Project Summary Adult digestive epithelia depend on adjacent mesenchyme to sustain intestinal stem cells (ISCs) at the crypt base and drive cell maturation at the villus base. Precisely layered cells that provide redundant and partially overlapping “trophic” factors execute these essential polarized functions. Our lab’s contributions to this emerging understanding include deep characterization of small intestine (SI) and colonic mesenchyme, discovery of potent “trophocytes,” a cellular explanation for the crypt BMP signaling gradient, identification of the authentic source of canonical Wnt ligands, and discovery of niche self-organization. Importantly, work from several groups converges on nearly identical consensus cell populations in mouse and human intestines. Strategically localized fractions of the most abundant cells (which express low PDGFRA), including distinct CD81+ trophocytes, represent notable functional niche elements. In the developing gut mesoderm, PDGFRAlo cells seem to give rise to key structural elements, such as different smooth muscle (SM) compartments and unique intestinal capillaries, before the remaining PDGFRAlo cells generate the ISC niche. Against the backdrop of a complete census of adult mesenchymal cells, these findings pave the way to understand their embryonic origins in modern mechanistic terms. This simple but crucial tissue thus offers opportunities to address a broad, fundamental question in developmental biology: How does an embryonic anlage with limited external cell input achieve and retain its adult form? Because injured intestines must reconstitute the mesenchymal compartment, the answers have important implications for understanding and treating ulcerative and other forms of intestinal damage. Prior investigation of signaling in intestinal development elegantly implicates Hedgehog (Hh, from endoderm) and BMP (from mesoderm) in specifying at least SM and endothelial cells (EC), and possibly other compartments, but how these signals elicit distinct cell fates in ostensibly similar progenitors remains unclear. Discrete cell identities reflect opening and closing of thousands of different cis-regulatory elements (CREs); to deconstruct steps that lie between a mesodermal anlage and the functional tissue into which it develops, we propose to study the chromatin basis of SM (Aim 1) and EC (Aim 2) differentiation. We will map mesenchymal ontogeny rigorously with respect to signature CREs for each resident cell type (Aims 1A and 1B), then investigate in primary fetal cell cultures how Hh activity and BMP inhibition together induce the CRE complement necessary for naïve precursors to undergo SM differentiation (Aim 1C). We then ask how the same signals (albeit likely in different forms or concentrations) induce ECs in similar progenitors (Aim 2A). Finally, we propose studies for mechanistic insight into processes designed for intestinal capillary growth to match, but not exceed, resting tissue demands (Aim 2B). Tog...