2. Academic Validation
  3. 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

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

  • J Neuroinflammation. 2022 Jan 21;19(1):20. doi: 10.1186/s12974-022-02381-6.
Kai Zhou 1 2,  Jinming Han 3 4,  Harald Lund 3 5,  Nageswara Rao Boggavarapu 6,  Volker M Lauschke 5 7 8,  Shinobu Goto 6 9,  Huaitao Cheng 10,  Yuyu Wang 6,  Asuka Tachi 6 11,  Cuicui Xie 6,  Keying Zhu 3,  Ying Sun 12,  Ahmed M Osman 6,  Dong Liang 6,  Wei Han 6,  Kristina Gemzell-Danielsson 6 13,  Christer Betsholtz 12 14,  Xing-Mei Zhang 3,  Changlian Zhu 15 16,  Martin Enge 10,  Bertrand Joseph 17,  Robert A Harris 3,  Klas Blomgren 18 19 20
Affiliations

Affiliations

  • 1 Henan Neurodevelopment Engineering Research Center for Children, Children's Hospital Affiliated to Zhengzhou University, Zhengzhou, China. [email protected].
  • 2 Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden. [email protected].
  • 3 Applied Immunology and Immunotherapy, Department of Clinical Neuroscience, Karolinska Institutet, Center for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden.
  • 4 Neuroimmunology Center, Department of Neurology, Xuanwu Hospital, Capital Medical University, National Center for Neurological Disorders, Beijing, China.
  • 5 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
  • 6 Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden.
  • 7 Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
  • 8 University of Tuebingen, Tuebingen, Germany.
  • 9 Department of Obstetrics and Gynecology, Nagoya City University Hospital, Nagoya, Japan.
  • 10 Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden.
  • 11 Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • 12 Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
  • 13 WHO-Centre, Karolinska University Hospital, Stockholm, Sweden.
  • 14 Department of Medicine Huddinge, Karolinska Institutet, Campus Flemingsberg, Huddinge, Sweden.
  • 15 Centre for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, University of Gothenburg, Gothenburg, Sweden.
  • 16 Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center, The Third Affiliated Hospital and Institute of Neuroscience, Zhengzhou University, Zhengzhou, China.
  • 17 Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden.
  • 18 Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden. [email protected].
  • 19 Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center, The Third Affiliated Hospital and Institute of Neuroscience, Zhengzhou University, Zhengzhou, China. [email protected].
  • 20 Pediatric Oncology, Karolinska University Hospital, Stockholm, Sweden. [email protected].
PMID: 35062962 DOI: 10.1186/s12974-022-02381-6
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 CX3CR1CreER-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 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.

Results: We 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.

Conclusions: Our 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.

Keywords

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

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