Regulatory Mechanism for Cellular Dedifferentiation in C. elegans Germline

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Throughout their lifespan, stem cells make several critical fate decisions: the initial decision between self-renewal (remaining pluripotent) and differentiation, and then the subsequent decision between whether to continue in the differentiation path or to return to an undifferentiated state, i.e., to engage in a form of dedifferentiation. Defects in cellular machineries governing differentiation decisions can result in loss of a specific cell type, overpopulation of undifferentiated cells, or overt growth of cancer cells. Over the past decade, the molecular mechanisms related to the initial decision have been described in major model organisms. However, an understanding of the underlying determinants of the subsequent decisions is vague at best. The long-term research objectives are to elucidate the mechanisms that cause differentiating cells to return to an undifferentiated state, and to develop an animal model to interrogate these mechanisms in the living organism. A molecular understanding of this cellular event holds promise for the development of powerful tools in stem cell biology, regenerative medicine, and oncotherapy. The project also includes a component in which traditionally underserved populations of students in North Carolina (minority high school students, community college and undergraduate students) will be actively involved in the basic research.This project addresses three key questions concerning the fundamental molecular controls of a known dedifferentiation mechanism: (1) How do differentiating cells maintain their state or return to undifferentiated cells? (2) What regulatory network is involved in this cellular process in vivo? (3) Is aberrant regulation of this network associated with oncogenesis? Using the nematode C. elegans germline as a model system, the investigators have previously reported that PUF-8 (an RNA-binding protein) and LIP-1 (a dual-specificity phosphatase) are required to (a) maintain the meiosis (differentiating state) of spermatocytes, and (b) prevent their dedifferentiation into mitotic cells (undifferentiating state) by inhibiting MPK-1 (an ERK homolog) signaling. Further studies found that the differentiation/dedifferentiation decision is regulated in part by cell cycle regulators (CYB-1 and X-ray) and mRNA decay factors (CGH-1 and CAR-1). In this project, the researchers will investigate two hypotheses: In the absence of PUF-8 and LIP-1, (1) spermatocyte dedifferentiation requires cell cycle arrest and the activation of cell survival pathways (Objective 1), and (2) inactivation of mRNA decay factors induces the ectopic translation of mRNAs that initiate spermatocyte dedifferentiation (Objective 2). To accomplish this goal, the researchers will take multidisciplinary approaches, including RNAi screening, genetic/functional analysis, CRISPR/Cas9-based genome editing, X-ray-based biophysical analysis, and high-resolution polysome profiling. Findings from the study will not only elucidate the fundamental mechanisms of the differentiation/dedifferentiation decision in vivo, but they will also provide a novel working platform for the identification of therapeutic targets for dedifferentiation-mediated cellular regeneration and tumorigenesis.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.