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Astudy on reulatory mechanisms of glial basement membrane formation and neuronal migration using a fish model

鱼模型研究胶质细胞基底膜形成和神经元迁移的调控机制

基本信息

批准号：
18500264
负责人：
KIKKAWA Satoshi
金额：
$ 2.61万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

We cloned and sequenced the complete coding region of the zebrafish fukutin gene. Zebrafish fukutin showed a significant degree of sequence homology to mouse and human fukutin. By whole mount in situ hybridization, fukutin mRNA was detected in a wide variety of tissues, including the brain and the spinal cord, as early as 4 hours post-fertilization. We also examined expression patterns of zebrafish orthologs of other muscular dystrophy genes causing central nervous system defects, namely LARGE and POMGnT1. They were also expressed in a variety of tissues from the early developmental stage, implying that sugar-modifying enzymes play important roles in zebrafish CNS development as well.Next, we analyzed effects of knocking-down Fukutin protein expression on embryogenesis by morpholino antisense oligonucleotide (MO) microinjection into fertilized zebrafish eggs. Injected embryos (morphants) showed a broad range of developmental defects including skeletal and cardiac muscle defects in a dose-dependent manner, reflecting the broad expression of fukutin. We then looked into CNS defects in fukutin morphants using transgenic zebrafish expressing a fluorescent protein either in motor neurons (isl-GFP) or the entire differentiated neuronal pool (HuC-Kaede). Contrary to our expectations, we have not yet been able to find apparent defects of CNS development in both transgenic embryos injected with fukutin MO in spite of gross developmental defects. Further study in more detail is being undertaken for histological analysis.

我们克隆并测序了斑马鱼fukutin基因的完整编码区。斑马鱼的fukutin与小鼠和人的fukutin序列具有显著的同源性。通过全载原位杂交，早在受精后4小时，就在包括脑和脊髓在内的多种组织中检测到fukutin mRNA。我们还检测了斑马鱼中其他引起中枢神经系统缺陷的肌肉萎缩症基因的同源基因，即LARGE和POMGnT1的表达模式。它们也在早期发育阶段的多种组织中表达，这意味着糖修饰酶在斑马鱼中枢神经系统发育中也起着重要作用。接下来，我们通过将morpholino反义寡核苷酸（morolino anti - sense oligonucleotide， MO）微量注射到斑马鱼受精卵中，分析了敲低Fukutin蛋白表达对胚胎发生的影响。注射胚胎（变形体）表现出广泛的发育缺陷，包括骨骼和心肌缺陷，并呈剂量依赖性，反映了fukutin的广泛表达。然后，我们利用在运动神经元（is - gfp）或整个分化神经元池（HuC-Kaede）中表达荧光蛋白的转基因斑马鱼，研究了fukutin变形体的中枢神经系统缺陷。与我们预期相反的是，尽管有明显的发育缺陷，但在注射了fukutin MO的转基因胚胎中，我们尚未发现明显的中枢神经系统发育缺陷。正在进行更详细的组织学分析。