Feedback between a retinoid-related nuclear receptor and the <i>let-7</i> microRNAs controls the pace and number of molting cycles in <i>C. elegans</i>.

Abstract

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying timing mechanisms are not well understood. <i>Caenorhabditis elegans</i> undergoes four molts at regular 8 to 10 hour intervals. The pace of the cycle is governed by PERIOD/<i>lin-42</i> and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the <i>let-7</i> family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian <u>r</u>etinoid-related <u>o</u>rphan <u>r</u>eceptors (RORs), and the <i>let-7</i> family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in <i>nhr-23</i> knockdowns and accelerated in <i>let-7(-)</i> mutants, but timed similarly in <i>let-7(-) nhr-23(-)</i> double mutants and wild-type animals. NHR-23 binds response elements (ROREs) in the <i>let-7</i> promoter and activates transcription. In turn, <i>let-</i>7 dampens <i>nhr-23</i> expression across development via a complementary <i>let-7</i>-binding site (LCS) in the <i>nhr-23</i> 3' UTR. The molecular interactions between NHR-23 and <i>let-7</i> hold true for other <i>let-7</i> family microRNAs. Either derepression of <i>nhr-23</i> transcripts by LCS deletion or high gene dosage of <i>nhr-23</i> leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and <i>let-7</i> also coregulate scores of genes required for execution of the molts, including <i>lin-42</i>. In addition, ROREs and LCSs isolated from mammalian <i>ROR</i> and <i>let-7</i> genes function in <i>C. elegans</i>, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network, possibly by functioning as a cycle counter.