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从其在视网膜中的起源沿着生长到其在顶盖中的最终目标时，它们由细胞外基质中的复杂信号阵列以及与其它视网膜轴突的相互作用引导。这一过程不仅对视觉功能的发展至关重要，而且通常作为轴突引导的可访问模型。该提案将在斑马鱼胚胎中使用遗传学、胚胎学和成像方法来分析体内视网膜轴突引导，研究Slit- Robo信号传导和轴突-轴突相互作用如何作用以塑造视网膜顶盖投射。我们将解决有关Slit-Robo信令的三个主要问题。第一，当生长锥从一个部分传递到另一个部分时，裂缝是否会在不同的环境中从排斥性变为吸引性？表达研究，时间推移分析，体外试验，并在体内靶向的错误表达的新方法将明确解决这个问题。第二，Robo 2受体的不同胞质结构域是否具有不同的所需功能？第三，我们在robo 2基因中看到的选择性剪接在功能上有意义吗？我们将解决这两个问题，采用剪接阻断吗啉代寡核苷酸的一个强大的新策略，有针对性的外显子删除。在视网膜内，我们的初步数据显示，早期出生的中央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的视网膜轴突寻路，然后使用时间推移和移植实验来询问早期RGC如何引导后期轴突。最后，我们将测试哪些细胞粘附分子介导视网膜轴突成束，以及它们在引导视网膜内、视交叉和视束中的视网膜轴突中起什么作用。与公共卫生的相关性。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，了解共同组装斑马鱼视觉系统的分子机制与人类发育直接相关。此外，我们的数据显示，轴突轴突在发展过程中的相互作用的重要性，突出了在轴突再生过程中考虑这种相互作用的必要性，以应对损伤或疾病。

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.