Theses Doctoral

Regulation of EGFR signal transduction and cell division in quiescent Vulval Precursor Cells during dauer developmental arrest

O'Keeffe, Catherine

Quiescent adult stem cells are important for both tissue maintenance and for responding to stress. C. elegans provides an ideal context to dissect pathways involved in the maintenance of long-term cellular quiescence. In favorable environmental conditions, larvae develop continuously into reproductive adults. In adverse environmental conditions, larvae can undergo an alternative life history and enter dauer diapause, a long-lived state with characteristics that promote survival and dispersal. Animals that enter dauer can survive for months, many times the normal life span of an animal that develops continuously. Entry into dauer interrupts the development of the vulva and is associated with a reprogramming-like event that ensures that Vulval Precursor Cells (VPCs) remain multipotent and quiescent until conditions improve.

In this work, I aim to understand how the pathways that regulate dauer entry dictate cellular outcomes that oppose VPC fate acquisition and cell division.VPC specification is initiated when an inductive EGF signal from the somatic gonad activates EGFR-Ras-ERK signaling in the nearest VPC, P6.p. Using an ERK activity biosensor, we found that EGFR signal transduction is activated in P6.p prior to dauer entry. However, during the molt into dauer, EGFR signal transduction itself is downregulated and ERK activity remains low in P6.p throughout dauer. To understand how the VPCs are maintained as multipotent precursors in dauer larvae, we investigated the level at which negative regulation of EGFR signaling occurs. We found that dauer VPCs are desensitized to both endogenous and ectopic expression of EGF despite the presence and correct localization of the EGFR. A constitutively active allele of Ras, but not the EGFR, was sufficient to increase ERK activity in the VPCs. This suggests that during dauer, regulation of EGFR signal transduction occurs at or above the level of Ras. We conclude that EGFR signaling is opposed within the VPCs themselves at the level of membrane associated events.

Entry into dauer is regulated by Insulin (IIS), TGF-β and Nuclear Hormone Receptor (NHR) signaling pathways. To understand how these pathways might act to block VPC specification and cell division, we characterized mutants acting in either the IIS or NHR pathway that show inappropriate VPC developmental progression in dauer larvae. We found that the phosphatase DAF-18/PTEN, a modulator of IIS, is required to maintain VPC quiescence during dauer. We created an endogenously floxed daf-18 allele and used tissue-specific Cre recombinase drivers to determine the cellular focus of DAF-18/PTEN in regulating VPCs. Our data is consistent with DAF-18/PTEN acting nonautonomously to prevent VPC division and to maintain competence in dauer.

DAF-16/FOXO, the major downstream effector of IIS, and DIN-1S/SHARP, which acts in NHR signaling, were previously implicated in regulation of the VPCs during dauer. We looked at null mutants over time in dauer life history and found that each transcription factor opposes VPC division during distinct stages in dauer development. While DIN-1S/SHARP appears to be required to maintain quiescence at the end of the L2d-dauer molt, DAF-16/FOXO is required to maintain quiescence in dauer itself. This suggests that regulation of the VPCs during dauer life history is dynamic and occurs in phases with each stage having distinct regulatory mechanisms, which is like what has been described for dauer exit. Our research provides insights into robust protective mechanisms that maintain multipotency and quiescence over long periods of time. While the pathways required to enact the dauer program are well defined, the downstream consequences of these pathways on individual or groups of cells are less understood. Future work will aim to link dauer regulating pathways to the downregulation of EGFR signaling in the VPCs.


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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Greenwald, Iva S.
Ph.D., Columbia University
Published Here
December 7, 2022