During vulval induction,
lin-15 acts as a negative regulator of vulval cell fate specification. In
lin-15 hermaphrodites, the vulval precursor cells that normally have a hypodermal fate instead have vulval cell fates, causing the worm to have ectopic vulval differentiation and the multivulva (Muv) phenotype. This phenotype is inductive-signal independent, and has been shown to be cell non- autonomous [Herman & Hedgecock (1990) Nature, 348,169-171]. Iin-15 has three different classes of mutations: two silent classes of mutations, A and B, that can contribute to a synthetic (syn) Muv phenotype, as well as mutations that are defective in both A and B function and cause a visible Muv phenotype [Ferguson & Horvitz (1989) Genetics, 123, 109-121]. Although there are other syn Muv genes,
lin-15 is the only known locus which has class A, class B, and visible Muv alleles. Using a visible Muv allele, we tried an Fl non-complementation screen to obtain null alleles of
lin-16 and were unsuccessful after screening 30, 000 Fls. The molecular analysis of
lin-15 provides a hint as to why our screen failed. We have rescued both class A and class B
lin-15 mutations with a 15 kb plasmid. Deletions (>3 kb) of the
lin-15 region are associated with all four N2 derived EMS visible Muv alleles. Furthermore, only one of nine EMS-derived
lin-15 syn Muv alleles shows a small 100 bp deletion whereas the other eight do not show detectable polymorphisms. Also, when we screened for new syn Muv alleles of lin- 15 starting with a syn Muv of the opposite class, our lab has observed that new
lin-15 syn Muv alleles are obtained at about 1/3,600, a frequency consistent with their being loss-of-function alleles. The surprising correlation of the visible Muv phenotype with large deletions, along with the frequencies of obtaining
lin-16 syn Muv alleles, leads us to propose the Two Hit Model. We believe that the
lin-16 A and B syn Muv alleles are defective in independently mutable functional regions. To get a visible Muv phenotype, an animal must either have two hits (one A and one B) or a deletion that removes both the A and the B region. We have identified two classes of cDNAs corresponding to the
lin-15 rescuing region and are currently working on further molecular analysis of the locus.