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Sall1 is a transcriptional regulator defining microglia identity and function

Nature Immunology volume 17, pages 1397–1406 (2016)Cite this article

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An Erratum to this article was published on 19 January 2017

This article has been updated

Abstract

Microglia are the resident macrophages of the central nervous system (CNS). Gene expression profiling has identified Sall1, which encodes a transcriptional regulator, as a microglial signature gene. We found that Sall1 was expressed by microglia but not by other members of the mononuclear phagocyte system or by other CNS-resident cells. Using Sall1 for microglia-specific gene targeting, we found that the cytokine receptor CSF1R was involved in the maintenance of adult microglia and that the receptor for the cytokine TGF-β suppressed activation of microglia. We then used the microglia-specific expression of Sall1 to inducibly inactivate the murine Sall1 locus in vivo, which resulted in the conversion of microglia from resting tissue macrophages into inflammatory phagocytes, leading to altered neurogenesis and disturbed tissue homeostasis. Collectively, our results show that transcriptional regulation by Sall1 maintains microglial identity and physiological properties in the CNS and allows microglia-specific manipulation in vivo.

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Figure 1: Sall1 expression is restricted mainly to CNS-resident microglia.
Figure 2: CNS-infiltrating myeloid cells and BM-derived microglia and/or macrophages do not express Sall1.
Figure 3: Microglia-specific targeting using Sall1CreER mice.
Figure 4: Gene-expression profile of Sall1-deficient microglia.
Figure 5: Deletion of Sall1 in microglia leads to a reactive phenotype.

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  • 28 November 2016

    In the HTML version of this article initially published, the scale bar was missing from the inset in the top right image in Figure 2d; the bottom left plot in Figure 2e was incorrectly a duplicate of the adjacent plot at right; and the designations in Figure 4b (Sall1fl and Sall1creER/fl) should have been Sall1fl/fl and Sall1CreER/fl (respectively). Also, the arrows in the designations above and below the plots in Supplementary Figure 3b were rendered as boxes; these should have been as follows: Sall1+/+→Cx3cr1CreER-iDTR and Sall1GFP/+→Cx3cr1CreER-iDTR. Finally, in Supplementary Figure 4f, the red (Ki67+) cells in the right set of images were not visible. These errors have been corrected for the HTML version of this article.

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Acknowledgements

We thank V. Tosevski and F. Mair for technical help and flow cytometry sorting; J. Jaberg, S. Nemetz and J. Candreia for technical support; A.L. Croxford for critical reading of the manuscript; S. Jessberger, B. Schreiner and C. Raposo for critical discussions; J.K. Georgijevic and W. Qi for performing the next generation sequencing analysis at the Functional Genomics Centre Zurich (FGCZ); Plexxikon (P. Singh and B. West) for PLX5622-containing and control diets; and the Neuroscience Center Zürich, and the Microbiology and Immunology PhD program, University of Zurich and ETH, Zurich, Switzerland. Cx3cr1CreER were kindly provided by S. Jung (Weizmann Institute of Science). Csf1rfl/fl mice were kindly provided by J. Pollard (Albert Einstein College of Medicine). Supported by the Swiss National Science Foundation (BSSGI0_155832, PP00P3_144781, 316030 150768, 310030 146130, and CRSII3 136203 for M.G. and B.B.), the Swiss Multiple Sclerosis Society (M.G. and B.B.), the European Union FP7 project TargetBraIn, NeuroKine, ATECT (B.B.), the National Agency of Research (LIPOCAMD and MACLEAR for E.L.G.) and the Fondation de France (00056835 for E.L.G.).

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Authors and Affiliations

  1. Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland

    Anne Buttgereit, Iva Lelios, Xueyang Yu, Melissa Vrohlings, Natalie R Krakoski, Burkhard Becher & Melanie Greter

  2. INSERM UMR_S 1166, Sorbonne Universités, UPMC Univ Paris 06, Pitié-Salpêtrière Hospital, Paris, France

    Emmanuel L Gautier

  3. Department of Kidney Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan

    Ryuichi Nishinakamura

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Contributions

A.B. and M.G. designed the study, performed experiments and wrote the manuscript; R.N. provided the Sall1 strains; M.V., X.Y., N.R.K. and I.L. performed experiments; E.L.G. analyzed microarray data (Immgen); and B.B. designed experiments and co-wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Sall1 expression is restricted to microglia within the hematopoietic system.

(a) Flow cytometry plots show representative pre-gating strategy for CD45+ cells (shown are CNS cells). (b) Flow cytometry analysis of GFP (Sall1) expression in different organs of Sall1GFP/+ and Sall1+/+ (control) mice (pre-gated on CD45+ cells as in a. (c) qPCR analysis of Sall1 mRNA in sorted cell populations derived from WT mice; results were normalized to Pol2 expression. Alveolar MFs: CD45+Siglec-F+CD11c+, Lung CD11b+ DCs: CD45+Siglec-F−CD11c+MHCII+CD11b+, Lung CD103+ DCs: CD45+Siglec-F−CD11c+MHCII+CD103+, SP MF (spleen macrophages): F4/80hiCD11b+, SP NPs (spleen neutrophils): Ly6G+SSChi, BM Mo (BM monocytes): Lin−CD11b+Ly6C+CD115+, microglia: CD45loLy6C−Ly6G−CD11b+F4/80+, Per. B cells (peritoneal B cells): B220+, Per. (peritoneal) MFs: CD115+CD11b+F4/80+. (d) qPCR analysis of Sall1 mRNA in total tissue lysates of different organs; results were normalized to Pol2 expression. (e) Representative flow cytometry plots of kidney, liver and heart of Sall1GFP/+ mice (pre-gated on CD45− cells). (f) Quantification of results in e, presented as frequency of GFP+ (Sall1) cells; each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). Data are representative of 2-4 mice per genotype, 2 experiments (b); 2 samples per population pooled from 2-3 WT mice, 2 experiments (c; mean ± s.e.m.); 13 (spleen), 12 (brain), 11 (kidney, liver), 9 (spinal cord), 7 (lung, heart), 4 (skin), 3 (lymph node) WT mice, 2-5 experiments, 1 experiment (lymph node) (d; mean ± s.e.m.); 6 (liver), 5 (kidney, heart) Sall1GFP/+ mice, 2 experiments (e,f).

Supplementary Figure 2 Sall1 expression is specific to resident microglia within the adult CNS.

(a) IHC of brain sections of Sall1GFP/+ mice, showing GFP (green), DAPI (blue), and GFAP (radial-glia-like stem cells), DCX (neuroblasts), Calbindin (Purkinje neurons), S100B (astrocytes) or MBP (oligodendrocytes) (red); insets (without DAPI; top right) are enlargements of the outlined areas in the main images. Scale bars, 20 μm (main image) or 5 μm (insets). (b) Gating strategy of non-hematopoietic (CD45−) CNS-resident cells and representative flow cytometry plots for their GFP expression in Sall1GFP/+ mice. (c) Quantification of results in b, presented as frequency of GFP+ cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). *P < 0.0001 (one-way ANOVA). (d) Gating strategy of CNS-resident myeloid cells and separately isolated choroid plexus (CP) cells. Representative flow cytometry plots display the percentage of GFP+ cells in Sall1GFP/+ mice. MF: Macrophage. (e) IHC of brain sections of Sall1GFP/+ mice, showing GFP (green), DAPI (blue), Iba-1 or F4/80 (microglia and CNS-MF) (red) and CD31 (endothelial cells) (gray); arrowheads indicate Iba-1 and GFP or F4/80 and GFP double-positive microglia; insets (without DAPI; top right) are enlargements of the outlined areas in the main images. Scale bars, 20 μm (main image) or 5 μm (insets). Data are representative of 2-3 mice per staining, 2 experiments (a); 3 mice, 1 experiment (b,c); 6 mice, 2 experiments (d); 2-3 mice per staining, 2 experiments (e).

Supplementary Figure 3 CNS-infiltrating myeloid cells and BM-derived microglia and/or macrophages do not express Sall1.

(a) Frequency of GFP+ microglia and CD45hi MF of Sall1GFP/+ and Cx3cr1GFP/+ reporter mice and of YFP+ or RFP+ microglia and CD45hi MFs in tamoxifen treated Sall1CreERR26-YFP or Cx3cr1CreERR26-RFP mice. Microglia: CD45loCD11b+F4/80+Ly6C−Ly6G−, CD45hi MFs: CD45hiCD11b+F4/80+Ly6C−Ly6G−. (b) Flow cytometry analysis and quantification of the frequency of GFP+ microglia in tamoxifen- and diphtheria toxin-treated Sall1+/+(CD45.1)→Cx3cr1CreER-iDTR(CD45.2) or Sall1GFP/+(CD45.1)→Cx3cr1CreER-iDTR(CD45.2) BM chimeras on day 0, 11 and 21 after treatment (pre-gated on CD11b+F4/80+CD45.1loLy6C−Ly6G− cells) or in untreated Sall1GFP/+ (control) mice. (c) Representative histograms and quantification of GFP expression in monocyte-derived cells (MCs) (gated on CD45hiCD11b+CD11c+MHCII+Ly6C+), neutrophils (gated on CD45hiCD11b+ Ly6G+), CD4+ T cells (gated on CD45hiCD11b-CD4+) and microglia (gated on CD45loLy6C-Ly6G-CD11b+) in Sall1GFP/+ mice at peak disease of MOG35-55/CFA-induced EAE. Each symbol (a-c) represents an individual mouse; small horizontal lines (a-c) indicate the mean (± s.e.m.). *P < 0.0001 (one-way ANOVA). Data are representative of 15 (Sall1GFP/+), 10 (Sall1CreERR26-YFP, Cx3cr1CreERR26-RFP), 4 (Cx3cr1GFP/+) mice, at least 2 experiments (a); 4 mice (d11, d21), 2 experiments and 1 mouse (d0, untreated Sall1GFP/+) (b); 7 (microglia), 5 (MCs, Neutrophils, CD4+ T cells) mice, 3 experiments (c).

Supplementary Figure 4 Microglia-specific targeting utilizing Sall1CreER mice.

(a) Flow cytometry analysis of Sall1CreERR26-YFP mice and R26-YFP (control) littermates on day 3 after 5 consecutive days of tamoxifen treatment, showing the frequency of YFP+ microglia (pre-gated on CD45loLy6G−Ly6C−CD11b+). (b) IHC of cortical brain sections of Sall1CreERCsf1rfl/fl and Csf1rfl/fl mice at day 0, 7 and 14 after 5 consecutive days of tamoxifen treatment, showing Iba-1 (microglia) (green) and DAPI (blue). Scale bar, 20 μm. Quantification shows microglia counts in different brain areas at day 0 after tamoxifen treatment. (c) qPCR analysis of Tgfbr2 mRNA in microglia sorted from Sall1CreERTgfbr2fl/fl and Tgfbr2fl/fl mice on day 0 after three consecutive days of tamoxifen treatment; results were normalized to Pol2 expression. (d) Histograms display the expression of different surface markers vs. FMO on microglia of Sall1CreERTgfbr2fl/fl and Tgfbr2fl/fl mice as in c on day 6 after tamoxifen treatment. (e) Quantification of microglia numbers in Sall1CreERTgfbr2fl/fl and Tgfbr2fl/fl mice as in c on day 0, 3 and 6 after tamoxifen treatment. Numbers are displayed as ratios to control (Tgfbr2fl/fl) mice. (f) IHC of brain sections from Sall1CreERTgfbr2fl/fl and Tgfbr2fl/fl mice as in c analyzed on day 3 after tamoxifen treatment, showing Iba-1 (green), Ki67 (red) and DAPI (blue). Arrowheads indicate Iba-1 and Ki67 double-positive cells. Scale bar, 50 μm. Each symbol (b,e) represents an individual mouse. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired t-test). Data are representative of 10 mice, 5 experiments (a); 3-5 sections of 2 mice, 2 experiments (b; mean ± s.e.m.); 1 pooled sample of 3-4 mice per genotype (c); 2-5 mice per genotype, at least 2 experiments (d); 4 (d0), 3 (d3), 5-6 (d6) mice, 2 experiments (e; mean ± s.e.m.); 1-2 mice per genotype (f).

Supplementary Figure 5 Gene expression profile of Sall1-deficient microglia.

(a-c) Gene expression analysis of microglia sorted from Sall1CreER/fl and Sall1fl/fl mice on day 1 after 5 times of tamoxifen treatment every second day as described in Figure 4. (a) Venn diagram of differentially expressed genes. (b) Volcano plot showing log2 ratios vs. p values (log10) of all 12,673 detected genes. Genes with highest significance values are annotated. (c) Expression (log2 ratio) of Sall1-regulated genes clustered according to their indicated GO-pathways; IS, Immune system; (bold indicates genes discussed in Results). (d) Multiplex immunoassays show levels (pg/mg) of IL-1, IL-6, TNF-α and IL-10 in serum and whole tissue lysates of spleen, kidney and liver of untreated (control) mice and Sall1CreER/fl and Sall1fl/fl mice at day 6 after start of tamoxifen treatment. (e) Graph displays cell counts of DCX+ neuroblasts in hippocampal brain sections of tamoxifen treated Cx3cr1CreERSall1fl/fl and Cre− (control) littermates; each symbol represents an individual mouse; small horizontal lines indicate the mean (± s.e.m.). * p = 0.0006 (unpaired t-test). Data are representative of 3-5 mice pooled per genotype and biological replicate, 3 experiments (a-c); 3 (Sall1CreER/fl, Sall1fl/fl), 2 (untreated) mice, 1 experiment (d; mean ± s.e.m.); 3 (Cx3cr1CreERSall1fl/fl), 4 (control) mice, 2 experiments (e).

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Buttgereit, A., Lelios, I., Yu, X. et al. Sall1 is a transcriptional regulator defining microglia identity and function. Nat Immunol 17, 1397–1406 (2016). https://doi.org/10.1038/ni.3585

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