Understanding the molecular mechanisms by which specific cell types develop from a common primordium in the anterior pituitary gland represents a basic question in molecular and developmental biology. Under this Grant, we analyzed the roles of Pit-1, cloned and characterized Prop-1 as the hypomorphic allele causing the Ames genetic dwarfism, and identified disease alleles in human pituitary combined hormone deficiency syndrome; identified the signaling molecules from ventral diencephalon and opposing signaling gradients that dictated the positional commitments of organ and cell-type determination events, and provided the initial evidence of the roles of induced transcription factors as the """"""""molecular memory"""""""" of the transient signaling gradients. In this competitive renewal application, we propose to investigate the role of a series of transcription factors, including the members of the Six, Pax, GATA and Pitx families, and of specific co-activators, in nearly and late developmental events. The roles of specific protein-protein interactions, and co-activators and co-repressors, on their actions will be considered, and we propose to identify artificial target genes that mediate their biological actions. The role of signaling factors, including Shh, BMP, Wnt and retinoic acid, will be further explored using in vivo and in vitro approaches. The mechanism of Pit-1 lineage determination will be explored, with experiments directed at understanding the activation of the early Pit-1 gene enhancer, regulation of the Pit-1 lineage by Prop-1, and the mechanisms of control by Pit-1 of the terminal differentiation of three pituitary cell types. The allosteric effects of cognate Pit-1 DNA binding sites on these events will be explored structurally, and in vitro, and the role of co-repressor complex assembly will be investigated. This research proposal extends the approach used over the current grant period, which has been the most successful in our 22 years under this grant, combining biochemical and genetic approaches to investigate potentially important regulatory proteins, and systematically exploring the functional role of the novel proteins. We believe that these experimental approaches will provide insights into the intriguing problems of organ and cell-type differentiation, and cell-specific patterns of gene activation, with clear implications for human diseases that are caused by abnormalities of these developmental events.
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