Roles of LIN-28 and LIN-46 in the Developmental Timing Pathway of Caenorabditis elegans

Bhaskar Vadla, Rowan University

Awarded by University of Medicine and Dentistry of New Jersey (prior to becoming part of Rowan University in 2013)


Cell fates specification over time is of paramount importance during development. The heterochronic pathway of C. elegans comprises a hierarchical gene regulatory pathway that specifies time-dependent stage-specific cell fate choices of hypodermal stem-cell like cells, termed seam cells, during its larval development. This pathway consists of at least five microRNAs and more than twelve protein coding genes that control a succession of cell fate decisions during the worm’s postembryonic development. MicroRNAs comprise a class of short non-coding RNAs that regulate genes crucial for timing cell fate decisions. Specifically, lin-28 and lin-46 are two members of this pathway, which have important roles in regulating a microRNA-mediated switch to control the second-larval-stage (L2) to subsequent cell fate transitions during the animal’s development. However, their exact functions in specifying cell fates, and their precise roles in the C. elegans developmental timing pathway are mostly unknown.

LIN-28 is an evolutionarily conserved RNA binding protein that was first identified in worms, and which specifies the second larval stage specific seam cell fates. In mammals, LIN28(A) is thought to regulate stem-cell differentiation by repressing the let-7 family of microRNAs post-transcriptionally. Whether LIN-28 in worms also represses let-7 microRNA production, and if it does, the consequence of this regulatory connection in the temporal cell fate specification, has not been addressed. Here, using a yeast three hybrid assay we show that worm LIN-28, similar to its mammalian homolog, specifically binds to precursor-microRNAs of four out of nine let-7 family microRNAs: let-7, miR-48, miR-84 and miR-241, which have known roles in the pathway. In lin-28 mutants, using a Taqman microRNA qRT-PCR assay, I have observed high let-7 levels at the L1 and L2 stages, suggesting that LIN-28 represses the accumulation of let-7 early in C. elegans. The miR-48, miR-84 and miR-241 levels are mostly unaffected in lin-28 mutants, suggesting that let-7 is the most relevant microRNA target of LIN-28. Because LIN-28 controls the L2 cell fates, we tested whether LIN-28 requires let-7 and its three sisters to control these cell-fates. Genetic and epistasis analyses reveal that that let-7 and its three relatives are dispensable for lin-28’s activity in specifying L2 cell fates, indicating that LIN-28 has a let-7-independent activity. However, LIN-28 does require let-7 to control later cell fates, specifically for the L3 to L4 cell fate transition. By assessing stage-specific seam cell behavior and heterochronic phenotypes, coupled with the analyses of male tail morphogenesis, I identified that LIN-28 regulation of let-7 is relevant for the third-larval-stage (L3) to fourth-larval-stage (L4) cell fate transition; placing let-7’s role a stage earlier than proposed in the temporal specification of cell fates. Furthermore, genetic epistasis analyses indicate that lin-41 and hbl-1, two downstream targets of let-7, control different stage-specific cell fates, and that let-7’s primary role is to repress lin-41 for the normal L3 to L4 cell fate transition. Additionally, based on reporter expression analyses, I identified a relevant downstream target of LIN-28, the HBL-1 transcription factor.

lin-46, a genetic suppressor of lin-28, is a periodically expressed factor and is one of the key players controlling the L2 to L3 cell fate transition. However, how LIN-46 acts to control cell fates is not known. Fine time scale temperature shift experiments indicate that LIN-46 activity is required two hours prior to the L2 molt for the L2 to L3 cell fate transition. Genetic interaction studies and reporter expression analyses suggest that LIN-46 acts together with the microRNA pathway, but does not act in the pathway per se, to negatively regulate hbl-1 expression and activity. These results are in agreement with genetic studies showing that lin-46 and lin-28 act opposingly to regulate a downstream target. Whether this target is hbl-1 and if it is, then how exactly LIN-46 inhibits its activity is not known.