Relatively little is known about the molecular pathways defining the epithelial progenitor cells of the embryonic and adult lung and their role in lung development, steady state homeostasis and repair after damage caused by environmental and other agents. In this competitive renewal we will build on previous discoveries to obtain new information about basic mechanisms controlling the specification, proliferation, and differentiation of lung epithelial progenitor/stem cells, using the mouse as a model genetic organism. In Aim 1 we will test the hypothesis that the developmental potential of epithelial progenitor cells in the distal tips of the embryonic lung changes in an orderly way over time so that early born descendants give rise to cells in the proximal airways while late born daughters give rise to alveolar cells. For this aim we have already generated two new lines of genetically engineered mice, Id2-GFP and Id2-CreER. These will enable us to follow the developmental fate of distal tip cells in vivo, isolate the cells at different times using flow cytometry, analyze their transcriptome using gene arrays, and test the role of candidate regulatory genes using gain and loss of function experiments. In Aim 2 we will focus on the adult trachea and main bronchi which has a pseudostratified epithelium, as in a large proportion of the human airways. We will test the relative contribution of basal stem cells and secretory (Clara) cells in maintaining this region and in repairing it after injury by exposure to naphthalene or sulfur dioxide. This will be achieved using new K5-CreEr and Scgb1a1-CreEr alleles for fate mapping and gene manipulation. We will also elucidate basic mechanisms regulating the specification, self-renewal and differentiation of basal stem cells, focusing initially on the role of the transcription factor, Sox2. Finally, in a high risk Aim 3, we will test the hypothesis that small non-coding RNAs known as microRNAs can reversibly enhance the self-renewal potential of progenitor cells in the adult lung, either in the steady state or during repair. To do this we will initially inducibly express in basal and Clara cells the miR17-92 gene that encodes a conserved cluster of six microRNAs. We have already shown that this cluster enhances the proliferation of epithelial progenitors in the embryonic lung at the expense of their differentiation and we will now test this in adult progenitors. We will then test the activity of individual miR17-92 members or combinations of them. In the long term we will ask whether this activity can be obtained by direct application of double stranded small RNAs linked to cell-specific aptamers. PROJECT NARRATIVE. This work will increase our understanding of basic mechanisms regulating the embryonic development of the lung and the ability of the epithelial cells to undergo repair in the adult. In the long term the work may lead to potential therapies or predictive assays for pathological conditions related to defects in the supply, proliferation or differentiation of progenitor/stem cells in the human lung.