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.

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.

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.

Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart.

Previous studies have shown that during avian heart development, epicardial and coronary vascular smooth muscle precursors are derived from the proepicardium, a derivative of the developing liver. This finding led to a model of coronary vascular development in which epicardial cells migrate over the postlooped heart, followed by migration of committed endothelial and smooth muscle precursors from the proepicardium through the subepicardial matrix where the coronary arteries develop. Here we show that epicardial cells undergo epithelial-mesenchymal transformation to become coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts. We began by establishing primary cultures of quail epicardial cells that retain morphologic and antigenic identity to epicardial cells in vivo. Quail epicardial monolayers stimulated with serum or vascular growth factors produced invasive mesenchyme in collagen gels. Chick epicardial cells labeled in ovo with DiI invaded the subepicardial extracellular matrix, demonstrating that mesenchymal transformation of epicardium occurs in vivo. To determine the fates of epicardially derived mesenchymal cells, quail epicardial cells labeled in vitro with LacZ were grafted into the pericardial space of E2 chicks. These cells attached to the heart, formed a chimeric epicardium, invaded the subepicardial matrix and myocardial wall, and became coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts, demonstrating the common epicardial origin of these cell types. A general model of coronary vascular development should now include epicardial-mesenchymal transformation and direct participation of mesenchyme derived from the epicardium in coronary morphogenesis.

Lipofection of a cDNA plasmid containing the dystrophin gene lowers intracellular free calcium and calcium leak channel activity in mdx myotubes.

Muscle cells from Duchenne muscular dystrophy (DMD) patients and the dystrophic mdx mouse lack the protein dystrophin. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myofibers and cultured myotubes and is correlated with abnormally active calcium-specific leak channels. Higher [Ca2+]i results in greater calcium-dependent proteolysis, which may eventually lead to necrosis. We performed liposome-mediated transfection of a cDNA plasmid containing the full-length dystrophin gene into mdx myoblasts and examined the resting [Ca2+]i and leak channel activity of the resulting differentiated myotubes. Many myotubes from transfected cultures expressed dystrophin at levels similar to normal myotubes as determined by immunostaining. The intracellular free calcium, measured by emission ratio microfluorimetry using the calcium indicator fura-PE3, was significantly lower in the dystrophin-positive mdx myotubes than in untransfected control mdx myotubes. The mean open probability of the calcium leak channel was also reduced to a level similar to normal myotubes and significantly less than that for untransfected mdx myotubes. These results show that introduction of extrachromosomal copies of the full dystrophin gene to originally dystrophic muscle cells can correct the defect in calcium homeostasis that is hypothesized to lead to the muscle cell necrosis seen in DMD.

Location and growth of epaxial myotome precursor cells.

The skeletal muscle progenitor cells of the vertebrate body originate in the dermomyotome epithelium of the embryonic somites. To precisely locate myotome precursor cells, fluorescent vital dyes were iontophoretically injected at specific sites in the dermomyotome in ovo and the fates of dye-labeled cells monitored by confocal microscopy. Dye-labeled myotome myofibers were generated from cells injected along the entire medial boundary and the medial portion of the cranial boundary of the dermomyotome, regions in close proximity to the dorsal region of the neural tube where myogenic-inducing factors are thought to be produced. Other injected regions of the dermomyotome did not give rise to myotome fibers. Analysis of nascent myotome fibers showed that they elongate along the embryonic axis in cranial and caudal directions, or in both directions simultaneously, until they reach the margins of the dermomyotome. Finally, deposition of myotome fibers and expansion of the dermomyotome epithelium occurs in a lateral-to-medial direction. This new model for early myotome formation has implications for myogenic specification and for growth of the epaxial domain during early embryonic development.

A critical evaluation of resting intracellular free calcium regulation in dystrophic mdx muscle.

There are conflicting reports regarding whether resting free calcium levels ([Ca2+]i) are elevated in dystrophic mouse (mdx) myotubes and adult myofibers. We reinvestigated this question and found several lines of evidence supporting the hypothesis that increased calcium influx via leak channels leads to increases in resting [Ca2+]i. 1) Step calibration of fura 2/free acid in myofibers with use of microinjected Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffers revealed greater [Ca2+]i in dystrophic cells. Careful calibration of fura PE3-AM, a compartmentalization-resistant derivative of fura 2, also showed elevated [Ca2+]i in mdx myotubes. 2) Chronic, but not acute, application of tetrodotoxin reduced resting [Ca2+]i in dystrophic myotubes, suggesting that elevated resting [Ca2+]i is a consequence of previous long-term contractile activity. 3) Rates of manganese quenching of fura 2 fluorescence, an indirect indicator of calcium influx, were significantly higher in mdx myotubes and were increased by nifedipine, a calcium leak channel agonist. 4) Calcium leak channel activity, measured using patch clamping, was greater in the sarcolemma of adult non-enzyme-treated mdx myofibers.

Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control.

Duchenne and mdx muscle tissues lack dystrophin where it normally interacts with glycoproteins in the sarcolemma. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myotubes and is correlated with abnormally active calcium-specific leak channels in dystrophic myotubes. We fused Duchenne human and normal mouse myoblasts and identified heterokaryon myotubes by Hoechst 33342 staining to measure the degree to which dystrophin introduced by normal nuclei could incorporate throughout the myotube at the sarcolemma and restore normal calcium homeostasis. Dystrophin expression in myotubes was determined by immunofluorescence and confocal laser scanning microscopy. Dystrophin was expressed at the sarcolemma in normal mouse and heterokaryon myotubes, but not in Duchenne myotubes. In heterokaryons, extensive dystrophin localization occurred at the sarcolemma even where only Duchenne nuclei were present, indicating that dystrophin does not exhibit nuclear domains. Heterokaryon, normal mouse and Duchenne myotube [Ca2+]i was measured using fura-2 and fluorescence ratio imaging. Heterokaryon and normal mouse myotubes were found to maintain similar levels of [Ca2+]i. In contrast, Duchenne myotubes had significantly higher [Ca2+]i (p < 0.001). Furthermore, the ability of heterokaryons to maintain normal [Ca2+]i did not depend on greater numbers of normal nuclei than Duchenne being present in the myotube. These results support the view that dystrophin expression in heterokaryons allows for efficient control of [Ca2+]i.

Increased calcium influx in dystrophic muscle.

We examined pathways which might result in the elevated resting free calcium [( Ca2+]i) levels observed in dystrophic mouse (mdx) skeletal muscle fibers and myotubes and human Duchenne muscular dystrophy myotubes. We found that mdx fibers, loaded with the calcium indicator fura-2, were less able to regulate [Ca2+]i levels in the region near the sarcolemma. Increased calcium influx or decreased efflux could lead to elevated [Ca2+]i levels. Calcium transient decay times were identical in normal and mdx fibers if resting [Ca2+]i levels were similar, suggesting that calcium-sequestering mechanisms are not altered in dystrophic muscle, but are slowed by the higher resting [Ca2+]i. The defect appears to be specific for calcium since resting free sodium levels and sodium influx rates in the absence of Na+/K(+)-ATPase activity were identical in normal and dystrophic cells when measured with sodium-binding benzofuran isophthalate. Calcium leak channels, whose opening probabilities (Po) were voltage independent, could be the major calcium influx pathway at rest. We have shown previously that calcium leak channel Po is significantly higher in dystrophic myotubes. These leak channels were selective for calcium over sodium under physiological conditions. Agents that increased leak channel activity also increased [Ca2+]i in fibers and myotubes. These results suggest that increased calcium influx, as a result of increased leak channel activity, could result in the elevated [Ca2+]i in dystrophic muscle.

Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin.

Elevated free Ca2+ concentrations found in adult dystrophic muscle fibers result in enhanced protein degradation. Since the difference in concentrations may reflect differences in entry, Ca2+ leak channels in cultures of normal and Duchenne human myotubes, and normal and mdx murine myotubes, have been identified and characterized. The open probability of leak channels is markedly increased in dystrophic myotubes. Other channel properties, such as mean open times, single channel conductance, ion selectivity, and behavior in the presence of pharmacological agents, were similar among myotube types. Compared to the Ca2+ concentrations in normal human and normal mouse myotubes, intracellular resting free Ca2+ concentrations ([Ca2+]i) in myotubes of Duchenne and mdx origin were significantly higher at a time when dystrophin is first expressed in normal tissue. Taken together, these findings suggest that the increased open probability of Ca2+ leak channels contributes to the elevated free intracellular Ca2+ concentration in Duchenne human and mdx mouse myotubes.