Throughout development, precise regulation of transcriptional timing and dosage is necessary for coordinating sequential cell fate specification and developmental checkpoints across multiple cell types and tissues. Previous reports from our lab indicate that
lin-42, the C. elegans homolog of the Period gene implicated in controlling circadian rhythms, negatively regulates the transcriptional output of cyclically expressed heterochronic microRNAs (including
lin-4, miR-48, miR-241, miR-84 and
let-7). Loss of
lin-42 function results in dramatic alterations in the post-embryonic cell lineages where late-staged developmental events are skipped due to the inappropriate and early down-regulation of the mRNA targets of heterochronic miRNAs. Because C. elegans lacks the direct orthologs of the core clock transcription factors (Clock and Bmal) that Period functions through, the mechanism by which LIN-42 regulates the transcriptional output of heterochronic miRNAs, and therefore temporal patterning, are unknown. Here, we demonstrate that LIN-42 regulates heterochronic miRNA transcriptional dosage through direct physical interactions with multiple conserved nuclear hormone receptors that are expressed in the hypodermis. More specifically, through yeast-2-hybrid, biochemical, and in vivo imaging of transcriptional reporters, we demonstrate that the LIN-42 protein modulates the transcriptional activity of the REV-ERb ortholog, NHR-85, and that NHR-85 also binds to the defined regulatory regions of multiple LIN-42 target genes. Consistent with a function interaction between LIN-42 and NHR-85, loss-of-function alleles of
nhr-85 suppresses precocious developmental phenotypes of
lin-42 mutants. We present evidence that the rudimentary developmental clock composed of NHR-85 and LIN-42 functions in the hypodermis to orchestrate the cynical transcription of temporal cell fate specifying microRNAs. Specifically, we demonstrate that LIN-42 auto-regulates its own expression as the expression of and NHR-85 target,
lin-4, in hypodermal cell types. Together our results establish the negative arm of the developmental molecular clock and demonstrate the mechanism of how LIN-42 regulates the transcriptional output of heterochronic miRNAs required to establish sequential cell fates and temporal patterning during post-embryonic development.