Ozz-E3, a muscle-specific ubiquitin ligase, regulates beta-catenin degradation during myogenesis

The identities of the ubiquitin-ligases active during myogenesis are largely unknown. Here we describe a RING-type E3 ligase complex specified by the adaptor protein, Ozz, a novel SOCS protein that is developmentally regulated and expressed exclusively in striated muscle. In mice, the absence of Ozz results in overt maturation defects of the sarcomeric apparatus. We identified beta-catenin as one of the target substrates of the Ozz-E3 in vivo. In the differentiating myofibers, Ozz-E3 regulates the levels of sarcolemma-associated beta-catenin by mediating its degradation via the proteasome. Expression of beta-catenin mutants that reduce the binding of Ozz to endogenous beta-catenin leads to Mb-beta-catenin accumulation and myofibrillogenesis defects similar to those observed in Ozz null myocytes. These findings reveal a novel mechanism of regulation of Mb-beta-catenin and the role of this pool of the protein in myofibrillogenesis, and implicate the Ozz-E3 ligase in the process of myofiber differentiation.     

Vitamin A inhibits chondrogenesis but not myogenesis

Limb bud cells were isolated from HH stage 22–23 chick embryos and were grown as a ‘spot culture’ in in vitro conditions which support their differentiation into chondrocytes and myotubes. By day 4 of culture, numerous chondrocyte nodules developed and were scattered mainly in the very centre of the cell spot. In contrast, multinucleated myotubes formed at both the centre and the periphery of the cell spot. Treatment with vitamin A starting on day 1, inhibited chondrogenesis in these cultures, and by day 4–6 chondrocyte nodules could not be detected histologically. In contrast, no dose of vitamin A tested was effective in suppressing the development of multi-nucleated myotubes. These data show that vitamin A selectively inhibits chondrogenesis but not myogenesis in limb bud cell cultures.     

Notch2, but not Notch1, is required for proximal fate acquisition in the mammalian nephron

The Notch pathway regulates cell fate determination in numerous developmental processes. Here we report that Notch2 acts non-redundantly to control the processes of nephron segmentation through an Rbp-J-dependent process. Notch1 and Notch2 are detected in the early renal vesicle. Genetic analysis reveals that only Notch2 is required for the differentiation of proximal nephron structures (podocytes and proximal convoluted tubules) despite the presence of activated Notch1 in the nuclei of putative proximal progenitors. The inability of endogenous Notch1 to compensate for Notch2 deficiency may reflect sub-threshold Notch1 levels in the nucleus. In line with this view, forced expression of a gamma-secretase-independent form of Notch1 intracellular domain drives the specification of proximal fates where all endogenous, ligand-dependent Notch signaling is blocked by a gamma-secretase inhibitor. These results establish distinct (non-redundant), instructive roles for Notch receptors in nephron segmentation.     

Syndecan-3 and Notch cooperate in regulating adult myogenesis

Skeletal muscle postnatal growth and repair depend on satellite cells and are regulated by molecular signals within the satellite cell niche. We investigated the molecular and cellular events that lead to altered myogenesis upon genetic ablation of Syndecan-3, a component of the satellite cell niche. In the absence of Syndecan-3, satellite cells stall in S phase, leading to reduced proliferation, increased cell death, delayed onset of differentiation, and markedly reduced numbers of Pax7⁺ satellite cells accompanied by myofiber hypertrophy and an increased number of centrally nucleated myofibers. We show that the aberrant cell cycle and impaired self-renewal of explanted Syndecan-3–null satellite cells are rescued by ectopic expression of the constitutively active Notch intracellular domain. Furthermore, we show that Syndecan-3 interacts with Notch and is required for Notch processing by ADAM17/tumor necrosis factor-α–converting enzyme (TACE) and signal transduction. Together, our data support the conclusion that Syndecan-3 and Notch cooperate in regulating homeostasis of the satellite cell population and myofiber size.     

Numb regulates the balance between Notch recycling and late-endosome targeting in Drosophila neural progenitor cells

The Notch signaling pathway plays essential roles in both animal development and human disease. Regulation of Notch receptor levels in membrane compartments has been shown to affect signaling in a variety of contexts. Here we used steady-state and pulse-labeling techniques to follow Notch receptors in sensory organ precursor cells in Drosophila. We find that the endosomal adaptor protein Numb regulates levels of Notch receptor trafficking to Rab7-labeled late endosomes but not early endosomes. Using an assay we developed that labels different pools of Notch receptors as they move through the endocytic system, we show that Numb specifically suppresses a recycled Notch receptor subpopulation and that excess Notch signaling in numb mutants requires the recycling endosome GTPase Rab11 activity. Our data therefore suggest that Numb controls the balance between Notch receptor recycling and receptor targeting to late endosomes to regulate signaling output after asymmetric cell division in Drosophila neural progenitors.     

Myogenin is required for late but not early aspects of myogenesis during mouse development

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.     

Founder cells regulate fiber number but not fiber formation during adult myogenesis in Drosophila

During insect myogenesis, myoblasts are organized into a pre-pattern by specialized organizer cells. In the Drosophila embryo, these cells have been termed founder cells and play important roles in specifying muscle identity and in serving as targets for myoblast fusion. A group of adult muscles, the dorsal longitudinal (flight) muscles, DLMs, is patterned by persistent larval scaffolds; the second set, the dorso-ventral muscles, DVMs is patterned by mono-nucleate founder cells (FCs) that are much larger than the surrounding myoblasts. Both types of organizer cells express Dumbfounded, which is known to regulate fusion during embryonic myogenesis. The role of DVM founder cells as well as the DLM scaffolds was tested in genetic ablation studies using the UAS/Gal4 system of targeted transgene expression. In both cases, removal of organizer cells prior to fusion, causes formation of supernumerary fibers, suggesting that cells in the myoblast pool have the capacity to initiate fiber formation, which is normally inhibited by the organizers. In addition to the large DVM FCs, some (smaller) cells in the myoblast pool also express Dumbfounded. We propose that these cells are responsible for seeding supernumerary fibers, when DVM FCs are eliminated prior to fusion. When these cells are also eliminated, myogenesis fails to occur. In the second set of studies, targeted expression of constitutively active Ras^V12 also resulted in the appearance of supernumerary fibers. In this case, the original DVM FCs are present, suggesting alterations in cell fate. Taken together, these data suggest that DVM myoblasts are able to respond to cues other than the original founder cell, to initiate fusion and fiber formation. Thus, the role of the large DVM founder cells is to generate the correct number of fibers, but they are not required for fiber formation itself. We also present evidence that the DVM FCs may arise from the leg imaginal disc.     

Bid, but not Bax, regulates VDAC channels

During apoptosis, cytochrome c is released from mitochondria into the cytosol, where it participates in caspase activation. Various and often conflicting mechanisms have been proposed to account for the increased permeability of the mitochondrial outer membrane that is responsible for this process. The voltage-dependent anion channel (VDAC) is the major permeability pathway for metabolites in the mitochondrial outer membrane and therefore is a very attractive candidate for cytochrome c translocation. Here, we report that properties of VDAC channels reconstituted into planar phospholipid membranes are unaffected by addition of the pro-apoptotic protein Bax under a variety of conditions. Contrary to other reports (Shimizu, S., Narita, M., and Tsujimoto, Y. (1999) Nature 399, 483-487; Shimizu, S., Ide, T., Yanagida, T., and Tsujimoto, Y. (2000) J. Biol. Chem. 275, 12321-12325; Shimizu, S., Konishi, A., Kodama, T., and Tsujimoto, Y. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 3100-3105), we found no electrophysiologically detectable interaction between VDAC channels isolated from mammalian mitochondria and either monomeric or oligomeric forms of Bax. We conclude that Bax does not induce cytochrome c release by acting on VDAC. In contrast to Bax, another pro-apoptotic protein (Bid) proteolytically cleaved with caspase-8 affected the voltage gating of VDAC by inducing channel closure. We speculate that by decreasing the probability of VDAC opening, Bid reduces metabolite exchange between mitochondria and the cytosol, leading to mitochondrial dysfunction.     

Heat-shock protein beta 1 regulates androgen-mediated bovine myogenesis

To elucidate the functional significance of heat-shock protein beta 1 (HSPB1) in androgen-mediated myogenesis of bovine cells, we conducted ‘loss and gain of function of HSPB1’ assays by siRNA inhibition and gene overexpression. siRNA inhibition of HSPB1 expression reduced the expression of desmin (a myogenic marker) and repressed the formation of myotubes in cells induced for myogenic differentiation. In contrast, overexpression of HSPB1 enhanced the expression of desmin and accelerated formation of myotubes. The loss and gain of HSPB1 function was closely associated with the expression level of androgen receptor (AR). Our findings suggest that HSPB1 mediates androgen signaling by binding directly to AR and then enhancing androgen-mediated myogenesis in myogenic cells.     

Chemokine-like receptor 1 regulates skeletal muscle cell myogenesis

The chemokine-like receptor-1 (CMKLR1) is a G protein-coupled receptor that is activated by chemerin, a secreted plasma leukocyte attractant and adipokine. Previous studies identified that CMKLR1 is expressed in skeletal muscle in a stage-specific fashion during embryogenesis and in adult mice; however, its function in skeletal muscle remains unclear. Based on the established function of CMKLR1 in cell migration and differentiation, we investigated the hypothesis that CMKLR1 regulates the differentiation of myoblasts into myotubes. In C(2)C(12) mouse myoblasts, CMKLR1 expression increased threefold with differentiation into multinucleated myotubes. Decreasing CMKLR1 expression by adenoviral-delivered small-hairpin RNA (shRNA) impaired the differentiation of C(2)C(12) myoblasts into mature myotubes and reduced the mRNA expression of myogenic regulatory factors myogenin and MyoD while increasing Myf5 and Mrf4. At embryonic day 12.5 (E12.5), CMKLR1 knockout (CMKLR1(-/-)) mice appeared developmentally delayed and displayed significantly lower wet weights and a considerably diminished myotomal component of somites as revealed by immunolocalization of myosin heavy chain protein compared with wild-type (CMKLR1(+/+)) mouse embryos. These changes were associated with increased Myf5 and decreased MyoD protein expression in the somites of E12.5 CMKLR1(-/-) mouse embryos. Adult male CMKLR1(-/-) mice had significantly reduced bone-free lean mass and weighed less than the CMKLR1(+/+) mice. We conclude that CMKLR1 is essential for myogenic differentiation of C(2)C(12) cells in vitro, and the CMKLR1 null mice have a subtle skeletal muscle deficit beginning from embryonic life that persists during postnatal life.     

Knock-in of integrin beta 1D affects primary but not secondary myogenesis in mice

Integrins are extracellular matrix receptors composed of alpha and beta subunits involved in cell adhesion, migration and signal transduction. The beta1 subunit has two isoforms, beta 1A ubiquitously expressed and beta 1D restricted to striated muscle. They are not functionally equivalent. Replacement of beta 1A by beta 1D (beta 1D knock-in) in the mouse leads to midgestation lethality on a 50% Ola/50% FVB background [Baudoin, C., Goumans, M. J., Mummery, C. and Sonnenberg, A. (1998). Genes Dev. 12, 1202-1216]. We crossed the beta 1D knock-in line into a less penetrant genetic background. This led to an attenuation of the midgestation lethality and revealed a second period of lethality around birth. Midgestation death was apparently not caused by failure in cell migration, but rather by abnormal placentation. The beta 1D knock-in embryos that survived midgestation developed until birth, but exhibited severely reduced skeletal muscle mass. Quantification of myotube numbers showed that substitution of beta 1A with beta 1D impairs primary myogenesis with no direct effect on secondary myogenesis. Furthermore, long-term primary myotube survival was affected in beta 1D knock-in embryos. Finally, overexpression of beta 1D in C2C12 cells impaired myotube formation while overexpression of beta 1A primarily affected myotube maturation. Together these results demonstrate for the first time distinct roles for beta1 integrins in primary versus secondary myogenesis and that the beta 1A and beta 1D variants are not functionally equivalent in this process.     

RhoA regulates initiation of invagination, but not convergent extension, during sea urchin gastrulation

During gastrulation, the archenteron is formed using cell shape changes, cell rearrangements, filopodial extensions, and convergent extension movements to elongate and shape the nascent gut tube. How these events are coordinated remains unknown, although much has been learned from careful morphological examinations and molecular perturbations. This study reports that RhoA is necessary to trigger archenteron invagination in the sea urchin embryo. Inhibition of RhoA results in a failure to initiate invagination movements, while constitutively active RhoA induces precocious invagination of the archenteron, complete with the actin rearrangements and extracellular matrix secretions that normally accompany the onset of invagination. Although RhoA activity has been reported to control convergent extension movements in vertebrate embryos, experiments herein show that RhoA activity does not regulate convergent extension movements during sea urchin gastrulation. Instead, the results support the hypothesis that RhoA serves as a trigger to initiate invagination, and once initiation occurs, RhoA activity is no longer involved in subsequent gastrulation movements.     

D1, but not D2, dopamine receptor regulates steroid levels during the final stages of pikeperch gametogenesis

In pikeperch, Sander lucioperca, aquaculture hormonal treatment is usually applied to synchronize ovulation. However, the effect of dopamine (DA) receptor antagonists, in particular those blocking the D1 DA receptors, remains unknown. Thus, the aim of the present study was to investigate and compare the effects of D1 and D2 DA receptor antagonists on the sex-steroid production and reproductive performance of the species. Two experiments were performed during which mature pikeperch females were injected with different molecules: NaCl 0.9% (negative control) or human chorionic gonadotropin 500 IU/kg (positive control) in both experiments, metoclopramide (a D2 receptor antagonist; 4 mg/kg or 20 mg/kg) or SCH23390 (a D1 receptor antagonist; 0.8 mg/kg or 4 mg/kg) alone (experiment 1) or in combination with a salmon gonadotropin-releasing hormone analogue (sGnRHa at 25 µg/kg; experiment 2). In experiment 2, fish were also injected with sGnRHa (25 µg/kg) as positive control. Samplings of oocytes and blood were performed on the day of injection and after 24 h (both experiments), after 48 h (experiment 2) and at the time of ovulation (both experiments). In non-ovulating fish, samplings were performed 7 days (experiment 1) or 14 days (experiment 2) after injection. In experiment 2, various zootechnical parameters of fertilized eggs were recorded (survival, hatching and malformation rates). The two antagonists alone were ineffective in inducing the final stages and regulating sex-steroid (testosterone, 11 ketotestosterone, 17β estradiol and 17,20β-dihydroxy-4-pregnen-3-one) production. When administered with sGnRHa, both SCH23390 and metoclopramide induced the final stages. However, only SCH23390 stimulated testosterone (4 mg/kg) and 17β estradiol (0.8 mg/kg) production compared with sGnRHa alone. None of the treatments affected the survival, hatching or malformation rates. This is the first report suggesting that in pikeperch the D1, but not the D2, DA receptor antagonist would be involved in the testosterone and 17β estradiol production as a potentiator of the sGnRHa effect.     

Myostatin regulates cell survival during C2C12 myogenesis

During the myogenic process in vitro, proliferating myoblasts withdraw irreversible from the cell cycle, acquire an apoptosis-resistant phenotype, and fuse into mature myotubes. The key factor regulating both myocyte cell cycle exit and viability during this transition is the the cyclin-dependent kinase inhibitor p21(cip1). Here we show that the expression of myostatin, a TGF-beta superfamily member known to act as a negative regulator of muscle growth, is upregulated in the course of C2C12 cells myogenesis. We also show that transient transfection of C2C12 myobasts with an expression vector encoding mouse myostatin cDNA efficiently inhibits cell proliferation. Paradoxically, myostatin cDNA overexpression also enhances the survival of differentiating C2C12 myocytes, probably by a mechanism involving, at least in part, upregulation of p21(cip1) mRNA. Our results suggest that myostatin role in myogenesis is more complex than initially suggested and involves another level of regulation apart from inhibition of myoblast proliferation.     

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.     

Vav regulates activation of Rac but not Cdc42 during FcgammaR-mediated phagocytosis

Phagocytosis is the process whereby cells direct the spatially localized, receptor-driven engulfment of particulate materials. It proceeds via remodeling of the actin cytoskeleton and shares many of the core cytoskeletal components involved in adhesion and migration. Small GTPases of the Rho family have been widely implicated in coordinating actin dynamics in response to extracellular signals and during diverse cellular processes, including phagocytosis, yet the mechanisms controlling their recruitment and activation are not known. We show herein that in response to ligation of Fc receptors for IgG (FcgammaR), the guanine nucleotide exchange factor Vav translocates to nascent phagosomes and catalyzes GTP loading on Rac, but not Cdc42. The Vav-induced Rac activation proceeds independently of Cdc42 function, suggesting distinct roles for each GTPase during engulfment. Moreover, inhibition of Vav exchange activity or of Cdc42 activity does not prevent Rac recruitment to sites of particle attachment. We conclude that Rac is recruited to Fcgamma membrane receptors in its inactive, GDP-bound state and that Vav regulates phagocytosis through subsequent catalysis of GDP/GTP exchange on Rac.     

The N-BAR domain protein,Bin3, regulates Rac1- and Cdc42-dependent processes in myogenesis

Actin dynamics are necessary at multiple steps in the formation of multinucleated muscle cells. BAR domain proteins can regulate actin dynamics in several cell types, but have been little studied in skeletal muscle. Here, we identify novel functions for the N-BAR domain protein, Bridging integrator 3 (Bin3), during myogenesis in mice. Bin3 plays an important role in regulating myofiber size in vitro and in vivo. During early myogenesis, Bin3 promotes migration of differentiated muscle cells, where it colocalizes with F-actin in lamellipodia. In addition, Bin3 forms a complex with Rac1 and Cdc42, Rho GTPases involved in actin polymerization, which are known to be essential for myotube formation. Importantly, a Bin3-dependent pathway is a major regulator of Rac1 and Cdc42 activity in differentiated muscle cells. Overall, these data classify N-BAR domain proteins as novel regulators of actin-dependent processes in myogenesis, and further implicate BAR domain proteins in muscle growth and repair.