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Roles of LIN-28 and LIN-46 in the developmental timing pathway of Caenorabditis elegans

by
Bhaskar Vadla
Bachelor of Technology, 2003
Anna University, India

Thesis Advisor: Eric Moss, Ph.D.

Cell and Molecular Biology Program

Science Center, Room 290

Monday, June 13, 2011
12 pm


Abstract

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 wormís post-embryonic 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 animalís 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, which specifies the second larval stage specific seam cell fates. In mammals, LIN28 is thought to regulate stem-cell differentiation by repressing the let-7 family of microRNAs expression post-transcriptionally. Whether LIN-28 in worms also represses let-7 microRNAs production, and if it does, the consequence of this regulatory connection in the temporal cell fate specification has not been addressed yet. 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. Using a Taqman microRNA qRT-PCR assay, we observed high let-7 levels at the L1 and L2 stages, in lin-28 mutants, suggesting LIN-28 represses the accumulation of let-7 early in C. elegans. The miR-48, -84 and -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-28ís activity in specifying L2 cell fates, indicating that LIN-28 has a let-7-independent activity in controlling cell fates. However, LIN-28 does require let-7 for the later cell fates. By assessing stage-specific seam cell behavior and heterochronic phenotypes manifested, coupled with the analyses of male tail morphogenesis, we 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-7ís role a stage earlier than predicted in the temporal specification of cell fates. Furthermore, genetic epistasis analyses indicate that lin-41 and hbl-1, two down-stream targets of let-7, control different stage-specific cell fates, and that let-7 primary role is to repress lin-41 for normal L3 to L4 cell fate transition. Additionally, based on reporter expression analyses, we identified a relevant downstream target of LIN-28 which is hbl-1, a putative 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 not acting in the pathway per se, in negatively regulating hbl-1 expression and activity. These results are in agreement with genetic studies that lin-46 and lin-28 act opposingly in regulating a downstream target, which might be hbl-1.


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