FOXL1 positive telocytes in intestinal development and homeostasis Abstract The intestinal stem cell niche plays an essential role in enabling the self-renewal of the columnar gut epithelium, a process that occurs continuously throughout life. Over the past five years we have established that subepithelial fibroblasts resembling telocytes that express the DNA-binding transcription factor Foxl1 are a critical component of the intestinal stem cell niche. Removal of Wnt signals emanating from Foxl1+ cells or elimination of these specialized cells themselves causes cessation of epithelial proliferation, crypt failure, and rapid death of mutant mice. In addition, Foxl1+ cells demarcate critical signaling centers during intestinal development, and Foxl1 itself is required for villus development in fetal life. Based on these exciting findings, we have built several crucial tools to further explore the function of Foxl1+ telocytes and fetal telocyte precursors in three independent but interrelated specific aims. In specific aim 1, we will ablate critical components of the planar cell polarity pathway in Foxl1+ telocyte precursors to determine their contribution to villus formation during the epithelial transition in midgestation, based on our findings from single cell RNAseq analysis that these factors are highly enriched in the Foxl1+ telocyte progenitor populations during villus formation, and the fact that villification is abnormal in Foxl1 null embryos. In specific aim 2, we will test the hypothesis that the closely linked Foxl1 and Foxf1 genes function cooperatively in telocytes or their progenitors to control both gut development and the function of the adult stem cell niche. We will employ an innovative ‘double floxed’ mutant allele to ablate both genes simultaneously in the Foxl1+ lineage and analyze fetal gut development and adult tissue homeostasis using histology, immuno-staining, and molecular assays. Finally, based on our discovery that GLP-2R, the receptor for the intestinal mitogen GLP- 2, and its downstream signal IGF1 are highly enriched in Foxl1+ telocytes, we will test the hypothesis that telocytes are the GLP-2 target cell using inducible conditional gene ablation of both Glp2r and Igf1 and evaluate GLP-2 action in multiple paradigms. Together, these linked yet independent aims will dramatically increase our understanding of intestinal telocyte biology, a cell type we have shown to be a critical component of the intestinal stem cell niche.