Summary: Modeling EA/TEF In Human PSC-Derived Embryonic Tissues During development of the vertebrate embryo, a common foregut tube gives rise to the esophagus and respiratory tract and this involves an array of complex molecular and morphological processes. The dorsal foregut tube forms the esophagus and the ventral domain forms the respiratory tract, and failure to do so can result in tracheaesophageal birth defects such as esophageal atresia and tracheoesophageal fistula (EA/TEF). As discussed in project 2, much is known about how Wnt and BMP signaling promote a respiratory fate by activation of the transcription factor Nkx2.1. In contrast, little is known about pro-esophageal factors. Mouse and human studies demonstrate that the HMG-box transcription factor Sox2 is involved in segregation of the esophageal and respiratory lineages, however whether Sox2 promotes an esophageal fate or acts predominantly to repress respiratory-inducing pathways the dorsal foregut is unclear. We hypothesize that both mechanisms are involved in normal esophageal development. In humans, most genes that cause EA/TEF remain unidentified. However, heterozygous mutations in SOX2 can cause of EA and TEF, which is in contrast to mice with heterozygous loss of Sox2, which are normal. Complete loss of Sox2 from the foregut endoderm of mouse embryos results in esophageal agenesis, however Sox2 is also expressed during development of the enteric nervous system (ENS) of the esophagus. Given that patients with EA can have motility defects, we hypothesize some EA-associated genes may affect ENS development. However, a study of how EA-associated mutations differentially affect the epithelium and/or ENS of the esophagus has never been done in any species, let alone humans. We propose several novel PSC- based approaches to study how Sox2 and other EA-associated genes impact Human esophagus specification, epithelial morphogenesis, and functional innervation using human pluripotent stem cell-derived esophageal organoids with an enteric nervous system. In this project we aim to identify the mechanisms underlying esophageal specification and development in humans by first focusing on the key esophageal factor Sox2. We hypothesize that SOX2 acts both to repress the respiratory lineage, and promote an esophageal fate via an unidentified gene regulatory network. We will use a human PSC-derived foregut model in combination with SOX2 gain- and loss- of-function to identify a respiratory GRN that is repressed by SOX2 and an esophageal GRN that is SOX2- dependant. Conversely we will determine if NKX2.1 represses the esophageal fate. We will take advantage of the expandable nature of human foregut cultures to identify direct transcriptional targ...