In vivo biosensor screen for mutants in lipid metabolism

描述：(申请者摘要)虽然基于形态的基因筛查 已经确定了影响斑马鱼规格和图案的突变 胚胎结构，很少有试剂被应用于评估生理 在体内发挥作用。我们生产了荧光脂质，作为底物用于 磷脂酶A2(PLA2)是一种重要的信号转导和消化酶 肠上皮的细胞质和刷状边缘。这些卵裂 PLA2的脂质增加或改变了它们的荧光发射波长， 从而揭示活体动物的局部酶活性。什么时候 给光学透明的斑马鱼幼体注射，这些脂类提供了 PLA2活性的实时目测分析。 斑马鱼仔鱼受精后第5天摄取的荧光脂质 产生强烈的胆汁荧光，这一发现反映了 肠道磷脂酶A2对脂质的处理和随后通过 肝胆系统。由于胆囊壁荧光的速度和程度是 在斑马鱼幼体中很容易观察到，这些试剂提供了一个灵敏的读数 在突变筛选的背景下可以 找出影响脂肪加工的基因。 我们在受精后5天给诱变剂注射荧光脂类。 斑马鱼幼体并发现了一种扰乱肠道的突变 磷脂酶活性。这份建议书中描述的工作概述了我们的计划 使用这些试剂进行大规模诱变筛选。通过识别 扰乱脂代谢的突变，我们希望恢复调控的基因 肠道和肝胆发育以及PLA2，一个与 印记在炎症、止血和细胞增殖中的作用。遗传 这些发育和生理过程的特征 对儿童先天性疾病相关研究的重要意义 肠道和肝脏、癌症、心血管和炎症性疾病。

DESCRIPTION: (Applicant's Abstract) Although morphology-based genetic screens have identified mutations that affect specification and patterning of zebrafish embryonic structures, few reagents have been applied to assess physiological functions in vivo. We produced fluorescent lipids that are substrates for phospholipase A2 (PLA2), an important signaling and digestive enzyme present in the cytoplasm and brush border of the intestinal epithelium. Cleavage of these lipids by PLA2 increases or shifts their wavelength of fluorescent emission, thereby revealing localized enzymatic activity in live animals. When administered to optically transparent zebrafish larvae, these lipids provide a real-time visual assay of PLA2 activity. Fluorescent lipids ingested by day 5 post-fertilization zebrafish larvae produce intense gallbladder fluorescence, a finding we have shown reflects lipid processing by intestinal PLA2 and subsequent transport through the hepatobiliary system. Since the rate and degree of gallbladder fluorescence are easily observed in zebrafish larvae, these reagents provide a sensitive readout of digestive physiology that in the context of a mutagenesis screen can identify genes that influence lipid processing. We administered fluorescent lipids to mutagenized 5-day post-fertilization zebrafish larvae and have identified one mutation that perturbs intestinal phospholipase activity. The work described in this proposal outlines our plan for a large scale mutagenesis screen using these reagents. By identifying mutations that perturb lipid metabolism, we hope to recover genes that regulate intestinal and hepatobiliary development as well as PLA2, a gene with an imprint role in inflammation, hemostasis and cell proliferation. Genetic characterization of these developmental and physiological processes has important implications for research related to congenital diseases of the intestine and liver, cancer, and cardiovascular and inflammatory diseases.