The heterochronic genes of C. elegans are global temporal regulators that control the relative timing and sequence of diverse events during post-embryonic development. One of these events is the terminal differentiation of the lateral hypodermal "seam" cells, an event that occurs during the final (L4) molt in wild-type animals. During the L4 molt, seam cells exit the cell cycle, fuse and synthesize a morphologically distinct adult cuticle. Mutations in heterochronic genes advance or retard the timing of hypodermal terminal differentiation, resulting in larvae with adult-type hypodermis or adults with larval-type hypodermis. Hypomorphic mutations of
lin-57, as well as post-embryonic RNAi depletion, cause seam cell fusion and adult cuticle synthesis one stage early, during the L3 molt. Cloning of
lin-57 revealed that it is allelic to
hbl-1, a gene previously identified by Fay et al. (1999 Developmental Biology 205: 240-253) based on its sequence homology to Drosophila hunchback.
lin-57::gfp fusions are down-regulated during the L4 stage and this down-regulation is mediated by the
lin-57 3'UTR. Examination of the
lin-57 3'UTR reveals binding sites for multiple microRNAs, including
let-7, and the temporal expression pattern of the
let-7 microRNA (late L3 through adult) is consistent with it playing a role in
lin-57 down-regulation. However,
let-7 is not absolutely required for
lin-57 inactivation as
lin-57::gfp fusions are still down-regulated in a
let-7(null) background. One possibility is that other gene products function redundantly with
let-7 to ensure proper
lin-57 expression. The recent identification of a large family of microRNAs in C. elegans (Lau et al. 2001 Science 294:858-862, Lee and Ambros, 2001 Science 294:862-864) provides a possible explanation for these observations. Three potential
let-7 paralogs have been found (Lau et al. 2001 and these abstracts). All three have sequence similarity to
let-7 and are expressed with the same temporal specificity as
let-7. Conceivably, some or all of these
let-7 homologues could participate in
lin-57 down-regulation. Examination of the
lin-57 3'UTR suggests that it might serve as a landing pad for multiple miRNAs that act in concert to inactivate
lin-57 expression during the late L3-L4 stage. Mutations in these miRNAs are needed in order to understand their role in
lin-57 regulation and other developmental processes. Two
let-7 homologues are situated in tandem (~1.7 kb apart) on chromosome V in the interval where
lin-58, a heterochronic gene identified as a
lin-4 suppressor, maps.
lin-58 has a weak precocious phenotype and interacts genetically with the heterochronic gene
lin-42. To test whether one of these miRNAs is allelic to
lin-57, we sequenced in the region of the microRNAs. Each
lin-58 allele contained a single, independent, point mutation in a palindromic sequence in the 1.7 kb region between these two miRNAs (~200 bp 5' to the downstream RNA). One possible scenario is that these mutations disrupt a repressor binding site such that one or both miRNAs are expressed prematurely, resulting in precocious inactivation of target genes. Consistent with this idea, one
lin-58 allele is weakly dominant. Northern blot analysis of RNA from synchronized populations of
lin-58 mutants is underway to further test this hypothesis. When expressed from an extrachromosomal array, these RNAs cause diverse developmental defects, including hypodermal abnormalities. Preliminary analysis indicates that transgenic arrays can be used to recapitulate the
lin-42 genetic interaction.