ABSTRACT: Introduction:The basement membrane (BM) is a sheet-like extracellular matrix (ECM) lining the basal side of epithelial and endothelial cells. The molecular composition of the BM diversifies as embryonic development proceeds, providing optimized microenvironments for individual cell types. In post-implantation stage embryos, the embryonic BMs are essential for differentiation of the epiblast, a layer of multipotent embryonic stem cells, and subsequent embryogenesis. To better understand the role of BMs and cell-BM interactions in early embryogenesis, it is imperative to accumulate information on the molecular entities of the embryonic BMs. Methods:We analyzed the expressions and localizations of 20 major BM proteins (11 laminin subunits, 6 type IV collagen subunits, nidogen-1 and -2, and perlecan) and other ECM-related proteins such as fibronectin and integrins in post-implantation stage embryos by immunohistochemistry. Results:We found that a set of BM proteins, laminin ?5, ?1, and ?1 (comprising laminin-511), type IV collagen ?1 and ?2 (yielding type IV collagen ?12?2 [IV]), nidogen-1 and -2, and perlecan, were consistently present in the epiblast/ectoderm BMs throughout the early post-implantation stages. In contrast, laminin ?1 was detected in the epiblast BM at E5.5 but decreased in later stages, suggesting that laminin-511 is a major laminin isoform in the early embryonic BM. In addition, fibronectin, a mesenchymal ECM protein, was enriched in the endoderm BM, indicating that the BM compositions differ between the ectoderm and the endoderm. Consistent with these observations, integrin ?5, a high-affinity receptor for fibronectin, was localized in the endoderm, while integrin ?6, a receptor for laminin-511, was localized in the ectoderm. Conclusions:The embryonic BMs underlying the epiblast/ectoderm contain a common toolkit comprising laminin-511, type IV collagen (?12?2 [IV]), nidogen-1 and -2, and perlecan, providing a physiological basis for the utility of laminin-511 as a culture substrate for pluripotent stem cells. The distinctive association of laminin-511 and fibronectin with endodermal and ectodermal cells, together with the differential expression of integrin ?5 and ?6 in these cells, suggests that the ectodermal and endodermal cells rely on their integrin-dependent interactions with laminin-511 and fibronectin, respectively, to ensure their fate specification in embryonic development.
