Worm Breeder's Gazette 10(2): 81
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.
The let-23 locus was originally defined by three larval lethal alleles [Herman, 1980; Sigurdson et al., 1984] and one cold-sensitive semi-lethal vulvaless allele, n1045 [Ferguson & Horvitz, 1985]. Our interest in let-23 was sparked by the observation that n1045 causes a novel phenotype at 25 C -- hyperinduction of the vulva, in which four or even five of the vulval precursor cells (VPCs) are induced. (In wild type, three of six VPCs are induced; in Vulvaless (Vul) animals, zero of six are induced.) This Hyperinduced or Hin phenotype requires a signal from the anchor cell and thus is distinct from the classic Multivulva (Muv) phenotype. That let-23 could be mutated to give opposite phenotypes -- n1045 at 15 C is Vul, but at 25 C is Hin -- led us to believe that let-23 plays a critical role in the specification of VPC fate in response the inductive signal from the anchor cell. We are therefore genetically and molecularly analyzing the let-23 locus. Our first genetic screen to obtain new let-23 alleles made use of the fact that let-23 is the only 'Determination' gene known to suppress the Muv phenotype of lin-15(n309) [Ferguson et al., 1987]. We therefore EMS-mutagenized lin-15(n309) and looked in the F2 for phenotypic revertants that result in a Vulvaless (Vul) phenotype when crossed away from n309. Using this screen, we isolated sy1, which maps to the let-23 locus and fails to complement n1045 for the Vul phenotype. 93% of homozygous sy1 hermaphrodites are egglaying- defective, a result of the Vul phenotype. All sy1 homozygotes are viable, and sy1 complements all known let-23 alleles for the Lethal phenotype. From additional lin-15 reversion screens, we have obtained another Vul mutation linked to let-23.n1045 is recessive and results in a Vul phenotype in trans to a deficiency for the locus at all temperatures, suggesting that n1045 results in a decreased level of let-23 activity at all temperatures. There are two reasonable hypotheses for the Hin phenotype. n1045 might be a neomorph that is dose-dependent. Alternatively, although levels are down with respect to wild type, n1045 has more activity at 25 C than at 15 C, and a particular level of let-23 activity might result in a Hin phenotype due to a bizarre dose-response curve. Complementation tests with sy1 have provided evidence favoring the hypothesis that while a severe decrease in let-23 activity results in a Vul phenotype, a less severe decrease in activity results in a Hin phenotype. The key observation is that the Hin phenotype can be recreated without using n1045, using instead sy1 and a lethal allele, mn224. In particular, 20% of sy1/mn224 heterozygotes are Hin; the remaining 80% are wild-type. We interpret the Hin phenotype as resulting from a failure to induce the 'Lateral Inhibitory Signal' [LIS] described in the last newsletter [Sternberg WBG 10(1)]. Specifically, we propose that in wild type the LIS is activated in VPCs in response to let-23, which in turn is activated by the signal from the anchor cell, but at a higher level of let-23 activity than required for generating vulval sublineages. In a Hin animal, we argue, VPCs are induced to generate vulval sublineages but fail to inhibit their neighbors from also participating in vulval development; hence more than three VPCs can generate vulval sublineages. Since sy1/Df hermaphrodites are highly penetrant for the Vul phenotype but are nonetheless viable, we screened for new EMS-induced mutations that fail to complement sy1. We have isolated fifteen new let-23 alleles after screening 20,000 F1 chromosomes. Each mutation is tightly linked to let-23 and fails to complement sy1 and n1045 for the Vul phenotype. Two of these alleles, sy10 and sy12, are incompletely penetrant lethals, causing 80% and 90% of larvae to die, respectively. The surviving worms show two new phenotypes: all hermaphrodites are sterile, and all males have short, crumpled spicules. The cellular bases of these phenotypes are under investigation because we are dying to know if let-23 is involved in the specification of other cell fates besides the VPCs and P11/P12. We are pursuing two approaches to clone the let-23 locus: we are screening for transposon-induced alleles that fail to complement sy1, and will use the over 20 existing alleles to identify RFLPs within a chromosomal walk