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.     

Differential susceptibility of midbrain and spinal cord patterning to floor plate defects in the talpid2 mutant

The chick talpid2 mutant displays polydactylous digits attributed to defects of the Hedgehog (HH) signaling pathway. We examined the talpid2 neural tube and show that patterning defects in the spinal cord and the midbrain are distinct from each other and from the limb. Unlike the Sonic Hedgehog (SHH) source in the limb, the SHH-rich floor plate (FP) is reduced in the talpid2 midbrain. This is accompanied by a severe depletion of medial cell populations that encounter high concentrations of SHH, an expansion of lateral cell populations that experience low concentrations of SHH and a broad deregulation of HH's principal effectors (PTC1, GLI1, GLI2, GLI3). Together with the failure of SHH misexpression to rescue the talpid2 phenotype, these results suggest that talpid2 is likely to have a tissue-autonomous, bidirectional (positive and negative) role in HH signaling that cannot be attributed to the altered expression of several newly cloned HH pathway genes (SUFU, DZIP1, DISP1, BTRC). Strikingly, FP defects in the spinal cord are accompanied by relatively normal patterning in the talpid2 mutant. We propose that this differential FP dependence may be due to the prolonged apposition of the notochord to the spinal cord, but not the midbrain during development.     

Analysis of the L1-deficient mouse phenotype reveals cross-talk between Sema3A and L1 signaling pathways in axonal guidance

In humans, defects of the corticospinal tract have been attributed to mutations in the gene encoding L1 CAM, a phenotype that is reproduced in L1-deficient mice. Using coculture assays, we report that Sema3A secreted from the ventral spinal cord repels cortical axons from wild-type but not from L1-deficient mice. L1 and neuropilin-1 (NP-1) form a stable complex, and their extracellular domains can directly associate. Thus, L1 is a component of the Sema3A receptor complex, and L1 mutations may disrupt Sema3A signaling in the growth cone, leading to guidance errors. Addition of soluble L1Fc chimeric molecules does not restore Sema3A responsiveness of L1-deficient axons; instead, it converts the repulsion of wild-type axons into an attraction, further supporting a function for L1 in the Sema3A transducing pathways within the growth cone.     

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.     

Zebrafish slow muscle cell migration induces a wave of fast muscle morphogenesis

The specification and morphogenesis of slow and fast twitch muscle fibers are crucial for muscle development. In zebrafish, Hedgehog is required for slow muscle fiber specification. However, less is known about signals that promote development of fast muscle fibers, which constitute the majority of somitic cells. We show that when Hedgehog signaling is blocked, fast muscle cell elongation is disrupted. Using genetic mosaics, we show that Hedgehog signal perception is required by slow muscle cells but not by fast muscle cells for fast muscle cell elongation. Furthermore, we show that slow muscle cells are sufficient to pattern the medial to lateral wave of fast muscle fiber morphogenesis even when fast muscle cells cannot perceive the Hedgehog signal. Thus, the medial to lateral migration of slow muscle fibers through the somite creates a morphogenetic signal that patterns fast muscle fiber elongation in its wake.     

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.     

Scube2 mediates Hedgehog signalling in the zebrafish embryo

The Hedgehog family of secreted morphogens specifies the fate of a large number of different cell types within invertebrate and vertebrate embryos, including the muscle cell precursors of the embryonic myotome of zebrafish. Formation of Hedgehog-sensitive muscle fates is disrupted within homozygous zebrafish mutants of the "you"-type class, the majority of which disrupt components of the Hedgehog (HH) signal transduction pathway. We have undertaken a phenotypic and molecular characterisation of one of these mutants, you, which we show results from mutations within the zebrafish orthologue of the mammalian gene scube2. This gene encodes a member of the Scube family of proteins, which is characterised by several protein motifs including EGF and CUB domains. Epistatic and molecular analyses position Scube2 function upstream of Smoothened (Smoh), the signalling component of the HH receptor complex, suggesting that Scube2 may act during HH signal transduction prior to, or during, receipt of the HH signal at the plasma membrane. In support of this model we show that scube2 has homology to cubilin, which encodes an endocytic receptor involved in protein trafficking suggesting a possible mode of function for Scube2 during HH signal transduction.     

Fish-Specific Duplicated dmrt2b Contributes to a Divergent Function through Hedgehog Pathway and Maintains Left-Right Asymmetry Establishment Function

Gene duplication is thought to provide raw material for functional divergence and innovation. Fish-specific dmrt2b has been identified as a duplicated gene of the dmrt2a/terra in fish genomes, but its function has remained unclear. Here we reveal that Dmrt2b knockdown zebrafish embryos display a downward tail curvature and have U-shaped somites. Then, we demonstrate that Dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway, because Dmrt2b knockdown reduces target gene expression of Hedgehog signaling, and also impairs slow muscle development and neural tube patterning through Hedgehog signaling. Moreover, the Dmrt2b morphants display defects in heart and visceral organ asymmetry, and, some lateral-plate mesoderm (LPM) markers expressed in left side are randomized. Together, these data indicate that fish-specific duplicated dmrt2b contributes to a divergent function in somitogenesis through Hedgehog pathway and maintains the common function for left-right asymmetry establishment.     

Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis.

Several heritable forms of myotonia and hyperkalemic periodic paralysis (HyperPP) are caused by missense mutations in the alpha subunit of the skeletal muscle Na channel (SkM1). These mutations impair fast inactivation or shift activation toward hyperpolarized potentials, inducing persistent Na currents that may cause muscle depolarization, myotonia, and onset of weakness. It has been proposed that the aberrant Na current and resulting weakness will be sustained only if Na channel slow inactivation is also impaired. We therefore measured slow inactivation for wild-type and five mutant Na channels constructed in the rat skeletal muscle isoform (rSkM1) and expressed in HEK cells. Two common HyperPP mutations (T698M in domain II-S5 and M1585V in IV-S6) had defective slow inactivation. This defect reduced use-dependent inhibition of Na currents elicited during 50-Hz stimulation. A rare HyperPP mutation (M1353V in IV-S1) and mutations within the domain III-IV linker that cause myotonia (G1299E) or myotonia plus weakness (T1306M) did not impair slow inactivation. We also observed that slow inactivation of wild-type rSkM1 was incomplete; therefore it is possible that stable membrane depolarization and subsequent muscle weakness may be caused solely by defects in fast inactivation or activation. Model simulations showed that abnormal slow inactivation, although not required for expression of a paralytic phenotype, may accentuate muscle membrane depolarization, paralysis, and sensitivity to hyperkalemia.     

Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems

Constitutive activation of the Notch pathway can promote gliogenesis by peripheral (PNS) and central (CNS) nervous system progenitors. This raises the question of whether physiological Notch signaling regulates gliogenesis in vivo. To test this, we conditionally deleted Rbpsuh (Rbpj) from mouse PNS or CNS progenitors using Wnt1-Cre or Nestin-Cre. Rbpsuh encodes a DNA-binding protein (RBP/J) that is required for canonical signaling by all Notch receptors. In most regions of the developing PNS and spinal cord, Rbpsuh deletion caused only mild defects in neurogenesis, but severe defects in gliogenesis. These resulted from defects in glial specification or differentiation, not premature depletion of neural progenitors, because we were able to culture undifferentiated progenitors from the PNS and spinal cord despite their failure to form glia in vivo. In spinal cord progenitors, Rbpsuh was required to maintain Sox9 expression during gliogenesis, demonstrating that Notch signaling promotes the expression of a glial-specification gene. These results demonstrate that physiological Notch signaling is required for gliogenesis in vivo, independent of the role of Notch in the maintenance of undifferentiated neural progenitors.     

Mouse Dispatched homolog1 is required for long-range,but not juxtacrine,Hh signaling

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh.     

Hedgehog signaling regulates expansion of pancreatic epithelial cells

Embryonic Hedgehog signaling is essential for proper tissue morphogenesis and organ formation along the developing gastrointestinal tract. Hedgehog ligands are expressed throughout the endodermal epithelium at early embryonic stages but excluded from the region that will form the pancreas. Ectopic activation of Hedgehog signaling at the onset of pancreas development has been shown to inhibit organ morphogenesis. In contrast, Hedgehog signaling components are found within pancreatic tissue during subsequent stages of development as well as in the mature organ, indicating that a certain level of pathway activation is required for normal organ development and function. Here, we ectopically activate the Hedgehog pathway midway through pancreas development via expression of either Sonic (Shh) or Indian Hedgehog (Ihh) under control of the human Pax4-promoter. Similar pancreatic defects are observed in both Pax4-Shh and Pax4-Ihh transgenic lines, suggesting that regulation of the overall level of Hedgehog activity is critical for proper pancreas development. We also show that Hedgehog signaling controls mesenchymal vs. epithelial tissue differentiation and that pathway activation impairs formation of epithelial progenitors. Thus, tight control of Hedgehog pathway activity throughout embryonic development ensures proper pancreas organogenesis.     

Distinct and cooperative roles for Nodal and Hedgehog signals during hypothalamic development

Despite its evolutionary conservation and functional importance, little is known of the signaling pathways that underlie development of the hypothalamus. Although mutations affecting Nodal and Hedgehog signaling disrupt hypothalamic development, the time and site of action and the exact roles of these pathways remain very poorly understood. Unexpectedly, we show here that cell-autonomous reception of Nodal signals is neither required for the migration of hypothalamic precursors within the neural plate, nor for further development of the anterior-dorsal hypothalamus. Nodal signaling is, however, cell-autonomously required for establishment of the posterior-ventral hypothalamus. Conversely, Hedgehog signaling antagonizes the development of posterior-ventral hypothalamus, while promoting anterior-dorsal hypothalamic fates. Besides their distinct roles in the regionalization of the diencephalon, we reveal cooperation between Nodal and Hedgehog pathways in the maintenance of the anterior-dorsal hypothalamus. Finally we show that it is the prechordal plate and not the head endoderm that provides the early signals essential for establishment of the hypothalamus.     

Genetic screens for genes controlling motor nerve-muscle development and interactions

Motor growth cones navigate long and complex trajectories to connect with their muscle targets. Experimental studies have shown that this guidance process critically depends on extrinsic cues. In the zebrafish embryo, a subset of mesodermal cells, the adaxial cells, delineates the prospective path of pioneering motor growth cones. Genetic ablation of adaxial cells causes profound pathfinding defects, suggesting the existence of adaxial cell derived guidance factors. Intriguingly, adaxial cells are themselves migratory, and as growth cones approach they migrate away from the prospective axonal path to the lateral surface of the myotome, where they develop into slow-twitching muscle fibers. Genetic screens in embryos stained with an antibody cocktail identified mutants with specific defects in differentiation and migration of adaxial cells/slow muscle fibers, as well as mutants with specific defects in axonal pathfinding, including exit from the spinal cord and pathway selection. Together, the genes underlying these mutant phenotypes define pathways essential for nerve and muscle development and interactions between these two cell types.