The EGFR/RAS/MAPK pathway is used reiteratively to control cell differentiation during C. elegans development. We are focusing on the role of this pathway during vulval development. In this process, the gonadal anchor cell (AC) secretes the inductive signal LIN-3 EGF to specify the vulval cell fates. The six equivalent Vulval Precursor Cells (VPCs) express the LET-23 EGFR that binds LIN-3 EGF on the basolateral surface of the VPCs. P6.p, the VPC most proximal to the AC, receives most of the inductive signal and adopts the 1 fate. In turn, P6.p sends a lateral signal to its neighbours P5.p and P7.p to activate the LIN-12 NOTCH signalling pathway. The lateral signal prevents P5.p and P7.p from adopting the 1 fate and specifies the 2 fate. The three remaining VPCs (P3.p, P4.p and P8.p) that do not receive enough inductive or lateral signals adopt the 3 fate, they divide once and fuse with the hypodermis. The three induced VPCs (P5- 7.p) then divide to generate 22 vulval cells that form a functional vulva. To clone new regulators of the EGFR/RAS/MAPK pathway we performed a genetic screen in a
gap-1(lf) sensitised background. The
gap-1(lf) mutation slightly activates the EGFR/RAS/MAPK pathway but not enough to cause a visible phenotype. When a second mutation that further increases the activity of the EGFR/RAS/MAPK pathway is generated in the
gap-1(lf) background, a multivulva (Muv) phenotype may be observed. From this screen, we isolated the
zh8 mutation that appears to increase EGFR/RAS/MAPK pathway activity.
zh8 suppresses the vulvaless (Vul) phenotype of
lin-3 and
lin-25 reduction-of-function mutations and increases the penetrance of the Muv phenotype of
lin-10(lf);
gap-1(lf) double mutants and of gaIs36[HS-mpk- 1,
mek-2(gf)] animals. Furthermore, the
zh8 mutation appears to bypass the lateral inhibition of the 1 fate by LIN-12 NOTCH, since adjacent VPCs can adopt the 1 fate in
zh8;
gap-1(lf) or
zh8; gaIs36[HS-
mpk-1,
mek-2(gf)] animals.
zh8 was mapped between the cosmids C52A11 and M106 using SNPs and a point mutation was found in the T19E10.1/let-21 gene.
let-21 is the C. elegans ortholog of the Drosophila pebble gene and the mouse
ect2 proto-oncogene. Pebble and Ect2 are guanine nucleotide exchange factors (GNEFs) for the Rho family of small GTPases, and they play an important role in the assembly of the actomyosin contractile ring essential for cytokinesis (Prokopenko et al. 1999, Genes & Devel. 13:2301; Tatsumoto et al. 1999, J. Cell Biol. 147: 921). The
zh8 mutation changes a conserved amino acid in the second of the two BRCT domains in the N-terminal portion of LET-21, leaving the putative GNEF domain intact. Feeding RNAi against T19E10.1 suppressed the Muv phenotype of
let-21(
zh8);
gap-1(lf) animals (the vulval induction index was reduced from 4.5 to 3.1), suggesting that
let-21(
zh8) could be a gain-of-function mutation. Consistent with this idea,
let-21 loss-of-function mutations cause a pachytene arrest phenotype in the germ cells, similar to mutations that inactivate RAS/MAPK signaling (Ohmachi et al., 2000 Midwest meeting abstract 85). To test the hypothesis of a gain-of-function mutation, we are performing rescue experiments examining if a
let-21(
zh8) genomic DNA fragment is able to rescue the
let-21(lf) phenotype and cause a Muv phenotype in a
gap-1(lf) background.