ZFIN ID: ZDB-PUB-171222-7
Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis
Kulkeaw, K., Inoue, T., Ishitani, T., Nakanishi, Y., Zon, L.I., Sugiyama, D.
Date: 2017
Source: British journal of haematology   180(3): 420-431 (Journal)
Registered Authors: Ishitani, Tohru, Zon, Leonard I.
Keywords: DRAQ5TM, flow cytometry, primitive erythrocyte, zebrafish
MeSH Terms:
  • Animals
  • Biomarkers
  • Cell Separation/methods
  • Erythrocytes/cytology*
  • Erythrocytes/metabolism*
  • Flow Cytometry
  • Gene Expression Regulation
  • Hematopoiesis*/genetics
  • Immunophenotyping
  • Organ Specificity/genetics
  • Zebrafish
PubMed: 29265183 Full text @ Br. J. Haematol.
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 (DRAQ5TM ), 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 DRAQ5high and DRAQ5low populations. DRAQ5high cells contained haemoglobin, lacked myeloperoxidase activity and expressed high levels of embryonic globin (hbae3 and hbbe1.1) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5TM analysis of gata1:dsRed transgenic embryos, we purified primitive DRAQ5high 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 DRAQ5TM -based flow cytometry enables purification of primitive zebrafish erythrocytes.