Project summary Congenital anomalies of the trachea, including tracheoesophageal fistula (TEF), are common, affecting around 1:3000 live births. TEF is defined as the inappropriate maintenance of a connection between the trachea and esophagus, and arises as a result of a failure of the complete separation of these structures from their common origin, the foregut endoderm tube. Normally, induction of respiratory identity in the ventral foregut endoderm and initiation of lung bud outgrowth is followed immediately by a morphogenetic separation of the ventral trachea from the dorsal esophagus. Despite the common occurrence of TEF, our understanding of the pathways that control this developmental process is incomplete, and almost no knowledge exists regarding the molecular effectors of separation. Further, the cell biological mechanisms by which the trachea separates from the esophagus are currently not known. Our proposal incorporates genome-wide approaches including RNA sequencing and chromatin immunoprecipitation sequencing, and integrates them with mouse genetics and imaging approaches to better understand how control of tracheal cell fate specification is coupled to the cell biological drivers of separation. This work will provide new insights into how early tracheal development is regulated, improve our understanding of TEF, and provide a resource for future regenerative and tissue engineering approaches that require a detailed understanding of early tracheal gene regulatory networks.