mixing-type movements were observed. Thus, cell types representative of all three germ layer contributions developed, and in addition, the gut, being mainly free, was able to elongate and bend (unlike on solid support cultures), while retaining its rostrocaudal identity.

    Developmental dynamics : an official publication of the American Association of Anatomists 1999;214;3;239-47

  • Reduced differentiation potential of primary MyoD-/- myogenic cells derived from adult skeletal muscle.

    Sabourin LA, Girgis-Gabardo A, Seale P, Asakura A and Rudnicki MA

    Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.

    To gain insight into the regeneration deficit of MyoD-/- muscle, we investigated the growth and differentiation of cultured MyoD-/- myogenic cells. Primary MyoD-/- myogenic cells exhibited a stellate morphology distinct from the compact morphology of wild-type myoblasts, and expressed c-met, a receptor tyrosine kinase expressed in satellite cells. However, MyoD-/- myogenic cells did not express desmin, an intermediate filament protein typically expressed in cultured myoblasts in vitro and myogenic precursor cells in vivo. Northern analysis indicated that proliferating MyoD-/- myogenic cells expressed fourfold higher levels of Myf-5 and sixfold higher levels of PEA3, an ETS-domain transcription factor expressed in newly activated satellite cells. Under conditions that normally induce differentiation, MyoD-/- cells continued to proliferate and with delayed kinetics yielded reduced numbers of predominantly mononuclear myocytes. Northern analysis revealed delayed induction of myogenin, MRF4, and other differentiation-specific markers although p21 was upregulated normally. Expression of M-cadherin mRNA was severely decreased whereas expression of IGF-1 was markedly increased in MyoD-/- myogenic cells. Mixing of lacZ-labeled MyoD-/- cells and wild-type myoblasts revealed a strict autonomy in differentiation potential. Transfection of a MyoD-expression cassette restored cytomorphology and rescued the differentiation deficit. We interpret these data to suggest that MyoD-/- myogenic cells represent an intermediate stage between a quiescent satellite cell and a myogenic precursor cell.

    The Journal of cell biology 1999;144;4;631-43

  • Abrogation of the Cripto gene in mouse leads to failure of postgastrulation morphogenesis and lack of differentiation of cardiomyocytes.

    Xu C, Liguori G, Persico MG and Adamson ED

    The Burnham Institute, La Jolla Cancer Research Center, N. Torrey Pines Road, La Jolla, CA 92037, USA.

    Cripto-1(Cr1) protein encoded by the tdgf1 gene, is a secreted growth factor that is expressed early in embryonic development and is re-expressed in some tumors of the breast and colon. During embryonic development, Cr1 is expressed in inner cell mass cells and the primitive streak, and later is restricted to the developing heart. To investigate the role of Cr1 during mouse development, mice were generated that contain a null mutation of both Cr1 genes, derived from homologous recombination in embryonic stem cells. No homozygous Cr1-/- mice were born, indicating that Cr1 is necessary for embryonic development. Embryos initiated gastrulation and some embryos produced mesoderm up to day E7.5. Increasingly aberrant morphogenesis gave rise to disordered neuroepithelium that failed to produce a recognizable neural tube, or head-fold. Although some biochemical markers of differentiating ectoderm, mesoderm and endoderm were expressed, all the cardiac-specific markers were absent from day E8.7 embryos: (&agr;)MHC, betaMHC, MLC2A, MLC2V and ANF, whereas they were expressed in wild-type embryos. The yolk sac and placental tissues continued development in the absence of the embryo until day E9.5 but lacked large yolk sac blood vessels. Chimeric mice were constructed by microinjection of double targeted Cr1(-/- )embryonic stem cells into normal C57BL/6 blastocysts. The Cr1 produced by the normal C57BL/6 cells fully rescued the phenotype of Cr1(-/-) cells, indicating that Cr1 protein acted in a paracrine manner. Cells derived from the embryo proliferated and migrated poorly and had different adhesion properties compared to wild type. Therefore, lethality in the absence of Cr1, likely resulted largely from defective precardiac mesoderm that was unable to differentiate into functional cardiomyocytes.

    Development (Cambridge, England) 1999;126;3;483-94

  • Strain-dependent embryonic lethality in mice lacking the retinoblastoma-related p130 gene.

    LeCouter JE, Kablar B, Whyte PF, Ying C and Rudnicki MA

    Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada L8S 4K1.

    The retinoblastoma-related p130 protein is a member of a conserved family, consisting of Rb, p107 and p130, which are believed to play important roles in cell-cycle control and cellular differentiation. We have generated a null mutation in p130 by gene targeting and crossed the null allele into Balb/cJ and C57BL/6J strains of mice. In an enriched Balb/cJ genetic background, p130(-/-) embryos displayed arrested growth and died between embryonic days 11 and 13. Histological analysis revealed varying degrees of disorganization in neural and dermamyotomal structures. Immunohistochemistry with antibody reactive with Islet-1 indicated markedly reduced numbers of neurons in the spinal cord and dorsal root ganglia. Immunohistochemistry with antibody reactive with desmin indicated a similar reduction in the number of differentiated myocytes in the myotome. The myocardium of mutant embryos was abnormally thin and resembled an earlier staged two-chambered heart consisting of the bulbus cordis and the ventricular chamber. TUNEL analysis indicated the presence of extensive apoptosis in various tissues including the neural tube, the brain, the dermomyotome, but not the heart. Immunohistochemistry with antibody reactive with PCNA revealed increased cellular proliferation in the neural tube and the brain, and decreased proliferation in the heart. The placentas of p130(-/-) embryos did not display elevated apoptosis and were indistinguishable from wild type suggesting that the phenotype was not due to placental failure. Following a single cross with the C57BL/6 mice, p130(-/-) animals were derived that were viable and fertile. These results indicate that p130 in a Balb/cJ genetic background plays an essential role that is required for normal development. Moreover, our experiments establish that second-site modifier genes exist that have an epistatic relationship with p130.

    Development (Cambridge, England) 1998;125;23;4669-79

  • Basal lamina molecules are concentrated in myogenic regions of the mouse limb bud.

    Godfrey EW and Gradall KS

    Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee 53225, USA. egodfrey@mcw.edu

    Molecular components of basal lamina, such as laminin, stimulate the differentiation of skeletal muscle cells in culture, while interstitial matrix components such as fibronectin are inhibitory. However, the role of extracellular matrix (ECM) molecules in muscle cell differentiation in the embryo is less well understood. As a first step toward understanding the role of the ECM in embryonic myogenesis, the localization of basal lamina molecules in the mouse limb bud before and during muscle cell differentiation was determined by immunofluorescence. Laminin, collagen type IV and nidogen (entactin) were concentrated in myogenic regions of the limb bud both before and during differentiation of skeletal muscle cells. Punctate immunofluorescence for basal lamina molecules was concentrated in dorsal and ventral premuscle and muscle masses, when compared with other regions of limb mesenchyme. In contrast, immunofluorescence for fibronectin, an interstitial extracellular matrix molecule, was decreased in premuscle and muscle masses. These results suggest that basal lamina components play an important stimulatory role in early stages of skeletal muscle differentiation in the developing mouse limb bud.

    Funded by: NICHD NIH HHS: HD20743

    Anatomy and embryology 1998;198;6;481-6

  • Sox4-deficiency syndrome in mice is an animal model for common trunk.

    Ya J, Schilham MW, de Boer PA, Moorman AF, Clevers H and Lamers WH

    Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, and the Department of Immunology, University of Utrecht, The Netherlands.

    Embryonic mice lacking functional Sox4 transcription factor die from cardiac failure at embryonic day (ED) 14. Heart morphogenesis in these embryos was analyzed in hematoxylin-azophlochsin or immunohistochemically stained, 3-dimensionally reconstructed serial sections between ED12 and ED14. Although Sox4 is expressed in the endocardially derived tissue of both the outflow tract and atrioventricular canal, Sox4-deficient hearts only suffer from defective transformation of the endocardial ridges into semilunar valves and from lack of fusion of these ridges, usually resulting in common trunk, although the least affected hearts should be classified as having a large infundibular septal defect. The more serious cases are, in addition, characterized by an abnormal number and position of the semilunar valve-leaflet anlagen, a configuration of the ridges typical for transposition of the great arteries (with linear rather than spiral course of both ridges and posterior position of the pulmonary trunk at the level of the valve), and variable size of the aorta relative to the pulmonary trunk. The coronary arteries always originated from the aorta, irrespective of its position relative to the pulmonary trunk. The restriction of the malformations to the arterial pole implies that the interaction between the endocardially derived tissue of the outflow tract and the neural crest-derived myofibroblasts determines proper development of the arterial pole.

    Circulation research 1998;83;10;986-94

  • Muscle-specific locus control region activity associated with the human desmin gene.

    Raguz S, Hobbs C, Yagüe E, Ioannou PA, Walsh FS and Antoniou M

    UMDS, Guy's Hospital, London Bridge, London, SE1 9RT, United Kingdom.

    We describe the reproduction of the full pattern of expression of the muscle-specific desmin gene in transgenic mice using a 240-kb genomic clone spanning the human desmin locus. Analysis of RNA from adult tissues demonstrated that this fragment possesses all the necessary genetic regulatory elements required to provide reproducible, site-of-integration-independent, physiological levels of tissue-specific expression that is directly proportional to transgene copy number in all muscle cell types. In situ hybridization revealed that in marked contrast to murine desmin which is strongly expressed in the myotome of the somites, skeletal muscles, the heart, and smooth muscle of the vasculature by 9.5 days postcoitum, human desmin transgene expression was completely absent from smooth muscles, was very weak and restricted to the atrium and outflow tract within the heart, and was expressed at only 5% of murine desmin mRNA levels within the myotome of the somites. The spatial distribution and levels of human and mouse desmin expression were not coincident until 14.5 days postcoitum. Immunohistochemical analysis of human embryos at comparable stages of development showed that this transgene faithfully reproduces the human and not the mouse developmental expression pattern for this gene in transgenic mice. These results indicate that the 240-kb desmin genomic clone is capable of establishing an independent, chromatin domain in transgenic mice and provides the first definitive data for muscle-specific locus control region activity. In addition, our results demonstrate that the behavior of human transgenes in mice should, whenever possible, be compared to expression patterns for that gene in human embryonic as well as adult tissues.

    Funded by: Wellcome Trust

    Developmental biology 1998;201;1;26-42

  • Paracrine PDGF-B/PDGF-Rbeta signaling controls mesangial cell development in kidney glomeruli.

    Lindahl P, Hellström M, Kalén M, Karlsson L, Pekny M, Pekna M, Soriano P and Betsholtz C

    Department of Medical Biochemistry, Göteborg University, PO Box 440, SE-405 30 Göteborg, Sweden.

    Kidney glomerulus mesangial cells fail to develop in mice carrying targeted null mutations in the platelet-derived growth factor (PDGF)-B or PDGF-Rbeta genes. We have examined the pattern of expression of these genes and smooth muscle markers during kidney development, to address the possible mechanisms underlying the mutant phenotypes. In wild-type embryos, PDGF-B was expressed in vascular endothelial cells, particularly in capillary endothelial cells in the developing glomeruli, whereas PDGF-Rbeta was found in perivascular mesenchymal cells in the developing renal cortex. In the course of glomerular development, small groups of PDGF-Rbeta and desmin-expressing cells collected in the 'S'-shaped and early cup-shaped vesicles, and at later stages such cells were found in the glomerular mesangium. In PDGF-B or -Rbeta null embryos, some PDGF-Rbeta/desmin or desmin-positive cells, respectively, were seen in early cup-shaped vesicles, but fewer than in the wild type, and further development of the mesangium failed. In mouse chimeras composed of PDGF-Rbeta +/+ and -/- cells, the Rbeta-/- cells failed to populate the glomerular mesangium. Our results show that while the mesangial cell lineage is specified independently of PDGF-B/Rbeta, these molecules provide critical permissive signals in mesangial cell development. We propose a model in which mesangial cells originate from PDGF-Rbeta-positive progenitors surrounding the developing glomerular afferent and efferent arterioles, and are co-recruited in response to PDGF-B during angiogenic formation of the glomerular capillary tuft.

    Development (Cambridge, England) 1998;125;17;3313-22

  • Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis.

    Yang Y, Palmer KC, Relan N, Diglio C and Schuger L

    Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, USA.

    Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.

    Funded by: NHLBI NIH HHS: HL48730-01

    Development (Cambridge, England) 1998;125;14;2621-9

  • Induced expression of myoD, myogenin and desmin during myoblast differentiation in embryonic mouse tongue development.

    Yamane A, Takahashi K, Mayo M, Vo H, Shum L, Zeichner-David M and Slavkin HC

    Department of Pharmacology, School of Dental Medicine, Tsurumi University, Yokohama, Japan.

    Significant progress has been made in defining mechanisms governing myogenesis at the transcriptional levels, but the extracellular signal-transduction pathways involved in myogenesis are not as yet defined. The developing mouse tongue provides a model for the regulation of myogenesis during precise time periods in embryogenesis. The molecular cues that regulate the close-range autocrine and/or paracrine signalling processes required for the fast-twitch complex tongue musculature are not known. This study was designed to test the hypothesis that transforming growth factor-alpha (TGF alpha) controls myogenesis in embryonic mouse tongue through the induction of myogenic regulatory factors such as myoD, myf5, myogenin and MRF4/myf6/herculin. To test this hypothesis, the effects of exogenous TGF alpha on the transcription of myoD, myf5, myogenin, MRF4 and desmin were examined in tongue samples from embryonic day-10.5 mandibular explants cultured in serum-free, chemically defined medium and then processed for competitive, reverse transcription-polymerase chain reaction. TGF alpha induced myoD, myogenin and desmin expression. Treatment with 20 and 40 ng/ml TGF alpha decreased or downregulated myf5 mRNA. MRF4 was not detected in the explants. TGF alpha apparently induces the early developmental stages of myogenesis through sequential upregulation of myoD and myogenin, downregulation of myf5 and corresponding significant increases in muscle-specific gene expression such as desmin transcription.

    Funded by: NIAMS NIH HHS: Z01-AR 41114-02-BCTB; NIDCR NIH HHS: DE-02824

    Archives of oral biology 1998;43;5;407-16

  • Heart defects in connexin43-deficient mice.

    Ya J, Erdtsieck-Ernste EB, de Boer PA, van Kempen MJ, Jongsma H, Gros D, Moorman AF and Lamers WH

    Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, The Netherlands.

    Cardiac malformation in connexin43 (CX43)-disrupted mice is restricted to the junction between right ventricle and outflow tract, even though CX43 is also expressed abundantly elsewhere. We analyzed cardiac morphogenesis in immunohistochemically and hybridohistochemically stained and three-dimensionally reconstructed serial sections of CX43-deficient embryos between embryonic day (ED) 10 and birth. The establishment of the D configuration in the ascending loop of CX43-deficient hearts is markedly retarded, so that the right ventricle retains a craniomedial position and is connected with the outflow tract by a more acute bend in ED10 and ED11 embryos. Because of the subsequent growth of the right ventricle, this condition usually evolves into a D loop, but when it persists, a "crisscross" configuration develops, with the atrioventricular cushions rotated 90 degrees, a horizontal muscular ventricular septum, and a parallel course of the endocardial ridges of the outflow tract. After ED12, large intertrabecular pouches develop at the ventricular side of both shelflike myocardial structures that support the endocardial ridges of the outflow tract, ie, at the location that was earlier characterized by the acute bend between the right ventricle and the outflow tract and that subsequently develops into the anterosuperior leaflet of the tricuspid valve. Retarded development of the D configuration in the ascending loop of the embryonic heart predisposes the myocardium at the junction of the right ventricle and outflow tract to excessive development of intertrabecular pouches during subsequent development.

    Circulation research 1998;82;3;360-6

  • Isoform transitions of the myosin binding protein C family in developing human and mouse muscles: lack of isoform transcomplementation in cardiac muscle.

    Gautel M, Fürst DO, Cocco A and Schiaffino S

    European Molecular Biology Laboratory, Heidelberg, Germany. Gautel@EMBL-Heidelberg.de

    Mutations in the gene for the cardiac isoform of myosin binding protein C (MyBP-C) have been identified as the cause of chromosome 11-associated autosomal-dominant familial hypertrophic cardiomyopathy (FHC). Most mutations produce a truncated polypeptide that lacks the sarcomeric binding region. We have now investigated the expression pattern of the cardiac and skeletal isoforms of cMyBP-C in mice and humans by in situ hybridization and immunofluorescence microscopy using specific antibodies and probes. We demonstrate that the cardiac isoform is expressed only in cardiac muscle throughout development. The slow and fast isoforms of MyBP-C remain specific for skeletal muscle, where they can be coexpressed. Immunological evidence also suggests that an embryonic isoform of MyBP-C precedes the expression of slow MyBP-C in developing skeletal muscle. This suggests that transcomplementation of MyBP-C isoforms is possible in skeletal but not cardiac muscle.

    Circulation research 1998;82;1;124-9

  • MyoD and Myf-5 define the specification of musculature of distinct embryonic origin.

    Kablar B, Asakura A, Krastel K, Ying C, May LL, Goldhamer DJ and Rudnicki MA

    Institute for Molecular Biology and Biotechnology, Cancer Research Group, McMaster University, Hamilton, ON, Canada.

    Mounting evidence supports the notion that Myf-5 and MyoD play unique roles in the development of epaxial (originating in the dorso-medial half of the somite, e.g. back muscles) and hypaxial (originating in the ventro-lateral half of the somite, e.g. limb and body wall muscles) musculature. To further understand how Myf-5 and MyoD genes cooperate during skeletal muscle specification, we examined and compared the expression pattern of MyoD-lacZ (258/2.5lacZ and MD6.0-lacZ) transgenes in wild-type, Myf-5, and MyoD mutant embryos. We found that the delayed onset of muscle differentiation in the branchial arches, tongue, limbs, and diaphragm of MyoD-/- embryos was a consequence of a reduced ability of myogenic precursor cells to progress through their normal developmental program and not because of a defect in migration of muscle progenitor cells into these regions. We also found that myogenic precursor cells for back, intercostal, and abdominal wall musculature in Myf-54-/- embryos failed to undergo normal translocation or differentiation. By contrast, the myogenic precursors of intercostal and abdominal wall musculature in MyoD-/- embryos underwent normal translocation but failed to undergo timely differentiation. In conclusion, these observations strongly support the hypothesis that Myf-5 plays a unique role in the development of muscles arising after translocation of epithelial dermamyotome cells along the medial edge of the somite to the subjacent myotome (e.g., back or epaxial muscle) and that MyoD plays a unique role in the development of muscles arising from migratory precursor cells (e.g., limb and branchial arch muscles, tongue, and diaphragm). In addition, the expression pattern of MyoD-lacZ transgenes in the intercostal and abdominal wall muscles of Myf-5-/- and MyoD-/- embryos suggests that appropriate development of these muscles is dependent on both genes and, therefore, these muscles have a dual embryonic origin (epaxial and hypaxial).

    Biochemistry and cell biology = Biochimie et biologie cellulaire 1998;76;6;1079-91

  • MyoD and Myf-5 differentially regulate the development of limb versus trunk skeletal muscle.

    Kablar B, Krastel K, Ying C, Asakura A, Tapscott SJ and Rudnicki MA

    Institute for Molecular Biology and Biotechnology, Cancer Research Group, McMaster University, Hamilton, Ontario, Canada.

    The myogenic progenitors of epaxial (paraspinal and intercostal) and hypaxial (limb and abdominal wall) musculature are believed to originate in dorsal-medial and ventral-lateral domains, respectively, of the developing somite. To investigate the hypothesis that Myf-5 and MyoD have different roles in the development of epaxial and hypaxial musculature, we further characterized myogenesis in Myf-5- and MyoD-deficient embryos by several approaches. We examined expression of a MyoD-lacZ transgene in Myf-5 and MyoD mutant embryos to characterize the temporal-spatial patterns of myogenesis in mutant embryos. In addition, we performed immunohistochemistry on sectioned Myf-5 and MyoD mutant embryos with antibodies reactive with desmin, nestin, myosin heavy chain, sarcomeric actin, Myf-5, MyoD and myogenin. While MyoD(-/-) embryos displayed normal development of paraspinal and intercostal muscles in the body proper, muscle development in limb buds and brachial arches was delayed by about 2.5 days. By contrast, Myf-5(-/-) embryos displayed normal muscle development in limb buds and brachial arches, and markedly delayed development of paraspinal and intercostal muscles. Although MyoD mutant embryos exhibited delayed development of limb musculature, normal migration of Pax-3-expressing cells into the limb buds and normal subsequent induction of Myf-5 in myogenic precursors was observed. These results suggest that Myf-5 expression in the limb is insufficient for the normal progression of myogenic development. Taken together, these observations strongly support the hypothesis that Myf-5 and MyoD play unique roles in the development of epaxial and hypaxial muscle, respectively.

    Development (Cambridge, England) 1997;124;23;4729-38

  • 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

  • Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development.

    Lindahl P, Karlsson L, Hellström M, Gebre-Medhin S, Willetts K, Heath JK and Betsholtz C

    Department of Medical Biochemistry and Microbiology, University of Göteborg, Sweden.

    PDGF-A(-/-) mice lack lung alveolar smooth muscle cells (SMC), exhibit reduced deposition of elastin fibres in the lung parenchyma, and develop lung emphysema due to complete failure of alveogenesis. We have mapped the expression of PDGF-A, PDGF receptor-alpha, tropoelastin, smooth muscle alpha-actin and desmin in developing lungs from wild type and PDGF-A(-/-) mice of pre- and postnatal ages in order to get insight into the mechanisms of PDGF-A-induced alveolar SMC formation and elastin deposition. PDGF-A was expressed by developing lung epithelium. Clusters of PDGF-Ralpha-positive (PDGF-Ralpha+) mesenchymal cells occurred at the distal epithelial branches until embryonic day (E) 15.5. Between E16.5 and E17.5, PDGF-Ralpha+ cells multiplied and spread to acquire positions as solitary cells in the terminal sac walls, where they remained until the onset of alveogenesis. In PDGF-A(-/-) lungs PDGF-Ralpha+ cells failed to multiply and spread and instead remained in prospective bronchiolar walls. Three phases of tropoelastin expression were seen in the developing lung, each phase characterized by a distinct pattern of expression. The third phase, tropoelastin expression by developing alveolar SMC in conjunction with alveogenesis, was specifically and completely absent in PDGF-A(-/-) lungs. We propose that lung PDGF-Ralpha+ cells are progenitors of the tropoelastin-positive alveolar SMC. We also propose that postnatal alveogenesis failure in PDGF-A(-/-) mice is due to a prenatal block in the distal spreading of PDGF-Ralpha+ cells along the tubular lung epithelium during the canalicular stage of lung development.

    Funded by: Wellcome Trust

    Development (Cambridge, England) 1997;124;20;3943-53

  • Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system.

    Delorme B, Dahl E, Jarry-Guichard T, Briand JP, Willecke K, Gros D and Théveniau-Ruissy M

    Laboratoire de Génétique et Physiologie du Développement, UMR C9943,Institut de Biologie du Développement de Marseille, France.

    The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ-hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.

    Circulation research 1997;81;3;423-37

  • Mice lacking the ski proto-oncogene have defects in neurulation, craniofacial, patterning, and skeletal muscle development.

    Berk M, Desai SY, Heyman HC and Colmenares C

    Department of Cancer Biology, Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.

    The c-ski proto-oncogene has been implicated in the control of cell growth and skeletal muscle differentiation. To determine its normal functions in vivo, we have disrupted the mouse c-ski gene. Our results show a novel role for ski in the morphogenesis of craniofacial structures and the central nervous system, and confirm its proposed function as a player in skeletal muscle development. Homozygous mutant mice show perinatal lethality resulting from exencephaly, a defect caused by failed closure of the cranial neural tube during neurulation. The timing of the neural tube defect in ski -/- embryos coincides with excessive apoptosis in the cranial neuroepithelium, as well as in the cranial mesenchyme. Homozygous ski mutants also exhibit a dramatic reduction in skeletal muscle mass, consistent with a defect in expansion of a myogenic precursor population. Nestin is an intermediate filament expressed in highly proliferative neuroepithelial stem cells and in myogenic precursors. Interestingly, we find decreased nestin expression in both the cranial neural tube and the somites of ski -/- embryos, compared with their normal littermates, but no reduction of nestin in the caudal neural tube. These results are consistent with a model in which ski activities are required for the successful expansion of a subset of precursors in the neuroepithelial or skeletal muscle lineages.

    Funded by: NICHD NIH HHS: HD30728

    Genes & development 1997;11;16;2029-39

  • Persistent expression of MNF identifies myogenic stem cells in postnatal muscles.

    Garry DJ, Yang Q, Bassel-Duby R and Williams RS

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, 75235, USA.

    Skeletal muscles contain an undifferentiated myogenic stem cell pool (satellite cells) that can be mobilized to regenerate myofibers in response to injury. We have determined that the winged helix transcription factor MNF is expressed selectively in quiescent satellite cells, which do not express known regulators of the myogenic program. Following muscle injury, MNF is present transiently in proliferating satellite cells and in centralized nuclei of regenerating myofibers, but expression declines as these fibers mature, until only the residual stem cell pool continues to express detectable levels of MNF. MNF also is expressed selectively but transiently at embryonic stages of myogenesis in the developing myotome, limb bud precursors, and heart tube, but by late fetal stages of development, MNF is down-regulated within differentiated cardiac and skeletal myocytes, and persistently high expression is observed only in satellite cells. These data identify MNF as a marker of quiescent satellite cells and suggest that downstream genes controlled by MNF serve to modulate proliferative growth or differentiation in this unique cell population.

    Funded by: NHLBI NIH HHS: HL03231; NIAMS NIH HHS: R01 AR40849

    Developmental biology 1997;188;2;280-94

  • Null mutation in the desmin gene gives rise to a cardiomyopathy.

    Thornell L, Carlsson L, Li Z, Mericskay M and Paulin D

    Department of Anatomy, Umeâ University, Umeâ, Sweden.

    A null mutation in the desmin gene has been introduced into the germ line of mice. Such mice develop and reproduce normally proving that desmin is not needed either for the formation of the heart or the alignment of functioning myofibrils. However, cardiovascular lesions and a skeletal myopathy were observed in growing and adult mice. In the present study we have carried out a detailed analysis of these cardiac lesions. Homozygous mutant mice, which were confirmed to lack expression of desmin mRNA and desmin protein in the heart, were revealed by electron microscopy to contain degenerating cardiomyocytes as early as 5 days post-partum. At 10 days post-partum and onwards the degeneration of cardiomyocytes gave rise to areas with an accumulation of macrophages, fibrosis and calcification preferentially in the inter-ventricular septum and the free wall of the right ventricle. The localization of the lesions mainly to these sites suggested that it is not the work load and contractions per se which were the pathogenic events leading to the cardiomyopathy. It might be that stress related to lengthening of the myocytes occur more in the right ventricle than in the left. At the ultrastructural level changes in the intercalated discs, disruption of the sarcolemma and supercontraction of myofibrils seemed to be the key events leading to cardiomyocyte death. Thus, the intermediate filaments are required to maintain the basic integrity of cardiomyocytes and especially the link between the intermediate filaments and the sarcolemma appear more important than previously realized.

    Journal of molecular and cellular cardiology 1997;29;8;2107-24

  • TGF-alpha, EGF, and their cognate EGF receptor are co-expressed with desmin during embryonic, fetal, and neonatal myogenesis in mouse tongue development.

    Yamane A, Mayo ML, Bringas P, Chen L, Huynh M, Thai K, Shum L and Slavkin HC

    Center for Craniofacial Molecular Biology, School of Dentistry, University of Southern California, Los Angeles, USA.

    The developing mouse tongue provides a model for discrete patterns of morphogenesis during short periods of embryonic development. Occipital somite-derived myogenic cells interact with cranial neural crest-derived ecto-mesenchymal cells to form the musculature of the tongue. The biochemical signals that control close range autocrine and/or paracrine signaling processes required to establish the fast-twitch complex tongue musculature are not known. The present study was designed to test the hypothesis that desmin, epidermal growth factor (EGF), and transforming growth factor-alpha (TGF alpha) and their cognate receptor, epidermal growth factor receptor (EGFr), are co-expressed during tongue myogenesis and define specific developmental stages of tongue muscle cell differentiation. To test this hypothesis, we performed studies to analyze the timing, position, and concentration of desmin, TGF alpha, EGF, and EGFr from embryonic day 9 (E9) through birth in Swiss Webster mouse tongue development. Desmin, TGF alpha, EGF, and EGFr co-localized to cells of myogenic lineage in the four occipital somites and subsequently in myoblasts and myotubes from E9 through E17. By newborn stage, desmin is localized to discrete regions in myofibers corresponding to Z-line delimiting sarcomeres, and A-band within sarcomeres; immunostaining for desmin, TGF alpha, and EGF persisted in differentiated myotubes and striated skeletal muscle. Desmin increased from 0.01% at E11 to 0.51% of the total protein by E17 and at birth. Concomitantly, the patterns and increases in TGF alpha, EGF, and EGFr showed significant increases during the same developmental period. The temporal and positional co-localization of TGF alpha, EGF, and EGFr support the hypothesis that autocrine and paracrine regulation of desmin by actions of growth factor ligand and receptor defines critical stages of tongue myogenesis.

    Funded by: NIDCR NIH HHS: DE-02848

    Developmental dynamics : an official publication of the American Association of Anatomists 1997;209;4;353-66

  • A potential role of R-cadherin in striated muscle formation.

    Rosenberg P, Esni F, Sjödin A, Larue L, Carlsson L, Gullberg D, Takeichi M, Kemler R and Semb H

    Department of Microbiology, Umeå University, Sweden.

    We have examined the murine embryonic expression pattern of the cell adhesion molecule R-cadherin in muscle, kidney, thymus, and lung. In developing muscle, R-cadherin was first seen at 10.5-11.5 days postcoitum in the somitic myotome. Consistently, we found R-cadherin expressed at the highest levels in the myotome, early skeletal muscle, and smooth muscle (both vascular and visceral), while very low levels of R-cadherin were detected in the heart. The expression pattern and subcellular localization of R-cadherin in developing skeletal muscle indicate a possible role in myoblast cell-cell interactions during both primary and secondary myogenesis. In the developing kidney, R-cadherin was first detected at 10.5 days postcoitum in the mesonephric epithelial tubule cells. In the metanephric kidney, it was specifically expressed in the pretubular aggregates, comma- and S-shaped bodies, proximal tubules, and collecting ducts. Thus, in the kidney, R-cadherin was associated with the mesenchymal-epithelial transition. R-cadherin was also found in other developing epithelia, for example in the thymic epithelial cells. In the lung, R-cadherin was expressed at the highest levels in the smooth muscle surrounding the lung epithelial tubules. To test whether R-cadherin can direct formation of tissues, we constitutively expressed R-cadherin in E-cadherin-/- ES cells and examined histogenesis in teratomas derived from these cells. R-cadherin exclusively rescued formation of striated muscle and epithelia in the teratomas. R-cadherin's ability to form epithelia in vivo was substantiated by its ability to rescue formation of cystic embryoid bodies in vitro. By comparing our data with the previously reported embryonic expression patterns and histogenetic activities of E- and N-cadherin, we suggest that R-cadherin plays an important role in the formation of striated muscle and possibly also of epithelia.

    Developmental biology 1997;187;1;55-70

  • Redefining the genetic hierarchies controlling skeletal myogenesis: Pax-3 and Myf-5 act upstream of MyoD.

    Tajbakhsh S, Rocancourt D, Cossu G and Buckingham M

    Department of Molecular Biology, Centre National de la Recherche Scientifique, Unité de Recherche Associe 1947, Pasteur Institute, Paris, France.

    We analyzed Pax-3 (splotch), Myf-5 (targeted with nlacZ), and splotch/Myf-5 homozygous mutant mice to investigate the roles that these genes play in programming skeletal myogenesis. In splotch and Myf-5 homozygous embryos, myogenic progenitor cell perturbations and early muscle defects are distinct. Remarkably, splotch/Myf-5 double homozygotes have a dramatic phenotype not seen in the individual mutants: body muscles are absent. MyoD does not rescue this double mutant phenotype since activation of this gene proves to be dependent on either Pax-3 or Myf-5. Therefore, Pax-3 and Myf-5 define two distinct myogenic pathways, and MyoD acts genetically downstream of these genes for myogenesis in the body. This genetic hierarchy does not appear to operate for head muscle formation.

    Funded by: Telethon: A.067

    Cell 1997;89;1;127-38

  • Desmin in muscle formation and maintenance: knockouts and consequences.

    Capetanaki Y, Milner DJ and Weitzer G

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

    Desmin, the muscle-specific member of the intermediate filament (IF) family, is one of the earliest known myogenic markers in both skeletal muscle and heart. Its expression precedes that of all known muscle proteins including the members of the MyoD family of myogenic helix-loop-helix (mHLH) regulators with the exception of myf5. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. In vitro studies using both antisense RNA and homologous recombination techniques in embryonic stem (ES) cells demonstrated that desmin plays a crucial role during myogenesis, as inhibition of desmin expression blocked myoblast fusion and myotube formation. Both in C2C12 cells and differentiating embryoid bodies, the absence of desmin interferes with the normal myogenic program, as manifested by the inhibition of the mHLH transcription regulators. To investigate the function of desmin in all muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, a considerable number of these mice are viable and fertile, potentially due to compensation by vimentin, nestin or synemin. However, desmin null mice demonstrate a multisystem disorder involving cardiac, skeletal and smooth muscle, beginning early in their postnatal life. Histological and electron microscopic analysis in both heart and skeletal muscle tissues reveals severe disruption of muscle architecture and degeneration. Structural abnormalities include loss of lateral alignment of myofibrils, perturbation of myofibril anchorage to the sarcolemma, abnormal mitochondrial number and organization, and loss of nuclear shape and positioning. Loose cell adhesion and increased intercellular space are prominent defects. The consequences of these abnormalities are most severe in the heart, which exhibits progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. There is a direct correlation between severity of damage and muscle usage, possibly due to increased susceptibility to normal mechanical damage and/or to repair deficiency in the absence of desmin. In conclusion, the studies so far have demonstrated that though desmin is absolutely necessary for muscle differentiation in vitro, muscle development can take place in vivo in the absence of this intermediate filament protein. However, desmin seems to play an essential role in the maintenance of myofibril, myofiber and whole muscle tissue structural and functional integrity.

    Funded by: NIAMS NIH HHS: AR 39617-01

    Cell structure and function 1997;22;1;103-16

  • Distribution of nestin in the developing mouse limb bud in vivo and in micro-mass cultures of cells isolated from limb buds.

    Wroblewski J, Engström M, Edwall-Arvidsson C, Sjöberg G, Sejersen T and Lendahl U

    Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.

    Early skeletal muscle development is accompanied by changes in the composition of the cytoskeleton. In this report we analyze the distribution of the intermediate filament nestin in the developing mouse limb buds in vivo and in mesenchymal cells isolated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cells was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing limb bud. Later, nestin mRNA and protein were down-regulated in the mesenchymal condensations undergoing chondrogenesis (E12 and E13), but remained expressed predominantly in the ectodermal cells and in the differentiating myoblasts. At E18, only muscle fibres, endothelial cells and nerves were nestin positive. This shift in expression was reproduced in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultures, nestin protein was initially expressed in all cells. Upon formation of cartilage foci (after 2-3 days in culture), nestin immunoreactivity was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucleated cells, peripheral to the cartilage nodules, expressed the muscle-specific intermediate filament desmin protein together with high levels of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin-positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mRNA is predominantly located in the ends of the muscle cell, whereas the protein is found in the central region. Intracellular localization of nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development.

    Differentiation; research in biological diversity 1997;61;3;151-9

  • 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

  • Distinct alpha 7A beta 1 and alpha 7B beta 1 integrin expression patterns during mouse development: alpha 7A is restricted to skeletal muscle but alpha 7B is expressed in striated muscle, vasculature, and nervous system.

    Velling T, Collo G, Sorokin L, Durbeej M, Zhang H and Gullberg D

    Department of Animal Physiology, Uppsala University, Sweden.

    The laminin binding alpha 7 beta 1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of alpha 7 integrin undergoes alternative splicing to generate two major forms, denoted alpha 7A and alpha 7B. In the current paper, we have examined the developmental expression patterns of the alpha 7A and alpha 7B splice variants in the mouse. The alpha 7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin-alpha 1 and laminin-alpha 2 chains. Alpha 7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for alpha 7B integrin and in situ hybridization revealed the presence of alpha 7 mRNA and alpha 7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, alpha 7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of alpha 7B in the myocardium. In addition to striated muscle, alpha 7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the alpha 7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, alpha 7 integrin, and laminin-alpha 2, but not laminin-alpha 1, in the forming skeletal muscle. We conclude that the alpha 7A and alpha 7B integrin variants are expressed in a developmentally regulated, tissue-specific pattern suggesting different functions for the two splice forms.

    Developmental dynamics : an official publication of the American Association of Anatomists 1996;207;4;355-71

  • Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.

    Milner DJ, Weitzer G, Tran D, Bradley A and Capetanaki Y

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

    Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystem disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed severe disruption of muscle architecture and degeneration. Structural abnormalities included loss of lateral alignment of myofibrils and abnormal mitochondrial organization. The consequences of these abnormalities were most severe in the heart, which exhibited progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. The present data demonstrate the essential role of desmin in the maintenance of myofibril, myofiber, and whole muscle tissue structural and functional integrity, and show that the absence of desmin leads to muscle degeneration.

    Funded by: NIAMS NIH HHS: AR 39617-01

    The Journal of cell biology 1996;134;5;1255-70

  • A high-resolution linkage map of the tight skin 2 (Tsk2) locus: a mouse model for scleroderma (SSc) and other cutaneous fibrotic diseases.

    Christner PJ, Siracusa LD, Hawkins DF, McGrath R, Betz JK, Ball ST, Jimenez SA and Peters J

    Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Mammalian genome : official journal of the International Mammalian Genome Society 1996;7;8;610-2

  • Cardiovascular lesions and skeletal myopathy in mice lacking desmin.

    Li Z, Colucci-Guyon E, Pinçon-Raymond M, Mericskay M, Pournin S, Paulin D and Babinet C

    Biologie moléculaire de la différenciation, SCME, Paris, France.

    In order to further our understanding of the biological role of desmin, the muscle-specific intermediate filament protein, a null mutation in the desmin gene was introduced into the germ line of mice. Despite the complete lack of desmin, these mice developed and reproduced. Since we show that skeletal, cardiac, and smooth muscles form in the Des-/- mice, it is reasonable to propose that desmin is not essential for myogenic commitment or for myoblast fusion or differentiation in vivo. However, morphological abnormalities were observed in the diaphragm of adult mice; these were demonstrated by disorganized, distended, and nonaligned fibers. The heart presented areas of hemorrhaging in which fibrosis and ischemia were observed. We have also shown that the absence of desmin produces specific defects in smooth muscles. In conclusion, our results have demonstrated that desmin is not required for the differentiation of skeletal, cardiac, and smooth muscles but is essential to strengthen and maintain the integrity of these tissues.

    Developmental biology 1996;175;2;362-6

  • Analysis of skeletal and cardiac muscle from desmin knock-out and normal mice by high resolution separation of myosin heavy-chain isoforms.

    Agbulut O, Li Z, Mouly V and Butler-Browne GS

    CNRS-URA 1448, UFR Biomedical de St-Pères, Paris, France.

    In this study, using a modified electrophoretic technique, we have defined in the mouse the myosin heavy-chain composition of both newborn and adult skeletal and cardiac muscles. Using this high resolution technique it was possible to detect modifications in the myosin heavy-chain expression in both cardiac and skeletal muscles of desmin knock-out mice.

    Biology of the cell / under the auspices of the European Cell Biology Organization 1996;88;3;131-5

  • Report and abstracts of the Fourth International Workshop on Human Chromosome 2 Mapping 1996.

    Spur NK, Bashir R, Bushby K, Cox A, Cox S, Hilde Brandt F, Hill N, Kao FT, Krols L, Marzella R, Miller N, Nothwang HG, Rocchi M, Sarfarazi M, Stratakis CA, Wallgren-Petterson C and Naylor S

    Cytogenetics and cell genetics 1996;73;4;255-73

  • Cytoskeletal control of myogenesis: a desmin null mutation blocks the myogenic pathway during embryonic stem cell differentiation.

    Weitzer G, Milner DJ, Kim JU, Bradley A and Capetanaki Y

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

    A differentiating system based on embryonic stem (ES) cell-derived embryoid bodies (EBs) which recapitulates the in vivo cardiac, skeletal, and smooth muscle myogenesis of mouse embryos was developed and used to investigate the effects of the disruption of the desmin gene on muscle cell differentiation. Wild-type, heterozygous, and homozygous cell lines with the mutated desmin allele were evaluated. Skeletal myogenesis was totally inhibited in desmin null mutant EBs, as manifested by the absence of myotube formation, contractility, and myoD, myogenin, myf5, and myosin heavy chain expression. Smooth muscle formation was also completely blocked in the absence of desmin. On the other hand, there were no obvious effects on cardiomyocyte differentiation in these desmin null mutant EBs. However, reduced desmin expression in EBs heterozygous for the desmin mutation leads to partial inhibition of cardiac muscle formation. These data suggest that in contrast to early cardiocyte differentiation, desmin is indispensable for skeletal and smooth muscle formation.

    Funded by: NIAMS NIH HHS: AR 39617-01

    Developmental biology 1995;172;2;422-39

  • Expression of laminin isoforms in mouse myogenic cells in vitro and in vivo.

    Schuler F and Sorokin LM

    Institute for Experimental Medicine, University of Edangen-Nuremberg, Germany.

    The expression of laminin-1 (previously EHS laminin) and laminin-2 (previously merosin) isoforms by myogenic cells was examined in vitro and in vivo. No laminin alpha 2 chainspecific antibodies react with mouse tissues, 50 rat monoclonal antibodies were raised against the mouse laminin alpha 2 chain: their characterization is described here. Myoblasts and myotubes from myogenic cell lines and primary myogenic cultures express laminin beta 1 and gamma 1 chains and form a complex with a 380 kDa alpha chain identified as laminin alpha 2 by immunofluorescence, immunoprecipitation and PCR. PCR from C2C12 myoblasts and myotubes for the laminin alpha 2 chain gene (LamA2) provided cDNA sequences which were used to investigate the in vivo expression of mouse LamA2 mRNA in embryonic tissues by in situ hybridization. Comparisons were made with specific probes for the laminin alpha 1 chain gene (LamA1). LamA2 but not LamA1 mRNA was expressed in myogenic tissues of 14- and 17-day-old mouse embryos, while the laminin alpha 2 polypeptide was localized in adjacent basement membranes in the muscle fibres. In situ hybridization also revealed strong expression of the LamA2 mRNA in the dermis, indicating that laminin alpha 2 is expressed other than by myogenic cells in vivo. Immunofluorescence studies localized laminin alpha 2 in basement membranes of basal keratinocytes and the epithelial cells of hair follicles, providing new insight into basement membrane assembly during embryogenesis. In vitro cell attachment assays revealed that C2C12 and primary myoblasts adhere to laminin-1 and -2 isoforms in a similar manner except that myoblast spreading was significantly faster on laminin-2. Taken together, the data suggest that laminins 1 and 2 play distinct roles in myogenesis.

    Journal of cell science 1995;108 ( Pt 12);3795-805

  • Myogenin's functions do not overlap with those of MyoD or Myf-5 during mouse embryogenesis.

    Rawls A, Morris JH, Rudnicki M, Braun T, Arnold HH, Klein WH and Olson EN

    Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.

    The four myogenic basic helix-loop-helix proteins, MyoD, myogenin, Myf-5, and MRF4, can each activate skeletal muscle differentiation when introduced into nonmuscle cells. During embryogenesis, each of these genes is expressed in a unique but overlapping pattern in skeletal muscle precursors and their descendants. Gene knockout experiments have shown that MyoD and Myf-5 play seemingly redundant roles in the generation of myoblasts. However, the role of either of these genes during differentiation in vivo has not been determined. In contrast, a myogenin-null mutation blocks differentiation and results in a dramatic decrease in muscle fiber formation, yet the role of myogenin in the generation or maintenance of myoblast populations is not known. Because myogenin possesses the same myogenic activity as MyoD and Myf-5 in vitro and the expression patterns of these three genes overlap in vivo, we sought to determine if myogenin shares certain functions with either MyoD or Myf-5 in vivo. We therefore generated mice with double homozygous null mutations in the genes encoding MyoD and myogenin or Myf-5 and myogenin. These mice showed embryonic and perinatal phenotypes characteristic of the combined defects observed in mice mutant for each gene alone. As shown by histological analysis and expression of muscle-specific genes, the numbers of undifferentiated myoblasts and residual myofibers were comparable between myogenin-mutant homozygotes and the double-mutant homozygotes. Myoblasts isolated from neonates of the combined mutant genotypes underwent myogenesis in tissue culture, indicating that no more than two of the four myogenic factors are required to support muscle differentiation. These results demonstrate that the functions of myogenin do not overlap with those of MyoD or Myf-5 and support the view that myogenin acts in a genetic pathway downstream of MyoD and Myf-5.

    Developmental biology 1995;172;1;37-50

  • Genes for tensin, villin and desmin are linked on mouse chromosome 1.

    Jankowski SA and Gumucio DL

    Department of Anatomy and Cell Biology, University of Michigan, Ann Arbor 48109-0616, USA.

    Funded by: NICHD NIH HHS: R29-HD28620

    Mammalian genome : official journal of the International Mammalian Genome Society 1995;6;10;744-5

  • Essential role for the c-met receptor in the migration of myogenic precursor cells into the limb bud.

    Bladt F, Riethmacher D, Isenmann S, Aguzzi A and Birchmeier C

    Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany.

    Limb muscles develop from cells that migrate from the somites. The signal that induces migration of myogenic precursor cells to the limb emanates from the mesenchyme of the limb bud. Here we report that the c-met-encoded receptor tyrosine kinase is essential for migration of myogenic precursor cells into the limb anlage and for migration into diaphragm and tip of tongue. In c-met homozygous mutant (-/-) mouse embryos, the limb bud and diaphragm are not colonized by myogenic precursor cells and, as a consequence, skeletal muscles of the limb and diaphragm do not form. In contrast, development of the axial skeletal muscles proceeds in the absence of c-met signalling. The specific ligand of the c-met protein, the motility and growth factor scatter factor/hepatocyte growth factor, is expressed in limb mesenchyme and can thus provide the signal for migration which is received by c-met. We have therefore identified a paracrine signalling system that regulates migration of myogenic precursor cells.

    Nature 1995;376;6543;768-71

  • Inactivation of Myf-6 and Myf-5 genes in mice leads to alterations in skeletal muscle development.

    Braun T and Arnold HH

    Department of Cell and Molecular Biology, University of Braunschweig, Germany.

    Myf-6, alternatively called MRF4 or herculin, is a member of a group of muscle-specific transcription factors which also comprises Myf-5, myogenin and MyoD. All family members show distinct expression patterns during skeletal muscle development and can convert a variety of cell lines to myocytes. We disrupted the Myf-6 gene in mice to investigate its functional role in the network of regulatory factors controlling myogenesis. Homozygous mice carrying the disrupted Myf-6 gene show pronounced down-regulation of Myf-5 transcription for reasons presently unknown. Consequently, these mice represent a double knock-out model for Myf-6 and Myf-5. The mutants resemble most of the Myf-5 phenotype with aberrant and delayed early myotome formation and lack of distal rib structures. In addition, we find a reduction in the size of axial muscles in the back. Apart from changes in the pattern of some contractile protein isoforms, the existing myofibers appear fairly normal. This suggests that Myf-6 has no major role in the maturation of myotubes, as previously proposed. Our results provide evidence that skeletal myogenesis can proceed in the absence of two myogenic factors, Myf-5 and Myf-6, therefore they must exert largely non-redundant functions in vivo.

    The EMBO journal 1995;14;6;1176-86

  • Myogenin is required for late but not early aspects of myogenesis during mouse development.

    Venuti JM, Morris JH, Vivian JL, Olson EN and Klein WH

    Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.

    Mice with a targeted mutation in the myogenic basic helix-loop-helix regulatory protein myogenin have severe muscle defects resulting in perinatal death. In this report, the effect of myogenin's absence on embryonic and fetal development is investigated. The initial events of somite differentiation occurred normally in the myogenin-mutant embryos. During primary myogenesis, muscle masses in mutant embryos developed simultaneously with control siblings, although muscle differentiation within the mutant muscle masses was delayed. More dramatic effects were observed when secondary myofibers form. During this time, very little muscle formation took place in the mutants, suggesting that the absence of myogenin affected secondary myogenesis more severely than primary myogenesis. Monitoring mutant neonates with fiber type-specific myosin isoforms indicated that different fiber types were present in the residual muscle. No evidence was found to indicate that myogenin was required for the formation of muscle in one region of the embryo and not another. The expression patterns of a MyoD-lacZ transgene in myogenin-mutant embryos demonstrated that myogenin was not essential for the activation of the MyoD gene. Together, these results indicate that late stages of embryogenesis are more dependent on myogenin than early stages, and that myogenin is not required for the initial aspects of myogenesis, including myotome formation and the appearance of myoblasts.

    Funded by: NCI NIH HHS: CA-16672

    The Journal of cell biology 1995;128;4;563-76

  • 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

  • Expression of M-cadherin protein in myogenic cells during prenatal mouse development and differentiation of embryonic stem cells in culture.

    Rose O, Rohwedel J, Reinhardt S, Bachmann M, Cramer M, Rotter M, Wobus A and Starzinski-Powitz A

    Institut der Anthropologie und Humangenetik für Biologen, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.

    Molecules regulating morphogenesis by cell-cell interactions are the cadherins, a class of calcium-dependent adhesion molecules. One of its members, M-cadherin, has been isolated from a myoblast cell line (Donalies et al. [1991] Proc. Natl. Acad. Sci. U.S.A. 88:8024-8028). In mouse development, expression of M-cadherin mRNA first appears at day 8.5 of gestation (E8.5) in somites and has been postulated to be down-regulated in developing muscle masses (Moore and Walsh [1993] Development 117:1409-1420). Affinity-purified polyclonal M-cadherin antibodies, detecting a protein of approximately 120 kDa, were used to study the cell expression pattern of M-cadherin protein. It was first visualized in somites at E10 1/3 and could be confined to desmin positive, myotomal cells. At all subsequent prenatal stages, M-cadherin was only found in myogenic cells of somitic origin. The detection of the protein at E10 1/3 suggests a translational delay of M-cadherin mRNA of 1 to 2 days (E8.5 vs. E10 1/3). This was further supported by the finding that during differentiation of ES cell line BLC6 into skeletal muscle cells in culture, expression of M-cadherin mRNA can be detected 2 days prior to M-cadherin protein. During prenatal development, the pattern of M-cadherin expression changes: In E10 1/3 embryos and also in myotomal cells of later stages, M-cadherin is evenly distributed on the cell surface. In developing muscle masses (tested at E16 to E18), however, M-cadherin protein becomes clustered most likely at sites of cell-cell contact as indicated by double-labelling experiments: M-cadherin-staining is the positive image of laminin negative areas excluding the presence of a basal lamina at M-cadherin positive sites. Furthermore, M-cadherin is coexpressed with the neuronal cell adhesion molecule N-CAM which has been shown to mediate cell-cell contact in myogenic cells. In summary, our results are in line with the idea that M-cadherin might play a central role in myogenic morphogenesis.

    Developmental dynamics : an official publication of the American Association of Anatomists 1994;201;3;245-59

  • MyoD expression marks the onset of skeletal myogenesis in Myf-5 mutant mice.

    Braun T, Bober E, Rudnicki MA, Jaenisch R and Arnold HH

    Department of Cell and Molecular Biology, University of Braunschweig, FRG.

    The expression pattern of myogenic regulatory factors and myotome-specific contractile proteins was studied during embryonic development of Myf-5 mutant mice by in situ hybridization and immunohistochemistry. In contrast to somites in wild-type embryos, no expression of myogenin and Myf-6 (MRF4), or any other myotomal markers was detected in mutant animals at E9.0 and E10.0 indicating that Myf-5 plays a crucial role during this developmental period. Significantly, the onset of MyoD expression in rostral somites of E10.5 embryos was unaffected by the Myf-5 mutation suggesting that the activation of the MyoD gene occurs independently of Myf-5 at the correct developmental time. Immediately after the activation of MyoD myogenin transcripts and protein accumulated within the myotome. The first contractile proteins of the sarcomeric apparatus appeared slightly later. By E11.5 the expression of muscle markers were indistinguishable between wild-type and Myf-5 mutant mice. The migration of muscle precursor cells that leave the somites to form limb musculature was monitored in Myf-5-mutant mice by Pax-3 expression. Pax-3-positive cells were equally found in somites and limbs of E10.0 wild-type and mutant mice indicating that myogenic factor expression at the level of somites is not a prerequisite for determination and subsequent migration of limb precursor cells.

    Development (Cambridge, England) 1994;120;11;3083-92

  • An E box in the desmin promoter cooperates with the E box and MEF-2 sites of a distal enhancer to direct muscle-specific transcription.

    Li H and Capetanaki Y

    Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.

    The first 85 nt upstream of the transcription initiation site of the mouse desmin gene, which contain an E box (E1), the binding site of the helix-loop-helix myogenic regulators, are sufficient to confer low level muscle-specific expression. High levels of desmin expression are due to an enhancer, located between nucleotides -798 and -976, which contains an additional E box (E2) and a muscle-specific enhancer factor-2 (MEF-2) binding site. We have previously shown that both myoD and myogenin can bind to the proximal (E1) and distal (E2) boxes. Here we demonstrate that MEF-2C, a myocyte-restricted member of the MEF-2 family, can bind to the desmin MEF-2 site. Functional units for the enhancer activity required intact E2 and MEF-2 elements. The desmin enhancer can function relatively well with either the E2 box or the MEF-2 site and only mutation of both eliminates transcriptional enhancement; the presence of both of these elements is required for maximum enhancer activity. On the other hand, mutagenesis of just the proximal E1 box showed that this element is essential for desmin gene expression. Double mutations of E1 with E2 or MEF-2 sites suggested that, to achieve high levels of desmin gene expression, E1 serves most possibly as an intermediary for either E2 or MEF-2 enhancer elements to function. The location of the E1 site relative to the TATA box is crucial. Its activity is DNA turn- and distance-dependent. Furthermore, this box seems to be the main element for desmin transactivation by myoD and myogenin in 10T1/2 cells. Its inactivation diminishes the transactivation by these factors; MRF4 and Myf5, however, can still partially function, possibly by using the distal E2 box.

    Funded by: NIAMS NIH HHS: AR 39617-01

    The EMBO journal 1994;13;15;3580-9

  • Inhibition of desmin expression blocks myoblast fusion and interferes with the myogenic regulators MyoD and myogenin.

    Li H, Choudhary SK, Milner DJ, Munir MI, Kuisk IR and Capetanaki Y

    Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

    The muscle-specific intermediate filament protein, desmin, is one of the earliest myogenic markers whose functional role during myogenic commitment and differentiation is unknown. Sequence comparison of the presently isolated and fully characterized mouse desmin cDNA clones revealed a single domain of polypeptide similarity between desmin and the basic and helix-loop-helix region of members of the myoD family myogenic regulators. This further substantiated the need to search for the function of desmin. Constructs designed to express anti-sense desmin RNA were used to obtain stably transfected C2C12 myoblast cell lines. Several lines were obtained where expression of the anti-sense desmin RNA inhibited the expression of desmin RNA and protein down to basal levels. As a consequence, the differentiation of these myoblasts was blocked; complete inhibition of myoblast fusion and myotube formation was observed. Rescue of the normal phenotype was achieved either by spontaneous revertants, or by overexpression of the desmin sense RNA in the defective cell lines. In several of the cell lines obtained, inhibition of desmin expression was followed by differential inhibition of the myogenic regulators myoD and/or myogenin, depending on the stage and extent of desmin inhibition in these cells. These data suggested that myogenesis is modulated by at least more than one pathway and desmin, which so far was believed to be merely an architectural protein, seems to play a key role in this process.

    Funded by: NIAMS NIH HHS: AR39617-01

    The Journal of cell biology 1994;124;5;827-41

  • Tenascin is induced at implantation sites in the mouse uterus and interferes with epithelial cell adhesion.

    Julian J, Chiquet-Ehrismann R, Erickson HP and Carson DD

    UT M.D. Anderson Cancer Center, Department of Biochemistry and Molecular Biology, Houston 77030.

    Expression of tenascin, an extracellular matrix protein associated with morphogenetic events and altered states of cellular adhesion, was examined in mouse uterus during the peri-implantation period. A uniform low level expression of tenascin was detected in stromal extracellular matrix during the estrous cycle and days 1 through 4 of early pregnancy. During the period of blastocyst attachment (day 4.5), an intense deposition of tenascin fibrils was located in the extracellular matrix of stroma immediately subjacent to the uterine epithelium surrounding the attaching blastocyst. This localized intensity of tenascin expression was both spatially and temporally restricted. By day 5.5, differentiation of stroma in the immediate area around the embryo to form the primary decidual zone was accompanied by a reduced amount of tenascin expression in the form of fragmented fibrils. Tenascin also could be induced by an artificial stimulus in uterine stroma of mice that had been hormonally prepared for implantation. The ability of artificial stimuli to induce tenascin expression suggested that the tenascin-inducing signals were derived from uterine cells, presumably lumenal epithelium, rather than embryonic cells. Consistent with this, conditioned medium from primary cultures of uterine epithelium was found to induce tenascin expression (2- to 4-fold) in isolated uterine stroma. Artificial stimuli generated a temporal pattern of tenascin expression similar to that observed during early pregnancy; however, in the artificially induced model, tenascin was induced in stroma immediately subjacent to lumenal epithelium along the entire length of the uterus. Purified tenascin and a recombinant tenascin fragment consisting of alternatively spliced fibronectin type III repeats, interfered with maintenance of uterine epithelial cell adhesion to Matrigel. In contrast, other recombinant tenascin fragments or fibronectin had no effect in this regard. Tenascin had no effect on adhesion of uterine stroma. Collectively, these results suggest that stimulation of TN expression in stromal extracellular matrix in vivo occurs via hormonally regulated, epithelial-mesenchymal interactions and serves as an early marker for uterine receptivity and the attachment phase of implantation. Furthermore, tenascin may facilitate embryo penetration by disrupting uterine epithelial cell adhesion to underlying basal lamina.

    Funded by: NICHD NIH HHS: HD 25235, HD 29963

    Development (Cambridge, England) 1994;120;3;661-71

  • Early skeletal muscle development proceeds normally in parthenogenetic mouse embryos.

    Fundele R, Bober E, Arnold HH, Grim M, Bender R, Wilting J and Christ B

    Institut für Biologie III, Universität Freiburg, Germany.

    In mouse chimeras with parthenogenetic cell contribution, the skeletal musculature appears to be largely devoid of parthenogenetically derived cells. To analyze the appearance and early distribution of myotomal cells in parthenotes, we determined the expression of the muscle-specific transcription factors myogenin, MYF-5, and MYF-6 by in situ hybridization in somites of Day 10 and 11 embryos. Here, we report that these myogenic regulatory proteins are expressed in parthenogenetic animals together with desmin, one of the early muscle-specific structural proteins. We also show that parthenogenetic cells contribute equally to dermatome, sclerotome, and myotome in Day 10 and 11 chimeras. These results suggest that early myotomal cells expressing the myogenic control proteins develop and allocate normally in parthenogenetic embryos and in parthenogenetic<==>normal chimeras. The underrepresentation in older chimeras may therefore be due to selective elimination. These data also argue against imprinting of the myogenic factor genes myogenin, Myf-5, and Myf-6.

    Developmental biology 1994;161;1;30-6

  • Abnormal incisor-tooth differentiation in transgenic mice expressing the muscle-specific desmin gene.

    Berteretche MV, Dunia I, Devilliers G, van der Kemp A, Pieper F, Bloemendal H, Benedetti EL and Forest N

    Laboratoire de Biologie-Odontologie, Institut Biomédical des Cordeliers, Université Paris VII, France.

    Immunocytochemistry and electron microscopic observations on the incisor-tooth organ of transgenic mice expressing the muscle-specific desmin gene under the direction of the vimentin promoter, reveal that the expression of the hybrid transgene occurs both in mesenchymal cells and differentiating odontoblasts. The muscle-specific desmin, as estimated by fluorescence intensity, is more expressed in immature mesenchymal cells than in postmitotic differentiated odontoblasts. The expression of the transgene generates alteration of the odontoblast-intermediate filament network and interferes with the secretory activity of both odontoblasts and ameloblasts. Our results are consistent with the hypothesis that odontoblasts have inductive properties on the differentiation of ameloblasts and that intermediate filaments among other factors play the role of cell and tissue organizer.

    European journal of cell biology 1993;62;2;183-93

  • Physical delineation of the minimal chromosomal segment encompassing the murine host resistance locus Bcg.

    Malo D, Vidal S, Lieman JH, Ward DC and Gros P

    Department of Biochemistry, McGill University, Montreal, Canada.

    The host resistance locus Bcg determines resistance of mice to infection with intracellular pathogens such as certain species of Mycobacteria, Salmonella typhimurium, and Leishmania donovani. Bcg maps on the proximal portion of mouse chromosome 1, very tightly linked to villin (Vil), with the gene order and intergene distances Tp-1-(1 cM)-D1Mcg105-(0.1 cM)-lambda Mm1C165/Vil/Bcg-(0.2 cM)-lambda Mm1C136-(0.3 cM)-Des-(0.1 cM)-Inha. In an effort to clone genomic sequences overlapping Bcg, we have used pulsed-field gel electrophoresis (PFGE) and fluorescence in situ hybridization to construct a physical map of the 3.9-Mb segment of proximal mouse chromosome 1, near Bcg. In situ hybridization to metaphase mouse chromosomes indicates that the mapped region is within band C5. Physical mapping of the Tp-1-Vil and lambda Mm1C136-Inha intervals was carried out by PFGE analysis, whereas the Vil-Des interval was estimated by in situ hybridization to interphase nuclei. Results of these combined analyses indicate the following locus order and maximal interlocus distances: Tp-1-(1000 kb)-D1Mcg105-(160 kb)-lambda Mm1C165-(180 kb)-Vil-(800 kb)-lambda Mm1C136-(290 kb)-Des-(130 kb)-Inha. Detailed restriction mapping of this region identifies numerous putative CpG islands, suggesting that several transcription units are present in the vicinity of Bcg.

    Genomics 1993;17;3;667-75

  • High-resolution linkage map in the vicinity of the host resistance locus Bcg.

    Malo D, Vidal SM, Hu J, Skamene E and Gros P

    Department of Biochemistry, McGill University, Montreal, Quebec, Canada.

    The mouse chromosome 1 locus Bcg determines natural resistance/susceptibility of inbred mouse strains to infection with antigenically unrelated intracellular parasites, including several Mycobacterium species, Salmonella typhimurium, and Leishmania donovani. In our effort to clone Bcg, we have constructed a high-resolution genetic linkage map in the vicinity of the gene. We have developed eight new highly polymorphic markers (simple sequence repeats) corresponding to cloned genes (Vil, Inha, Des), microdissected chromosome 1 anonymous probes (lambda Mm1C136, lambda Mm1C163, lambda Mm1C165), or novel DNA markers from the region obtained by chromosome walking (D1Mcg101 and D1Mcg105). We have followed the cosegregation of these markers with respect to Bcg in a novel panel of 1000 (C57L/J x C57BL/6J) x C57BL/6J segregating backcross mice. Additional segregation analyses were carried out in preexisting panels of intra- and interspecific backcross mice and recombinant inbred strains. Three of these markers were found to be very tightly linked to Bcg: lambda Mm1C165 did not show recombination with Bcg in 1424 meioses analyzed, while D1Mcg105 and lambda Mm1C136 were located 0.1 cM proximal and 0.2 cM distal to Bcg, respectively. This analysis enabled us to define further the proximal and distal boundaries of the Bcg interval: the proximal limit was defined by a single crossover occurring between D1Mcg105 and Bcg/lambda Mm1C165/Vil, and the distal limit by 1 cross-over between Bcg/lambda Mm1C165/Vil and lambda Mm1C136 in 1683 and 575 informative meioses, respectively, for a maximal interval of 0.3 cM.(ABSTRACT TRUNCATED AT 250 WORDS)

    Genomics 1993;16;3;655-63

  • Regulation of the mouse desmin gene: transactivated by MyoD, myogenin, MRF4 and Myf5.

    Li H and Capetanaki Y

    Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030, USA.

    Desmin, the muscle specific intermediate filament (IF) protein, is expressed at low levels in myoblasts and at the onset of differentiation its expression increases several fold. In an effort to explore the mechanism involved in the tissue-specific and developmentally regulated expression of desmin, we have isolated the mouse desmin gene. Sequence analysis of 976 bp 5' flanking region revealed several potential cis-acting elements: 1) Three E boxes (MyoD binding sites), namely, E1, E2 and E3, located at -79, -832 and -936, respectively; 2) one MEF2 binding site at -864; 3) a region with homology to M-CAT motif at -587; 4) several GC boxes. Transient transfections with various 5' flank deletion mutants into C2C12 muscle cells have revealed both positive and negative elements that seem to be involved in the expression of desmin. The first 81 bp upstream of the transcription initiation site, including E1 box, were sufficient to confer muscle specific expression of the desmin gene. The maximal level of expression was achieved by the construct containing up to -897 base pairs. The region between -578 to -976 behaves as a classical enhancer in the absence of which the region between -578 and -81 suppresses CAT activity. Gel electrophoretic mobility shift assays using both C2C12 muscle cell nuclear extracts as well as in vitro translated myoD/E12 and myogenin/E12 heterodimers, showed that both myoD and myogenin bind to the proximal E1 and the distal E2 boxes of the desmin promoter and enhancer respectively. Co-transfection of myoD, myogenin, MRF4 and Myf5, with the desmin-CAT construct into 10T-1/2 cells demonstrated that all these factors could transactivate desmin gene expression.

    Funded by: NIAMS NIH HHS: AR39617-01

    Nucleic acids research 1993;21;2;335-43

  • Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3.

    Epstein DJ, Vekemans M and Gros P

    Department of Biology, McGill University, Montreal, Canada.

    The molecular basis of the mouse mutation splotch (Sp), which is associated with spina bifida and exencephaly, was analyzed at three of its alleles, Sp, Sp2H, and Spr. We mapped the paired box gene Pax-3 within the Inha to Akp3 interval, near or at the Sp locus on chromosome 1, and found Pax-3 to be deleted in heterozygous Spr/+ mice. Analysis of genomic DNA and cDNA clones constructed from Sp2H/Sp2H embryos identified a deletion of 32 nucleotides in the Pax-3 mRNA transcript and gene. This deletion maps within the paired homeodomain of PAX-3 and is predicted to create a truncated protein as a result of a newly created termination codon at the deletion breakpoint. Our study provides evidence for a causal link between deletion of the paired homeodomain of Pax-3 and the Sp2H mutation, and infers that Pax-3 plays a key role in normal neural development.

    Cell 1991;67;4;767-74

  • The host resistance locus Bcg is tightly linked to a group of cytoskeleton-associated protein genes that include villin and desmin.

    Malo D, Schurr E, Epstein DJ, Vekemans M, Skamene E and Gros P

    Department of Biochemistry, McGill University, Montreal, Canada.

    In the mouse, innate resistance or susceptibility to infection with a group of unrelated intracellular parasites which includes, Mycobacteria, Salmonella, and Leishmania is determined by the expression of a single dominant autosomal gene designated Bcg located on the proximal portion of chromosome 1. The gene is expressed at the level of the mature tissue macrophage and influences its capacity to restrict intracellular proliferation of the parasites. We have used restriction fragment length polymorphism analysis in segregating populations of inter- and intraspecific backcross mice and in recombinant inbred strains to position four new marker genes, transition protein 1 (Tp-1), desmin (Des), the alpha subunit of inhibin (Inha), and retinal S-antigen (Sag), in the vicinity of the host resistance locus, Bcg. The gene order for Tp-1, Des, Inha, and Sag was established in an eight-point testcross with respect to anchor loci previously assigned to that portion of mouse chromosome 1 and was found to be centromere-Fn-1-Tp-1-(Vil,Bcg)-Des-Inha-Akp-3-Acrg+ ++-Sag. Two of these new marker genes were found very tightly linked to Bcg: Des was located 0.3 +/- 0.3 cM distal from (Vil,Bcg) and 0.3 +/- 0.3 cM proximal to Inha. Tp-1 mapped 0.8 +/- 0.8 cM proximal and Sag 12.8 +/- 1.7 cM distal to (Vil,Bcg). Tp-1, Des, Inha, and Sag all fall within a large mouse chromosome 1 segment homologous with the telomeric region of the long arm of human chromosome 2 (2q). Our findings indicate that the two closest markers to the host resistance locus, Bcg, encode cytoskeleton-associated proteins which are capable of interaction with actin filaments.

    Genomics 1991;10;2;356-64

  • Molecular characterization of a deletion encompassing the splotch mutation on mouse chromosome 1.

    Epstein DJ, Malo D, Vekemans M and Gros P

    Department of Biology, McGill University, Montreal, Quebec, Canada.

    We have used a set of markers newly assigned to the proximal portion of mouse chromosome 1 to characterize the chromosomal segment deleted in the splotch-retarded (Spr) mouse mutant. Among nine markers tested in the heterozygote Spr/+mouse, we have identified four genes, Vil, Des, Inha, and Akp-3, which map within the Spr deletion. The closest distal marker to the deletion is the Acrg gene, with the distal deletion breakpoint mapping within the 0.8-cM segment separating Akp-3 and Acrg. The most proximal gene to the Spr deletion is Tp1. The proximal deletion breakpoint maps within the 0.8-cM segment separating Tp1 and Vil. The minimum size of the Spr deletion would therefore be limited to 14 cM, the genetic distance between Vil and Akp-3. The maximum size of the Spr deletion is estimated to be 16 cM, the genetic distance between Tp1 and Acrg.

    Genomics 1991;10;1;89-93

  • Assignment of the mouse desmin gene to chromosome 1 band C3.

    Li ZL, Mattei MG, Mattei JF and Paulin D

    Laboratoire de Biologie Moléculaire de la Différenciation de l'Université Paris.

    The chromosomal localization of the mouse gene coding for desmin, one of the muscle-specific intermediate filament subunits, was determined by in situ hybridization using a specific 3H-labelled DNA probe. There is only one copy of the desmin gene and it is located on chromosome 1 in the band C3. This result adds an eleventh locus to a conserved gene cluster and confirms the partial homology that exists between the long arm of human chromosome 2 and chromosome 1 of the mouse.

    Genetical research 1990;55;2;101-5

  • 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 (5)

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
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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