? Using pituitary pro-opiomelanocortin (POMC) expression as an entry point, the original K08 proposal aims to study genetic and molecular mechanisms underlying corticotroph early ontogeny. Transparent zebrafish embryos expressing green fluorescent protein (GFP) specifically targeted to pituitary corticotroph lineage driven by Pomc gene promoter allows one to follow the dynamic process of pituitary ontogeny in live animals. Ongoing studies in the K08 award isolate mutants using ethylnitrosourea (ENU) mutagenesis of transgenic zebrafish harboring multiple fluorescent tissues including pituitary POMC cells, then classify and categorize these mutants for future systematic studies that aim to identify molecular and genetic mechanisms governing POMC lineage development using gene mapping/cloning and in vivo developmental characterization. Due to known genetic parallels between fish and human, such study will ultimately lead to further insights for human pituitary disease development. This supplemental R03 application focuses on one mutant line generated in pilot ENU mutagenesis screens. Line 1023-5 shows overall normal development at early stages, however, aberrant hypothalamic and pituitary POMC expression pattern at 48 hours post fertilization by whole mount in situ hybridization, thus representing a potentially unique model for studying molecular and genetic regulations of hypothalamic-pituitary/corticotroph axis. Based on preliminary studies, this R03 application will test a single hypothesis: Defective hypothalamic development in mutant 1023-5 (caused by single gene mutation) elicits altered extrinsic signals on pituitary lineage maintenance leading to aberrant POMC cell growth after terminal differentiation. There are two Specific Aims: 1) To identify a single gene defect leading to aberrant POMC phenotype of line 1023-5 using positional cloning; and 2) To characterize hypothalamic and pituitary/corticotroph ontogeny in mutant 1023-5 using RNA whole mount in situ hybridization and in vivo time-lapse confocal microscopy analysis. ? ? Molecular and genetic determinants mediating hypothalamic regulation of corticotroph development are yet to be understood. This study is complimentary to the primary goals of the original K08 proposal. The proposed experiments will validate the experimental approach that will continue with future studies of mutants generated from the K08 award. More importantly, data generated from this study will lead to additional hypotheses enabling development of a R01 application focusing on the function of identified putative gene and associated pathway regulating development of hypothalamus and pituitary. ? ?
