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Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis
Author(s) -
Kulkeaw Kasem,
Inoue Tomoko,
Ishitani Tohru,
Nakanishi Yoichi,
Zon Leonard I.,
Sugiyama Daisuke
Publication year - 2018
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/bjh.15048
Subject(s) - gata1 , zebrafish , haematopoiesis , biology , embryonic stem cell , microbiology and biotechnology , flow cytometry , embryo , gene , stem cell , biochemistry
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 ( DRAQ 5 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 DRAQ 5 high and DRAQ 5 low populations. DRAQ 5 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 DRAQ 5 TM analysis of gata1 : dsRed transgenic embryos, we purified primitive DRAQ 5 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 DRAQ 5 TM ‐based flow cytometry enables purification of primitive zebrafish erythrocytes.