Microglia are resident immune cells that play critical roles in maintaining normal physiology of the central nervous system. Remarkably, microglia have intrinsic capacity to replenish after being acutely ablated. However, the underlying mechanisms that drive such microglial restoration remain elusive. Here, we removed microglia via CSF1R inhibitor PLX5622 and characterized repopulation both spatially and temporally. We also investigated the cellular origin of repopulated microglia and report that microglia are replenished via self-renewal, with little contribution from non-microglial lineage progenitors, including nestin+ progenitors and the circulating myeloid population. Interestingly, spatial analyses with multi-color labeling reveal that newborn microglia recolonize the parenchyma by forming distinctive clusters that maintain stable territorial boundaries over time, indicating proximal expansion nature of adult microgliogenesis and stability of microglia tiling. Temporal transcriptome profiling from newborn microglia at different repopulation stages revealed that the adult newborn microglia gradually regain steady-state maturity from an immature state that is reminiscent of neonatal stage and follow a series of maturation programs that include NF-kB activation, interferon immune activation and apoptosis, etc. Importantly, we show that the restoration of microglial homeostatic density requires NF-kB signaling as well as apoptotic egress of excessive cells. In summary, our study reports key events that take place from microgliogenesis to homeostasis re-establishment.

Proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain