Deletion of a Csf1r Enhancer Selectively Impacts CSF1R Expression and Development of Tissue Macrophage Populations

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Deletion of a Csf1r Enhancer Selectively Impacts CSF1R Expression and Development of Tissue Macrophage Populations ARTICLE https://doi.org/10.1038/s41467-019-11053-8 OPEN Deletion of a Csf1r enhancer selectively impacts CSF1R expression and development of tissue macrophage populations Rocío Rojo 1,2, Anna Raper 1, Derya D. Ozdemir1, Lucas Lefevre 1, Kathleen Grabert 1,3, Evi Wollscheid-Lengeling1, Barry Bradford 1, Melanie Caruso 1, Iveta Gazova 1, Alejandra Sánchez1, Zofia M. Lisowski 1, Joana Alves 1, Irene Molina-Gonzalez 4, Hayk Davtyan 5, Rebecca J. Lodge6, James D. Glover1, Robert Wallace7, David A.D. Munro8, Eyal David9, Ido Amit 9, Véronique E. Miron4, Josef Priller8, Stephen J. Jenkins 6, Giles E. Hardingham8,10, Mathew Blurton-Jones 5, Neil A. Mabbott 1, 1234567890():,; Kim M. Summers 11, Peter Hohenstein1,12, David A. Hume11,14 & Clare Pridans 6,13,14 The proliferation, differentiation and survival of mononuclear phagocytes depend on signals from the receptor for macrophage colony-stimulating factor, CSF1R. The mammalian Csf1r locus contains a highly conserved super-enhancer, the fms-intronic regulatory element (FIRE). Here we show that genomic deletion of FIRE in mice selectively impacts CSF1R expression and tissue macrophage development in specific tissues. Deletion of FIRE ablates macrophage development from murine embryonic stem cells. Csf1rΔFIRE/ΔFIRE mice lack macrophages in the embryo, brain microglia and resident macrophages in the skin, kidney, heart and peri- toneum. The homeostasis of other macrophage populations and monocytes is unaffected, but monocytes and their progenitors in bone marrow lack surface CSF1R. Finally, Csf1rΔFIRE/ΔFIRE mice are healthy and fertile without the growth, neurological or developmental abnormalities reported in Csf1r−/− rodents. Csf1rΔFIRE/ΔFIRE mice thus provide a model to explore the homeostatic, physiological and immunological functions of tissue-specific macrophage populations in adult animals. 1 The Roslin Institute & Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Midlothian EH25 9RG, UK. 2 Tecnologico de Monterrey, Escuela de Medicina y Ciencias de la Salud, Av. Ignacio Morones Prieto 3000 Pte, Col. Los Doctores, C.P. 64710 Monterrey, N.L., Mexico. 3 Department of Environmental Medicine, Toxicology Unit, Karolinska Institutet, Box 210SE-171 77 Stockholm, Sweden. 4 The MRC University of Edinburgh Centre for Reproductive Health, The Queen’s Medical Research Institute, Edinburgh BioQuarter, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. 5 Department of Neurobiology and Behavior, University of California Irvine, 3014 Gross Hall 845 Health Sciences Rd, Irvine, CA 92697-1705, USA. 6 University of Edinburgh Centre for Inflammation Research, The Queen’s Medical Research Institute, Edinburgh BioQuarter, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. 7 The Department of Orthopedic Surgery, University of Edinburgh, Chancellor’s Building, Edinburgh BioQuarter, 49 Little France Crescent, Edinburgh EH16 4SB, UK. 8 UK Dementia Research Institute, The University of Edinburgh, Chancellor’s Building, Edinburgh BioQuarter, 49 Little France Crescent, Edinburgh EH16 4SB, UK. 9 Department of Immunology, Weizmann Institute of Science, 234 Herzl St., Rehovot 7610001, Israel. 10 Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, 15 George Square, Edinburgh EH8 9XD, UK. 11 Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, QLD 4102, Australia. 12 Leiden University Medical Center, P.O. Box 96002300 RC Leiden, The Netherlands. 13 Simons Initiative for the Developing Brain, Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK. 14These authors jointly supervised this work: David A. Hume, Clare Pridans. Correspondence and requests for materials should be addressed to D.A.H. (email: [email protected]) or to C.P. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:3215 | https://doi.org/10.1038/s41467-019-11053-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11053-8 he proliferation, differentiation, and survival of vertebrate kidney, and peritoneum, Csf1rΔFIRE/ΔFIRE mice are healthy and Tmacrophages is controlled by signals from the macrophage fertile. Aside from highlighting the likely function of other reg- colony-stimulating factor (CSF1) receptor (CSF1R), enco- ulatory elements in the Csf1r locus, these mice provide a model to ded by the c-fms protooncogene1, now known as Csf1r. The explore tissue-specific macrophage transcriptional regulation and function of the receptor and its two ligands, CSF1 and interleukin function. 34 (IL34), in macrophage differentiation is conserved in all 2,3 amniotes (i.e. reptiles, birds, and humans) . Deletion of the Results Csf1r locus in the mouse or rat germ line produces a global In vitro validation of CRISPRs targeting the FIRE sequence. deficiency in most tissue macrophage populations4,5. Mutant Guide RNAs (gRNAs) designed to delete FIRE (Fig. 1a) were first animals are osteopetrotic (associated with loss of osteoclasts) and validated in the RAW 264.7 macrophage cell line and in E14 exhibit severe postnatal growth retardation and multiple pleio- mouse embryonic stem cells (ESC). The macrophage cell line tropic impacts on development. Tissue macrophages remain expresses Csf1r mRNA and was used in all of the transfection dependent upon CSF1R in adult mice and can be depleted by studies that previously led to the characterization of the role of treatment with an inhibitory anti-CSF1R antibody6 or treatment FIRE14. Both cell types were co-transfected with pairs of Cas9-2A- with orally available inhibitors of CSF1R kinase activity7.In EGFP constructs each expressing single gRNAs (US1+DS1 or humans, dominant-negative mutations in the tyrosine kinase + DS2), and pools of EGFP cells were screened via PCR to detect domain are associated with an adult-onset neurodegenerative the deletion of FIRE mediated by the CRISPR/Cas9 system. Both disease8. pairs of gRNAs produced deletions in E14 ESC and RAW 264.7 The expression of Csf1r mRNA is restricted to myeloid cells. cells (Supplementary Fig. 1a). The deletion of FIRE and the ab- During embryonic development, Csf1r mRNA is expressed in the sence of mismatches in the remaining Csf1r sequence in E14 ESC earliest macrophages identifiable in the yolk sac and transcription were confirmed by Sanger sequencing (Supplementary Fig. 1b). In of this gene in pluripotent bone marrow (BM) progenitors is the Δ Δ RAW 264.7 macrophage cells, individual Csf1r FIRE/ FIRE clones hallmark of commitment to the monocyte–macrophage lineage. carried slightly different deletions of the FIRE sequence (Supple- Accordingly, the molecular basis for myeloid-restricted tran- mentary Fig. 1c). RAW 264.7 cells are not CSF1R-dependent and scription regulation of Csf1r has been studied in considerable most of the cellular CSF1R protein is retained in the Golgi detail (reviewed in ref. 9). The second intron, downstream of the apparatus. We therefore analyzed cell-associated CSF1R by flow first coding exon, contains a conserved 337bp sequence known as cytometry in permeabilized cells. The deletion of FIRE completely the fms-intronic regulatory element (FIRE), identified as a super- ablates detectable intracellular CSF1R (Fig. 1b). There is no impact enhancer in genome-wide analysis of chromatin in mouse mac- of the FIRE mutation on cell morphology (Fig. 1c) or phagocytic rophages. The mouse FIRE sequence contains binding sites for capacity of cells (Fig. 1d). Neither the frequency nor the median numerous macrophage-expressed transcription factors9. The fluorescence intensity (MFI) of the macrophage surface marker combination of a 3.5 kb Csf1r promoter and intron 2, containing + + Δ Δ F4/80 distinguishes between Csf1r / and Csf1r FIRE/ FIRE clones FIRE, is sufficient to direct reproducible transgenic reporter gene (Fig. 1e). We conclude that FIRE is required specifically for the expression in progenitors, monocytes, granulocytes, classical expression of CSF1R in the RAW 264.7 macrophage cell line. dendritic cells, and tissue macrophages that also express Csf1r E14 mouse ESC can be cultured in the absence of feeders and mRNA10,11. Removal of FIRE from the reporter construct abol- form embryoid bodies (EB)18 with the potential to generate cells ishes expression10. The same Csf1r construct was used to drive belonging to all the primary embryonic germ layers (i.e. conditional Cre-recombinase in lineage-trace studies that dis- endoderm, mesoderm, and ectoderm). Macrophage differentia- sected the role of the yolk sac in macrophage development12.In tion from EB was induced by supplementing culture medium mammals, the FIRE sequence is more highly conserved than any with murine IL3 (mIL3) and recombinant human CSF1 of the exons of the Csf1r gene13. A FIRE sequence is present in the Δ Δ (rhCSF1). Csf1r FIRE/ FIRE mouse ESC generated using the same relative intronic location of the Csf1r locus in all reptile and gRNA pair US1+DS2 are able to produce EB that are bird species, and a core element required for enhancer activity14 indistinguishable from the controls (Fig. 1f, Day 0). By day 7, is perfectly conserved13. The chicken CSF1R promoter and FIRE they give rise to cells that attach to culture plates, indicating that sequences are also sufficient to direct macrophage-specific the deletion of FIRE does not affect responsiveness to IL3. By expression in chicken transgenic lines and to highlight the 2 weeks post-differentiation, a
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