Fatty acid metabolism and signaling during zebrafish development

描述(由申请人提供)：脂类代谢对所有细胞以及最有可能的每个发育过程都是必不可少的，但关于脂类处理和运输的机制仍有许多问题。Farber实验室使用发育中的斑马鱼来研究长链酰辅酶A合成酶(ACSLs)和脂肪酸运输蛋白(Fatps)的作用，这两种酶处理和运输长链和超长链脂肪酸。我的长期目标是了解其中一种蛋白质--脂肪酸运输蛋白2a(Fatp2a)在斑马鱼发育过程中的作用，并最终在人类生理中发挥作用。Fatp2a是人类Fatp2的同源物，人类Fatp2是一种酶，既能跨膜运输长链脂肪酸，又能激活超长链脂肪酸进入脂肪酰-COA。我的初步研究表明，斑马鱼幼体中Fatp2a的缺失会严重削弱原始红细胞的发育(红细胞生成)。有趣的是，FATP2a转录本主要不是在原始红细胞生成部位表达，而是在卵黄合胞层(YSL)中表达。由于YSL包围着卵黄细胞，并表达许多对脂肪吸收、新陈代谢和包装重要的基因(Marza等人2005年；Farber等人，未发表)，我们认为YSL作用于吸收卵黄中的脂肪并将它们包装起来输送到发育中的胚胎。拟议中的研究将检验这样的假设，即Fatp2a激活或运输蛋黄中的一种特定脂肪酸，这是正常原始红细胞生成所必需的。以下特定目的将有助于阐明表型的性质，并揭示Fatp2a激活和/或输入影响红细胞生成的底物。我将通过不同的染色技术和体内时间推移显微镜来探索Fatp2a在红系细胞增殖、迁移和成熟中的作用。我将通过以组织特异性的方式显微注射反义试剂和信使核糖核酸，以及创造转基因鱼类品系来探索细胞自主或非自主方式对Fatp2a的需求。最后，我将用质谱液相色谱/质谱仪揭示Fatp2a的底物。初步数据，再加上拟议的实验，将解决Fatp2活性在调节血细胞发育中的一个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，而且可能对某些白血病等破坏性血液疾病的治疗具有临床意义。

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and expresses many genes important for lipid absorption, metabolism, and packaging (Marza et al 2005; Farber et al, unpublished), we believe the YSL acts to absorb lipids from the yolk and package them for delivery to the developing embryo.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.