Fatty acid metabolism and signaling during zebrafish development

描述（由申请人提供）：脂质代谢对所有细胞和最有可能的每个发育过程都是必不可少的，但关于脂质加工和运输的机制仍存在许多问题。Farber实验室使用发育中的斑马鱼研究长链酰基辅酶A合成酶（Acsls）和脂肪酸转运蛋白（Fatps）的作用，这些酶处理和转运长链和超长链脂肪酸。我的长期目标是了解这些蛋白质之一，脂肪酸转运蛋白2a（Fatp 2a），在斑马鱼发育过程中的作用，并最终在人类生理学。Fatp 2a是人Fatp 2的直系同源物，Fatp 2是一种既能跨膜转运长链脂肪酸又能将极长链脂肪酸活化为脂肪酰辅酶A的酶。我的初步研究表明，斑马鱼幼鱼中Fatp 2a的缺失大大减弱了原始红细胞的发育（红细胞生成）。有趣的是，fatp 2a转录本主要在卵黄合胞体层（YSL）中表达，而不是在原始红细胞生成部位。由于YSL围绕在卵黄细胞周围，并表达许多对脂质吸收、代谢和包装重要的基因（Marza et al 2005; Farber et al，未发表），我们认为YSL的作用是从卵黄中吸收脂质，并将其包装，以输送到发育中的胚胎。拟议的研究将测试Fatp 2a激活或转运蛋黄中的特定脂肪酸的假设，这是适当的原始红细胞生成所必需的。以下具体目标将有助于阐明表型的性质，并揭示Fatp 2a激活和/或输入以影响红细胞生成的底物。我将通过使用不同的染色技术和体内延时显微镜来探索Fatp 2a在红系细胞增殖、迁移和成熟中的作用。我将探索在细胞自主或非自主的方式，通过显微注射反义试剂和mRNA在组织特异性的方式，以及创建转基因鱼线的Fatp 2a的需求。最后，我将揭示Fatp 2a的底物使用质量液相色谱/质谱。初步数据，加上拟议的实验将解决Fatp 2活性在调节血细胞发育中的新的和意想不到的作用。这些结果不仅将增加我们对这一重要发育途径的了解，而且可能对治疗某些白血病等毁灭性血液疾病具有临床意义。

DESCRIPTION (provided by applicant): Lipid metabolism is essential for all cells and most likely every developmental process, and yet many questions remain regarding mechanisms of lipid processing and transport. The Farber lab uses the developing zebrafish to study the roles of long-chain acyl-CoA synthetases (Acsls) and fatty acid transport proteins (Fatps), enzymes that process and transport long-chain and very long-chain fatty acids. My long term goal is to understand the role of one of these proteins, fatty acid transport protein 2a (Fatp2a), during zebrafish development and ultimately in human physiology. Fatp2a is an ortholog of human Fatp2, an enzyme that both transports long-chain fatty acids across membranes and activates very long-chain fatty acids into fatty acyl-CoAs. My preliminary studies demonstrate that loss of Fatp2a in zebrafish larvae profoundly attenuates primitive red blood cell development (erythropoiesis). Interestingly, fatp2a transcript is primarily expressed not in the site of primitive erythropoiesis, but in the yolk syncytial layer (YSL). Because the YSL surrounds the yolk cell and expresses many genes important for lipid absorption, metabolism, and packaging (Marza et al 2005; Farber et al, unpublished), we believe the YSL acts to absorb lipids from the yolk and package them for delivery to the developing embryo. The proposed studies will test the hypothesis that Fatp2a activates or transports a specific fatty acid from the yolk that is necessary for proper primitive erythropoiesis. The following specific aims will help elucidate the nature of the phenotype and reveal the substrate that Fatp2a activates and/or imports to influence erythropoiesis. I will explore the role of Fatp2a in proliferation, migration and maturation of erythroid cells by using different staining techniques and in vivo time-lapse microscopy. I will explore the requirement for Fatp2a in a cell autonomous or non-autonomous manner by microinjecting antisense reagents and mRNA in a tissue specific manner as well as creating transgenic fish lines. Finally I will reveal Fatp2a's substrate using mass liquid chromatography/mass spectroscopy. The preliminary data, coupled with the proposed experiments will address a new and unexpected role for Fatp2 activity in regulating blood cell development. These results will not only increase our knowledge of this important developmental pathway, but may have clinical implications for the treatment of devastating blood diseases like some leukemias.