Proliferation and Differentiation of Thrombocyte Progenitors In the Liver and the Spleen In Xenopus Laevis Under the Stimulation of Thrombopoietin

• Published in: Blood 2010-11, Vol.116 (21), p.2012-2012
• Main Authors: Tanizaki, Yuta, Tahara, Ayaka, Kinoshita, Sayaka, Yamauchi, Motoki, Meguro, Mizue, Maekawa, Shun, Nagasawa, Kazumichi, Ishida-Iwata, Takako, Obuchi-Shimoji, Miyako, Nogawa-Kosaka, Nami, Kato, Takashi
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V116.21.2012.2012

Abstract 2012 In the biology of thrombopoiesis, several challenging issues such as polyploidy induction, proplatelet formation with endomitotic maturation and tubular cytoplasmic projections, and ability of cell division as reported in human platelets, have not been elucidated sufficiently. Comparative characterization of thrombocyte developments in animals may bring about a new perspective. Characteristics of thrombocyte precursors as megakaryocytes (MKs) and mature thrombocytes in most vertebrates, however, remain poorly defined. Most non-mammalian vertebrates have nucleated and spindle thrombocytes instead of platelets. Since african clawed frog, Xenopus laevis, is one of the most popular species providing various animal models in embryology and physiology, we attempt to establish an adult Xenopus model for analyses of hematopoiesis. We clarified peripheral thrombocytes by various staining methods, and searched immature thrombocytic cells in Xenopus organs. When peripheral blood cells were subjected to acetylcholinesterase staining, thrombocytes in the circulation, i.e. mature thrombocytes were positively identified. The size of elliptical mature thrombocytes was approx. 20.5±0.6 μm by 7.6±1.1 μm in diameters on cytocentrifuge preparations. We produced monoclonal antibody to Xenopus mature thrombocytes (T12) previously. The subsequent flow cytometry with a FACSAria II cell sorter revealed that the proportion of the peripheral T12-positive thrombocytes in lower FSC and SSC ranges were 1.5±0.3% of whole peripheral blood cells, and the expression of Xenopus c-Mpl (xlMpl) mRNA in the sorted cells was detected by RT-PCR. The mRNA expressions of Xenopus TPO (xlTPO) and xlMpl were also detected predominantly in the spleen and the liver, indicating that the sites of thrombocyte progenitor-residing organ and thrombopoietic activity-releasing organ were coincident in adult Xenopus. This resembled the relationship between Xenopus erythropoietin (EPO) and EPO receptor-expressing erythrocytic progenitors, as we have reported (Nogawa-Kosaka et al, 2010, Exp Hematol). Next, immunohistochemical analysis with T12 antibody revealed that thrombocytic cells were localized in sinusoid of the liver and the spleen. We then performed a thrombocytic colony assay in the presence of recombinant xlTPO expressed in E. coli. Hepatic and splenic cells composed of respective 80,000 cells in 1mL were incubated in 35mm dishes at 23°C under 5% CO2 with 0.87% methylcellulose-based semi-so