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ABSTRACT: The cytoskeletal composition of the ‘primitive streak’ stage of mouse embryos, i.e. at late day 8 (‘day 8.5′) of gestation, has been examined by electron microscopy, using thin sections of fixed and embedded embryos, and by immunofluorescence microscopy, using cryostat sections of frozen embryos. At this stage primary mesenchymal cells are observed in the posterior part of the embryo which seem to migrate toward the anterior region. For most of the embryo, these mesenchymal cells are separated from the embryonic ectoderm by a continuous basal lamina. Frequently mesenchymal cells form cytoplasmic projections many of which make contact with this basal lamina, with surfaces of proximal endoderm cells, or with other mesenchymal cells. Primary mesenchymal cells contain sparse individual intermediate-sized filaments (IF), but closely packed IF bundles as they occur as tonofibrils in both embryonic epithelia, ectoderm and proximal endoderm, have not been found. Mesenchymal cells also can form junctions of the fascia adhaerens-type but appear to be devoid of desmosomes.Antibodies to cytokeratins reveal strong fibrillar fluorescence in cells of the proximal endoderm and weak, predominantly subapical staining in embryonic ectoderm. Correspondingly, antibodies to desmoplakins, the major proteins of the desmosomal plaque, show punctate fluorescence in both embryonic epithelia. These epithelial cells are not significantly stained with antibodies to other IF proteins such as vimentin and desmin. However, antibodies to vimentin show positive fluorescence, often in fibrillar tangles, in primary mesenchymal cells which in turn are negative with cytokeratin and desmin antibodies. This first detection of expression of vimentin in embryogenesis has been confirmed by two-dimensional gel electrophoresis of cytoskeletal proteins from 35S-methionine-labelled embryos.The observations indicate that during embryogenesis synthesis of vimentin occurs, for the first time, in the primitive streak stage and is restricted to the primary mesenchymal cells. Concomitantly, these cells cease to produce cytokeratins and desmoplakin. Possible mechanisms effective in this rapid change from epithelial to mesenchymal character, i.e. from cytokeratin IF to vimentin IF, are discussed.
Differentiation 07/2006; 23(1‐3):43 - 59. · 2.81 Impact Factor
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Differentiation 07/2006; 17(1‐3):161 - 179. · 2.81 Impact Factor
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ABSTRACT: Following our study on the expression of cytokeratin filaments in preimplantation mouse embryos [30], we have examined the organization of cytoskeletal elements in early postimplantation embryos up to day 8 of gestation, employing electron microscopy, immunofluorescence microscopy and two-dimensional gel electrophoresis of cytoskeletal proteins labelled by incorporation of 35S-methionine. The characteristic epithelia formed by the embryonic ectoderm and proximal (visceral) endoderm present well-developed junctional complexes and various differentiated membrane structures. Several apical differentiations of the proximal endodermal cells, such as brush border-like microvilli, the endocytotic labyrinthum, and the supranuclear vacuoles resemble the organization of epithelial cells of the ileum of neonatal mammals. Both embryonic epithelia show typical desmosomes and attached intermediate sized filaments of the cytokeratin type. Other types of intermediate-sized filaments, such as vimentin and desmin filaments, have not been detected in any of the cells of embryos of days 6 and 7, but filaments of the vimentin type can be seen, by immunofluorescence microscopy, late in day 8 in certain cells located in the forming mesoderm. Gel electrophoresis has further revealed that the major cytoskeletal proteins synthesized during days 6–8 in both extraembryonic and embryonic tissue are similar to those characteristic of preimplantation blastocysts and include a major polypeptide corresponding to cytokeratin A described in some internal organs of adult rodents. By the same techniques, synthesis of another cytoskeletal protein, vimentin, has first been found late in day 8. It is concluded that early postimplantation embryonic development, up to mesoderm formation, is characterized by the exclusive presence, in both embryonic ectoderm and proximal endoderm, of differentiated epithelial cells containing desmosome-cytokeratin filament complexes and that other types of intermediate-sized filaments are not yet expressed.
Differentiation 07/2006; 20(1‐3):203 - 216. · 2.81 Impact Factor
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ABSTRACT: 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:1.The first intermediate filaments formed during mouse embryogenesis are cytokeratin-type filaments present in the trophectodermal cells of blastocysts.2.Their formation is closely associated in time and topography with the appearance of desmosomal structures.3.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.
Differentiation.
