Chemical communication between cells plays a central role in guiding the early development of vertebrate embryos. Such communication often involves three molecularly distinct steps. First, ligands (such as peptide growth factors) are released from transmitting cells. Second, information concerning the binding of these ligands at surface receptors is transduced across the plasma membrane and throughout the cytoplasm of receiving cells. Third, this information is translated into differential gene expression, thus achieving discrete patterns of cell determination and differentiation in the embryo. The first and third steps in this process have, thus far, received the most attention from developmental biologists. Our long-term goal is to characterize the second step -- signal transduction during embryogenesis -- at the same level of molecular detail already achieved for these other steps. Success in this goal will contribute significantly to our insight into the molecular mechanisms of development, and thus define loci of both chemical teratogenesis and inborn errors of development which give rise to birth defects. The recent demonstration that the polyphosphoinositide (PI) cycle signal transduction pathway plays an important role in embryonic mesoderm induction provides us an unparalleled opportunity to begin achieving this goal, as the PI cycle is arguably one of the best-understood signaling pathways (in somatic cells). We propose to exploit this knowledge to molecularly characterize this cycle's actions in mesoderm induction. Specifically, the proposed studies will (1) employ novel spatially resolved assays of PI cycle-mediated second messenger responses (intracellular free Ca2+ changes and inositol 1,4,5-trisphosphate production) to determine which (if any) putative mesoderm inducers modulate embryonic PI cycle activity in a spatiotemporally dynamic manner; (2) utilize well-established immunochemical and affinity chromatographic techniques to identify the specific proteins involved (G proteins, phospholipase C isozymes, plasma membrane receptors) and the molecular mechanisms regulating their activities; and (3) bring to bear both approaches in a strategy to detect and molecularly characterize as-yet unidentified ligand receptors which may be involved in this aspect of mesoderm induction.
