The role of GABAB signaling during axon guidance in the developing zebrafish

描述（由申请人提供）：在神经系统发育过程中，轴突被引导通过化学和物理复杂的环境，以响应排斥和吸引的线索。精确的轴突寻路是建立功能神经网络的关键。阐明其引导机制将为神经发育和神经退行性疾病的发病机制和治疗提供有价值的见解。 本提案的目的是测试的假设，即通过代谢型3-氨基丁酸（GABA）受体，GABAB信号减少轴突反应排斥指导线索，这种活动是必要的，在体内正确的轴突指导。GABAB受体在轴突导向中的作用将在体内使用斑马鱼视网膜投射作为模型进行探索，并在体外使用来自胚胎鸡视网膜神经节细胞（RGC）的原代神经元培养物进行探索。结合Roundabout-2（Robo 2）受体的排斥性Slit 2在垂直于中线的条带中表达，并且被认为通过环绕排斥来确定视交叉的位置。在交叉处，轴突必须穿过Slit 2表达的区域。GABAB激活可能是一种机制，通过该机制，对Slit 2的敏感性降低，尽管存在排斥信号，仍允许交叉。 这项建议包括三个目标。目的1是利用免疫组织化学和原位杂交技术研究斑马鱼胚胎发育过程中GABA和GABAB受体的表达模式。转基因斑马鱼，其中视网膜神经节细胞标记有绿色荧光蛋白，将用于识别视网膜投影。目的2是使用两种独立的方法确定GABAB信号传导是否是体内适当轴突寻路所必需的：使用UAS-GAL 4系统在RGC中表达显性阴性GABAB受体（R1 C）和反义吗啉代敲低。目的3是确定GABA/GABAB活性是否通过充当抗驱避剂或通过激活吸引信号通路来降低对Slit 2的敏感性。这将在体外通过用鸡RGC进行生长锥转动测定来解决，并在体内通过在具有减少的或没有Slit/Robo信号传导的突变体斑马鱼中表达R1 C来解决。斑马鱼的错误基因编码Robo 2。无效等位基因和亚纯等位基因都导致视网膜投影中的引导误差。如果GABAB信号传导降低了对Slit 2的敏感性，那么GABAB信号传导的损失应该增加敏感性，使得由于减少的Slit/Robo信号传导而导致的引导错误被挽救。如果GABA/GABAB信号传导产生抗驱避反应，那么其拯救引导错误的能力将取决于Slit/Robo信号传导的存在，并且R1 C的表达将拯救误入歧途的亚型中的缺陷，而不是无效的斑马鱼。涉及斑马鱼的实验将通过整个胚胎的共聚焦显微镜进行分析。 公共卫生相关性：参与轴突引导的许多分子与神经系统疾病有关，例如精神发育迟滞、癫痫、阅读障碍、自闭症、帕金森病和肌萎缩侧索硬化症（综述于Yaron和Zheng，2006; Anitha等人，2008; Lesnick等人，2008年）。通过研究GABAB受体在轴突导向中的作用，我们可以阐明正常的发育过程，并为神经发育和神经退行性疾病的发病机制和治疗提供有价值的见解。

DESCRIPTION (provided by applicant): During nervous system development, axons are guided through a chemically and physically complex environment in response to repulsive and attractive cues. Accurate axon pathfinding is critical for the establishment of functional neural networks. The elucidation of guidance mechanisms will provide valuable insight into the pathogenesis and treatment of neurodevelopment and neurodegenerative diseases. The objective of this proposal is to test the hypothesis that signaling through the metabotropic 3- aminobutyric acid (GABA) receptor, GABAB reduces axonal responsiveness to repellent guidance cues and that this activity is essential for proper axon guidance in vivo. The role of GABAB receptors in axon guidance will be explored in vivo using the zebrafish retinal projection as a model and in vitro using primary neuronal cultures derived from embryonic chick retinal ganglion cells (RGCs). The repellent Slit2, which binds the Roundabout-2 (Robo2) receptor, is expressed in bands perpendicular to the midline and is believed to determine the location of the optic chiasm via surround repulsion. At the chiasm, axons must travel through areas of Slit2 expression. GABAB activation may be a mechanism through which sensitivity to Slit2 is reduced, permitting crossing despite the presence of a repulsive signal. This proposal consists of three aims. Aim1 is to characterize the expression patterns of GABA and GABAB receptors during zebrafish development using immunohistochemistry and in situ hybridization in whole embryos. Transgenic zebrafish, in which retinal ganglion cells are labeled with green fluorescent protein, will be used to identify the retinal projection. Aim2 is to determine if GABAB signaling is necessary for proper axonal pathfinding in vivo using two independent methods: expression of a dominant negative GABAB receptor (R1C) in RGCs using the UAS-GAL4 system and antisense morpholino knockdown. Aim3 is to determine if GABA/GABAB activity reduces sensitivity to Slit2 by acting as an antirepellent or by activating an attractive signaling pathway. This will be addressed in vitro by performing growth cone turning assays with chick RGCs and in vivo by expressing R1C in mutant zebrafish with either reduced or no Slit/Robo signaling. The zebrafish astray gene encodes Robo2. Both null and hypomorphic alleles result in guidance errors in the retinal projection. If GABAB signaling reduces sensitivity to Slit2, then loss of GABAB signaling should increase sensitivity such that guidance errors due to reduced Slit/Robo signaling are rescued. If GABA/GABAB signaling produces an antirepellent response, then its ability to rescue guidance errors will depend on the presence of Slit/Robo signaling and expression of R1C would rescue defects in the astray hypomorphs but not the null zebrafish. Experiments involving zebrafish will be analyzed via confocal microscopy of whole embryos. PUBLIC HEALTH RELEVANCE: Many of the molecules that are involved in axon guidance have been linked to neurological diseases such as mental retardation, epilepsy, dyslexia, autism, Parkinson's, and Amyotrophic lateral sclerosis (reviewed in Yaron and Zheng, 2006; Anitha et al., 2008; Lesnick et al., 2008). By investigating the role the GABAB receptor plays in axon guidance, we may elucidate normal developmental processes and provide valuable insight into the pathogenesis and treatment of neurodevelopmental and neurodegenerative diseases.