An overlooked subset of Cx3cr1(wt/wt) microglia in the Cx3cr1(CreER-Eyfp/wt) mouse has a repopulation advantage over Cx3cr1(CreER-Eyfp/wt) microglia following microglial depletion

BACKGROUND: Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1(CreER-Eyfp/wt) mouse strain for studies of microglia. METHODS: Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1(CreER-Eyfp/wt) mouse brains. Genetically mediated microglia depletion using Cx3cr1(CreER-Eyfp/wt)Rosa26(DTA/wt) mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies. RESULTS: We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1(CreER-Eyfp/wt)Cre(+)Eyfp(+) microglia in Cx3cr1(CreER-Eyfp/wt) mouse brains, thus termed Cx3cr1(high)Cre(−)Eyfp(−) microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1(high)Cre(−)Eyfp(−) microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1(high)Cre(−)Eyfp(−) microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1(high)Cre(−)Eyfp(−) microglia are Cx3cr1(wt/wt)Cre(−)Eyfp(−). Finally, we demonstrated that CX3CL1–CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro. CONCLUSIONS: Our results raise a cautionary note regarding the use of Cx3cr1(CreER-Eyfp/wt) mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12974-022-02381-6.