The mouse blastocyst expresses a 240,000-mol-wt polypeptide that cross-reacts with antibody to avian erythrocyte c~-spectrin. Immunofluorescence localization showed striking changes in the distribution of the putative embryonic spectrin during preimplantation and early postimplantation development. There was no detectable spectrin in either the unfertilized or fertilized egg. The first positive reaction was observed in the early 2-cell stage when a bright band of fluorescence delimited the region of cell-cell contact. The blastomeres subsequently developed continuous cortical layers of spectrin and this distribution was maintained throughout the cleavage stages. A significant reduction in fluorescence intensity occurred before implantation in the apical region of the mural trophoblast and the trophoblast outgrowths developed linear arrays of spectrin spots that were oriented in the direction of spreading. In contrast to the alterations that take place in the periphery of the embryo, spectrin was consistently present in the cortical cytoplasm underlying regions of contact between the blastomeres and between cells of the inner cell mass. The results suggest a possible role for spectrin in cell-cell interactions during early development.The cortical cytoskeleton of the preimplantation mouse embryo contains actin microfilaments and microtubules (7), intermediate filaments of the cytokeratin type (15), myosin (31), and a-actinin (20). A developmental function for these components has been inferred from the changes in cortical organization and from the effects of cytoskeletal disrupting agents on major developmental transitions during preimplantation development (7,27,35,36). Essentially nothing is known, however, about the actual mechanisms whereby the cytoskeleton influences cell shape and polarization, locomotion, and contact interaction between the cells.The recent discovery of spectrin-like proteins in many nonerythroid cells and the possibility that nonerythroid spectrin may function in a manner analogous to erythroid spectrin (for reviews see references 6,8,12,18) is of potential significance for an understanding of developmental processes because spectrin, in association with actin and other components of the membrane cytoskeleton, is a major determinant of cell shape, deformability and fusogenicity, and the mobility of membrane proteins (1,3,23,25). The present study was undertaken to see when spectrin appears during embryonic development. Using immunoblotting procedures, a 240,000-mol-wt polypeptide was identified in the mouse blastocyst that cross-reacts with antibody to avian erythrocyte a-spectrin.Immunofluorescence localization, moreover, shows changes in the distribution of the putative embryonic spectrin that suggest a role for spectrin in cell-cell interactions during preimplantation development. MATERIALS AND METHODS Collection of Embryos:Preimplantation mouse embryos were obtained from 5-7-wk-old Institute for Cancer Research (ICR) females (West Seneca Breeding Facility, Roswell Park Memori...

The relationship between myosin organization and cell spreading in the preimplantation mouse embryo was studied by indirect immunofluorescence in embryos cultured on lectin-coated substrates. Binding of cell surface polysaccharides to substrate-bound concanavalin A and wheat germ agglutinin induced changes in myosin distribution that resembled those which occur during cell-cell contact interaction. This involved an initial loss of myosin from the contact region that was associated with the development of stable cell-substrate attachments. In addition, a ring of myosin was formed along the edge of the cells' contact to the substrate. The presence of such a ring may be related to the potential for subsequent cell spreading. A myosin ring was also identified in the apical junctional region of the outer morula cells where it similarly separated the cell periphery into contacted and free peripheral domains. Following these changes in myosin organization the embryos spread on the substrate by extension of lamellipodia. These movements were coupled to the dissolution of the myosin ring and the reorganization of myosin into filament bundles. The sequence of changes in the pattern of myosin distribution suggests that contact regulation of myosin organization plays an important role in controlling the spreading behavior of blastomeres and perhaps more generally in the organization of cells into epithelia.A major morphological change takes place in the eight-cell mouse embryo when the rounded blastomeres spread and flatten against each other. This process of compaction has an important influence on blastocyst formation and differentiation of the epithelial trophoblast. It involves active cell movement (15) and appears to be related to changes in adhesiveness (14,15,25) and to cytoskeletal reorganization (6, 7).The cortex of compacting blastomeres contains a network of microfilaments (7) and immunofluorescent localization shows a continuous layer of cortical actin (16). Myosin, on the other hand, exhibits a polarized cortical distribution (22). It forms a continuous cortical layer in the uncontacted apical region of the cells and is not detectable in regions of cell contact. The loss of myosin from the contact regions is mediated by contacted interaction between the blastomeres and may play a role in the development of stable cellular associations (23). Contact modulated loss of myosin begins in the two-cell embryo and continues through the morula stages (23). It is therefore unlikely to serve as the only indication of myosin involvement during compaction. If myosin has a specific role in compaction one might expect to see a new pattern of myosin organization in which one element would be the missing patch of cortical myosin.The present study was designed to see if spreading is associated with such a specific pattern of myosin organization. Blastomeres spread on lectin coated substrates (14) and this system was adapted to examine the temporal relationship between myosin organization and spreading. The results show ...

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The role of spectrin and its association with calmodulin in spreading mouse blastomeres was investigated. Embryonic spectrin binds 125I-calmodulin in a calcium-dependent fashion in the blot overlay technique. Double-labeling experiments show coordinate redistribution of spectrin and calmodulin in blastomeres preparing to undergo active spreading movement. At this stage cortical spectrin staining is lost from the region of cell-substrate contact and spectrin and calmodulin become concentrated in two structures closely associated with the contacted region: a group of spherical bodies located on the cytoplasmic side of the cortical layer and a subcortical ring that marks the perimeter of the contacted region. The localization pattern of spectrin and calmodulin is also coordinated with that of actin and myosin. The results suggest that spectrin plays a role in the spreading of blastomeres and that this function may involve linkage of spectrin, calmodulin, and the cortical contractile apparatus.

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