PROJECT 2 | PROJECT SUMMARY Failure to separate the fetal foregut into distinct trachea and esophagus (TE) can result in a spectrum of life- threatening tracheoesophageal defects (TEDs). The genetic etiology of TEDs is poorly understood, and risk variants are known in only 12% of TED patients. Even in cases where the causative mutations are known such as in developmental transcription factors (TFs), how they result in TEDs is unclear because up until recently the mechanisms of TE morphogenesis were ill defined. We made significant progress in the previous award. Using a combination of Xenopus and mouse embryology, we elucidated the conserved cellular events driving TE morphogenesis and showed that disrupting any step can result in TED-like phenotypes. We used Xenopus CRISPR screens to validate potential TED-causing variants from patient genome sequences. One major discovery was that downstream of Hedgehog (HH) signaling and developmental TFs, endosome-mediated membrane remodeling is required to separate the foregut into distinct TE tubes. Consistent with this, genome sequencing of 185 TED patients from Project 1 revealed an enrichment of damaging variants in membrane/vesicular trafficking genes including the endocytic adaptor ITSN1, which in preliminary data we demonstrated is required for Xenopus TE morphogenesis. The goal of the CLEAR consortium Project 2 is to define the developmental basis of TED with cellular resolution in animal models. We will: Determine the mechanism by which endosome trafficking regulates TE morphogenesis (Aim1). Test the hypothesis that developmental TFs control the cell-specific expression of effector proteins such as endosome trafficking machinery or cargo (Aim2, in collaboration with Project 3). Assess the pathogenicity of patient variants in animals testing the provocative hypothesis that endosomeopathies might be a major cause of TEDs (Aim3).