We would like to understand how Pn.p cell fates are determined. At least 18 genes have already been identified that specify Pn.p cell fates, and we would like to expand this genetic pathway by identifying new genes. In particular, we wanted to identify genes that interact with
lin-10, and so we isolated mutations that suppress the
lin-10 vulvaless phenotype. The screen for
lin-10 suppressors is powerful because
lin-10 animals do not have a vulva and thus do not lay eggs, whereas one suppressed animal lays many eggs. Thus, mutants are easily isolated by picking eggs laid by the F2 generation of mutagenized
lin-10 animals. We have identified over 60 suppressors of the
lin-10(
n1390) vulvaless phenotype after screening the equivalent of 150,000 EMS-mutagenized haploid genomes. One mutation is a gain-of- function allele of
lin-34 that has a semi-dominant multivulva phenotype (G. Beitel, personal communication). Twenty-seven of the remaining suppressors have been analyzed, and they define five new genes (termed suv for suppressor of the
lin-10 vulvaless phenotype):
suv-1(12 alleles),
suv-2(7 alleles),
suv-3(4 alleles),
suv-4(2 alleles) and
suv-5(2 alleles).
suv-1 and
suv-2 map on LGX;
suv-1 maps very close to
dpy-3, while
suv-2 maps between
sdc-2 and
sma-5.
suv-1, which has been studied in the most detail, is interesting because it interacts with many other vulval determination genes.
suv-1(
n1329) suppresses the vulvaless phenotype of
lin-2(
e1309),
lin-3(
n378),
lin-7(
e1413),
lin-10(
e1439,
n1299,
n1390) and
let-23(
n1045), while it enhances the multivulva phenotype of
lin-15(
n309,
n765,
n767). Thus,
suv-1(
n1329) increases the expression of vulval cell lineages (1 and 2 ) in both vulvaless and multivulva mutants. The fact that
suv-1 interacts with all of the determination genes tested indicates that
suv-1 is also involved in determining Pn.p cell fates. However, the
suv-1(
n1329) mutation results in a wild-type phenotype by itself, indicating that mutations such as this cannot be isolated directly and must rather be isolated in a second level genetic screen, e.g. as lin- 10 suppressors. Using an RNAse protection assay, we found that
lin-10 transcripts were five times more abundant in mixed stage RNA from
suv-1 animals than from wild-type animals (using
act-1 as a control), indicating that
suv-1 is involved in regulating
lin-10 expression, either directly or indirectly. Increased levels of
lin-10 expression can partly explain how
suv-1 acts as a suppressor, since overexpression of a partial loss-of-function
lin-10 allele could restore gene activity. However,
suv-1(
n1329) suppresses all three of the
lin-10 alleles that were tested (all of which are candidates for null alleles), suggesting that
suv-1(
n1329) must affect something else in addition to increasing
lin-10 expression. One possibility is that
suv-1 also regulates the expression of other determination genes, such as
lin-2,
lin-3,
lin-7,
let-23 or
lin-15; the misregulation of these other determination genes in
suv-1 mutants may be able to bypass the functional requirement for
lin-10 gene activity. This possibility can be tested once molecular probes for these other genes have been isolated.