No abstract

The development of mammalian megakaryocytes (MKs) and platelets, which are thought to be absent in non-mammals, is primarily regulated by the thrombopoietin (TPO)/Mpl system. Although non-mammals possess nucleated thrombocytes instead of platelets, the features of nucleated thrombocyte progenitors remain to be clarified. Here, we provide the general features of TPO using Xenopus laevis TPO (xlTPO). Platelets are generated from the cytoplasm of polyploid megakaryocytes (MKs). In humans, MKs differentiate from haematopoietic stem cells (HSCs) and constitute only a small fraction of bone marrow cells (0.1%-0.5%) 1 . MKs are unique cells that undergo DNA replication, giving rise to polyploid cells that undergo proplatelet formation 2 . The proliferation and maturation of MKs by thrombopoietin (TPO), a ligand for the receptor encoded by the c-mpl proto-oncogene (Mpl) 3-5 , has been well characterized. TPO has been independently identified and purified from different species in mammals 6,7 . In contrast, the origin and development of circulating nucleated thrombocytes in most non-mammalian vertebrates, including fish 8-10 , amphibians 11 , reptiles 12 and aves 13 , remain unknown 14,15 . The evolutionary advantage of deriving platelets from MKs has been previously discussed 16 . Circulating thrombocytes mediate haemostasis and blood coagulation, and result in the activation and cytoskeletal changes of non-mammalian nucleated thrombocytes, similar to those of platelets 17 . In zebrafish, thrombin activates nucleated thrombocytes produced by TPO stimulation 18 . Nevertheless, it is not clear whether polyploid MKs are the precursors of mature nucleate thrombocytes.In humans, HSCs develop into committed multipotent progenitors, which in turn differentiate to produce lymphocyte progenitors, granulocyte/monocyte progenitors, and MK/erythroid progenitors (MEPs). MEPs committed to the formation of erythroid and megakaryocytic progeny then produce mature erythrocytes or platelets 19 . Although TPO is one of the most important inducers of MK maturation, high concentrations of TPO inhibit proplatelet formation in vitro 20 . Recently, Nishimura et al. reported that the IL-1α also stimulates platelet production in response to acute platelet needs 21 . Newly released peripheral platelets exhibit bipolar morphology of round cells and multi-bodied proplatelets 22 . Proplatelet formation and platelet release are accelerated by shear forces in vitro 23 .

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-solid medium containing 20% FCS and xlTPO (5 ng/ml). The xlTPO-induced colonies derived from the spleen, including T12 positive thrombocytic colonies, emerged after 2 days, and the number reached to 65±2 in the culture (1 mL). The number of liver-derived colonies was smaller than that of spleen-derived ones, indicating that the density of thrombocyte progenitors in Xenopus was higher in the spleen, but the total mass of thrombocyte progenitors in the body is mostly distributed in the liver based on ratio by organ weights. In Xenopus, moderate thrombocytopenia, as well as anemia, was induced by phenylhydrazine (PHZ). The nadir of circulating thrombocyte counts was observed 4 days after PHZ-administration. When we culture cells of the liver or the spleen in the presence of the PHZ-induced thrombocytopenic serum, colonies composed of white cells and red cells were developed, suggesting that multiple or bipotent hematopoietic progenitors existed. When the hepatic cells were stimulated by xlTPO (5 ng/ml) for 2 days in the liquid culture, T12-positive megakaryocytic larger cells with multinucleated spherical shapes (approx. 30 ±3 μm in diameter) appeared, and such cells did not appear under EPO stimulation. On the other hand, the size of megakaryocytic cells derived from the spleen was smaller. Regardless of the origin of the thrombocyte progenitors, the cells stimulated by xlTPO in the liquid cultures expressed mRNAs of c-Mpl, CD41 and Fli-1, demonstrating that thrombocyte progenitors at different development stages resided in the liver and the spleen. It is still a missing piece of the puzzle whether Xenopus thrombocyte progenitors or mature thrombocytes undergo endomitosis to generate higher polyploid cells under the stimulation by TPO; however the unique megakaryocytic cells observed in this study have a clue to reveal the cellular evolution of platelets/MKs. Disclosures: No relevant conflicts of interest to declare.

In primates and rodents, platelets originate from the bone marrow megakaryocytes through a unique differentiation process with nuclear polyploidization, cytoplasmic maturation and proplatelet formation. In contrast, circulating thrombocytes of most non-mammalian vertebrates are particularly distinctive; the cells are large and nucleated. Adult Xenopus laevis may be an useful non-mammalian model for analyzing dynamic hematopoiesis because they are individually tolerable for time lapse analysis in vivo with sequential blood sampling, whereas classification of cell types has not been established yet. Microstructures of Xenopus thrombocytes observed with electron microscope exhibited structural characteristics largely resembling zebrafish thrombocytes with nucleated spindle cellular features (Thattaliyath et al., Blood 2005), and they had lobulated nuclear chromatin, granules, microparticles and open canalicular system-like-structures as in mammalian megakaryocytes. Since thrombocyte identification based on the morphological aspect was not sufficient, chemical staining with acetylecholinesterase and thiazole orange were performed. Additionally, mice were immunized by Xenopus peripheral blood cells to generate monoclonal antibodies, and two hybridomas producing IgG, respectively T12 and T5, were screened. T12+ (T12 positive) cells were morphologically typical thrombocytes. Flow cytometric analysis revealed that T12+ cells were also positive to anti-human GpIIb/IIIa polyclonal antibodies, and approximately 2-3% of whole peripheral blood cells were T12+/GpIIb/IIIa+ that distributed in FSClow/SSClow fraction. When T12 was injected into Xenopus to deplete T12+ cells in vivo, the detectable level of T12 in the circulation lasted for more than several weeks. Peripheral thrombocyte counts predominantly began to decrease immediately and reached their nadir at day 3, but white blood cell counts were not changed. RNA-rich blood cells considered as younger cells were then increasingly appeared, and finally the cell counts recovered to normal levels at day 10–15, indicating that in vivo depletion of T12+ cells induced thrombopoiesis and/or release of mature thrombocytes from the pool. T5 recognizing cells were classified into two populations by immunostaining and flow cytometry; T5+/GpIIb/IIIa+ cells were morphologically thrombocytic as the cells recognized by T12, while T5+/GpIIb/IIIa− cells were spherical and similar appearance to lymphocytic cells. These observations raised some possibilities e.g.; antigen of T5 was a membrane protein common to both lymphocytes and thrombocytes, or T5+/GpIIb/IIIa− cells were thrombocyte progenitors at earlier development stage than T12+/GpIIb/IIIa+ cells. Nevertheless only a few percent of T12+ and T5+ cells resided in peripheral blood, immunostaining revealed that the proportions of T12+/T5+ and T5+ cells in spleen were 10% and 70%, and T12+/T5+ and T5+ cells in liver were 5% and 20%, respectively. These suggest that spleen is predominantly involved in thrombopoiesis and/or thrombocyte storage in adult Xenopus. As T12 and T5 can be used successfully in flow cytometry and magnetic cell sorting, they should contribute us directly to elucidate the origin of circulating Xenopus thrombocytes and their cellular development process.