SUMMARY The enteric nervous system (ENS) is a complex network of neural crest-derived neurons and glia responsible for regulating key intestinal functions including motility, sensation, and secretion. Unfortunately, the ENS is frequently subject to injury leading to motor and other abnormalities. Often, this leads to debilitating disorders with few available treatment options. Excitingly, there is now mounting evidence of postnatal ENS injury-induced neurogenesis. Importantly, through work on adult animal models we have shown that Schwann cells (SC) can enter the gut alongside the extrinsic nerves and then differentiate into specific neuronal and glial subtypes (enteric neuro-gliogenesis). Thus, SC provide an unexpected source of cells to repopulate injured neurons and enteric glia. Furthermore, we have found that microbiome manipulation is a powerful method to induce Schwann cell-mediated enteric neuro-gliogenesis leading to functional recovery of the ENS and that this is mediated via the serotonin 5HT4 pathway. However, many aspects of postnatal ENS neuro-gliogenesis are not fully understood, including the functional impact of the neuro-gliogenesis from the SC, and the therapeutic potential for 5HT4 manipulation in human disease aiming for an enhanced SC-induced neuro-glial regeneration. Building on our published and preliminary results from mice and humans, our overarching hypothesis is that SC migrating into the gut from the gut’s extrinsic innervation are an important source for postnatal enteric neuro-gliogenesis, and that this ENS regenerative response is regulated by the microbiome via 5HT4. To test this novel hypothesis, we propose: Aim 1 will characterize postnatal SC-derived enteric neuro- gliogenesis after microbiome eradication/re-establishment using inducible, fluorescently labeled mice. We will also determine the functional effects of SC neuro-gliogenesis through extensive in vivo assays of motility and permeability and ex vivo characterization of cellular function using calcium imaging. Additionally, we will determine the functional effect of eliminating the SC entering the gut using a diphtheria toxin mouse model. In Aim 2, we will use two knockout mouse lines: (1) P0CreER/tdT::Tph1-/- and (2) P0CreER/tdT::Tph2-/- to determine the source of serotonin and the possible clinical applications of our findings by evaluating the SC response to a 5HT4 agonist, prucalopride. We will also identify specific metabolomic and transcriptomic profiles of the GI tract (mucosal and myenteric compartments). Finally in Aim 3, We will determine components of human microbiome-host crosstalk regulating SC-derived enteric neuro-gliogenesis in patients with slow colonic transit/dysmotility including the effect of 5HT4 agonists (i.e., prucalopride, tegaserod) on the ENS integrity/neuro- glial regeneration and function and determine metagenomic profiles in our patient cohort. Last, we will perform fecal transplants from these subjects into germ-free (GF) m...