Project Summary/Abstract Abnormal lung development results in a variety of congenital respiratory diseases contributing to neonatal morbidity and mortality. Although BMP signaling is known to be important in lung organogenesis, understanding of BMP in lung development and pulmonary diseases is incomplete. In particular, BMP signaling in developing lung mesenchyme has not been studied due to embryonic lethality in mice with global abrogation of BMP pathway and the lack of genetic tools to target lung mesenchyme. Using our new Tbx4 lung enhancer- driven loxP/Cre system, lung mesenchymal BMP receptor IA (Bmpr1a) has been specifically deleted. The knockout mice have abnormal airway branching and cystic malformation, which resembles the pathology of congenital pulmonary airway malformation seen in human patients. Deficiencies of airway smooth muscle cells and subepithelial elastin fibers, abnormal mesenchymal expression of Bmp ligands, as well as airway epithelial perturbation of Sox2-Sox9 progenitor axis are found in the cystic lungs of the Bmpr1a knockout mice. In addition, we found that Bmpr1a-mediated Smad-independent signaling regulated airway smooth muscle cell growth and elastin expression in vitro and in vivo. Thus, we hypothesize that mesenchymal Bmpr1a-mediated Smad-independent signaling is essential for airway smooth muscle cell differentiation and subepithelial elastin production as well as suppression of Bmp ligand expression. Combined disruption of these processes results in abnormal airway development and subsequent prenatal airway cystic malformation. Two specific aims are proposed: (1) To determine the pathogenic mechanisms by which defective mesenchymal BMP signaling causes congenital lung malformation through disruption of the airway smooth muscle and subepithelial elastin layer. Mesenchymal Bmpr1a will be deleted in embryonic mouse lung. The lung mesenchymal abnormality and its contribution to airway cystic malformation will be determined from histologic structure to cellular and molecular changes. The underlying molecular mechanisms from BMP intracellular signaling (Smad- independent pathway) to decreased Myocd expression/SMC differentiation and deficiency of subepithelial elastin production will then be studied using both in vivo and in vitro approaches. (2) To determine the mechanisms by which mesenchymal deficiency of Bmpr1a indirectly results in abnormal airway epithelial growth and overall airway abnormality. The airway epithelial abnormality resulting from mesenchymal Bmpr1a deletion will be analyzed. The mechanisms by which mesenchymal deficiency of Bmpr1a signaling results in abnormal mesenchymal-epithelial crosstalk through mesenchymal misexpression of Bmp ligands will be studied. The role of the resultant airway epithelial Sox2+ progenitor deficiency and Sox9+ progenitor expansion in contributing to the lung abnormality will also be investigated.