Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis

Kasem Kulkeaw, Tomoko Inoue, Tohru Ishitani, Yoichi Nakanishi, Leonard I. Zon, Daisuke Sugiyama

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1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)420-431
Number of pages12
JournalBritish Journal of Haematology
Volume180
Issue number3
DOIs
Publication statusPublished - Feb 1 2018

All Science Journal Classification (ASJC) codes

  • Hematology

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    Kulkeaw, K., Inoue, T., Ishitani, T., Nakanishi, Y., Zon, L. I., & Sugiyama, D. (2018). Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis. British Journal of Haematology, 180(3), 420-431. https://doi.org/10.1111/bjh.15048