This project seeks to continue activities focused to the elucidation of mechanisms which determine, 1) commitment of foregut endoderm to form the embryonic lung, 2) proliferation and differentiation of distinct subsets for respiratory cells in the developing lung and 3) proliferation and remodeling of the respiratory epithelium following lung injury. This application is based on progress made in the last four years of funding which demonstrated critical, interacting roles of three distinct families of transcription factors, namely TTF-1 (homeodomain), HNF-3beta and HFH-4 (winged helix) and GATA-6 (zinc finger) in respiratory epithelial cell lineage commitment and lung morphogenesis. The present proposal will determine the precise temporal/spatial requirements for two of these genes (HNF-3beta and TTF-1) in formation of the lung and will determine their influence on respiratory epithelial cell differentiation, gene expression and branching morphogenesis. The research plan is based on the successful development of technology for conditional, lung-specific cre-recombinase mediated gene deletion and addition, technology that will enable respiratory epithelial cell lineage analysis and site-specific gene targeting. Genes encoding TTF-1 and HNF-3beta will be subjected to cre-recombinase mediated deletion at precise times during development, inducing cre-recombinase with a doxycycline-controlled lung-specific conditional system. Temporal and spatial effects of recombination mediated deletion of TTF-1 and HNF-3beta on lung morphogenesis will be discerned. Respiratory epithelial cell differentiation and the expression of HNF-3beta and TTF-1 target genes will be assessed in vivo and in vitro. Cre-recombinase and retroviral mediated cell marking of progenitor cells will be used to define a lineage map of the respiratory epithelium in the normal developing lung in vivo. Effects of TTF-1 and HNF-3beta deletion on lung morphogenesis and respiratory epithelial cell differentiation will be discerned prenatally and postnatally. Effects of decreased TTF-1 and HNF-3beta on cell proliferation and differentiation during repair from oxygen injury will be discerned. Potential roles of TTF-1 and HNF-3beta in the pathogenesis of lung malformation and bronchopulmonary dysplasia (BPD) and other forms of chronic lung disease will be sought in human studies.
