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的一种基本模式形成机制 系统。细胞死亡是否以及如何塑造星形胶质细胞数量尚不清楚。这里的目标是 通过学习自然产生的星形胶质细胞的机制来洞察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 到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图案。其基本原理是 这项工作是视网膜星形细胞决定了血管系统的发育模式。对星形胶质细胞死亡的认识 机制将使研究塑造星形胶质细胞最终模式的新因素和 血管网络--在正常和病理发育环境中都是如此。为此目的，规定如下： 其目的是：1)确定视网膜星形细胞发育性死亡的细胞机制； 细胞。初步研究表明，视网膜星形胶质细胞最初是过度生产的，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。这将在体内使用补充解剖学进行测试- 化学和化学发生方法。2)确定星形胶质细胞消除的分子机制- 在开发过程中。初步数据显示，细胞凋亡不能解释发育丧失的原因。 原始星形胶质细胞。相反，一个由磷脂酰丝氨酸组成的三方跨细胞分子复合体 星形胶质细胞表面、可溶性脂结合蛋白MFGE8和小胶质细胞上的αvβ5整合素-与 星形胶质细胞死亡的关键调节因子。这一工作假说将在体内使用小鼠遗传工具进行验证。3) 在疾病模型中确定发育性死亡对星形胶质细胞构型的贡献。在两只小鼠中 与人类相比，新生儿缺氧暴露会扰乱视网膜血管的形成。因为星形胶质细胞 作为发育血管的构图模板，星形胶质细胞构图缺陷可能导致缺氧- 诱导血管病变。为了研究这一问题，人们开发了一种新的小鼠模型。初步数据来自 这一模型导致了一种工作假说，即小胶质细胞介导的星形胶质细胞死亡受到缺氧的损害，因为... 星形胶质细胞和血管构图缺陷。这将通过比较两个小鼠品系来测试：低氧- 敏感菌株，以及从最初的低氧诱导的病理中恢复的弹性菌株。完成这些工作 AIMS有望：1)提供对发育性星形胶质细胞死亡的第一个机制的理解；2) 开始揭示死亡在形成视网膜星形细胞群中的作用。这笔捐款将签署为- 这是因为它有望阐明特定的图案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。这个项目具有创新性，因为它具有很强的潜力 揭示一种全新的小胶质细胞介导的自然细胞死亡机制；这一新机制 除了星形胶质细胞外，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.