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 ?v?5 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.
The project is relevant to public health because, throughout the nervous system, astrocyte spatial arrange- ments relate to their essential functions in synaptic and vascular biology. Therefore, astrocyte functions may become compromised when they are not properly patterned during development. Through identification of as- trocyte spatial patterning mechanisms in the retina, the proposed research is expected to pinpoint mechanisms that may become dysfunctional in childhood vision diseases and that may one day be manipulated for thera- peutic benefit.
