Morphogenesis of the male tail tip in C. elegans provides an ideal system to study the integration of sexual, spatial and temporal cues in the regulation of a developmental process. During the L4 stage, the male tail tip cells retract, resulting in the rounded tail of the adult. A master regulator of tail tip retraction, the DM-domain protein DMD-3, is both necessary and sufficient to specify this process (Mason et al., 2008). In WT males,
dmd-3 is expressed in the tail tip starting in early L4 and its expression is quickly extinguished as retraction completes during late L4. Thus, temporal, sexual and spatial signals act to control male tail morphogenesis by regulating
dmd-3 expression. We are studying
lep-5, a novel gene that controls the timing of
dmd-3 and tail tip retraction. In loss-of-function (
fs8) and deletion (
ny10) mutants of
lep-5,
dmd-3 expression and tail tip retraction are delayed.
dmd-3 is not expressed in L4 tail tips, but is expressed in the unretracted tail tips of adults, which are pointed (called Lep, for leptoderan). Males continue tail tip retraction during adulthood, and males and hermaphrodites display supernumerary molts as adults. Similar to other heterochronic genes, the mutant phenotype of
lep-5 is suppressed by transit through the dauer stage. To understand the exact position of
lep-5 in the heterochronic pathway, we are determining the epistatic relationships between it and known heterochronic genes, such as
lin-41 and
let-7. In
lin-41(lf) mutants, tail tip retraction occurs precociously in the L3 and continues throughout L4, resulting in the over-retracted (Ore) adult tail tips (Del Rio-Albrechtsen et al., 2006).
lin-41(lf);
lep-5 double mutants do not have Lep tails, consistent with
lep-5 being upstream of and repressing
lin-41. In support of these data, expression of a
lin-41 transcriptional reporter (which includes the
lin-41 3'UTR) extends into adulthood in the
lep-5(
ny10) background, whereas it shuts off during L4 in WT animals. We are currently testing the relationship between
lep-5 and
let-7. PCR fragments that include a gene with unknown function, H36L18.2, rescues both
lep-5 mutants. Also, injection of RNAi against H36L18.2 leads to a low-penetrant Lep phenotype. Sequencing this region in the
fs8 mutant revealed a G-to-A change close to the SL1 acceptor site. Further experiments are underway to examine the expression of this gene and better understand its role in the heterochronic pathway.