Project Summary/Abstract This goal of this project is to identify the mechanisms by which Hedgehog/Gli signaling regulates tracheal development and how disruptions in this process can lead to perinatal tracheal defects. Errors in tracheal development may range from tracheal atresia and tracheoesophageal (TE) clefts, where the fetal foregut tube fails to separate properly, to tracheomalacia due to impaired tracheal chondrogenesis. The etiology of tracheal defects is poorly understood and new diagnoses and therapies are needed to address these relatively common yet potentially lethal defects. Human patient exome sequencing as well as murine mutant models have begun to identify a few developmental pathways that regulate tracheal development, including the Hedgehog/Gli pathway. For example, in murine models a total loss of Gli2 and Gli3 results in a complete failure of tracheal development, while some compound heterozygous Gli2;Gli3 mutants exhibit a TE cleft with tracheomalacia. In addition, our preliminary data suggests that transgenic expression of a Gli3 mutant that mimics the genetic lesion found in human Pallister-Hall syndrome patients also leads to impaired TE separation and virtually absent tracheal cartilage. The specific aims of this project are to identify Gli target genes in the developing trachea, to determine how Gli regulates TE separation and tracheal cartilage differentiation, and to model the mechanism of human tracheal defects. Preliminary RNA-seq studies of control and mutant foreguts have begun to define Gli targets during TE development. I will test the hypothesis that these candidate Gli target genes are misexpressed and/or mislocalized during TE development in Gli mutants. Our preliminary further data suggest that Wnt and BMP pathways act downstream of HH/Gli, and we will use a combination of mouse genetics and fetal foregut cultures to test the hypothesis that defective Wnt and BMP signaling is responsible for the defects in tracheal chondrogenesis. The results of these experiments will inform the basis of tracheal defects in human patients and as well as guide differentiation of tracheal iPSCs towards new therapeutic possibilities. This project will also provide valuable training in current clinical diagnosis and treatment strategies for these patients. !