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 PMID: 35062962

ADC Targets in Oncology

2026-04-10

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

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].

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.