Zebrafish Myotome Research Articles

Slow muscles guide fast myocyte fusion to ensure robust myotome formation despite the high spatiotemporal stochasticity of fusion events.

Skeletal myogenesis is dynamic, and it involves cell-shape changes together with cell fusion and rearrangements. However, the final muscle arrangement is highly organized with striated fibers. By combining live imagingwith quantitative analyses, we dissected fast-twitch myocyte fusion within the zebrafish myotome in toto. We found a strong mediolateral bias in fusion timing; however, at a cellular scale, there was heterogeneity in cell shape and the relationship between initial position of fast myocytes and resulting fusion partners. We show that the expression of the fusogen myomaker is permissive, but not instructive, in determining the spatiotemporal fusion pattern. Rather, we observed a close coordination between slow muscle rearrangements and fast myocyte fusion. In mutants that lack slow fibers, the spatiotemporal fusion pattern is substantially noisier. We propose a model in which slow muscles guide fast myocytes by funneling them close together, enhancing fusion probability. Thus, despite fusion being highly stochastic, a robust myotome structure emerges at the tissue scale.

Shaping the zebrafish myotome by intertissue friction and active stress

Organ formation is an inherently biophysical process, requiring large-scale tissue deformations. Yet, understanding how complex organ shape emerges during development remains a major challenge. During zebrafish embryogenesis, large muscle segments, called myotomes, acquire a characteristic chevron morphology, which is believed to aid swimming. Myotome shape can be altered by perturbing muscle cell differentiation or the interaction between myotomes and surrounding tissues during morphogenesis. To disentangle the mechanisms contributing to shape formation of the myotome, we combine single-cell resolution live imaging with quantitative image analysis and theoretical modeling. We find that, soon after segmentation from the presomitic mesoderm, the future myotome spreads across the underlying tissues. The mechanical coupling between the future myotome and the surrounding tissues appears to spatially vary, effectively resulting in spatially heterogeneous friction. Using a vertex model combined with experimental validation, we show that the interplay of tissue spreading and friction is sufficient to drive the initial phase of chevron shape formation. However, local anisotropic stresses, generated during muscle cell differentiation, are necessary to reach the acute angle of the chevron in wild-type embryos. Finally, tissue plasticity is required for formation and maintenance of the chevron shape, which is mediated by orientated cellular rearrangements. Our work sheds light on how a spatiotemporal sequence of local cellular events can have a nonlocal and irreversible mechanical impact at the tissue scale, leading to robust organ shaping.

Naa15 knockdown enhances c2c12 myoblast fusion and induces defects in zebrafish myotome morphogenesis.

The understanding of muscle tissue formation and regeneration is essential for the development of therapeutic approaches to treat muscle diseases or loss of muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres. To identify new genes controlling myoblast fusion, we performed a siRNA screen in c2c12 myoblasts. The genes identified during this screen were then studied in vivo by knockdown in zebrafish using morpholino. We found that N-alpha-acetyltransferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion, suggesting that Naa15 negatively regulates myogenic cell fusion. We identified two Naa15 orthologous genes in the zebrafish genome: Naa15a and Naa15b. These two orthologs were expressed in the myogenic domain of the somite. Knockdown of zebrafish Naa15a and Naa15b genes induced a "U"-shaped segmentation of the myotome and alteration of myotome boundaries, resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together, these results show that Naa15 regulates myotome formation and myogenesis in fish.

Muscle Stem Cells Undergo Extensive Clonal Drift during Tissue Growth via Meox1-Mediated Induction of G2 Cell-Cycle Arrest

Organ growth requires a careful balance between stem cell self-renewal and lineage commitment to ensure proper tissue expansion. The cellular and molecular mechanisms that mediate this balance are unresolved in most organs, including skeletal muscle. Here we identify a long-lived stem cell pool that mediates growth of the zebrafish myotome. This population exhibits extensive clonal drift, shifting from random deployment of stem cells during development to reliance on a small number of dominant clones to fuel the vast majority of muscle growth. This clonal drift requires Meox1, a homeobox protein that directly inhibits the cell-cycle checkpoint gene ccnb1. Meox1 initiates G2 cell-cycle arrest within muscle stem cells, and disrupting this G2 arrest causes premature lineage commitment and the resulting defects in muscle growth. These findings reveal that distinct regulatory mechanisms orchestrate stem cell dynamics during organ growth, beyond the G0/G1 cell-cycle inhibition traditionally associated with maintaining tissue-resident stem cells.

Interplay between mechanical forces and cell differentiation in the zebrafish myotome morphogenesis

Interplay between mechanical forces and cell differentiation in the zebrafish myotome morphogenesis

On the Growth and Form of the Zebrafish Myotome

On the Growth and Form of the Zebrafish Myotome

Myomaker mediates fusion of fast myocytes in zebrafish embryos

Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

Scube3 Is Expressed in Multiple Tissues during Development but Is Dispensable for Embryonic Survival in the Mouse

The vertebrate Scube family consists of three independent members Scube1-3; which encode secreted cell surface-associated membrane glycoproteins that share a domain organization of at least five recognizable motifs and the ability to both homo- and heterodimerize. There is recent biochemical evidence to suggest that Scube2 is directly involved in Hedgehog signaling, acting co-operatively with Dispatched to mediate the release in soluble form of cholesterol and palmitate-modified Hedgehog ligand during long-range activity. Indeed, in the zebrafish myotome, all three Scube proteins can subtly promote Hedgehog signal transduction in a non-cell autonomous manner. In order to further investigate the role of Scube genes during development, we have generated mice with targeted inactivation of Scube3. Despite a dynamic developmental expression pattern, with transcripts present in neuroectoderm, endoderm and endochondral tissues, particularly within the craniofacial region; an absence of Scube3 function results in no overt embryonic phenotype in the mouse. Mutant mice are born at expected Mendelian ratios, are both viable and fertile, and seemingly retain normal Hedgehog signaling activity in craniofacial tissues. These findings suggest that in the mouse, Scube3 is dispensable for normal development; however, they do not exclude the possibility of a co-operative role for Scube3 with other Scube members during embryogenesis or a potential role in adult tissue homeostasis over the long-term.

The zebrafish myotome contains tonic muscle fibers: Morphological characterization and time course of formation

It is long known that the skeletal muscle of teleost fish contains muscle fibers which are in all probability of a tonic type according to morphological criteria. However, the evidence for the existence of teleost tonic fibers is still confined to a very small number of species, and knowledge concerning their ontogeny and possible functions is even more restricted. A remarkable deficit in this context is that it is not even exactly known whether the zebrafish, which is widely used to study vertebrate developmental biology, has such fibers, or how they arise. The present study demonstrates the existence of tonic fibers in the zebrafish myotome. They are identical with a fiber population previously termed "red muscle rim" fibers. A combined histochemical, immunocytochemical, and ultrastructural approach is used to characterize the morphology and development of these fibers. This study provides a basis for using the zebrafish model system in the future research on the developmental regulation and the functions of tonic fibers.

Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome

One of the central questions of developmental biology is how cells of equivalent potential—an equivalence group—come to adopt specific cellular fates. In this study we have used a combination of live imaging, single cell lineage analyses, and perturbation of specific signaling pathways to dissect the specification of the adaxial cells of the zebrafish embryo. We show that the adaxial cells are myogenic precursors that form a cell fate equivalence group of approximately 20 cells that consequently give rise to two distinct sub-types of muscle fibers: the superficial slow muscle fibers (SSFs) and muscle pioneer cells (MPs), distinguished by specific gene expression and cell behaviors. Using a combination of live imaging, retrospective and indicative fate mapping, and genetic studies, we show that MP and SSF precursors segregate at the beginning of segmentation and that they arise from distinct regions along the anterior-posterior (AP) and dorsal-ventral (DV) axes of the adaxial cell compartment. FGF signaling restricts MP cell fate in the anterior-most adaxial cells in each somite, while BMP signaling restricts this fate to the middle of the DV axis. Thus our results reveal that the synergistic actions of HH, FGF, and BMP signaling independently create a three-dimensional (3D) signaling milieu that coordinates cell fate within the adaxial cell equivalence group.

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

Integration of Hedgehog and BMP signalling by the <i>engrailed2a</i> gene in the zebrafish myotome

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

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.

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

Laminins, via heparan sulfate proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues, such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analysed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmp-responding cells are excluded from the central domain and that this pattern is modulated by laminins, but not by Hedgehog signalling. Knockdown of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos. Last, extracellular matrix-associated heparan sulfate proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that laminins, via heparan sulfate proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

O6. Cell fate specification in the zebrafish myotome: A paradigm for cross-talk between the Hedgehog and BMP signalling pathways

The morphogen like properties of Hedgehog (Hh) family proteins have been well documented in a number of contexts. In vertebrates, the best characterized example of such a role is in the developing neural tube, where different ventral neuronal identities are specified in response to the strength and duration of Shh signaling emanating from the notochord and floorplate. In an analogous fashion, members of the BMP family secreted by the lateral non neural ectoderm and the roofplate of the neural tube act to specify dorsal neural cell fates. The effects of Shh and BMP on cell fate specification in the neural tube are mutually antagonistic but how cells integrate the effects of these opposing influences is not well understood. Several lines of evidence have shown that the specification of distinct cell types in the zebrafish myotome is similarly mediated by varying levels of Hh signalling activity, and that BMP likely plays a role in modulating the response of these cells to Hh activity. Through analysis of the regulation of the eng2a gene, a key marker of muscle cell types induced in response to high threshold levels of Shh, we have now identified a clear link between BMP signaling and expression of a putative Hh target gene. We show that Smad activity represses eng2a expression and that activated forms of Smads bind to specific Hh responsive enhancer elements upstream of the eng2a promoter: moreover, we show that nuclear accumulation of pSmads is abrogated in cells exposed to the highest levels of Hh signaling activity. Our findings provide a mechanistic basis for the response of cells to different levels of a morphogen (Shh) while at the same time revealing a novel example of “cross-talk” between the two signaling pathways.

S21-05. Hedgehog signalling and cell diversity in the zebrafish myotome

S21-05. Hedgehog signalling and cell diversity in the zebrafish myotome

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 4×5 array of cuboidal cells to a 1×20 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.

Dynamic somite cell rearrangements lead to distinct waves of myotome growth

The myogenic precursors responsible for muscle growth in amniotes develop from the dermomyotome, an epithelium at the external surface of the somite. In teleosts, the myogenic precursors responsible for growth have not been identified. We have used single cell lineage labeling in zebrafish to show that anterior border cells of epithelial somites are myogenic precursors responsible for zebrafish myotome growth. These cells move to the external surface of the embryonic myotome and express the transcription factor Pax7. Some remain on the external surface and some incorporate into the fast myotome, apparently by moving between differentiated slow fibres. The posterior cells of the somite, by contrast, elongate into medial muscle fibres. The surprising movement of the anterior somite cells to the external somite surface transforms a segmentally repeated arrangement of myogenic precursors into a medio-lateral arrangement similar to that seen in amniotes.

Cadherin-Mediated Differential Cell Adhesion Controls Slow Muscle Cell Migration in the Developing Zebrafish Myotome

Slow-twitch muscle fibers of the zebrafish myotome undergo a unique set of morphogenetic cell movements. During embryogenesis, slow-twitch muscle derives from the adaxial cells, a layer of paraxial mesoderm that differentiates medially within the myotome, immediately adjacent to the notochord. Subsequently, slow-twitch muscle cells migrate through the entire myotome, coming to lie at its most lateral surface. Here we examine the cellular and molecular basis for slow-twitch muscle cell migration. We show that slow-twitch muscle cell morphogenesis is marked by behaviors typical of cells influenced by differential cell adhesion. Dynamic and reciprocal waves of N-cadherin and M-cadherin expression within the myotome, which correlate precisely with cell migration, generate differential adhesive environments that drive slow-twitch muscle cell migration through the myotome. Removing or altering the expression of either protein within the myotome perturbs migration. These results provide a definitive example of homophilic cell adhesion shaping cellular behavior during vertebrate development.