Mechanisms of naturally-occurring astrocyte death during development

摘要 自然发生的发育细胞死亡是神经系统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。这里的目标是 通过了解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响应杀死并吞噬视网膜星形胶质细胞 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。理由 这项工作是视网膜星形胶质细胞决定脉管系统的发育模式。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——在正常和病理发育环境中。为此，特制定以下规定： 提出的 ic 目标是： 1) 确定视网膜星形细胞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过量产生，然后在 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的工作假设 在此期间杀死和消除星形胶质细胞。这将使用互补的解剖学进行体内测试 化学和化学遗传学方法。 2) 确定星形胶质细胞消除的分子机制 开发过程中重刑。初步数据表明，细胞凋亡不能解释 ret- 的发育损失。 最终的星形胶质细胞。相反，三部分跨细胞分子复合物 - 包含磷脂酰丝氨酸 星形胶质细胞表面、小胶质细胞上的可溶性脂质结合蛋白 MFGE8 和 αvβ5 整合素 - 被认为是 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。 3） 确定发育死亡对疾病模型中星形胶质细胞模式的贡献。在两只小鼠中 和人类一样，新生儿缺氧会扰乱视网膜脉管系统的形成。因为星形胶质细胞 作为发育中血管的图案模板，星形胶质细胞图案缺陷可能会导致缺氧 诱发血管病理学。开发了一种新型小鼠模型来研究这个问题。初步数据来自 该模型得出了这样的假设：缺氧会损害小胶质细胞介导的星形胶质细胞死亡，导致 星形胶质细胞和血管模式缺陷。这将通过比较两种小鼠品系进行测试：缺氧小鼠 敏感菌株和从最初缺氧引起的病理中恢复的弹性菌株。完成这些 预计目标是：1）提供对发育性星形胶质细胞死亡的第一个机制理解；和 2) 开始揭示死亡在视网膜星形胶质细胞群体模式中的功能。这笔捐款将被签署 意义重大，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发挥作用 视网膜和整个神经系统。该项目具有创新性，因为它具有强大的潜力 揭示一种全新的小胶质细胞介导的自然发生细胞死亡机制；这个新机制 除星形胶质细胞外，还可能影响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.