Ultrastructural and morphometric analysis of the separation of two thigh muscles in the chick

Abstract

Limb muscles separate from one another in a complex but highly stereotyped sequence and spatial pattern. The process of separation is characterized by the progression of a region of increased extracellular space, the cleavage zone, along the proximodistal axis between the individual muscle anlagen. We analyzed ultrastructurally the muscles and cleavage zone during the separation of two representative muscles, the developing sartorius and iliotibialis in the chick thigh, to establish an accurate baseline for an analysis of the mechanisms of separation. Comparisons of the morphology and distribution of cells before and after separation show no evidence that muscles became separated by the massive influx of an exterior cell population; if populations invade the cleavage zone, they are small. We do find characteristic transitions within the cell population of the cleavage zone in situ that could accomplish cleavage without invoking massive cell movements. These progressive transitions within the cleavage zone include a loss of close cell-cell interactions, an increase in extracellular space, the assumption of a more stellate morphology by mesenchyme cells, and a gradual alteration in the composition of the extracellular matrix from one typical of early muscle to one typical of loose connective tissue. Myotubes do differentiate between the incipient muscles, ruling out the possibility that the location where muscles will separate is defined by sites where myotubes fail to differentiate. Instead, the myotubes in the cleavage zone gradually diminish in number and appear to be specifically recognized and removed from the cleavage zone by phagocytes. We suggest that the transitions within the cleavage zone, including the loss of muscle cells, are a result of the progressive differentiation of loose connective tissue. If so, then the spatial pattern and process of cleavage is a consequence of spatially programmed cell differentiation.

Hox Genes and Limb Musculoskeletal Development

“…Finally, tendon primordia arise in the dorsal and ventral limb mesenchyme directly from the lateral plate mesoderm then align between the muscle masses and the skeletal elements (Fig III) [41, 42]. The dorsal and ventral muscle bundles segregate into individual anatomical groups as muscle connective tissue cells align along future sites of splitting and initiate cell death [41, 43, 44]. This process generally proceeds in a proximal to distal and dorsal to ventral fashion along the limb axis.…”

Spatial and temporal patterns of muscle cleavage in the chick thigh and their value as criteria for homology

“…Twenty-three embryos were fixed in Carnoy's and Bouin's fixatives (Humason, 1972) and stained with Alcian blue, cresyl violet, and eosin. Serial, 25 pm plastic sections of thighs prepared as in Schroeter and Tosney (1991) were also examined; cellular detail was revealed in these sections by postfixation in osmium.…”

“…We see no morphological evidence of migrating cells, and experimental manipulations suggest that there is little cell migration in the limb when muscles are separating (see, for instance, Tickle et al, 1978). In addition, condensed myogenic regions do not retain their size and move apart as the less densely packed cleavage zone emerges between two muscle primordia (Schroeter and Tosney, 1991). The positional changes of the muscles during their morphogenesis is most likely due to differential growth of individual muscles, chondrification of the skeleton, and mutual growth differences of single muscle primordia rather than to migration of entire muscle anlagen (see also Cihak, 1972;Sullivan, 1962).…”

“…The basis for the ability to describe the formation of individual muscles with precision is derived from the results of our previous ultrastructural study (Schroeter and Tosney, 1991). We described a sequence of cellular changes that accompanies the progression of one particular cleavage zone along the proximodistal axis of the limb.…”

Involvement of vessels and PDGFB in muscle splitting during chick limb development

“…Gradual diminution in the number of the myogenic cells in the cleavage zone has been observed using electron microscopy (Schroeter and Tosney, 1991b). The residual myotubes in the cleavage zones are then thought to be selectively removed by phagocytic cells via an unknown mechanism (Schroeter and Tosney, 1991b).…”