fem-3 acts as a developmental switch gene in the C. elegans sex dtermination pathway; its activity is necessary and limiting for male development in all tissues. We are interested in understanding how FEM-3 protein interactions and FEM-3 subcellular localization control
fem-3 activity so that male development occurs appropriately in XX and XO animals. FEM-3 is a novel protein that interacts with the cytoplasmic domain of the predicted transmembrane receptor TRA-2A. This interaction leads to the inhibition of
fem-3 activity in the XX soma (Mehra et. al , 1999). FEM-3 also binds to FEM-2, a type 2C protein phosphatase (Chin-Sang and Spence, 1996).
fem-3 and
fem-2 act at the same genetic level and both are required for male somatic development in XO animals and for spermatogenesis in XX and XO animals. Therefore, we hypothesize that the FEM-3/FEM-2 interaction is required for male development in all tissues. Using a modified version of the yeast 2-hybrid system we isolated a FEM-3 mutant that is unable to bind to FEM-2 but retains the ability to bind to TRA-2A. If our hypthesis about the biological significance of the FEM-3/FEM-2 interaction is correct then a
fem-3 transgene carrying this mutation will fail to rescue male development in
fem-3(0) animals. We are currently testing this prediction. We have also made a
fem-3::GFP transgene in order to look at the subcellular localization of FEM-3 in cells that are specified wtih female versus male fates. One model is that TRA-2A sequesters FEM-3 at the cell membrane thereby preventing FEM-3 from gaining access to its downstream targets. In this model, when
tra-2 activity is low then FEM-3 is able to act with FEM-2 in the cytoplasm to direct male development. We are making transgenic animals that carry the
fem-3::GFP reporter transgene to look at FEM-3 distribution in all tissues of XX and XO animals at different developmental stages. By introducing mutations that interfere with FEM-3 binding into the
fem-3::GFP transgene, we will be able to dtermine how FEM-3 binding activities control FEM-3 localization.