There is currently a critical need for a genetically tractable model system to rigorously delineate the molecular function of the Lin-28 pluripotency factor in stem cell metabolism. Defining Lin-28's underlying molecular role in controlling adult stem cell behavior currently appears nearly intractable using an in vivo vertebrate model. Lack of such a model will slow the pace of development of regenerative therapies and medical interventions that target the human Lin-28 pathway. Accomplishing the long-term goal of determining the conserved, Lin-28-related, RNA based mechanism that controls stem cell expansion in adult tissues in response to environmental cues will provide a molecular framework for prospective interventions designed to modulate human stem cell behaviors underlying tissue homeostasis and regeneration. Preliminary work has identified the fly as a powerful and relevant model for human Lin-28 function in adult stem cells, since fly and human Lin-28 are both enriched in pluripotent cell types and share similar subcellular expression profiles, binding partners, and function in stem cell expansion. The objective of this exploratory R21 proposal is to use adult fly intestinal stem cells (ISCs) as an in vivo model to identify new mRNAs, proteins and metabolites involved in the conserved process of stem cell expansion. This will be accomplished by high throughput genetic analysis at single cell resolution in ISC lineages of top candidates identified from genome-wide transcriptomic, proteomic, and metabolomics screens - a rigorous and rapid approach that is uniquely suited to the strengths of the fly system and not currently possible in other stem cell model systems. Subsequent work will confirm the functional conservation of these molecules in mouse lin-28a and lin-28b knockouts through collaboration with vertebrate colleagues. To this end, the two aims proposed here are to 1) identify functional mRNA targets and protein co-factors of Lin-28 in adult ISCs, and 2) identify metabolic pathways involved in ISC symmetric self-renewal. Successful completion of these two aims will molecularly delineate a conserved pathway that promotes tissue regeneration in mammals as well as develop a model system in which to test the efficiency of therapeutic prototypes that control tissue homeostasis and regeneration by targeting this pathway.