Using the Zebrafish as an Approach to Examine the Mechanisms of Vertebrate Erythropoiesis
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 wordsMicroinjection 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.).
- 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
- 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.O’Brien BA (1961) Identification of haemoglobin by its catalase reaction with peroxide and O-dianisidine. Biotech Histochem 36:57–61Google Scholar