p2ry12-creer mouse line  (Jackson Laboratory)

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

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

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

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

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

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:

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.

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:

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 .

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: Expressing

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.

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: Quantitative RT-PCR

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.

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: Expressing, Construct, Labeling

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.

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: Expressing

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: