Members of the
let-7 microRNA (miRNA) family are well-known for their role in the heterochronic pathway, which times cell proliferation and differentiation in the hypodermal seam cells. In addition to retarded hypodermal phenotypes,
let-7 mutants show lethal vulva bursting. The underlying cause of vulva bursting and the developmental role(s) of the
let-7 family in the vulva are largely unknown. As
let-60/RAS, a major regulator of vulva cell fates, is a well-established
let-7 target, we sought to determine
let-7's role in vulva precursor cell (VPC) fate specification. Whereas
let-60 deregulation would be predicted to cause mis-specification, we unexpectedly find that early vulva cell fate specification at the L3 stage is executed normally in
let-7 or
mir-84 animals as assessed by the 1 deg and 2 deg cell fate markers,
egl-17::cfp and
lin-11::gfp. To resolve this issue, we examined post-transcriptional regulation of the
let-60 3'UTR using a single-copy integrated reporter. We observed repression of this reporter in the hypodermis and in the anchor cell at the L4 stage, but not in the VPCs in earlier stages. Furthermore, expression of a functional
let-60 rescue construct without a 3'UTR results in normal number of apparently wild-type vulvae, indicating that
let-60 is not a relevant
let-7 target in the vulva. To determine whether
let-7 activity in the vulva is necessary at all, we expressed a
let-7 rescue fragment in selected tissues from single-copy integrated transgenes in the
let-7(null) background. Exclusively hypodermal expression of
let-7 alone was not sufficient, but required presence of
let-7 in VPCs to rescue lethality of the
let-7 mutation. Indeed, we find high expression of
mir-48/84 and
let-7 in the vulva at the L4 stage as assessed by single-copy integrated promoter::GFP reporters. Taken together, these results suggest that the vulva morphogenesis defects and bursting observed in
let-7 and
let-7 family mutants are caused by altered gene expression during vulva morphogenesis at the L4 stage. We are currently characterizing in detail vulva morphogenesis in
let-7 family mutants using time lapse imaging and testing which genes must be down-regulated by
let-7 to ensure formation of a wild-type vulva.