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- Title
Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis.
- Authors
Kulkeaw, Kasem; Inoue, Tomoko; Ishitani, Tohru; Nakanishi, Yoichi; Zon, Leonard I.; Sugiyama, Daisuke
- 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 (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 (<italic>hbae3</italic> and <italic>hbbe1.1</italic>) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5TM analysis of <italic>gata1</italic>:<italic>dsRed</italic> 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.
- Subjects
REGULATION of hematopoiesis; ERYTHROCYTES; ZEBRA danio embryos; ZEBRA danio; FISH blood proteins; FISH embryos
- Publication
British Journal of Haematology, 2018, Vol 180, Issue 3, p420
- ISSN
0007-1048
- Publication type
Article
- DOI
10.1111/bjh.15048