Mechanisms of retinal axon pathfinding

描述（由申请人提供）：当视网膜轴突沿着视网膜顶盖通路生长时，它们受到细胞外基质中一系列复杂信号的引导，并与其他视网膜轴突相互作用。这一过程不仅对视觉功能的发展至关重要，而且作为一般轴突引导的可访问模型。本研究将利用斑马鱼胚胎的遗传、胚胎学和成像方法分析视网膜轴突在体内的引导，研究Slit- Robo信号和轴突-轴突相互作用如何影响视网膜顶突投影。我们将解决关于slit - robot信号的三个主要问题。首先，当生长锥从其路径的一个部分传递到另一个部分时，裂缝在不同的环境下是否会从排斥变为吸引？表达研究、时间延迟分析、体外分析和一种新的靶向错误表达的体内方法将最终解决这个问题。其次，Robo2受体的不同细胞质结构域是否具有不同的所需功能？第三，我们在robo2基因中看到的选择性剪接在功能上是否重要？我们将通过使用剪接阻断的morpholino寡核苷酸来解决这两个问题，这是一种强大的新策略，靶向外显子删除。在视网膜内，我们的初步数据显示，早期出生的中枢rgc是后来出生的外周轴突离开眼睛所必需的。我们将首先全面分析视网膜内轴突寻路，然后使用时间推移和移植实验来了解早期RGCs如何指导后期轴突。最后，我们将测试哪些细胞粘附分子介导视网膜轴突的束状，以及它们在视网膜内、视交叉和视束中引导视网膜轴突的作用。与公共卫生有关。视觉系统解剖学和遗传控制机制在从鱼类到人类的脊椎动物中都是高度保守的。因此，了解共同组成斑马鱼视觉系统的分子机制与人类发展直接相关。此外，我们的数据显示了轴突-轴突相互作用在发育过程中的重要性，强调了在响应损伤或疾病的轴突再生过程中考虑这种相互作用的必要性。

DESCRIPTION (provided by applicant): As retinal axons grow along the retinotectal pathway from their origins in the retina to their final targets in the tectum, they are guided by a complex array of signals in the extracellular matrix, and by interactions with other retinal axons. This process is not only critical for the development of visual function, but serves as an accessible model for axon guidance in general. This proposal will use genetic, embryological, and imaging approaches in the zebrafish embryo to analyze retinal axon guidance in vivo, studying how Slit- Robo signaling and axon-axon interactions act to shape the retinotectal projection. We will address three main questions about Slit-Robo signaling. First, can Slits change from repulsive to attractive in different contexts as growth cones pass from one part of their pathway to another? Expression studies, time lapse analysis, in vitro assays, and a novel in vivo method for targeted misexpression will address this question definitively. Second, do different cytoplasmic domains of the Robo2 receptor have different required functions? And third, is the alternative splicing that we have seen in the robo2 gene functionally significant? We will address these two questions by employing splice-blocking morpholino oligonucleotides for a powerful new strategy, targeted exon deletion. Within the retina, our preliminary data show that early-born central RGCs are required for later-born peripheral axons to exit the eye. We will first comprehensively analyze retinal axon pathfinding within the retina, then use time lapse and transplant experiments to ask how early RGCs act to guide later axons. Finally, we will test which cell-adhesion molecules mediate retinal axon fasciculation, and what role they play in guiding retinal axons within the retina, in the chiasm, and in the optic tract. Relevance to public health. Visual system anatomy and genetic control mechanisms are both highly conserved across the vertebrates from fish to humans. Understanding the molecular mechanisms that work together to assemble the zebrafish visual system is thus directly relevant to human development. Furthermore, our data showing the importance of axon-axon interactions during development highlight the necessity of considering such interactions during axon regeneration in response to injury or disease.