The sub-lip domain--a distinct pathway for myotome precursors that demonstrate rostral-caudal migration

We have previously reported that the myotome is formed by a first wave of pioneer cells generated from all along the dorsomedial portion of the epithelial somite and a second wave of cells issued from all four edges of the dermomyotome. Cells from the extreme rostral and caudal edges directly generate myofibers that elongate towards the opposite pole of each segment and along the pre-existing myotomal scaffold. In contrast, cells from the dorsomedial and ventrolateral lips first reach the extreme edges and then contribute to myofiber formation. The mechanism by which these epithelial cells translocate remained unknown and was the goal of the present study. We have found that epithelial cells along the dorsomedial and ventrolateral lips of the dermomyotome first delaminate into the immediate underlayer of the corresponding lips, the sub-lip domain, then migrate longitudinally along this pathway until reaching the extreme edges from which they differentiate into myofibers. Cells of the sub-lip domain are negative for Pax3 and desmin but express MyoD, Myf5 and FREK, suggesting that they are specific myogenic progenitors.     

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.     

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.     

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.     

Sclerotome-derived Slit1 drives directional migration and differentiation of Robo2-expressing pioneer myoblasts

Pioneer myoblasts generate the first myotomal fibers and act as a scaffold to pattern further myotome development. From their origin in the medial epithelial somite, they dissociate and migrate towards the rostral edge of each somite, from which differentiation proceeds in both rostral-to-caudal and medial-to-lateral directions. The mechanisms underlying formation of this unique wave of pioneer myofibers remain unknown. We show that rostrocaudal or mediolateral somite inversions in avian embryos do not alter the original directions of pioneer myoblast migration and differentiation into fibers, demonstrating that regulation of pioneer patterning is somite-intrinsic. Furthermore, pioneer myoblasts express Robo2 downstream of MyoD and Myf5, whereas the dermomyotome and caudal sclerotome express Slit1. Loss of Robo2 or of sclerotome-derived Slit1 function perturbed both directional cell migration and fiber formation, and their effects were mediated through RhoA. Although myoblast specification was not affected, expression of the intermediate filament desmin was reduced. Hence, Slit1 and Robo2, via RhoA, act to pattern formation of the pioneer myotome through the regulation of cytoskeletal assembly.     

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.     

Somite subdomains, muscle cell origins, and the four muscle regulatory factor proteins

We show by immunohistology that distinct expression patterns of the four muscle regulatory factor (MRF) proteins identify subdomains of mouse somites. Myf-5 and MyoD are, at specific stages, each expressed in both myotome and dermatome cells. Myf-5 expression is initially restricted to dorsal cells in all somites, as is MyoD expression in neck somites. In trunk somites, however, MyoD is initially expressed in ventral cells. Myogenin and MRF4 are restricted to myotome cells, though the MRF4-expressing cells are initially less widely distributed than the myogenin-expressing cells, which are at all stages found throughout the myotome. All somitic myocytes express one or more MRFs. The transiently distinct expression patterns of the four MRF proteins identify dorsal and ventral subdomains of somites, and suggest that skeletal muscle cells in somites originate at multiple sites and via multiple molecular pathways.     

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.     

A comparative analysis of Meox1 and Meox2 in the developing somites and limbs of the chick embryo

We have examined the expression pattern of the avian Meox1 homeobox gene during early development and up to late limb bud stages. Its expression pattern indicates that it is involved in somite specification and differentiation. The domains of expression are similar but different to those of Meox2. Meox1 is expressed from stage 6 in the pre-somitic mesoderm and as development proceeds, in the tail bud, the dermomyotome of the rostral somites and in the dermomyotome and sclerotome of the caudal somites, the lateral rectus muscle, truncus arteriosus of the heart and the limb buds. Unlike Meox1, Meox2 is not expressed in the pre-somitic mesoderm, but is expressed first in somites formed from stage 11 onwards. In the developing limb, both genes are expressed in the dorsal and ventral limb mesoderm in adjacent domains with a small region of overlap. In the limb bud, Meox1 is co-expressed with Meox2 but neither Meox gene is co-expressed with MyoD. These expression patterns suggest that these two genes have overlapping and distinct functions in development.     

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.     

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.     

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.     

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.