In vivo analysis of the developing vertebrate retina

描述(由申请人提供)：由于先天缺陷，在发育过程中可能会出现神经元丢失和功能障碍，成熟时可能会因为损伤和疾病而出现神经元丢失和功能障碍。如果不能产生正确的细胞类型及其准确的连接模式，将会产生严重的功能后果。因此，我们的广泛目标是促进我们对以下方面的理解：(I)确保神经元及其电路在发育过程中正确生成的细胞过程，以及(Ii)允许或限制神经元群体在损伤或疾病后重新建立其起始电路的过程。尽管干细胞治疗是恢复功能的主要策略，但目前尚不清楚置于受损或患病细胞环境中的新神经元是否能够形成原始的连接模式。由于种植在中枢神经系统内的细胞存活仍然具有挑战性，我们将利用斑马鱼这一研究发育和疾病的重要遗传模型系统来实现我们的目标。这是因为斑马鱼具有再生神经元的先天能力。我们建议将重点放在视觉系统中锥体光感受器与其靶双极细胞之间的第一个突触上，因为这些细胞的结构、功能和连通性已经得到了深入的研究，而它们之间的联系受到干扰会导致视力受损。我们将使用基因工具和最先进的成像方法来回答神经元和视觉发育和修复领域的三个突出问题。在目标1中，我们将确定在单一的神经元类型--视锥细胞感受器中是否存在针对产生和再生特定亚型的内源性细胞发生途径。在目标2中，我们将确定在先天性疾病模型中，突触后双极细胞如何补偿发育过程中首选的突触前锥体类型的缺失。在目标3中，我们将在体内选择性地消融视锥感光细胞或双极细胞，并评估神经元再生时回路重组的特异性和准确性。总之，我们的发现将显著增加我们对在体内招募的生成和再生过程的理解，这些过程被招募来建立复杂的电路，如发育和修复中的锥体通路。

DESCRIPTION (provided by applicant): Neuronal loss and dysfunction can arise both during development due to congenital defects, and at maturity because of injury and disease. Failure to produce the correct cell types and their precise connectivity patterns have severe functional consequences. Thus, our broad goal is to advance our understanding of: (i) the cellular processes that ensure the proper generation of neurons and their circuits during development, and (ii) the processes that enable or limit neuronal populations from re-establishing their origina circuitry after injury or disease. Although stem-cell therapy represents a major strategy for restoring function, it is not yet known whether new neurons placed in injured or diseased cellular environments are able to form their original connectivity patterns. Because survival of cells seeded within the central nervous system remains challenging, we will take advantage of zebrafish, an important genetic model system for investigating development and disease, to achieve our goals. This is because zebrafish have an inherent ability to regenerate its neurons. We propose to focus on the first synapse in the visual system between cone photoreceptors and their target bipolar cells because structure, function and connectivity of these cell types are heavily studied, and perturbations to their connections result in impaired vision. We will use genetic tools and state-of-the-art imaging approaches to answer three outstanding questions in the fields of neuronal and visual development and repair. In Aim 1, we will determine whether there are endogenous cell-genesis pathways directed at producing and regenerating specific subtypes within a single neuronal type, the cone photoreceptors. In Aim 2, we will ascertain how postsynaptic bipolar cells compensate for the absence of a preferred presynaptic cone type during development, in models of congenital disease. In Aim 3, we will selectively ablate cone photoreceptors or bipolar cells in vivo and assess the specificity and accuracy of circuit reassembly upon neuronal regeneration. Together, our findings will significantly increase our understanding of the generative and regenerative processes that are recruited in vivo to establish complex circuits, such as the cone pathways, in development and in repair.