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Unifying Mechanisms of Developmental Timing: Genetic and Molecular Analysis of the Molting Cycle Timer of Caenorhabditis elegans

  • Author(s): Monsalve, Gabriela Carolina
  • Advisor(s): Frand, Alison R
  • et al.
Abstract

Chronobiologists focus on understanding at least two ways of regulating the time of life: biological clocks that anticipate environmental conditions, and temporal switches that regulate sequential events. Of the former, the best-characterized biological clocks regulate circadian rhythms in metazoans. These self sustaining oscillators involve transcriptional-translational feedback loops that affect the physiology of the organism. In contrast, progression through development requires the proper temporal coordination of factors that control sequential events. However, the timing mechanisms that coordinate both cyclical and successive events in development are not generally well understood.

The molting cycle of C. elegans is an example of both a periodic and sequential developmental program. Molting of the exoskeleton involves the coordinated synthesis, secretion, and assembly of a new skeleton, as well as degradation of the old one. Quiescent behaviors also accompany the molting cycle. Worms molt four times, approximately once every eight hours when cultivated at room temperature. The molts are coordinated with successive transitions in the temporal fates of epidermal stem cells, which are programmed by genes in the heterochronic regulatory network. Every molt is a critical developmental transition and mis-coordination of the either the sequential or reiterative events can lead to larval arrest and death.

This thesis describes how the C. elegans heterochronic gene lin-42a, homologous to the mammalian circadian clock protein PERIOD, is required for cyclical and sequential progression of development. The oscillatory expression of lin-42a in the epidermis peaks during the molts. Inactivation of lin-42a results in arrhythmic molts and continuously abnormal epidermal stem cell dynamics. In contrast, forced expression of lin-42a leads to anachronistic larval molts and lethargy in adults. These results suggest that rising and falling levels of LIN-42A allow the start and completion, respectively, of larval molts. Ancillary factors of the molting timer likely include the conserved nuclear hormone receptors NHR-23/RORα and NHR-25/SF1, and the family of let-7 microRNAs. Thus, interconnected positive and negative regulatory interactions among LIN-42A, NHR-23 and -25, and let-7 may drive transitions through each molt. I propose that these factors regulate molting cycles in much the same way that PERIOD-based oscillators drive rhythmic behaviors and metabolic processes in mature mammals.

The LIN-42-based timer may synchronize the periodic molts with fluctuations in external or internal conditions, in much the same way that circadian clocks synchronize rhythmic biological processes of higher metazoans with recurrent fluctuations in zeitgebers. To identify novel factors that regulate these molting cues, we designed a forward genetic screen to identify novel factors that regulate the timing of the molt. I identified that the LRP-2/Megalin receptor is required for

the cessation of larval molting cycles. The lrp-2 transcripts are detected predominately in the anterior head neurons, suggesting that that major site of action of LRP-2 is in the nervous system. LRP-2 may function to sequester neuroendocrine cues from other tissues to control the terminal fate of larval molts.

By analyzing the genetic and molecular mechanisms of that regulate the molting cycle, we anticipate the discovery of the broader mechanisms that couple biological periodicity and rapid development in higher organisms.

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