Genetics of Pituitary Development in Zebrafish

描述(由申请人提供)： 以垂体促阿片黑素原(POMC)的表达为切入点，最初的K08方案旨在研究促肾上腺皮质激素早期个体发育的遗传和分子机制。在pomc基因启动子的驱动下，表达针对垂体促肾上腺皮质激素谱系的绿色荧光蛋白(GFP)的透明斑马鱼胚胎可以跟踪活体动物垂体个体发育的动态过程。正在进行的K08大奖研究利用乙基亚硝胺(ENU)诱变含有多种荧光组织(包括垂体POMC细胞)的转基因斑马鱼，然后对这些突变体进行分类和分类，用于未来的系统研究，旨在通过基因定位/克隆和体内发育特征确定支配POMC谱系发育的分子和遗传机制。由于已知鱼类和人类之间的遗传相似之处，这种研究最终将导致对人类脑下垂体疾病发展的进一步洞察。这个补充的R03应用程序专注于在试点ENU突变筛选中产生的一个突变系。1023-5系在早期发育基本正常，而在受精后48小时出现下丘脑和垂体POMC的异常表达，为研究下丘脑-垂体/促肾上腺皮质激素轴的分子和遗传调控提供了一个潜在的独特模型。在初步研究的基础上，R03的应用将检验一个单一的假设：突变1023-5的下丘脑发育缺陷(由单基因突变引起)会引起垂体系维持的外部信号改变，导致POMC细胞在终末分化后异常生长。有两个特定的目的：1)利用定位克隆鉴定导致1023-5系POMC表型异常的单基因缺陷；2)利用RNA整体原位杂交和活体时间推移共聚焦显微镜分析鉴定突变体1023-5的下丘脑和垂体/促肾上腺皮质激素个体发育。 调节下丘脑促肾上腺皮质激素发育的分子和遗传决定因素尚不清楚。这项研究是对最初K08提案的主要目标的补充。拟议的实验将验证实验方法，该方法将继续对K08奖产生的突变进行未来研究。更重要的是，这项研究产生的数据将导致其他假设，使R01应用程序的开发能够专注于已识别的推定基因的功能以及调节下丘脑和垂体发育的相关途径。

DESCRIPTION (provided by applicant): 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.