In the central nervous system, endothelial cells (ECs) and pericytes (PCs) of blood vessel walls cooperatively form a physical and chemical barrier to maintain neural homeostasis. However, in diabetic retinopathy (DR), the loss of PCs from vessel walls is assumed to cause breakdown of the blood-retina barrier (BRB) and subsequent vision-threatening vascular dysfunctions. Nonetheless, the lack of adequate DR animal models has precluded disease understanding and drug discovery. Here, by using an anti-PDGFRβ antibody, we show that transient inhibition of the PC recruitment to developing retinal vessels sustained EC-PC dissociations and BRB breakdown in adult mouse retinas, reproducing characteristic features of DR such as hyperpermeability, hypoperfusion, and neoangiogenesis. Notably, PC depletion directly induced inflammatory responses in ECs and perivascular infiltration of macrophages, whereby macrophage-derived VEGF and placental growth factor (PlGF) activated VEGFR1 in macrophages and VEGFR2 in ECs. Moreover, angiopoietin-2 (Angpt2) upregulation and Tie1 downregulation activated FOXO1 in PC-free ECs locally at the leaky aneurysms. This cycle of vessel damage was shut down by simultaneously blocking VEGF, PlGF, and Angpt2, thus restoring the BRB integrity. Together, our model provides new opportunities for identifying the sequential events triggered by PC deficiency, not only in DR, but also in various neurological disorders.

An array of lipidic compounds that constitute the cell wall of mycobacteria is recognized by host receptors. Examples include trehalose dimycolate (TDM), which is a major surface-exposed glycolipid of mycobacteria, that interacts with the macrophage inducible C-type lectin, Mincle, and exerts its highly potent adjuvant functions. Recent evidence has suggested that glycerol monomycolate (GroMM), another mycolate-containing lipid species produced by mycobacteria, can stimulate innate immune cells; however, its specific host receptors have yet to be identified. We here demonstrated that cell transfectants expressing human Mincle (hMincle) reacted to both TDM and GroMM, while those expressing mouse Mincle (mMincle) only reacted to TDM and failed to recognize GroMM. Studies using domain swap chimeras confirmed that the ectodomain of hMincle, but not that of mMincle, interacted with GroMM, and site-directed mutagenesis analyses revealed that short stretches of amino acid residues at positions 174–176 and 195–196 were involved in GroMM recognition. To further substantiate the differential recognition of GroMM by hMincle and mMincle, hMincle transgenic/mMincle knock-out mice (i.e. hMincle⁺ mice) were established and compared with non-transgenic mice (i.e. mMincle⁺ mice). We showed that macrophages derived from hMincle⁺ mice were activated by GroMM and produced inflammatory cytokines, whereas those derived from mMincle⁺ mice did not exhibit any reactivity to GroMM. Furthermore, local inflammatory responses were elicited in the GroMM-injected skin of hMincle⁺, but not mMincle⁺ mice. These results demonstrated that GroMM is a unique ligand for hMincle that is not recognized by mMincle.

The relationships between tissue-resident microglia and early macrophages, especially their lineage segregation outside the yolk sac, have been recently explored, providing a model in which a conversion from macrophages seeds microglia during brain development. However, spatiotemporal evidence to support such microglial seeding in situ and to explain how it occurs has not been obtained. By cell tracking via slice culture, intravital imaging, and Flash tag-mediated or genetic labeling, we find that intraventricular CD206⁺ macrophages, which are abundantly observed along the inner surface of the mouse cerebral wall, frequently enter the pallium at embryonic day 12. Immunofluorescence of the tracked cells show that postinfiltrative macrophages in the pallium acquire microglial properties while losing the CD206⁺ macrophage phenotype. We also find that intraventricular macrophages are supplied transepithelially from the roof plate. This study demonstrates that the "roof plate→ventricle→pallium" route is an essential path for microglial colonization into the embryonic mouse brain.

In the developing cortex, postmigratory neurons accumulate in the cortical plate (CP) to properly differentiate consolidating subtype identities. Microglia, despite their extensive surveying activity, temporarily disappear from the midembryonic CP. However, the mechanism and significance of this absence are unknown. Here, we show that microglia bidirectionally migrate via attraction by CXCL12 released from the meninges and subventricular zone and thereby exit the midembryonic CP. Upon nonphysiological excessive exposure to microglia in vivo or in vitro, young postmigratory and in vitro-grown CP neurons showed abnormal differentiation with disturbed expression of the subtype-associated transcription factors and genes implicated in functional neuronal maturation. Notably, this effect is primarily attributed to interleukin 6 and type I interferon secreted by microglia. These results suggest that “sanctuarization” from microglia in the midembryonic CP is required for neurons to appropriately fine-tune the expression of molecules needed for proper differentiation, thus securing the establishment of functional cortical circuit.

Microglia are the resident immune cells of the central nervous system. Microglial progenitors are generated in the yolk sac during the early embryonic stage. Once microglia enter the brain primordium, these cells colonize the structure through migration and proliferation during brain development. Microglia account for a minor population among the total cells that constitute the developing cortex, but they can associate with many surrounding neural lineage cells by extending their filopodia and through their broad migration capacity. Of note, microglia change their distribution in a stage-dependent manner in the developing brain: microglia are homogenously distributed in the pallium in the early and late embryonic stages, whereas these cells are transiently absent from the cortical plate (CP) from embryonic day (E) 15 to E16 and colonize the ventricular zone (VZ), subventricular zone (SVZ), and intermediate zone (IZ). Previous studies have reported that microglia positioned in the VZ/SVZ/IZ play multiple roles in neural lineage cells, such as regulating neurogenesis, cell survival and neuronal circuit formation. In addition to microglial functions in the zones in which microglia are replenished, these cells indirectly contribute to the proper maturation of post-migratory neurons by exiting the CP during the mid-embryonic stage. Overall, microglial time-dependent distributional changes are necessary to provide particular functions that are required in specific regions. This review summarizes recent advances in the understanding of microglial colonization and multifaceted functions in the developing brain, especially focusing on the embryonic stage, and discuss the molecular mechanisms underlying microglial behaviors.