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 cypher is important for somite formation and heart development

Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.     

Slow muscle regulates the pattern of trunk neural crest migration in zebrafish

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration; normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.     

Zebrafish periostin is required for the adhesion of muscle fiber bundles to the myoseptum and for the differentiation of muscle fibers

The myoseptum of fishes, composed of dense collagen, is a connective tissue layer that forms in the embryo, dividing somites from the trunk, and its structure and function are similar to those of the mammalian tendon. Both the myoseptum and tendon serve as the transmitter of muscular contractility to bones and adjoining muscles, and their structure is indispensable for movement of vertebrate animals. We cloned the zebrafish periostin gene and examined its expression and function in the myoseptum. The expression in embryos started in the rostral part of each segmented somite in the early segmentation stage; and consequently, metameric stripes were observed. At the end of segmentation, the expression region shifted to the transverse myoseptum and the myotome-epidermis boundary, and each myotome was surrounded by periostin. Using a polyclonal antibody, we found that the periostin protein was localized to the transverse myoseptum. Consistently, periostin morpholino antisense oligonucleotide led to defects in myoseptum formation, a delay in the differentiation of myofibers, and disorder of connection between myofibrils and myoseptum. We demonstrated here that periostin is the first molecule involved in myoseptum formation and propose that periostin secretion on the surface of the myoseptum is required for the adhesion of muscle fiber bundles to the myoseptum and the differentiation of muscle fibers.     

Hedgehog regulation of superficial slow muscle fibres in Xenopus and the evolution of tetrapod trunk myogenesis

In tetrapod phylogeny, the dramatic modifications of the trunk have received less attention than the more obvious evolution of limbs. In somites, several waves of muscle precursors are induced by signals from nearby tissues. In both amniotes and fish, the earliest myogenesis requires secreted signals from the ventral midline carried by Hedgehog (Hh) proteins. To determine if this similarity represents evolutionary homology, we have examined myogenesis in Xenopus laevis, the major species from which insight into vertebrate mesoderm patterning has been derived. Xenopus embryos form two distinct kinds of muscle cells analogous to the superficial slow and medial fast muscle fibres of zebrafish. As in zebrafish, Hh signalling is required for XMyf5 expression and generation of a first wave of early superficial slow muscle fibres in tail somites. Thus, Hh-dependent adaxial myogenesis is the likely ancestral condition of teleosts, amphibia and amniotes. Our evidence suggests that midline-derived cells migrate to the lateral somite surface and generate superficial slow muscle. This cell re-orientation contributes to the apparent rotation of Xenopus somites. Xenopus myogenesis in the trunk differs from that in the tail. In the trunk, the first wave of superficial slow fibres is missing, suggesting that significant adaptation of the ancestral myogenic programme occurred during tetrapod trunk evolution. Although notochord is required for early medial XMyf5 expression, Hh signalling fails to drive these cells to slow myogenesis. Later, both trunk and tail somites develop a second wave of Hh-independent slow fibres. These fibres probably derive from an outer cell layer expressing the myogenic determination genes XMyf5, XMyoD and Pax3 in a pattern reminiscent of amniote dermomyotome. Thus, Xenopus somites have characteristics in common with both fish and amniotes that shed light on the evolution of somite differentiation. We propose a model for the evolutionary adaptation of myogenesis in the transition from fish to tetrapod trunk.     

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.     

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.     

New insights into skeletal muscle development and growth in teleost fishes

Recent research has significantly broadened our understanding of how the teleost somite is patterned to achieve embryonic and postembryonic myogenesis. Medial (adaxial) cells and posterior cells of the early epithelial somite generate embryonic superficial slow and deep fast muscle fibers, respectively, whereas anterior somitic cells move laterally to form an external cell layer of undifferentiated Pax7-positive myogenic precursors surrounding the embryonic myotome. In late embryo and in larvae, some of the cells contained in the external cell layer incorporate into the myotome and differentiate into new muscle fibers, thus contributing to medio-lateral expansion of the myotome. This supports the suggestion that the teleost external cell layer is homologous to the amniote dermomyotome. Some of the signalling molecules that promote lateral movement or regulate the myogenic differentiation of external cell precursors have been identified and include stromal cell-derived factor 1 (Sdf1), hedgehog proteins, and fibroblast growth factor 8 (Fgf8). Recent studies have shed light on gene activations that underlie the differentiation and maturation of slow and fast muscle fibers, pointing out that both adaxially derived embryonic slow fibers and slow fibers formed during the myotome expansion of larvae initially and transiently bear features of the fast fiber phenotype.     

Control of morphogenetic cell movements in the early zebrafish myotome

As the vertebrate myotome is generated, myogenic precursor cells undergo extensive and coordinated movements as they differentiate into properly positioned embryonic muscle fibers. In the zebrafish, the "adaxial" cells adjacent to the notochord are the first muscle precursors to be specified. After initially differentiating into slow-twitch myosin-expressing muscle fibers, these cells have been shown to undergo a remarkable radial migration through the lateral somite, to populate the superficial layer of slow-twitch muscle of the mature myotome. Here we characterize an earlier set of adaxial cell behaviors; the transition from a roughly 4x5 array of cuboidal cells to a 1x20 stack of elongated cells, prior to the migration event. We find that adaxial cells display a highly stereotypical series of behaviors as they undergo this rearrangement. Furthermore, we show that the actin regulatory molecule, Cap1, is specifically expressed in adaxial cells and is required for the progression of these behaviors. The requirement of Cap1 for a cellular apical constriction step is reminiscent of similar requirements of Cap during apical constriction in Drosophila development, suggesting a conservation of gene function for a cell biological event critical to many developmental processes.     

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.     

Distinct mechanisms regulate slow-muscle development

Vertebrate muscle development begins with the patterning of the paraxial mesoderm by inductive signals from midline tissues [1, 2]. Subsequent myotome growth occurs by the addition of new muscle fibers. We show that in zebrafish new slow-muscle fibers are first added at the end of the segmentation period in growth zones near the dorsal and ventral extremes of the myotome, and this muscle growth continues into larval life. In marine teleosts, this mechanism of growth has been termed stratified hyperplasia [3]. We have tested whether these added fibers require an embryonic architecture of muscle fibers to support their development and whether their fate is regulated by the same mechanisms that regulate embryonic muscle fates. Although Hedgehog signaling is required for the specification of adaxial-derived slow-muscle fibers in the embryo [4, 5], we show that in the absence of Hh signaling, stratified hyperplastic growth of slow muscle occurs at the correct time and place, despite the complete absence of embryonic slow-muscle fibers to serve as a scaffold for addition of these new slow-muscle fibers. We conclude that slow-muscle-stratified hyperplasia begins after the segmentation period during embryonic development and continues during the larval period. Furthermore, the mechanisms specifying the identity of these new slow-muscle fibers are different from those specifying the identity of adaxial-derived embryonic slow-muscle fibers. We propose that the independence of early, embryonic patterning mechanisms from later patterning mechanisms may be necessary for growth.     

Integration of Hedgehog and BMP signalling by the engrailed2a gene in the zebrafish myotome

Different levels and timing of Hedgehog (Hh) signalling activity have been proposed to specify three distinct cell types in the zebrafish myotome. Two of these, the medial fast-twitch fibres (MFFs) and the slow-twitch muscle pioneers (MPs) are characterised by expression of eng1a, -1b and -2a and require the highest levels of Hh for their specification. We have defined a minimal eng2a element sufficient to drive reporter expression specifically in MPs and MFFs. This element binds both Gli2a, a mediator of Hh signalling, and activated Smads (pSmads), mediators of bone morphogenic protein (BMP) signalling, in vivo. We found a strict negative correlation between nuclear accumulation of pSmad, and eng2a expression in myotomal cells and show that abrogation of pSmad accumulation results in activation of eng2a, even when Hh signalling is attenuated. Conversely, driving nuclear accumulation of pSmad suppresses the induction of eng expression even when Hh pathway activity is maximal. Nuclear accumulation of pSmads is depleted by maximal Hh pathway activation. We show that a synthetic form of the Gli2 repressor interacts with Smad1 specifically in the nuclei of myotomal cells in the developing embryo and that this interaction depends upon BMP signalling activity. Our results demonstrate that the eng2a promoter integrates repressive and activating signals from the BMP and Hh pathways, respectively, to limit its expression to MPs and MFFs. We suggest a novel basis for crosstalk between the Hh and BMP pathways, whereby BMP-mediated repression of Hh target genes is promoted by a direct interaction between Smads and truncated Glis, an interaction that is abrogated by Hh induced depletion of the latter.