Persistent myogenic capacity of the dermomyotome dorsomedial lip and restriction of myogenic competence

The dorsomedial lip (DML) of the somite dermomyotome is the source of cells for the early growth and morphogenesis of the epaxial primary myotome and the overlying dermomyotome epithelium. We have used quail-chick transplantation to investigate the mechanistic basis for DML activity. The ablated DML of chick wing-level somites was replaced with tissue fragments from various mesoderm regions of quail embryos and their capacity to form myotomal tissue assessed by confocal microscopy. Transplanted fragments from the epithelial sheet region of the dermomyotome exhibited full DML growth and morphogenetic capacity. Ventral somite fragments (sclerotome), head paraxial mesoderm or non-paraxial (lateral plate) mesoderm tested in this assay were each able to expand mitotically in concert with the surrounding paraxial mesoderm, although no myogenic potential was evident. When ablated DMLs were replaced with fragments of the dermomyotome ventrolateral lip of wing-level somites or pre-somitic mesoderm (segmental plate), myotome development was evident but was delayed or otherwise limited in some cases. Timed DML ablation-replacement experiments demonstrate that DML activity is progressive throughout the embryonic period (to at least E7) and its continued presence is necessary for the complete patterning of each myotome segment. The results of serial transplantation and BrdU pulse-chase experiments are most consistent with the conclusion that the DML consists of a self-renewing population of progenitor cells that are the primary source of cells driving the growth and morphogenesis of the myotome and dermomyotome in the epaxial domain of the body.     

Morphogenetic cell movements in the middle region of the dermomyotome dorsomedial lip associated with patterning and growth of the primary epaxial myotome

The morphogenetic cell movements responsible for growth and morphogenesis in vertebrate embryos are poorly understood. Myotome precursor cells undergo myotomal translocation; a key morphogenetic cell movement whereby myotomal precursor cells leave the dermomyotome epithelium and enter the subjacent myotome layer where myogenic differentiation ensues. The precursors to the embryonic epaxial myotome are concentrated in the dorsomedial lip (DML) of the somite dermomyotome (W. F. Denetclaw, B. Christ and C. P. Ordahl (1997) Development 124, 1601-1610), a finding recently substantiated through surgical transplantation studies (C. P. Ordahl, E. Berdougo, S. J. Venters and W. F. Denetclaw, Jr (2001) Development 128, 1731-1744). Confocal microscopy was used here to analyze the location and pattern of myotome cells whose precursors had earlier been labeled by fluorescent dye injection into the middle region of the DML, a site that maximizes the potential to discriminate among experimental outcomes. Double-dye injection experiments conducted at this site demonstrate that cells fated to form myotome do not involute around the recurved epithelium of the DML but rather are displaced laterally where they transiently intermingle with cells fated to enter the central epithelial sheet region of the dermomyotome. Time- and position-dependent labeling experiments demonstrated that myotome precursor cells translocate directly from the middle region of the DML without prior intra-epithelial 'translational' movements of precursor cells to either the cranial or caudal lips of the dermomyotome epithelium, nor were any such translational movements evident in these experiments. The morphogenetic cell movements demonstrated here to be involved in the directional growth and segmental patterning of the myotome and dermomyotome bear interesting similarities with those of other morphogenetic systems.     

LGN-dependent orientation of cell divisions in the dermomyotome controls lineage segregation into muscle and dermis

The plane of cell divisions is pivotal for differential fate acquisition. Dermomyotome development provides an excellent system with which to investigate the link between these processes. In the central sheet of the early dermomyotome, single epithelial cells divide with a planar orientation. Here, we report that in the avian embryo, in addition to self-renewing, a subset of progenitors translocates into the myotome where they generate differentiated myocytes. By contrast, in the late epithelium, individual progenitors divide perpendicularly to produce both mitotic myoblasts and dermis. To examine whether spindle orientations influence fate segregation, early planar divisions were randomized and/or shifted to a perpendicular orientation by interfering with LGN function or by overexpressing inscuteable. Clones derived from single transfected cells exhibited an enhanced proportion of mixed dermomyotome/myotome progeny at the expense of `like' daughter cells in either domain. Loss of LGN or Gαi1 function in the late epithelium randomized otherwise perpendicular mitoses and favored muscle development at the expense of dermis. Hence, LGN-dependent early planar divisions are required for the proper allocation of progenitors into either dermomyotome or myotome, whereas late perpendicular divisions are necessary for the normal balance between muscle and dermis production.     

Coherent development of dermomyotome and dermis from the entire mediolateral extent of the dorsal somite

We have previously shown that overall growth of the myotome in the mediolateral direction occurs in a coherent and uniform pattern. We asked whether development of the dermomyotome and resultant dermis follow a similar pattern or are, alternatively, controlled by restricted pools of stem cells driving directional growth. To this end, we studied cellular events that govern dermomyotome development and the regional origin of dermis. Measurements of cell proliferation, nuclear density and cellular rearrangements revealed that the developing dermomyotome can be subdivided in the transverse plane into three distinct and dynamic regions: medial, central and lateral, rather than simply into epaxial and hypaxial domains. To understand how these temporally and spatially restricted changes affect overall dermomyotome growth, lineage tracing with CM-DiI was performed. A proportional pattern of growth was measured along the entire epithelium, suggesting that mediolateral growth of the dermomyotome is coherent. Hence, they contrast with a stem cell view suggesting focal and inversely oriented sources of growth restricted to the medial and lateral edges. Consistent with this uniform mediolateral growth, lineage tracing experiments showed that the dermomyotome-derived dermis originates from progenitors that reside along the medial as well as the lateral halves of somites, and whose contribution to dermis is regionally restricted. Taken together, our results support the view that all derivatives of the dorsal somite (dermomyotome, myotome and dermis) keep a direct topographical relationship with their epithelial ascendants.     

Generality of vertebrate developmental patterns: evidence for a dermomyotome in fish

The somitic compartment that gives rise to trunk muscle and dermis in amniotes is an epithelial sheet on the external surface of the somite, and is known as the dermomyotome. However, despite its central role in the development of the trunk and limbs, the evolutionary history of the dermomyotome and its role in nonamniotes is poorly understood. We have tested whether a tissue with the morphological and molecular characteristics of a dermomyotome exists in nonamniotes. We show that representatives of the agnathans and of all major clades of gnathostomes each have a layer of cells on the surface of the somite, external to the embryonic myotome. These external cells do not show any signs of terminal myogenic or dermogenic differentiation. Moreover, in the embryos of bony fishes as diverse as sturgeons (Chondrostei) and zebrafish (Teleostei) this layer of cells expresses the pax3 and pax7 genes that mark myogenic precursors. Some of the pax7-expressing cells also express the differentiation-promoting myogenic regulatory factor Myogenin and appear to enter into the myotome. We therefore suggest that the dermomyotome is an ancient and conserved structure that evolved prior to the last common ancestor of all vertebrates. The identification of a dermomyotome in fish makes it possible to apply the powerful cellular and genetic approaches available in zebrafish to the understanding of this key developmental structure.     

The growth of the dermomyotome and formation of early myotome lineages in thoracolumbar somites of chicken embryos

Myotome formation in the epaxial and hypaxial domains of thoraco-lumbar somites was analyzed using fluorescent vital dye labeling of dermomyotome cells and cell-fate assessment by confocal microscopy. Muscle precursor cells for the epaxial and hypaxial myotomes are predominantly located in the dorsomedial and ventrolateral dermomyotome lips, respectively, and expansion of the dermomyotome is greatest along its mediolateral axis coincident with the dorsalward and ventralward growth directions of the epaxial and hypaxial myotomes. Measurements of the dermomyotome at different stages of development shows that myotome growth begins earlier in the epaxial than in the hypaxial domain, but that after an initial lag phase, both progress at the same rate. A combination of dye injection and/or antibody labeling of early and late-expressed muscle contractile proteins confirms the myotome mediolateral growth directions, and shows that the myotome thickness increases in a superficial (near dermis) to deep (near sclerotome) growth direction. These findings also provide a basis for predicting the following gene expression sequence program for the earliest muscle precursor lineages in mouse embryos: Pax-3 (stem cells), myf-5 (myoblast cells) and myoD (myocytes). The movements and mitotic activity of early muscle precursor cells lead to the conclusion that patterning and growth in the myotome specifically, and in the epaxial and hypaxial domains of the body generally, are governed by morphogenetic cell movements.     

Characterization of the early development of specific hypaxial muscles from the ventrolateral myotome.

We have previously found that the myotome is formed by a first wave of pioneer cells generated along the medial epithelial somite and a second wave emanating from the dorsomedial lip (DML), rostral and caudal edges of the dermomyotome (Kahane, N., Cinnamon, Y. and Kalcheim, C. (1998a) Mech. Dev. 74, 59-73; Kahane, N., Cinnamon, Y. and Kalcheim, C. (1998b) Development 125, 4259-4271). In this study, we have addressed the development and precise fate of the ventrolateral lip (VLL) in non-limb regions of the axis. To this end, fluorescent vital dyes were iontophoretically injected in the center of the VLL and the translocation of labeled cells was followed by confocal microscopy. VLL-derived cells colonized the ventrolateral portion of the myotome. This occurred following an early longitudinal cell translocation along the medial boundary until reaching the rostral or caudal dermomyotome lips from which fibers emerged into the myotome. Thus, the behavior of VLL cells parallels that of their DML counterparts which colonize the opposite, dorsomedial portion of the myotome. To precisely understand the way the myotome expands, we addressed the early generation of hypaxial intercostal muscles. We found that intercostal muscles were formed by VLL-derived fibers that intermingled with fibers emerging from the ventrolateral aspect of both rostral and caudal edges of the dermomyotome. Notably, hypaxial intercostal muscles also contained pioneer myofibers (first wave) showing for the first time that lateral myotome-derived muscles contain a fundamental component of fibers generated in the medial domain of the somite. In addition, we show that during myotome growth and evolution into muscle, second-wave myofibers progressively intercalate between the pioneer fibers, suggesting a constant mode of myotomal expansion in its dorsomedial to ventrolateral extent. This further suggests that specific hypaxial muscles develop following a consistent ventral expansion of a 'compound myotome' into the somatopleure.     

A two-step mechanism for myotome formation in chick

The study of the morphogenetic cell movements underlying myotome formation in the chick embryo has led to the emergence of highly controversial models. Here we report a real-time cell lineage analysis of myotome development using electroporation of a GFP reporter in newly formed chick somites. Confocal analysis of cell movements demonstrates that myotome formation involves two sequential steps. In a first phase, incremental myotome growth results from a contribution of myocytes derived solely from the medial border of the dermomyotome. In a second phase, myocytes are produced from all four borders of the dermomyotome. The relative distribution of myocytes demonstrates that the medial and the lateral borders of the somite generate exclusively epaxial and hypaxial muscles. This analysis also identified five myotomal regions, characterized by the origin of the myocytes that constitute them. Together, our results provide a comprehensive model describing the morphogenesis of the early myotome in higher vertebrates.     

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.     

New insights into skeletal muscle development and growth in teleost fishes

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

The expression of the homeobox gene Msx1 reveals two populations of dermal progenitor cells originating from the somites

Experimental manipulation in birds has shown that trunk dermis has a double origin: dorsally, it derives from the somite dermomyotome, while ventrally, it is formed by the somatopleure. Taking advantage of an nlacZ reporter gene integrated into the mouse Msx1 locus (Msx1(nlacZ) allele), we detected segmental expression of the Msx1 gene in cells of the dorsal mesenchyme of the trunk between embryonic days 11 and 14. Replacing somites from a chick host embryo by murine Msx1(nlacZ )somites allowed us to demonstrate that these Msx1-(beta)-galactosidase positive cells are of somitic origin. We propose that these cells are dermal progenitor cells that migrate from the somites and subsequently contribute to the dorsalmost dermis. By analysing Msx1(nlacZ) expression in a Splotch mutant, we observed that migration of these cells does not depend on Pax3, in contrast to other migratory populations such as limb muscle progenitor cells and neural crest cells. Msx1 expression was never detected in cells overlying the dermomyotome, although these cells are also of somitic origin. Therefore, we propose that two somite-derived populations of dermis progenitor cells can be distinguished. Cells expressing the Msx1 gene would migrate from the somite and contribute to the dermis of the dorsalmost trunk region. A second population of cells would disaggregate from the somite and contribute to the dermis overlying the dermomyotome. This population never expresses Msx1. Msx1 expression was investigated in the context of the onset of dermis formation monitored by the Dermo1 gene expression. The gene is downregulated prior to the onset of dermis differentiation, suggesting a role for Msx1 in the control of this process.     

Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates

The dermomyotome develops into myotome and dermis. We previously showed that overall growth of the dermomyotome and myotome in the mediolateral direction occurs in a uniform pattern. While myofibers arise from all four dermomyotome lips, the dermis derives from both medial and lateral halves of the dermomyotome sheet. Here we mapped the fate of this epithelial sheet by analyzing cell types that arise from its central region. We found that these precursors give rise not only to dermis, as expected, but also to a population of proliferating progenitors in the myotome that maintain expression of PAX7, PAX3 and FREK. Given this dual fate, we asked whether single dermomyotome precursors generate both dermal and mitotic myoblast precursors, or alternatively, whether these cell types derive from distinct epithelial founders. Inovo clonal analysis revealed that single dermomyotome progenitors give rise to both derivatives. This is associated with a sharp change in the plane of cell division from the young epithelium, in which symmetrical divisions occur parallel to the mediolateral plane of the dermomyotome, to the dissociating dermomyotome, in which cell divisions become mostly perpendicular. Taken together with clonal analysis of the dermomyotome sheet, this suggests that a first stage of progenitor self-renewal, accounting for dermomyotomal expansion, is followed by fate segregation, which correlates with the observed shift in mitotic spindle orientation.     

Reptilian myotomal myogenesis-lessons from the sand lizard Lacerta agilis L. (Reptilia, Lacertidae)Update

Reptilian myotomal myogenesis is poorly understood. This paper reports on structural, ultrastructural and immunocytochemical studies of muscle differentiation in sand lizard (Lacerta agilis) embryos. During somitogenesis, the somites are composed of epithelial vesicles with a centrally located somitocoel. At later developmental stages the ventral portion of the somite cortex disaggregates into the sclerotome mesenchyme, while the dorsal wall of the somite differentiates into dermomyotome. At these developmental stages, mononucleated cells of the dermomyotome are Pax3-positive. The dermomyotome layer forms the dorsomedial and ventromedial lips. The myotome is first composed of mono- and then of multinucleated myotubes and small mononucleated cells that occur in the vicinity of the myotubes. These mononucleated cells exhibit low proliferative potential as revealed by the use of PCNA antibody. At subsequent stages of myogenesis the mononucleated cells express Pax7 protein, a marker of satellite cells, and assume ultrastructural features characteristic of satellite cells. Some of the mononucleated cells contribute to muscle growth, being involved in fusion with differentiating muscle fibers. This study revealed similarities of myotomal myogenesis in reptiles to that of other vertebrates.     

Myotome formation: a multistage process

The epaxial muscles of the body are localized in a dorsomedial position with respect to the axial structures, attach to the vertebral column and are concerned with maintenance of posture and movements of the vertebral column. The epaxial musculature derives from the myotome, a transient embryonic structure whose formation is initiated at the epithelial somite stage and is accomplished following complete dissociation of the epithelial dermomyotome. Recent results suggest that myotome development is a multistage process, characterized by addition of sequential waves of muscle progenitors. A first wave originates along the medial part of the epithelial somite and gives rise to a primary myotomal structure; a second wave arises from the rostral and caudal lips of the epithelial dermomyotome and from the dorsomedial lip, which contributes indirectly through the rostral and caudal edges, and a third wave which is composed of mitotically active resident progenitors accounts for significant growth of the myotomal mass and for its transition into epaxial muscle. In this review we discuss the origin, migration and known cellular and molecular features that characterize each wave of progenitors that colonize the myotome.     

Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.     

Medial pioneer fibers pattern the morphogenesis of early myoblasts derived from the lateral somite

The first wave of myoblasts which constitutes the post-mitotic myotome stems from the medial epithelial somite. Whereas medial pioneers extend throughout the entire mediolateral myotome at cervical and limb levels, at flank regions they are complemented laterally by a population of early myoblasts emerging from the lateral epithelial somite. These myoblasts delaminate underneath the nascent dermomyotome and become post-mitotic. They are Myf5-positive but express MyoD and desmin only a day later while differentiating into fibers. Overexpression of Noggin in the lateral somite triggers their premature differentiation suggesting that lateral plate-BMP4 maintains them in an undifferentiated state. Moreover, directly accelerating their differentiation by MyoD overexpression prior to arrival of medial fibers, generates a severely mispatterned lateral myotome. This is in contrast to medial pioneers that have the capacity for self-organization. Furthermore, inhibiting differentiation of medial pioneers with dominant-negative MyoD also disrupts lateral myoblast patterning and differentiation. Thus, we propose that medial pioneers are needed for proper morphogenesis of the lateral population which is kept as undifferentiated mesenchyme by BMP4 until their arrival. In addition, medial pioneers also organize dermomyotome lip-derived fibers suggesting that they have a general role in patterning myotome development.     

Clonal separation and regionalisation during formation of the medial and lateral myotomes in the mouse embryo.

In vertebrates, muscles of the back (epaxial) and of the body wall and limbs (hypaxial) derive from precursor cells located in the dermomyotome of the somites. In this paper, we investigate the mediolateral regionalisation of epaxial and hypaxial muscle precursor cells during segmentation of the paraxial mesoderm and myotome formation, using mouse LaacZ/LacZ chimeras. We demonstrate that precursors of medial and lateral myotomes are clonally separated in the mouse somite, consistent with earlier studies in birds. This clonal separation occurs after segmentation of the paraxial mesoderm. We then show that myotome precursors are mediolaterally regionalised and that this regionalisation precedes clonal separation between medial and lateral precursors. Strikingly, the properties of myotome precursors are remarkably similar in the medial and lateral domains. Finally, detailed analysis of our clones demonstrates a direct spatial relationship between the myocytes in the myotome and their precursors in the dermomyotome, and earlier in the somite and presomitic mesoderm, refuting several models of myotome formation, based on permanent stem cell systems or extensive cell mingling. This progressive mediolateral regionalisation of the myotome at the cellular level correlates with progressive changes in gene expression in the dermomyotome and myotome.