Regulation of stem cell fate by FOXO and RNA binding proteins

A fundamental gap exists in the understanding of how developmental pathways are regulated to maintain stem cell multipotency during extended periods of quiescence, or non-division. The FOXO family of transcription factors are key regulators of stem cell maintenance and quiescence. However, the mechanisms by which FOXO proteins impact developmental pathways to control cell fate are poorly understood. This application capitalizes on the power of the C. elegans system to address the mechanisms by which the single FOXO ortholog, daf-16, regulates conserved developmental pathways to preserve stem cell multipotency during quiescence. To model stem cell quiescence, we will use the quiescent dauer larva stage, adopted midway through development in response to adverse environmental conditions. This approach is innovative because the C. elegans system allows us to study quiescent, multipotent cells in vivo at single cell resolution, complementing mammalian studies. The long-term goal of this lab is to decipher the mechanisms that promote multipotency during dauer. Epidermal seam cells, the stem cell model, are multipotent and undergo a characteristic pattern of self-renewing cell divisions at each larval stage until differentiating at adulthood. During dauer, seam cells are quiescent and active mechanisms maintain multipotency. Preliminary data establish that during dauer, FOXO/daf-16 blocks adult cell fate by positively regulating the expression of three genes that encode RNA-binding proteins (RBPs). The orthologs of these RBPs regulate the proliferation and function of stem and progenitor cells in flies and mammals. The objective of this application is to unravel the mechanisms by which FOXO/daf-16 acts via RBPs to regulate adult cell fate during the quiescent dauer stage. Three specific aims are proposed to meet this objective. 1) Determine the genetic relationship between FOXO/daf-16 and RBPs. Loss-of-function and gain-of-function experiments will establish the regulation of RBPs by FOXO/daf-16, a novel mechanism to control cell fate. 2) Identify direct RBP targets that block adult cell fate during quiescence. Direct mRNA targets of RBPs will be identified by iCLIP. Functional testing will determine which targets are involved in the regulation of seam cell fate. Together these experiments will elucidate the connection between FOXO/daf-16-regulated RBPs and adult cell fate. 3) Dissect the transcriptional regulation of an adult cell fate marker during quiescence. Preliminary data establish that during dauer, FOXO/daf-16 and the three RBPs block expression of a transcriptional reporter of an adult-specific gene, widely used to mark adult cell fate. Promoter dissection and functional testing of candidate transcription factors will decipher the quiescence-specific regulation of a key adult cell fate marker. The proposed work is significant because it will illuminate how the regulation of developmental pathways is coordinated with the regulation of quiescence in multipotent cells.