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A A mouse model and experimental timeline for targeting microglial Alk5 and analyzing microglia homeostatic marker expression and microgliosis 3 weeks post tamoxifen. B , C Representative images of the hippocampus stained for IBA1, TMEM119, <t>P2RY12</t> (BioLegend Ab), and GFAP in B Cx3cr1 CreER -ALK5 wt/wt or C Cx3cr1 CreER -ALK5 fl/fl mice D FACS-sorted GFP+ microglia were analyzed for mRNA levels using qRT-PCR showing E Alk5 (indicating gene deletion efficiency), F Tmem119, G P2ry12, H Tnf, and I Igf1 expression levels in control and iKO microglia ( E – I ) ( n = 3 for control, n = 3 for Alk5 KO, * p = 0.0146 for panel E, *** p = 0.0005 for panel F, *** p = 0.0004 for panel G, * p = 0.0372 for panel H, * p = 0.0167 for panel I, two-sided student’s t-test was used for statistical analysis for panels E-I). Representative immunohistochemistry images of IBA1, Ki67, and DAPI in the cortex of J Cx3cr1 CreER -ALK5 wt/wt or K Cx3cr1 CreER -ALK5 fl/fl at 3 weeks after TAM treatment. Arrows show Ki67+ cells (yellow arrows show Ki67+ cells not co-localized with IBA1+ cells and white arrows show Ki67+ cells co-localizing with IBA1+ cells). Unbiased stereological quantification for L Microglia density, M Astrocyte density, N total number of Ki67+ cells, and O the number of IBA1 + /Ki67+ cells ( L – O ) ( n = 4 for control, n = 3 for Alk5 KO, ** p = 0.002, *** p < 0.001, and ** p = 0.001 for panel L, ns = not significant for panel M , ** p < 0.001, p < 0.00, and p = 0.011 for panel N , ** p < 0.001, *** p < 0.001, and ** p = 0.006 for panel O , two-sided 2-way ANOVA with Tukey post hoc pairwise comparison was used for statistical analysis for panels L – O ). Each data point represents the average of a single animal ( > 3 brain sections per mouse for each brain region). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/0xildk2 . Source data are provided as a source data file.

P2ry12 Creer, supplied by Jackson Laboratory, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice"

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

Journal: Nature Communications

doi: 10.1038/s41467-026-68885-4

Figure Legend Snippet: A A mouse model and experimental timeline for targeting microglial Alk5 and analyzing microglia homeostatic marker expression and microgliosis 3 weeks post tamoxifen. B , C Representative images of the hippocampus stained for IBA1, TMEM119, P2RY12 (BioLegend Ab), and GFAP in B Cx3cr1 CreER -ALK5 wt/wt or C Cx3cr1 CreER -ALK5 fl/fl mice D FACS-sorted GFP+ microglia were analyzed for mRNA levels using qRT-PCR showing E Alk5 (indicating gene deletion efficiency), F Tmem119, G P2ry12, H Tnf, and I Igf1 expression levels in control and iKO microglia ( E – I ) ( n = 3 for control, n = 3 for Alk5 KO, * p = 0.0146 for panel E, *** p = 0.0005 for panel F, *** p = 0.0004 for panel G, * p = 0.0372 for panel H, * p = 0.0167 for panel I, two-sided student’s t-test was used for statistical analysis for panels E-I). Representative immunohistochemistry images of IBA1, Ki67, and DAPI in the cortex of J Cx3cr1 CreER -ALK5 wt/wt or K Cx3cr1 CreER -ALK5 fl/fl at 3 weeks after TAM treatment. Arrows show Ki67+ cells (yellow arrows show Ki67+ cells not co-localized with IBA1+ cells and white arrows show Ki67+ cells co-localizing with IBA1+ cells). Unbiased stereological quantification for L Microglia density, M Astrocyte density, N total number of Ki67+ cells, and O the number of IBA1 + /Ki67+ cells ( L – O ) ( n = 4 for control, n = 3 for Alk5 KO, ** p = 0.002, *** p < 0.001, and ** p = 0.001 for panel L, ns = not significant for panel M , ** p < 0.001, p < 0.00, and p = 0.011 for panel N , ** p < 0.001, *** p < 0.001, and ** p = 0.006 for panel O , two-sided 2-way ANOVA with Tukey post hoc pairwise comparison was used for statistical analysis for panels L – O ). Each data point represents the average of a single animal ( > 3 brain sections per mouse for each brain region). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/0xildk2 . Source data are provided as a source data file.

Techniques Used: Marker, Expressing, Staining, Quantitative RT-PCR, Control, Immunohistochemistry, Comparison

A Mouse model for targeting microglial ( P2ry12 CreER/CreER ) Alk5 in adult mice to examine adult neurogenesis in SGZ. B – C Representative images showing immunohistochemistry staining for IBA1, TMEM119, and P2RY12 (Clone P2YM 1E5) in B control mice and C P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. D , E Representative images showing immunohistochemistry staining for DCX and Ki67 in D control mice and E P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. F – I Quantification for F TMEM119 immunoreactivity, G P2RY12 immunoreactivity, H DCX+ cells quantification compared to the wildtype mean, and I quantification of dendritic arborization (% of immunoreactive positive area) compared to the wildtype mean. F ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); G ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); H ( n = 6 for WT and n = 6 for KO, * p = 0.0359); I ( n = 6 for WT and n = 6 for KO, *** p = 0.0009). Each data point represents the average of a single animal (3–6 brain sections analyzed per mouse) and n = 6 for each group (wildtype or knockout). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Two-sided Student’s t-test for all panels except Welch’s t-test was used for panel G. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/1834acf . Source data are provided as a source data file.

Figure Legend Snippet: A Mouse model for targeting microglial ( P2ry12 CreER/CreER ) Alk5 in adult mice to examine adult neurogenesis in SGZ. B – C Representative images showing immunohistochemistry staining for IBA1, TMEM119, and P2RY12 (Clone P2YM 1E5) in B control mice and C P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. D , E Representative images showing immunohistochemistry staining for DCX and Ki67 in D control mice and E P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. F – I Quantification for F TMEM119 immunoreactivity, G P2RY12 immunoreactivity, H DCX+ cells quantification compared to the wildtype mean, and I quantification of dendritic arborization (% of immunoreactive positive area) compared to the wildtype mean. F ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); G ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); H ( n = 6 for WT and n = 6 for KO, * p = 0.0359); I ( n = 6 for WT and n = 6 for KO, *** p = 0.0009). Each data point represents the average of a single animal (3–6 brain sections analyzed per mouse) and n = 6 for each group (wildtype or knockout). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Two-sided Student’s t-test for all panels except Welch’s t-test was used for panel G. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/1834acf . Source data are provided as a source data file.

Techniques Used: Immunohistochemistry, Staining, Control, Knock-Out

A mouse model used to B micro-dissect the hippocampus and process for single cell 10x Genomic Flex sequencing. C UMAP clustering of cells with annotation for immature neurons and microglia. D Feature plot of Aif1 (coding IBA1 protein) marker to denote microglia cluster. E Feature plot of Dcx to denote DCX+ immature neuroblasts/neuron cluster. F – I Feature plots of Tmem119, P2ry12, Aldh1l1 and Gfap to denote enrichment of microglial signature genes but not astrocytic signature genes in the microglia cluster. J DEGs in MG- Alk5 iKO derived microglia compared to control microglia presented as a volcano plot. K DEGs in MG- Alk5 iKO derived neuroblasts compared to control neuroblasts presented as a volcano plot. L LIANA was used to identify cell-cell interactions and show strong interaction between microglia-endothelial cells and microglia-neuroblasts with identified potential ligand-receptor pairs. M , N EnrichR pathway analysis of DEGs from neuroblasts in MG- Alk5 or Tgfb1 iKO mice with tables denoting top altered disease/cellular processes and molecular pathways. (DEGs) p values were calculated using the Wald test with adjustments for multiple comparisons corrected using the Benjamini-Hochberg method, and (GO Process) p values were calculated using Fisher’s Exact test with adjustments for multiple comparisons were made using Benjamini-Hochberg method. Panel A and B was created in BioRender. Luo, A. (2026) https://BioRender.com/10eqn6o . Source data are provided as a source data file.

Figure Legend Snippet: A mouse model used to B micro-dissect the hippocampus and process for single cell 10x Genomic Flex sequencing. C UMAP clustering of cells with annotation for immature neurons and microglia. D Feature plot of Aif1 (coding IBA1 protein) marker to denote microglia cluster. E Feature plot of Dcx to denote DCX+ immature neuroblasts/neuron cluster. F – I Feature plots of Tmem119, P2ry12, Aldh1l1 and Gfap to denote enrichment of microglial signature genes but not astrocytic signature genes in the microglia cluster. J DEGs in MG- Alk5 iKO derived microglia compared to control microglia presented as a volcano plot. K DEGs in MG- Alk5 iKO derived neuroblasts compared to control neuroblasts presented as a volcano plot. L LIANA was used to identify cell-cell interactions and show strong interaction between microglia-endothelial cells and microglia-neuroblasts with identified potential ligand-receptor pairs. M , N EnrichR pathway analysis of DEGs from neuroblasts in MG- Alk5 or Tgfb1 iKO mice with tables denoting top altered disease/cellular processes and molecular pathways. (DEGs) p values were calculated using the Wald test with adjustments for multiple comparisons corrected using the Benjamini-Hochberg method, and (GO Process) p values were calculated using Fisher’s Exact test with adjustments for multiple comparisons were made using Benjamini-Hochberg method. Panel A and B was created in BioRender. Luo, A. (2026) https://BioRender.com/10eqn6o . Source data are provided as a source data file.

Techniques Used: Single Cell, Sequencing, Marker, Derivative Assay, Control

A , B In vivo animal models used and experimental timeline. C Representative images of Cx3cr1 CreER -ALK5 WT/WT or Cx3cr1 CreER -ALK5 fl/fl treated with either Vehicle (VEH) or Rapamycin (RAPA) showing immunohistochemistry staining for DCX and IBA1 3 weeks after Tamoxifen treatment. Yellow Arrowhead shows DCX+ cells migrated out of the SGZ inner layer. D Percentage of total DCX+ cells compared to the wildtype mean and E percentage area of dendritic arborization compared to the wildtype mean. F Percentage of migrated DCX+ cells relative to the total number of DCX+ cells per mouse. G , H Representative images of WT or iKO mice treated with either VEH or RAPA showing immunohistochemistry staining for DCX and pS6 3 weeks after TAM treatment. D – G WT VEH n = 7, KO VEH n = 4, WT RAPA n = 5, and KO RAPA n = 4 (ns=not significant ( D – G ); *** p < 0.001, ** p = 0.01 D ; ** p = 0.003, *** p < 0.001 E ; * p = 0.042, *** p < 0.001 F ; * p = 0.048, ** p = 0.01, *** p < 0.001 G ). The sex of each animal is represented by open circles (females) and open triangles (males). I In vitro experimental model and timeline for primary adult NSCs and microglia ( P2ry12 CreER/CreER - tdTomato) 3D co-culture. J Representative images of a differentiated neuroimmune 3D coculture sphere showing microglial tdTomato, GFAP and TUJI immunostaining (maximum projection from Z stacked confocal images). K , L Representative images of the 3D coculture spheres quantification of the TUJI immunoreactive density in the spheres after differentiation with indicated microglia genotypes and VEH or RAPA treatment (WT VEH n = 4, iKO VEH n = 6, WT RAPA n = 6, iKO RAPA n = 4) (ns=not significant, * p = 0.0352, *** p = 0.001 K ). M A proposed model of mechanistic action through microglia onto adult-born neurons. Mean ± SE. Scale bar = 100 µm, Two-way ANOVA test, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis for all panels. Panel A , B , I and M were created in BioRender. Luo, A. (2026) https://BioRender.com/sxk2w6x . Source data are provided as a source data file.

Figure Legend Snippet: A , B In vivo animal models used and experimental timeline. C Representative images of Cx3cr1 CreER -ALK5 WT/WT or Cx3cr1 CreER -ALK5 fl/fl treated with either Vehicle (VEH) or Rapamycin (RAPA) showing immunohistochemistry staining for DCX and IBA1 3 weeks after Tamoxifen treatment. Yellow Arrowhead shows DCX+ cells migrated out of the SGZ inner layer. D Percentage of total DCX+ cells compared to the wildtype mean and E percentage area of dendritic arborization compared to the wildtype mean. F Percentage of migrated DCX+ cells relative to the total number of DCX+ cells per mouse. G , H Representative images of WT or iKO mice treated with either VEH or RAPA showing immunohistochemistry staining for DCX and pS6 3 weeks after TAM treatment. D – G WT VEH n = 7, KO VEH n = 4, WT RAPA n = 5, and KO RAPA n = 4 (ns=not significant ( D – G ); *** p < 0.001, ** p = 0.01 D ; ** p = 0.003, *** p < 0.001 E ; * p = 0.042, *** p < 0.001 F ; * p = 0.048, ** p = 0.01, *** p < 0.001 G ). The sex of each animal is represented by open circles (females) and open triangles (males). I In vitro experimental model and timeline for primary adult NSCs and microglia ( P2ry12 CreER/CreER - tdTomato) 3D co-culture. J Representative images of a differentiated neuroimmune 3D coculture sphere showing microglial tdTomato, GFAP and TUJI immunostaining (maximum projection from Z stacked confocal images). K , L Representative images of the 3D coculture spheres quantification of the TUJI immunoreactive density in the spheres after differentiation with indicated microglia genotypes and VEH or RAPA treatment (WT VEH n = 4, iKO VEH n = 6, WT RAPA n = 6, iKO RAPA n = 4) (ns=not significant, * p = 0.0352, *** p = 0.001 K ). M A proposed model of mechanistic action through microglia onto adult-born neurons. Mean ± SE. Scale bar = 100 µm, Two-way ANOVA test, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis for all panels. Panel A , B , I and M were created in BioRender. Luo, A. (2026) https://BioRender.com/sxk2w6x . Source data are provided as a source data file.

Techniques Used: In Vivo, Immunohistochemistry, Staining, In Vitro, Co-Culture Assay, Immunostaining, Comparison

A The mouse model for single Alk5 iKO or double Alk5/Igf1 iKO and experimental timeline (right) for targeting microglial Igf1 and/or Alk5 and analyzing immature neurons 3 weeks post tamoxifen. Representative images of immunohistochemistry staining of the hippocampus from tamoxifen-treated (3 weeks post) A Cx3Cr1 CreER(WT/WT) , B Cx3Cr1 CreER -Igf1 fl/fl , C Cx3Cr1 CreER -Alk5 fl/fl , D Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl tissue showing IBA1, TMEM119, P2RY12 (P2YM 1E5) staining. E – G Representative DCX staining in SGZ in above mouse lines. Quantification of mRNA levels from Cx3Cr1 CreER(WT/WT) , Cx3Cr1 CreER -Alk5 fl/fl , or Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl sorted microglia (normalized to Hmbs1) showing levels of H Hprt1 , I Iba1 , J Alk5 , and K Igf1 relative to Cx3Cr1 CreER(WT/WT) mice( n = 3 for all groups in panels ( H – K )) (ns=not significant ( H – I ); **** p < 0.0001 and ns=not significant J ; *** p = 0.0001 and p = 0.0001 and ns=not significant K ). L Quantification of the number of DCX+ cells and M dendritic arborization of DCX+ cells compared to WT mean ( n = 12 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO L ; n = 10 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO M ) (** p = 0.0071 and p = 0.0011 and ns=not significant for panel L ; **** p < 0.0001 *** p = 0.0005 and ns=not significant M ). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. One-way ANOVA, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis in all panels ( H –M). Scale bar = 100 µm. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/egkiitm . Source data are provided as a source data file.

Figure Legend Snippet: A The mouse model for single Alk5 iKO or double Alk5/Igf1 iKO and experimental timeline (right) for targeting microglial Igf1 and/or Alk5 and analyzing immature neurons 3 weeks post tamoxifen. Representative images of immunohistochemistry staining of the hippocampus from tamoxifen-treated (3 weeks post) A Cx3Cr1 CreER(WT/WT) , B Cx3Cr1 CreER -Igf1 fl/fl , C Cx3Cr1 CreER -Alk5 fl/fl , D Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl tissue showing IBA1, TMEM119, P2RY12 (P2YM 1E5) staining. E – G Representative DCX staining in SGZ in above mouse lines. Quantification of mRNA levels from Cx3Cr1 CreER(WT/WT) , Cx3Cr1 CreER -Alk5 fl/fl , or Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl sorted microglia (normalized to Hmbs1) showing levels of H Hprt1 , I Iba1 , J Alk5 , and K Igf1 relative to Cx3Cr1 CreER(WT/WT) mice( n = 3 for all groups in panels ( H – K )) (ns=not significant ( H – I ); **** p < 0.0001 and ns=not significant J ; *** p = 0.0001 and p = 0.0001 and ns=not significant K ). L Quantification of the number of DCX+ cells and M dendritic arborization of DCX+ cells compared to WT mean ( n = 12 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO L ; n = 10 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO M ) (** p = 0.0071 and p = 0.0011 and ns=not significant for panel L ; **** p < 0.0001 *** p = 0.0005 and ns=not significant M ). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. One-way ANOVA, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis in all panels ( H –M). Scale bar = 100 µm. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/egkiitm . Source data are provided as a source data file.

Techniques Used: Immunohistochemistry, Staining, Comparison

Image Search Results

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A A mouse model and experimental timeline for targeting microglial Alk5 and analyzing microglia homeostatic marker expression and microgliosis 3 weeks post tamoxifen. B , C Representative images of the hippocampus stained for IBA1, TMEM119, P2RY12 (BioLegend Ab), and GFAP in B Cx3cr1 CreER -ALK5 wt/wt or C Cx3cr1 CreER -ALK5 fl/fl mice D FACS-sorted GFP+ microglia were analyzed for mRNA levels using qRT-PCR showing E Alk5 (indicating gene deletion efficiency), F Tmem119, G P2ry12, H Tnf, and I Igf1 expression levels in control and iKO microglia ( E – I ) ( n = 3 for control, n = 3 for Alk5 KO, * p = 0.0146 for panel E, *** p = 0.0005 for panel F, *** p = 0.0004 for panel G, * p = 0.0372 for panel H, * p = 0.0167 for panel I, two-sided student’s t-test was used for statistical analysis for panels E-I). Representative immunohistochemistry images of IBA1, Ki67, and DAPI in the cortex of J Cx3cr1 CreER -ALK5 wt/wt or K Cx3cr1 CreER -ALK5 fl/fl at 3 weeks after TAM treatment. Arrows show Ki67+ cells (yellow arrows show Ki67+ cells not co-localized with IBA1+ cells and white arrows show Ki67+ cells co-localizing with IBA1+ cells). Unbiased stereological quantification for L Microglia density, M Astrocyte density, N total number of Ki67+ cells, and O the number of IBA1 + /Ki67+ cells ( L – O ) ( n = 4 for control, n = 3 for Alk5 KO, ** p = 0.002, *** p < 0.001, and ** p = 0.001 for panel L, ns = not significant for panel M , ** p < 0.001, p < 0.00, and p = 0.011 for panel N , ** p < 0.001, *** p < 0.001, and ** p = 0.006 for panel O , two-sided 2-way ANOVA with Tukey post hoc pairwise comparison was used for statistical analysis for panels L – O ). Each data point represents the average of a single animal ( > 3 brain sections per mouse for each brain region). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/0xildk2 . Source data are provided as a source data file.

Article Snippet: We generated cell-type-specific inducible knockout models of critical TGF-β signaling components using Cx3cr1 CreER(Jung) (JAX:020940) , Cx3cr1 CreER(Littman) (JAX:021160) , and P2ry12 CreER (JAX:034727) , to delete Alk5 , Tgfbr2 , Igf1 in microglia or Tnfα constitutive knockout.

Techniques: Marker, Expressing, Staining, Quantitative RT-PCR, Control, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A Mouse model for targeting microglial ( P2ry12 CreER/CreER ) Alk5 in adult mice to examine adult neurogenesis in SGZ. B – C Representative images showing immunohistochemistry staining for IBA1, TMEM119, and P2RY12 (Clone P2YM 1E5) in B control mice and C P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. D , E Representative images showing immunohistochemistry staining for DCX and Ki67 in D control mice and E P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. F – I Quantification for F TMEM119 immunoreactivity, G P2RY12 immunoreactivity, H DCX+ cells quantification compared to the wildtype mean, and I quantification of dendritic arborization (% of immunoreactive positive area) compared to the wildtype mean. F ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); G ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); H ( n = 6 for WT and n = 6 for KO, * p = 0.0359); I ( n = 6 for WT and n = 6 for KO, *** p = 0.0009). Each data point represents the average of a single animal (3–6 brain sections analyzed per mouse) and n = 6 for each group (wildtype or knockout). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Two-sided Student’s t-test for all panels except Welch’s t-test was used for panel G. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/1834acf . Source data are provided as a source data file.

Techniques: Immunohistochemistry, Staining, Control, Knock-Out

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A mouse model used to B micro-dissect the hippocampus and process for single cell 10x Genomic Flex sequencing. C UMAP clustering of cells with annotation for immature neurons and microglia. D Feature plot of Aif1 (coding IBA1 protein) marker to denote microglia cluster. E Feature plot of Dcx to denote DCX+ immature neuroblasts/neuron cluster. F – I Feature plots of Tmem119, P2ry12, Aldh1l1 and Gfap to denote enrichment of microglial signature genes but not astrocytic signature genes in the microglia cluster. J DEGs in MG- Alk5 iKO derived microglia compared to control microglia presented as a volcano plot. K DEGs in MG- Alk5 iKO derived neuroblasts compared to control neuroblasts presented as a volcano plot. L LIANA was used to identify cell-cell interactions and show strong interaction between microglia-endothelial cells and microglia-neuroblasts with identified potential ligand-receptor pairs. M , N EnrichR pathway analysis of DEGs from neuroblasts in MG- Alk5 or Tgfb1 iKO mice with tables denoting top altered disease/cellular processes and molecular pathways. (DEGs) p values were calculated using the Wald test with adjustments for multiple comparisons corrected using the Benjamini-Hochberg method, and (GO Process) p values were calculated using Fisher’s Exact test with adjustments for multiple comparisons were made using Benjamini-Hochberg method. Panel A and B was created in BioRender. Luo, A. (2026) https://BioRender.com/10eqn6o . Source data are provided as a source data file.

Techniques: Single Cell, Sequencing, Marker, Derivative Assay, Control

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A , B In vivo animal models used and experimental timeline. C Representative images of Cx3cr1 CreER -ALK5 WT/WT or Cx3cr1 CreER -ALK5 fl/fl treated with either Vehicle (VEH) or Rapamycin (RAPA) showing immunohistochemistry staining for DCX and IBA1 3 weeks after Tamoxifen treatment. Yellow Arrowhead shows DCX+ cells migrated out of the SGZ inner layer. D Percentage of total DCX+ cells compared to the wildtype mean and E percentage area of dendritic arborization compared to the wildtype mean. F Percentage of migrated DCX+ cells relative to the total number of DCX+ cells per mouse. G , H Representative images of WT or iKO mice treated with either VEH or RAPA showing immunohistochemistry staining for DCX and pS6 3 weeks after TAM treatment. D – G WT VEH n = 7, KO VEH n = 4, WT RAPA n = 5, and KO RAPA n = 4 (ns=not significant ( D – G ); *** p < 0.001, ** p = 0.01 D ; ** p = 0.003, *** p < 0.001 E ; * p = 0.042, *** p < 0.001 F ; * p = 0.048, ** p = 0.01, *** p < 0.001 G ). The sex of each animal is represented by open circles (females) and open triangles (males). I In vitro experimental model and timeline for primary adult NSCs and microglia ( P2ry12 CreER/CreER - tdTomato) 3D co-culture. J Representative images of a differentiated neuroimmune 3D coculture sphere showing microglial tdTomato, GFAP and TUJI immunostaining (maximum projection from Z stacked confocal images). K , L Representative images of the 3D coculture spheres quantification of the TUJI immunoreactive density in the spheres after differentiation with indicated microglia genotypes and VEH or RAPA treatment (WT VEH n = 4, iKO VEH n = 6, WT RAPA n = 6, iKO RAPA n = 4) (ns=not significant, * p = 0.0352, *** p = 0.001 K ). M A proposed model of mechanistic action through microglia onto adult-born neurons. Mean ± SE. Scale bar = 100 µm, Two-way ANOVA test, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis for all panels. Panel A , B , I and M were created in BioRender. Luo, A. (2026) https://BioRender.com/sxk2w6x . Source data are provided as a source data file.

Techniques: In Vivo, Immunohistochemistry, Staining, In Vitro, Co-Culture Assay, Immunostaining, Comparison

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A The mouse model for single Alk5 iKO or double Alk5/Igf1 iKO and experimental timeline (right) for targeting microglial Igf1 and/or Alk5 and analyzing immature neurons 3 weeks post tamoxifen. Representative images of immunohistochemistry staining of the hippocampus from tamoxifen-treated (3 weeks post) A Cx3Cr1 CreER(WT/WT) , B Cx3Cr1 CreER -Igf1 fl/fl , C Cx3Cr1 CreER -Alk5 fl/fl , D Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl tissue showing IBA1, TMEM119, P2RY12 (P2YM 1E5) staining. E – G Representative DCX staining in SGZ in above mouse lines. Quantification of mRNA levels from Cx3Cr1 CreER(WT/WT) , Cx3Cr1 CreER -Alk5 fl/fl , or Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl sorted microglia (normalized to Hmbs1) showing levels of H Hprt1 , I Iba1 , J Alk5 , and K Igf1 relative to Cx3Cr1 CreER(WT/WT) mice( n = 3 for all groups in panels ( H – K )) (ns=not significant ( H – I ); **** p < 0.0001 and ns=not significant J ; *** p = 0.0001 and p = 0.0001 and ns=not significant K ). L Quantification of the number of DCX+ cells and M dendritic arborization of DCX+ cells compared to WT mean ( n = 12 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO L ; n = 10 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO M ) (** p = 0.0071 and p = 0.0011 and ns=not significant for panel L ; **** p < 0.0001 *** p = 0.0005 and ns=not significant M ). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. One-way ANOVA, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis in all panels ( H –M). Scale bar = 100 µm. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/egkiitm . Source data are provided as a source data file.

Techniques: Immunohistochemistry, Staining, Comparison

(A) Diagram of transgenes used to express CreER in Cx3cr1 YFP – CreER (Litt) , Cx3cr1 CreER (Jung) , Tmem119 CreER , Hexb CreER , and P2ry12 CreER mice. (B) Diagram of the Rosa26 mTmG allele and corresponding cellular fluorescence before and after Cre/ loxP DNA recombination. loxP sites are indicated by yellow triangles. (C) Diagram of experimental protocol used to assess tamoxifen (TAM)-induced Cre/ loxP recombination of Rosa26 mTmG/ + in microglia by flow cytometry. (D–H) Representative flow cytometry results show the percentage of mGFP + (mG + ) and mTomato + (mT + ) microglia from individual animals from each group. (I) Quantification of the percentage of recombined mGFP + microglia in microglial CreER lines showed increased recombination in TAM vs. oil for all five CreER lines (Student’s t tests: Cx3cr1 YFP – CreER/+ (Litt) :n = 5 oil, 5 TAM mice; Cx3cr1 CreER/+ (Jung) :n = 5 oil, 4 TAM mice; Tmem119 CreER/ + :n = 8 oil, 7 TAM mice; Hexb CreER/ + : n = 9 oil, 10 TAM mice; P2ry12 CreER/ + : n = 4 oil, 4 TAM mice; ****p < 0.0001). All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also and and .

Journal: Cell reports

Article Title: A comparative analysis of microglial inducible Cre lines

doi: 10.1016/j.celrep.2023.113031

Figure Lengend Snippet: (A) Diagram of transgenes used to express CreER in Cx3cr1 YFP – CreER (Litt) , Cx3cr1 CreER (Jung) , Tmem119 CreER , Hexb CreER , and P2ry12 CreER mice. (B) Diagram of the Rosa26 mTmG allele and corresponding cellular fluorescence before and after Cre/ loxP DNA recombination. loxP sites are indicated by yellow triangles. (C) Diagram of experimental protocol used to assess tamoxifen (TAM)-induced Cre/ loxP recombination of Rosa26 mTmG/ + in microglia by flow cytometry. (D–H) Representative flow cytometry results show the percentage of mGFP + (mG + ) and mTomato + (mT + ) microglia from individual animals from each group. (I) Quantification of the percentage of recombined mGFP + microglia in microglial CreER lines showed increased recombination in TAM vs. oil for all five CreER lines (Student’s t tests: Cx3cr1 YFP – CreER/+ (Litt) :n = 5 oil, 5 TAM mice; Cx3cr1 CreER/+ (Jung) :n = 5 oil, 4 TAM mice; Tmem119 CreER/ + :n = 8 oil, 7 TAM mice; Hexb CreER/ + : n = 9 oil, 10 TAM mice; P2ry12 CreER/ + : n = 4 oil, 4 TAM mice; ****p < 0.0001). All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also and and .

Article Snippet: Cx3cr1 YFP–CreER(Litt) (Stock# 021160, B6.129P2(Cg)- Cx3cr1 tm2.1(cre/ERT2)Litt /WganJ), Cx3cr1 CreER (Jung) (Stock# 020940, B6.129P2(Cg)- Cx3cr1 tm2.1(cre/ERT2)Jung /J), Tmem119 CreER (Stock# 031820, C57BL/6- Tmem119 em1(cre/ERT2)Gfng /J), P2ry12 CreER (Stock# 034727, B6(129S6)- P2ry12 em1(icre/ERT2)Tda /J), Rosa26 mTmG (Stock# 007676, B6.129(Cg)- Gt(ROSA) 26Sortm4(ACTB-tdTomato,-EGFP)Luo /J), Cx3cr1 EGFP (Stock# 005582, B6.129P2(Cg)- Cx3cr1 tm1Litt /J), Rosa26 Ai9 (Stock# 007909, B6.Cg- Gt(ROSA) 26Sortm9(CAG-tdTomato)Hze /J), C1qa Flox (Stock# 031261, B6(SJL)- C1qa tm1c(EUCOMM)Wtsi /TennJ), and Becn1 Flox (Stock# 028794, STOCK Becn1 tm1.1Yue /J) mice were obtained from Jackson Laboratories (Bar Harbor, ME).

Techniques: Fluorescence, Flow Cytometry

(A, C, E, G, and I) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected mice used for flow cytometry analysis in . Sections were immunolabeled for anti-P2RY12 (Alexa Fluor 647 [AF647] pseudo-colored red) to identify microglia and anti-GFP (green) to identify recombined cells. Scale bars, 50 μm. (B, D, F, H, and J) Quantifications of the percentage of P2RY12 + microglia that are mGFP + in the cortex, hippocampus, thalamus, cerebellum, and brainstem of each CreER line after exposure to oil or TAM (B: Cx3cr1 YFP – CreER (Litt) , n = 3, 3 mice; D: Cx3cr1 CreER (Jung) , n = 4, 3 mice; H: Hexb CreER , n = 4, 4 mice; J: P2ry12 CreER , n = 4, 4 mice; two-way repeated-measures ANOVA with Tukey’s post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (K–O) Quantifications of the number of P2RY12 − cells per section that are mGFP + in the cortex, hippocampus, thalamus, cerebellum, and brainstem of each CreER line after exposure to oil or TAM (K: Cx3cr1 YFP – CreER (Litt) , n = 3, 3 mice; L: Cx3cr1 CreER (Jung) , n = 4, 3 mice; M: Tmem119 CreER , n = 4, 3 mice; N: Hexb CreER : n = 4, 4 mice; O: P2ry12 CreER : n = 4, 4 mice; two-way repeated measures ANOVA with Tukey’s post hoc test). All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also .

Journal: Cell reports

Article Title: A comparative analysis of microglial inducible Cre lines

doi: 10.1016/j.celrep.2023.113031

Figure Lengend Snippet: (A, C, E, G, and I) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected mice used for flow cytometry analysis in . Sections were immunolabeled for anti-P2RY12 (Alexa Fluor 647 [AF647] pseudo-colored red) to identify microglia and anti-GFP (green) to identify recombined cells. Scale bars, 50 μm. (B, D, F, H, and J) Quantifications of the percentage of P2RY12 + microglia that are mGFP + in the cortex, hippocampus, thalamus, cerebellum, and brainstem of each CreER line after exposure to oil or TAM (B: Cx3cr1 YFP – CreER (Litt) , n = 3, 3 mice; D: Cx3cr1 CreER (Jung) , n = 4, 3 mice; H: Hexb CreER , n = 4, 4 mice; J: P2ry12 CreER , n = 4, 4 mice; two-way repeated-measures ANOVA with Tukey’s post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (K–O) Quantifications of the number of P2RY12 − cells per section that are mGFP + in the cortex, hippocampus, thalamus, cerebellum, and brainstem of each CreER line after exposure to oil or TAM (K: Cx3cr1 YFP – CreER (Litt) , n = 3, 3 mice; L: Cx3cr1 CreER (Jung) , n = 4, 3 mice; M: Tmem119 CreER , n = 4, 3 mice; N: Hexb CreER : n = 4, 4 mice; O: P2ry12 CreER : n = 4, 4 mice; two-way repeated measures ANOVA with Tukey’s post hoc test). All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also .

Techniques: Injection, Flow Cytometry, Immunolabeling

(A) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected Rosa26 mTmG/ + ;Cre-Driver CreER/ + mice used for flow cytometry analysis in . Sections were immunolabeled for anti-LYVE1 (AF647 pseudo-colored red) to identify border-associated macrophages and anti-GFP (green) to identify recombined cells. Arrows indicate mGFP + border-associated macrophages. Asterisks indicate mGFP + cells along the meninges immunonegative for anti-LYVE1. Scale bars, 50 μm. (B) Quantification of the percentage of recombined mGFP + border-associated macrophages shows increased recombination in TAM-injected Cx3cr1 CreER lines compared with Tmem119 CreER , Hexb CreER , and P2ry12 CreER lines (two-way ANOVA with Tukey’s post hoc test; n = 3 oil Cx3cr1 YFP – CreER (Litt) , 3 TAM Cx3cr1 YFP – CreER (Litt) , 4 oil Cx3cr1 CreER (Jung) , 3 TAM Cx3cr1 CreER (Jung) , 4 oil Tmem119 CreER , 3 TAM Tmem119 CreER , 4 oil Hexb CreER , 4 TAM Hexb CreER , 4 oil P2yr12 CreER , and 4 TAM P2ry12 CreER mice; ***p < 0.001, ****p < 0.0001). (C) Quantification of the frequency of recombined mGFP + cells immunonegative for anti-LYVE1 along the meningeal border shows increased recombination in Tmem119 CreER mice (two-way ANOVA with Tukey’s post hoc test; n = 3 oil Cx3cr1 YFP–CreER (Litt) , 3 TAM Cx3cr1 YFP–CreER (Litt) , 4 oil Cx3cr1 CreER (Jung) , 3 TAM Cx3cr1 CreER (Jung) , 4 oil Tmem119 CreER , 3 TAM Tmem119 CreER , 4 oil Hexb CreER , 4 TAM Hexb CreER , 4 oil P2yr12 CreER , and 4 TAM P2ry12 CreER mice; ****p < 0.0001). (D and E) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected Rosa26 mTmG ; Tmem119 CreER mice used for flow cytometry analysis. Sections were immunolabeled for anti-GFP (green) to identify recombined cells and (D) anti-ALDH1A2 (AF647 pseudo-colored red) to identify arachnoid meningeal fibroblasts or (E) anti-S100A6 (AF647 pseudo-colored red) to identify pial meningeal fibroblasts. Arrows indicate mGFP + cells along the meninges. Scale bars, 50 μm. All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles).

Journal: Cell reports

Article Title: A comparative analysis of microglial inducible Cre lines

doi: 10.1016/j.celrep.2023.113031

Figure Lengend Snippet: (A) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected Rosa26 mTmG/ + ;Cre-Driver CreER/ + mice used for flow cytometry analysis in . Sections were immunolabeled for anti-LYVE1 (AF647 pseudo-colored red) to identify border-associated macrophages and anti-GFP (green) to identify recombined cells. Arrows indicate mGFP + border-associated macrophages. Asterisks indicate mGFP + cells along the meninges immunonegative for anti-LYVE1. Scale bars, 50 μm. (B) Quantification of the percentage of recombined mGFP + border-associated macrophages shows increased recombination in TAM-injected Cx3cr1 CreER lines compared with Tmem119 CreER , Hexb CreER , and P2ry12 CreER lines (two-way ANOVA with Tukey’s post hoc test; n = 3 oil Cx3cr1 YFP – CreER (Litt) , 3 TAM Cx3cr1 YFP – CreER (Litt) , 4 oil Cx3cr1 CreER (Jung) , 3 TAM Cx3cr1 CreER (Jung) , 4 oil Tmem119 CreER , 3 TAM Tmem119 CreER , 4 oil Hexb CreER , 4 TAM Hexb CreER , 4 oil P2yr12 CreER , and 4 TAM P2ry12 CreER mice; ***p < 0.001, ****p < 0.0001). (C) Quantification of the frequency of recombined mGFP + cells immunonegative for anti-LYVE1 along the meningeal border shows increased recombination in Tmem119 CreER mice (two-way ANOVA with Tukey’s post hoc test; n = 3 oil Cx3cr1 YFP–CreER (Litt) , 3 TAM Cx3cr1 YFP–CreER (Litt) , 4 oil Cx3cr1 CreER (Jung) , 3 TAM Cx3cr1 CreER (Jung) , 4 oil Tmem119 CreER , 3 TAM Tmem119 CreER , 4 oil Hexb CreER , 4 TAM Hexb CreER , 4 oil P2yr12 CreER , and 4 TAM P2ry12 CreER mice; ****p < 0.0001). (D and E) Representative immunofluorescent images of brain sections from right hemispheres of oil- and TAM-injected Rosa26 mTmG ; Tmem119 CreER mice used for flow cytometry analysis. Sections were immunolabeled for anti-GFP (green) to identify recombined cells and (D) anti-ALDH1A2 (AF647 pseudo-colored red) to identify arachnoid meningeal fibroblasts or (E) anti-S100A6 (AF647 pseudo-colored red) to identify pial meningeal fibroblasts. Arrows indicate mGFP + cells along the meninges. Scale bars, 50 μm. All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles).

Techniques: Injection, Flow Cytometry, Immunolabeling

(A) Timeline of neonatal TAM injection for images in (B) and (C). (B) Fluorescent images of brain sections from Cx3cr1 YFP – CreER/+ (Litt) , Tmem119 CreER/ + , and Cx3cr1 EGFP/ + mice injected with TAM neonatally, immunolabeled with anti-P2RY12. Large regions of the cortex are devoid of anti-P2RY12 immunofluorescence in Cx3cr1 YFP – CreER/+ (Litt) mice, but not Tmem119 CreER/ + or Cx3cr1 EGFP/ + mice. Scale bars, 500 mm; 200 μm (insets). (C) Fluorescent images of brain sections from Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM neonatally, immunolabeled with anti-P2RY12 (red) and anti-GFP to identify Cx3cr1 YFP + cells (green). Regions of the cortex devoid of anti-P2RY12 immunofluorescence (dotted outline in inset) contain Cx3cr1 YFP + cells with a morphology characteristic of reactive microglia. Scale bars, 500 μm; 100 μm (insets). (D) Diagram of experimental protocol used to assess the effect of TAM injection at P28 on anti-P2RY12 immunofluorescence in Cx3cr1 YFP–CreER/+ (Litt) ; Rosa26 mTmG/ + mice. (E and F) Fluorescent images of brain sections from Cx3cr1 YFP–CreER/+ (Litt) ; Rosa26 mTmG/ + mice used for flow cytometry analysis in , immunolabeled with anti-P2RY12 (imaged using AF647). No patches are devoid of anti-P2RY12 immunofluorescence. Scale bars, 500 μm (E); 200 μm (F). (G) Quantification of the percentage of mice with patches devoid of anti-P2RY12 immunofluorescence shows higher prevalence in Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM as neonates vs. Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM at P28 (chi-squared test: n = 4, 4 mice; **p < 0.01). (H) Diagram of experimental protocol used to perform RNA sequencing on Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM or oil at P28. (I) Smear plot of TAM-vs. oil-injected Cx3cr1 YFP – CreER/+ (Litt) mice depicting log fold change (FC) on the y axis against log counts per million (CPM) on the x axis ( n = 4, 5 mice). Differentially expressed genes with false discovery rate (FDR) < 0.05 are annotated in red (upregulated by TAM) or blue (downregulated by TAM). See also .

Journal: Cell reports

Article Title: A comparative analysis of microglial inducible Cre lines

doi: 10.1016/j.celrep.2023.113031

Figure Lengend Snippet: (A) Timeline of neonatal TAM injection for images in (B) and (C). (B) Fluorescent images of brain sections from Cx3cr1 YFP – CreER/+ (Litt) , Tmem119 CreER/ + , and Cx3cr1 EGFP/ + mice injected with TAM neonatally, immunolabeled with anti-P2RY12. Large regions of the cortex are devoid of anti-P2RY12 immunofluorescence in Cx3cr1 YFP – CreER/+ (Litt) mice, but not Tmem119 CreER/ + or Cx3cr1 EGFP/ + mice. Scale bars, 500 mm; 200 μm (insets). (C) Fluorescent images of brain sections from Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM neonatally, immunolabeled with anti-P2RY12 (red) and anti-GFP to identify Cx3cr1 YFP + cells (green). Regions of the cortex devoid of anti-P2RY12 immunofluorescence (dotted outline in inset) contain Cx3cr1 YFP + cells with a morphology characteristic of reactive microglia. Scale bars, 500 μm; 100 μm (insets). (D) Diagram of experimental protocol used to assess the effect of TAM injection at P28 on anti-P2RY12 immunofluorescence in Cx3cr1 YFP–CreER/+ (Litt) ; Rosa26 mTmG/ + mice. (E and F) Fluorescent images of brain sections from Cx3cr1 YFP–CreER/+ (Litt) ; Rosa26 mTmG/ + mice used for flow cytometry analysis in , immunolabeled with anti-P2RY12 (imaged using AF647). No patches are devoid of anti-P2RY12 immunofluorescence. Scale bars, 500 μm (E); 200 μm (F). (G) Quantification of the percentage of mice with patches devoid of anti-P2RY12 immunofluorescence shows higher prevalence in Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM as neonates vs. Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM at P28 (chi-squared test: n = 4, 4 mice; **p < 0.01). (H) Diagram of experimental protocol used to perform RNA sequencing on Cx3cr1 YFP – CreER/+ (Litt) mice injected with TAM or oil at P28. (I) Smear plot of TAM-vs. oil-injected Cx3cr1 YFP – CreER/+ (Litt) mice depicting log fold change (FC) on the y axis against log counts per million (CPM) on the x axis ( n = 4, 5 mice). Differentially expressed genes with false discovery rate (FDR) < 0.05 are annotated in red (upregulated by TAM) or blue (downregulated by TAM). See also .

Techniques: Injection, Immunolabeling, Immunofluorescence, Flow Cytometry, RNA Sequencing

(A) Diagram of experimental protocol used to assess spontaneous Cre/ loxP recombination of Rosa26 mTmG/ + in microglia by flow cytometry and immunofluorescence. (B) Representative flow cytometry results show the percentage of recombined mGFP + (mG) and mTomato + (mT) microglia from individual animals from each group from and uninjected (no oil) Cx3cr1 YFP – CreER/+ (Litt) mice. (C) Quantification of the percentage of recombined mGFP + microglia shows increased spontaneous recombination of the Rosa26 mTmG allele in the Cx3cr1 YFP – CreER (Litt) line compared with the Cx3cr1 CreER (Jung) , Tmem119 CreER , Hexb CreER , and P2ry12 CreER lines (one-way ANOVA with Tukey’s post hoc test; n = 5 Cx3cr1 YFP – CreER/+ (Litt) , 5 Cx3cr1 CreER/+ (Jung) , 8 Tmem119 CreER/ + , 9 Hexb CreER/ + , and 4 P2ry12 CreER/ + mice; *p < 0.05, ****p < 0.0001). (D) Representative immunofluorescent images of brain sections from right hemispheres of oil-injected mice used for flow cytometry analysis in (B) and (C). Sections were immunolabeled for anti-P2RY12 (AF647 pseudo-colored red) to identify microglia and anti-GFP (green) to identify recombined cells. The number of recombined mGFP + microglia (white arrows) matches the results observed by flow cytometry. In the Cx3cr1 YFP – CreER/+ (Litt) line, the soma of non-recombined microglia are also immunolabeled by anti-GFP because of the constitutive expression of YFP (asterisks), but it can be distinguished from recombined mGFP + microglia by fluorescence intensity and membrane labeling. Scale bars, 50 μm. All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also .

Journal: Cell reports

Article Title: A comparative analysis of microglial inducible Cre lines

doi: 10.1016/j.celrep.2023.113031

Figure Lengend Snippet: (A) Diagram of experimental protocol used to assess spontaneous Cre/ loxP recombination of Rosa26 mTmG/ + in microglia by flow cytometry and immunofluorescence. (B) Representative flow cytometry results show the percentage of recombined mGFP + (mG) and mTomato + (mT) microglia from individual animals from each group from and uninjected (no oil) Cx3cr1 YFP – CreER/+ (Litt) mice. (C) Quantification of the percentage of recombined mGFP + microglia shows increased spontaneous recombination of the Rosa26 mTmG allele in the Cx3cr1 YFP – CreER (Litt) line compared with the Cx3cr1 CreER (Jung) , Tmem119 CreER , Hexb CreER , and P2ry12 CreER lines (one-way ANOVA with Tukey’s post hoc test; n = 5 Cx3cr1 YFP – CreER/+ (Litt) , 5 Cx3cr1 CreER/+ (Jung) , 8 Tmem119 CreER/ + , 9 Hexb CreER/ + , and 4 P2ry12 CreER/ + mice; *p < 0.05, ****p < 0.0001). (D) Representative immunofluorescent images of brain sections from right hemispheres of oil-injected mice used for flow cytometry analysis in (B) and (C). Sections were immunolabeled for anti-P2RY12 (AF647 pseudo-colored red) to identify microglia and anti-GFP (green) to identify recombined cells. The number of recombined mGFP + microglia (white arrows) matches the results observed by flow cytometry. In the Cx3cr1 YFP – CreER/+ (Litt) line, the soma of non-recombined microglia are also immunolabeled by anti-GFP because of the constitutive expression of YFP (asterisks), but it can be distinguished from recombined mGFP + microglia by fluorescence intensity and membrane labeling. Scale bars, 50 μm. All data are presented as mean ± SEM. Individual data points indicate males (squares) and females (circles). See also .

Techniques: Flow Cytometry, Immunofluorescence, Injection, Immunolabeling, Expressing, Fluorescence, Membrane, Labeling

A ) Analysis of Itgb8 expression in the E14.5 mouse embryo in neural progenitor cells (NPCs) and radial glia, microglia, endothelial cells, and mural cells . B) Itgb8-TdT reporter expression confirms strong Itgb8 expression in SOX9+NESTIN+ radial progenitors at E14.5. Open arrowhead marks radial glia fibers; closed arrowhead marks ramified radial glia endfeet at the surface of the neuroepithelium. C) Model describing developmental expression of Itgb8 in neuroepithelium and radial glia, and correlation with sequential timing of Cre recombination in Emx1 Cre , Nestin Cre and hGFAP Cre lines. (see also Supplementary Figures 2 ). D-F) Deletion of Itgb8 from neuroepithelial and radial progenitors using indicated Cre lines. Coronal brain sections stained for tdT (Cre recombination, red), vascular endothelium (CD31, cyan), and macrophages/microglia (IBA1, yellow); hemorrhage (red blood cells marked by TER119 (yellow) observed outside of vascular lumen (CD31, cyan) and microglia precursors (CD206, yellow) and committed/homeostatic microglia (P2RY12, cyan). G ) E14.5 brain section from Emx1 Cre ;RG-brainbow mouse stained for membranous GFP (individual recombined radial glia; endfeet, green), microglia precursors (CD206, magenta), and committed/homeostatic microglia (P2RY12, yellow). Arrowheads indicate foot process of radial glia contacting presumptive pial-associated CD206+ microglia precursor (model to right). Scale bar in B=500μm, D=200μm, G=25 μm.

Journal: bioRxiv

Article Title: Radial glia control microglial differentiation via integrin avb8-dependent trans-activation of TGFB1

doi: 10.1101/2023.07.13.548459

Figure Lengend Snippet: A ) Analysis of Itgb8 expression in the E14.5 mouse embryo in neural progenitor cells (NPCs) and radial glia, microglia, endothelial cells, and mural cells . B) Itgb8-TdT reporter expression confirms strong Itgb8 expression in SOX9+NESTIN+ radial progenitors at E14.5. Open arrowhead marks radial glia fibers; closed arrowhead marks ramified radial glia endfeet at the surface of the neuroepithelium. C) Model describing developmental expression of Itgb8 in neuroepithelium and radial glia, and correlation with sequential timing of Cre recombination in Emx1 Cre , Nestin Cre and hGFAP Cre lines. (see also Supplementary Figures 2 ). D-F) Deletion of Itgb8 from neuroepithelial and radial progenitors using indicated Cre lines. Coronal brain sections stained for tdT (Cre recombination, red), vascular endothelium (CD31, cyan), and macrophages/microglia (IBA1, yellow); hemorrhage (red blood cells marked by TER119 (yellow) observed outside of vascular lumen (CD31, cyan) and microglia precursors (CD206, yellow) and committed/homeostatic microglia (P2RY12, cyan). G ) E14.5 brain section from Emx1 Cre ;RG-brainbow mouse stained for membranous GFP (individual recombined radial glia; endfeet, green), microglia precursors (CD206, magenta), and committed/homeostatic microglia (P2RY12, yellow). Arrowheads indicate foot process of radial glia contacting presumptive pial-associated CD206+ microglia precursor (model to right). Scale bar in B=500μm, D=200μm, G=25 μm.

Article Snippet: The P2ry12-CreER mouse line will be deposited at Jackson Labs (Stock #034727) and at the Mutant Mouse Resource and Research Center (MMMRC).

Techniques: Expressing, Staining

A) Comparison of the transcriptional properties of adult Itgb8 f/f ; Emx1 Cre mutant and control microglia to stage specfic developmental markers reveals that dysmature microglia retain the gene expression profiles of early embryonic microglia. B) Analysis of developmental gene cluster expression reveals enrichment for progenitor (cluster 1) and early embryonic phase (clusters 3-5) enriched gene sets. C) Whole brain sagittal immunostaining of adult Itgb8 f/f ; Emx1 Cre mice revealed anatomically restricted maintenance of the microglial precursor marker CD206 in the cortex and hippocampus (asterisk), accompained by loss of the homeostatic marker P2RY12. D) Increased expression of the MGnD marker LGALS3 in a subset of cortical and hippocampal microglia in Itgb8 f/f ; Emx1 Cre mice (asterisk). Closed and open arrowheads in E-G) mark cortical and striatal microglia respectively. E) Downregulation of the microglial homeostatic marker TMEM119 in the cortex of a Itgb8 f/f ; Emx1 Cre mouse. F) Cortex-restricted upregulation of the microglial reactive marker APOE in IBA1+ cells of the cortex (green cells). D) Cortex-restricted upregulation of the microglial reactive marker CLEC7a. Cx= cerebral cortex; Cc= corpus callosum; Str= striatum; Dashed line= cortical/striatal boundary. Scale bar in C= 2mm, E=150μm.

Journal: bioRxiv

Article Title: Radial glia control microglial differentiation via integrin avb8-dependent trans-activation of TGFB1

doi: 10.1101/2023.07.13.548459

Figure Lengend Snippet: A) Comparison of the transcriptional properties of adult Itgb8 f/f ; Emx1 Cre mutant and control microglia to stage specfic developmental markers reveals that dysmature microglia retain the gene expression profiles of early embryonic microglia. B) Analysis of developmental gene cluster expression reveals enrichment for progenitor (cluster 1) and early embryonic phase (clusters 3-5) enriched gene sets. C) Whole brain sagittal immunostaining of adult Itgb8 f/f ; Emx1 Cre mice revealed anatomically restricted maintenance of the microglial precursor marker CD206 in the cortex and hippocampus (asterisk), accompained by loss of the homeostatic marker P2RY12. D) Increased expression of the MGnD marker LGALS3 in a subset of cortical and hippocampal microglia in Itgb8 f/f ; Emx1 Cre mice (asterisk). Closed and open arrowheads in E-G) mark cortical and striatal microglia respectively. E) Downregulation of the microglial homeostatic marker TMEM119 in the cortex of a Itgb8 f/f ; Emx1 Cre mouse. F) Cortex-restricted upregulation of the microglial reactive marker APOE in IBA1+ cells of the cortex (green cells). D) Cortex-restricted upregulation of the microglial reactive marker CLEC7a. Cx= cerebral cortex; Cc= corpus callosum; Str= striatum; Dashed line= cortical/striatal boundary. Scale bar in C= 2mm, E=150μm.

Article Snippet: The P2ry12-CreER mouse line will be deposited at Jackson Labs (Stock #034727) and at the Mutant Mouse Resource and Research Center (MMMRC).

Techniques: Mutagenesis, Expressing, Immunostaining, Marker

A) Analysis of the embryonic expression of Tgfb1 in E14.5 neural progenitors, microglia, endothelial and mural cells (from ) B,C) E14.5 coronal brain sections from control ( Tgfb +/- ) embryos, or embryos with global ( Tgfb1 -/- ) or cell-lineage specific deletion of Tgfb1 ( Tgfb1 fl/fl ) in D) macrophages/microglia ( Cx3cr1 Cre ), E) endothelial cells and microglia/macrophages ( Tie2 Cre ), or F) vascular mural cells ( Pdgfbrb Cre ) were stained for hemorrhage (TER119, magenta) and vasculature (CD31, green); microglia/macrophage (IBA1, magenta) association with blood vessels (CD31, green); and committed/hemostatic microglia (P2RY12, magenta). Only Tgfb1 -/- mutants have consistent evidence of vascular dysplasia (marked by X) and hemorrhage (asterisk), whereas mice with microglia/macrophage deletion of Tgfb1 ( Tgfb -/- , Tgfb1 fl/fl ;Cx3cr1 Cre and Tgfb1 fl/fl ; Tie2Cre mutants) have presence of dysmature microglia (open arrowheads, blowups to right). Scale bar in B=150μm.

Journal: bioRxiv

Article Title: Radial glia control microglial differentiation via integrin avb8-dependent trans-activation of TGFB1

doi: 10.1101/2023.07.13.548459

Figure Lengend Snippet: A) Analysis of the embryonic expression of Tgfb1 in E14.5 neural progenitors, microglia, endothelial and mural cells (from ) B,C) E14.5 coronal brain sections from control ( Tgfb +/- ) embryos, or embryos with global ( Tgfb1 -/- ) or cell-lineage specific deletion of Tgfb1 ( Tgfb1 fl/fl ) in D) macrophages/microglia ( Cx3cr1 Cre ), E) endothelial cells and microglia/macrophages ( Tie2 Cre ), or F) vascular mural cells ( Pdgfbrb Cre ) were stained for hemorrhage (TER119, magenta) and vasculature (CD31, green); microglia/macrophage (IBA1, magenta) association with blood vessels (CD31, green); and committed/hemostatic microglia (P2RY12, magenta). Only Tgfb1 -/- mutants have consistent evidence of vascular dysplasia (marked by X) and hemorrhage (asterisk), whereas mice with microglia/macrophage deletion of Tgfb1 ( Tgfb -/- , Tgfb1 fl/fl ;Cx3cr1 Cre and Tgfb1 fl/fl ; Tie2Cre mutants) have presence of dysmature microglia (open arrowheads, blowups to right). Scale bar in B=150μm.

Article Snippet: The P2ry12-CreER mouse line will be deposited at Jackson Labs (Stock #034727) and at the Mutant Mouse Resource and Research Center (MMMRC).

Techniques: Expressing, Staining

A) Sorted bulk-Seq analysis of embryonic microglia and BAMs reveals that Tgfb1 is expressed in both microglia and BAMs during embryonic develpoment, whereas P2ry12 and Pf4 are specific markers of these two respective populations. B,C) Analysis of control (B) and conditional Cx3cr1 CreER mediated deletion of Tgfb1 deletion in the E14.5 forebrain following E11.5, 12.5 and 13.5 tamoxifen induction. Analysis revealed no hemorrhage (CD31 in green, TER119 in magenta), no change in macrophage/blood vessel association (IBA1 in magenta, CD31 in green), loss of the homeostatic marker P2RY12 (in magenta, IBA1 in green), and loss of Tgfb1 (cyan) in Isolectin B4 (green) and Tdt (red) labeled microglia, but not in IB4 + labeled blood vessels. D) Analysis of conditional P2ry12 CreER mediated deletion of Tgfb1 deletion in E14.5 microglia the following E11.5, 12.5 and 13.5 tamoxifen induction. Analysis revealed no brain hemorrhage (CD31 in green, TER119 in magenta), no change in macrophage/blood vessel association (IBA1 in magenta, CD31 in green), and loss of the homeostatic marker P2RY12 (magenta), in IBA1+ (green) microglia. E) Analysis of conditional Pf4 Cre mediated deletion of Tgfb1 deletion in the E14.5 forebrain. Analysis revealed no brain hemorrhage (CD31 in green, TER119 in magenta), no change in macrophage/blood vessel association (IBA1 in magenta, CD31 in green), and no loss of the homeostatic marker P2RY12 (magenta), in IBA1+ (green) microglia. F) Bulk-seq analysis of Tgfb1 expression in the adult mouse brain . Analysis revealed enrichment for Tgfb1 expression primarily in microglia and vascular cells. G) Analysis of conditional Cx3cr1 CreER mediated deletion of Tgfb1 deletion in the P30 mouse brain following neonatal tamoxifen induciton at P4,5 and 6. Analysis revealed a “patchy” distribution of Tgfb1 negative microglia with altered morphology and loss of P2RY12 (open arrowheads in 1) adjacent to patches of microglia with no loss of Tgfb1 or P2RY12. Scale bar in B=150μm, G=100μm.

Journal: bioRxiv

Article Title: Radial glia control microglial differentiation via integrin avb8-dependent trans-activation of TGFB1

doi: 10.1101/2023.07.13.548459

Figure Lengend Snippet: A) Sorted bulk-Seq analysis of embryonic microglia and BAMs reveals that Tgfb1 is expressed in both microglia and BAMs during embryonic develpoment, whereas P2ry12 and Pf4 are specific markers of these two respective populations. B,C) Analysis of control (B) and conditional Cx3cr1 CreER mediated deletion of Tgfb1 deletion in the E14.5 forebrain following E11.5, 12.5 and 13.5 tamoxifen induction. Analysis revealed no hemorrhage (CD31 in green, TER119 in magenta), no change in macrophage/blood vessel association (IBA1 in magenta, CD31 in green), loss of the homeostatic marker P2RY12 (in magenta, IBA1 in green), and loss of Tgfb1 (cyan) in Isolectin B4 (green) and Tdt (red) labeled microglia, but not in IB4 + labeled blood vessels. D) Analysis of conditional P2ry12 CreER mediated deletion of Tgfb1 deletion in E14.5 microglia the following E11.5, 12.5 and 13.5 tamoxifen induction. Analysis revealed no brain hemorrhage (CD31 in green, TER119 in magenta), no change in macrophage/blood vessel association (IBA1 in magenta, CD31 in green), and loss of the homeostatic marker P2RY12 (magenta), in IBA1+ (green) microglia. E) Analysis of conditional Pf4 Cre mediated deletion of Tgfb1 deletion in the E14.5 forebrain. Analysis revealed no brain hemorrhage (CD31 in green, TER119 in magenta), no change in macrophage/blood vessel association (IBA1 in magenta, CD31 in green), and no loss of the homeostatic marker P2RY12 (magenta), in IBA1+ (green) microglia. F) Bulk-seq analysis of Tgfb1 expression in the adult mouse brain . Analysis revealed enrichment for Tgfb1 expression primarily in microglia and vascular cells. G) Analysis of conditional Cx3cr1 CreER mediated deletion of Tgfb1 deletion in the P30 mouse brain following neonatal tamoxifen induciton at P4,5 and 6. Analysis revealed a “patchy” distribution of Tgfb1 negative microglia with altered morphology and loss of P2RY12 (open arrowheads in 1) adjacent to patches of microglia with no loss of Tgfb1 or P2RY12. Scale bar in B=150μm, G=100μm.

Article Snippet: The P2ry12-CreER mouse line will be deposited at Jackson Labs (Stock #034727) and at the Mutant Mouse Resource and Research Center (MMMRC).

Techniques: Marker, Labeling, Expressing

A) Schematic of TGFβ, with mutant mouse models analyzed by bulk and microglial flow cytometry noted by color ( Itgb8 =red; Tgfb1 =cyan; Lrrc33 =orange; Tgfb2 =blue, Smad2/3 =green). B) Correlation of bulk-seq gene expression across TGFβ mutant models. C) Compensatory transcriptional changes of key TGFβ signaling genes in different TGFβ mutant models. D) Bulk-seq analysis of microglial homeostatic and disease associated (MgND/DAM) microglial markers across TGFβ mutant models. E) Bulk-seq analysis of astrocytosis-associated markers across TGFβ mutant models. F-G) Comparison of control (F) Tgfb1 fl+l ;Cx3cr1 Cre , (G) Tgfb1 fl/fl ;Cx3cr1 Cre and (H) Smad2/3 f/f ;Cx3cr1 Cre adult mice. Analysis revealed loss of the homeostatic marker P2RY12 in both conditional Tgfb1 and Smad2/3 mutants, but significantly higher upregulation of the MGnD-associated microglial marker LGALS3 (see arrowheads). LGALS3 upregulation in Tgfb1 conditional mutants was significantly higher in white matter (asterisk in G and H ) and was only seen in the white matter of Smad2/3 conditional mutants ( H ). Scale bar in F=50μm.

Journal: bioRxiv

Article Title: Radial glia control microglial differentiation via integrin avb8-dependent trans-activation of TGFB1

doi: 10.1101/2023.07.13.548459

Figure Lengend Snippet: A) Schematic of TGFβ, with mutant mouse models analyzed by bulk and microglial flow cytometry noted by color ( Itgb8 =red; Tgfb1 =cyan; Lrrc33 =orange; Tgfb2 =blue, Smad2/3 =green). B) Correlation of bulk-seq gene expression across TGFβ mutant models. C) Compensatory transcriptional changes of key TGFβ signaling genes in different TGFβ mutant models. D) Bulk-seq analysis of microglial homeostatic and disease associated (MgND/DAM) microglial markers across TGFβ mutant models. E) Bulk-seq analysis of astrocytosis-associated markers across TGFβ mutant models. F-G) Comparison of control (F) Tgfb1 fl+l ;Cx3cr1 Cre , (G) Tgfb1 fl/fl ;Cx3cr1 Cre and (H) Smad2/3 f/f ;Cx3cr1 Cre adult mice. Analysis revealed loss of the homeostatic marker P2RY12 in both conditional Tgfb1 and Smad2/3 mutants, but significantly higher upregulation of the MGnD-associated microglial marker LGALS3 (see arrowheads). LGALS3 upregulation in Tgfb1 conditional mutants was significantly higher in white matter (asterisk in G and H ) and was only seen in the white matter of Smad2/3 conditional mutants ( H ). Scale bar in F=50μm.

Article Snippet: The P2ry12-CreER mouse line will be deposited at Jackson Labs (Stock #034727) and at the Mutant Mouse Resource and Research Center (MMMRC).

Techniques: Mutagenesis, Flow Cytometry, Expressing, Marker

Evaluation of the TAM-independent leakiness and the efficiency of TAM-dependent cre recombination in the four different creER driver lines using either the Ai9 (tdTomato) or R26-YFP reporter mouse lines. The experimental timeline is shown in panel (A). Representative images from each cre driver and reporter line (B-V for VEH treatment and b-v for TAM treatment). Cre driver and the reporter line are indicated on the left side of the panels. Quantification of reporter+ cells in the IBA1+ populations in the brain is shown in panel W (for VEH treatment) and w (for TAM treatment). Representative images are taken from the cortical region which reflects the general and homogenous trend in the whole parenchyma. Each data point represents the average of 1 animal (the average for each animal is obtained by quantifying multiple brain sections at similar anatomical location) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, for Two way ANOVA analysis, Tukey post-hoc pair wise analysis. Ai9 vs R26-YFP is significantly different as a factor (p<0.001). Data were combined from 2 independent cohorts of mice. Scale bar: 100 μm. Compared to the two Cx3cr1 CreER lines (Littman and Jung), TMEM119 CreER and P2ry12 CreER show less leakiness in the absence of TAM but a decreased recombination efficiency and mosaic recombination in microglia.

Journal: bioRxiv

Article Title: Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

doi: 10.1101/2023.04.17.536878

Figure Lengend Snippet: Evaluation of the TAM-independent leakiness and the efficiency of TAM-dependent cre recombination in the four different creER driver lines using either the Ai9 (tdTomato) or R26-YFP reporter mouse lines. The experimental timeline is shown in panel (A). Representative images from each cre driver and reporter line (B-V for VEH treatment and b-v for TAM treatment). Cre driver and the reporter line are indicated on the left side of the panels. Quantification of reporter+ cells in the IBA1+ populations in the brain is shown in panel W (for VEH treatment) and w (for TAM treatment). Representative images are taken from the cortical region which reflects the general and homogenous trend in the whole parenchyma. Each data point represents the average of 1 animal (the average for each animal is obtained by quantifying multiple brain sections at similar anatomical location) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, for Two way ANOVA analysis, Tukey post-hoc pair wise analysis. Ai9 vs R26-YFP is significantly different as a factor (p<0.001). Data were combined from 2 independent cohorts of mice. Scale bar: 100 μm. Compared to the two Cx3cr1 CreER lines (Littman and Jung), TMEM119 CreER and P2ry12 CreER show less leakiness in the absence of TAM but a decreased recombination efficiency and mosaic recombination in microglia.

Article Snippet: To evaluate cre recombinase specificity and efficiency, we utilized four different microglia CreER lines: Cx3cr1 GFPCreER(Litt) Line (JAX stock number 021160) ; Cx3cr1 CreER(Jung ) Line (JAX stock number 020940) ; Tmem119 CreER line (JAX stock number 031820) and the P2ry12 CreER line (JAX stock number 034727) .

Techniques:

FACS analysis of reporter-positive cells in total brain single cell resuspension confirms the reporter recombination efficiency differences among the three investigated CreER lines. tdTomato+ cell percentage from all three different lines show similar high-efficiency recombination and the YFP+ cells show higher recombination efficiency in the CX3CR1CreERJung-R26-YFP mice but significantly lower recombination efficiency in the P2RY12 CreER -R26-YFP mice and TMEM119 CreER -R26-YFP line. Each data point represents data from one animal. **p < 0.01 and ***p < 0.001, for Two way ANOVA analysis, Tukey post-hoc pairwise analysis. Data were combined from 2-3 independent cohorts of mice for each line.

Journal: bioRxiv

Article Title: Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

doi: 10.1101/2023.04.17.536878

Figure Lengend Snippet: FACS analysis of reporter-positive cells in total brain single cell resuspension confirms the reporter recombination efficiency differences among the three investigated CreER lines. tdTomato+ cell percentage from all three different lines show similar high-efficiency recombination and the YFP+ cells show higher recombination efficiency in the CX3CR1CreERJung-R26-YFP mice but significantly lower recombination efficiency in the P2RY12 CreER -R26-YFP mice and TMEM119 CreER -R26-YFP line. Each data point represents data from one animal. **p < 0.01 and ***p < 0.001, for Two way ANOVA analysis, Tukey post-hoc pairwise analysis. Data were combined from 2-3 independent cohorts of mice for each line.

Techniques:

Independent recombination of the two floxed alleles on a single cell level in the P2RY12 CreER double reporter (Ai9:R26-YFP) mouse line. Evaluation of the TAM-dependent recombination of either Ai9-tdTomato or R26-YFP allele which are both located in the ROSA26 loci in a double reporter mouse in the P2RY12CreER line suggests that although on a populational level, Ai9-tdTomato reporter has a higher probability of being recombined than the R26-YFP allele, on a single cell level, the recombination of each individual allele can be independent and does not always follow the size of the floxed region rule. (A), experimental timeline. (B-E), IHC evaluation of the recombination of microglia on either of the reporter expression. Note tdTomato+:YFP+ double positive microglia (orange arrow), more abundant tdTomato+:YFP-microglia (white arrow) and the less abundant YFP+:tdTomato-microglia (white arrowhead). (F-G), representative FACS plots for no color control or the double reporter flow analysis. For quantification of % of each population, see .

Journal: bioRxiv

Article Title: Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

doi: 10.1101/2023.04.17.536878

Figure Lengend Snippet: Independent recombination of the two floxed alleles on a single cell level in the P2RY12 CreER double reporter (Ai9:R26-YFP) mouse line. Evaluation of the TAM-dependent recombination of either Ai9-tdTomato or R26-YFP allele which are both located in the ROSA26 loci in a double reporter mouse in the P2RY12CreER line suggests that although on a populational level, Ai9-tdTomato reporter has a higher probability of being recombined than the R26-YFP allele, on a single cell level, the recombination of each individual allele can be independent and does not always follow the size of the floxed region rule. (A), experimental timeline. (B-E), IHC evaluation of the recombination of microglia on either of the reporter expression. Note tdTomato+:YFP+ double positive microglia (orange arrow), more abundant tdTomato+:YFP-microglia (white arrow) and the less abundant YFP+:tdTomato-microglia (white arrowhead). (F-G), representative FACS plots for no color control or the double reporter flow analysis. For quantification of % of each population, see .

Techniques: Expressing

Evaluation of the gene deletion efficiency on distinct homozygous floxed target gene alleles in the Cx3cr1CreER(Jung) and P2Ry12CreER drivers using quantitative RT-PCR. (A-D). Animal genotype and experimental flow. Total mRNA levels are evaluated for the floxed exon in the TGFb1 gene in YFP+ microglia sorted from the Cx3cr1 CreER(Jung) or P2RY12 CreER —TGFb1 fl/fl -R26-YFP mice at 3 weeks after TAM treatment. (E-G). Total mRNA levels are evaluated for the floxed exon in the ALK5 gene in either YFP+ microglia sorted from the Cx3cr1 CreER(Jung) or P2RY12 CreER —ALK5 fl/fl -R26-YFP mice or tdTomato+ microglia from the P2RY12 CreER —ALK5 fl/fl -Ai9 mice at 3 weeks after TAM treatment. Each data point represents the average of 1 animal (the average for each animal is obtained by averaging 3 technical replication of the qRT-PCR reaction for that animal) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, **** p<0.0001 for Two way ANOVA analysis, Tukey post-hoc pairwise analysis.

Journal: bioRxiv

Article Title: Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

doi: 10.1101/2023.04.17.536878

Figure Lengend Snippet: Evaluation of the gene deletion efficiency on distinct homozygous floxed target gene alleles in the Cx3cr1CreER(Jung) and P2Ry12CreER drivers using quantitative RT-PCR. (A-D). Animal genotype and experimental flow. Total mRNA levels are evaluated for the floxed exon in the TGFb1 gene in YFP+ microglia sorted from the Cx3cr1 CreER(Jung) or P2RY12 CreER —TGFb1 fl/fl -R26-YFP mice at 3 weeks after TAM treatment. (E-G). Total mRNA levels are evaluated for the floxed exon in the ALK5 gene in either YFP+ microglia sorted from the Cx3cr1 CreER(Jung) or P2RY12 CreER —ALK5 fl/fl -R26-YFP mice or tdTomato+ microglia from the P2RY12 CreER —ALK5 fl/fl -Ai9 mice at 3 weeks after TAM treatment. Each data point represents the average of 1 animal (the average for each animal is obtained by averaging 3 technical replication of the qRT-PCR reaction for that animal) and the average for each animal was used as a single data point for statistical analysis. **p < 0.01 and ***p < 0.001, **** p<0.0001 for Two way ANOVA analysis, Tukey post-hoc pairwise analysis.

Techniques: Quantitative RT-PCR

iSuRe-Cre mouse line successfully induces constitutive cre-P2A-MbTomato expression in DCX CreER mouse line but not in the P2ry12 CreER mouse line after TAM treatment. (A) Illustration of mouse transgene constructs and experimental timeline. (B-E) in the absence of TAM, there is no MbTomato expression in microglia with ectopic MbTomato expression in cells that demonstrates typical neuron morphology in cortex and striatum. (F-G), treatment of TAM in mice does not induce MbTomato expression in microglia and presents with similar neuronal ectopic expression of MbTomato. (J-R). In contrast, in the DCXC reER -iSuReCre mice that are treated with TAM, at 5 days post TAM treatment, DCX+ immature neuroblasts are labeled with MbTomato protein (white arrows) and at 30 days post TAM treatment, MbTomato expression are mostly detected in DCX-NeuN+ mature neurons (yellow arrows), supporting that the iSuRe-Cre construct is able to be induced in a cohort of immature neuroblasts which mature later into NeuN+ neurons in the dentate gyrus of adult mice. Scale bar=100 µm.

Journal: bioRxiv

Article Title: Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

doi: 10.1101/2023.04.17.536878

Figure Lengend Snippet: iSuRe-Cre mouse line successfully induces constitutive cre-P2A-MbTomato expression in DCX CreER mouse line but not in the P2ry12 CreER mouse line after TAM treatment. (A) Illustration of mouse transgene constructs and experimental timeline. (B-E) in the absence of TAM, there is no MbTomato expression in microglia with ectopic MbTomato expression in cells that demonstrates typical neuron morphology in cortex and striatum. (F-G), treatment of TAM in mice does not induce MbTomato expression in microglia and presents with similar neuronal ectopic expression of MbTomato. (J-R). In contrast, in the DCXC reER -iSuReCre mice that are treated with TAM, at 5 days post TAM treatment, DCX+ immature neuroblasts are labeled with MbTomato protein (white arrows) and at 30 days post TAM treatment, MbTomato expression are mostly detected in DCX-NeuN+ mature neurons (yellow arrows), supporting that the iSuRe-Cre construct is able to be induced in a cohort of immature neuroblasts which mature later into NeuN+ neurons in the dentate gyrus of adult mice. Scale bar=100 µm.

Techniques: Expressing, Construct, Labeling

Evaluation of the dyshomeostatic microglia in different icroglia-specific CreER drivers after TAM treatment at different ages. P2Ry12 expression is used as a measure of dyshomeostasis in microglia. (A), consistent with previous studies, we observe dyshomeostasis of microglia (indicated by loss of P2RY12 expression) across many region in the neonatal Cx3cr1 CreER(Litt) (+/WT) mice treated with TAM. This phenotype is not observed in (B) the adolescent (3wk old) Cx3cr1 CreER(Litt) (+/WT) mice that received TAM treatment or (C) neonatal Cx3cr1 CreER(Jung) (+/WT) and P2RY12CreER (+/WT) mice that received TAM at the similar time frame. Scale bar= 100µm.

Journal: bioRxiv

Article Title: Finding the right tool: a comprehensive evaluation of microglial inducible cre mouse models

doi: 10.1101/2023.04.17.536878

Figure Lengend Snippet: Evaluation of the dyshomeostatic microglia in different icroglia-specific CreER drivers after TAM treatment at different ages. P2Ry12 expression is used as a measure of dyshomeostasis in microglia. (A), consistent with previous studies, we observe dyshomeostasis of microglia (indicated by loss of P2RY12 expression) across many region in the neonatal Cx3cr1 CreER(Litt) (+/WT) mice treated with TAM. This phenotype is not observed in (B) the adolescent (3wk old) Cx3cr1 CreER(Litt) (+/WT) mice that received TAM treatment or (C) neonatal Cx3cr1 CreER(Jung) (+/WT) and P2RY12CreER (+/WT) mice that received TAM at the similar time frame. Scale bar= 100µm.

Techniques: Expressing

a Scheme of tamoxifen-triggered sparse labeling of microglia for investigation of the microglial engulfment capacity of microglial debris in CX3CR1-CreER::Ai14 and P2Y12-CreER-GFP::Ai14 mice. b tdTomato - microglia do not engulf tdTomato + microglial debris under physiological conditions (D30) or upon CSF1R inhibition (D32) in CX3CR1-CreER::Ai14 mice. c Quantification of the tdTomato + microglial percentage in CX3CR1-CreER::Ai14 mice. N = 5 mice for each group. Two-tailed independent t test. d tdTomato - microglia do not engulf tdTomato + microglial debris under physiological conditions (D20) or upon CSF1R inhibition (D22) in P2Y12-CreER-GFP::Ai14 mice. e Quantification of the tdTomato + microglial percentage in P2Y12-CreER-GFP::Ai14 mice. N = 4 mice for each group. Two-tailed independent t test. f Quantification of tdTomato + microglial debris engulfment by microglia and astrocytes under physiological conditions. The data from the microglial group were adjusted based on the sparse labeling percentage at D30 (CX3CR1-CreER::Ai14) and D20 (P2Y12-CreER-GFP::A14). The data from the astrocytes are obtained from the cortex results shown in Fig. . N = 5 CX3CR1-CreER::Ai14 mice, N = 4 P2Y12-CreER-GFP::Ai14 mice and N = 6 ALDH1L1-CreER::Ai14 mice. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). g Scheme of the in vitro assay of microglia-to-microglial debris engulfment using pHrodo-labeled microglial debris. h IBA1 + microglia engulf pHrodo-labeled microglial debris in the cell culture. pHrodo is illustrated by the green pseudocolor for better visualization. i Quantification of the microglia-to-microglial debris engulfment after exposure to pHrodo-labeled microglial debris for 24 h. N = 4 biological replicates of each group. Two-tailed independent t test. j The territory-dependent competition model (hypothesis) explains that microglia do not phagocytose microglial debris in vivo. (I) Under normal conditions and (II) when a microglial cell dies. PLX5622: PLX5622-formulated AIN-76A diet; IP intraperitoneal. Data are presented as mean ± SD. The source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Microglial debris is cleared by astrocytes via C4b-facilitated phagocytosis and degraded via RUBICON-dependent noncanonical autophagy in mice

doi: 10.1038/s41467-022-33932-3

Figure Lengend Snippet: a Scheme of tamoxifen-triggered sparse labeling of microglia for investigation of the microglial engulfment capacity of microglial debris in CX3CR1-CreER::Ai14 and P2Y12-CreER-GFP::Ai14 mice. b tdTomato - microglia do not engulf tdTomato + microglial debris under physiological conditions (D30) or upon CSF1R inhibition (D32) in CX3CR1-CreER::Ai14 mice. c Quantification of the tdTomato + microglial percentage in CX3CR1-CreER::Ai14 mice. N = 5 mice for each group. Two-tailed independent t test. d tdTomato - microglia do not engulf tdTomato + microglial debris under physiological conditions (D20) or upon CSF1R inhibition (D22) in P2Y12-CreER-GFP::Ai14 mice. e Quantification of the tdTomato + microglial percentage in P2Y12-CreER-GFP::Ai14 mice. N = 4 mice for each group. Two-tailed independent t test. f Quantification of tdTomato + microglial debris engulfment by microglia and astrocytes under physiological conditions. The data from the microglial group were adjusted based on the sparse labeling percentage at D30 (CX3CR1-CreER::Ai14) and D20 (P2Y12-CreER-GFP::A14). The data from the astrocytes are obtained from the cortex results shown in Fig. . N = 5 CX3CR1-CreER::Ai14 mice, N = 4 P2Y12-CreER-GFP::Ai14 mice and N = 6 ALDH1L1-CreER::Ai14 mice. One-way ANOVA with Holm‒Sidak’s multiple comparisons test (post hoc). g Scheme of the in vitro assay of microglia-to-microglial debris engulfment using pHrodo-labeled microglial debris. h IBA1 + microglia engulf pHrodo-labeled microglial debris in the cell culture. pHrodo is illustrated by the green pseudocolor for better visualization. i Quantification of the microglia-to-microglial debris engulfment after exposure to pHrodo-labeled microglial debris for 24 h. N = 4 biological replicates of each group. Two-tailed independent t test. j The territory-dependent competition model (hypothesis) explains that microglia do not phagocytose microglial debris in vivo. (I) Under normal conditions and (II) when a microglial cell dies. PLX5622: PLX5622-formulated AIN-76A diet; IP intraperitoneal. Data are presented as mean ± SD. The source data are provided as a Source Data file.

Article Snippet: P2Y12-CreER-GFP ( P2ry12 -p2A-CreER-p2A-EGFP) mice were generated by Beijing Biocytogen through CRISPR/Cas9-based extreme genome editing (EGE) according to the authors’ design as a “fee-for-service”.

Techniques: Labeling, Inhibition, Two Tailed Test, In Vitro, Cell Culture, In Vivo

An animated GIF of optical sections obtained from a P2ry12-CreER; Rosa26 Ai14 whole-mount cerebral cortex. The whole-mount sample was immunostained for CD206 (green), which marks meningeal and perivascular macrophages. TdTomato expression marks microglia.

Journal: eLife

Article Title: A new genetic strategy for targeting microglia in development and disease

doi: 10.7554/eLife.54590

Figure Lengend Snippet: An animated GIF of optical sections obtained from a P2ry12-CreER; Rosa26 Ai14 whole-mount cerebral cortex. The whole-mount sample was immunostained for CD206 (green), which marks meningeal and perivascular macrophages. TdTomato expression marks microglia.

Article Snippet: The P2ry12-CreER mouse line will be deposited at Jackson Labs (Stock #034727) and at the Mutant Mouse Resource and Research Center (MMMRC).

Techniques:

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