Abstract
Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.
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References
Murayama E et al (2006) Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development. Immunity 25:963–975
Dzierzak E, Speck NA (2008) Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat Immunol 9:129–136
Bertrand JY, Kim AD, Teng S, Traver D (2008) CD41+ cmyb + precursors colonize the zebrafish pronephros by a novel migration route to initiate adult hematopoiesis. Development 135:1853–1862
Kissa K et al (2008) Live imaging of emerging hematopoietic stem cells and early thymus colonization. Blood 111:1147–1156
Ciau-Uitz A, Monteiro R, Kirmizitas A, Patient R (2014) Developmental hematopoiesis: ontogeny, genetic programming and conservation. Exp Hematol 42:669–683
Carroll KJ, North TE (2014) Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish. Exp Hematol 42:684–696
North TE et al (2002) Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity 16:661–672
Burns CE et al (2002) Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators. Exp Hematol 30:1381–1389
Kissa K, Herbomel P (2010) Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464:112–115
Chen MJ, Yokomizo T, Zeigler BM, Dzierzak E, Speck NA (2009) Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature 457:887–891
Iwanami N (2014) Zebrafish as a model for understanding the evolution of the vertebrate immune system and human primary immunodeficiency. Exp Hematol 42:697–706
Zhang P, Liu F (2011) In vivo imaging of hematopoietic stem cell development in the zebrafish. Front Med 5:239–247
Stachura DL et al (2011) Clonal analysis of hematopoietic progenitor cells in the zebrafish. Blood 118:1274–1282
Stachura DL, Traver D (2011) Cellular dissection of zebrafish hematopoiesis. Methods Cell Biol 101:75–110
Lam EYN et al (2009) Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors. Blood 113:1241–1249
Lin H-F et al (2005) Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 106:3803–3810
North TE et al (2007) Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis. Nature 447:1007–1011
Zhu H et al (2005) Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish. Dev Biol 281:256–269
Kikuchi K et al (2011) Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration. Dev Cell 20:397–404
Traver D et al (2003) Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol 4:1238–1246
Lepilina A et al (2006) A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration. Cell 127:607–619
Svoboda O et al (2014) Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin. Blood 124:220–228
Akitake CM, Macurak M, Halpern ME, Goll MG (2011) Transgenerational analysis of transcriptional silencing in zebrafish. Dev Biol 352:191–201
Roederer M (2002) Compensation in flow cytometry. Curr Protoc Cytom Chapter 1, Unit 1.14–1.14.20
Acknowledgments
We thank David Traver (University of California, San Diego) and David Stachura (California State University, Chico) for sharing their protocols, assays, and reagents; and P. Crozier, R. Handin, and K. Poss for providing runx1:eGFP, CD41:eGFP and flk1:dsRed transgenic lines, respectively. We also thank David Stachura and Wolfram Goessling for critical reading of the manuscript. This work was supported by the Harvard Stem Cell Institute, the American Society of Hematology, and National Institutes of Health NIDDK 1R01DK098241-01A1 and 3R01DK098241-03S1.
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Esain, V., Cortes, M., North, T.E. (2016). Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos. In: Kawakami, K., Patton, E., Orger, M. (eds) Zebrafish. Methods in Molecular Biology, vol 1451. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3771-4_13
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DOI: https://doi.org/10.1007/978-1-4939-3771-4_13
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