Mechanisms of naturally-occurring astrocyte death during development

摘要 自然发生的发育性细胞死亡是神经系统中的基本模式形成机制， 系统细胞死亡是否以及如何塑造星形胶质细胞群体尚不清楚。我们的目标是 通过学习自然发生的星形胶质细胞的机制，深入了解星形胶质细胞的模式 小鼠视网膜死亡。中心假设是小胶质细胞杀死并吞噬视网膜星形胶质细胞作为反应 星形胶质细胞衍生的“吃我”信号，从而调节星形胶质细胞的数量和模式。的理由 这项工作是视网膜星形胶质细胞决定了血管系统发育的模式。星形胶质细胞死亡的知识 机制将使研究塑造星形胶质细胞最终模式的新因素成为可能， 血管网络-在正常和病理发育的情况下。为此，以下具体- 目的：1）确定视网膜星形细胞瘤发育性细胞死亡的细胞机制; 细胞。初步研究表明，视网膜星形胶质细胞最初是过度生产，然后淘汰之间 出生后第5天和第14天。这些研究进一步提出了小胶质细胞负责 在此期间杀死和消除星形胶质细胞。这将在体内进行测试，使用互补的anatom- 化学和化学方法。2)确定负责星形胶质细胞消除的分子机制- 发展过程中。初步数据表明，细胞凋亡不能解释视网膜发育的损失， 星形胶质细胞相反，一种三重跨细胞分子复合物--在细胞膜上包含磷脂酰丝氨酸 星形胶质细胞表面，可溶性脂质结合蛋白MFGE 8和小胶质细胞上的αvβ5整合素-被认为是一种 星形胶质细胞死亡的关键介质。这一工作假设将使用小鼠体内遗传工具进行测试。第三章 在疾病模型中确定发育性死亡对星形胶质细胞模式的贡献。在两种小鼠中 和人类，新生儿缺氧暴露可扰乱视网膜血管系统的形成。因为星形胶质细胞 作为血管发育的图案模板，星形胶质细胞图案缺陷可能导致缺氧- 诱发血管病变。开发了一种新的小鼠模型来研究这个问题。的初步数据 该模型导致了工作假设，即小胶质细胞介导的星形胶质细胞死亡受到缺氧的损害， 星形胶质细胞和血管图案缺陷。这将通过比较两种小鼠品系来测试：缺氧- 敏感品系和从初始缺氧诱导的病理恢复的弹性品系。完成这些 目的是：1）提供发育星形胶质细胞死亡的第一个机制的理解; 2） 开始揭示了死亡在视网膜星形胶质细胞群形成中的作用。这一贡献将被签署- 重要的是，因为它有望阐明特定的模式形成机制如何使星形胶质细胞功能， 在视网膜和整个神经系统中。该项目具有创新性，因为它具有强大的潜力 揭示了一种全新的小胶质细胞介导的自然细胞死亡机制;这种新机制 可能影响除星形胶质细胞外的许多细胞类型和组织的发育。

ABSTRACT Naturally-occurring developmental cell death is a fundamental pattern formation mechanism in the nervous system. Whether and how cell death sculpts the astrocyte population is not known. The objective here is to gain insight into astrocyte patterning by learning the mechanisms underlying naturally-occurring astrocyte death in the mouse retina. The central hypothesis is that microglia kill and engulf retinal astrocytes in response to astrocyte-derived “eat-me” signals, thereby regulating astrocyte numbers and patterning. The rationale for this work is that retinal astrocytes dictate the pattern of developing vasculature. Knowledge of astrocyte death mechanisms will make it possible to study novel factors that shape the ultimate pattern of the astrocyte and vascular networks – in both normal and pathological developmental contexts. To this end, the following Specif- ic Aims are proposed: 1) Determine cellular mechanisms for developmental cell death of retinal astro- cytes. Preliminary studies show that retinal astrocytes are initially overproduced and then culled between postnatal days 5 and 14. These studies further suggest the working hypothesis that microglia are responsible for killing and eliminating astrocytes during this period. This will be tested in vivo using complementary anatom- ical and chemogenetic approaches. 2) Identify molecular mechanisms responsible for astrocyte elimina- tion during development. Preliminary data show that apoptosis cannot account for developmental loss of ret- inal astrocytes. Instead, a tripartite trans-cellular molecular complex – comprising phosphatidylserine on the astrocyte surface, the soluble lipid-binding protein MFGE8, and αvβ5 integrins on microglia – is implicated as a key mediator of astrocyte death. This working hypothesis will be tested using mouse genetic tools in vivo. 3) Determine contribution of developmental death to astrocyte patterning in a disease model. In both mice and humans, neonatal hypoxia exposure can perturb formation of retinal vasculature. Because astrocytes serve as a patterning template for developing vessels, astrocyte patterning defects might contribute to hypoxia- induced vascular pathology. A novel mouse model was developed to study this issue. Preliminary data from this model led to the working hypothesis that microglia-mediated astrocyte death is impaired by hypoxia, caus- ing astrocyte and vessel patterning defects. This will be tested by comparing two mouse strains: a hypoxia- sensitive strain, and a resilient strain that recovers from initial hypoxia-induced pathology. Completion of these aims is expected to: 1) provide the first mechanistic understanding of developmental astrocyte death; and 2) begin to reveal the function of death in patterning the retinal astrocyte population. This contribution will be sig- nificant because it is expected to illuminate how specific pattern formation mechanisms enable astrocyte func- tions, in the retina and throughout the nervous system. The project is innovative because it has strong potential to unveil an entirely new microglia-mediated mechanism for naturally-occurring cell death; this new mechanism may impact development of many cell types and tissues in addition to astrocytes.