The microRNA let-7 is a critical regulator of developmental timing events at the larval-to-adult transition in C. elegans. Recently, microRNAs with sequence similarity to let-7 have been identified. We find that doubly mutant animals lacking the let-7 family microRNA genes mir-48 and mir-84 exhibit retarded molting behavior and retarded adult gene expression in the hypodermis. Triply mutant animals lacking mir-48, mir-84, and mir-241 exhibit repetition of L2-stage events in addition to retarded adult-stage events. mir-48, mir-84, and mir-241 function together to control the L2-to-L3 transition, likely by base pairing to complementary sites in the hbl-1 3' UTR and downregulating hbl-1 activity. Genetic analysis indicates that mir-48, mir-84, and mir-241 specify the timing of the L2-to-L3 transition in parallel to the heterochronic genes lin-28 and lin-46. These results indicate that let-7 family microRNAs function in combination to affect both early and late developmental timing decisions.

MicroRNAs (miRNAs), a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs.

With increasing time after ovulation, mammalian eggs become more sensitive to agonists of activation in vitro or may undergo spontaneous activation in vivo. We have tested the hypothesis that postovulatory eggs undergo time-dependent cell cycle and cytoplasmic changes that result in a partially activated state, accounting for their time-dependent susceptibility to activate. In vivo changes in key activation markers in mouse eggs were quantified at 13, 16, and 22 h post-hCG (1, 4, and 10 h postovulation). Spontaneous activation was first detected at 16 h, with a 20-25% decrease in the activities of histone H1 and mitogen-activated protein (MAP) kinases and with 3% of eggs undergoing both anaphase onset and a partial loss of cortical granules. By 22 h, more than 60% of eggs were in anaphase, H1 and MAP kinase activities had decreased 40-50%, the extent of zona pellucida modification had increased, and proteins normally synthesized after fertilization had appeared. Pronuclear formation in response to inositol 1,4,5-trisphosphate injection increased dramatically from 10% at 13 h to about 40% and 90% at 16 h and 22 h, respectively. The partial decreases (less than those after fertilization) in H1 and MAP kinase activities provide a likely biochemical basis for the increased sensitivity of eggs to agonists, seen over time, that results in pronuclear formation. Also, all of these time-dependent changes caution against the use of mouse eggs > 16 h after hCG administration in studying the mechanism of normal fertilization and have implications for animal and human in vitro fertilization.

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