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.     

The dermomyotome dorsomedial lip drives growth and morphogenesis of both the primary myotome and dermomyotome epithelium

The cellular and molecular mechanisms that govern early muscle patterning in vertebrate development are unknown. The earliest skeletal muscle to organize, the primary myotome of the epaxial domain, is a thin sheet of muscle tissue that expands in each somite segment in a lateral-to-medial direction in concert with the overlying dermomyotome epithelium. Several mutually contradictory models have been proposed to explain how myotome precursor cells, which are known to reside within the dermomyotome, translocate to the subjacent myotome layer to form this first segmented muscle tissue of the body. Using experimental embryology to discriminate among these models, we show here that ablation of the dorsomedial lip (DML) of the dermomyotome epithelium blocks further primary myotome growth while ablation of other dermomyotome regions does not. Myotome growth and morphogenesis can be restored in a DML-ablated somite of a host embryo by transplantation of a second DML from a donor embryo. Chick-quail marking experiments show that new myotome cells in such recombinant somites are derived from the donor DML and that cells from other regions of the somite are neither present nor required. In addition to the myotome, the transplanted DML also gives rise to the dermomyotome epithelium overlying the new myotome growth region and from which the mesenchymal dermatome will later emerge. These results demonstrate that the DML is a cellular growth engine that is both necessary and sufficient to drive the growth and morphogenesis of the primary myotome and simultaneously drive that of the dermomyotome, an epithelium containing muscle, dermis and possibly other potentialities.     

Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo

We addressed the potential role of cell-laminin interactions during epaxial myotome formation in the mouse embryo. Assembly of the myotomal laminin matrix occurs as epaxial myogenic precursor cells enter the myotome. Most Myf5-positive and myogenin-negative myogenic precursor cells localise near assembled laminin, while myogenin-expressing cells are located either away from this matrix or in areas where it is being assembled. In Myf5(nlacZ/nlacZ) (Myf5-null) embryos, laminin, collagen type IV and perlecan are present extracellularly near myogenic precursor cells, but do not form a basement membrane and cells are not contained in the myotomal compartment. Unlike wild-type myogenic precursor cells, Myf5-null cells do not express the alpha6beta1 integrin, a laminin receptor, suggesting that integrin alpha6beta1-laminin interactions are required for myotomal laminin matrix assembly. Blocking alpha6beta1-laminin binding in cultured wild-type mouse embryo explants resulted in dispersion of Myf5-positive cells, a phenotype also seen in Myf5(nlacZ/nlacZ) embryos. Furthermore, inhibition of alpha6beta1 resulted in an increase in Myf5 protein and ectopic myogenin expression in dermomyotomal cells, suggesting that alpha6beta1-laminin interactions normally repress myogenesis in the dermomyotome. We conclude that Myf5 is required for maintaining alpha6beta1 expression on myogenic precursor cells, and that alpha6beta1 is necessary for myotomal laminin matrix assembly and cell guidance into the myotome. Engagement of laminin by alpha6beta1 also plays a role in maintaining the undifferentiated state of cells in the dermomyotome prior to their entry into the myotome.     

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.     

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.     

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.     

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.     

Dorsoventral axis determination in the somite: a re-examination

We have repeated classic dorsoventral somite rotation experiments (Aoyama and Asamoto, 1988, Development 104, 15-28) and included dorsal and ventral gene expression markers for the somitogenic tissue types, myotome and sclerotome, respectively. While the histological results are consistent with those previously published, gene expression analysis indicates that cells previously thought to be 'sclerotome' no longer express Pax1 mRNA, a sclerotome marker. These results, together with recent quail-chick transplantation experiments indicating that even very late sclerotome tissue fragments are multipotential (Dockter and Ordahl, 1998, Development 125, 2113-2124), lead to the conclusion that sclerotome tissue remains phenotypically and morphogenetically plastic during early embryonic somitogenesis. Myotome precursor cells, by contrast, appear to be determined within hours after somite epithelization; a finding consistent with recent reports (Williams and Ordahl, 1997, Development 124, 4983-4997). Therefore, while these findings support a central conclusion of Aoyama and Asamoto, that axis determination begins to occur within hours after somite epithelialization, the identity of the responding tissues, myotome versus sclerotome, differs. A simple model proposed to reconcile these observations supports the general hypothesis that determinative aspects of early paraxial mesoderm growth and morphogenesis occur in early and late phases that are governed principally by the myotome and sclerotome, respectively.     

The third wave of myotome colonization by mitotically competent progenitors: regulating the balance between differentiation and proliferation during muscle development

The myotome is formed by a first wave of pioneer cells originating from the entire dorsomedial region of epithelial somites and a second wave that derives from all four lips of the dermomyotome but generates myofibers from only the rostral and caudal edges. Because the precedent progenitors exit the cell cycle upon myotome colonization, subsequent waves must account for consecutive growth. In this study, double labeling with CM-DiI and BrdU revealed the appearance of a third wave of progenitors that enter the myotome as mitotically active cells from both rostral and caudal dermomyotome edges. These cells express the fibroblast growth factor (FGF) receptor FREK and treatment with FGF4 promotes their proliferation and redistribution towards the center of the myotome. Yet, they are negative for MyoD, Myf5 and FGF4, which are, however, expressed in myofibers. The proliferating progenitors first appear around the 30-somite stage in cervical-level myotomes and their number continuously increases, making up 85% of total muscle nuclei by embryonic day (E)4. By this stage, generation of second-wave myofibers, which also enter from the extreme lips is still under way. Formation of the latter fibers peaks at 30 somites and progressively decreases with age until E4. Thus, cells in these dermomyotome lips generate simultaneously distinct types of muscle progenitors in changing proportions as a function of age. Consistent with a heterogeneity in the cellular composition of the extreme lips, MyoD is normally expressed in only a subset of these epithelial cells. Treatment with Sonic hedgehog drives most of them to become MyoD positive and then to become myofibers, with a concurrent reduction in the proportion of proliferating muscle precursors.     

The roles of cell migration and myofiber intercalation in patterning formation of the postmitotic myotome

We have previously found that the postmitotic myotome is formed by two successive waves of myoblasts. A first wave of pioneer cells is generated from the dorsomedial region of epithelial somites. A second wave originates from all four edges of the dermomyotome but cells enter the myotome only from the rostral and caudal lips. We provide new evidence for the existence of these distinctive waves. We show for the first time that when the somite dissociates, pioneer myotomal progenitors migrate as mesenchymal cells from the medial side towards the rostral edge of the segment. Subsequently, they generate myofibers that elongate caudally. Pioneer myofiber differentiation then progresses in a medial-to-lateral direction with fibers reaching the lateralmost region of each segment. At later stages, pioneers participate in the formation of multinucleated fibers during secondary myogenesis by fusing with younger cells. We also demonstrate that subsequent to primary myotome formation by pioneers, growth occurs by uniform cell addition along the dorsoventral myotome. At this stage, the contributing cells arise from multiple sources as the myotome keeps growing even in the absence of the dorsomedial lip. Moreover, as opposed to suggestions that myotome growth is driven primarily and directly by the medial and lateral edges, we demonstrate that there is no direct fiber generation from the dorsomedial lip. Instead, we find that added fibers elongate from the extreme edges. Altogether, the integration between both myogenic waves results in an even pattern of dorsoventral growth of the myotome which is accounted for by progressive cell intercalation of second wave cells between preexisting pioneer fibers.     

The epaxial-hypaxial subdivision of the avian somite

In all jaw-bearing vertebrates, three-dimensional mobility relies on segregated, separately innervated epaxial and hypaxial skeletal muscles. In amniotes, these muscles form from the morphologically continuous dermomyotome and myotome, whose epaxial-hypaxial subdivision and hence the formation of distinct epaxial-hypaxial muscles is not understood. Here we show that En1 expression labels a central subdomain of the avian dermomyotome, medially abutting the expression domain of the lead-lateral or hypaxial marker Sim1. En1 expression is maintained when cells from the En1-positive dermomyotome enter the myotome and dermatome, thereby superimposing the En1-Sim1 expression boundary onto the developing musculature and dermis. En1 cells originate from the dorsomedial edge of the somite. Their development is under positive control by notochord and floor plate (Shh), dorsal neural tube (Wnt1) and surface ectoderm (Wnt1-like signalling activity) but negatively regulated by the lateral plate mesoderm (BMP4). This dependence on epaxial signals and suppression by hypaxial signals places En1 into the epaxial somitic programme. Consequently, the En1-Sim1 expression boundary marks the epaxial-hypaxial dermomyotomal or myotomal boundary. In cell aggregation assays, En1- and Sim1-expressing cells sort out, suggesting that the En1-Sim1 expression boundary may represent a true compartment boundary, foreshadowing the epaxial-hypaxial segregation of muscle.     

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.     

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.     

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.     

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.