Vertebral pattern and morphology is determined during embryonic segmentation.

BACKGROUND: The segmented nature of the adult vertebral column is based on segmentation of the paraxial mesoderm during early embryogenesis. Disruptions to embryonic segmentation, whether caused by genetic lesions or environmental stress, result in adult vertebral pathologies. However, the mechanisms linking embryonic segmentation and the details of adult vertebral morphology are poorly understood. RESULTS: We induced border defects using two approaches in zebrafish: heat stress and misregulation of embryonic segmentation genes tbx6, mesp-ba, and ripply1. We assayed vertebral length, regularity, and polarity using microscopic and radiological imaging. In population studies, we find a correlation between specific embryonic border defects and specific vertebral defects, and within individual fish, we trace specific adult vertebral defects to specific embryonic border defects. CONCLUSIONS: Our data reveal that transient disruptions of embryonic segment border formation led to significant vertebral anomalies that persist through adulthood. The spacing of embryonic borders controls the length of the vertebra. The positions of embryonic borders control the positions of ribs and arches. Embryonic borders underlie fusions and divisions between adjacent spines and ribs. These data suggest that segment borders have a dominant role in vertebral development.

Characterizing the diverse cells that associate with the developing commissures of the zebrafish forebrain.

During embryonic development of bilaterally symmetrical organisms, neurons send axons across the midline at specific points to connect the two halves of the nervous system with a commissure. Little is known about the cells at the midline that facilitate this tightly regulated process. We exploit the conserved process of vertebrate embryonic development in the zebrafish model system to elucidate the identity of cells at the midline that may facilitate postoptic (POC) and anterior commissure (AC) development. We have discovered that three different gfap+ astroglial cell morphologies persist in contact with pathfinding axons throughout commissure formation. Similarly, olig2+ progenitor cells occupy delineated portions of the postoptic and anterior commissures where they act as multipotent, neural progenitors. Moreover, we conclude that both gfap+ and olig2+ progenitor cells give rise to neuronal populations in both the telencephalon and diencephalon; however, these varied cell populations showed significant developmental timing differences between the telencephalon and diencephalon. Lastly, we also showed that fli1a+ mesenchymal cells migrate along the presumptive commissure regions before and during midline axon crossing. Furthermore, following commissure maturation, specific blood vessels formed at the midline of the POC and immediately ventral and parallel to the AC. This comprehensive account of the cellular populations that correlate with the timing and position of commissural axon pathfinding has supported the conceptual modeling and identification of the early forebrain architecture that may be necessary for proper commissure development.

Anterior trunk muscle shows mix of axial and appendicular developmental patterns.

BACKGROUND: Skeletal muscle in the trunk derives from the somites, paired segments of paraxial mesoderm. Whereas axial musculature develops within the somite, appendicular muscle develops following migration of muscle precursors into lateral plate mesoderm. The development of muscles bridging axial and appendicular systems appears mixed. RESULTS: We examine development of three migratory muscle precursor-derived muscles in zebrafish: the sternohyoideus (SH), pectoral fin (PF), and posterior hypaxial (PHM) muscles. We show there is an anterior to posterior gradient to the developmental gene expression and maturation of these three muscles. SH muscle precursors exhibit a long delay between migration and differentiation, PF muscle precursors exhibit a moderate delay in differentiation, and PHM muscle precursors show virtually no delay between migration and differentiation. Using lineage tracing, we show that lateral plate contribution to the PHM muscle is minor, unlike its known extensive contribution to the PF muscle and absence in the ventral extension of axial musculature. CONCLUSIONS: We propose that PHM development is intermediate between a migratory muscle mode and an axial muscle mode of development, wherein the PHM differentiates after a very short migration of its precursors and becomes more anterior primarily by elongation of differentiated muscle fibers.

Regulatory Network of the Scoliosis-Associated Genes Establishes Rostrocaudal Patterning of Somites in Zebrafish.

Gene regulatory networks govern pattern formation and differentiation during embryonic development. Segmentation of somites, precursors of the vertebral column among other tissues, is jointly controlled by temporal signals from the segmentation clock and spatial signals from morphogen gradients. To explore how these temporal and spatial signals are integrated, we combined time-controlled genetic perturbation experiments with computational modeling to reconstruct the core segmentation network in zebrafish. We found that Mesp family transcription factors link the temporal information of the segmentation clock with the spatial action of the fibroblast growth factor signaling gradient to establish rostrocaudal (head to tail) polarity of segmented somites. We further showed that cells gradually commit to patterning by the action of different genes at different spatiotemporal positions. Our study provides a blueprint of the zebrafish segmentation network, which includes evolutionarily conserved genes that are associated with the birth defect congenital scoliosis in humans.

Osmotic and Heat Stress Effects on Segmentation.

During vertebrate embryonic development, early skin, muscle, and bone progenitor populations organize into segments known as somites. Defects in this conserved process of segmentation lead to skeletal and muscular deformities, such as congenital scoliosis, a curvature of the spine caused by vertebral defects. Environmental stresses such as hypoxia or heat shock produce segmentation defects, and significantly increase the penetrance and severity of vertebral defects in genetically susceptible individuals. Here we show that a brief exposure to a high osmolarity solution causes reproducible segmentation defects in developing zebrafish (Danio rerio) embryos. Both osmotic shock and heat shock produce border defects in a dose-dependent manner, with an increase in both frequency and severity of defects. We also show that osmotic treatment has a delayed effect on somite development, similar to that observed in heat shocked embryos. Our results establish osmotic shock as an alternate experimental model for stress, affecting segmentation in a manner comparable to other known environmental stressors. The similar effects of these two distinct environmental stressors support a model in which a variety of cellular stresses act through a related response pathway that leads to disturbances in the segmentation process.

Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord.

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial-derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later-born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter-driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap-negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood-brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170-1189.

Tbx6, Mesp-b and Ripply1 regulate the onset of skeletal myogenesis in zebrafish.

During embryonic development, the paraxial mesoderm becomes segmented into somites, within which proliferative muscle progenitors and muscle fibers establish the skeletal musculature. Here, we demonstrate that a gene network previously implicated in somite boundary formation, involving the transcriptional regulators Tbx6, Mesp-b and Ripply1, also confers spatial and temporal regulation to skeletal myogenesis in zebrafish. We show that Tbx6 directly regulates mesp-b and ripply1 expression in vivo, and that the interactions within the regulatory network are largely conserved among vertebrates. Mesp-b is necessary and sufficient for the specification of a subpopulation of muscle progenitors, the central proportion of the Pax3(+)/Pax7(+) dermomyotome. Conditional ubiquitous expression indicates that Mesp-b acts by inhibiting myogenic differentiation and by inducing the dermomyotome marker meox1. By contrast, Ripply1 induces a negative-feedback loop by promoting Tbx6 protein degradation. Persistent Tbx6 expression in Ripply1 knockdown embryos correlates with a deficit in dermomyotome and myotome marker gene expression, suggesting that Ripply1 promotes myogenesis by terminating Tbx6-dependent inhibition of myogenic maturation. Together, our data suggest that Mesp-b is an intrinsic upstream regulator of skeletal muscle progenitors and that, in zebrafish, the genes regulating somite boundary formation also regulate the development of the dermomyotome in the anterior somite compartment.

Immunocytochemistry to study myogenesis in zebrafish.

During myogenesis, cells gradually transition from mesodermal precursors to myoblasts, myocytes, and then to muscle fibers. The molecular characterization of this process requires the ability to identify each of these cell types and the factors that regulate the transitions between them. The most versatile technique for assaying cell identities in situ is immunocytochemistry, because multiple independent molecular markers of differentiation can be assayed simultaneously. The zebrafish has developed into a popular model for the study of myogenesis, and immunocytochemical techniques have been critical. We have adapted existing protocols to optimize immunocytochemistry in zebrafish, and have tested many antibodies developed against mouse, chick, and frog muscle antigens for their cross-reactivity in zebrafish. Here, we present protocols for whole mount immunocytochemistry on both formaldehyde and Carnoy's fixed embryos as well as on sectioned zebrafish tissue. We include a table of antibodies useful for experiments on the molecular biology of myogenesis in zebrafish.

BMP regulation of myogenesis in zebrafish.

In amniotes, BMP signaling from lateral plate and dorsal neural tube inhibits differentiation of muscle precursors in the dermomyotome. Here, we show that BMPs are expressed adjacent to the dermomyotome during and after segmentation in zebrafish. In addition, downstream BMP pathway members are expressed within the somite during dermomyotome development. We also show that zebrafish dermomyotome is responsive to BMP throughout its development. Ectopic overexpression of Bmp2b increases expression of the muscle precursor marker pax3, and changes the time course of myoD expression. At later stages, overexpression increases the number of Pax7+ myogenic precursors, and delays muscle differentiation, as indicated by decreased numbers of MEF2+ nuclei, decreased number of multi-nucleated muscle fibers, and an increased myotome angle. In addition, we show that while BMP overexpression is sufficient to delay myogenic differentiation, inhibition of BMP does not detectably affect this process, suggesting that other factors redundantly inhibit myogenic differentiation.

Growth in the larval zebrafish pectoral fin and trunk musculature.

After initial patterning, muscle in the trunk and fins of teleosts grows extensively. Here, we describe muscle growth in zebrafish, with emphasis on the pectoral fin musculature. In the trunk, slow muscle fibers differentiate first. In contrast, slow muscle does not appear in the pectoral fin until the beginning of the juvenile period. Mosaic hyperplasia contributes to trunk muscle growth, and new fibers are apparent within the muscle as early as 6 mm standard length. In the pectoral fin muscle, mosaic hyperplasia is not evident at any examined stage. Instead, the predominant mode of hyperplasia is stratified. In larval pectoral fin muscle new fibers appear subjacent to the skin, and this correlates with the expression of myogenic genes such as muscle regulatory factors and Pax7. Our results suggest that regulation of fiber type development and muscle growth may differ in the pectoral fin and trunk.

The teleost dermomyotome.

Recent work in teleosts has renewed interest in the dermomyotome, which was initially characterized in the late 19th century. We review the evidence for the teleost dermomyotome, comparing it to the more well-characterized amniote dermomyotome. We discuss primary myotome morphogenesis, the relationship between the primary myotome and the dermomyotome, the differentiation of axial muscle, appendicular muscle, and dermis from the dermomyotome, and the signaling molecules that regulate myotome growth from myogenic precursors within the dermomyotome. The recognition of a dermomyotome in teleosts provides a new perspective on teleost muscle growth, as well as a fruitful approach to understanding the vertebrate dermomyotome.

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.

Hedgehog acts directly on the zebrafish dermomyotome to promote myogenic differentiation.

Vertebrate myogenesis is regulated by signaling proteins secreted from surrounding tissues. One of the most important, Sonic hedgehog, has been proposed to regulate myogenic precursor cell survival, proliferation, and differentiation in a variety of vertebrates. In zebrafish, Hedgehog signaling is both necessary and sufficient for the development of embryonic slow muscle fibers-the earliest differentiating muscle fibers. Here we investigated the function of Hedgehog signaling in another zebrafish myogenic lineage, a dermomyotomal population of cells defined by somitic pax3/7 expression. We found that Hedgehog negatively regulates the number of myogenic precursors expressing pax3/7. Hh also positively regulates the growth of embryonic fast muscle. Unlike Hedgehog's function in regulating the elongation of fast muscle fibers, this regulation is not mediated by embryonic slow muscle fibers. Instead, it is a direct Hedgehog response, cell autonomous to myogenic precursors. The regulation of myogenic precursors and their differentiation into fast fibers have a different critical time period for Hh signaling, and different requirements for specific gli gene family members of Hh activated transcription factors from the earlier promotion of embryonic slow muscle fiber differentiation. We propose that Hedgehog signaling acts at multiple times on different lineages, through different downstream pathways, to promote myogenic differentiation.

The development of muscle fiber type identity in zebrafish cranial muscles.

Cranial skeletal muscles underlie breathing, eating, and eye movements. In most animals, at least two types of muscle fibers underlie these critical functions: fast and slow muscle fibers. We describe here the anatomical distribution of slow and fast twitch muscle in the zebrafish (Danio rerio) head in the adult and at an early larval stage just after feeding has commenced. We found that all but one of the cranial muscles examined contain both slow and fast muscle fibers, but the relative proportion of slow muscle in each varies considerably. As in the trunk, slow muscle fibers are found only in an anatomically restricted zone of each muscle, usually on the periphery. The relative proportion of slow and fast muscle in each cranial muscle changes markedly with development, with a pronounced decrease in the proportion of slow muscle with ontogeny. We discuss our results in relation to the functional roles of each muscle in larval and adult life and compare findings among a variety of vertebrates.

Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors.

In zebrafish, skeletal muscle precursors can adopt at least three distinct fates: fast, non-pioneer slow, or pioneer slow muscle fibers. Slow muscle fibers develop from adaxial cells and depend on Hedgehog signaling. We analyzed when precursors become committed to their fates and the step(s) along their differentiation pathway affected by Hedgehog. Unexpectedly, we find that embryos deficient in Hedgehog signaling still contain postmitotic adaxial cells that differentiate into fast muscle fibers instead of slow. We show that by the onset of gastrulation, slow and fast muscle precursors are already spatially segregated but uncommitted to their fates until much later, in the segmental plate when slow precursors become independent of Hedgehog. In contrast, pioneer and non-pioneer slow muscle precursors share a common lineage from the onset of gastrulation. Our results demonstrate that slow muscle precursors form independently of Hedgehog signaling and further provide direct evidence for a multipotent muscle precursor population whose commitment to the slow fate depends on Hedgehog at a late stage of development when postmitotic adaxial cells differentiate into slow muscle fibers.

Functional morphology and developmental biology of zebrafish: reciprocal illumination from an unlikely couple.

Functional morphology has benefited greatly from the input of techniques and thinking from other disciplines. This has been especially productive in situations where each discipline has made significant contributions to a particular research topic. A combination of methodologies from functional morphology and developmental biology has allowed us to characterize feeding mechanics of first-feeding larval zebrafish (Danio rerio). Contrary to kinematic patterns commonly seen in adult teleosts, larval zebrafish showed no lateral abduction during the expansive phase of a suction-feeding event. Instead, dorsoventral expansion of the buccal chamber, more typical of patterns seen in primitive fishes, characterized the expansive phase. Moreover, a pronounced preparatory phase during which the buccal chamber is constricted by the protractor hyoideus was consistently seen in first-feeding larval kinematics. Key kinematic variables associated with first feeding correlated significantly with the hydrodynamic regime as measured by the Reynolds number. Using the tools of both functional morphology and developmental biology we have not only determined which cranial muscles are important for successful feeding but also uncovered important physiological differences in muscle structure. Muscles necessary for the rapid dorsoventral expansion of the head are composed primarily of fast-twitch fibers while those involved in more tonic contractions such as hyoid protraction have more slow-twitch muscle fibers. While most evolutionary developmental studies have examined mechanisms responsible for large evolutionary changes in morphology, we propose that the type of data uncovered in functional studies can lead to the generation of hypotheses concerning the developmental mechanisms responsible for smaller intra- and/or interspecific changes.