The C. elegans hermaphrodite vulva is an ectodermal organ that connects the internal gonad with the external environment. Vulval morphogenesis involves cell-fate specification, cell adhesion, cell fusion, cell migration, and cell invasion. While much progress towards understanding signaling pathways has been made, an understanding of later morphogenetic events and how they are connected to cell fate specification is limited. The different fates of the six VPCs are determined in response to several extracellular signals and divide three times in a particular pattern to produce 22 descendants that begin as a linear array, then a tube, and finally a stack of seven toroids, with the anchor cell at the apex of this tube connecting to the uterus. The anchor cell then fuses to the utse uterine multinucleated cell, causing the opening of the tube. While the biochemical events that transduce the signal in the Ras/MAPK pathway are well characterized, it is not well understood how two transcription factors downstream of MAPK (LIN-31 and LIN-1) activate their downstream targets and play a role in vulval development. Specifically, the latter events (cell arrangements, invagination, toroid formation, cell migration, and cell-cell fusion events) are still not well understood.
LIN-31 contains a winged-helix (WH) DNA-binding domain (DBD), a small acidic domain (AD), a putative MAPK D-domain docking site (MAPKDS), four MAPK consensus phosphorylation sites (MAPK sites), and a homology region (RII). Site-directed mutagenesis revealed that the DBD, AD, and RII are important for LIN-31’s function in vulval development. In addition, heterologous transcription assays in yeast indicated that that both the AD and RII function can act as transactivation domains. In contrast, we observed no measurable effect on LIN-31 function when we disrupted all four MAPK sites or the MAPKDS.
Since LIN-1 is also phosphorylated by MAPK during vulval development, we tested the effect of the loss of LIN-31 phosphorylation in a genetic background containing a point mutation in the LIN-1 MAPK docking site. In this sensitized genetic background, this mutation by itself does not display a strong Egl phenotype, however, when both LIN-31 and LIN-1 are defective for phosphorylation, we observe a strong Egl phenotype. The identification of a sensitized background in which to study LIN-31 phosphorylation has also allowed us to identify a putative MAPK docking site on LIN-31, and preliminary confocal analysis of the lin-31; lin-1 double phosphorylation-defective mutant suggests that the strong Egl phenotype is due to defective toroid formation.