The goal of this project is to contribute to the understanding of the molecular basis of an embryonic cell guidance information system. Studies on pronephric duct (PND) cell migration in salamander (amphibian) embryos indicate that the information system guiding these cells displays the formal properties of a gradient of adhesiveness. A """"""""Cell Guidance Associated Molecule"""""""" (CGAM) is predicted to map to the putative gradient. A candidate CGAM has been found. A second region rich in this molecule also preferentially supports cell migration. The molecule is identified as the cell-surface enzyme alkaline phosphatase (ALP). A prime focus of this project is to understand the role of ALP in guiding migrating embryonic cells. Inhibition studies will be conducted to determine whether ALP is actively involved in cell guidance. Selected reagents will be tested in embryos for their ability to interfere with both ALP activity and directed cell migration. Agents to be tested will include: (1) Low MW ALP inhibitors: (2) Fab fragments to antibodies directed against ALP and against its active sites; (3) Phosphatidylinositol-specific phospholipase C, an enzyme know to release ALP from cell surfaces; (4) Exogenous ALP. A survey of other ALP-rich regions of the embryo will be conducted, and transplantation experiments will be used to investigate further the correlation between the presence of ALP and selection by migrating cells as a preferred migration substratum. If initial experiments indicate only a spatial correlation but not a causal connection between ALP distribution and PND cell guidance information, a search for other candidate CGAM's will be conducted by generating and screening monoclonal antibodies. Plasma membranes will be purified from salamander embryos and further fractionated into ALP-rich and ALP-poor fractions. ALP-poor fractions will be used in conjunction with an immunosuppressant while ALP-rich fractions will be used as a subsequent immunogen. Hybridoma supernatants will then be screened for antigen localization patterns on whole-mount embryos. If initial experiments suggest a functional involvement of ALP in guidance information, ALP will be tested for its effects on cell behavior. For example, will cells preferentially adhere to and/or migrate upon an ALP-coated substratum? This work has important medical implications. Many birth defects result from the failure of cells to undergo their normal morphogenetic movements. Invasion in malignancy involves the migration of cells to abnormal locations. Understanding guidance mechanisms in normal cell migration may help shed light upon these pathological situations.
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