The genes of the heterochronic pathway regulate temporal patterning during post-embryonic development. Specifically, they direct when cell-fate programs appropriate to specific stages are initiated in their respective tissues. Interestingly, many of the heterochronic genes are themselves regulated post-transcriptionally by microRNAs (miRNA), namely
lin-4 and
let-7, via sites in the 3'UTRs of their mRNAs. We have previously identified
hbl-1, the Drosophila hunchback (hb) homologue, as a heterochronic gene. Like
lin-41,
hbl-1 regulates the larval to adult transition and is a likely target of miRNA regulation. The
hbl-1 3'UTR is necessary and sufficient to down-regulate reporter gene during development and at least two miRNAs (
lin-4 and
let-7) are required for that down-regulation. Saliently, multiple elements in the
hbl-1 3'UTR show complementarity to known heterochronic miRNAs and to other temporally regulated miRNAs. Many of these elements are conserved in C. briggsae, underscoring their probable relevance. We are currently investigating the interaction between
hbl-1 and
lin-41, each of which can genetically enhance the other's heterochronic phenotypes.
lin-41 is a homologue of Drosophila Brain Tumor; Brain Tumor, Nanos, and Pumilio, form a protein complex that regulates hb expression through NREs (Nanos Response Element) in the 3'UTR. There are conserved, potential NRE sites in the
hbl-1 3'UTR, suggesting that LIN-41 may interact with the
hbl-1 mRNA. Interestingly, preliminary genetic evidence suggests that one C. elegans Pumilio homologue,
puf-9, is a negative regulator of
hbl-1. We are also examining the mechanism by which
hbl-1 expression is post-transcriptionally regulated. Other heterochronic targets of miRNA regulation show constant mRNA levels during development;
hbl-1 is unusual because mRNA levels change over time, implying that
hbl-1 may be regulated at the level of mRNA stability or degradation. We are determining whether the change in mRNA levels is miRNA-dependent. Our work supports the probability that there are conserved genetic and mechanistic similarities between hb and
hbl-1 regulation. miRNAs may have an heretofore unrealized role in hb regulation; indeed, we have found potential complementary elements to multiple Drosophila miRNAs in the hb 3'UTR. Our work will shed light on how expression of hb family members is post-transcriptionally regulated, whether by miRNAs, proteins, or both.