An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion

Abstract

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^highCre^-Eyfp^- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1^highCre^-Eyfp^- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1^highCre^-Eyfp^- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1^highCre^-Eyfp^- microglia are Cx3cr1^wt/wtCre^-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.

Keywords: Cre; Cx3cr1; Diphtheria toxin subunit A (DTA); GFP; Homologous recombination; Loss of heterozygosity (LOH); Microglia; Microglial depletion; Microglial repopulation; YFP.