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.     

Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.     

Expression of patched, prdm1 and engrailed in the lamprey somite reveals conserved responses to Hedgehog signaling

SUMMARY In the zebrafish embryo, expression of the prdm1 and patched1 genes in adaxial cells is indicative of their specification to give rise to slow twitch muscle fibers in response to Hedgehog (Hh) signaling. Subsets of these slow twitch muscle progenitors activate engrailed ( eng ) strongly in response to high-level Hh signaling, and differentiate into muscle pioneer cells, which are important for subsequent development of the horizontal myoseptum. In addition, eng is expressed more weakly in medial fast fibers in response to lower Hh levels. Somite morphology in the lamprey, an agnathan (jawless) vertebrate, differs significantly from that of teleosts. In particular, the lamprey does not have clear epaxial/hypaxial domains, lacks a horizontal myoseptum, and does not appear to possess distinct populations of fast and slow fibers in the embryonic somite. Nevertheless, Hh is expressed in the midline of the lamprey embryo, and we report here that, as in zebrafish, homologues of patched and prdm1 are expressed in adaxial regions of the lamprey somite, and an eng homologue is also expressed in the somite. However, the lamprey adaxial region does not exhibit the same distinct adaxial cell morphology as in the zebrafish. In addition, the expression of follistatin is not excluded from the adaxial region, and eng is not detected in discrete muscle pioneer-like cells. These data suggest the presence of conserved responses to Hh signaling in lamprey somites, although the full range of effects elicited by Hh in the zebrafish somite is not recapitulated.     

A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (Ci) the only known target of Hh signaling. Hh promotes the entry of Sxl into the nucleus in the wing disc. In the anterior compartment, the Hh receptor Patched (Ptc) is required for this effect, revealing Ptc as a positive effector of Hh. Some of the downstream components of the Hh signaling pathway also alter the rate of Sxl nuclear entry. Mutations in Suppressor of Fused or Fused with altered ability to anchor Ci are also impaired in anchoring Sxl in the cytoplasm. The levels, and consequently, the ability of Sxl to translationally repress downstream targets in the sex determination pathway, can also be adversely affected by mutations in Hh signaling genes. Conversely, overexpression of Sxl in the domain that Hh patterns negatively affects wing patterning. These data suggest that the Hh pathway impacts on the sex determination process and vice versa and that the pathway may serve more functions than the regulation of Ci.     

Restricted expression of the zebrafish hsp90alpha gene in slow and fast muscle fiber lineages

Members of the heat shock protein 90 (Hsp90) family of molecular chaperones play important roles in allowing a select group of intracellular signaling molecules reach and maintain functionally active conformations. We have previously shown that hsp90alpha gene expression in early zebrafish embryos is restricted to a subgroup of paraxial-mesoderm derived somitic cells prior to muscle formation and that the gene is downregulated in mature trunk and tail muscle fibers. Here we have compared the expression of the hsp90alpha gene to muscle regulatory genes during development of slow and fast muscle fibers in normal embryos and in embryos carrying mutations which affect somitic muscle formation. We show that hsp90alpha is first expressed early during the development of slow somitic muscle progenitors shortly following myoD activation and at a point prior to or co-incident with the expression of other known muscle regulatory genes. Expression of hsp90alpha is also activated in the midline of flh mutants when these cells switch from a notochord to a muscle fate. Conversely, expression is not detectable in cells of the paraxial mesoderm lineage which fail to converge in spt mutants and which do not activate expression of other muscle specific marker genes. Finally, expression of hsp90alpha is downregulated in slow muscle fibers by 24 h of age but becomes detectable in the later developing fast fibers at this time. Thus, hsp90alpha is expressed in developing muscle progenitors during short temporal and spatial windows of both slow and fast fiber lineages in the zebrafish somite.     

Cdkn1c drives muscle differentiation through a positive feedback loop with Myod

Differentiation often requires conversion of analogue signals to a stable binary output through positive feedback. Hedgehog (Hh) signalling promotes myogenesis in the vertebrate somite, in part by raising the activity of muscle regulatory factors (MRFs) of the Myod family above a threshold. Hh is known to enhance MRF expression. Here we show that Hh is also essential at a second step that increases Myod protein activity, permitting it to promote Myogenin expression. Hh acts by inducing expression of cdkn1c (p57^Kip2) in slow muscle precursor cells, but neither Hh nor Cdkn1c is required for their cell cycle exit. Cdkn1c co-operates with Myod to drive differentiation of several early zebrafish muscle fibre types. Myod in turn up-regulates cdkn1c, thereby providing a positive feedback loop that switches myogenic cells to terminal differentiation.     

Mys Protein Regulates Protein Kinase A Activity by Interacting with Regulatory Type I�� Subunit during Vertebrate Development

During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type Iα subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells.     

The zebrafish mutants dre, uki, and lep encode negative regulators of the hedgehog signaling pathway

Proliferation is one of the basic processes that control embryogenesis. To identify factors involved in the regulation of proliferation, we performed a zebrafish genetic screen in which we used proliferating cell nuclear antigen (PCNA) expression as a readout. Two mutants, hu418B and hu540A, show increased PCNA expression. Morphologically both mutants resembled the dre (dreumes), uki (ukkie), and lep (leprechaun) mutant class and both are shown to be additional uki alleles. Surprisingly, although an increased size is detected of multiple structures in these mutant embryos, adults become dwarfs. We show that these mutations disrupt repressors of the Hedgehog (Hh) signaling pathway. The dre, uki, and lep loci encode Su(fu) (suppressor of fused), Hip (Hedgehog interacting protein), and Ptc2 (Patched2) proteins, respectively. This class of mutants is therefore unique compared to previously described Hh mutants from zebrafish genetic screens, which mainly show loss of Hh signaling. Furthermore, su(fu) and ptc2 mutants have not been described in vertebrate model systems before. Inhibiting Hh activity by cyclopamine rescues uki and lep mutants and confirms the overactivation of the Hh signaling pathway in these mutants. Triple uki/dre/lep mutants show neither an additive increase in PCNA expression nor enhanced embryonic phenotypes, suggesting that other negative regulators, possibly Ptc1, prevent further activation of the Hh signaling pathway. The effects of increased Hh signaling resulting from the genetic alterations in the uki, dre, and lep mutants differ from phenotypes described as a result of Hh overexpression and therefore provide additional insight into the role of Hh signaling during vertebrate development.     

Hedgehog signaling plays a cell-autonomous role in maximizing cardiac developmental potential

Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, our understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. Here, we show that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, we find that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells.     

Hedgehog signaling regulates the amount of hypaxial muscle development during Xenopus myogenesis

Hedgehog (Hh) signaling is proposed to have different roles on differentiation of hypaxial myoblasts of amniotes. Within the somitic environment, Hh signals restrict hypaxial development and promote epaxial muscle formation. On the other hand, in the limb bud, Hh signaling represses hypaxial myoblast differentiation. This poses the question of whether differences in response to Hh signaling are due to variations in local environment or are intrinsic differences between pre- and post-migratory hypaxial myoblasts. We have approached this question by examining the role of Hh signaling on myoblast development in Xenopus laevis, which, due to its unique mode of hypaxial muscle development, allows us to examine myoblast development in vivo in the absence of the limb environment. Cyclopamine and sonic hedgehog (shh) mRNA overexpression were used to inhibit or activate the Hh pathway, respectively. We find that hypaxial myoblasts respond similarly to Hh manipulations regardless of their location, and that this response is the same for epaxial myoblasts. Overexpression of shh mRNA causes a premature differentiation of the dermomyotome, subsequently inhibiting all further growth of the epaxial and hypaxial myotome. Cyclopamine treatment has the opposite effect, causing an increase in dermomyotome and a shift in myoblast fate from epaxial to hypaxial, eventually leading to an excess of hypaxial body wall muscle. Cyclopamine treatment before stage 20 can rescue the effects of shh overexpression, indicating that early Hh signaling plays an essential role in maintaining the balance between epaxial and hypaxial muscle mass. After stage 20, the premature differentiation of the dermomyotome caused by shh overexpression cannot be rescued by cyclopamine, and no further embryonic muscle growth occurs.     

Hedgehog signaling controls homeostasis of adult intestinal smooth muscle

The Hedgehog (Hh) pathway plays multiple patterning roles during development of the mammalian gastrointestinal tract, but its role in adult gut function has not been extensively examined. Here we show that chronic reduction in the combined epithelial Indian (Ihh) and Sonic (Shh) hedgehog signal leads to mislocalization of intestinal subepithelial myofibroblasts, loss of smooth muscle in villus cores and muscularis mucosa as well as crypt hyperplasia. In contrast, chronic over-expression of Ihh in the intestinal epithelium leads to progressive expansion of villus smooth muscle, but does not result in reduced epithelial proliferation. Together, these mouse models show that smooth muscle populations in the adult intestinal lamina propria are highly sensitive to the level of Hh ligand. We demonstrate further that Hh ligand drives smooth muscle differentiation in primary intestinal mesenchyme cultures and that cell-autonomous Hh signal transduction in C3H10T1/2 cells activates the smooth muscle master regulator Myocardin (Myocd) and induces smooth muscle differentiation. The rapid kinetics of Myocd activation by Hh ligands as well as the presence of an unusual concentration of Gli sties in this gene suggest that regulation of Myocd by Hh might be direct. Thus, these data indicate that Hh is a critical regulator of adult intestinal smooth muscle homeostasis and suggest an important link between Hh signaling and Myocd activation. Moreover, the data support the idea that lowered Hh signals promote crypt expansion and increased epithelial cell proliferation, but indicate that chronically increased Hh ligand levels do not dampen crypt proliferation as previously proposed.     

Endodermal Hedgehog signals modulate Notch pathway activity in the developing digestive tract mesenchyme

The digestive tract epithelium and its adjoining mesenchyme undergo coordinated patterning and growth during development. The signals they exchange in the process are not fully characterized but include ligands of the Hedgehog (Hh) family, which originate in the epithelium and are necessary for mesenchymal cells to expand in number and drive elongation of the developing gut tube. The Notch signaling pathway has known requirements in fetal and adult intestinal epithelial progenitors. We detected Notch pathway activity in the embryonic gut mesenchyme and used conditional knockout mice to study its function. Selective disruption of the Notch effector gene RBP-Jκ (Rbpj) in the mesenchyme caused progressive loss of subepithelial fibroblasts and abbreviated gut length, revealing an unexpected requirement in this compartment. Surprisingly, constitutive Notch activity also induced rapid mesenchymal cell loss and impaired organogenesis, probably resulting from increased cell death and suggesting the need for a delicate balance in Notch signaling. Because digestive tract anomalies in mouse embryos with excess Notch activity phenocopy the absence of Hh signaling, we postulated that endodermal Hh restrains mesenchymal Notch pathway activity. Indeed, Hh-deficient embryos showed Notch overactivity in their defective gut mesenchyme and exposure to recombinant sonic hedgehog could override Notch-induced death of cultured fetal gut mesenchymal cells. These results reveal unexpected interactions between prominent signals in gastrointestinal development and provide a coherent explanation for Hh requirements in mesenchymal cell survival and organ growth.     

Ci antagonizes Hippo signaling in the somatic cells of the ovary to drive germline stem cell differentiation

Many stem cell populations are tightly regulated by their local microenvironment (niche), which comprises distinct types of stromal cells. However, little is known about mechanisms by which niche subgroups coordinately determine the stem cell fate. Here we identify that Yki, the key Hippo pathway component, is essential for escort cell (EC) function in promoting germline differentiation in Drosophila ovary. We found that Hedgehog (Hh) signals emanating primarily from cap cells support the function of ECs, where Cubitus interruptus (Ci), the Hh signaling effector, acts to inhibit Hippo kinase cascade activity. Mechanistically, we found that Ci competitively interacts with Hpo and impairs the Hpo-Wts signaling complex formation, thereby promoting Yki nuclear localization. The actions of Ci ensure effective Yki signaling to antagonize Sd/Tgi/Vg-mediated default repression in ECs. This study uncovers a mechanism explaining how subgroups of niche cells coordinate to determine the stem cell fate via Hh-Hippo signaling crosstalk, and enhances our understanding of mechanistic regulations of the oncogenic Yki/YAP signaling.     

The adventures of Sonic Hedgehog in development and repair. I. Hedgehog signaling in gastrointestinal development and disease

Hedgehog (Hh) proteins are members of a family of secreted signaling factors that orchestrate the development of many organs and tissues including those of the gastrointestinal (GI) tract. The requirement for Hh activity is not limited to early development but underlies the homeostasis of a number of tissues, and abnormal activity of the Hh pathway is associated with several GI malignancies. Understanding the roles and mechanisms of action of Hh signaling both in development and postnatally should thus give novel insights into potential treatments for these diseases. Here we focus on the Hh signaling pathway and its role in GI tract development and maintenance and consider the diseases resulting from aberrant Hh activity.     

Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling

Graded Hedgehog (Hh) signaling patterns the spinal cord dorsoventral axis by inducing and positioning distinct precursor domains, each of which gives rise to a different type of neuron. These domains also generate glial cells, but the full range of cell types that any one precursor population produces and the mechanisms that diversify cell fate are unknown. By fate mapping and clonal analysis in zebrafish, we show that individual ventral precursor cells that express olig2 can form motoneurons, interneurons and oligodendrocytes. However, olig2+ precursors are not developmentally equivalent, but instead produce subsets of progeny cells in a spatially and temporally biased manner. Using genetic and pharmacological manipulations, we provide evidence that these biases emerge from Hh acting over time to set, maintain, subdivide and enlarge the olig2+ precursor domain and subsequently specify oligodendrocyte development. Our studies show that spatial and temporal differences in Hh signaling within a common population of neural precursors can contribute to cell fate diversification.     

Competition between two phosphatases fine-tunes Hedgehog signaling

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. How its signaling activity is fine-tuned in response to fluctuated Hh gradient is less known. Here, we identify protein phosphatase V (PpV), the catalytic subunit of protein phosphatase 6, as a homeostatic regulator of Hh signaling. PpV is genetically upstream of widerborst (wdb), which encodes a regulatory subunit of PP2A that modulates high-level Hh signaling. We show that PpV negatively regulates Wdb stability independent of phosphatase activity of PpV, by competing with the catalytic subunit of PP2A for Wdb association, leading to Wdb ubiquitination and subsequent proteasomal degradation. Thus, regulated Wdb stability, maintained through competition between two closely related phosphatases, ensures graded Hh signaling. Interestingly, PpV expression is regulated by Hh signaling. Therefore, PpV functions as a Hh activity sensor that regulates Wdb-mediated PP2A activity through feedback mechanisms to maintain Hh signaling homeostasis.