The trachea and esophagus (TE) arise from a single foregut tube in early fetal development. Defects in TE morphogenesis result in a spectrum of life-threatening congenital tracheo-esophageal birth defects (TEDs) that prevent proper breathing or feeding in newborns. The goal of this project is to determine the molecular and cellular basis of TEDs using animal models. Corrected surgically in the neonatal period, TEDs are often associated with long-term co-morbidity. Occurring in ~1:3500 births, the etiology of TEDs is poorly understood. Although evidence indicates a major genetic component, known mutations in 14 genes account for only 12% of patients with esophageal atresia and/or tracheoesophageal fistula (EA/TEF) [1], while the genetic basis of more rare and lethal tracheal atresia (TA) is unknown. Sporadic mutations in ~25 additional genes have been associated with EA/TEF patients, but these remain to be validated. Mouse has proven to be effective for modeling TEDs, and indicates a key role for the Hedgehog (HH) and BMP pathways, with mutants exhibiting defects similar to human patient. Despite this progress there are a number of limitations in the field. Mouse is a relatively low throughput model and only a few of candidate mutations from patients have been validated to date. Second, while HH and BMP are implicated TE morphogenesis the cellular mechanisms they regulate, to control separation of the foregut tube into esophagus and trachea are unknown. This is in part because these events occur early in fetal development when internally developing mouse embryos are challenging to manipulate and visualize. In preliminary data we have established Xenopus embryos as an innovative high-throughput model to complement mouse genetics, and have begun to identify novel and conserved cellular mechanisms controlling TE morphogenesis. These studies lead us to hypothesize that HH and BMP interact to regulate the cellular processes of TE morphogenesis and that mutations in these pathways result in a spectrum of phenotypes that model human TEDs. This project will define the molecular and cellular mechanisms of TE development, define the structural basis of TEDs and test putative TED-causing mutations from patients (project-1). Ultimately this will improve diagnosis, enhance patient care, and inform strategies to generate TE tissue from human pluripotent stem cells (hPSCs) (project-3). Aim 1 Characterize the cellular mechanisms of TE morphogenesis in animals. Aim 2 Determine how defects in HH-Gli and BMP-Sox2 pathways disrupt TE morphogenesis. Aim 3 Validate candidate TED-causing mutations in Xenopus and mouse.