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.     

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.     

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 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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Divergent regulation of Wnt-mediated development of the dorsomedial and ventrolateral dermomyotomal lips

The dermomyotome is the dorsal compartment of the somite which gives rise to multiple cell fates including skeletal muscle, connective tissue, and endothelia. It consists of a pseudostratified, roughly rectangular epithelial sheet, the margins of which are called the dermomyotomal lips. The dermomyotomal lips are blastema-like epithelial growth zones, which continuously give rise to resident dermomyotomal cells and emigrating muscle precursor cells, which populate the subjacent myotomal compartment. Wnt signaling has been shown to regulate both dermomyotome formation and maintenance of the dermomyotomal lips. Whereas the epithelialization of the dermomyotome is regulated via canonical, β-catenin-dependent Wnt signaling, the downstream signaling mechanisms suppressing epithelial-mesenchymal transition (EMT) in the mature dermomyotomal lips have been unknown. Here, we present evidence that dermomyotomal lip sustainment is differentially regulated. Whereas the dorsomedial dermomyotomal lip is maintained by canonical Wnt signaling, development of the ventrolateral dermomyotomal lip is regulated by non-canonical, PCP-like Wnt signaling. We discuss our results in the light of the different developmental prerequisites in the dorsomedial and ventrolateral lips, respectively, thus providing a new perspective on the regulation of dermomyotomal EMT.