Subectodermal microfibrillar bundles are organized into a distinct parallel array in the developing chick limb bud

Abstract

In this study, a unique fiber system in the subectodermal mesenchyme of the chick limb bud was visualized immunohistochemically with the use of a novel monoclonal antibody termed "FB1." This antibody stained a subset of extracellular fibers in the embryonic mesenchyme. Among the fibers visualized, those running perpendicularly to the limb bud ectoderm became progressively prominent in their thickness and length, and organized into a parallel array in the subectodermal region. This fiber system was distinct from that of major collagens, fibronectin, or tenascin. A molecule immunoprecipitated with FB1 comigrated with JB3 antigen, or chicken fibrillin-2. The fibers visualized immunohistochemically by FB1 and JB3 were indistinguishable from each other, and ultrastructurally appeared to be bundles composed of tubular-like microfibrils that originated directly from the ectodermal basal lamina. They lacked the amorphous deposits that are characteristic of elastin. A similar array of subectodermal fibers was also found in the developing axilla and some truncal regions, again well before the development of a definitive dermis. These findings suggest that a parallel array of subectodermal FB1-positive fibers constitutes a precocious fiber system in the presumptive dermis prior to the substantial formation of collagenous fibers. These fibers could be developmentally linked to oxytalan fibers, which are known to be present in the papillary dermis in mature cutaneous tissue.

Late Deposition of Elastin to Vertical Microfibrillar Fibers in the Presumptive Dermis of the Chick Embryonic Tarsometatarsus

“…Hurle et al (1988Hurle et al ( , 1989 reported unique argyrophilic fibrils oriented dorsoventrally in the digital and toe plates of the developing limb bud. We have further characterized those fibrils ultrastructurally and immunohistochemically and showed that the fibrils consisted of tubular-appearing microfibrils immunoreactive for fibrillin (Isokawa et al, 2004). It was also suggested in the latter report that MFs with similar immunohistochemical characteristics emerge differentially in the presumptive dermis of the avian embryo, probably depending on local mechanophysiological demands.…”

“…Immunoreactivity for fibrillin is known to be positive in the limb bud in its initial budding phase (Isokawa et al, 2004). Subectodermal fibrils immunoreactive for fibrillin became aligned perpendicularly to the covering ectoderm and organized into a characteristic array of vertical MFs by ED4 ( Fig.…”

“…After blocking in 1% bovine serum albumin (BSA)-PBS for 1 hr, sections were reacted for 1 hr with mouse anti-chick fibrillin-2 IgG 1 (undiluted FB1 hybridoma conditioned medium; Isokawa et al, 2004) or mouse anti-chick tropoelastin IgG (1:100 dilution of 10B8, Chemicon; Hurle et al, 1994) and washed in PBS; some, for double staining, were further incubated for 1 hr with rabbit antiserum to bovine epidermal cytokeratin (1:100 dilution of Z622; Dako), which crossreacts with avian embryonic keratins (Virágh et al, 1993;Sejima et al, 2001). Controls included omission of these antibodies or the use of normal, nonimmune mouse IgG for the antibody incubation.…”

“…Ultrathin sections prepared from Lowicryl K4M (Polysciences, Inc.) water-miscible resin-embedded specimens were stained with 10B8 anti-chick tropoelastin antibody according to the postembedding immunogold labeling procedure (Isokawa et al, 2004). Control sections were prepared by omitting 10B8 or the use of normal, nonimmune mouse IgG for the antibody incubation.…”

“…MFs and elastin deposition were visualized immunohistochemically with monoclonal antibodies termed FB1 (Isokawa et al, 2004) and 10B8 (Hurle et al, 1994), respectively. Accumulation of collagenous fibers was evaluated by a picrosirius-polarization method.…”