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富丁蛋白基因的完整编码区域。斑马鱼富丁蛋白与小鼠和人富丁蛋白具有很大程度的序列同源性。在整个原位杂交中，在施肥后4小时，在包括大脑和脊髓在内的各种组织中检测到富丁素mRNA。我们还检查了其他肌肉营养不良基因的斑马鱼直系同源物的表达模式，这些基因引起了中枢神经系统缺陷，即大和pomgnt1。它们还在早期发育阶段的各种组织中表达，这表明糖改性酶在斑马鱼中枢神经系统发育中也起着重要作用。我们分析了敲击粉刺富丁蛋白蛋白表达对morpholino抗溶质寡核苷酸的胚胎发生对胚胎的作用的影响。 Mo）将其显微注射侵蚀性斑马鱼卵。注射的胚胎（形态）以剂量依赖性方式显示出广泛的发育缺陷，包括骨骼肌和心脏肌肉缺陷，反映了Fukutin的广泛表达。然后，我们使用转基因斑马鱼在运动神经元（ISL-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（HUC-KAEDE）。与我们的期望相反，尽管发育严重缺陷，但我们尚未能够在两种转基因胚胎中发现CNS发育的明显缺陷。正在进行进一步的研究以进行组织学分析。