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
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE186700 | GEO | 2022/02/01
REPOSITORIES: GEO
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Project description: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
| PRJNA775631 | ENA
Project description: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.
| S-EPMC8783445 | biostudies-literature
Project description:ER71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an ER71-EYFP transgenic mouse model by fusing the 3.9 kb ER71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP+ populations of cells in ER71 mutant and wildtype littermates. In the absence of ER71, we observed an increase in the number of EYFP expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program compared to the wildtype littermate controls. We have also generated a novel ER71-Cre transgenic mouse model using the same 3.9 kb ER71 promoter. Genetic fate mapping studies revealed that the ER71 expressing cells daughter hematopoietic and endothelial lineages in the wildtype background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages including the cardiac lineage. To extend these analyses, we used an inducible ES/EB system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions, acting to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis. 12samples were analyzed, including triplicates of WT; EYFP positive, WT EYFP negative, ER71 MT; EYFP positive and ER71 MT; EYFP negative cells
2011-11-02 | E-GEOD-32223 | biostudies-arrayexpress
Project description:C/EBPalpha is a transcription factor critically involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor (LSK) cells, which express C/EBPalpha, we developed a mouse model expressing Cre recombinase from the Cebpa promoter and an inducible EYFP allele. We show that Cebpa/EYFP+ cells represent a significant subset of LSK cells, which predominantly give rise to myeloid cells in steady state hematopoiesis. C/EBPalpha induced a robust myeloid gene expression signature and downregulated E2A-induced regulators of early lymphoid development. In addition, Cebpa/EYFP+ cells comprise a fraction of early thymic progenitors (ETP) with robust myeloid potential. However, Cebpa/EYFP+ LSK and ETP cells retained the ability to develop into erythroid and T-lymphoid lineages, respectively. These findings support an instructive, but argue against a lineage restrictive role of C/EBPalpha in multipotent hematopoietic and thymic progenitors. We performed global gene expression profiling of double-sorted Cebpa/EYFP+ and Cebpa/EYFP- LSK cells of pooled Cebpa Cre/wt R26 EYFP reporter mice to identify differentially regulated genes in Cebpa+ versus Cebpa- LSK cells. RNA was isolated from three biological replicates of Cebpa/EYFP+ LSK cells and two biological replicates of Cebpa/EYFP- LSK cells. To determine if the identified genes were truly dependent on Cebpa expression, we also performed global gene expression profilling of Cebpa/EYFP+ and Cebpa/EYFP- fetal liver LSK cells of Cebpa Cre/fl R26 EYFP mice. Induction of Cebpa/Cre expression in these mice leads to Cre-mediated recombination of the floxed wt Cebpa allele resulting in a complete Cebpa knock-out. In this case, RNA was isolated from two biological replicates of either Cebpa/EYFP+ and Cebpa/EYFP- LSK cells. In addition, we included one biological replicate of Cebpa/EYFP+ and Cebpa/EYFP- fetal liver LSK cells of Cebpa Cre/wt R26 EYFP mice to determine the correlation of differentially regulated genes in bone marrow and fetal liver LSK cells.
2010-08-26 | E-GEOD-23800 | biostudies-arrayexpress
Project description:ER71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an ER71-EYFP transgenic mouse model by fusing the 3.9 kb ER71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP+ populations of cells in ER71 mutant and wildtype littermates. In the absence of ER71, we observed an increase in the number of EYFP expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program compared to the wildtype littermate controls. We have also generated a novel ER71-Cre transgenic mouse model using the same 3.9 kb ER71 promoter. Genetic fate mapping studies revealed that the ER71 expressing cells daughter hematopoietic and endothelial lineages in the wildtype background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages including the cardiac lineage. To extend these analyses, we used an inducible ES/EB system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions, acting to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis.
2011-11-02 | GSE32223 | GEO
Project description:C/EBPalpha is a transcription factor critically involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor (LSK) cells, which express C/EBPalpha, we developed a mouse model expressing Cre recombinase from the Cebpa promoter and an inducible EYFP allele. We show that Cebpa/EYFP+ cells represent a significant subset of LSK cells, which predominantly give rise to myeloid cells in steady state hematopoiesis. C/EBPalpha induced a robust myeloid gene expression signature and downregulated E2A-induced regulators of early lymphoid development. In addition, Cebpa/EYFP+ cells comprise a fraction of early thymic progenitors (ETP) with robust myeloid potential. However, Cebpa/EYFP+ LSK and ETP cells retained the ability to develop into erythroid and T-lymphoid lineages, respectively. These findings support an instructive, but argue against a lineage restrictive role of C/EBPalpha in multipotent hematopoietic and thymic progenitors.
2010-08-26 | GSE23800 | GEO
Project description:Although most tissue macrophages are embryonically derived it is evident that circulating monocytes can compete for virtually any macrophage niche. Monocytes can thus become long-lived replacements of tissue macrophages that are indistinguishable from their embryonic counterparts, but the factors regulating this process are incompletely understood. To study niche competition in the CNS we depleted microglia with >95% efficiency using CX3CR1CreER/+R26DTA/+ mice and monitored long-term repopulation. The microglial niche was repopulated within weeks by a combination of local microglia proliferation giving rise to CX3CR1+F4/80lowClec12a– microglia, as well as infiltration of CX3CR1+F4/80hiClec12a+ macrophages. Adoptive transfer experiments demonstrated that peripherally-derived macrophages arose directly from Ly6Chi monocytes without contribution from hematopoietic progenitors and was independent of BBB breakdown. After repopulation we sorted microglia and monocyte-derived macrophages and performed transcriptional, epigenetic (DNA methylation) and ex vivo functional profiling. This revealed that Ly6Chi monocytes upregulated microglia gene expression and adopted microglia DNA methylation signatures. However, in contrast to proliferating microglia, which rapidly regained their homeostatic gene signature, monocyte-derived macrophages retained a distinct gene signature associated with antigen presentation, interferon signaling and chemotaxis. This translated into functional changes in monocyte-derived macrophages, as demonstrated by differences in surface marker expression, phagocytosis and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct. This may have implications for neuroinflammatory disease states and direct design of novel therapies.
2018-10-19 | GSE121409 | GEO
Project description:Cx3cr1CreER driven Cre-recombinase (Cre) is a widely used genetic tool for enabling gene manipulation in microglia and macrophages. However, an in-depth analysis for the possible detrimental effects of Cre-activity in microglia, surprisingly remains missing. Here we demonstrate an age-dependent sensitivity of microglia to Cx3cr1-Cre-toxicity, wherein Cre-induction specifically in early postnatal microglia is detrimental for microglial development, proliferation and function. Tamoxifen (TAM) induced Cre-activity leads to microglial activation, type1-interferon (IFN-1) signaling and increased phagocytosis, causing aberrant synaptic pruning during early postnatal period and anxiety behavior in later age. The detrimental effects of Cre-induction are caused due to DNA-damage induced toxicity in microglia, and is limited to the early postnatal period, showing no detrimental effects in adult microglia. Thus, our study reveals the age-dependent vulnerability of microglia to Cre-activity, thereby highlighting age-dependencies of Cre-action, which could be especially applicable in the broader context of environment-responsive cell-types.
2021-12-08 | GSE190207 | GEO
Project description:Although most tissue macrophages are embryonically derived it is evident that circulating monocytes can compete for virtually any macrophage niche. Monocytes can thus become long-lived replacements of tissue macrophages that are indistinguishable from their embryonic counterparts, but the factors regulating this process are incompletely understood. To study niche competition in the CNS we depleted microglia with >95% efficiency using CX3CR1CreER/+R26DTA/+ mice and monitored long-term repopulation. The microglial niche was repopulated within weeks by a combination of local microglia proliferation giving rise to CX3CR1+F4/80lowClec12a– microglia, as well as infiltration of CX3CR1+F4/80hiClec12a+ macrophages. Adoptive transfer experiments demonstrated that peripherally-derived macrophages arose directly from Ly6Chi monocytes without contribution from hematopoietic progenitors and was independent of BBB breakdown. After repopulation we sorted microglia and monocyte-derived macrophages and performed transcriptional, epigenetic (DNA methylation) and ex vivo functional profiling. This revealed that Ly6Chi monocytes upregulated microglia gene expression and adopted microglia DNA methylation signatures. However, in contrast to proliferating microglia, which rapidly regained their homeostatic gene signature, monocyte-derived macrophages retained a distinct gene signature associated with antigen presentation, interferon signaling and chemotaxis. This translated into functional changes in monocyte-derived macrophages, as demonstrated by differences in surface marker expression, phagocytosis and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct. This may have implications for neuroinflammatory disease states and direct design of novel therapies. Data was processed using the mEPIC tool PMID:29141580
2018-10-19 | GSE121483 | GEO