This protocol describes the ex vivo characterization of zebrafish hematopoietic progenitors. We show how to isolate zebrafish hematopoietic cells for cultivation and differentiation in colony assays in semi-solid media. We also describe procedures for the generation of recombinant zebrafish cytokines and for the isolation of carp serum, which are essential components of the medium required to grow zebrafish hematopoietic cells ex vivo. The outcome of these clonal assays can easily be evaluated using standard microscopy techniques after 3-10 d in culture. In addition, we describe how to isolate individual colonies for further imaging and gene expression profiling. In other vertebrate model organisms, ex vivo assays have been crucial for elucidating the relationships among hematopoietic stem cells (HSCs), progenitor cells and their mature progeny. The present protocol should facilitate such studies on cells derived from zebrafish.

Department of Biological Sciences California State University Chico Chico California USA

Department of Cell Differentiation Institute of Molecular Genetics AS CR v v i Prague Czech Republic

Department of Cellular and Molecular Medicine University of California San Diego La Jolla California USA

Division of Hematology Oncology Dana Farber Boston Children's Boston Massachusetts USA

Howard Hughes Medical Institute Harvard Stem Cell Institute Harvard Medical School Boston Massachusetts USA

Stem Cell Program Dana Farber Boston Children's Boston Massachusetts USA

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de Jong JL, Zon LI. Use of the zebrafish system to study primitive and definitive hematopoiesis. Annu Rev Genet. 2005;39:481–501. PubMed

Carroll KJ, North TE. Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish. Exp Hematol. 2014;42:684–696. PubMed PMC

Boatman S, et al. Assaying hematopoiesis using zebrafish. Blood Cells Mol Dis. 2013;51:271–276. PubMed PMC

Stewart AM, Braubach O, Spitsbergen J, Gerlai R, Kalueff AV. Zebrafish models for translational neuroscience research: from tank to bedside. Trends Neurosci. 2014;37:264–278. PubMed PMC

Goessling W, North TE. Repairing quite swimmingly: advances in regenerative medicine using zebrafish. Dis Model Mech. 2014;7:769–776. PubMed PMC

White R, Rose K, Zon L. Zebrafish cancer: the state of the art and the path forward. Nat Rev Cancer. 2013;13:624–636. PubMed PMC

Yen J, White RM, Stemple DL. Zebrafish models of cancer: progress and future challenges. Curr Opin Genet Dev. 2014;24:38–45. PubMed PMC

Bertrand JY, et al. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature. 2010;464:108–111. PubMed PMC

Espin-Palazon R, et al. Proinflammatory signaling regulates hematopoietic stem cell emergence. Cell. 2014;159:1070–1085. PubMed PMC

Kim AD, et al. Discrete Notch signaling requirements in the specification of hematopoietic stem cells. EMBO J. 2014;33:2363–2373. PubMed PMC

Kim AD, Stachura DL, Traver D. Cell signaling pathways involved in hematopoietic stem cell specification. Exp Cell Res. 2014;329:227–233. PubMed PMC

Bock TA. Assay systems for hematopoietic stem and progenitor cells. Stem Cells. 1997;15:185–195. PubMed

Beug H, Steinlein P, Bartunek P, Hayman MJ. Avian hematopoietic cell culture: in vitro model systems to study oncogenic transformation of hematopoietic cells. Methods Enzymol. 1995;254:41–76. PubMed

Alexander WS, Begley CG. Thrombopoietin in vitro and in vivo. Cytokines Cell Mol Ther. 1998;4:25–34. PubMed

Stachura DL, et al. Zebrafish kidney stromal cell lines support multilineage hematopoiesis. Blood. 2009;114:279–289. PubMed PMC

Stachura DL, et al. Clonal analysis of hematopoietic progenitor cells in the zebrafish. Blood. 2011;118:1274–1282. PubMed PMC

Stachura DL, Traver D. Cellular dissection of zebrafish hematopoiesis. Methods Cell Biol. 2011;101:75–110. PubMed

Svoboda O, et al. Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin. Blood. 2014;124:220–228. PubMed PMC

Moore MA, Williams N, Metcalf D. In vitro colony formation by normal and leukemic human hematopoietic cells: characterization of the colony-forming cells. J Natl Cancer Inst. 1973;50:603–623. PubMed

McCulloch EA. Stem cells in normal and leukemic hemopoiesis (Henry Stratton Lecture, 1982) Blood. 1983;62:1–13. PubMed

Coulombel L. Identification of hematopoietic stem/progenitor cells: strength and drawbacks of functional assays. Oncogene. 2004;23:7210–7222. PubMed

Quelen C, et al. Identification of a transforming MYB-GATA1 fusion gene in acute basophilic leukemia: a new entity in male infants. Blood. 2011;117:5719–5722. PubMed

Stachura DL, et al. The zebrafish granulocyte colony-stimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance. Blood. 2013;122:3918–3928. PubMed PMC

Surdziel E, et al. Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways. Blood. 2011;117:4338–4348. PubMed

Gerlach GF, Schrader LN, Wingert RA. Dissection of the adult zebrafish kidney. J Vis Exp. 2011;54:2839–2844. PubMed PMC

Traver D, et al. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol. 2003;4:1238–1246. PubMed

Lin HF, et al. Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood. 2005;106:3803–3810. PubMed PMC

Renshaw SA, et al. A transgenic zebrafish model of neutrophilic inflammation. Blood. 2006;108:3976–3978. PubMed

Davidson AJ, Zon LI. The ‘definitive’ (and ‘primitive’) guide to zebrafish hematopoiesis. Oncogene. 2004;23:7233–7246. PubMed

Murtha JM, Qi W, Keller ET. Hematologic and serum biochemical values for zebrafish (Danio rerio) Comp Med. 2003;53:37–41. PubMed

Pedroso GL, et al. Blood collection for biochemical analysis in adult zebrafish. J Vis Exp. 2012:3865–3868. PubMed PMC

Napier I, Ponka P, Richardson DR. Iron trafficking in the mitochondrion: novel pathways revealed by disease. Blood. 2005;105:1867–1874. PubMed

Katzenback BA, Belosevic M. Molecular and functional characterization of kita and kitla of the goldfish (Carassius auratus L.) Dev Comp Immunol. 2009;33:1165–1175. PubMed

Liongue C, Hall CJ, O’Connell BA, Crosier P, Ward AC. Zebrafish granulocyte colony-stimulating factor receptor signaling promotes myelopoiesis and myeloid cell migration. Blood. 2009;113:2535–2546. PubMed

Paffett-Lugassy N, et al. Functional conservation of erythropoietin signaling in zebrafish. Blood. 2007;110:2718–2726. PubMed PMC

Santos MD, Yasuike M, Hirono I, Aoki T. The granulocyte colony-stimulating factors (CSF3s) of fish and chicken. Immunogenetics. 2006;58:422–432. PubMed

Meyer A, Schartl M. Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions. Curr Opin Cell Biol. 1999;11:699–704. PubMed

Meyer A, Van de Peer Y. From 2R to 3R: evidence for a fish-specific genome duplication (FSGD) Bioessays. 2005;27:937–945. PubMed

Chen K, et al. The evaluation of rapid cooling as an anesthetic method for the zebrafish. Zebrafish. 2014;11:71–75. PubMed

Wilson JM, Bunte RM, Carty AJ. Evaluation of rapid cooling and tricaine methanesulfonate (MS222) as methods of euthanasia in zebrafish (Danio rerio) J Am Assoc Lab Anim Sci. 2009;48:785–789. PubMed PMC

Pugach EK, Li P, White R, Zon L. Retro-orbital injection in adult zebrafish. J Vis Exp. 2009:1645–1648. PubMed PMC

Hall C, Flores MV, Storm T, Crosier K, Crosier P. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev Biol. 2007;7:42. PubMed PMC

Ellett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood. 2011;117:49–56. PubMed PMC

Bertrand JY, Kim AD, Teng S, Traver D. CD41+ cmyb+ precursors colonize the zebrafish pronephros by a novel migration route to initiate adult hematopoiesis. Development. 2008;135:1853–1862. PubMed PMC

Balla KM, et al. Eosinophils in the zebrafish: prospective isolation, characterization, and eosinophilia induction by helminth determinants. Blood. 2010;116:3944–3954. PubMed PMC

Wittamer V, Bertrand JY, Gutschow PW, Traver D. Characterization of the mononuclear phagocyte system in zebrafish. Blood. 2011;117:7126–7135. PubMed

Gardiner MR, Gongora MM, Grimmond SM, Perkins AC. A global role for zebrafish klf4 in embryonic erythropoiesis. Mech Dev. 2007;124:762–774. PubMed

Tiedke J, Gerlach F, Mitz SA, Hankeln T, Burmester T. Ontogeny of globin expression in zebrafish (Danio rerio) J Comp Physiol B. 2011;181:1011–1021. PubMed

Kondo H, Watabe S. Growth promoting effects of carp serum components on goldfish culture cells. Fisheries Sci. 2006;72:4.

M-CSFR/CSF1R signaling regulates myeloid fates in zebrafish via distinct action of its receptors and ligands

Zebrafish Kit ligands cooperate with erythropoietin to promote erythroid cell expansion