An overlooked subset of Cx3cr1wt/wt microglia lacking Cre and EYFP expression in Cx3cr1CreER-Eyfp/wt mouse

Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1 percent of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further showed that GFP- microglia could also be detected in the Cx3cr1Gfp/wt mouse strain. Finally, we demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Our results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt and Cx3cr1Gfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.