This application is to study pattern formation during development of the nematode vulva. Previous work has shown that the product of the lin-3 gene, an EGF homolog, acts as a secreted morphogen in this system. A morphogen is defined as a molecule whose spatial distribution contributes to development of biological pattern by dictating alternate cell responses in a concentration or dose-dependent manner. How this comes about is not known in any growth factor system. One general aim of the experiments proposed here is to understand the mechanism by which different levels of lin-3 activity determine three alternative responses in vulva precursor cells (VPC's). A second general aim is to understand how lin-3 signalling acts in the context of a network of several additional patterning forming mechanisms known to act in the vulva. One ultimate goal is to explain the precision with which VPC cell fates are specified. The role of the lin-3 ligand itself in induction of two different cell fates will be examined by determining whether there is more than one form of the lin-3 ligand, each with a different effect on VPC's, and by testing whether concentration, timing, or duration of lin- 3 activity is important in VPC response. The level at which different qualitative or quantitative signals have their effect in the responding cell will be examined by analyzing mutant forms of the let-23 receptor, to determine whether the signalling pathway branches at this point, and by examining the effects of altering dosage or activity of known downstream components such as ras and raf. To determine whether the patterning function of the lin-3-let-23 signal is reinforced by differing responsiveness of VPC's (that is, by a prepattern), the effects on VPC response of mutations in HOM-C genes will be determined. The time during which VPC's are competent to respond to signal will be determined, as well as the effects of additional signalling pathways on VPC responsiveness, including the lin-12 pathway and several known negative regulators of let- 23. Finally, experiments will be carried out to separate the effects on VPC's of the lin-3 signal and the lin-12-mediated lateral signal. VPC induction will be studied in double lin- 12/glp-1 mutants in mosaics to test whether differing levels of lin-3 activity alone are sufficient to dictate alternate VPC fates. Likewise, the patterning functions of the lin-12 pathway in the absence of lin-3 induction will be studied. Finally, experiments to test competition between these pathways are proposed.
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