Hedgehog signaling is required for primary motoneuron induction in zebrafish

Sonic hedgehog (Shh) is crucial for motoneuron development in chick and mouse. However, zebrafish embryos homozygous for a deletion of the shh locus have normal numbers of motoneurons, raising the possibility that zebrafish motoneurons may be specified differently. Unlike other vertebrates, zebrafish express three hh genes in the embryonic midline: shh, echidna hedgehog (ehh) and tiggywinkle hedgehog (twhh). Therefore, it is possible that Twhh and Ehh are sufficient for motoneuron formation in the absence of Shh. To test this hypothesis we have eliminated, or severely reduced, all three Hh signals using mutations that directly or indirectly reduce Hh signaling and antisense morpholinos. Our analysis shows that Hh signals are required for zebrafish motoneuron induction. However, each of the three zebrafish Hhs is individually dispensable for motoneuron development because the other two can compensate for its loss. Our results also suggest that Twhh and Shh are more important for motoneuron development than Ehh.     

Zebrafish ftz-f1a (nuclear receptor 5a2) functions in skeletal muscle organization

Fushi-tarazu factor 1a (Ftz-F1a, Ff1a, Nr5a2) is a nuclear receptor with diverse functions in many tissues. Here, we report the function of ff1a in zebrafish muscle differentiation. In situ hybridization revealed that ff1a mRNA was present in the adaxial and migrating slow muscle precursors and was down-regulated when slow muscle cells matured. This expression was under the control of hedgehog genes, expanded when hedgehog was increased and missing in mutants defective in genes in the Hedgehog pathway like you-too (yot), sonic you (syu), and u-boot (ubo). Blocking ff1a activity by injecting a deleted form of ff1a or an antisense ff1a morpholino oligo into fish embryos caused thinner and disorganized fibers of both slow and fast properties. Transient expression of ff1a in syu, ubo, and yot embryos led to more fibril bundles, even when slow myoblasts were transfated into fast properties. We showed that ff1a and prox1 complemented each other in slow myofibril assembly, but they did not affect the expression of each other. These results demonstrate that ff1a functions in both slow and fast muscle morphogenesis in response to Hedgehog signaling, and this function parallels the activity of another slow muscle gene, prox1.     

The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity

Hedgehog proteins mediate many of the inductive interactions that determine cell fate during embryonic development. Hedgehog signaling has been shown to regulate slow muscle fiber type development. We report here that mutations in the zebrafish slow-muscle-omitted (smu) gene disrupt many developmental processes involving Hedgehog signaling. smu(-/-) embryos have a 99% reduction in the number of slow muscle fibers and a complete loss of Engrailed-expressing muscle pioneers. In addition, mutant embryos have partial cyclopia, and defects in jaw cartilage, circulation and fin growth. The smu(-/-) phenotype is phenocopied by treatment of wild-type embryos with forskolin, which inhibits the response of cells to Hedgehog signaling by indirect activation of cAMP-dependent protein kinase (PKA). Overexpression of Sonic hedgehog (Shh) or dominant negative PKA (dnPKA) in wild-type embryos causes all somitic cells to develop into slow muscle fibers. Overexpression of Shh does not rescue slow muscle fiber development in smu(-/-) embryos, whereas overexpression of dnPKA does. Cell transplantation experiments confirm that smu function is required cell-autonomously within the muscle precursors: wild-type muscle cells rescue slow muscle fiber development in smu(-/-) embryos, whereas mutant muscle cells cannot develop into slow muscle fibers in wild-type embryos. Slow muscle fiber development in smu mutant embryos is also rescued by expression of rat Smoothened. Therefore, Hedgehog signaling through Slow-muscle-omitted is necessary for slow muscle fiber type development. We propose that smu encodes a vital component in the Hedgehog response pathway.     

Zebrafish smoothened functions in ventral neural tube specification and axon tract formation

Sonic hedgehog (Shh) signaling patterns many vertebrate tissues. shh mutations dramatically affect mouse ventral forebrain and floor plate but produce minor defects in zebrafish. Zebrafish have two mammalian Shh orthologs, sonic hedgehog and tiggy-winkle hedgehog, and another gene, echidna hedgehog, that could have overlapping functions. To examine the role of Hedgehog signaling in zebrafish, we have characterized slow muscle omitted (smu) mutants. We show that smu encodes a zebrafish ortholog of Smoothened that transduces Hedgehog signals. Zebrafish smoothened is expressed maternally and zygotically and supports specification of motoneurons, pituitary cells and ventral forebrain. We propose that smoothened is required for induction of lateral floor plate and a subpopulation of hypothalamic cells and for maintenance of medial floor plate and hypothalamic cells.     

Hedgehog can drive terminal differentiation of amniote slow skeletal muscle

Background

Secreted Hedgehog (Hh) signalling molecules have profound influences on many developing and regenerating tissues. Yet in most vertebrate tissues it is unclear which Hh-responses are the direct result of Hh action on a particular cell type because Hhs frequently elicit secondary signals. In developing skeletal muscle, Hhs promote slow myogenesis in zebrafish and are involved in specification of medial muscle cells in amniote somites. However, the extent to which non-myogenic cells, myoblasts or differentiating myocytes are direct or indirect targets of Hh signalling is not known.

Results

We show that Sonic hedgehog (Shh) can act directly on cultured C2 myoblasts, driving Gli1 expression, myogenin up-regulation and terminal differentiation, even in the presence of growth factors that normally prevent differentiation. Distinct myoblasts respond differently to Shh: in some slow myosin expression is increased, whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells, yet simultaneously enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres, however, do not express extra slow myosin. Conversely, Hh loss of function in the limb bud, caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody, reduces early slow muscle formation and differentiation, but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis.

Conclusions

Taken together, the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires Hh for normal differentiation and slow myosin expression. We propose a simple model of how direct and indirect effects of Hh regulate early limb myogenesis.

     

Hedgehog signaling induces cardiomyogenesis in P19 cells

Sonic Hedgehog (Shh) is a critical signaling factor for a variety of developmental pathways during embryogenesis, including the specification of left-right asymmetry in the heart. Mice that lack Hedgehog signaling show a delay in the induction of cardiomyogenesis, as indicated by a delayed expression of Nkx2-5. To further examine a role for Shh in cardiomyogenesis, clonal populations of P19 cells that stably express Shh, termed P19(Shh) cells, were isolated. In monolayer P19(Shh) cultures the Shh pathway was functional as shown by the up-regulation of Ptc1 and Gli1 expression, but no cardiac muscle markers were activated. However, Shh expression induced cardiomyogenesis following cellular aggregation, resulting in the expression of factors expressed in cardiac muscle including GATA-4, MEF2C, and Nkx2-5. Furthermore, aggregated P19 cell lines expressing Gli2 or Meox1 also up-regulated the expression of cardiac muscle factors, leading to cardiomyogenesis. Meox1 up-regulated the expression of Gli1 and Gli2 and, thus, can modify the Shh signaling pathway. Finally, Shh, Gli2, and Meox1 all up-regulated BMP-4 expression, implying that activation of the Hedgehog pathway can regulate bone morphogenetic protein signals. Taken together, we propose a model in which Shh, functioning via Gli1/2, can specify mesodermal cells into the cardiac muscle lineage.     

Two distinct cell populations in the floor plate of the zebrafish are induced by different pathways

The floor plate is a morphologically distinct structure of epithelial cells situated along the midline of the ventral spinal cord in vertebrates. It is a source of guidance molecules directing the growth of axons along and across the midline of the neural tube. In the zebrafish, the floor plate is about three cells wide and composed of cuboidal cells. Two cell populations can be distinguished by the expression patterns of several marker genes, including sonic hedgehog (shh) and the fork head-domain gene fkd4: a single row of medial floor plate (MFP) cells, expressing both shh and fkd4, is flanked by rows of lateral floor plate (LFP) cells that express fkd4 but not shh. Systematic mutant searches in zebrafish embryos have identified a number of genes, mutations in which visibly reduce the floor plate. In these mutants either the MFP or the LFP cells are absent, as revealed by the analysis of the shh and fkd4 expression patterns. MFP cells are absent, but LFP cells are present, in mutants of cyclops, one-eyed pinhead, and schmalspur, whose development of midline structures is affected. LFP cells are absent, but MFP cells are present, in mutants of four genes, sonic you, you, you-too, and chameleon, collectively called the you-type genes. This group of mutants also shows defects in patterning of the paraxial mesoderm, causing U- instead of V-shaped somites. One of the you-type genes, sonic you, was recently shown to encode the zebrafish Shh protein, suggesting that the you-type genes encode components of the Shh signaling pathway. It has been shown previously that in the zebrafish shh is required for the induction of LFP cells, but not for the development of MFP cells. This conclusion is supported by the finding that injection of shh RNA causes an increase in the number of LFP, but not MFP cells. Embryos mutant for iguana, detour, and umleitung share the lack of LFP cells with you-type mutants while somite patterning is not severely affected. In mutants that fail to develop a notochord, MFP cells may be present, but are always surrounded by LFP cells. These data indicate that shh, expressed in the notochord and/or the MFP cells, induces the formation of LFP cells. In embryos doubly mutant for cyclops (cyc) and sonic you (syu) both LFP and MFP cells are deleted. The number of primary motor neurons is strongly reduced in cyc;syu double mutants, while almost normal in single mutants, suggesting that the two different pathways have overlapping functions in the induction of primary motor neurons.     

Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream ( Sparus aurata)

Members of the myogenic regulatory gene family, including MyoD, Myf5, Myogenin and MRF4, are specifically expressed in myoblast and skeletal muscle cells and play important roles in regulating skeletal muscle development and growth. They are capable of converting a variety of non-muscle cells into myoblasts and myotubes. To better understand their roles in the development of fish muscles, we have isolated the MyoD genomic genes from gilthead seabream (Sparus aurata), analyzed the genomic structures, patterns of expression and the regulation of muscle-specific expression. We have demonstrated that seabream contain two distinct non-allelic MyoDgenes, MyoD1 and MyoD2. Sequence analysis revealed that these two MyoD genes shared a similar gene structure. Expression studies demonstrated that they exhibited overlapping but distinct patterns of expression in seabream embryos and adult slow and fast muscles. MyoD1 was expressed in adaxial cells that give rise to slow muscles, and lateral somitic cells that give rise to fast muscles. Similarly, MyoD2 was initially expressed in both slow and fast muscle precursors. However, MyoD2 expression gradually disappeared in the adaxial cells of 10- to 15-somite-stage embryos, whereas its expression in fast muscle precursor cells was maintained. In adult skeletal muscles, MyoD1 was expressed in both slow and fast muscles, whereas MyoD2 was specifically expressed in fast muscles. Treating seabream embryos with forskolin, a protein kinase A activator, inhibited MyoD1 expression in adaxial cells, while expression in fast muscle precursors was not affected. Promoter analysis demonstrated that both MyoD1 and MyoD2 promoters could drive green fluorescence protein expression in muscle cells of zebrafish embryos. Together, these data suggest that the two non-allelic MyoD genes are functional in seabream and their expression is regulated differently in fast and slow muscles. Hedgehog signaling is required for induction of MyoDexpression in adaxial cells.