Isolation and Analysis of
lin-3 Suppressors Jing Liu and Paul W. Sternberg HHMI and Division of Biology 156-29 Caltech, Pasadena, CA 91125
lin-3 is the vulval inducing signal made by the anchor cell during C. elegans vulval development. It is a novel member of the epidermal growth factor (EGF) family and encodes a transmembrane protein with one extracellular EGF repeat. The extracellular region is predicted to be processed to form a soluble signal, which is sufficient to induce the vulva. Reduction of
lin-3 activity causes a Vulvaless (Vul) phenotype, overexpression of
lin-3 in transgenic animals results in a Multivulva (Muv) phenotype with a large abnormal vulva and smaller pseudo vulvae. Iin-3 expression in the somatic gonad is restricted to the anchor cell, beginning at the stage when vulva induction initiates. Using genetic screens, we are looking for genes that are required for proper expression and function of
lin-3, for example, transcription factors, proteins required for
lin-3 processing into a secreted signal, and cofactors. We may also recover new components of the vulva induction pathway downstream of
let-23 in these screens. syIsl is a chromosomal integrant of a
lin-3 transgene and has a dominant and fully penetrant Muv phenotype. We screened for suppressors of syIsl by EMS mutagenesis. 23 independent suppressor mutants were obtained in a screen of 56,000 haploid genomes. These suppressors fall into several classes: 1).
sy272 and 12 other alleles define a locus on LGV between
dpy-11 and
rol-3. They suppress syIsl to close to wild type and do not confer a vulva defect in an otherwise wild type background. The suppression of syIsl is temperature- sensitive.
sy272 is recessive when syIsl is homozygous but dominant when syIsl is heterozygous.
sy272 does not suppress
lin-15(1f), suggesting it acts prior to
let-23. 2).
sy274 and 2 other alleles define a locus on LGX between
dpy-8 and dpy- 6. They also suppress syIsl to close to wild type and are recessive. The suppression is also temperature sensitive. 3). M3.13 defines a locus on LGX. It is a recessive suppressor of syIsl and suppresses it to close to wild type. It also has interactions with both
sy272 snd
sy274. 4).
sy286 confers a highly penetrant Vul phenotype in syIsl background. It also has male spicule defect, moderate sterility and slight lethality. In more than 50% of the animals P7.p and P8.p remsin undivided. 5). M6.13 also confers a Vul phenotype in syIsl background. 6). We recovered two strains with
lin-12(d) like phenotype, that is, five to six pseudovulvae and no anchor cell. We also recovered one
let-23 allele. We did not recover deletion of syIsl, and this suggests that integrated transgene can be used for suppressor screen. We also EMS mutagenized
lin-3(
n378)/lin-3(nlO59), a highly penetrant Vul strain. From a screen of 30,000 haploid genomes, 7 independent suppressors were recovered. They suppress the Vul phenotype to various extent. Some of them are dominant and may be linked to the original
lin-3 mutation. At least three are extragenic and recessive.