A Combination of Ontogeny and CNS Environment Establishes Microglial Identity

Abstract

Microglia, the brain's resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies.

Keywords: Csf1r; Tmem119; brain macrophage; glial biology; microglia; neuroimmunity; ontogeny; transplantation.

Figure 1. Transplanted microglia engraft in the Csf1r−/− brain and express Tmem119

A) Schematic of microglial transplantation system. B) Immunostaining showing ramified IBA1+(green)/TMEM119+(red) microglia in Csf1r+/+ (WT) and microglia-transplanted Csf1r−/−hosts, and untransplanted Csf1r−/− control. Scale bar = 50μm. C) Expression heat map (log₂(FC/WT)) of microglia (blue), myeloid (orange) and reactivity (red) genes by microglia after culture or transplantation into Csf1r−/− CNS. Detailed inventory of experimental replicates is listed in methods. * = FDR <0.05 compared with WT D) Overlaid volcano plots show reduced differential gene expression of ICT-cells (blue) compared to non-transplanted cultured MGs (orange), represented by reduced spread of volcano. See also Figure S1.

Figure 2. Diverse myeloid populations engraft in the Csf1r−/− brain, ramify, and are Tmem119+

A) IBA1+(green) MLCs ramify and are TMEM119+ (red) in the brain parenchyma 14 days after ICT into Csf1r−/− hosts. Bottom row depicts TMEM119+ MLCs at high magnification. Scale bar = 36.5μm (top 3 rows), 25μm (bottom row). B) Engrafted GFP+ MLCs 14 days after bone marrow ICT into P1 Csf1r−/− host. Arrowheads indicate non-parenchymal donor cells in the ventricles and choroid plexus, of which virtually all are IBA1 positive (see Figure S2F). Scale bar = 900μm. C) Histograms show Tmem119 expression in MLCs 14 days after transplantation by flow cytometry, including reduced staining in HSC-derived MLCs. See also Figure S2.

Figure 3. Peripheral bone marrow injection leads to widespread engraftment of donor-derived cells and results in partial rescue of the Csf1r−/− phenotype; both purified monocytes and Ccr2 −/− bone marrow cells engraft in the Csf1r −/− brain and express Tmem119

A) Engrafted MLCs 1 month after intraperitoneal (IP) bone marrow injection into P1 host. Scale bar = 900μm. B) Hippocampal section of Csf1r−/− brain stained for IBA1 8 months after IP BMT. Scale bar = 400μm. C) Typical Csf1r−/− mouse showing abnormal head shape, small size (left), compared to 3 months after intraperitoneal bone marrow injection at P2 (right). D) Untransplanted Csf1r−/− mouse lacks teeth (top), while transplanted Csf1r−/− mouse shows tooth growth (bottom). E) Both Ccr2 Rfp/Rfp BM and F) purified BM monocytes engraft in the Csf1r −/− brain and express TMEM119 at T=21 days. Scale bar = 100μm. See also Figure S3.

Figure 4. Ontogeny shapes adoption of microglial transcriptional identity; BM-derived cells show highly similar transcriptomes at 2 weeks compared to 2-3 months of brain residence

A) Heatmap showing log2(FC versus WT) expression of microglial (blue), myeloid (orange) and reactivity (red) genes across MLC types. * = FDR<0.05 compared to WT. Grey box indicates that edgeR algorithm could not compute log2(FC) due to low read abundance. B) Plot of largest principal components for cultured microglia (purple), WT microglia (dark blue), pooled ICT MG (blue), YS-MLCs (ICT yolk sac, fetal brain; lighter shades of blue), fetal liver MLCs (light orange), and HSC-MLCs (ICT Blood, BM, and BMT; orange/red), using top 2500 most variant genes. Ellipses demarcate 95% confidence interval for assigned clusters. C) Unsupervised hierarchical clustering of microglia, pooled ICT microglia and MLCs by Spearman coefficients using 1000 most variant genes, AU = approximately unbiased p-value using PVclust package, bootstrap n=10000 D) Venn diagram showing differential gene expression between pooled YS- and HSC-MLCs, both compared to pooled ICT-MGs (2-fold cutoff, FPKM>20, FDR <0.05). See also Figure S5A. E) Volcano plot overlay showing differential gene expression of YS- and HSC-MLCs types compared to ICT-MGs, measured as log2(pMLC/pMG). F) Volcano plot overlay comparing MLCs derived from ICT BM at 14 days (red), to MLCs from IP BM at 2-3 months (orange) showing no gross shift in transcriptome difference from MGs. G) MG identity genes do not change between ICT BM (red) and IP BM (orange), ns, FDR >0.05 by edgeR comparison. See also Figure S5C. MG= microglia, MLC = microglia-like cell, ICT = intracerebral transplant, HSC = hematopoietic stem cells, YS = Yolk Sac, pYS= pooled YS, Fetal Br = fetal brain, Fetal Liv = fetal liver, IP BM = intraperitoneal bone marrow transplant. See also Figure S4, S5.

Figure 5. HSC-MLCs resemble microglia lacking identity genes and in disease states

A) Normalized Enrichment Scores (NES) from GSEA comparing YS- to HSC-MLCs for enrichment in genes upregulated in Sall1−/− (Sall1 KO UP), Nrros−/− (Nrros KO UP), Amyotrophic Lateral Sclerosis (ALS UP from), AD (AD UP), after LPS treatment (LPS UP1, 2), changed in during development (DEV UP 1, 2 or DEV DOWN1,2), in culture (CULT DOWN) and MHCII genes. * = FDR <0.05. B) Expression plot comparing HSC-MLCs and Sall1−/− microglia, both expressed as log2(FC/WT). Red dots highlight genes of interest. r = correlation coefficient, p = p-value for linear regression analysis. C) Apoe gene expression in MGs/MLCs. * = FDR < 0.05. D) RNA in situ hybridization for Apoe (red) with IBA1 counterstain in Csf1r−/− brains 14D post-transplantation with BM or MGs. Arrows indicate Apoe+ MLCs. Scale bar = 100μm. See also Figure S5.

Figure 6. Ontogeny markers discriminate HSC- from YS-MLCs and microglia

A) FPKM values for HSC- and YS-MLC/MG enriched genes (red-orange and blue bars, respectively). Error bars represent SEM, FDR < 0.005 for all genes between YS/MG and HSC groups. B) RNA in situ hybridization showing expression of Ms4a7 or Clec12a (red) in ICT BM- but not YS-MLCs nor ICT MGs. Arrowheads highlight BM-MLCs. Scale bar = 50μm. C) Fluorescent RNA in situ hybridization showing expression of Ms4a7 or Clec12a (red) in ICT BM- but not YS-MLCs nor ICT MGs, co-stained for IBA1 (green). Scale bar = 50μm. D) RNA in situs show persistent expression of Ms4a7 (red) in IBA1+ cells (green) six months after transplantation in HSC-MLCs, but no expression in WT microglia. Scale bar = 50μm. See also Figure S5, 6.

Figure 7. Macrophage transplantation, origin markers, and anti-TMEM119 monoclonal antibodies for the study of primary human macrophages in vivo

A) Pervasively engrafted TMEM119+ (red) cells from human blood in the Rag2−/−Il2rg−/−hMCSF+/+Csf1r−/− brain parenchyma, co-stained with human cytoplasm marker (hCyto, green). Arrowhead indicates TMEM119- cells at edge of engraftment territory. Scale bar = 200μm. (B-D) Representative images of MLCs from human blood (B), adult microglia (C), and cultured fetal brain (D), immunostained for human TMEM119 (red), IBA1 (yellow) and human cytoplasmic marker (green). Arrowheads in (B) identify human TMEM119 low/- cells. Scale bar = 50μm. (E) A custom anti-human TMEM119 antibody identifies CD45+/CD11B+ fetal human MLCs 14 days after transplantation into the mouse CNS. Tmem119 staining (blue) is shown compared to isotype control (red). (F-H) Representative images of MLCs from blood (F), fetal brain (G) and primary human microglia (H) immunostained for human MS4A7 (red) and IBA1 (green). Arrowheads (B,F-H) mark location IBA1+ cell bodies. Scale bar = 100μm. (I-K) Representative images from RNA in situ hybridization of post-mortem Alzheimer’s disease brain samples, showing (I) rare MS4A7+(red)/TMEM119+(green)/IBA1 protein+ (white) macrophages, (J) abundant MS4A7-/TMEM119+/IBA1+ macrophages, and (K) MS4A7+/TMEM119-/IBA1+ perivascular macrophages. Arrowheads show examples of positive puncta, given abundant autofluorescent signal (puncta in perfect registration) Scale bars = 12.5μm. See also Figure S7.