Zebrafish Models to Study Adult Forebrain Neurogenesis

描述（由申请人提供）：本提案的目标是使用斑马鱼条件基因表达和脑损伤模型来检查成年出生的端脑神经元的整合和再生能力。神经干/祖细胞（npc）和神经发生在成年脊椎动物脑室下区(SVZ)-嗅球通路中持续存在，但对成年出生的神经元的调控和生物学功能知之甚少。哺乳动物的这种神经源性通路受到中风和其他脑损伤的刺激。SVZ神经母细胞从其正常嗅球目标转移到受损纹状体，并分化为具有明显纹状体表型的神经元。因此，成人的npc似乎有助于再生反应，如果增强，可能会改善前脑损伤的恢复。然而，损伤诱导的SVZ神经发生的后果仍然不清楚，因为在确定脑损伤后成年出生的神经元的长期存活和功能整合方面几乎没有进展。斑马鱼是一个有吸引力的，尽管未充分利用的模型系统，在完整或受伤的前脑成人神经发生的研究。成年硬骨鱼（包括斑马鱼）的中枢神经系统（CNS）再生反应比哺乳动物强得多。了解斑马鱼脑损伤后再生是如何实现的，可能有助于了解哺乳动物中枢神经系统再生受到限制的原因，以及如何逆转这种限制以实现恢复性鼻炎治疗。斑马鱼系统的这些和其他优势使我们的团队和其他人开始表征成年斑马鱼的svz -嗅球神经发生。我们的数据表明，成年出生的神经元在球内整合，并且兴奋毒素诱导的端脑损伤刺激了SVZ神经发生。我们也已经开始开发可诱导的转基因斑马鱼系，用于命运定位，以研究成年出生的端脑神经元的长期整合。利用这些细胞系和兴奋性毒性前脑损伤模型，我们提出验证以下假设：1)成年出生的SVZ神经母细胞迁移到嗅球并产生与先前存在的网络整合的嗅觉神经元；2)兴奋毒性远端脑损伤刺激成体SVZ神经发生，取代受损神经元。为了验证这些假设，提出了两个具体的目标。在Aim 1中，在npc特异性启动子的控制下，带有可诱导Cre重组酶的转基因斑马鱼系将与一个报告系杂交，以有条件地标记端脑SVZ中成年出生的神经元，并检查其结构和功能整合。在目的2中，将对鱼进行兴奋毒性远端脑损伤，以确定损伤是否刺激有丝分裂标记或转基因方法鉴定的成年出生的神经元的增殖、迁移和长期整合。这些研究将揭示成年出生的前脑npc的长期命运和再生潜力，并将为研究完整和受伤的脊椎动物前脑中的成年神经发生提供有价值的工具。

DESCRIPTION (provided by applicant): The goals of this proposal are to examine the integration and regenerative capacity of adult-born telencephalic neurons using zebrafish models of conditional gene expression and brain injury. Neural stem/progenitor cells (NPCs) and neurogenesis persist throughout life in the adult vertebrate subventricular zone (SVZ)-olfactory bulb pathway, but the regulation and biological function of adult-born neurons are poorly understood. This neurogenic pathway in mammals is stimulated by stroke and other brain injuries. SVZ neuroblasts are diverted from their normal olfactory bulb target to the injured striatum and differentiate into neurons with an apparent striatal phenotype. NPCs in the adult therefore appear to contribute to a regenerative response that, if augmented, may improve recovery from forebrain injuries. The consequences of injury-induced SVZ neurogenesis remain obscure, however, as little progress has been made in establishing the long-term survival and functional integration of adult-born neurons after brain insults. Zebrafish is an attractive, albeit underutilized, model system for the study of adult neurogenesis in the intact or injured forebrain. The central nervous system (CNS) regenerative response of adult teleost fish, including zebrafish, is much more robust than in mammals. Understanding how regeneration is achieved after brain injury in the zebrafish is likely to provide insight into why mammalian CNS regeneration is limited, and how this limitation might be reversed to achieve restorative NPC therapies. These and other advantages of the zebrafish system have led our group and others to begin characterizing SVZ-olfactory bulb neurogenesis in adult zebrafish. Our data suggest that adult-born neurons integrate in the bulb and that SVZ neurogenesis is stimulated by excitotoxin-induced telencephalic injury. We also have begun developing inducible transgenic zebrafish lines for fate mapping to examine the long-term integration of adult-born telencephalic neurons. Using these lines and the excitotoxic forebrain injury model, we propose to test the following hypotheses: 1) Adult-born SVZ neuroblasts migrate to the olfactory bulb and generate olfactory neurons that integrate into preexisting networks; and 2) Excitotoxic telencephalic injury stimulates adult SVZ neurogenesis to replace damaged neurons. Two specific aims are proposed to test these hypotheses. In Aim 1, transgenic zebrafish lines with inducible Cre recombinase under the control of NPC-specific promoters will be crossed with a reporter line to conditionally label adult-born neurons in the telencephalic SVZ and examine their structural and functional integration. In Aim 2, fish will undergo excitotoxic telencephalic lesioning to determine whether injury stimulates the proliferation, migration, and long-term integration of adult-born neurons identified by mitotic labeling or transgenic approaches. These studies will shed light on the long-term fate and regenerative potential of adult-born forebrain NPCs and will provide valuable tools to study adult neurogenesis in the intact and injured vertebrate forebrain. PUBLIC HEALTH RELEVANCE: Neural stem cells and the birth of new nerve cells persist in the adult brain. These cells have therapeutic potential and may be stimulated to repair the brain after injury. The reasons why repair is often incomplete after acute brain insults are unknown, but zebrafish have a greater nervous system regenerative capacity than mammals and may shed light on the factors that limit repair. Because the same pathways of nerve cell birth are present in the forebrains of zebrafish and mammals, progress in understanding neural stem cell behavior in the intact or injured fish brain may lead to therapies for neural repair after stroke or other brain insults.