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Blood. 2014 Jul 10;124(2):220-8. doi: 10.1182/blood-2014-03-564682. Epub 2014 May 28.

Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin.

Author information

1
Department of Cell Differentiation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic;
2
Department of Cellular and Molecular Medicine, and Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA; and.
3
Stem Cell Program and Division of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Stem Cell Institute, Harvard Medical School, Boston, MA.

Abstract

In nonmammalian vertebrates, the functional units of hemostasis are thrombocytes. Thrombocytes are thought to arise from bipotent thrombocytic/erythroid progenitors (TEPs). TEPs have been experimentally demonstrated in avian models of hematopoiesis, and mammals possess functional equivalents known as megakaryocyte/erythroid progenitors (MEPs). However, the presence of TEPs in teleosts has only been speculated. To identify and prospectively isolate TEPs, we identified, cloned, and generated recombinant zebrafish thrombopoietin (Tpo). Tpo mRNA expanded itga2b:GFP(+) (cd41:GFP(+)) thrombocytes as well as hematopoietic stem and progenitor cells (HSPCs) in the zebrafish embryo. Utilizing Tpo in clonal methylcellulose assays, we describe for the first time the prospective isolation and characterization of TEPs from transgenic zebrafish. Combinatorial use of zebrafish Tpo, erythropoietin, and granulocyte colony stimulating factor (Gcsf) allowed the investigation of HSPCs responsible for erythro-, myelo-, and thrombo-poietic differentiation. Utilizing these assays allowed the visualization and differentiation of hematopoietic progenitors ex vivo in real-time with time-lapse and high-throughput microscopy, allowing analyses of their clonogenic and proliferative capacity. These studies indicate that the functional role of Tpo in the differentiation of thrombocytes from HSPCs is well conserved among vertebrate organisms, positing the zebrafish as an excellent model to investigate diseases caused by dysregulated erythro- and thrombo-poietic differentiation.

PMID:
24869937
PMCID:
PMC4093681
DOI:
10.1182/blood-2014-03-564682
[Indexed for MEDLINE]
Free PMC Article

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