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

doi:10.1186/s12974-022-02381-6

Abstract

BackgroundFluorescent 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.MethodsImmunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies.ResultsWe unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre−Eyfp− microglia. This subpopulation constituted less than 1% 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 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−. Finally, we demonstrated that CX3CL1–CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro.ConclusionsOur results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

Highlights

Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cel‐ lular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-enhanced yellow fluorescent protein gene (Eyfp)/wt mouse strain for studies of microglia
Flow cytometric analysis confirmed the existence of E YFP− cells in Cx3cr1CreER-Eyfp/wt mice, accounting for less than 1% of total CD11b+CD45+ Ly6C–Ly6G– cells at 3–15 weeks, with no significant differences between ages
To confirm that Cx3cr1highCre−Eyfp−microglia were not an anomaly of one specific mouse strain, we investigated the microglial populations in Cx3cr1GFP/wt mice (JAX, 005582) by flow cytometry and immunohistochemistry

Summary

Introduction

Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cel‐ lular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. Microglia are derived from the yolk sac during early embryonic development and represent approximately 10% of the healthy adult brain’s total cell population [1, 2] They play critical roles in maintaining brain development and function [3, 4]. Cx3cr1Cre/wt and Cx3cr1CreER-Eyfp/wt mouse strains are potent, commonly used tools for studies of microglial fate mapping [1, 24], microglial depletion [25,26,27,28] and modification of the microglial genome by leveraging floxed target genes [29,30,31] These methods have greatly increased our understanding of microglia in CNS homeostasis and disease conditions

Methods

Results

Discussion

Conclusion