The anterior lobe of the pituitary releases hormones that are critical for developmental and physiological functions. During embryogenesis, the actions of transcription factors govern the establishment of the hormone-secreting pituitary cell lineages. Heritable mutations in these regulatory genes cause combined pituitary hormone deficiency diseases in children. However, the molecular defects involved in the majority of pituitary disorders remain unknown. LHX3, a LIM homeodomain transcription factor, is essential for pituitary development and function. Although mutations in the human LHX3 gene cause a severe endocrine disease that features compound hormone deficiencies, the biochemical mechanism by which the LHX3 gene exerts its critical functions is not understood. The human LHX3 gene encodes protein isoforms with distinct expression patterns and gene regulatory activities. These isoforms are LHX3a, LHX3b, and a novel protein, M2-LHX3. To test the hypothesis that the human LHX3 gene produces isoforms with unique functions in pituitary development, the protein domains required for the function of the LHX3 isoforms will be characterized and partner proteins that mediate or modulate LHX3 activity by interacting with these domains will be identified. The functions of the LHX3a, LHX3b, and M2-LHX3 proteins will be analyzed by overexpressing each isoform in specific pituitary cell types during development in transgenic mice. In addition, the transcriptional mechanisms that generate the human LHX3a and LHX3b transcripts will be determined using in vitro and transgenic animal approaches. Further, to test the in vivo functions of LHX3a and LHX3b, gene-targeting techniques will be used to selectively prevent their individual expression in mice. These experiments will provide an increased understanding of the genetic pathways that control pituitary organogenesis and will, therefore, improve our ability to treat pituitary disease. Identified proteins and gene regulatory regions will provide candidates for pituitary diseases of unknown etiology. Finally, these studies will demonstrate how single genes can produce protein isoforms with different activities, providing an example of how mammals have evolved mechanisms to increase the proteome-encoding capacity of their genomes.
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