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

GABAB 信号在斑马鱼轴突引导过程中的作用

• 批准号: 7917000
• 负责人: Alemji Taku
• 金额: $ 4.14万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917000
• 关键词: 3-aminobutyric acid; Address; Alleles; Amyotrophic Lateral Sclerosis; Area; Autistic Disorder; Axon; Binding; Biological Assay; Biological Neural Networks; Complex; Confocal Microscopy; Cues; Defect; Developmental Process; Dominant-Negative Mutation; Dyslexia; Embryo; Environment; Epilepsy; GABA Receptor; Genes; Green Fluorescent Proteins; Growth Cones; Immunohistochemistry; In Situ Hybridization; In Vitro; Label; Link; Location; Mental Retardation; Methods; Neurodegenerative Disorders; Neurons; Optic Chiasm; Parkinson Disease; Pathogenesis; Pattern; Play; Retinal; Retinal Ganglion Cells; Role; Signal Pathway; Signal Transduction; System; Testing; Transgenic Organisms; Travel; Zebrafish; axon guidance; axonal pathfinding; gamma-Aminobutyric Acid; in vitro Model; in vivo; insight; mutant; nervous system development; nervous system disorder; neurodevelopment; public health relevance; receptor; research study; response; zebrafish development

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.

描述(申请人提供)：在神经系统发育期间，轴突被引导穿过化学和物理复杂的环境，对令人厌恶和吸引的提示做出反应。准确的轴突寻径是建立功能神经网络的关键。引导机制的阐明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。这一建议的目的是验证这样一种假设，即通过代谢性3-氨基丁酸(GABA)受体GABAB发出的信号降低了轴突对排斥引导线索的反应性，并且这种活动对于体内正确的轴突引导是必不可少的。GABAB受体在轴突导向中的作用将以斑马鱼视网膜投射为模型在体内和体外利用胚胎鸡视网膜神经节细胞(RGCs)的原代神经元培养来探索。排斥性Slit2与Robo2受体结合，在垂直于中线的条带中表达，被认为通过环绕斥力决定视交叉的位置。在交叉时，轴突必须穿过Slit2表达的区域。GABAB的激活可能是一种降低对Slit2敏感性的机制，即使存在排斥信号也允许交叉。这项提议包括三个目标。目的利用免疫组织化学和原位杂交技术研究斑马鱼发育过程中GABA和GABAB受体的表达模式。转基因斑马鱼中的视网膜神经节细胞被标记了绿色荧光蛋白，将被用于识别视网膜投射。AIM2将使用两种独立的方法来确定GABAB信号是否对体内正确的轴突路径是必需的：使用UAS-GAL4系统在RGC中表达显性负性GABAB受体(R1C)和反义吗啉基因敲除。AIM3旨在确定GABA/GABAB活性是否通过作为抗排斥剂或通过激活吸引人的信号通路来降低对SLIT2的敏感性。这将在体外通过对小鸡视网膜神经节细胞进行生长锥翻转试验来解决，并通过在体内通过在突变斑马鱼中表达R1C来解决，这些突变斑马鱼的Sit/Robo信号减少或没有。斑马鱼迷途基因编码Robo2。零等位基因和亚形等位基因都会导致视网膜投射中的引导误差。如果GABAB信令降低了对SLIT2的敏感度，那么GABAB信令的丢失应该会提高敏感度，从而挽救由于减少的时隙/ROBO信令而导致的制导错误。如果GABA/GABAB信号产生抗排斥反应，那么它拯救引导错误的能力将取决于Sit/Robo信号的存在，而R1C的表达将拯救迷失亚型斑马鱼的缺陷，而不是零斑马鱼。涉及斑马鱼的实验将通过对整个胚胎的共聚焦显微镜进行分析。 与公共卫生相关：许多参与轴突引导的分子都与神经疾病有关，如智力低下、癫痫、阅读障碍、自闭症、帕金森氏症和肌萎缩侧索硬化症(在Yaron和Zeng，2006年；Anitha等人，2008年；Lesnick等人，2008年)。通过研究GABAB受体在轴突引导中的作用，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。