Radial glia promote microglial development through integrin αVβ8 -TGFβ1 signaling

Abstract

Microglia diversity emerges from interactions between intrinsic genetic programs and environment-derived signals, but how these processes unfold and interact in the developing brain remains unclear. Here, we show that radial glia-expressed integrin beta 8 (ITGB8) expressed in radial glia progenitors activates microglia-expressed TGFβ1, permitting microglial development. Domain-restricted deletion of Itgb8 in these progenitors establishes complementary regions with developmentally arrested "dysmature" microglia that persist into adulthood. In the absence of autocrine TGFβ1 signaling, we find that microglia adopt a similar dysmature phenotype, leading to neuromotor symptoms almost identical to Itgb8 mutant mice. In contrast, microglia lacking the TGFβ signal transducers Smad2 and Smad3 have a less polarized dysmature phenotype and correspondingly less severe neuromotor dysfunction. Finally, we show that non-canonical (Smad-independent) signaling partially suppresses disease and development associated gene expression, providing compelling evidence for the adoption of microglial developmental signaling pathways in the context of injury or disease.

Keywords: Microglia; TGFb signaling; development; differentiation; homeostasis; integrin αVβ8; neuroinflammation; radial glia; stem cells.

Figure 1.. Deletion of Itgb8 in early embryonic radial glia disrupts microglial maturation.

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. 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); 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 pial-associated CD206+ presumptive microglia precursor (model to right). Scale bar in B=500μm, D=200μm, G=25 μm.

Figure 2.. Emx1-Cre deletion of Itgb8 results in anatomically restricted blockage of microglial differentiation.

A) Comparison of the transcriptional properties of adult Itgb8^fl/fl; Emx1^Cre mutant and control microglia to stage specific 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^fl/fl; Emx1^Cre mice revealed anatomically restricted maintenance of the microglial precursor marker CD206 in the cortex and hippocampus (asterisk), accompanied 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^fl/fl; 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^fl/fl; Emx1^Cre mouse. F) Cortex-restricted upregulation of the microglial reactive marker APOE in IBA1+ cells of the cortex (green cells). G) 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.

Figure 3.. Dysmature microglia show pervasive epigenetic changes associated with MGnD and BAM gene upregulation.

A) Tracks of ATAC-seq and H3K9ac ChIP-seq from Itgb8^fl/fl; Emx1^Cre microglia illustrating key homeostatic, border associated macrophage (BAM) and MgND marker gene bodies. B) Comparison between DEGs (RNAseq) and DEPs (ATAC-seq) in cortical and hippocampal microglia from Itgb8^fl/fl; Emx1^Cre vs control brains (Padj < 0.05). Linear correlation between overlapping DEGs and DEPs, Log2FC. C) Comparison between DEGs (RNA-seq) and DEPs (H3K9ac ChIP-seq) in Itgb8^fl/fl; Emx1^Cre (Padj < 0.05) vs control brains. Linear correlation between overlapping DEPs and DEGs Log2FC. D) Heatmap of ATAC-seq showing top 100 Differentially accessible peaks (DEPs) comparing Itgb8^fl/fl; Emx1^Cre to control microglia (n=6, Padj < 0.05). E) Heatmap of H3K9ac ChIP-seq showing top 100 DEGs comparing Itgb8^fl/fl; Emx1^Cre to WT microglia. Padj < 0.05). F) Motif enrichment (HOMER) for positive or negative enriched peaks.

Figure 4.. Microglia provide their own TGFb1 to promote and maintain homeostasis.

A) Sorted bulk-Seq analysis of embryonic microglia and BAMs reveals that Tgfb1 is expressed in both microglia and BAMs during embryonic development, whereas B) P2ry12 and Pf4 are specific markers of these two respective populations. C,D) Analysis of control (C) and conditional Cx3cr1^CreER mediated deletion (D) 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. E) 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. F) P2ry12^CreER recombination, as shown by ROSA-TDT (Ai14) Cre reporter expression, was restricted to microglia (closed arrowheads), and was rarely seen in the overlying meninges (open arrowheads). G) 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. H) Pf4-Cre recombination, as shown by ROSA-TDT (Ai14) Cre reporter expression, was restricted to the embryonic meninges (open arrowheads), and was not seen in microglia (open arrowheads). Scale bar in A=150μm, F-50μm.

Figure 5.. Vascular Tgfb1 is not required for microglial development.

E14.5 coronal brain sections from (A) control (Tgfb^+/−) embryos, (B) embryos with global (Tgfb1^−/−) or cell-lineage specific deletion of Tgfb1 (Tgfb1^fl/fl) in (C) endothelial cells (Cdh5Cre^ER) (D,E) vascular mural cells (Pdgfrb^Cre), or (F,G) endothelial cells and microglia/macrophages (Tie2^Cre). Sections were stained for hemorrhage (TER119, magenta) and vasculature (CD31, green) or for committed/hemostatic microglia (IBA1, magenta and P2RY12, green), or to study P2RY12 expression in the context of hemorrhage (P2RY12, cyan and TER119, yellow). Only Tgfb1^−/− mutants have consistent evidence of vascular dysplasia (marked by X) and hemorrhage (asterisk), whereas mice with microglia/macrophage deletion of Tgfb1 (Tgfb^−/−, and Tgfb1^fl/fl;Tie2Cre mutants) have presence of dysmature microglia (open arrowheads, blowups to right). Panels in E) and G) show ROSA-TdT (Ai14) recombination pattern of Pdgfrb^Cre and Tie2Cre mouse lines respectively, and P2RY12 expression (or lack thereof) in the sporadic hemorrhage (arrowheads) seen when Tgfb1 is deleted with these lines. Cx=Cortex, Str=Striatum. Scale bar in A=150μm.

Figure 6.. TGFb1 is required postnatally for microglial homeostasis.

F) Bulk-seq analysis of Tgfb1 expression in the adult mouse brain. Analysis revealed enrichment for Tgfb1 expression primarily in microglia and vascular cells. B-E) Analysis of conditional Cx3cr1^CreER mediated deletion of Tgfb1 deletion in the P30 mouse brain following neonatal tamoxifen induction at P4,5 and 6. Analysis revealed a “patchy” distribution of dysmature microglia characterized by altered morphology and A) Tgfb1 loss; B) Upregulation of CD206 and loss of pSmad3 staining; D) Loss of the homeostatic marker TMEM119 and E) loss of the homeostatic marker P2RY12. Open arrowheads in B-G mark dysmature microglia, closed arrowheads mark dysmature microglia. Postnatal deletion of Tgfb1 in adulthood (P30-P60) using P2ry12^CreER resulted in isolated production of microglia with altered morphology (F), and loss of pSMAD3 staining (F) and downregulation of the homeostatic marker TMEM119 (G), upregulation of CD206 (H) Scale bar in B=100μm.

Figure 7.. Disruption of non-canonical TGFβ signaling in microglia drives disease-associated gene expression.

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) Cumulative incidence of motor dysfunction seen in different conditional TGFβ pathway mutants as a function of age. Incidence included any of detriment to gait, appearance, or tremor, based on a 0,1,2 rating scale (see for details). C) Correlation of bulk-seq gene expression across TGFβ mutant models. D) Compensatory transcriptional changes of key TGFβ signaling genes in different TGFβ mutant models. E) Bulk-seq analysis of microglial homeostatic and disease associated (MGnD/DAM) microglial 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^fl/fl;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 (asterisks in G and H) and was only seen in the white matter of Smad2/3 conditional mutants (H). Scale bar in F=50μm.