Worm Breeder's Gazette 14(4): 44 (October 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Developmental Biology, Stanford University School of Medicine, Stanford CA, 94305
During vulval development, the anchor cell signal normally induces P6.p to express the primary cell fate by activating a conserved receptor tyrosine kinase/Ras/Map Kinase signaling pathway. Here we describe the genetic analysis and the molecular cloning of gap-1 (previously known as suv-1; Kim,S.K. and Horvitz,H.R.,WBG 11(3),68), a component of this signaling pathway. The mutant phenotype of gap-1 suggests that it inhibits vulval induction by the let-60 ras signaling pathway. Null mutations in gap-1 (see below) suppress the vulvaless phenotype caused by mutations that reduce but do not eliminate signaling activity at a step upstream of lin-45 raf. For example, gap-1(ga133) suppresses the vulvaless phenotype caused by partial reduction-of-function alleles of lin-3 which encodes the anchor cell signal, let-23 which encodes a receptor tyrosine kinase, sem-5 which encodes a GRB-2 homolog and let-60 which encodes Ras (Table). In addition, gap-1(ga133) suppresses the vulvaless phenotype of null alleles of lin-2, lin-7 and lin-10, three genes required for receptor activation. However, gap-1(ga133) does not suppress the vulvaless phenotype of a weak allele of lin-45, which encodes Raf and acts downstream of let-60 ras. Finally, gap-1(ga133) does not exhibit a vulval phenotype in a single mutant. These results suggest that gap-1 mutations do not allow vulval induction independently of the anchor cell signal, but rather appear to increase the sensitivity of the Pn.p cells towards the anchor cell signal. We were able to define a 9kb DNA fragment of the cosmid T24C12 that contains gap-1, first by using RFLP mapping, then by finding DNA rearrangements associated with gap-1 alleles and finally by transformation rescue experiments. The only predicted open reading frame on this DNA fragment encodes a protein of 629 amino acids that is similar to the Drosophila GTPase activating protein GAP1 (27% identical and 53% similar) and to human GAP1(m) (25% identical and 42% similar). Sequence analysis of strong gap-1 alleles has identified four potential null alleles. The first (ga133) is a 334 bp deletion causing a frameshift after amino acid 163 and the others (n1691, ga14 and ga12) are stop mutations at positions 151, 174 and 182, respectively. All four alleles are predicted to express non-functional proteins because they each lack the putative catalytic domain. GAP proteins inhibit the signaling activity of Ras by stimulating its intrinsic GTPase activity, which results in a decrease in the levels of Ras in the active, GTP-bound form. Thus, GAP-1 may directly inhibit the signaling activity of LET-60 RAS during vulval induction. Interestingly, double mutants containing gap-1(ga133) in combination with mutations that reduce let-23 receptor activity such as let-23(sy1), lin-2(n397), lin-7(e1449) or lin-10(n1390) cause a hyperinduced phenotype in up to 80% of the animals (a gonad-dependent multivulva phenotype). This phenotype is not observed in double mutants containing gap-1(ga133) and mutations in genes that act at other steps in the signaling pathway (Table). These observations are paradoxical since single mutants in either lin-2, lin-7, lin-10 or let-23 have a vulvaless phenotype due to reduced receptor activity but double mutants containing gap-1(ga133) and the same mutations have a hyperinduced phenotype indicating increased signaling activity. One explanation for this reversal of phenotypes, previously proposed by Aroian et al. (Genetics (1991) 128,251), is that let-23 may have two functions; one function is to activate the let-60 ras signaling pathway, and the other function might be to inhibit vulval induction through a pathway that is yet to be determined. In single mutants, the vulvaless phenotype is the result of reduced activation by let-23 whereas in double mutants with gap-1(ga133), the hyperinduced phenotype might be due to reduced inhibition by let-23. genotype % Egl % WT % Hin n gap-1(ga133) 0 100 0 280 lin-3(e1417) 88+/-5 12+/-5 0 189 lin-3(e1417);gap-1(ga133) 42+/-6 8+/-6 0 238 let-23(sy1) 80+/-4 0+/-4 0 427 let-23(sy1);gap-1(ga133) 3+/-2 4+/-5 73+/-6 238 lin-2(n397) 93+/-4 7+/-4 0 185 gap-1(ga133)lin-2(n397) 4+/-3 2+/-6 74+/-6 197 lin-7(e1449) 85+/-4 3+/-4 1+/-1 357 lin-7(e1449);gap-1(ga133) 2+/-2 7+/-5 81+/-5 228 lin-10(n1390) 94+/-3 6+/-3 0 203 lin-10(n1390);gap-1(ga133) 6+/-3 31+/-7 63+/-7 177 sem-5(n2019) 91+/-3 9+/-3 0 292 gap-1(ga133)sem-5(n2019) 41+/-6 59+/-6 0 306 let-60(n1876)* 100 0 0 >200** let-60(n1876);gap-1(ga133)* 14+/-14 86+/-14 0 20 lin-45(n2018ts) @14C 76+/-6 24+/-6 0 186 lin-45(n2018ts);gap-1(ga133) 72+/-5 28+/-5 0 341 @14C * the progeny of n1876/+ animals was scored ** from Beitel et al.,(1990) Nature 348:503