As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include border-associated meningeal, perivascular and choroid plexus macrophages. Using immunofluorescent labeling, flow cytometry and Cre-dependent ribosomal immunoprecipitations, we describe P2ry12-CreER, a new tool for the genetic targeting of microglia. We use this new tool to track microglia during embryonic development and in the context of ischemic injury and neuro-inflammation. Because of the specificity and robustness of microglial recombination with P2ry12-CreER, we believe that this new mouse line will be particularly useful for future studies of microglial function in development and disease.

As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. Developmentally, microglia originate from the embryonic yolk sac and serve important roles in the sculpting of neonatal neural circuits. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include borderzone (meningeal and perivascular) and choroid plexus macrophages. To distinguish between resident microglia and these other CNS macrophage subtypes, we generated a P2ry12-CreER mouse line. P2RY12 is a microglialspecific nucleotide sensing GPCR that is important for microglial response to tissue damage. Using immunofluorescent labeling and flow cytometry experiments, we show that P2ry12-CreER recombination is exceptionally specific to parenchymal microglia. We also perform ribosome immunoprecipitations and transcriptional profiling of P2ry12-CreER recombined cells, using a Cre-dependent Rpl22-HA mouse line. By identifying genes enriched in this dataset that are not correspondingly enriched in a broader Cx3CR1-CreER; Rpl22 dataset, we isolate a number of borderzone macrophage-specific transcripts, including the gene PF4. Using a PF4-Cre mouse line, we show that PF4 expression robustly marks borderzone macrophages. Together, we demonstrate two new methods to genetically target distinct CNS macrophage subtypes.

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia progenitor-expressed integrin beta 8 activates microglia-expressed TGFB1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary domains of developmentally arrested dysmature microglia and homeostatic microglia that persist into adulthood. In the absence of autocrine TGFB1 signaling, we find that microglia adopt a similar reactive microglial phenotype, leading to astrogliosis and neuromotor symptoms almost identical to Itgb8 mutant mice. By comparing mice with genetic deletions in critical components downstream of Itgb8, we show that non-canonical (Smad-independent) signaling partially suppresses the dysmature microglia phenotype, associated neuromotor dysfunction and expression of disease-associated genes, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

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As the resident macrophage cells of the brain, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. Developmentally, microglia originate from the embryonic yolk sac and serve important roles in the sculpting of neonatal neural circuits. In the context of severe brain injury or infection, bone‐marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. Although bone‐marrow derived macrophages can adopt some features of endogenous microglia, they are not able to fully recapitulate all of their properties, suggesting that there may be important functional differences between these two populations of cells. To distinguish between resident microglia and circulating myelogenous macrophages, we generated a P2ry12‐CreER mouse line. P2RY12 is a microglial‐specific nucleotide sensing GPCR that is important for microglial response to tissue damage. The P2ry12‐CreER line was generated by CRISPR‐facilitated homologous recombination and insertion of a 2A‐CreER sequence into the 3’ end of the P2RY12 coding sequence. Using immunofluorescent labeling and flow cytometry, we determined whether this recombination was specific to parenchymal microglia, as compared to meningeal, choroid plexus or perivascular macrophages. We also describe preliminary experiments that involve transcriptional profiling of P2ry12‐CreER recombined cells, using a Cre‐dependent Rpl22‐HA mouse line. Finally, using mouse models of neonatal and adult stroke, we used the P2ry12‐CreER line to fate map true brain microglia in the context of brain injury. Altogether, these results demonstrate the utility of this novel mouse line for genetic recombination, gene expression and fate‐mapping studies of brain microglia.Support or Funding InformationNIH K08NS096192This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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