Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis. (19th December 2017)

• Record Type: Journal Article
• Authors: Kulkeaw, Kasem; Inoue, Tomoko; Ishitani, Tohru; Nakanishi, Yoichi; Zon, Leonard I.; Sugiyama, Daisuke
• Is Part Of: British journal of haematology. Volume 180:Number 3(2018)
• Page Start: 420
• Page End: 431
• Publication Date: 2017-12-19
• Subjects: zebrafish -- primitive erythrocyte -- DRAQ5TM -- flow cytometry; Hematology -- Periodicals; Blood -- Diseases -- Periodicals; 616.15
• DOI: 10.1111/bjh.15048
• Languages: English
• ISSNs: 0007-1048

Abstract

Summary: Zebrafish embryos are useful to study haematopoietic gene function in vertebrates, although lack of antibodies to zebrafish proteins has limited the purification of specific cell populations. Here, we purified primitive zebrafish erythrocytes using 1, 5‐bis{[2‐(di‐methylamino)ethyl]amino}‐4, 8‐dihydroxyanthracene‐9, 10‐dione (DRAQ5 TM ), a DNA‐staining fluorescent dye. At 48‐h post‐fertilization, we sorted small‐sized cells from embryos using forward scatter and found that they consisted of DRAQ5 high and DRAQ5 low populations. DRAQ5 high cells contained haemoglobin, lacked myeloperoxidase activity and expressed high levels of embryonic globin ( hbae3 and hbbe1.1 ) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5 TM analysis of gata1 : dsRed transgenic embryos, we purified primitive DRAQ5 high dsRed+ erythrocytes from haematopoietic progenitor cells. Using this method, we identified docking protein 2 (Dok2) as functioning in differentiation of primitive erythrocytes. We conclude that DRAQ5 TM ‐based flow cytometry enables purification of primitive zebrafish erythrocytes.