Formation of Cytoskeletal Elements During Mouse Embryogenesis

Different tissues of vertebrates may contain various types of intermediate-sized (7–11 nm) filaments, but little is known about their structural and functional properties during early mammalian development. We have studied the time and mode of formation of cytoskeletal elements in relation to cells and tissue differentiation during mouse embryogenesis. Here we describe the first appearance and formation of intermediate filament proteins and structures in preimplantation embryos using: (1) electron microscopy; (2) immunofluorescence microscopy with antibodies to the different types of intermediate filament proteins; and (3) two-dimensional gel electrophoresis of embryonic proteins and high salt-resistant cytoskeletal preparations. Neither intermediate filament structures nor known intermediate filament proteins have been detected between the two-cell and early-morula stage. In late morulae and blastocysts, however, three major insoluble cytoskeletal proteins (molecular weights: 46,000; 54,000; 61,000) are produced and tentatively identified as prekeratinlike proteins. Two other important cytoskeletal proteins, desmin and vimentin, are not detected. In the outer cells of morulae and in the trophectoderm of early blastocysts we have observed special junctional complexes interpreted as ‘nascent’ desmosomes because of their small size, their incomplete desmosomal plaques and midline structures, and their short tufts of associated intermediate (tonofilamentlike) filaments. In the trophectodermal cells, the number of typical (‘mature’) desmosomes and the length of bundles of densely fasciated tonofilaments extending throughout the cytoplasm increases during blastocyst growth (days 4 and 5). The prekeratinlike nature of constituent proteins of these intermediate-sized filaments has been demonstrated by their specific decoration with antibodies to bovine epidermal prekeratin and their insensitivity to colcemid treatment. Conversely, in cells of the inner cell mass of blastocysts, neither desmosomes nor intermediate-sized filaments have been observed. In the outgrowths of trophectodermal cells of blastocysts allowed to attach to and develop on cover slips for 48 h we have noticed a remarkable increase in the number of desmosomes and intermediate filaments, most of which are arranged into bundles of variable thickness. Such filament bundles are strongly stained with antibodies to prekeratin and, upon treatment of the cells with colcemid, are not aggregated into perinuclear whorls. Our results show that: The first intermediate filaments formed during mouse embryogenesis are cytokeratin-type filaments present in the trophectodermal cells of blastocysts. Their formation is closely associated in time and topography with the appearance of desmosomal structures. We conclude from the absence of desmin and vimentin that intermediate filaments of both these types are not essential for the development of the preimplantation embryo. We further suggest that the trophectoderm resembles a differentiated cytokeratin-rich epithelium, and that the desmosome-tonofilament complex is involved in epithelial differentiation during early murine embryogenesis.