lin-3 encodes the only member of the epidermal growth factor (EGF) family in C. elegans. LIN-3 has a number of essential functions during larval development, hermaphrodite fertility and behavior. One of the best studied functions is the role of LIN-3 during primary (1°) vulval fate specification. After vulval induction, lin-3 is also expressed in the inner-most 1° VulF cells to determine the uv1 subfate of cells in the ventral uterus. However, due to the many essential functions of lin-3 during early larval development, it is difficult to detect potential later functions of LIN-3 after vulva induction. lin-3 is a large locus with three different transcriptional start sites and many splice variants, making it difficult to create transcriptional reporters that reproduce the wild-type expression pattern. Using CRISPR/CAS9, we inserted an mNeongreen reporter into the lin-3 locus to create an endogenous and fully functional LIN-3 reporter. This reporter has enabled us to analyze the expression pattern and subcellular localization of LIN-3.
Tissue-specific RNAi data has indicated that LIN-3 secreted by the VulF cells is required for dorsal lumen formation during vulval morphogenesis. To verify these RNAi data we generated a tissue-specific knockout strain by inserting into the lin-3 locus two FRT recombination sites flanking part of the EGF domain. By expressing the flp recombinase under control of the heat-shock promoter we can now induce the loss of LIN-3 function at different developmental stages. In addition, we are expressing the flp recombinase under the control of different promoters to investigate the tissue-specific functions of LIN-3. This system will allow us to explore novel functions of LIN-3 in a highly specific manner.