Standardization of zebrafish drug testing parameters for muscle diseases.

Skeletal muscular diseases predominantly affect skeletal and cardiac muscle, resulting in muscle weakness, impaired respiratory function and decreased lifespan. These harmful outcomes lead to poor health-related quality of life and carry a high healthcare economic burden. The absence of promising treatments and new therapies for muscular disorders requires new methods for candidate drug identification and advancement in animal models. Consequently, the rapid screening of drug compounds in an animal model that mimics features of human muscle disease is warranted. Zebrafish are a versatile model in preclinical studies that support developmental biology and drug discovery programs for novel chemical entities and repurposing of established drugs. Due to several advantages, there is an increasing number of applications of the zebrafish model for high-throughput drug screening for human disorders and developmental studies. Consequently, standardization of key drug screening parameters, such as animal husbandry protocols, drug compound administration and outcome measures, is paramount for the continued advancement of the model and field. Here, we seek to summarize and explore critical drug treatment and drug screening parameters in the zebrafish-based modeling of human muscle diseases. Through improved standardization and harmonization of drug screening parameters and protocols, we aim to promote more effective drug discovery programs.

Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne muscular dystrophy.

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.

Lysosomal Function Impacts the Skeletal Muscle Extracellular Matrix.

Muscle development and homeostasis are critical for normal muscle function. A key aspect of muscle physiology during development, growth, and homeostasis is modulation of protein turnover, the balance between synthesis and degradation of muscle proteins. Protein degradation depends upon lysosomal pH, generated and maintained by proton pumps. Sphingolipid transporter 1 (spns1), a highly conserved gene encoding a putative late endosome/lysosome carbohydrate/H+ symporter, plays a pivotal role in maintaining optimal lysosomal pH and spns1-/- mutants undergo premature senescence. However, the impact of dysregulated lysosomal pH on muscle development and homeostasis is not well understood. We found that muscle development proceeds normally in spns1-/- mutants prior to the onset of muscle degeneration. Dysregulation of the extracellular matrix (ECM) at the myotendinous junction (MTJ) coincided with the onset of muscle degeneration in spns1-/- mutants. Expression of the ECM proteins laminin 111 and MMP-9 was upregulated. Upregulation of laminin 111 mitigated the severity of muscle degeneration, as inhibition of adhesion to laminin 111 exacerbated muscle degeneration in spns1-/- mutants. MMP-9 upregulation was induced by tnfsf12 signaling, but abrogation of MMP-9 did not impact muscle degeneration in spns1-/- mutants. Taken together, these data indicate that dysregulated lysosomal pH impacts expression of ECM proteins at the myotendinous junction.

NAD+ improves neuromuscular development in a zebrafish model of FKRP-associated dystroglycanopathy.

BACKGROUND: Secondary dystroglycanopathies are muscular dystrophies that result from mutations in genes that participate in Dystroglycan glycosylation. Glycosylation of Dystroglycan is essential for muscle fibers to adhere to the muscle extracellular matrix (myomatrix). Although the myomatrix is disrupted in a number of secondary dystroglycanopathies, it is unknown whether improving the myomatrix is beneficial for these conditions. We previously determined that either NAD+ supplementation or overexpression of Paxillin are sufficient to improve muscle structure and the myomatrix in a zebrafish model of primary dystroglycanopathy. Here, we investigate how these modulations affect neuromuscular phenotypes in zebrafish fukutin-related protein (fkrp) morphants modeling FKRP-associated secondary dystroglycanopathy. RESULTS: We found that NAD+ supplementation prior to muscle development improved muscle structure, myotendinous junction structure, and muscle function in fkrp morphants. However, Paxillin overexpression did not improve any of these parameters in fkrp morphants. As movement also requires neuromuscular junction formation, we examined early neuromuscular junction development in fkrp morphants. The length of neuromuscular junctions was disrupted in fkrp morphants. NAD+ supplementation prior to neuromuscular junction development improved length. We investigated NMJ formation in dystroglycan (dag1) morphants and found that although NMJ morphology is disrupted in dag1 morphants, NAD+ is not sufficient to improve NMJ morphology in dag1 morphants. Ubiquitous overexpression of Fkrp rescued the fkrp morphant phenotype but muscle-specific overexpression only improved myotendinous junction structure. CONCLUSIONS: These data indicate that Fkrp plays an early and essential role in muscle, myotendinous junction, and neuromuscular junction development. These data also indicate that, at least in the zebrafish model, FKRP-associated dystroglycanopathy does not exactly phenocopy DG-deficiency. Paxillin overexpression improves muscle structure in dag1 morphants but not fkrp morphants. In contrast, NAD+ supplementation improves NMJ morphology in fkrp morphants but not dag1 morphants. Finally, these data show that muscle-specific expression of Fkrp is insufficient to rescue muscle development and homeostasis.

How muscle contraction strengthens tendons.

The force generated by muscles leads to signaling that helps to shape nearby tendon precursor cells.

Ethanol Exposure Causes Muscle Degeneration in Zebrafish.

Alcoholic myopathies are characterized by neuromusculoskeletal symptoms such as compromised movement and weakness. Although these symptoms have been attributed to neurological damage, EtOH may also target skeletal muscle. EtOH exposure during zebrafish primary muscle development or adulthood results in smaller muscle fibers. However, the effects of EtOH exposure on skeletal muscle during the growth period that follows primary muscle development are not well understood. We determined the effects of EtOH exposure on muscle during this phase of development. Strikingly, muscle fibers at this stage are acutely sensitive to EtOH treatment: EtOH induces muscle degeneration. The severity of EtOH-induced muscle damage varies but muscle becomes more refractory to EtOH as muscle develops. NF-kB induction in muscle indicates that EtOH triggers a pro-inflammatory response. EtOH-induced muscle damage is p53-independent. Uptake of Evans blue dye shows that EtOH treatment causes sarcolemmal instability before muscle fiber detachment. Dystrophin-null sapje mutant zebrafish also exhibit sarcolemmal instability. We tested whether Trichostatin A (TSA), which reduces muscle degeneration in sapje mutants, would affect EtOH-treated zebrafish. We found that TSA and EtOH are a lethal combination. EtOH does, however, exacerbate muscle degeneration in sapje mutants. EtOH also disrupts adhesion of muscle fibers to their extracellular matrix at the myotendinous junction: some detached muscle fibers retain beta-Dystroglycan indicating failure of muscle end attachments. Overexpression of Paxillin, which reduces muscle degeneration in zebrafish deficient for beta-Dystroglycan, is not sufficient to rescue degeneration. Taken together, our results suggest that EtOH exposure has pleiotropic deleterious effects on skeletal muscle.

A need for NAD+ in muscle development, homeostasis, and aging.

Skeletal muscle enables posture, breathing, and locomotion. Skeletal muscle also impacts systemic processes such as metabolism, thermoregulation, and immunity. Skeletal muscle is energetically expensive and is a major consumer of glucose and fatty acids. Metabolism of fatty acids and glucose requires NAD+ function as a hydrogen/electron transfer molecule. Therefore, NAD+ plays a vital role in energy production. In addition, NAD+ also functions as a cosubstrate for post-translational modifications such as deacetylation and ADP-ribosylation. Therefore, NAD+ levels influence a myriad of cellular processes including mitochondrial biogenesis, transcription, and organization of the extracellular matrix. Clearly, NAD+ is a major player in skeletal muscle development, regeneration, aging, and disease. The vast majority of studies indicate that lower NAD+ levels are deleterious for muscle health and higher NAD+ levels augment muscle health. However, the downstream mechanisms of NAD+ function throughout different cellular compartments are not well understood. The purpose of this review is to highlight recent studies investigating NAD+ function in muscle development, homeostasis, disease, and regeneration. Emerging research areas include elucidating roles for NAD+ in muscle lysosome function and calcium mobilization, mechanisms controlling fluctuations in NAD+ levels during muscle development and regeneration, and interactions between targets of NAD+ signaling (especially mitochondria and the extracellular matrix). This knowledge should facilitate identification of more precise pharmacological and activity-based interventions to raise NAD+ levels in skeletal muscle, thereby promoting human health and function in normal and disease states.

"Muscling" Throughout Life: Integrating Studies of Muscle Development, Homeostasis, and Disease in Zebrafish.

The proper development and function of skeletal muscle is vital for health throughout the lifespan. Skeletal muscle function enables posture, breathing, and locomotion; and also impacts systemic processes-such as metabolism, thermoregulation, and immunity. Diseases of skeletal muscle (myopathies, muscular dystrophies) and even some neurological, age-related, and metabolic diseases compromise muscle function and negatively affect health span and quality of life. There have been numerous, recent examples of studies on skeletal muscle development with exciting, therapeutic implications for muscle diseases. The zebrafish (Danio rerio) is a vertebrate model organism well accepted for developmental biology and biomedical research and thus an ideal system in which to elucidate the translational implications of mechanisms regulating skeletal muscle development and homeostasis. Muscle fiber types (slow- vs fast-twitch) are spatially segregated in zebrafish allowing for the opportunity to identify distinct mechanisms regulating fiber type specification during development as well as observe fiber type-specific effects in zebrafish models of muscle diseases. Accessible genetics coupled with transparent zebrafish embryos has enabled in vivo cell biology experiments allowing for the visualization and understanding of never-before-seen cellular processes occurring in muscle development, regeneration, and disease. In addition, high-throughput drug screening provides a platform for efficient drug discovery. The purpose of this chapter is to review the studies in zebrafish that significantly contributed to our understanding of cellular and molecular mechanisms regulating skeletal muscle development, homeostasis, or disease in vertebrates, with a particular emphasis on the basic developmental biology studies with promising therapeutic implications.

Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction.

BACKGROUND: Remodeling of the extracellular matrix (ECM) regulates cell adhesion as well as signaling between cells and their microenvironment. Despite the importance of tightly regulated ECM remodeling for normal muscle development and function, mechanisms underlying ECM remodeling in vivo remain elusive. One excellent paradigm in which to study ECM remodeling in vivo is morphogenesis of the myotendinous junction (MTJ) during zebrafish skeletal muscle development. During MTJ development, there are dramatic shifts in the primary components comprising the MTJ matrix. One such shift involves the replacement of Fibronectin (Fn)-rich matrix, which is essential for both somite and early muscle development, with laminin-rich matrix essential for normal function of the myotome. Here, we investigate the mechanism underlying this transition. RESULTS: We show that laminin polymerization indirectly promotes Fn downregulation at the MTJ, via a matrix metalloproteinase 11 (Mmp11)-dependent mechanism. Laminin deposition and organization is required for localization of Mmp11 to the MTJ, where Mmp11 is both necessary and sufficient for Fn downregulation in vivo. Furthermore, reduction of residual Mmp11 in laminin mutants promotes a Fn-rich MTJ that partially rescues skeletal muscle architecture. CONCLUSIONS: These results identify a mechanism for Fn downregulation at the MTJ, highlight crosstalk between laminin and Fn, and identify a new in vivo function for Mmp11. Taken together, our data demonstrate a novel signaling pathway mediating Fn downregulation. Our data revealing new regulatory mechanisms that guide ECM remodeling during morphogenesis in vivo may inform pathological conditions in which Fn is dysregulated.

Hanging on for the ride: adhesion to the extracellular matrix mediates cellular responses in skeletal muscle morphogenesis and disease.

Skeletal muscle specification and morphogenesis during early development are critical for normal physiology. In addition to mediating locomotion, skeletal muscle is a secretory organ that contributes to metabolic homeostasis. Muscle is a highly adaptable tissue, as evidenced by the ability to increase muscle cell size and/or number in response to weight bearing exercise. Conversely, muscle wasting can occur during aging (sarcopenia), cancer (cancer cachexia), extended hospital stays (disuse atrophy), and in many genetic diseases collectively known as the muscular dystrophies and myopathies. It is therefore of great interest to understand the cellular and molecular mechanisms that mediate skeletal muscle development and adaptation. Muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction. This process requires carefully orchestrated interactions between cells and their extracellular matrix microenvironment. These interactions are dynamic, allowing muscle cells to sense biophysical, structural, organizational, and/or signaling changes within their microenvironment and respond appropriately. In many musculoskeletal diseases, these cell adhesion interactions are disrupted to such a degree that normal cellular adaptive responses are not sufficient to compensate for accumulating damage. Thus, one major focus of current research is to identify the cell adhesion mechanisms that drive muscle morphogenesis, with the hope that understanding how muscle cell adhesion promotes the intrinsic adaptability of muscle tissue during development may provide insight into potential therapeutic approaches for muscle diseases. Our objectives in this review are to highlight recent studies suggesting conserved roles for cell-extracellular matrix adhesion in vertebrate muscle morphogenesis and cellular adaptive responses in animal models of muscle diseases.

NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex- or integrin alpha7-deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies.

Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis.

Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not beta-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn plays roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis.

Dynamic interactions between cells and their extracellular matrix mediate embryonic development.

Cells and their surrounding extracellular matrix microenvironment interact throughout all stages of life. Understanding the continuously changing scope of cell-matrix interactions in vivo is crucial to garner insights into both congenital birth defects and disease progression. A current challenge in the field of developmental biology is to adapt in vitro tools and rapidly evolving imaging technology to study cell-matrix interactions in a complex 4-D environment. In this review, we highlight the dynamic modulation of cell-matrix interactions during development. We propose that individual cell-matrix adhesion proteins are best considered as complex proteins that can play multiple, often seemingly contradictory roles, depending upon the context of the microenvironment. In addition, cell-matrix proteins can also exert different short versus long term effects. It is thus important to consider cell behavior in light of the microenvironment because of the constant and dynamic reciprocal interactions occurring between them. Finally, we suggest that analysis of cell-matrix interactions at multiple levels (molecules, cells, tissues) in vivo is critical for an integrated understanding because different information can be acquired from all size scales.

Hedgehog signaling and laminin play unique and synergistic roles in muscle development.

Hedgehog (Hh) signaling and laminin-111, a basement membrane protein, are required for early muscle development. Hh signaling specifies different populations of muscle fibers and laminin-111 is critical for early muscle morphogenesis. However, additional requirements for Hh signaling and laminin during later phases of muscle development are not known. Furthermore, interactions between Hh signaling and laminin in this context are unknown. We used laminin gamma1 mutant zebrafish and cyclopamine to block Hh signal transduction separately and in combination to investigate their functions and interactions. We found that both Hh signaling and laminin are required for normal myosin chain expression. In addition, Hh signaling and laminin act synergistically during fast-twitch fiber elongation: fast muscle cells do not elongate in embryos deficient for both Hh signaling and laminin. Finally, we present evidence that suggests that Hh signaling is indirectly required via slow fiber specification for recovery of fast fiber elongation in laminin gamma1 mutant embryos.

Dynamic formation of microenvironments at the myotendinous junction correlates with muscle fiber morphogenesis in zebrafish.

Muscle development involves the specification and morphogenesis of muscle fibers that attach to tendons. After attachment, muscles and tendons then function as an integrated unit to transduce force to the skeletal system and stabilize joints. The attachment site is the myotendinous junction, or MTJ, and is the primary site of force transmission. We find that attachment of fast-twitch myofibers to the MTJ correlates with the formation of novel microenvironments within the MTJ. The expression or activation of two proteins involved in anchoring the intracellular cytoskeleton to the extracellular matrix, Focal adhesion kinase (Fak) and beta-dystroglycan is up-regulated. Conversely, the extracellular matrix protein Fibronectin (Fn) is down-regulated. This degradation of Fn as fast-twitch fibers attach to the MTJ results in Fn protein defining a novel microenvironment within the MTJ adjacent to slow-twitch, but not fast-twitch, muscle. Interestingly, however, Fak, laminin, Fn and beta-dystroglycan concentrate at the MTJ in mutants that do not have slow-twitch fibers. Taken together, these data elucidate novel and dynamic microenvironments within the MTJ and indicate that MTJ morphogenesis is spatially and temporally complex.

Time-lapse analysis and mathematical characterization elucidate novel mechanisms underlying muscle morphogenesis.

Skeletal muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction (MTJ). In vertebrates, a great deal is known about muscle specification as well as how somitic cells, as a cohort, generate the early myotome. However, the cellular mechanisms that generate long muscle fibers from short cells and the molecular factors that limit elongation are unknown. We show that zebrafish fast muscle fiber morphogenesis consists of three discrete phases: short precursor cells, intercalation/elongation, and boundary capture/myotube formation. In the first phase, cells exhibit randomly directed protrusive activity. The second phase, intercalation/elongation, proceeds via a two-step process: protrusion extension and filling. This repetition of protrusion extension and filling continues until both the anterior and posterior ends of the muscle fiber reach the MTJ. Finally, both ends of the muscle fiber anchor to the MTJ (boundary capture) and undergo further morphogenetic changes as they adopt the stereotypical, cylindrical shape of myotubes. We find that the basement membrane protein laminin is required for efficient elongation, proper fiber orientation, and boundary capture. These early muscle defects in the absence of either lamininbeta1 or laminingamma1 contrast with later dystrophic phenotypes in lamininalpha2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development. Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon. Taken together, our results define three phases of muscle fiber morphogenesis and show that the critical second phase of elongation proceeds by a repetitive process of protrusion extension and protrusion filling. Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.

Muscle development is disrupted in zebrafish embryos deficient for fibronectin.

After somitogenesis, skeletal muscle precursors elongate into muscle fibers that anchor to the somite boundary, which becomes the myotome boundary. Fibronectin (Fn) is a major component of the extracellular matrix in both boundaries. Although Fn is required for somitogenesis, effects of Fn disruption on subsequent muscle development are unknown. We show that fn knockdown disrupts myogenesis. Muscle morphogenesis is more disrupted in fn morphants than in a mutant where initial somite boundaries did not form, aei/deltaD. We quantified this disruption using the two-dimensional Wavelet-Transform Modulus Maxima method, which uses the variation of intensity in an image with respect to the direction considered to characterize the structure in a cell lattice. We show that fibers in fn morphants are less organized than in aei/deltaD mutant embryos. Fast- and slow-twitch muscle lengths are also more frequently uncoupled. These data suggest that fn may function to regulate fiber organization and limit fast-twitch muscle fiber length.

The role of the SPT6 chromatin remodeling factor in zebrafish embryogenesis.

Somitogenesis is a highly controlled process that results in segmentation of the paraxial mesoderm. Notch pathway activity in the presomitic mesoderm is fundamental for management of synchronized gene expression which is necessary for regulation of somitogenesis. We have isolated an embryonic lethal mutation, SBU2, that causes somite formation defects very similar to Notch pathway mutants. SBU2 mutants generate only 6-7 asymmetrically arranged somites. However, in contrast to Notch pathway mutants, these mutants do not maintain previously formed somite boundaries and by 24 hpf, almost no somite boundaries remain. Other developmental processes disrupted in SBU2 mutants include tail morphogenesis, muscle fiber elongation, pigmentation, circulatory system development and neural differentiation. We demonstrated that these defects are the result of a nonsense mutation within the spt6 gene. spt6 encodes a transcription elongation factor that genetically interacts with the Paf-1 chromatin remodeling complex. SBU2 mutant phenotypes could be rescued by microinjection of spt6 mRNA and microinjection of spt6 morpholinos phenocopied the mutation. Our real-time PCR analysis revealed that Spt6 is essential for the transcriptional response to activation of the Notch pathway. Analysis of sbu2;mib double mutants indicates that Spt6 deficiency suppresses the neurogenic effects of the mib. Altogether, these results demonstrate that Spt6 is critical for somite formation in zebrafish and suggest that some defects observed in spt6 mutants result from alterations in Notch signaling. However, additional Spt6 mutant phenotypes are likely caused by vital functions of Spt6 in other pathways.

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.

Regionally autonomous segmentation within zebrafish presomitic mesoderm.

Similar to chick, mouse, and Xenopus, a number of intercellular signaling pathways are required for zebrafish segmentation. However, the spatial scales over which these signaling pathways operate in zebrafish remain largely unknown. During zebrafish segmentation, waves of her1 transcription (a) initiate within the tailbud region, (b) propagate anteriorly through presomitic mesoderm (PSM), and (c) terminate in the anlage of newly-forming somites. These observations raise the question of whether the tailbud region serves as a "pacemaker" or "organizing center" for the initiation of propagating her1 expression waves. Microsurgical manipulations reveal that the anteriorly waves of her1 transcription are not perturbed by removal of the zebrafish tailbud. Furthermore, expression patterns of deltaD, paraxial protocadherin C (papc), and myoD within recently formed somites also appear to be relatively unperturbed by either removal of the tailbud, or by removal of lateral plate mesoderm. Although dynamic gene networks rapidly specify mesenchymal or epithelial cellular identities within forming somites, this specification is plastic. Time-lapse analysis has shown that the cellular progeny of mitotically-active epithelial border cells within newly formed somites can adopt different cellular identities than their precursor cells. Overall, these results indicate that sustained long-range intercellular communication with the tailbud, anterior somites, or lateral plate mesoderm is not necessary for segmentation or somitogenesis to proceed within the PSM. The basic segmentation and somitogenesis processes in zebrafish presomitic mesoderm appear to be largely regionally autonomous and governed by local morphogenetic cell behaviors.