Immunohistological and ultrastructural study of the developing tendons of the avian foot

Abstract

The aim of the present report is to provide a detailed description of the morphogenesis and initial differentiation of the long tendons of the chick foot, the long autopodial tendons (LAT), from day 6 to day 11 of development. The fine structure of the developing LAT was studied by light and transmission electron microscopy. The characterization by immunofluorescent techniques of the extracellular matrix was performed using laser scanning confocal (tenascin, elastin, fibrillin, emilin, collagen type I, II, III, IV and VI) or routine fluorescence (tenascin, 13F4) microscopy. In addition, cell proliferation in pretendinous blastemas was analyzed by the detection of BrdU incorporation by immunofluorescence. The light microscopic analysis permitted the identification of different stages during LAT morphogenesis. The first stage is the formation of a thick ectoderm-mesenchyme interface along the digital rays, followed by the differentiation of the "mesenchyme lamina", an extracellular matrix tendon precursor, and ending with the formation and differentiation of the cellular condensation that forms the tendon blastema around this lamina. The immunofluorescence study revealed the presence and arrangement of the different molecules analyzed. Tenascin and collagen type VI are precocious markers of the developing tendons and remain present during the whole process of tendon formation. Collagen type I becomes mainly restricted to the developing tendons from day 7.5. Collagens type II and IV are never detected in the developing tendons, while a faint labeling for collagen type III is first detected at day 7. The analysis of the distribution of the elastic matrix components in the developing tendons is a major contribution of our study. Elastin was detected in the periphery of the tendons from day 8 and also in fibrils anchoring the tendons to the skeletal elements. At the same stage, emilin strongly stains the core of the tendon rods, while fibrillin is detected a little later. Our study indicates the existence of an ectoderm-mesoderm interaction at the first stage of the tendon formation. In addition, our results show the different spatial and temporal pattern of distribution of extracellular matrix molecules in developing tendons. Of special importance are the findings concerning the tendinous elastic matrix and its possible role in tendon maturation and stabilization.

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

“…Nevertheless, we found that the FB1 epitope is localized immunoelectron-microscopically in the bundles of subectodermal microfibrils, and that the electrophoretic mobility and immunohistochemical distribution of the FB1 antigen are indistinguishable from those of JB3 antigen, or chicken fibrillin-2 Rongish et al, 1998). The spatial pattern of the fibers demonstrated in this study also overlaps in part the pattern of elastin immunoreactivity in the limb bud (Hurle et al, 1994;Ros et al, 1995;Hurle and Colombatti, 1996), but appears to be more comparable to the pattern of emilin (Hurle and Colombatti, 1996), which is known to be localized in the elastin-microfibril interface (Bressan et al, 1993). On the basis of these and other findings in this study, we discuss the functional and developmental significance of a parallel spatial array of extracellular subectodermal microfibrils.…”

Spatiotemporal protein distribution of TGF‐βs, their receptors, and extracellular matrix molecules during embryonic tendon development

“…Collagen type III is a member of the fibrillar collagen family and the second most abundant collagen molecule in tendon. Studies in skin, tendon, and other tissues indicate a role for collagen type III in regulating fibrillogenesis of collagen type I (Fleischmajer et al, 1988(Fleischmajer et al, , 1990Ros et al, 1995;Liu et al, 1997;Birk and Mayne, 1997). Decorin is a small chondroitin-dermatan sulfate proteoglycan consisting of a core protein and a single glycosaminoglycan chain, and is the most abundant proteoglycan in mature tendon (Goh et al, 2003).…”

Signals regulating tendon formation during chick embryonic development

“…Type I collagen is not specific to tendon and is a major component of many other extracellular matrices, because it is also synthesised by skin and cornea fibroblasts, osteoblasts, and odontoblasts. Type I collagen is detected in early chick limb buds and has been described as being associated with the developing limb tendons from E6 (Hurle et al, 1989;Ros et al, 1995). The low abundance fibrillar collagens types III and V are able to form heterotypic fibrils with type I collagen (Canty and Kadler, 2002) and, therefore, are also part of the precise architecture of extracellular matrix of the tendons (Table 1).…”

“…This finding can partly explain the small amount of research on the early steps of tendon development compared with those on muscle and bone development. The glycoprotein tenascin was classically used as a tendon marker during embryonic development (Hurle et al, 1989;Ros et al, 1995;Kardon, 1998); with the limitation that it labels other embryonic structures (such as nerves and perichondrium), making it difficult to follow early steps of tendon development. The discovery that the bHLH transcription factor scleraxis labels tendons, cells of tendon primordia, and probably the tendon progenitor cells (Schweitzer et al, 2001) constituted a breakthrough for research into the embryology of tendons.…”

“…In the chick, tendons are mature and functional at hatching. However, tenocytes start to differentiate at embryonic day (E) 7 by synthesising extracellular matrix (Ros et al, 1995), which leads to the progressive assembly of collagen early fibrils or fibril segments . The overall tendon architecture is established by E14.…”