Hirschsprung’s disease (HSCR) is a birth defect where the enteric nervous system (ENS) is absent from the distal bowel. Most HSCR patients have reduced activity of RET and many have reduced EDNRB (endothelin receptor 3) activity. Although only the distal colon is aganglionic (missing neurons and glia) in 80% of children with HSCR, proximal bowel regions (and the rest of the GI tract) innervated by enteric neurons harbor the same mutated genes. Since RET and EDNRB are needed for many aspects of ENS development, it seems likely enteric neurons in ganglionic regions of the colon and their connections (hereafter referred to as the ENS connectome) are abnormal. Consistent with this hypothesis, HSCR surgery to remove aganglionic bowel and reconnect “normal bowel” to the anal verge does not alleviate all HSCR symptoms; up to 50% of children have ongoing issues post-surgery including bowel distension, inflammation, explosive diarrhea, blood in the stool, lethargy and poor feeding. The long-term goal of this research project is to determine how mutations in HSCR-related genes affect the entire ENS and contribute to GI tract dysfunction. The short-term goals are: 1) to identify the gene-expression changes downstream of mutations in RET and EDNRB that occur in HSCR patients and in mice in that contain HSCR-relevant mutations, and 2) Perform anatomical and functional studies in HSCR mouse models to determine how these gene defects negatively impact the enteric nervous system. The overall hypothesis guiding these experiments is that for the innervated portion of the colon, different HSCR mutations produce defects in motility resulting from specific changes in communication between unique subsets of neurons (in the myenteric plexus and autonomic nervous system (ANS)), ICC, and glia. Aim 1: Conduct pooled and single cell RNA-Seq analysis on enteric neurons of wild type and HSCR mice models; compare murine data with RNA-Seq analysis from HSCR patients and controls. Aim 2: Determine the effect of HSCR-associated mutations on ENS/ICC/glia communication. Aim 3: Examine extrinsic parasympathetic and sympathetic drive of myenteric neurons, ICC, glia and associated smooth muscle contractions in HSCR mouse models. Impact: Transcriptomics indicate that the different HSCR mutations will negatively affect the function of different cell types in the ENS and ANS (neurons, glia and ICC). The consequence of the involvement of multiple cell types is that patients may appear similar symptomatically, but the underlying cause, and hence appropriate treatment, may be very different. This research program is designed to identify mutation-specific mechanisms of disease as a basis for development of patient-specific treatments.