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Morin-Kensicki, E.M. & Eisen, J.S. Sclerotome development and peripheral nervous system segmentation in embryonic zebrafish. Development 124, 159-167
Abstract
Vertebrate embryos display segmental patterns in many trunk structures, including somites and peripheral nervous system elements. Previous work in avian embryos suggests a role for somite-derived sclerotome in segmental patterning of the peripheral nervous system. We investigated sclerotome development and tested its role in patterning motor axons and dorsal root ganglia in embryonic zebrafish. Individual somite cells labeled with vital fluorescent dye revealed that some cells of a ventromedial cell cluster within each somite produced mesenchymal cells that migrated to positions expected for sclerotome. Individual somites showed anterior/posterior distinctions in several aspects of development: (1) anterior ventromedial cluster cells produced only sclerotome, (2) individual posterior ventromedial cluster cells produced both sclerotome and muscle, and (3) anterior sclerotome migrated earlier and along a more restricted path than posterior sclerotome. Vital labeling showed that anterior sclerotome colocalized with extending identified motor axons and migrating neural crest cells. To investigate sclerotome involvement in peripheral nervous system patterning, we ablated the ventromedial cell cluster and observed subsequent development of peripheral nervous system elements. Primary motor axons were essentially unaffected by sclerotome ablation, although in some cases outgrowth was delayed. Removal of sclerotome did not disrupt segmental pattern or development of dorsal root ganglia or peripheral nerves to axial muscle. We propose that peripheral nervous system segmentation is established through interactions with adjacent paraxial mesoderm which develops as sclerotome in some vertebrate species and myotome in others.
... The teleost fish somite is predominantly myotome, with very little sclerotome [96] (Fig 4 A, C). In zebrafish (Danio rerio), each somite separates from the PSM as an envelope of cells embracing the rounded cells within it [96]. ...
... The teleost fish somite is predominantly myotome, with very little sclerotome [96] (Fig 4 A, C). In zebrafish (Danio rerio), each somite separates from the PSM as an envelope of cells embracing the rounded cells within it [96]. Time-lapse imaging of zebrafish PSM revealed that prior to separation of somites from each other, the initially intermingled prospective border cells undergo segregation, alignment along the medio-lateral axis, and anterior-posterior elongation; however, cells between the border cells do not undergo those changes. ...
... In the zebrafish, the sclerotome is much smaller than its chick counterpart and forms ventromedially as an epithelial cell cluster after a somite separates from the PSM [96]. As in amniotes, somites are subdivided into rostral and caudal halves. ...
Vertebrate segmentation, the process that generates a regular arrangement of somites and thereby establishes the pattern of the adult body and of the musculoskeletal and peripheral nervous systems, was noticed many centuries ago. In the last few decades, there has been renewed interest in the process and especially in the molecular mechanisms that might account for its regularity and other spatial-temporal properties. Several models have been proposed but surprisingly, most of these do not provide clear links between the molecular mechanisms and the cell behaviours that generate the segmental pattern. Here we present a short survey of our current knowledge about the cellular aspects of vertebrate segmentation and the similarities and differences between different vertebrate classes in how they achieve their metameric pattern. Taking these variations into account should help to assess each of the models more appropriately.
... Somites then differentiate into three regions: myotome (muscle), dermomyotome, and sclerotome (skeletal structures). Each of these regions accounts for different percentages of the somite in different organisms, with tetrapods characterized by increased sclerotome (Morin-Kensicki and Eisen, 1997;Stickney et al., 2000;Morin-Kensicki et al., 2002;Scaal and Wiegreffe, 2006). The sclerotome then further segregates into anterior and posterior regions which migrate around the notochord to develop into vertebral elements (Morin-Kensicki and Eisen, 1997). ...
... Each of these regions accounts for different percentages of the somite in different organisms, with tetrapods characterized by increased sclerotome (Morin-Kensicki and Eisen, 1997;Stickney et al., 2000;Morin-Kensicki et al., 2002;Scaal and Wiegreffe, 2006). The sclerotome then further segregates into anterior and posterior regions which migrate around the notochord to develop into vertebral elements (Morin-Kensicki and Eisen, 1997). Our analyses investigating differential growth among species, among axial levels, and among vertebral elements allow us to narrow the focus of likely developmental and genetic players involved in the formation and diversification of the Weberian apparatus. ...
... The potentially limited role of the upstream sclerotomal genes in mediating morphological change in the teleostean vertebral column is likely due to its reduced contribution to the somite of teleosts. The increased need for locomotory muscles with a reduced need for a robust axial skeleton results in a greatly increased myotome and reduced sclerotome in fishes (Morin-Kensicki and Eisen, 1997;Stickney et al., 2000;Morin-Kensicki et al., 2002;Scaal and Wiegreffe, 2006;Stellabotte and Devoto, 2007). While important sclerotomal genes, such as Pax9 (and its regulators Bmp7 and Fgf8) and twist, will still be critical for sclerotomal differentiation, repression of muscle fate, and establishment of a skeletal fate in condensing mesenchyme before the start of chondrogenesis of the vertebral column (Nornes et al., 1996;Stickney et al., 2000;Jackman et al., 2004), their upstream position likely renders them of reduced importance in the fine-tuned changes necessary for the morphology seen in the Weberian apparatus (Fig. 4). ...
... In zebrafish, as in amniotes, spinal motor axons exit the spinal cord from a common ventral root into each hemisegment; however the substrate essential for migration has been shown to be myotome, not sclerotome (Eisen and Pike, 1991). Support for this came when researchers ablated sclerotome in the somite, and found axon outgrowth to be unaffected (MorinKensicki and Eisen, 1997). Further support followed when molecules, Tenascin-C, chondroitin sulfate proteoglycans, and Sema3A2 were found to be restricted to the posterior myotome (Bernhardt et al., 1998;Bernhardt and Schachner, 2000;Roos et al., 1999). ...
... The ventromedial myotome, composed of fast muscle, has been shown to be crucial for proper CaP axon extension. When the ventromedial cell cluster containing sclerotome and 20 myotome was ablated, CaP axons displayed a stunted appearance (MorinKensicki and Eisen, 1997). This stereotyped axon outgrowth along the ventromedial fast muscle implies the presence of ventrally located guidance cues, but none have yet been identified. ...
... The ventromedial myotome, composed of fast muscle, has been shown to be crucial for proper CaP axon extension. When the ventromedial cell cluster containing sclerotome and myotome was ablated, CaP axons displayed a stunted appearance (MorinKensicki and Eisen, 1997). This stereotyped axon outgrowth along the ventromedial fast muscle implies the presence of ventrally located guidance cues, but none have yet been identified. ...
... Therefore, in addition to tenocytes and perivascular fibroblasts associated with ISVs (Rajan et al., 2020) that we previously described, we identified new types of sclerotome-derived cells in the fin fold, associated with distinct vascular beds, as well as in the interstitial space. The contribution of muscles from the nkx3.1 lineage suggests that nkx3.1 labels some early bipotent somitic cells that generate both sclerotome progenitors and muscles, which is consistent with previous single-cell lineage analysis of the ventral somite (Morin-Kensicki and Eisen, 1997). In addition to the five groups of cells from the nkx3.1 lineage, the nkx3.1:Gal4 ...
... This R.C. Ma et al. Developmental Biology 498 (2023) 35-48 observation is in agreement with previous single-cell lineage analysis of the presumptive sclerotome in the ventral somite where a sub-population of these cells gives rise to both sclerotome derivatives and muscles (Morin-Kensicki and Eisen, 1997). We speculate that some bipotent somitic cells might transiently express nkx3.1, ...
Fibroblasts play an important role in maintaining tissue integrity by secreting components of the extracellular matrix and initiating response to injury. Although the function of fibroblasts has been extensively studied in adults, the embryonic origin and diversification of different fibroblast subtypes during development remain largely unexplored. Using zebrafish as a model, we show that the sclerotome, a sub-compartment of the somite, is the embryonic source of multiple fibroblast subtypes including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, fin mesenchymal cells, and interstitial fibroblasts. High-resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Long-term Cre-mediated lineage tracing reveals that the sclerotome also contributes to cells closely associated with the axial skeleton. Ablation of sclerotome progenitors results in extensive skeletal defects. Using photoconversion-based cell lineage analysis, we find that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single-cell clonal analysis combined with in vivo imaging suggests that the sclerotome mostly contains unipotent and bipotent progenitors prior to cell migration, and the fate of their daughter cells is biased by their migration paths and relative positions. Together, our work demonstrates that the sclerotome is the embryonic source of trunk fibroblasts as well as the axial skeleton, and local signals likely contribute to the diversification of distinct fibroblast subtypes.
... Three main fates were equally represented-stromal cells of the CHT (33%), DMCs (31%), and muscle fibers (31%), while the FMC fate was more marginal (5%) (Fig. 5E). This cell fate analysis thus confirmed our previous findings, but added the muscle fiber category, reminiscent of the pioneer study by Morin-Kensicki et al. 9 who found that the posterior part of each ventro-medial cell cluster gave rise to some muscle cells in addition to the typical sclerotome fates (Fig. 5F). Only SCP fated cells underwent mitosis during the observed period (4/14), and they did so on their way to or within the CHT; therefore all photoconverted cells showed only one fate during the tracked period, suggesting that cell fate within the VCs may already be restricted by somite stage S8 (the earliest stage at which Kaede was sufficiently expressed for photoconversion-based cell tracking). ...
... We identified the earliest morphological individualization of the caudal sclerotome as a distinct ventral cell cluster appearing at somite maturation stage S5 (in agreement with Morin-Kensicki et al. 9 for the trunk somites) and encompassing the whole mediolateral extent of the somite before these cells become mesenchymal and migrate both dorsal-and ventral-wards. Interestingly, Naganathan et al. 51 recently found that the antero-posterior length of freshly formed somites is somewhat variable and that over the next 1-2 h (i.e., until somite stage S4), they undergo a mechanical adjustment of this A-P length that is facilitated by somite surface tension, which requires the somite to be fully packed within an uninterrupted basal lamina. ...
Mesenchymal stromal cells are essential components of hematopoietic stem and progenitor cell (HSPC) niches, regulating HSPC proliferation and fates. Their developmental origins are largely unknown. In zebrafish, we previously found that the stromal cells of the caudal hematopoietic tissue (CHT), a niche functionally homologous to the mammalian fetal liver, arise from the ventral part of caudal somites. We have now found that this ventral domain is the sclerotome, and that two markers of mammalian mesenchymal stem/stromal cells, Alcam and Pdgfr-α, are distinctively expressed there and instrumental for the emergence and migration of stromal cell progenitors, which in turn conditions the proper assembly of the vascular component of the CHT niche. Furthermore, we find that trunk somites are similarly dependent on Alcam and Pdgfr-α to produce mesenchymal cells that foster HSPC emergence from the aorta. Thus the sclerotome contributes essential stromal cells for each of the key steps of developmental hematopoiesis.
... 10 In zebrafish, as in other teleosts, the majority of somite-derived cells are myotomal, with only a small ventrally localized sclerotome compartment forming. 150,151 Ablation of the zebrafish sclerotome does not disrupt the segmental pattern or subsequent development of dorsal root ganglia (DRG). 151 Instead, the slow muscle precursors, termed adaxial cells, regulate the pattern of neural crest migration on the medial pathway. ...
... 150,151 Ablation of the zebrafish sclerotome does not disrupt the segmental pattern or subsequent development of dorsal root ganglia (DRG). 151 Instead, the slow muscle precursors, termed adaxial cells, regulate the pattern of neural crest migration on the medial pathway. 150 This is perhaps not surprising as myotome cells are the first encountered by migrating NCCs and, in fact, the onset of neural crest migration coincides with contact between the two cell types. ...
Our understanding of the neural crest, a key vertebrate innovation, is built upon studies of multiple model organisms. Early research on neural crest cells (NCCs) was dominated by analyses of accessible amphibian and avian embryos, with mouse genetics providing complementary insights in more recent years. The zebrafish model is a relative newcomer to the field, yet it offers unparalleled advantages for the study of NCCs. Specifically, zebrafish provide powerful genetic and transgenic tools, coupled with rapidly developing transparent embryos that are ideal for high‐resolution real‐time imaging of the dynamic process of neural crest development. While the broad principles of neural crest development are largely conserved across vertebrate species, there are critical differences in anatomy, morphogenesis, and genetics that must be considered before information from one model is extrapolated to another. Here, our goal is to provide the reader with a helpful primer specific to neural crest development in the zebrafish model. We focus largely on the earliest events—specification, delamination, and migration—discussing what is known about zebrafish NCC development and how it differs from NCC development in non‐teleost species, as well as highlighting current gaps in knowledge.
... Hedgehog (Hh) signaling plays an essential role in somite patterning [6][7][8]. Sonic hedgehog (Shh) is expressed in the notochord and the floor plate, and patterns the surrounding tissues including the somite. In the canonical pathway, Shh functions by binding to its receptor Patched (Ptc), which releases the repression on Smoothened (Smo). ...
... In both zebrafish and medaka, expression of the sclerotomal markers pax9 and twist2 in the ventral region of the somite [23][24][25] suggests that basic sclerotome development is conserved between higher vertebrates and teleosts. Indeed, cell tracing experiments in zebrafish demonstrate that the presumptive sclerotome contributes to the formation of the vertebral column [7,26]. Morpholino knock-down of key sclerotome genes in medaka, such as pax1, pax9 and twist2, causes defects in vertebral column formation [25]. ...
Author summary
The coordinated generation of bones, muscles and tendons at the correct time and location is critical for the development of a functional musculoskeletal system. Although it is well known that tendon is the connective tissue that attaches muscles to bones, it is still poorly understood how tendon cells, or tenocytes, are generated during embryo development. Using the zebrafish model, we identify trunk tenocytes located along the boundary of muscle segments. Using cell tracing in live animals, we find that tenocytes originate from the sclerotome, an embryonic structure that is previously known to generate the trunk skeleton. In contrast to higher vertebrates, the zebrafish sclerotome consists of two separate domains, a ventral domain and a novel dorsal domain. Both domains give rise to trunk tenocytes in a dynamic and stereotypic manner. Hedgehog (Hh) signaling, an important cell signaling pathway, is not required for sclerotome induction but essential for the generation of sclerotome derived cells. Inhibition of Hh signaling leads to loss of tenocytes and increased sensitivity to muscle detachment. Thus, our work shows that tenocytes develop from the sclerotome and play an important role in maintaining muscle integrity.
... However, frontal sections through trout embryos showed that not all cells expressing col1a1 surrounded the axial structure (Fig. 4C); some of them, instead, were found to colonise, in an apparent medio-lateral progression, the space separating two adjacent myotomes (Fig. 4D-E and D9-E9). Thus, the apparent movement of collagen I expressing cells is compatible with the view that the fish sclerotome gives rise not only to cells contributing to the formation of the axial skeleton as previously reported [21], but also to cells that intercalate between myotomes to form the myoseptum. ...
... Using col1a1 as a marker gene, we found that its expression was first detected in disseminating cells ventral to the myotome before being observed in presumptive skeletogenic cells found along a path between axial structures and the myotome, and cells invading the intermyotomal space. This apparent movement of col1a1 expressing cells in early trout embryo opens the possibility that the sclerotome gives rise to a population of cells involved in the development of the axial skeleton (i.e., the formation of the neural and haemal arch rudiments) as previously reported [21], [32], as well as to a cell population that participates to the formation of the fish myoseptum. However, to definitely demonstrate that sclerotome in fish produces myoseptal cells it will be necessary to achieve genetic lineage labelling and time-lapse analysis [33]. ...
The trunk muscle in fish is organized as longitudinal series of myomeres which are separated by sheets of connective tissue called myoseptum to which myofibers attach. In this study we show in the trout that the myoseptum separating two somites is initially acellular and composed of matricial components such as fibronectin, laminin and collagen I. However, myoseptal cells forming a continuum with skeletogenic cells surrounding axial structures are observed between adjacent myotomes after the completion of somitogenesis. The myoseptal cells do not express myogenic markers such as Pax3, Pax7 and myogenin but express several tendon-associated collagens including col1a1, col5a2 and col12a1 and angiopoietin-like 7, which is a secreted molecule involved in matrix remodelling. Using col1a1 as a marker gene, we observed in developing trout embryo an initial labelling in disseminating cells ventral to the myotome. Later, labelled cells were found more dorsally encircling the notochord or invading the intermyotomal space. This opens the possibility that the sclerotome gives rise not only to skeletogenic mesenchymal cells, as previously reported, but also to myoseptal cells. We furthermore show that myoseptal cells differ from skeletogenic cells found around the notochord by the specific expression of Scleraxis, a distinctive marker of tendon cells in amniotes. In conclusion, the location, the molecular signature and the possible sclerotomal origin of the myoseptal cells suggest that the fish myoseptal cells are homologous to the axial tenocytes in amniotes.
... Our principal aim was to map the fate of the anterior-most Somites three to five in the Mexican axolotl. The technique of injecting a marker (DiI, fluorescein dextran) into single somites for cell tracing has been used successfully in the past (Bagnall, 1992;Morin-Kensicki and Eisen, 1997;Morin-Kensicki et al., 2002;Ferguson and Graham, 2004;Olsson, 2007, 2011). Embryos from late neurula to early tailbud stages (i.e., Stages 19-25) were used for individually labeling Somites three to five. ...
... Our results from fate mapping of somites in the axolotl clearly show the existence of resegmentation in a species with a small sclerotome. In addition, fate-mapping experiments in zebrafish revealed resegmentation in this anamniote with a small sclerotome and indistinct anterior and posterior sclerotomal halves (Morin-Kensicki and Eisen, 1997;Stickney et al., 2000;Morin-Kensicki et al., 2002;Scaal and Wiegreffe, 2006). Thus, there are two anamniote examples for the existence of resegmentation despite a small size of the sclerotome. ...
The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well-studied using the quail-chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.
... Some of these include the proportion of the somites, the segmentation pattern of the spine that relies on the segmentation clock patterning, and the segmental periodicity of vertebrae that is determined by the sclerotome instead of the notochord in some species. However, in teleost fish, the segmentation of the spine relies on the notochord, with contribution from the paraxial mesoderm (Morin-Kensicki and Eisen, 1997). Despite these differences, the general architecture of the spine is evolutionarily conserved among vertebrate groups and suggests the existence of multiscale and spatially defined signatures of its morphogenesis at the phenogenetic level (Boswell and Ciruna, 2017). ...
The vertebral column, or spine, provides mechanical support and determines body axis posture and motion. The most common malformation altering spine morphology and function is adolescent idiopathic scoliosis (AIS), a three-dimensional spinal deformity that affects approximately 4% of the population worldwide. Due to AIS genetic heterogenicity and the lack of suitable animal models for its study, the etiology of this condition remains unclear, thus limiting treatment options. We here review current advances in zebrafish phenogenetics concerning AIS-like models and highlight the recently discovered biological processes leading to spine malformations. First, we focus on gene functions and phenotypes controlling critical aspects of postembryonic aspects that prime in spine architecture development and straightening. Second, we summarize how primary cilia assembly and biomechanical stimulus transduction, cerebrospinal fluid components and flow driven by motile cilia have been implicated in the pathogenesis of AIS-like phenotypes. Third, we highlight the inflammatory responses associated with scoliosis. We finally discuss recent innovations and methodologies for morphometrically characterize and analyze the zebrafish spine. Ongoing phenotyping projects are expected to identify novel and unprecedented postembryonic gene functions controlling spine morphology and mutant models of AIS. Importantly, imaging and gene editing technologies are allowing deep phenotyping studies in the zebrafish, opening new experimental paradigms in the morphometric and three-dimensional assessment of spinal malformations. In the future, fully elucidating the phenogenetic underpinnings of AIS etiology in zebrafish and humans will undoubtedly lead to innovative pharmacological treatments against spinal deformities.
... While somitogenesis and muscle development has been studied extensively in both amniotes (Monsoro-Burq 2003;Stockdale et al. 2000;Tani et al. 2020;Saga 2012;Gridley 2006;Brand-Saberi et al. 1996) and zebrafish (Devoto et al. 1996;Weinberg et al. 1996;Stellabotte et al. 2007;Nguyen-Chi et al. 2012), the formation of sclerotome and the genetic regulatory networks involved have remained understudied in zebrafish. A sclerotomal compartment in the ventral somite has been described in zebrafish, albeit in the context of the influence of sclerotome development on segmentation of the peripheral nervous system (Morin-Kensicki and Eisen 1997) and the vertebral column (Morin-Kensicki et al. 2002). In zebrafish, a contribution of sclerotome cells to the neural and haemal arches has been established; however, whether the sclerotome contributes to the formation of centra is still being disputed (Bensimon-Brito et al. 2012;Fleming et al. 2001Fleming et al. , 2004Fleming et al. , 2015Inohaya et al. 2007;Renn et al. 2013;Willems et al. 2012). ...
Atonal homologue 8 ( atoh8 ) is a basic helix-loop-helix transcription factor expressed in a variety of embryonic tissues. While several studies have implicated atoh8 in various developmental pathways in other species, its role in zebrafish development remains uncertain. So far, no studies have dealt with an in-depth in situ analysis of the tissue distribution of atoh8 in embryonic zebrafish. We set out to pinpoint the exact location of atoh8 expression in a detailed spatio-temporal analysis in zebrafish during the first 24 h of development (hpf). To our surprise, we observed transcription from pre-segmentation stages in the paraxial mesoderm and during the segmentation stages in the somitic sclerotome and not—as previously reported—in the myotome. With progressing maturation of the somites, the restriction of atoh8 to the sclerotomal compartment became evident. Double in situ hybridisation with atoh8 and myoD revealed that both genes are expressed in the somites at coinciding developmental stages; however, their domains do not spatially overlap. A second domain of atoh8 expression emerged in the embryonic brain in the developing cerebellum and hindbrain. Here, we observed a specific expression pattern which was again in contrast to the previously published suggestion of atoh8 transcription in neural crest cells. Our findings point towards a possible role of atoh8 in sclerotome, cerebellum and hindbrain development. More importantly, the results of this expression analysis provide new insights into early sclerotome development in zebrafish—a field of research in developmental biology which has not received much attention so far.
... While a bona fide amniote-like sclerotome region cannot be ascribed during amphioxus embryogenesis, amphioxus homologs of known vertebrate sclerotome markers, Bapx (Nk3), FoxC, and Tbx1/10, are expressed in the myotome (Chapman et al., 1996;Winnier et al., 1997;Hiemisch et al., 1998;Tanaka et al., 1999). Hence, in amphioxus, the medial portion (be it the myotome or the lateral somite) displays the molecular features of both sclerotome and myotome, reminiscent of the posterior ventromedial cells of teleosts, which give rise to both sclerotome and muscle cells (Morin-Kensicki and Eisen, 1997). In addition, two other important sclerotome markers, Pax1/9 and Twist, are expressed in the dorsolateral and centrolateral portions of the somite, reminiscent of the recently identified dorsal sclerotome domain in teleosts (Mansfield et al., 2015;Ma et al., 2018). ...
Mineralized skeletal tissues of vertebrates are an evolutionary novelty within the chordate lineage. While the progenitor cells that contribute to vertebrate skeletal tissues are known to have two embryonic origins, the mesoderm and neural crest, the evolutionary origin of their developmental process remains unclear. Using cephalochordate amphioxus as our model, we found that cells at the lateral wall of the amphioxus somite express SPARC (a crucial gene for tissue mineralization) and various collagen genes. During development, some of these cells expand medially to surround the axial structures, including the neural tube, notochord and gut, while others expand laterally and ventrally to underlie the epidermis. Eventually these cell populations are found closely associated with the collagenous matrix around the neural tube, notochord, and dorsal aorta, and also with the dense collagen sheets underneath the epidermis. Using known genetic markers for distinct vertebrate somite compartments, we showed that the lateral wall of amphioxus somite likely corresponds to the vertebrate dermomyotome and lateral plate mesoderm. Furthermore, we demonstrated a conserved role for BMP signaling pathway in somite patterning of both amphioxus and vertebrates. These results suggest that compartmentalized somites and their contribution to primitive skeletal tissues are ancient traits that date back to the chordate common ancestor. The finding of SPARC-expressing skeletal scaffold in amphioxus further supports previous hypothesis regarding SPARC gene family expansion in the elaboration of the vertebrate mineralized skeleton.
... and fw/sf (Nornes et al., 1996), and migrate dorsally to encircle the notochord and neural tube (Morin-Kensicki and Eisen, 1997). It has been demonstrated that in zebrafish, leaky' resegmentation occurs (Morin-Kensicki et al., 2002). ...
Vertebrate somitogenesis involves the establishment of a segmental pattern of gene expression within the presomitic mesoderm (PSM) and the subsequent translation of this pattern into physical furrows and epithelial somites. In the first part of this thesis we have investigated the role of Eph/Ephrin signalling in this process. We show that in the fused somites mutant, lack of intersomitic boundaries and failure of paraxial mesodermal cells to undergo mesenchymal-to-epithelial transition is accompanied by a lack of Eph/Ephrin signalling interfaces. Using mosaic analysis, we provide evidence for a role for the Eph/Ephrin signalling pathway in somite boundary morphogenesis and epithelialisation. Restoration of the Eph/Ephrin interface in the paraxial mesoderm of fss/tbx24 embryos resulted in the rescue of morphological boundaries and many aspects of epithelialisation. In the second part of this thesis we took two approaches to try to understand how the early prepatterning mechanisms lead to the segmental expression of ephA4 and efnB2a in the rostral PSM. We screened a subtractive library enriched for clones expressed in the PSM. We have isolated two interesting genes from this library, a hes6 and meox1. We present here the analysis of hes6. It does not appear to have a role during somitogenesis but instead appears to regulate early cell-fate specification, gastrulation and neurogenesis. In a second approach we have analysed the promoter regions of the paralogs efnB2a and efnB2b. To facilitate this analysis we tested and then used the IScel meganuclease system. We established two transgenic lines that drive GFP under the control of efnB2a and efnB2b promoters. The efnB2a transgenic line recapitulates the endogenous expression in the PSM but the efnB2b line only drives expression during gastrulation. Based on this and a comprehensive comparison of efnB2a and efnB2b and EfnB2 expression, the evolution of efnB2 promoter regions is discussed.
... We also found uncx4.1-expressing cells on both sides of the notochord, perhaps corresponding to the fish sclerotome, the myogenic contribution to backbone formation (Morin- Kensicki and Eisen, 1997) (Fig. 3K, L). ...
The paired-type homeodomain transcription factor Uncx is involved in multiple processes of embryogenesis in vertebrates. Reasoning that zebrafish genes uncx4.1 and uncx are orthologs of mouse Uncx, we studied their genomic environment and developmental expression. Evolutionary analyses indicate the zebrafish uncx genes as being paralogs deriving from teleost-specific whole-genome duplication. Whole-mount in situ mRNA hybridization of uncx transcripts in zebrafish embryos reveals novel expression domains, confirms those previously known, and suggests sub-functionalization of paralogs. Using genetic mutants and pharmacological inhibitors, we investigate the role of signaling pathways on the expression of zebrafish uncx genes in developing somites. In identifying putative functional role(s) of zebrafish uncx genes, we hypothesized that they encode transcription factors that coordinate growth and innervation of somitic muscles.
... Besides Drosophila and human, members of the TWIST family have also been identified in different species such as frog [26], mouse [27], leech [28], zebrafish [29], lancelet [30], nematode [31], jellyfish [32] and chicken [33]. ...
TWIST1 is thought to be a novel oncogene. Understanding the molecular mechanisms regulating the TWIST1 gene expression profiles in tumor cells may give new insights regarding prognostic factors and novel therapeutic targets in veterinary oncology. In the present study we partially isolated the TWIST1 gene in Felis catus and performed comparative studies. Several primer combinations were used based on the alignments of homologous DNA sequences. After PCR amplification, three bands were obtained, purified and sequenced. Several bioinformatic tools were utilized to carry out the comparative studies. Higher similarity was found between the isolated TWIST1 gene in Felis catus and Homo sapiens (86%) than between Homo sapiens and Rattus norvegicus or Mus musculus (75%). Partial amino acid sequence showed no change in the four species analyzed. This confirmed that coding sequences presented high similarity (~96%) between man and cat. These results give the first insights regarding the TWIST1 gene in cat but further studies are required in order to establish, or not, its role in tumor formation and progression in veterinary oncology.
... The anatomy and development of the teleost spine is somewhat different to the situation in amniotes. The teleost sclerotome is much smaller, with most of the somite cells going on to form myotome (Morin-Kensicki and Eisen, 1997). The notochord also differs in the two vertebrate groups. ...
The vertebral column is segmented, comprising an alternating series of vertebrae and intervertebral discs along the head-tail axis. The vertebrae and outer portion (annulus fibrosus) of the disc are derived from the sclerotome part of the somites, whereas the inner nucleus pulposus of the disc is derived from the notochord. Here we investigate the role of the notochord in vertebral patterning through a series of microsurgical experiments in chick embryos. Ablation of the notochord causes loss of segmentation of vertebrae and discs. However, the notochord cannot segment in the absence of the surrounding sclerotome. To test whether the notochord dictates sclerotome segmentation, we grafted an ectopic notochord. We find that the intrinsic segmentation of the sclerotome is dominant over any segmental information the notochord may possess, and no evidence that the chick notochord is intrinsically segmented. We propose that the segmental pattern of vertebral bodies and discs in chick is dictated by the sclerotome, which first signals to the notochord to ensure that the nucleus pulposus develops in register with the somite-derived annulus fibrosus. Later, the notochord is required for maintenance of sclerotome segmentation as the mature vertebral bodies and intervertebral discs form. These results highlight differences in vertebral development between amniotes and zebrafish and some other teleosts, where the notochord dictates the segmental pattern. The relative importance of the sclerotome and notochord in vertebral patterning has changed significantly during evolution.
... In amniotes, it has been shown experimentally that the NC cells are permitted to migrate only within the rostral half of the somites (i.e. migration is inhibited by the posterior half of the somites); thereby, the dorsal root ganglia appear at the level of the anterior half of the somites (Keynes and Stern, 1984;Loring and Erickson, 1987;Tosney, 1988a;Tosney, 1988b;Kalcheim and Teillet, 1989;Kalcheim and Goldstein, 1991;Le Douarin and Dupin, 1993;Koblar et al., 2000;Bussen et al., 2004;Gammill et al., 2006;Schwarz et al., 2009;Kalcheim, 2011;Lumb et al., 2014; also see Detwiler, 1934; as for the zebrafish development, see Morin-Kensicki and Eisen, 1997;Honjo and Eisen, 2005). This migratory route of trunk NC cells has been called the ventrolateral pathway. ...
The migration and distribution patterns of neural crest (NC) cells reflect the distinct embryonic environments of the head and trunk: cephalic NC cells migrate predominantly along the dorsolateral pathway to populate the craniofacial and pharyngeal regions, whereas trunk crest cells migrate along the ventrolateral pathways to form the dorsal root ganglia. These two patterns thus reflect the branchiomeric and somitomeric architecture, respectively, of the vertebrate body plan. The so-called vagal NC occupies a postotic, intermediate level between the head and trunk NC. This level of NC gives rise to both trunk- and cephalic-type (circumpharyngeal) NC cells. The anatomical pattern of the amphioxus, a basal chordate, suggests that somites and pharyngeal gills coexist along an extensive length of the body axis, indicating that the embryonic environment is similar to that of vertebrate vagal NC cells and may have been ancestral for vertebrates. The amniote-like condition in which the cephalic and trunk domains are distinctly separated would have been brought about, in part, by anteroposterior reduction of the pharyngeal domain.
... In this way, embryonic cells of fish commit rapidly to the myogenic linage (at the end of gastrulation), whereas in amniotes myogenesis occurs until somitogenesis. These differences allow fish to move rapidly at hatch [10,31]. ...
... Each population of cells migrates along unique pathways and contributes to specific cell and tissue types that are specific to their axial level of origin. In the trunk region of most animals, neural crest cells travel in two pathways, a medial pathway through the somitic mesoderm or between the neural tube and somites and a dorsolateral pathway between the somites and the overlying ectoderm (Kensicki and Eisen 1997). There are, however, species-specific differences in neural crest cell formation and migration between the mouse and avian species (Nichols 1986; Serbedzija et al. 1992). ...
Draxin is a repulsive axon guidance protein that plays important roles in the formation of three commissures in the central nervous system and dorsal interneuron 3 (dI3) in the chick spinal cord. In the present study, we report the expression pattern of mouse draxin in the embryonic mouse trunk spinal cord. In the presence of draxin, the longest net migration length of a migrating mouse trunk neural crest cell was significantly reduced. In addition, the relative number of apolar neural crest cells increased as the draxin treatment time increased. Draxin caused actin cytoskeleton rearrangement in the migrating trunk neural crest cells. Our data suggest that draxin may regulate mouse trunk neural crest cell migration by the rearrangement of cell actin cytoskeleton and by reducing the polarization activity of these cells subsequently.
... Besides Drosophila and human, members of the TWIST family have also been identified in different species such as frog [26], mouse [27], leech [28], zebrafish [29], lancelet [30], nematode [31], jellyfish [32] and chicken [33]. ...
... Lineage of single cells in the ventral somite of zebrafish embryos has confirmed that they give rise to sclerotome, although some single cells can give rise to both myotome and sclerotome cells. 49 Further work has characterized the contribution of specific somites to the vertebrae; as has been shown for other vertebrates, cells from individual somites B178-Ch11 26/08/04, 2:12 PM 397 contribute to two adjacent vertebrae. 50 However, more recent work has revealed discrepancies between this accepted model of vertebral formation and the actual mechanisms in zebrafish. ...
The zebrafish, a species within the family Cyprinidae (minnows and carps), has emerged as an important vertebrate model for the study of development, including skeletal development, due to the availability of embryological, molecular, and genetic tools. Ichthyology has a long history, extending from Aristotle through present, fueled by a large number of species relative to most major vertebrate groups and by the considerable fossil record consisting of fish skeletons. The comparative adult osteology of fishes is a traditional area of study within the field, and many of the differences in skeletal anatomy closely track the paths of evolution within various clades. Developmental studies in zebrafish have the potential to clarify historically significant evolutionary questions pertaining to the evolution of the skeleton. We focus on the study of skeletogenesis in zebrafish, although our emphasis is on the extent to which findings can be generalized to other vertebrates. After a review of descriptive studies, we discuss the availability of molecular markers, and the mutational analysis of skeletal development in zebrafish. Finally, we conclude with a discussion of prospects for additional, targeted mutant screens, and studies to examine lineage relationships among skeletogenic cells in the embryo and the adult.
... Since secondary motor axons pioneer the ventral root in the absence of all primary motor axons (Pike et al., 1992), additional guidance cues must delineate the common path for secondary motor axons. Migratory neural crest cells and sclerotome cells share the common path and sclerotome ablation causes a retardation of the CaP axon that exhibits unusal branches (Kensicki and Eisen, 1997). Thus, the sclerotome might similarly influence secondary motor axons, but the influence of migratory neural crest cells on the pathfinding of spinal motor axons is not known. ...
Neurolin (zf DM-GRASP), a transmembrane protein with five extracellular immunoglobulin domains, is expressed by secondary but not primary motoneurons during zebrafish development. The spatiotemporally restricted expression pattern suggests that Neurolin plays a role in motor axon growth and guidance. To test this hypothesis, we injected zebrafish embryos with function-blocking Neurolin antibodies. In injected embryos, secondary motor axons form a broadened bundle along the common path and ectopic branches leave the common path at right angles. Moreover, the formation of the ventral and the rostral projection of secondary motor axons is inhibited during the second day of development. Pathfinding errors, resulting in secondary motor axons growing through ectopic regions of the somites, occur along the common path and in the dorsal and rostral projection. Our data are compatible with the view that Neurolin is involved in the recognition of guidance cues and acts as a receptor on secondary motor axons. Consistent with this idea is the binding pattern of a soluble Neurolin-Fc construct showing that putative ligands are distributed along the common path, the ventral projection, and in the area where the rostral projection develops.
... Anamniotes (fish and amphibians) differ in several important respects. Among the main differences, anamniote somites are mainly composed of myotome (future axial muscle) and the sclerotome is almost absent [70]. The vertebral column, especially in Teleost fish, may derive much more prominently from the notochord, which may even have an intrinsic segmental pattern [71,72]. ...
During development, vertebrate embryos produce serially repeated elements, the somites, on each side of the midline. These generate the vertebral column, skeletal musculature and dermis. They form sequentially, one pair at a time, from mesenchymal tissue near the tail. Somite development is a complex process. The embryo must control the number, size, and timing of somite formation, their subdivision into functional regions along three axes, regional identity such that somites develop in a region-specific way, and interactions with neighbouring tissues that coordinate them with nearby structures. Here we discuss many timing-related mechanisms that contribute to set up the spatial pattern.
Copyright © 2015. Published by Elsevier Ltd.
... The dermomyotome will give rise to the primary myotome, to muscle progenitors that sustain embryonic and fetal growth, and to muscle stem cells that mediate homeostasis and repair in the adult (Relaix et al., 2005;Ben-Yair and Kalcheim, 2005;Kassar-Duchossy et al., 2005;Gros et al., 2005;Schienda et al., 2006). In zebrafish, the majority of somitic cells give rise to the primary myotome, with the sclerotome and syndetome elements occupying a small proportion of the somite (Kimmel et al., 1995;Morin-Kensicki and Eisen, 1997). Although zebrafish lack a dermomyotome per se, there is a population of somitic cells that gives rise to an external cell layer (ECL) that covers the myotome Siegel et al., 2013;Stellabotte and Devoto, 2007) (Fig. 2). ...
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.
... They begin as balls of mesoderm that form in a rostrocaudal order. In lower vertebrates, such as fish, the somite mainly gives rise to muscle (Morin-Kensicki & Eisen, 1997). Zebrafish somites form at a rate of one every 20-30 minutes beginning at ~10 hpf and continuing on to about 24 hpf to create a total of 30-34 somites (Devoto et al., 1996). ...
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... By contrast, the vertical myosepta are believed to be of sclerotomal origin, making them analogous to mammalian axial tendon tissue (Bricard et al., 2014;Charvet et al., 2011). In the developing somite of zebrafish, which comprises predominantly myotomal cells, the sclerotomal cells form in the ventralmost domain and migrate dorsally to eventually surround the notochord and neural tube (Morin-Kensicki and Eisen, 1997;Stickney et al., 2000). Interestingly, we observed strong scxa expression in ventral myoseptal regions between 36 and 48 hpf ( Fig. 1B; data not shown). ...
Despite the importance of tendons and ligaments for transmitting movement and providing stability to the musculoskeletal system, their development is considerably less well understood than that of the tissues they serve to connect. Zebrafish have been widely used to address questions in muscle and skeletal development, yet few studies describe their tendon and ligament tissues. We have analyzed in zebrafish the expression of several genes known to be enriched in mammalian tendons and ligaments, including scleraxis (scx), collagen 1a2 (col1a2) and tenomodulin (tnmd), or in the tendon-like myosepta of the zebrafish (xirp2a). Co-expression studies with muscle and cartilage markers demonstrate the presence of scxa, col1a2 and tnmd at sites between the developing muscle and cartilage, and xirp2a at the myotendinous junctions. We determined that the zebrafish craniofacial tendon and ligament progenitors are neural crest derived, as in mammals. Cranial and fin tendon progenitors can be induced in the absence of differentiated muscle or cartilage, although neighboring muscle and cartilage are required for tendon cell maintenance and organization, respectively. By contrast, myoseptal scxa expression requires muscle for its initiation. Together, these data suggest a conserved role for muscle in tendon development. Based on the similarities in gene expression, morphology, collagen ultrastructural arrangement and developmental regulation with that of mammalian tendons, we conclude that the zebrafish tendon populations are homologous to their force-transmitting counterparts in higher vertebrates. Within this context, the zebrafish model can be used to provide new avenues for studying tendon biology in a vertebrate genetic system.
... This may reflect a reduced demand for the supporting skeleton and an increased locomotory requirement for axial, rather than appendicular, muscle associated with life in a supportive aqueous medium (Bone 1966). As a result, the positional relationships between tissues are altered, and the sclerotome, instead of forming adjacent to the underside of the notochord, arises at some distance from it (Morin-Kensicki and Eisen 1997). The notochord and neural tube are thus embedded entirely within the myotome, which constitutes the bulk of the somite (Currie and Ingham 1998). ...
Three phases of myogenesis have been identified in the myotomal muscles of larval teleosts. The commitment of embryonic slow and fast muscle lineages is determined prior to segmentation (embryonic myogenesis) and involves notochord and floorplate derived signaling pathways, which drive the adaxial cells to a slow muscle fate. The adaxial cells elongate to span the entire somite width and subsequently migrate through the myotome to form a superficial layer of slow muscle fibers. The remaining cells of the lateral mesoderm adopt the default fast muscle phenotype. The second phase of fiber expansion in the myotomes involves recruitment from discrete germinal zones for both slow and fast muscle fibers (stratified hyperplasia). Finally, myogenic precursor cells are activated throughout the myotome (mosaic hyperplasia). The progeny of these cells either fuse to form additional fibers on the surface of existing muscle fibers or are absorbed by fibers as they expand in diameter (hypertrophic growth). There is considerable species diversity with respect to the timingofinnervation of the embryonic muscle fibers in relation to other developmental events, the degree of maturation of the muscle fibers at hatching, and the onset and relative importance of stratified and mosaic hyperplasia to growth during larval life. A subset of myogenic cells specified by their position in the anterior myotomes are thought to migrate out and populate the pectoral fin buds leading to the differentiation of the pectoral fin muscles. Little is known about the mechanism of formation of the unpaired fin muscles, which occurs after the differentiation of the myotomes and is often delayed until relatively late in larval life. During ontogeny, embryonic isoforms of the myofibrillar proteins are replaced by larval and adult isoforms, and the adult multiterminal pattern of slow muscle innervation gradually develops, reflecting changes in swimming style and performance as body size increases. The body length at which particular protein isoforms are switched on varies for each myofibrillar component and with temperature. In general, early larval stages show a greater reliance on aerobic metabolic pathways and a lower capacity for anaerobic glycolysis than later larval and juvenile stages. Temperature has a marked effect on the ultrastructure, number, and phenotype of larval muscle fibers. Recent evidence suggests that egg incubation temperature can influence myogenic cell commitment, pro- ducing long-term consequences for fiber recruitment and growth performance during subsequent stages of the life cycle. The ecological significance of the phenotypic plasticity of muscle growth and some potential applications to fisheries science are briefly discussed.
... Exactly how long the ECL lasts for, whether it gives rise to all satellite-like cells or just a subset, and when and where these satellite-like cells gain their characteristic morphology and expression pattern, all remain unknown. By the end of primary myogenesis, the zebrafish somite contains a myotome, sclerotome and the dermomyotome-like ECL, which gives rise to mature muscle, skeleton and the myogenic progenitors responsible for subsequent growth, just as for the amniotes [57,58]. However, some differences in myogenesis have been observed at this early developmental time. ...
The cellular basis for mammalian muscle regeneration has been an area of intense investigation over the last few decades. Consensus has been reached that a specialized self-renewing stem cell termed the satellite cell plays the major role during the process of regeneration in amniotes. How broadly deployed this mechanism is within the vertebrate phylogeny remains an open question. This lack of information on the role of analogous cells to the satellite cell in other vertebrate systems is even more surprising given the fact that that satellite cells were first designated in frogs. An intriguing aspect of this debate is the fact that a number of amphibia and many fish species have been shown to exhibit epimorphic regenerative processes in specific tissues, whereby regeneration occurs by the dedifferentiation of the damaged tissue itself, and does not deploy specialized stem cell populations analogous to satellite cells. Hence it is feasible that a cellular process completely distinct to that deployed during mammalian muscle regeneration could operate in species capable of epimorphic regeneration. In this review we examine the evidence for or against the broad phylogenetic distribution of satellite cells. We conclude that in the vertebrates so far examined, epimorphosis does not appear to be deployed during muscle regeneration, and that analogous cells expressing similar marker genes to satellite cells appear to be deployed during the regenerative process. However, the functional definition of these cells as self-renewing muscle stem cells remains a final hurdle to the definition of the satellite cell as a generic vertebrate cell type. This article is protected by copyright. All rights reserved.
Somites are transitory metameric structures at the basis of the axial organization of vertebrate musculoskeletal system. During evolution, somites appear in the chordate phylum and compartmentalize mainly into the dermomyotome, the myotome, and the sclerotome in vertebrates. In this review, we summarized the existing literature about somite compartmentalization in Xenopus and compared it with other anamniote and amniote vertebrates. We also present and discuss a model that describes the evolutionary history of somite compartmentalization from ancestral chordates to amniote vertebrates. We propose that the ancestral organization of chordate somite, subdivided into a lateral compartment of multipotent somitic cells (MSCs) and a medial primitive myotome, evolves through two major transitions. From ancestral chordates to vertebrates, the cell potency of MSCs may have evolved and gave rise to all new vertebrate compartments, i.e., the dermomyome, its hypaxial region, and the sclerotome. From anamniote to amniote vertebrates, the lateral MSC territory may expand to the whole somite at the expense of primitive myotome and may probably facilitate sclerotome formation. We propose that successive modifications of the cell potency of some type of embryonic progenitors could be one of major processes of the vertebrate evolution.
The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair.
Hedgehog pathway components and select G protein-coupled receptors (GPCRs) localize to the primary cilium, an organelle specialized for signal transduction. We investigated whether cells distinguish between ciliary and extraciliary GPCR signaling. To test whether ciliary and extraciliary cyclic AMP (cAMP) convey different information, we engineered optogenetic and chemogenetic tools to control the subcellular site of cAMP generation. Generating equal amounts of ciliary and cytoplasmic cAMP in zebrafish and mammalian cells revealed that ciliary cAMP, but not cytoplasmic cAMP, inhibited Hedgehog signaling. Modeling suggested that the distinct geometries of the cilium and cell body differentially activate local effectors. The search for effectors identified a ciliary pool of protein kinase A (PKA). Blocking the function of ciliary PKA, but not extraciliary PKA, activated Hedgehog signal transduction and reversed the effects of ciliary cAMP. Therefore, cells distinguish ciliary and extraciliary cAMP using functionally and spatially distinct pools of PKA, and different subcellular pools of cAMP convey different information.
Crucial to an animal’s movement through their environment and to the maintenance of their homeostatic physiology is the integration of sensory information. This is achieved by axons communicating from organs, muscle spindles and skin that connect to the sensory ganglia composing the peripheral nervous system (PNS), enabling organisms to collect an ever-constant flow of sensations and relay it to the spinal cord. The sensory system carries an wide variety spectrum of sensory modalities – from sharp pain to cool refreshing touch – from the periphery to the spinal cord via dorsal root ganglia (DRG). This review covers the origins and development of the DRG and the cells that populate it, and focuses on how sensory connectivity to the spinal cord is achieved by the diverse developmental and molecular processes that control axon guidance in the trunk sensory system. We also describe convergences and differences in sensory neuron formation among different vertebrate species to gain insight into underlying developmental mechanisms.
Vertebrate somitogenesis involves the establishment of a segmental pattern of gene expression within the paraxial mesoderm and the subsequent translation of this pattern into physical furrows and epithelial somites. In this thesis I present first the development of an in vitro technique to culture embryo trunk explants. These explants develop and segment correctly despite the absence of overlying ectoderm, however no defects in somite formation were observed when treated with Eph signalling blocking reagents. Using mosaic analysis I provide evidence for a role for the Eph/ephrin signalling pathway in somite epithelialisation. A comparative analysis of the cellular behaviours and gene expression patterns within the segmental plate during somitogenesis was carried out in wild type zebrafish embryos and in fusedsomites (fss) mutants. In wild type embryos, the receptor EphA4 and the ligand ephrin-B2a are segmentally expressed in complementary non-overlapping domains in the anterior presomitic mesoderm and the forming somites. Somite boundaries form at the interface between cells expressing EphA4 and cells expressing ephrin-B2a. Cells at somite boundaries acquire epithelial morphology manifested by the re-localisation of adhesion molecules towards the apical pole and nuclear migration towards the basal pole. In fss embryos, expression of EphA4 is absent in the paraxial mesoderm and ephrin-B2a is expressed throughout the segmental plate. Somite boundaries fail to form and cells within the paraxial mesoderm remain mesenchymal failing to epithelialise. Restoration of the Eph/ephrin signalling in the paraxial mesoderm of fss mutants resulted in the rescue of ectopic physical boundaries (Durbin, 2000). Moreover, some aspects of epithelialisation such as the re-localisation of β-catenin to the apical pole and nuclear migration towards the basal pole were also rescued.
The work presented here addresses the question of how skeletal muscle formation is initiated in the mouse by dissecting the regulatory mechanisms that control the myogenic regulatory factor Myf-5. Myf-5 is expressed in the dorsal somite from E8, before the other MRFs, myogenin, MRF4 and MyoD, become activated. In the mouse Myf-5 is located 8.5kb downstream of MRF4. Previous results have shown that dispersed over the intergenic region and intragenic regions are the regulatory elements involved in directing Myf-5 expression to the different anatomical subdomains that make up its complete expression pattern. The regulatory element(s) controlling the dorsal somite expression of Myf-5 is contained in the intergenic region while ventral somite expression depends on elements in the Myf-5 gene itself. Because of the large size of this region I have isolated the MRF4 and Myf-5 genes of the teleost Fugu rubripes, which has a genome eight times smaller than that of the mouse. Although synteny is conserved in Fugu and the intergenic distance is only 3kb, noncoding sequence including the introns is poorly conserved. Focusing on the Myf-5 gene itself, sequence comparison between the mouse and human Myf-5 genes was employed sucessfully to eliminate more than 60% of the intron sequence by identifying conserved regions in the 3'half of each of the Myf-5 introns which, together with the 3'UTR can activate reporter gene expression in the ventral posterior part of the somites. EMSA analysis with embryonic protein extracts revealed several protein binding regions within the conserved intron fragments and subsequent transgenic analysis showed not only that separate genomic regions control individual anatomical domains of Myf-5 expression, but that within these regions multiple binding sites are found, adding a further level of complexity to the regulation of Myf-5. Analysis of the Fugu Myf-5 gene in transgenic mice showed remarkable similarities with the expression pattern of Myf-5 in another teleost, the zebrafish Danio rerio. Both are expressed in the presomitic mesoderm, as well as the somites, suggesting that the expression of the Fugu transgene is a reflection of its native expression pattern.
Fish axial muscle consists of a series of W-shaped muscle blocks, called myomeres, that are composed primarily of multinucleated contractile muscle cells (myofibres) gathered together by an intricate network of connective tissue that transmits forces generated by myofibre contraction to the axial skeleton. This review summarises current knowledge on the successive and overlapping myogenic waves contributing to axial musculature formation and growth in fish. Additionally, this review presents recent insights into muscle connective tissue development in fish, focusing on the early formation of collagenous myosepta separating adjacent myomeres and the late formation of intramuscular connective sheaths (i.e. endomysium and perimysium) that is completed only at the fry stage when connective fibroblasts expressing collagens arise inside myomeres. Finally, this review considers the possibility that somites produce not only myogenic, chondrogenic and myoseptal progenitor cells as previously reported, but also mesenchymal cells giving rise to muscle resident fibroblasts.
Rainbow trout has been considered for many years as a model of glucose intolerant species. The different hypothesis raised by many researchers to explain such phenomenon has been tested thoroughly in recent years without arriving at a clear explanation. One of the processes that could be involved in its inability to deal with increased levels of glucose could be the absence of glucosensing mechanisms similar to those found in mammals. However, several recent studies in rainbow trout have demonstrated the existence of glucosensor systems in hypothalamus, hindbrain and Brockmann bodies. The fact that this system has been characterized in a species whose natural diet contains less than 1% carbohydrate intake makes rainbow trout an attractive model for glucosensing studies, as so far it is the only vertebrate carnivorous species in which this system has been explored.The glucosensing system has been shown to be activated when glucose levels increase at the same time that food intake decreases while conversely, when glucose levels decrease, glucosensors are inactivated and food intake increases. The mechanisms through which these glucosensor systems operate are similar to those described in mammalian brain regions, though quite different in pancreatic cells. Information has also been reported regarding the molecular characterization of these systems, their specific location in the brain and their endocrine regulation.The present review will provide a general overview of the research carried out in this area in recent years and provide perspectives for future research in this field.
Hematopoietic stem cells (HSCs) replenish all types of blood cells. It is debating whether HSCs in adults solely originate
from the aorta-gonad-mesonephros (AGM) region, more specifically, the dorsal aorta, during embryogenesis. Here we report that
somite hematopoiesis, a previously unwitnessed hematopoiesis, can generate definitive HSCs (dHSCs) in zebrafish. By transgenic
lineage tracing, we found that a subset of cells within the forming somites emigrate ventromedially and mix with lateral plate
mesoderm-derived primitive hematopoietic cells before the blood circulation starts. These somite-derived hematopoietic precursors
and stem cells (sHPSCs) subsequently enter the circulation and colonize the kidney of larvae and adults. RNA-seq analysis
reveals that sHPSCs express hematopoietic genes with sustained expression of many muscle/skeletal genes. Embryonic sHPSCs
transplanted into wild-type embryos expand during growth and survive for life time with differentiation into various hematopoietic
lineages, indicating self-renewal and multipotency features. Therefore, the embryonic origin of dHSCs in adults is not restricted
to the AGM.
Zebrafish neural crest is amenable to study both as a population and at the level of the single cell. Zebrafish neural crest has many fewer cells relative to other vertebrates; in the trunk there are only 10–12 neural crest cells per body segment compared to hundreds in other species. Despite the relative simplicity of the zebrafish neural crest, almost all aspects of its development are similar to those of other vertebrates including the migration pathways it takes and types of derivatives it makes. This chapter examines when zebrafish trunk neural crest cells undergo fate restrictions and whether these restrictions are the result of restrictions in cell potential. It is important to know when cell fate restrictions occur by performing cell lineage analysis: labeling individual cells within the trunk neural crest, following them during development, recording cell divisions, and keeping track of all progeny. The chapter also reviews whether cell fate restrictions result from restrictions in cell potential by comparing the fates of individual cells from different crest subpopulations when transplanted to the same environmental conditions. These experiments have led researchers to propose a model of neural crest development in which regulative interactions among neural crest cells influence their fates.
Fibronectins (FNs) are major extracellular proteins in blood plasma and many tissues of vertebrates, and play important roles in adhesion, migration and differentiation of cells. We have identified a novel variant (FN3) of fibronectin in zebrafish. FN3 mRNA is abundant, as detected by whole-mount in situ hybridization, in the presomitic mesoderm and the newly formed somites, but less abundant in mature somites. Ectopic expression of Sonic Hedgehog (SHH) results in a decrease of FN3 expression, whereas the expression level of FN3 increases in the flh mutants that lack the notochord. Our results suggest that FN3 may be involved in the formation of somites, but during somite differentiation its expression needs to be downregulated by signals derived from the axial tissues.
Zebrafish (Danio rerio) has recently become a popular model to study skeletogenesis because it provides a unique system for both easy developmental analyses and powerful genetic screening. The transparent nature of the zebrafish embryo enables convenient observation of cartilage and bone development at the microscopic level. Genetic screens have generated large numbers of valuable mutants with developmental defects in craniofacial, axial, and fin skeletons. This chapter focuses primarily on recent studies on skeletogenesis in zebrafish, which have taken advantage of the simple embryology and genetic tractability of zebrafish to dissect the cellular and molecular mechanisms underlying skeletal development in vertebrates.
Much of the peripheral nervous system (PNS) is derived from trunk neural crest. Trunk neural crest is an initially multipotent cell population that must migrate and undergo cell fate decisions in order to generate the diversity of cell types that exists in the PNS. Here, we discuss the generation of the sympathetic chain ganglia and the dorsal root ganglia (DRG) from neural crest, specifically focusing on the gene regulatory network that contributes to their development. Neural crest is induced to develop as sympathetic neurons by BMP emanating from the aorta. This initiates a transcription factor cascade beginning with Ascl1. This network involves extensive cross-regulation terminating in the expression of tyrosine hydroxylase and dopamineβ-hydroxylase. DRG are induced to develop by unknown signals; expression of the Neurogenin transcription factors initiates a more sequential cascade ending in the expression of Trk receptors, which are restricted to neuronal subtypes carrying different sensory modalities.
This chapter summarizes current research on the molecular, genetic and cellular bases of skeletal muscle development and growth in the trout [i]Oncorhynchus mykiss[/i]. The myogenic transcriptional network is examined in relation with the transient sub-compartmentalization of the early somites that results in the formation of distinct myogenic cell populations involved in distinct phases of myogenesis. The importance of the endocrine and paracrine regulation of muscle growth is also discussed with special reference to the influence of environmental factors ont the GH-IGF system. Insights from [i]in vitro[/i] models ares also provided.
This chapter discusses the segmentation of the paraxial mesoderm and the delineation of tissue compartments. Segmentation is a fundamental developmental process that subdivides the body, or parts thereof, into a series of serially repeated subunits and thereby generates a segmental pattern. In the mouse embryo, like other vertebrate embryos, the earliest manifestation of tissue segmentation is the formation of somites during organogenesis. Somites are blocks of mesodermal cells located on either side of the neural tube and the notochord. Together with the mesenchyme that envelops the cephalic neural tube, they constitute the paraxial mesoderm of the embryo. The segmental arrangement of the somites along the anterior–posterior body axis prefigures and underlies the metarnerism of the somite derived vertebral column and epaxial muscles and also determines the segmented arrangement of parts of the peripheral nervous system.
Current evidence indicates that post-embryonic muscle growth and regeneration in amniotes is mediated almost entirely by stem cells derived from muscle progenitor cells (MPCs), known as satellite cells. Exhaustion and impairment of satellite cell activity is involved in the severe muscle loss associated with degenerative muscle diseases such as Muscular Dystrophies and is the main cause of age-associated muscle wasting. Understanding the molecular and cellular basis of satellite cell function in muscle generation and regeneration (myogenesis) is critical to the broader goal of developing treatments that may ameliorate such conditions.
Considerable knowledge exists regarding the embryonic stages of amniote myogenesis. Much less is known about how post-embryonic amniote myogenesis proceeds, how adult myogenesis relates to embryonic myogenesis on a cellular or genetic level. Of the studies focusing on post-embryonic amniote myogenesis, most are post-mortem and in vitro analyses, precluding the understanding of cellular behaviours and genetic mechanisms in an undisturbed in vivo setting. Zebrafish are optically clear throughout much of their post-embryonic development, facilitating their use in live imaging of cellular processes. Zebrafish also possess a compartment of MPCs, which appear similar to satellite cells and persist throughout the post-embryonic development of the fish, permitting their use in examining the contribution of these cells to muscle tissue growth and regeneration.
Medaka: Biology, Management, and Experimental Protocols, written by experienced researchers and reviewed by international leaders in the medaka field will provide details on how to set up and maintain medaka colonies in animal facilities, how to troubleshoot systems, how to handle the fish when applied to experimental methods, and most importantly it will introduce the researcher to cutting edge research in basic and applied biology using medaka as a model animal. The book will include well-written descriptions of experimental methods and protocols designed to educate the reader how to understand and handle medaka effectively. Medaka: Biology, Management, and Experimental Protocols will serve as the definitive reference on the species providing essential information on medaka biology, genetics, and genomics, practical guidance to maintenance of fish stocks, and valuable experimental protocols all in a single volume. This book will be a must have addition to the library of fish researchers and those using medaka as a model organism within their laboratories.
The early development of turbot (Scophthalmus maximus) from fertilization to hatching was described. Hatching occurred at 108 h post-fertilization (hpf) in 14 °C. Yolk syncytial layer and blastocoel formed at morula stage and low stage, respectively. Neural rod derived from the ectoderm appeared and the first somite formed in the middle of the embryonic body at 90 % epiboly stage, and notochord primordium formed at complete epiboly stage. Kupffer’s vesicle appeared at 59 h 35 min hpf and degenerated at 89 hpf. At 72 hpf, the digestive tract formed in the ventral side of the embryonic body, and the posterior digestive tract of embryo was ciliated at 89 hpf. Enzymes play a key role in the catabolism of yolk during embryogenesis of fishes. In this study, the main enzymes alkaline phosphatase (AP), leucine aminopeptidase N (LAP), pepsin, trypsin and Leucine-alanine peptidase (Leu-ala) were all observed in unfertilized eggs and embryo of S. maximus, but amylase was not detected, speculating that amino acids appear to be the main energy substrate during embryonic development of S. maximus, while carbohydrates is less essential. AP reached the lowest value at the gastrula stage and then increased rapidly reaching the highest value at hatching. LAP showed the highest value in unfertilized eggs and kept on decreasing until the blastula stage with the lowest value and then increased at the gastrula stage, followed by a gradual decline thereafter. Trypsin reached the lowest value at the blastula stage and then fluctuated with the maximal value at hatching. Pepsin reached the highest and the lowest values at the unfertilized eggs and the cleavage stage, respectively, but disappeared at hatching. Leu-ala had the maximum activity at the blastula stage and then declined to the minimum at the gastrula stage followed by a gradual increase thereafter.
This chapter describes the induction and patterning of embryonic skeletal muscle cells in the zebrafish. The optical clarity of the zebrafish embryo makes it especially well suited for the application of sophisticated cell labeling techniques, approaches that facilitate the direct visualization of muscle cell ontogeny. The identification of the genes that control this process is well underway following two large-scale mutant screens of the zebrafish genome and these have uncovered numerous mutants that disrupt muscle cell formation and differentiation in various ways. In fish, as in other vertebrates, the skeletal muscle of the trunk and tail derives from a specific embryological compartment, the myotome, which in amniotes has been shown to be induced within the segmented mesoderm of the somite. Somites condense from mesoderm immediately adjacent to the central body axis, the so-called “paraxial mesoderm,” and segment in a stereotypic rostral to caudal progression. The simultaneous development of both muscle layers in the free swimming larvae has been suggested to endow the organism with the abilities to search for food sources at low swimming velocity and to avoid predators by escape at high velocity.
The ontogeny of amphicoelous vertebrae was studied in Ptyodactylus hasselquistii and Hemidactylus turcicus, and that of procoelous vertebrae, in Sphaerodactylus argus. The embryos were assigned arbitrary stages, drawn to scale, and mostly studied in serial sections.
Resegmentation occurs as in all amniotes. A sclerocoel divides each sclerotome into an anterior “presclerotomite” and a denser posterior “postsclerotomite.” Tissue surrounding the intersegmental boundary forms the centrum, which is intersegmental. Tissue around the sclerocoel builds the intervertebral structures, which are midsegmental. In the trunk and neck, postsclerotomites form neural arches, and presclerotomites build zygapophyses.
The adult centrum consists of the perichordal primary centrum, plus neural arch bases (= secondary centrum). Between the latter and the arch proper, a neurocentral suture persists until obliterated in maturity. A dorso-ventral central canal persists on either side of the primary centrum, between the latter and the secondary centrum.
The notochord becomes true cartilage midvertebrally in all vertebrae, and elastic cartilage intervertebrally in the posterior caudal region. Elsewhere its characteristic tissue persists.
Intervertebrally, cervical hypapophyses, caudal chevrons and chevron-bases in the trunk are preformed early in cartilage. Directly ossifying median intercentra are added later in all regions.
The first cervical presclerotomite is absent: the hypapophysis (= corpus) of the atlas consists exclusively of postsclerotomitic tissue, there is no proatlas, and the odontoid lacks the apical half-centrum present in other lepidosaurians.
In the autotomous caudal region presclerotomites are as prominent as postsclerotomites. Both build neural arches, the two arches of each vertebra remaining distinct and ossifying separately, so that the intersegmental autotomy split persists between them.
The last sclerotome is complete, its postsclerotomite forming a half centrum which ossifies.
In Sphaerodactylus, while the vertebrae ossify, each intervertebral ring becomes concave anteriorly, convex posteriorly; it remains as a cushion between the condyle and a facet formed by differential growth of the centra. Thus these procoelous centra resemble the amphicoelous centra of Ptyodactylus and Hemidactylus, rather than the procoelus centra of other squamates.
The vertebral column of Gekkonoidea closely resembles in its development and microscopical structure that of Sphenodon.
The role of myogenic cells in accurate pathway selection and muscle nerve formation was studied in chick embryos. Myotubes were eliminated from the forelimb of the chick embryo by extirpating the somites, which give rise to myogenic cells. Other elements of the wing tissue, connective tissue, and cartilagenous elements derived from the somatopleure, were left intact. Injections of WGA-HRP were made into either dorsal or ventral nerve trunks in the wing and the positions of retrogradely labeled motoneurons determined. The positions of the motoneurons within the brachial lateral motor column were appropriate for the injection made. Thus, the accuracy of the motoneuronal projections was unaffected by the absence of muscle cells. The absence of myotubes was not correlated with the absence of muscle nerves. Muscle nerves were consistently observed in muscleless wings until stage 36, the oldest stage examined. Muscle nerves in muscleless wings differed from those in normal wings in that they were smooth and stubby, and lacked the normal pattern of intramuscular nerve branches. From these studies we conclude that muscle cells are not necessary for accurate motor axon guidance into the periphery along the routes of major nerve trunks, nor for the formation of muscle nerves. By inference, somatopleural derivatives provide sufficient cues for selection of specific axonal pathways and for patterning of muscle nerves within the chick limb.
To learn how neural segments are structured in a simple vertebrate, we have characterized the embryonic zebrafish hindbrain with a library of monoclonal antibodies. Two regions repeat in an alternating pattern along a series of seven segments. One, the neuromere centers, contains the first basal plate neurons to develop and the first neuropil. The other region, surrounding the segment boundaries, contains the first neurons to develop in the alar plate. The projection patterns of these neurons differ: those in the segment centers have descending axons, while those in the border regions form ventral commissures. A row of glial fiber bundles forms a curtain-like structure between each center and border region. Specific features of the individual hindbrain segments in the series arise within this general framework. We suggest that a cryptic simplicity underlies the eventual complex structure that develops from this region of the CNS.
It is known that both neural crest cell migration and motor axon outgrowth in most vertebrate embryos are segmented because of restrictions imposed upon their distribution by the neighbouring sclerotomes, each of which is divided into a rostral and a caudal half. The caudal half does not allow crest migration or axon outgrowth, while the rostral half does. In this paper, we investigate the expression of proteins and glycoproteins in the two halves of the sclerotome of the chick embryo at stages between 20 and 32 pairs of somites by two-dimensional SDS-polyacrylamide gel electrophoresis. We find that the patterns of expression are complex, and that polypeptides and glycoproteins vary both spatially and temporally: of those that are expressed differentially by the sclerotome, some differ quantitatively and others qualitatively. Some macromolecules change their spatial distribution with developmental age, and some appear or disappear as the embryos become older.
The first expression and distribution of acetylcholinesterase (AChE) activity was studied among a distinct population of early neurons in embryonic zebrafish by using histochemical and retrograde labeling techniques. AChE first appeared in the nervous system in the primary motoneurons of the rostral spinal cord when the embryo had nine somites, approximately 14 hours postfertilization. Subsequent expression of AChE activity in the spinal cord proceeded in a rostral-to-caudal sequence. Cranial neurons expressed AChE activity shortly after it appeared in the rostral spinal cord. Several hours later, near the end of the first day, primary neurons in the hind-brain and spinal cord all contained AChE, including sensory neurons, reticulospinal interneurons, and primary motoneurons. AChE activity was also detected in the nucleus of the medial longitudinal fasciculus. Presumptive cranial ganglia transiently expressed AChE activity between 14 and 24 hours of development. These results, combined with previous observations that examined the time of origin and axogenesis of primary neurons, suggest that primary neurons in the embryonic zebrafish contain AChE before they sprout axons. The primary neurons appear to follow a common sequence of development consisting of a withdrawal from the cell division cycle, the expression of AChE, and axogenesis. Although this sequence is followed by all primary neurons, lack of a rostral-to-caudal sequence in the time of birth and variability in the time of axon outgrowth demonstrate that the relative timing of these three events is not rigidly programmed in individual neurons. Moreover, the very early expression of AChE in such diverse cell types suggests that it may have a developmental role in addition to its function in transmitter metabolism.
Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.
We have investigated the interactions between the cells of the rostral and caudal halves of the chick somite by carrying out grafting experiments. The rostral halfsclerotome was identified by its ability to support axon outgrowth and neural crest cell migration, and the caudal half by the binding of peanut agglutinin and the absence of motor axons and neural crest cells. Using the chick-quail chimaera technique we also studied the fate of each half-somite.
It was found that when half-somites are placed adjacent to one another, their interactions obey a precise rule: sclerotome cells from like halves mix with each other, while those from unlike halves do not; when cells from unlike halves are adjacent to one another, a border is formed.
Grafting quail half-somites into chicks showed that the fates of the rostral and caudal sclerotome halves are similar: both give rise to bone and cartilage of the vertebral column, as well as to intervertebral connective tissue. We suggest that the rostrocaudal subdivision serves to maintain the segmental arrangement when the mesenchymal sclerotome dissociates, so that the nervous system, vasculature and possibly vertebrae are patterned correctly.
We have observed the development of live, fluorescently labeled motoneurons in the spinal cord of embryonic and larval zebrafish. There are 2 classes of motoneurons: primary and secondary. On each side of each spinal segment there are 3 individually identifiable primary motoneurons, named CaP, MiP, and RoP. The motoneurons of the embryo and larva are similar in morphology and projection pattern to those of the adult. During initial development, axons of primary motoneurons make cell-specific, divergent pathway choices and grow without error to targets appropriate for their adult functions. We observed no period of cell death, and except for one consistently observed case, there was no remodeling of peripheral arbors. We have observed a consistent temporal sequence of axonal outgrowth within each spinal segment. The CaP motor axon is the first to leave the spinal cord, followed by the axons of the other primary motoneurons. The Mauthner growth cone enters the spinal cord after all the primary motoneurons of the trunk spinal cord have begun axonal outgrowth. Secondary motor growth cones appear only after the Mauthner growth cone has passed by. Our results suggest that this stereotyped temporal sequence of axonal outgrowth may play a role in defining the contacts between the Mauthner axon and the motoneurons; the behavior of growth cones in the periphery suggests that interactions with the environment, not timing, may determine path-finding and peripheral connectivity of the motoneurons.
The organization of spinal cord motoneurons and their innervation of axial (white) muscles in the zebrafish were studied. Motoneurons can be divided into 2 classes, primary and secondary, on the basis of their cell-body sizes and positions. Each side of each spinal segment contains 3 primary motoneurons that are uniquely identifiable as individuals by their stereotyped cell-body positions and peripheral branching patterns. Moreover, these motoneurons precisely innervate cell-specific subsets of contiguous muscle fibers in mutually exclusive regions of their own body segment. Individual muscle fibers receive inputs from a single primary motoneuron and, in addition, from up to 3 secondary motoneurons. The results demonstrate that the precision of innervation previously described in invertebrates is also present in some vertebrates.
We have studied the pathway of migration of neural crest cells through the somites of the developing chick embryo, using the monoclonal antibodies NC-1 and HNK-1 to stain them. Crest cells, as they migrate ventrally from the dorsal aspect of the neural tube, pass through the lateral part of the sclerotome, but only through that part of the sclerotome which lies in the rostral half of each somite. This migration pathway is almost identical to the path which presumptive motor axons take when they grow out from the neural tube shortly after the onset of neural crest migration. In order to see whether the ventral root axons are guided along this pathway by neural crest cells, we surgically excised the neural crest from a series of embryos, and examined the pattern of axon outgrowth approximately 24 h later. In somites which contained no neural crest cells, ventral root axons were still found only in the rostral half of the somite, although axonal growth was slightly delayed. These axons were surrounded by sheath cells, which had presumably migrated out of the neural tube, to a point about 50 micron proximal to the growth cones. With appropriate antibodies we found that the extracellular matrix components fibronectin and laminin are evenly distributed between the rostral and caudal halves of the somite. Neither of these molecules therefore plays a critical role in determining the specific pathway of neural crest cells or motor axons through the rostral half of the somite.
Mesoderm formation is critical for the establishment of the animal body plan and in Drosophila requires the snail gene. This report concerns the cloning and expression pattern of the structurally similar gene snail1 from zebrafish. In situ hybridization shows that the quantity of snail1 RNA increases at the margin of the blastoderm in cells that involute during gastrulation. As gastrulation begins, snail1 RNA disappears from the dorsal axial mesoderm and becomes restricted to the paraxial mesoderm and the tail bud. snail1 RNA increases in cells that define the posterior border of each somite and then disappears when somitic cells differentiate. Later in development, expression appears in cephalic neural crest derivatives. Many snail1-expressing cells were missing from mutant spadetail embryos and the quantity of snail1 RNA was greatly reduced in mutant no tail embryos. The work presented here suggests that snail1 is involved in morphogenetic events during gastrulation, somitogenesis and development of the cephalic neural crest, and that no tail may act as a positive regulator of snail1.
Zebrafish floating head mutant embryos lack notochord and develop somitic muscle in its place. This may result from incorrect specification of the notochord domain at gastrulation, or from respecification of notochord progenitors to form muscle. In genetic mosaics, floating head acts cell autonomously. Transplanted wild-type cells differentiate into notochord in mutant hosts; however, cells from floating head mutant donors produce muscle rather than notochord in wild-type hosts. Consistent with respecification, markers of axial mesoderm are initially expressed in floating head mutant gastrulas, but expression does not persist. Axial cells also inappropriately express markers of paraxial mesoderm. Thus, single cells in the mutant midline transiently co-express genes that are normally specific to either axial or paraxial mesoderm. Since floating head mutants produce some floor plate in the ventral neural tube, midline mesoderm may also retain early signaling capabilities. Our results suggest that wild-type floating head provides an essential step in maintaining, rather than initiating, development of notochord-forming axial mesoderm.
We have examined the development of specific muscle fiber types in zebrafish axial muscle by labeling myogenic precursor cells with vital fluorescent dyes and following their subsequent differentiation and fate. Two populations of muscle precursors, medial and lateral, can be distinguished in the segmental plate by position, morphology and gene expression. The medial cells, known as adaxial cells, are large, cuboidal cells adjacent to the notochord that express myoD. Surprisingly, after somite formation, they migrate radially away from the notochord, becoming a superficial layer of muscle cells. A subset of adaxial cells develop into engrailed-expressing muscle pioneers. Adaxial cells differentiate into slow muscle fibers of the adult fish. We have named the lateral population of cells in the segmental plate, lateral presomitic cells. They are smaller, more irregularly shaped and separated from the notochord by adaxial cells; they do not express myoD until after somite formation. Lateral presomitic cells remain deep in the myotome and they differentiate into fast muscle fibers. Thus, slow and fast muscle fiber types in zebrafish axial muscle arise from distinct populations of cells in the segmental plate that develop in different cellular environments and display distinct behaviors.
The role of somitic mesoderm in the development of dorsal plumage in chick embryos. I. Origin, regulation capacity and determination
The role of somitic mesoderm in the development of the dorsal plumage has been studied in chick embryos. The operations were performed at 2–2·5 days of incubation.
The replacement of a portion of somitic mesoderm by somitic mesoderm labelled with [3H]thymidine or obtained from Japanese quail embryos (whose nuclei bear distinctive specific markers) showed that cells originating from the dermatomes build up the dermis of the dorsal skin only. They do not migrate farther than approximately midway down the flank. Beyond this limit, dermal cells originate from the somatopleural mesoderm.
The unsegmented somitic mesoderm is capable of extensive regulation, which leads to the development of a dorsal plumage, normal in the number and arrangement of its feathers according to the characteristic pattern of the spinal pteryla. Uni- or bilateral excision of segmented somitic mesoderm resulted in dorsal plumage deficiencies, the extent and frequency of which was related to the state of differentiation of the excised mesoderm. Thus, the excision of somites generally led to an incomplete spinal pteryla (absence of feather rows, apteria). However, the somitic mesoderm is still capable of regulation even though it has already undergone its differentiation into dermatome, myotome and sclerotome. These results show that somitic mesoderm retains its regulative capacity, even though it has already acquired its feather-forming determination.
The replacement of unsegmented somitic mesoderm by various implants (agar, tantalum, gut, neural tube, somatopleural mesoderm), intended to block the regulation processes, abolished the differentiation of the spinal feathers on the operated side. In some cases, the implantation of somatopleural mesoderm resulted in the formation of a supernumerary tract. No tissue other than somitic mesoderm – not even the somatopleural mesoderm, which is normally in part feather-forming – is able to give rise to region-specific spinal pteryla dermis.
The excision and replacement of somitic mesoderm prevented the differentiation of dense dermis, whereas these operations had no effect on the early histogenesis of the epidermis, with the formation of arches and anchor filaments.
We have exploited our ability to visualize and follow individual cells in situ, in the living embryo, to study the development of trunk neural crest in the embryonic zebrafish. In most respects, the development of zebrafish trunk neural crest is similar to the development of trunk neural crest in other species: zebrafish trunk neural crest cells segregate from the dorsal neural keel in a rostrocaudal sequence, migrate ventrally along two pathways, and give rise to neurons of the peripheral nervous system, Schwann cells, and pigment cells. However, some aspects of the development of zebrafish trunk neural crest differ from those of other vertebrates: zebrafish trunk neural crest cells are significantly larger and fewer in number than those in avian embryos and the locations of their migratory pathways are slightly different. This initial description of neural crest development in the zebrafish embryo provides the foundation for future experimental studies. © 1992 Wiley-Liss, Inc.
Application of horseradish peroxidase to lesions of the muscles and the central nervous system of larval zebrafish Brachydanio rerio was used to identify several types of neurons present in the spinal cord. The spinal cord was found to contain three distinct motoneuronal types: primary and secondary motoneurons that innervate the axial muscles, and pectoral fin motoneurons that innervate the muscles of the pectoral girdle. The cell types are similar to those described in larvae of other anamniote vertebrates. The axial muscles of t, given hemisegment are innervated by two or three primary motoneurons and a larger number of secondary motoneurons in the corresponding spinal segment, whereas fin muscles are innervated by a pool of motoneurons spanning several spinal segments.
We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
Myotome differentiation during Mexican axolotl (Ambystoma mexicanum) somitogenesis was analyzed by employing anti-actin and anti-myosin monoclonal antibodies as molecular probes. Myotome differentiation occurs after segmentation and proceeds in the cranial-to-caudal direction along the somite file. Within individual somites myotome differentiation displays distinct polarities. Examination of the somite file at the tailbud stage revealed that soon after segmentation, actin/myosin accumulate predominantly in the anterior and medial region of the myotome initially. Subsequently, cells within the myotome differentiate in an anterior-to-posterior and medial-to-lateral direction. Experimental analysis of presomitic paraxial mesoderm grafts before segmentation revealed that this transient myotome polarity is autonomous. Comparative analyses indicate that this myotome differentiation pattern is urodele specific. Cynops pyrrhogaster undergoes myotome differentiation like the axolotl, while two anurans, Xenopus laevis and Bombina orientalis, do not.
The cells of individual somites in 2-day-old chick embryos were marked by injecting a fluorescent dye into the somitocoele. This procedure permanently marked the cells and allowed their subsequent development and distribution to be followed. The cells were found to remain in close association with each other within limited boundaries and did not mix to any great extent with similar cells from adjacent somites. Fluorescent cells from single somites were found in the intervertebral disc, connective tissue surrounding two adjacent neural arches, all the tissues between the neural arches, the dermatome, and the associated myotome. No fluorescent cells were found in the notochord or in any nervous tissue apart from accompanying connective tissue. Surprisingly, the vertebral bodies and neural arches did not contain any fluorescent cells apart from those in the connective tissue surrounding them, but this absence of fluorescent cells was thought to be due to the dilution of the fluorescence following cell proliferation. These results provide further experimental support for the theory of resegmentation in vertebral formation, and also provide evidence of a compartmental method of development along the rostrocaudal axis in vertebrates, similar to that already discovered in insects. On the basis of cell lineage criteria, the sclerotome might be considered as a developmental compartment.
We have used microsurgical techniques to investigate the effects of embryonic mesodermal tissues on the pattern of chick neural crest cell migration in the trunk. Segmental plate or lateral plate mesenchyme was transplanted into regions encountered by neural crest cells. We found that neural crest cells are able to migrate through lateral plate mesenchyme but not through segmental plate tissue until this tissue differentiates into a sclerotome. After this stage, segmental migration is controlled by the subdivision of the sclerotome into a rostral and a caudal half; when the rostrocaudal orientation of the sclerotomes is reversed by rotating the segmental plate 180 degrees about its rostrocaudal axis, neural crest cells migrate through the portion of the sclerotome that was originally rostral.
We examined the arrangement and development of identified neurons in zebrafish embryos homozygous for the mutation spt-1, which acts autonomously and specifically to alter the development of precursors of trunk segmented mesoderm, resulting in muscle-deficient myotomes. We found that the mutation alters the morphology, number, and arrangement of identified motoneurons. By transplanting identified motoneurons between wild-type and mutant embryos, we found that the effect of the mutation was nonautonomous. We suggest that the segmental arrangement and proper axonal development of motoneurons may result from interactions with segmented mesoderm.
We have developed a program for the Macintosh computer to control a Panasonic Optical Memory Disk Recorder (OMDR) in order to generate time-lapse video recordings of growing neurons. The software, in addition to regulating the timing of a recording in a flexible way, can also digitize and pre-process images before writing them out to the optical disk. NeuroVideo includes a complete set of functions to enhance images, and provides both an easy-to-use graphical interface and a simple but powerful text-based scripting language.
The onset of myotome formation in somites of chick embryos was studied by use of a polyclonal antidesmin antibody and by histochemical demonstration of acetylcholine esterase activity. The myotome cells originate from the dermatome only; sclerotome cells do not contribute to the myotome. The formation of the myotome starts in the craniomedial corner of the dermatome. From there the myotome formation continues simultaneously along the medial and the cranial edge of the dermatome. It was found that only the already longitudinally oriented cells of the cranial dermatome edge give rise to the myotome; the cells of the dorsomedial dermatome edge do not contribute to the myotome. Myotome cells do not originate directly from the surface of the overlying dermatome by delamination.
Developing motoneurons in zebrafish embryos follow a stereotyped sequence of axonal outgrowth and accurately project their axons to cell-specific target muscles. During axonal pathfinding, an identified motoneuron pioneers the peripheral motor pathway. Growth cones of later motoneurons interact with the pioneer via contact, coupling, and axonal fasciculation. In spite of these interactions, ablation of the pioneer motoneuron does not affect the ability of other identified motoneurons to select the pathways that lead to appropriate target muscles. We conclude that interactions between these cells during pathfinding are not required for accurate pathway selection.
We have investigated dorsal root ganglion formation, in the avian embryo, as a function of the composition of the paraxial somitic mesoderm. Three or four contiguous young somites were unilaterally removed from chick embryos and replaced by multiple cranial or caudal halfsomites from quail embryos. Migration of neural crest cells and formation of DRG were subsequently visualized both by the HNK-1 antibody and the Feulgen nuclear stain.
At advanced migratory stages (as defined by Teillet et al. Devi Biol. 120, 329 – 347 1987), neural crest cells apposed to the dorsolateral faces of the neural tube were distributed in a continuous, nonsegmented pattern that was indistinguishable on unoperated sides and on sides into which either half of the somites had been grafted. In contrast, ventrolaterally, neural crest cells were distributed segmentally close to the neural tube and within the cranial part of each normal sclerotome, whereas they displayed a nonsegmental distribution when the graft involved multiple cranial half-somites or were virtually absent when multiple caudal half-somites had been implanted.
In spite of the identical dorsal distribution of neural crest cells in all embryos, profound differences in the size and segmentation of DRG were observed during gangliogenesis (E4 – 9) according to the type of graft that had been performed. Thus when the implant consisted of compound cranial half-somites, giant, coalesced ganglia developed, encompassing the entire length of the graft. On the other hand, very small, dorsally located ganglia with irregular segmentation were seen at the level corresponding to the graft of multiple caudal halfsomites. We conclude that normal morphogenesis of dorsal root ganglia depends upon the craniocaudal integrity of the somites.
The somitic involvement in the formation of the vertebral column was examined using the chick-quail chimaera model. Single cervical somites from quail donor embryos were transplanted into similarly staged chick host embryos. Following further incubation, serial sections of variously staged embryos were stained with the Feulgen reaction to distinguish the two cell populations.
Quail cells were generally located within a delimited region in one half of each of the two adjacent vertebrae, as well as in the intervening disc. The horizontal plane of division through each vertebra passed approximately through the centre of the body and divided the neural arch into rostral and caudal halves through the rostral border of the caudal notch. These results give support to the controversial theory of resegmentation, in which it was suggested that there is an apparent realignment of segmentation between the somite stage and the subsequent vertebral stage of development.
The present study explores the formation of the dorsal root and sympathetic ganglia in the trunk of the avian embryo. Particular emphasis was given to the timing of gangliogenesis and the relative positions of the neural crest-derived ganglia with respect to the somites. Neural crest cells and their derivatives were recognized by the HNK-1 antibody. The time at which neural crest cell coalesced to form ganglia was assessed by the state of cellular aggregation. The state of ganglionic differentiation was assessed by the expression of neurofilament proteins and the neural cell adhesion molecule (N-CAM). At the level of the 15th somite, neural crest cells were observed in the rostral half of the somite at stage 15, during active neural crest migration, and occupied the rostral two-thirds of the somite at progressive stages. HNK-1 positive cells appeared to be organized in three to four streams of cells oriented mediolaterally and dorsoventrally. The dorsal root ganglia and sympathetic ganglia were first detectable at stages 20 and 21, respectively. Both ganglionic rudiments were aligned with the rostral portion of the somite. The dorsal root ganglia occupied the rostral two-thirds of each somite, whereas cells in the sympathetic ganglia occupied a region corresponding to approximately one-third of each somite. At the time of condensation of the dorsal root ganglia, abundant neurofilament staining was observed within the ganglia. However, no N-CAM immunoreactivity was detected until three stages later at stage 23. In contrast, the sympathetic ganglia demonstrated both neurofilament and N-CAM immunoreactivity at the time of condensation. The observation that both dorsal root and sympathetic ganglia form in register with the rostral portion of somite suggests that cues localized at these axial levels, perhaps within the rostral somite, may influence the position where neural crest cells condense to form ganglia. In sensory ganglia, N-CAM expression does not correlate with the onset of gangliogenesis, suggesting that molecules other than N-CAM may play an important role in the aggregation of some neuronal populations.
The development of patterned axon outgrowth and dorsal root ganglion (DRG) formation was examined after partially or totally removing chick somitic mesoderm. Since the dermamyotome is not essential and a full complement of limb muscles developed, alterations in neural patterns could be ascribed to deletion of sclerotome. When somitic tissue was completely removed, axons extended and DRG formed, but in an unsegmented pattern. Therefore the somite does not elicit outgrowth of axons or migration of DRG precursors, it is not a manditory substratum and it is not required for DRG condensation. These results suggest that posterior sclerotome is relatively inhibitory to invasion, an inhibition that is released when sclerotome is absent. When somites were partially deleted, axonal segmentation was not lost proportionally with the amount of sclerotome removed, suggesting that properties that may vary with sclerotome volume (such as diffusible cues) do not play a primary role. Instead, spinal nerves lost segmentation only when ventral sclerotome was deleted, regardless of whether dorsal sclerotome was or was not removed. This strongly suggests that axonal segmentation is imposed by direct interactions between growth cones and extracellular matrices or surfaces sclerotome cells. While DRG tended to be normally segmented when ventral sclerotome was deleted and to lose segmentation when dorsomedial sclerotome was absent, a coordinate loss of DRG segmentation with sclerotome volume could not be ruled out. However it is clear that axonal and DRG segmentation are independent. Observations on a subset of embryos in which the notochord was displaced relative to the spinal cord suggest that the ventromedial sclerotome surrounding the notochord inhibits axon advance. Posterior and ventromedial sclerotome are hypothesized to act as barriers to axon outgrowth due to some feature of their common cartilaginous development. Specific innervation patterns were also examined. When the notochord was displaced toward the control limb, axons on this side made and corrected projection errors, suggesting that the notochord can influence the precision of axonal pathway selection. In contrast, motor axons that entered the limb on all operated sides innervated muscle with their normal precision despite the absence of the somite and axonal segmentation. Therefore, the somite and the process of spinal nerve segmentation are largely irrelevant to the specificity of motoneuron projection.
The role of the dermamyotome (the dorsal portion of the somite which gives rise to muscles and dermis) in the development of patterned axon outgrowth was examined under conditions where limb development was substantially undisturbed. One or more chick dermamyotomes were removed before or during early neurite outgrowth and subsequent development was examined. Several developmental processes suspected to depend on the dermamyotome were not altered by its removal: (1) Neural crest cells that form sensory ganglia migrated and condensed in their normal segmental pattern. (2) The distal progression, dorsal-ventral organization, and segmentation of spinal nerves were unaltered. (3) Motoneuron pathway selection and projection patterns in the limb were normal in all respects. The most interesting finding was that the formation of the dorsal ramus is dependent on the nearby dermamyotome which provides the targets for this nerve. When a single or two adjacent dermamyotomes were removed, the metameric epaxial muscles derived from each dermamyotome were absent and the dorsal ramus extended into epaxial muscle in the closest adjacent segment. However, when dermamyotomes in both adjacent segments had also been removed or substantially reduced, the dorsal ramus did not form. These results strongly suggest that the target provides a chemotactic signal for proper outgrowth of dorsal ramus axons.
How is the adult pattern of connections between motoneurones and the muscles that they innervate established during vertebrate development? Populations of motoneurones are thought to follow one of two patterns of development: (1) motor axons initially follow stereotyped pathways and project to appropriate regions of the developing muscle or (2) motor axons initially project to some regions that are incorrect, the inappropriate projections being eliminated subsequently. Here we observed individually identified motoneurones in live zebra fish embryos as they formed growth cones and as their growth cones navigated towards their targets. We report that from axogenesis, each motor axon followed a stereotyped pathway and projected only to the specific region of the muscle appropriate for its adult function. In addition, the peripheral arbor established by each motoneurone was restricted to a stereotyped region of its own segment and did not overlap with the peripheral arbor of the other motoneurones in that segment. We conclude that the highly stereotyped pattern of innervation seen in the adult is due to initial selection of the appropriate pathway, rather than elimination of incorrect projections.
The development of neuromasts and sensory neurons of the posterior lateral line was studied in zebrafish (Brachydanio rerio) in order to determine the relationship between growing axons of sensory neurons and the migratory cellular primordium of midbody line neuromasts. Scanning electron microscopy revealed that a primary system of six neuromasts develops during the second day after fertilization and evidence is presented that these arise from cells of a migratory primordium. The primordium is first detected in the postauditory region immediately adjacent to the developing sensory ganglion. Growth cones of posterior lateral line sensory neurons are found within the premigratory primordium when it is adjacent to the ganglion. At later times growth cones of these sensory neurons are found within the primordium as it migrates caudally along the midbody line.
These results demonstrate that although the growth cones of the sensory neurons grow over a considerable distance to their final destination, they are never very far from their target cells (or target cell precursors), which migrate with them and may even lead them.
Although there is good evidence that growing axons can be guided by specific cues during the development of the vertebrate peripheral nervous system, little is known about the cellular mechanisms involved. We describe here an example where axons make a clear choice between two neighbouring groups of cells. Zinc iodide-osmium tetroxide staining of chick embryos reveals that motor and sensory axons grow from the neural tube region through the anterior (rostral) half of each successive somite. 180 degrees antero-posterior rotation of a portion of the neural tube relative to the somites does not alter this relationship, showing that neural segmentation is not intrinsic to the neural tube. Furthermore, if the somitic mesoderm is rotated 180 degrees about an antero-posterior axis, before somite segmentation, axons grow through the posterior (original anterior) half of each somite. Some difference therefore exists between anterior and posterior cells of the somite, undisturbed by rotation, which determines the position of axon outgrowth. It is widespread among the various vertebrate classes.
The wing and wing-associated muscles of the shoulder and thorax in the bird all cleave from common myogenic masses in the developing wing bud and are referred to collectively as brachial muscles. In this study the precise embryonic origin of the brachial muscles was determined using chick-quail chimaeras. Such chimaeras consisted of a graft of one somite taken from a 2-day quail donor embryo transplanted to the equivalent location in a 2-day chick host embryo. The chimaeras were analysed at 9.5-10.0 days in ovo to determine the location of the grafted cells and therefore the structures that were derived from the transplanted somite. The somites that were studied in this matter were somites 13 to 23 inclusive. The results show that only somites 16 to 21 inclusive contribute cells to the brachial musculature; moreover, the cells from a given somite are not distributed randomly among the brachial muscles but populate specific muscles only: thus it has been possible to map the somitic origin of individual brachial muscles. Moreover, there is an indication that each somite plays a unique role in the development of the brachial muscles.
Myogenesis in Xenopus laevis and in Bombina variegata is similar despite differences in the structure of the nonsegmented mesoderm and in the formation of the myotomes. In X. laevis the nonsegmented mesoderm consists of two cell layers with the premyocoel between them. During somitogenesis the premyoblasts rotate covering subsequently the whole myotome length. In B. variegata the premyocoel is absent. The myotomal cells change their shape and elongate, attaining ultimately the whole myotome length. The morphologically mature mononuclear muscle cells in both species result from myogenesis beginning in similarly arranged myoblasts. The multinuclear myotubes arise in the swimming tadpole (stage 45). The structure of the nonsegmented mesoderm and of the newly formed myotomes in Pelobates fuscus is similar to that of B. variegata, while the process of myogenesis is different. It begins in the multinuclear myotubes. The stage of morphologically mature mononuclear muscle cells was not observed in the light microscope. The results suggest that myotomal myogenesis is related neither to any particular type of nonsegmented mesoderm structure nor to any specific mode of myotome formation.
If the somitic mesoderm of a 2-day chick embryo is destroyed by X-irradiation, the adjacent limb develops with a normal pattern of connective tissues, but is devoid of muscle. The innervation of muscleless wings produced in this way was examined in silver-stained whole mounts, fixed 3 to 8 days later. The main nerve trunks and their cutaneous branches developed normally; but the nerve branches which in a normal limb would lead to individual muscles were generally absent. In almost all those exceptional cases where muscle nerve branches were present, muscle was found to be present also, despite the X-irradiation. Where there was no muscle, the muscle nerve branches apparently did not even begin to form. As a control for side effects of the X-irradiation, wing buds were grafted from normal to irradiated embryos and vice-versa, and again analysed for their innervation. The results confirmed that the absence of muscle nerve branches was due to the absence of muscle cells in the limb. Thus
(1) the routes taken through a limb by the main mixed nerve trunks and by their cutaneous branches are determined by the connective tissues, and not by any mechanisms requiring muscle cells; but (2) muscle cells are necessary to provoke the formation of the side branches leading to the sites of individual muscles.
Detailed SEM observations of the changes in cellular morphology, arrangements, and contacts that occur during the process of somite formation were made in two species of urodele amphibians, Ambystoma mexicanum and Pleurodeles waitlii, and one species of anuran amphibian, Rana sphenocephala. After fixation, embryos were fractured transversely, horizontally, and parasagittally, and the intrasomitic cellular arrangement pattern was examined with the SEM. It was found that Ambystoma and Pleurodeles embryos followed exactly the same development sequence in rosette formation and myoblast fusion. Rana somites did not, however, appear to form rosettes. Those myotomal cells underwent fusion immediately after a few segmentations occurred.
Patterns of cellular rearrangement were also described during urodele rosette formation at the time of somite segmentation and during myoblast fusion. Extensive changes in cell shape and orientation appeared to occur during those processes. When cells changed their orientation, they often exhibited a triangular configuration. Probable roles of these triangular-shaped cells in rosette formation and myoblast fusion are discussed.
During the initial period of myoblast or myotomal cell fusion, cells first send out specialized cell processes and then establish their cell-cell contacts. The establishment of such contacts eventually leads to tight membrane appositions and fusion. Since myoblast fusion appeared to occur between two cells which were tandemly arranged in a rosette, the origin of multinuclearity in the fused cells is discussed.
Finally, comparative analyses of the pattern of somite formation and subsequent muscle development were made between different species of amphibians. The possibility is discussed that patterns of somitogenesis may provide useful indicators for determining how different families of amphibians evolved.
Somite formation in the mouse embryo begins with the recruitment of mesenchymal cells into the paraxial mesoderm. Cells destined for the paraxial mesoderm are recruited from a progenitor population found first in the embryonic ectoderm and later in the primitive streak and the tail bud. Experimental evidence suggests that the allocation of precursor cells to different mesodermal lineages may be related to the site at which the cells ingress through the primitive streak. An increasing number of genes, such as those encoding growth factor and transcription factors, are now known to be expressed in the primitive streak. It is not known whether the specification of mesodermal cell fate has any relationship with the activity of genes that are expressed in the restricted cell populations of the primitive streak. Somitomeres, which are spherical clusters of mesenchymal cells in the presomitic mesoderm, presage the segmentation of somites in the paraxial mesoderm. The somitomeric organization denotes a pre-pattern of segmentation that defines the physical boundary and the bilateral symmetry of the mesodermal segments in the body axis. The establishment of new somitomeres seems to require the interaction of a resident cell population in the presomitic mesoderm and the incoming primitive streak cells. Cell mixing, which occurs in the somitomeres prior to somite segmentation, poses problems in understanding the developmental role of the somitomere and the real significance of the partitioning of the node-derived and primitive streak-derived cells in the mesodermal segments. In the presomitic mesoderm, the expression of some genes that encode transcription factors, growth factors or tyrosine kinase receptor, and the localization of certain cell adhesion molecules are closely associated with distinct morphogenetic events, such as cell clustering in the presomitic mesoderm and the formation of epithelial somites. There is, however, very little direct relationship between the spatial pattern of gene expression and the somitomeric organization in the presomitic mesoderm. Results of somite transplantation experiments suggest that both the segmental address and the morphogenetic characteristics of the somite may be determined during somite segmentation. Regional identity of the paraxial mesodermal segment is conferred by the expression of a combination of Hox genes in the sclerotome and probably other lineage-specific genes that are subject to imprinting. Superimposed on the global metameric pattern, two orthogonal polarities of cell differentiation are endowed in each mesodermal segment. The rostro-caudal polarity is established prior to somite segmentation. This polarity is later manifested by the subdivision of the sclerotome and the alliance of the neural crest cells and motor axons with the rostral half-somite.(ABSTRACT TRUNCATED AT 400 WORDS)
This paper is concerned with the influences which cause undifferentiated mesoderm to become converted into somites in the chick embryo. The experiments that are reported were designed to test several theories which already exist in the literature. Each of these theories ascribes an essential, inductive role to one of the following regions:
1. The ‘somite centres’. Spratt (1955) put forward the idea that two ‘somite centres’ exist in the chick blastoderm and that these actively induce the formation of somites. His evidence is based mainly on a series of transection experiments.
2. Hensen's node. This theory is based on the results of experiments in which the node is damaged or extirpated (e.g., Peebles, 1898; Wetzel, 1929; Fraser, 1954). The literature already contains evidence against this theory.
3. The neural tissue. This theory, which has been put forward by various authors (e.g. Grünwald, 1936; Fraser, 1960), is based on the fact that neural tissue and somites are usually closely associated.
Metameric Relationship of the Somitic Sclerotome to Elements of the Peripheral Nervous System and the Vertebral Column During Zebrafish Development. Doctoral dissertation
- E M Morin-Kensicki
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Les premier stades de la différentiation interne du myotome et la formation des éléments sclérotomatiques chez les acraniens, les Sélaciens, et les Téléostéens
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The role of muscle pioneers in pathfinding of primary motoneurons in the embryonic zebrafish
- Melançon
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motoneurons in the embryonic zebrafish. Master's Thesis. Department of
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