Alkaline phosphatase (APase) is expressed in the preimplantation mouse embryo in a stage- and then a tissue-specific manner. It has been inferred that the form of APase present in the early murine embryo is the tissue non-specific APase, which has been detected in liver, kidney, bone, placenta and F9 teratocarcinoma. Preliminary studies from the PI's laboratory have demonstrated that the embryonic APase is not the tissue non-specific form, based on its thermal and inhibitor sensitivities. Unlike the F9 APase, the embryonic APase has also been found to cross-react with sera directed against human placental APase (a form not detected so far in mouse). It is, therefore, of considerable interest to further characterize the embryonic APase by immunological, electrophoretic, proteolytic, ultrastructural and biophysical approaches and to determine its relatedness to other murine and human APases. A variety of embryo-derived and teratocarcinoma cell lines will be screened for the presence of the embryonic APase, in addition to, or, in place of, the tissue-unspecific form believed to be present in these cell types. The expression of the mRNA encoding the embryonic APase will be studied by several molecular biological approaches (RNA dot blots, Northern blots and in situ hybridization) to determine the levels of APase mRNA in different stage embryos and tumor cells, thus, defining the temporal sequence for its expression and repression. The mouse embryonic APase gene will be cloned and analyzed for the size of the embryonic APase gene, the intron- exon structure of the gene and the structure of promotor and regulatory regions. To define the role and/or importance of APase in early mouse embryos and/or tumor cells, the expression of the embryonic APase will be suppressed by the injection of anti-sense APase RNA into 2-cell blastomeres and by then following the developmental capabilities of these injected cells. Additionally, inhibition of APase activity will be attempted with chemical inhibitors or by treatment of embryos with anti-peptide antibodies capable of abolishing APase activity. In this way it may be possible to assess the importance of APase in murine preimplantation development.
