Browsing by Author "Kay, Jeremy N"

Naturally-occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the central nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type – the astrocyte – has rarely been studied. It is presently unclear whether astrocytes are subject to significant amounts of developmental death, or how it occurs. Here we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia kill and engulf astrocytes to mediate their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally-occurring cell death, and demonstrate its importance for formation and integrity of the retinal gliovascular network.

In the central nervous system, billions of neurons interconnect with precision to form morphologically complex and functionally diverse neural circuits. The stereotypical fashion by which neurons assemble suggests that cell-surface molecular cues can act as identity tags during development. These cell surface receptors allow neurons to distinguish between circuit partners, incorrect connections and homotypic neighbors. Multiple EGF-like domains 10 (Megf10) was previously identified to mediate homotypic recognition of certain retinal cell types. Genetic evidence suggests that MEGF10 acts as both ligand and receptor to initiate cell-cell repulsion. Although its significance in cell-cell recognition has been demonstrated, the exact mechanism of how MEGF10 mediates mosaic formation remains unclear. Specifically, the biochemical basis of MEGF10-MEGF10 interaction is largely unknown nor do we have knowledge on what molecules are involved in signaling transduction. Further, MEGF10 is also expressed in glia cells, but it has not been tested if this MEGF10 recognition event is neuron specific.To address these questions, we decided to first characterize the molecular components of the MEGF10 complex. We determined MEGF10 complex composition through co-immunoprecipitation (co-IP) and chemical crosslinking and discovered that MEGF10 forms a lateral complex. Truncation and co-IP studies reveal that the interacting motifs are located on the ectodomain of MEGF10. Such binding is not restricted to MEGF10 as we also discovered hetero-multimers between MEGF10 and MEGF12. Next, to identify other molecules in the MEGF10 signaling pathway, we performed IP to isolate native MEGF10 interacting complexes and conducted proteomic analysis. We found previously known MEGF10-interacting molecules such as Dynamin1 and Traf4, as well as novel MEGF10 associating candidates. Our identification of interacting molecules that facilitate cytoskeletal and membrane rearrangement suggests that MEGF10 activates these cellular processes. Finally, we characterized Müller glia organization through a genetic-based labeling method. With a MEGF10 mutant mouse, we determined that MEGF10 is not necessary for glial array formation. We conclude that MEGF10 has distinct functions in neurons vs. glia. This study sets the stage to describe the molecular mechanism by which MEGF10 mediates cell-cell recognition, potentially uncovered novel MEGF10 interactions, and distinguishes MEGF10 neuronal function from its role in glia.

Developmental cell death is essential for nervous system development, sculpting the developing tissue by controlling cell numbers. While developmental neuron death has been studied extensively, the most abundant cell type of the nervous system – the astrocyte – has often been overlooked. Our lab recently showed that astrocytes in the developing retina undergo an unusual non-apoptotic form of death that eliminates a vast proportion of the original population. Further, we found that microglia are the major effectors of astrocyte death. However, the mechanisms that induce microglia to kill astrocytes remain mysterious. It is important to understand these astrocyte death mechanisms because astrocytes play a crucial role in patterning the retinal blood vessel network. Developmental perturbations to astrocyte number have large effects on their patterning, and in turn cause severe vascular patterning defects – some of which resemble vasculopathies typical of human blinding disorders. Because death has such a major impact on astrocyte number, it presumably has an outsized impact on this critical patterning process. We therefore sought to identify the non-apoptotic mechanisms that drive astrocyte death. Previously, we showed that astrocyte numbers modulate microglial phagocytic activity – increasing this activity as astrocyte numbers rise and decreasing it as astrocyte numbers decline. This observation suggested that astrocytes themselves are the source of cues that drive their own death via recruitment of phagocytic microglia. Here we identify the membrane lipid phosphatidylserine (PtdSer) as one such astrocyte-derived “eat-me” cue. PtdSer is best known as an “eat-me” signal expressed on the surface of apoptotic cells. We show that PtdSer is also externalized on the cell surface of apparently normal astrocytes during the developmental death period. Moreover, using a genetic approach to increase cell-surface PtdSer, we show that it is sufficient to drive astrocyte death. For these studies, we used an astrocyte-specific mouse knockout of Tmem30a, an obligate subunit of the flippase enzymes that normally remove PtdSer from the cell surface. In these knockout animals, microglia are recruited to Tmem30a mutant astrocytes, engulf them, and cause a significant acceleration of cell number decline. This excess astrocyte loss has functional consequences for the development of the vasculature: The astrocytic template for angiogenesis is overly sparse, which leads to vascular patterning defects and delayed angiogenesis. Interestingly, these defects can be rescued by blocking the function of a phagocytic signaling pathway that can recognize PtdSer exposure, suggesting that the excess PtdSer exposure in the Tmem30a knockout animals is responsible for the increase in astrocyte death. Altogether our findings highlight the broad impact of dysregulated astrocyte death. Understanding how astrocyte population size is controlled will provide new insights into death mechanisms that are crucial for development not only in the retina but may also sculpt glial populations elsewhere in the central nervous system.