Scanning electron microscopical and histochemical study of the structure and function of basement membranes in the early chick embryo

Abstract

A regional pattern exists in the structure and chemical composition of the basement membrane associated with the chick embryo ectoderm during and after gastrulation. The basement membrane consists of a thin amorphous sheet, separable from the associated cells by treatment with Triton X-100. A meshwork of fibrils is associated with the outer surface of this sheet, i. e. with the side facing away from the ectoderm cells. No obvious regional differences were seen in the appearance of the sheetlike component of the membrane. Four well marked zones could be demonstrated in the arrangement of the fibrils in all embryos examined between stage 3 and stage 6. The pattern was gradually lost after the formation of somites. A loose network of fine radial and concentric fibrils underlies the mesoderm of the area pellucida. The anterior boundary of the area pellucida as far back as the level of Hensen’s node is outlined by a band of thick parallel fibrils, lying just inside the area opaca. Between these two regions is the pro-amnion. This represents that portion of the area pel­lucida at any stage which has not yet been penetrated by mesoderm (Hamilton 1952; Patten 1958). There the basement membrane bears a mat of very short thick rod-like fibrils. In the area opaca beyond the fibrous band fine radial fibrils may be seen extending out to the outer margins of the area opaca. A similar regional pattern can be demonstrated histochemically. A particularly high concentration of glycosaminoglycan (GAG), both sulphated and non-sulphated, could be demonstrated at the area pellucida-area opaca boundary in the anterior half of the embryo by alcian blue staining. The greatly reduced staining conditions favouring the staining of sulphated GAG, with 0.25 mM MgCl 2 indicate that the majority of the GAG is not sulphated. In the same site there is a high concentration of large extracellular transformation sensitive (l. e. t. s.) protein demonstrated by immuno­fluorescence. This protein was present in the form of a fibrous band around the anterior area pellucida - area opaca boundary, and fine particulate staining between the fibres. It is proposed that this pattern relates to the function of the fibrils as a contact guidance system directing the movements of mesoderm cells. Cells from the primitive streak migrate outwards in the area pel­lucida guided by the loose mesh. On reaching the area opaca they spread posteriorly and laterally but reach the anterior half of the area opaca by migrating around the area pellucida boundary. It is suggested that the fibrous band in the area directs the movement of these cells. It is also suggested that the fibrous band is used as a migration pathway by the primordial germ cells as they leave the germinal crescent. Grafting experiments demonstrate that the fibrous band is laid down as the result of an interaction between the endoderm of the germinal crescent and its associated ectoderm. It also appears to have a constant relation to the antero-posterior axis of the embryo lying always anterior to Hensen’s node. Abnormalities of the structure and organization of the basement membrane pattern, whether naturally occurring or induced by chemical treatment result in gross morphological abnormalities of the embryo due to defective mesodermal migration.