Understanding how innexins direct stem cell niche morphology

Duke University, Durham NC

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Abstract

Background: Stem cells require support from a niche, which provides regulatory and housekeeping functions. In the C. elegans gonad, the stem cell niche is the distal tip cell (DTC). The germline stem cells induce their own enwrapment by long cellular processes of the DTC. When the gonad basement membrane ruptures, germ cells escape and spill into the body cavity. Escaped germ cells induce enwrapment by body wall muscle to form an ectopic niche. Broad, long-term objective: This system is ideal for studying a property of the niche that is not well understood?reciprocal signaling from stem cells to the niche. Preliminary results show that innexin proteins are necessary for DTC processes to form, and for ectopic enwrapment by muscle. The objective of this proposal is to discover how innexins and other genes mediate this interaction in endogenous and ectopic niches. Specific aims: Aim 1 of the study is to determine the precise role of innexins in normal germ cell-niche interactions. Aim 2 will determine their role in the ectopic niche. Aim 3 is an RNAi screen for genes necessary for the elaboration of DTC processes that also function in ectopic enwrapment. Method: Innexin genes will be endogenously tagged and mutated using CRISPR/ Cas9 genome-editing. Combined with traditional transgenic reporters for the relevant cells types, these genome-edited worms will allow for high resolution confocal microscopy in live animals to see the interactions between germ cells and the niche. RNAi knockdown of genes at precise developmental times and existing mutants will be used to analyze gene function. C. elegans are perfectly suited to genetic screens, and the RNAi screen will be performed according to established protocols. Health-relatedness: Enwrapment of stem cells by their niches and gap junctions between stem cells and their niches are both widespread but not previously known to be related. The proposed research will determine the mechanism by which gap junctions function in stem cells to induce enwrapment by the DTC and ectopic niche, with implications for better understanding stem cell- niche interactions in many contexts, including regenerative therapies and cancer metastasis. Gap junctions are known to be misregulated in cancer. The ectopic muscle niche constitutes a new model for the formation of secondary tumors seeded by cancer stem cells during cancer metastasis.