The long range goal of this research program is to identify the developmental mechanisms responsible for the formation, enlargement and union of the facial primordia. The present specific aims represent continuing analysis of the relationship between epithelial-mesenchymal interaction and patterns of cell communication and cell proliferation. Prior studies indicated that the presence of epithelium had a profound effect on the viability and proliferation of subjacent mesenchyme. Microinjection of fluorescent tracers into cells of explants of recombined tissues revealed patterns of gap junctional communication in subjacent mesenchyme. Other studies, employing immunofluorescent localization with antibodies to gap junction proteins (27kDa and 43kDa) demonstrated unique patterns of distribution in specific locations, such as the nasal placode (27kDa), and patterns of distribution associated with cell proliferation (43kDa). In light of these findings, the following will be done.
Specific Aim #1 : Communication-incompetent cell populations will be prepared and employed in organ culture systems to test the hypotheses: a) developmental signals between epithelium and mesenchyme are transmitted by gap junctions b) blockage of gap junctional communication at the epithelial-mesenchymal interface will affect proliferation of the adjacent communication-competent mesenchyme.
Specific Aim #2 : Immunocytochemistry, employing antibodies to the 43kDa gap junction protein, will be used to test the hypotheses a) that the 43kDa protein is present in the facial primordia and is distributed in a region-specific manner b) its distribution is correlated with previously determined rates of cell proliferation in the facial primordia c) the distribution is concordant (or discordant) with the distribution of the 27kDa gap junction protein.
Specific Aim #3 : The hypothesis that expression of the gene for the 27kDa is developmentally regulated will be tested by a) determining whether expression of the gene is region-specific in the nasal placode b) determining whether expression of the gene is temporally regulated during nasal placode formation c) determining whether expression of the gene is associated with a particular morphogenetic event during nasal placode development. Epithelial-mesenchymal separation and recombination, organ culture, microinjection, electroporation, flow cytometry, immunocytochemistry, in situ hybridization, and autoradiography will be utilized.
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