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Isolation of Tissue Macrophages in Adult Zebrafish

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Tissue-Resident Macrophages

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2713))

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Abstract

Tissue macrophages are essential components of the immune system that also play key roles in vertebrate development and homeostasis, including in zebrafish, which has gained popularity over the years as a translational model for human disease. Commonly, zebrafish macrophages are identified based on expression of fluorescent transgenic reporters, allowing for real-time imaging in living animals. Several of these lines have also proven instrumental to isolate pure populations of macrophages in the developing embryo and larvae using fluorescence-activated cell sorting (FACS). However, the identification of tissue macrophages in adult fish is not as clear, and robust protocols are needed that would take into account changes in reporter specificity as well as the heterogeneity of mononuclear phagocytes as fish reach adulthood. In this chapter, we describe the methodology for analyzing macrophages in various tissues in the adult zebrafish by flow cytometry. Coupled with FACS, these protocols further allow for the prospective isolation of enriched populations of tissue-specific mononuclear phagocytes that can be used in downstream transcriptomic and/or epigenomic analyses. Overall, we aim at providing a guide for the zebrafish community based on our expertise investigating the adult mononuclear phagocyte system.

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References

  1. Link BA, Megason SG (2008) Zebrafish as a model for development. In: Conn PM (ed) Sourcebook of models for biomedical research. Humana Press, Totowa, pp 103–112. https://doi.org/10.1007/978-1-59745-285-4_13

    Chapter  Google Scholar 

  2. Gore AV, Pillay LM, Venero Galanternik M, Weinstein BM (2018) The zebrafish: a fintastic model for hematopoietic development and disease. Wiley Interdisc Rev 7(3). https://doi.org/10.1002/wdev.312

  3. Stachura DL, Traver D (2016) Cellular dissection of zebrafish hematopoiesis. Methods Cell Biol 133:11–53. https://doi.org/10.1016/bs.mcb.2016.03.022

    Article  CAS  PubMed  Google Scholar 

  4. Wattrus SJ, Smith ML, Rodrigues CP, Hagedorn EJ, Kim JW, Budnik B, Zon LI (2022) Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality. Science 377:7. https://doi.org/10.1126/science.abo4837

    Article  CAS  Google Scholar 

  5. Cambier CJ, Takaki KK, Larson RP, Hernandez RE, Tobin DM, Urdahl KB, Cosma CL, Ramakrishnan L (2014) Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature 505(7482):218–222. https://doi.org/10.1038/nature12799

    Article  CAS  PubMed  Google Scholar 

  6. Volkman HE, Pozos TC, Zheng J, Davis JM, Rawls JF, Ramakrishnan L (2010) Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science 327(5964):466–469. https://doi.org/10.1126/science.1179663

    Article  CAS  PubMed  Google Scholar 

  7. Hughes AN, Appel B (2020) Microglia phagocytose myelin sheaths to modify developmental myelination. Nat Neurosci 23(9):1055–1066. https://doi.org/10.1038/s41593-020-0654-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Peri F, Nusslein-Volhard C (2008) Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell 133(5):916–927. https://doi.org/10.1016/j.cell.2008.04.037

    Article  CAS  PubMed  Google Scholar 

  9. Shwartz A, Goessling W, Yin C (2019) Macrophages in zebrafish models of liver diseases. Front Immunol 10:2840. https://doi.org/10.3389/fimmu.2019.02840

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Marques IJ, Lupi E, Mercader N (2019) Model systems for regeneration: zebrafish. Development 146(18). https://doi.org/10.1242/dev.167692

  11. Simoes FC, Cahill TJ, Kenyon A, Gavriouchkina D, Vieira JM, Sun X, Pezzolla D, Ravaud C, Masmanian E, Weinberger M, Mayes S, Lemieux ME, Barnette DN, Gunadasa-Rohling M, Williams RM, Greaves DR, Trinh LA, Fraser SE, Dallas SL, Choudhury RP, Sauka-Spengler T, Riley PR (2020) Macrophages directly contribute collagen to scar formation during zebrafish heart regeneration and mouse heart repair. Nat Commun 11(1):600. https://doi.org/10.1038/s41467-019-14263-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yan C, Brunson DC, Tang Q, Do D, Iftimia NA, Moore JC, Hayes MN, Welker AM, Garcia EG, Dubash TD, Hong X, Drapkin BJ, Myers DT, Phat S, Volorio A, Marvin DL, Ligorio M, Dershowitz L, McCarthy KM, Karabacak MN, Fletcher JA, Sgroi DC, Iafrate JA, Maheswaran S, Dyson NJ, Haber DA, Rawls JF, Langenau DM (2019) Visualizing engrafted human cancer and therapy responses in immunodeficient zebrafish. Cell 177(7):1903–1914. e1914. https://doi.org/10.1016/j.cell.2019.04.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bevan L, Lim ZW, Venkatesh B, Riley PR, Martin P, Richardson RJ (2020) Specific macrophage populations promote both cardiac scar deposition and subsequent resolution in adult zebrafish. Cardiovasc Res 116(7):1357–1371. https://doi.org/10.1093/cvr/cvz221

    Article  CAS  PubMed  Google Scholar 

  14. van Leeuwen LM, van der Kuip M, Youssef SA, de Bruin A, Bitter W, van Furth AM, van der Sar AM (2014) Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum. Dis Model Mech 7(9):1111–1122. https://doi.org/10.1242/dmm.015453

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Choe CP, Choi S-Y, Kee Y, Kim MJ, Kim S-H, Lee Y, Park H-C, Ro H (2021) Transgenic fluorescent zebrafish lines that have revolutionized biomedical research. Lab Anim Res 37(1). https://doi.org/10.1186/s42826-021-00103-2

  16. Ellett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ (2010) mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117(4):e49–e56. https://doi.org/10.1182/blood-2010-10-314120

    Article  CAS  PubMed  Google Scholar 

  17. Ferrero G, Gomez E, Lyer S, Rovira M, Miserocchi M, Langenau DM, Bertrand JY, Wittamer V (2020) The macrophage-expressed gene (mpeg) 1 identifies a subpopulation of B cells in the adult zebrafish. J Leukoc Biol 107(3):431–443. https://doi.org/10.1002/JLB.1A1119-223R

    Article  CAS  PubMed  Google Scholar 

  18. Moyse BR, Richardson RJ (2020) A population of injury-responsive lymphoid cells expresses mpeg1.1 in the adult zebrafish heart. Immunohorizons 4(8):464–474. https://doi.org/10.4049/immunohorizons.2000063

    Article  CAS  PubMed  Google Scholar 

  19. Kuil LE, Oosterhof N, Ferrero G, Mikulasova T, Hason M, Dekker J, Rovira M, van der Linde HC, van Strien PM, de Pater E, Schaaf G, Bindels EM, Wittamer V, van Ham TJ (2020) Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes. elife 9. https://doi.org/10.7554/eLife.53403

  20. Page DM, Wittamer V, Bertrand JY, Lewis KL, Pratt DN, Delgado N, Schale SE, McGue C, Jacobsen BH, Doty A, Pao Y, Yang H, Chi NC, Magor BG, Traver D (2013) An evolutionarily conserved program of B-cell development and activation in zebrafish. Blood 122(8):e1–e11. https://doi.org/10.1182/blood-2012-12-471029

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Wittamer V, Bertrand JY, Gutschow PW, Traver D (2011) Characterization of the mononuclear phagocyte system in zebrafish. Blood 117(26):7126–7135. https://doi.org/10.1182/blood-2010-11-321448

    Article  CAS  PubMed  Google Scholar 

  22. Traver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI (2003) Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol 4(12):1238–1246. https://doi.org/10.1038/ni1007

    Article  CAS  PubMed  Google Scholar 

  23. Sieger D, Moritz C, Ziegenhals T, Prykhozhij S, Peri F (2012) Long-range Ca2+ waves transmit brain-damage signals to microglia. Dev Cell 22(6):1138–1148. https://doi.org/10.1016/j.devcel.2012.04.012

    Article  CAS  PubMed  Google Scholar 

  24. Tang Q, Iyer S, Lobbardi R, Moore JC, Chen H, Lareau C, Hebert C, Shaw ML, Neftel C, Suva ML, Ceol CJ, Bernards A, Aryee M, Pinello L, Drummond IA, Langenau DM (2017) Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing. J Exp Med 214(10):2875–2887. https://doi.org/10.1084/jem.20170976

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Paolicelli RC, Sierra A, Stevens B, Tremblay M-E, Aguzzi A, Ajami B, Amit I, Audinat E, Bechmann I, Bennett M, Bennett F, Bessis A, Biber K, Bilbo S, Blurton-Jones M, Boddeke E, Brites D, Brône B, Brown GC, Butovsky O, Carson MJ, Castellano B, Colonna M, Cowley SA, Cunningham C, Davalos D, De Jager PL, de Strooper B, Denes A, Eggen BJL, Eyo U, Galea E, Garel S, Ginhoux F, Glass CK, Gokce O, Gomez-Nicola D, González B, Gordon S, Graeber MB, Greenhalgh AD, Gressens P, Greter M, Gutmann DH, Haass C, Heneka MT, Heppner FL, Hong S, Hume DA, Jung S, Kettenmann H, Kipnis J, Koyama R, Lemke G, Lynch M, Majewska A, Malcangio M, Malm T, Mancuso R, Masuda T, Matteoli M, McColl BW, Miron VE, Molofsky AV, Monje M, Mracsko E, Nadjar A, Neher JJ, Neniskyte U, Neumann H, Noda M, Peng B, Peri F, Perry VH, Popovich PG, Pridans C, Priller J, Prinz M, Ragozzino D, Ransohoff RM, Salter MW, Schaefer A, Schafer DP, Schwartz M, Simons M, Smith CJ, Streit WJ, Tay TL, Tsai L-H, Verkhratsky A, von Bernhardi R, Wake H, Wittamer V, Wolf SA, Wu L-J, Wyss-Coray T (2022) Microglia states and nomenclature: a field at its crossroads. Neuron 110(21):3458–3483. https://doi.org/10.1016/j.neuron.2022.10.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ferrero G, Mahony CB, Dupuis E, Yvernogeau L, Di Ruggiero E, Miserocchi M, Caron M, Robin C, Traver D, Bertrand JY, Wittamer V (2018) Embryonic microglia derive from primitive macrophages and are replaced by cmyb-dependent definitive microglia in zebrafish. Cell Rep 24(1):130–141. https://doi.org/10.1016/j.celrep.2018.05.066

    Article  CAS  PubMed  Google Scholar 

  27. Mazzolini J, Le Clerc S, Morisse G, Coulonges C, Kuil LE, van Ham TJ, Zagury JF, Sieger D (2019) Gene expression profiling reveals a conserved microglia signature in larval zebrafish. Glia. https://doi.org/10.1002/glia.23717

  28. Guilliams M, Bonnardel J, Haest B, Vanderborght B, Wagner C, Remmerie A, Bujko A, Martens L, Thone T, Browaeys R, De Ponti FF, Vanneste B, Zwicker C, Svedberg FR, Vanhalewyn T, Goncalves A, Lippens S, Devriendt B, Cox E, Ferrero G, Wittamer V, Willaert A, Kaptein SJF, Neyts J, Dallmeier K, Geldhof P, Casaert S, Deplancke B, Ten Dijke P, Hoorens A, Vanlander A, Berrevoet F, Van Nieuwenhove Y, Saeys Y, Saelens W, Van Vlierberghe H, Devisscher L, Scott CL (2022) Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches. Cell 185(2):379–396 e338. https://doi.org/10.1016/j.cell.2021.12.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to Valérie Wittamer .

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Rovira, M., Pozo, J., Miserocchi, M., Wittamer, V. (2024). Isolation of Tissue Macrophages in Adult Zebrafish. In: Mass, E. (eds) Tissue-Resident Macrophages. Methods in Molecular Biology, vol 2713. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3437-0_5

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  • DOI: https://doi.org/10.1007/978-1-0716-3437-0_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3436-3

  • Online ISBN: 978-1-0716-3437-0

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