We propose to study the early regulatory events that determine when and where the foregut endoderm is specified to become lung and to form buds. During early development, endodermal cells from the ventral foregut start a gene expression program that demarcates the territories of the future thyroid, lung, liver and pancreas. Once the endoderm is specified, bud formation is initiated. Little is known about when the endoderm is determined to become lung, which transcription factors direct lung specification prior to budding, and how diffusible signals from the mesoderm regulate the spatial and temporal differentiation of the lung primordium. We and others have shown that signaling by retinoic acid (RA) and fibroblast growth factor (FGF) is essential for primary lung bud morphogenesis. However, it is unclear whether RA and FGF regulate endoderm specification during development or how their signals interact with other signaling pathways active in the foregut. Our goals are to identify and characterize transcription factors involved in lung specification before the lung buds (Aim 1), to study the role of RA and FGF signaling in foregut gene expression and lung bud induction (Aim 2), to study interactions between the biological relevance of RA, FGF, and the factors identified in Aim 1 (Aim 3). To accomplish these goals we will define the molecular boundaries of the foregut lung field using laser capture microdissection, and quantitative real-time PCR. We will use cDNA arrays to identify transcription factors that characterize the region of the prospective lung. In vivo footprinting will be used to determine whether lung gene promoters are primed with transcription factors prior to initiation of organogenesis. These studies will be coupled with a new in vitro system that allows analysis and experimental manipulation of the foregut and lung bud during its initial stages of formation. This system will be used to assess the role that retinoids and fibroblast growth factors play in the earliest steps of lung formation.
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