title: Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin.
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.
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic;
authors.forename: Ondřej
authors.initials: O
authors.lastname: Svoboda
authors.affiliation: Department of Cellular and Molecular Medicine, and Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA; and.
authors.forename: David L
authors.initials: DL
authors.lastname: Stachura
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic;
authors.forename: Olga
authors.initials: O
authors.lastname: Machoňová
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic;
authors.forename: Petr
authors.initials: P
authors.lastname: Pajer
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic;
authors.forename: Jiří
authors.initials: J
authors.lastname: Brynda
authors.affiliation: 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.
authors.forename: Leonard I
authors.initials: LI
authors.lastname: Zon
authors.affiliation: Department of Cellular and Molecular Medicine, and Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA; and.
authors.forename: David
authors.initials: D
authors.lastname: Traver
authors.affiliation: Department of Cell Differentiation, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic v.v.i., Prague, Czech Republic;
authors.forename: Petr
authors.initials: P
authors.lastname: Bartůněk
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001792:Blood Platelets / Q000502:physiology; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D004625:Embryo, Nonmammalian; D006410:Hematopoiesis / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000502:physiology; D013926:Thrombopoietin / Q000235:genetics*; D015027:Zebrafish / Q000196:embryology / Q000502:physiology*
chemical_list: D013926:Thrombopoietin