Timing influences vulval precursor cell (VPC) competence and fate patterning. For example, VPCs respond to LIN-3 produced by the gonad in the L2 stage but are not induced until the L3 stage. LIN-12, the receptor for the lateral signal, is present in the L2 stage, but constitutively active LIN-12 does not specify the 2o vulval fate until the L3 stage. Heterochronic genes mediate many different timing events. In the canonical pathway, the microRNA
lin-4 downregulates its targets, LIN-14 and LIN-28, to allow stage-specific events to occur. Previous work suggested that VPCs in
lin-4(0) mutants are immature, as they express
lin-12 but do not display normal 1o or 2o fates (Euling and Ambros, 1996). Using markers and tools that have become available since then, we have re-examined VPC fates in
lin-4(
e912) null mutants. Our results suggest that there is a defect in lateral signaling. VPCs in
lin-4 respond to the inductive signal, as, a 1o fate marker,
egl-17p::gfp, is expressed in P6.p in a gonad-dependent manner. In addition, the lateral signal is likely to be produced, as
apx-1p::yfp and
lag-2p::yfp are expressed. However,
lin-4 mutants exhibit a phenotype indicative of a failure of LIN-12-mediated lateral signaling: ectopic expression of
egl-17p::gfp and loss of expression of two 2o fate markers,
lin-11p::gfp and
lst-5p::yfp, in P5.p and P7.p. Tissue-specific rescue experiments suggest that
lin-4 functions in the VPCs to promote the 2o fate. Furthermore, loss of
lin-4 can suppress constitutively active mutant forms of LIN-12, including LIN-12(intra). Our results suggest that in
lin-4 mutants, VPCs are not competent to respond to LIN-12 activation or that there is a block downstream of LIN-12 signal transduction. To understand the nature of the block in lateral signaling, we need to know the relevant
lin-4 target(s). Our data suggest that
lin-28 and
hbl-1 are not responsible for the lateral signaling defect in
lin-4(0), because
lin-28(
n719null) or
hbl-1(
ve18) cannot restore the 2o fate marker expression in
lin-4(0). However, the hypomorphic allele
lin-14(
n179) suppresses
lin-4(0), and
lin-14(
n355), a mutation the removes the binding sites for
lin-4, shows a similar phenotype as
lin-4(0). These results indicate that the lateral signaling defect in
lin-4 mutants is primarily due to inappropriate
lin-14 activity. We are currently testing the critical period during which LIN-14 accumulation blocks 2o fate specification. We also plan to do a genetic screen to look for targets or co-factors of LIN-14 that are involved in 2o fate specification.