lin-31 is required for the normal specification of vulval fates (Miller et al, Genes & Dev, 1993; 7(6):933-47). A
lin-31::gfp transgene is expressed in vulva precursor cells (Pn.p) cells beginning in the L2, and is down regulated once the Pn.p cells adopt their fates (Patrick Tan and Stuart Kim, personal comm.). We recently identified a 5.7Kb
lin-14 genomic DNA sequence,
lin-14R, which rescues
lin-14(
n179ts) precocious vulval and lateral hypodermal defects when expressed under the control of a
col-10 promoter. We constructed a
lin-31::
lin-14R transcriptional fusion to express
lin-14R specifically in Pn.p cells. Transgenic animals carrying the
lin-31::
lin-14R transgene are egg-laying-defective and exhibit vulval cell lineage defects like that of
lin-14(gf) or
lin-4(lf) mutants, presumably due to the expression of functional LIN-14 specifically in the vulval cell lineage.
lin-31::lin-14Rd, a derivative of
lin-31::
lin-14R in which 500bp containing Lin-4 RNA binding sequences was removed from
lin-14R 3'UTR, causes a more severe retarded vulva phenotype than
lin-31::
lin-14R. This is presumably a result of continuous expression independent of down-regulation by
lin-4. A strain (VT775) containing an integrated
lin-31::lin-14Rd transgenic array exhibits nearly 100% vul phenotype. Unlike the vulval defect of other heterochronic mutants, the
lin-31::lin-14Rd vulval phenotype is not suppressed by post-dauer development, probably as a result of post-dauer activity of the
lin-31 promoter. A gfp fusion derivative of
lin-31::lin-14Rd in which GFP is inserted into the carboxy end of LIN-14 results in similar vulva-specific retarded development, indicating that this LIN-14::GFP fusion protein is functional. Preliminary results show that in
lin-31::lin-14Rd::gfp lines retarded Pn.p cells express strong GFP up to the late L4. Further study will focus on: 1) analyzing vulva cell lineages in
lin-31::lin-14Rd::gfp lines; 2) testing the dependence of the
lin-31::lin-14Rd vul phenotype on other genes of the vulval and heterochronic gene pathways; and 3) genetic screening for the revertants from VT775 to identify genes acting downstream of
lin-14 in the vulval cell cycle and developmental pathways.