Functional Analysis and Receptor Identification of Nobel Membrane Protein CUBL in Floor Plate

以高度精确的模式书写大量的神经元对于脊椎动物神经系统复杂结构的形成至关重要。神经网络的形成主要依赖于生长锥（生长轴突的尖端）解释沿着通向目标区域的通路的环境线索的能力。最近发现的细胞外信号分子，作为化学吸引的线索，Netrin，和化学排斥的线索，Semaphorin，Ephrin，和Slit，和受体（DCC，Eph，Robo，Plexin，and Neuropilin）已经引导我们了解网络分子机制的基本方面，尽管由于神经系统的高度复杂性，它还远未完成。在本研究中，我鉴定了一种新的膜蛋白CUBL和趋化因子SDF-1，它们是在神经系统发育过程中在底板中表达的基因，并且在PC轴突和视网膜轴突的轴突导向中起作用。CUBL是一种新的膜蛋白，其在胞外区具有CUB结构域和LDL受体重复结构域。它在中脑、后脑和连合下器的底板中表达。本研究构建了转基因斑马鱼品系，该品系携带热激启动子控制下的转基因CUBL。在该系统中，转基因可以在热休克后在鱼的所有组织中异位表达。通过原位杂交或GFP荧光验证转基因的热诱导。我发现整个胚胎的CUBL的热诱导导致PC轴突的错误路由。这一结果表明CUBL在PC轴突的轴突导向中的作用。

Writing an enormous number of neurons in highly precise pattern is essential for the formation of the complex architecture of vertebrate nervous system. The formation of neural network primarily depends on the ability of growth cone, a tip of the growing axon, to interpret environmental cues along the pathway to the target region. Recent discovery of extracellular signaling molecules which act as chemoattractive cue, Netrin, and chemorepulsive cue, Semaphorin, Ephrin, and Slit, and the receptors (DCC, Eph, Robo, Plexin, and Neuropilin) has lead us to understanding of basic aspects of molecular mechanism of network, although it is far from complete because of high degree of complexity of the nervous system.In the present study, I identified a novel membrane protein CUBL and chemokine SDF-1 as genes that are expressed in the floor plate during nervous system development and play a role in the axon guidance of PC axons and retinal axons.CUBL is a novel membrane protein that has CUB domain and LDL receptor repeat domain in the extracellular region. It is expressed in the floor plate of midbrain, hindbrain and subcommissural organ. I generated transgenic zebrafish line that carries transgene CUBL under the control of heat shock promoter. In this system the transgene can be expressed ectopically in all tissues in the fish upon heat shock. The heat induction of the transgene was verified by either in situ hybridization or GFP fluorescence. I found that heat induction of CUBL in whole embryo leads to misrouting of PC axons. This result suggests role of CUBL in axon guidance of PC axons.