Using the Zebrafish as an Approach to Examine the Mechanisms of Vertebrate Erythropoiesis

  • Martin D. Kafina
  • Barry H. Paw
Part of the Methods in Molecular Biology book series (MIMB, volume 1698)


The zebrafish, Danio rerio, is a powerful model for the study of erythropoiesis and defining the genetic basis of hematological diseases. The mechanisms of erythroid differentiation are highly conserved in the zebrafish, permitting translational research studies and the modeling of erythropoiesis in higher vertebrates. An advantage of the system is the ability to manipulate gene expression and observe the effect on erythroid development in vivo, with relative ease and rapidity. The production of optically transparent embryos also makes it an attractive tool for visual analysis of circulating erythrocytes that can be used to study erythropoiesis. Through large-scale chemical mutagenesis screens, a variety of zebrafish blood mutants have been identified that are used for gene discoveries and the recapitulation of human diseases. Experimental techniques including in situ hybridization, o-dianisidine staining, flow cytometry, and microinjection are now commonly employed to study red blood cell biochemistry and erythropoiesis in the zebrafish. These techniques have been applied for identifying novel genes required for the hemoglobin synthesis, isolating blood cell lineages, visualizing genetic expression within erythroid tissues, and characterizing the phenotype of blood disorders. The applications of zebrafish methodology to the study of erythropoiesis and optimized step-by-step protocols are discussed in this chapter.

Key words

Microinjection Flow cytometry In situ hybridization o-dianisidine staining Hemoglobin Danio rerio 



We thank our colleagues for critical feedback and comments on this chapter: Eva Buys, Aaron Kithcart, Ludmila Flores, Brian Dulmovits, Lionel Blanc, Alex Cintolo, Thomas Pedulla, Lisa van der Vorm, Bryce Klehm, and Isy Mekler. The in situ methods were from Gabriele E. Ackermann. This work was supported by grants from the National Institutes of Health (R01 DK070838 and P01 HL032262 to B.H.P.).


  1. 1.
    Colle-Vandevelde A (1963) Blood anlage in teleostei. Nature 198:1223CrossRefPubMedGoogle Scholar
  2. 2.
    Shafizadeh E, Paw BP (2004) Zebrafish as a model of human hematologic disorders. Curr Opin Hematol 11:255–261CrossRefPubMedGoogle Scholar
  3. 3.
    Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132:631–644CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Jagannathan-Bogdan M, Zon LI (2013) Hematopoiesis. Development 140:2463–2467CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Traver D, Paw BH, Poss KD, Penberthy WT, Lin S et al (2003) Transplantation and in vivo imagine of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol 4:1238–1246CrossRefPubMedGoogle Scholar
  6. 6.
    Carradice D, Lieschke GJ (2008) Zebrafish in hematology: sushi or science? Blood 111:3331–3339CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Jing L, Durand EM, Ezzio C, Pagliuca SM, Zon LI (2012) In situ hybridization assay-based small molecule screening in zebrafish. Curr. Protocol Chem Biol 4:143–160Google Scholar
  8. 8.
    Reischauer S, Stone OA, Villasenor A, Chi N, Jin SW et al (2015) Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification. Nature 535:294–298CrossRefGoogle Scholar
  9. 9.
    Schulte-Merker S, Lee KJ, McMahon AP, Hammerschmidt M (1997) The zebrafish organizer requires chordino. Nature 387:862–863CrossRefPubMedGoogle Scholar
  10. 10.
    Chen C, Garcia-Santos D, Ishikawa Y, Seguin A, Li L et al (2013) Snx3 regulates recycling of the transferrin receptor and iron assimilation. Cell Metab 17:343–352CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Amigo JD, Ackerman GE, Cope JJ, Yu M, Cooney JD et al (2009) The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish. Blood 114:4654–4663CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Schulte-Merker S (2002) Looking at embryos. In: Nusslein-Volhard C, Dahm R (eds) Zebrafish: a practical approach. Oxford Univ Press, New York, NY, pp 41–43Google Scholar
  13. 13.
    O’Brien BA (1961) Identification of haemoglobin by its catalase reaction with peroxide and O-dianisidine. Biotech Histochem 36:57–61Google Scholar
  14. 14.
    Iuchi I, Yamamoto M (1983) Erythropoiesis in the developing rainbowtrout, Salmo gairdneri irideus: histochemical and immunochemical detection of erythropoietic organs. J Exp Zool 226:409–417CrossRefPubMedGoogle Scholar
  15. 15.
    Shaw GS, Cope JJ, Li L, Corson K, Hersey C et al (2006) Mitoferrin is essential for erythroid iron assimilation. Nature 440:96–100CrossRefPubMedGoogle Scholar
  16. 16.
    Wang Y, Langer NB, Shaw GC, Yang G, Li L et al (2011) Abnormal mitoferrin-1 expression in patients with erythropoietic protoporphyria. Exp Hematol 39:784–794CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Lin HF, Traver D, Zhu H, Dooley K, Paw BH (2005) Analysis of thrombocyte development in CD41-GFP transgenic zebrafish. Blood 106:3803–3810CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Ma D, Zhang J, Lin HF, Italiano J, Handin RI (2011) The identification and characterization of zebrafish hematopoietic stem cells. Blood 118:289–297CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Cooney JD, Hildick-Smith GJ, Shafizadeh E, McBride PF, Carroll KJ et al (2013) Teleost growth factor independence (gfi) genes differentially regulate successive waves of hematopoiesis. Dev Biol 373:431–441CrossRefPubMedGoogle Scholar
  20. 20.
    Ganis JJ, Hsia N, Trompouki E, de Jong JL, DiBiase A et al (2012) Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR. Dev Biol 366:185–194CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Morcos PA (2007) Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos. Biochem Biophys Res Commun 358:521–527CrossRefPubMedGoogle Scholar
  22. 22.
    Eisen JS, Smith JC (2008) Controlling morpholino experiments: don’t stop making antisense. Development 135:1735–1743CrossRefPubMedGoogle Scholar
  23. 23.
    Lichtenstein DA, Crispin AW, Sendamarai AK, Campagna DR, Schmitz-Abe K et al (2016) A recurring mutation in the respiratory complex 1 protein NDUFB11 is responsible for a novel form of X-linked sideroblastic anemia. Blood 128:1913–1917CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Hildick-Smith GJ, Cooney JD, Garone C, Kremer LS, Haack TB et al (2013) Macrocytic anemia and mitochondriopathy resulting from a defect in sideroflexin 4. Am J Hum Genet 93:906–914CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Kardon JR, Yien YY, Huston NC, Branco DS, Hildick-Smith GJ et al (2015) Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis. Cell 161:858–867CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Dang M, Fogley R, Zon LI (2016) Identifying novel cancer therapies using chemical genetics and zebrafish. Adv Exp Med Biol 916:103–124CrossRefPubMedGoogle Scholar
  27. 27.
    Dow LE (2015) Modeling disease in vivo with CRISPR/Cas9. Trends Mol Med 21:609–621CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Irion U, Krauss J, Nüsslein-Volhard C (2014) Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development 141:4827–4830CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Albain J, Durand EM, Yang S, Zhou Y, Zon LI (2015) A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish. Dev Cell 32:756–764CrossRefGoogle Scholar
  30. 30.
    Weinberg E (2007) A device to hold zebrafish embryos during microinjection, Chapter 5. In: The zebrafish book, 4th edn. University of Oregon Press, Eugene, OR. Google Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Hematology Division, Department of MedicineBrigham & Women’s HospitalBostonUSA
  2. 2.Department of MedicineHarvard Medical SchoolBostonUSA
  3. 3.Department of PediatricsHarvard Medical SchoolBostonUSA
  4. 4.Department of Pediatric OncologyDana-Farber Cancer InstituteBostonUSA
  5. 5.Hematology-Oncology Division, Department of MedicineBoston Children’s HospitalBostonUSA
  6. 6.BWH HematologyHarvard Institutes of MedicineBostonUSA

Personalised recommendations