PROJECT SUMMARY Intestinal secretory (Sec: goblet (Gob), enteroendocrine (EE), Paneth (Pan), and tuft) cells serve vital digestive, metabolic, barrier, and other physiologic functions. Intestinal stem cells (ISCs) replenish both absorptive and Sec epithelial cells, but the origins of Sec diversity and census are largely unknown. Because it has been difficult to isolate and characterize intermediate precursor cell states, much of the current understanding of the mouse and human Sec lineages rests on mouse genetic studies. On morphologic and functional grounds, the four mature Sec cell types are considered distinct and their origins trace to a common ATOH1-expressing crypt progenitor (Sec-Pro), although some mouse EE and tuft –but not Gob or Pan– cells arise in the absence of this Sec-specifying transcription factor (TF). Cell fates are generally dictated by cell- specific cis-regulatory elements (CREs – mainly enhancers) that drive particular transcriptional programs under the direction of cell-restricted TFs. To understand how primitive Sec-Pro execute subsequent CRE and cell fate decisions, we have developed two parallel and powerful experimental systems: (1) Capture of labeled early mouse Sec derivatives in vivo for deep characterization of gene and enhancer activity at single- cell resolution, and (2) Differentiation of 2D human ISC-like cells in vitro along the full Sec lineage. Both models yield large numbers of multi- and bi-potential precursors, hence allowing original mechanistic insights into the corresponding transcriptional and CRE states. Our three Specific Aims integrate findings from the two parallel models to decipher the epigenetic basis and regulatory TF logic of mouse and human Sec cell differentiation. Aim 1 uses mRNA and open-chromatin profiles of precursor and descendant Sec cells within a differentiation continuum to characterize the salient features of specific intermediate states, map previously unknown TF-driven transitions, distinguish cell-autonomous from -extrinsic factors, and thus derive a unifying model for mammalian Sec lineage diversity. Considered in the light of scattered published findings, our preliminary data suggest that Gob and Pan are not genuinely distinct cell types, but different versions of a versatile Sec end-product where specific local signals act on the same complement of open chromatin to drive Gob or Pan features. Aim 2 applies a suite of in vitro and in vivo experiments to test specific hypotheses that epidermal growth factor (EGF) and especially canonical Wnt signaling favor the Pan state, while bone morphogenetic protein (BMP) signaling drives the Gob state. We will examine how alternative recruitment of Gob- or Pan-selective CREs by specific signaling pathways might underlie these fluid cell states. Aim 3 uses gain- and loss-of-function strategies to address previously challenging questions about TF-driven specification of human Sec, especially EE and tuft, cells. Overall, a particular stren...