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
Kai Zhou, Jinming Han, Harald Lund, Nageswara Rao Boggavarapu, Volker M. Lauschke, Shinobu Goto, Huaitao Cheng, Yuyu Wang, Asuka Tachi, Cuicui Xie, Keying Zhu, Ying Sun, Ahmed M. Osman, Dong Liang, Wei Han, Kristina Gemzell‐Danielsson, Christer Betsholtz, Xing‐Mei Zhang, Changlian Zhu, Martin Enge, Bertrand Joseph, Robert A. Harris, Klas Blomgren
Abstract
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 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.