Yuta Tanizaki

Identification of erythroid progenitors induced by erythropoietic activity in Xenopus laevis

Oxygen is essential for the survival of animals. Red blood cells in the circulation, i.e. peripheral erythrocytes, are responsible for transporting oxygen to tissues. The regulation of erythropoiesis in vertebrates other than mammals is yet to be elucidated. Recently we identified erythropoietin, a primary regulator of erythropoiesis, in Xenopus laevis, which should enable us to identify target cells, including erythroid progenitors, and to investigate the production and development of erythroid cells in amphibians. Here, we established a semi-solid colony-forming assay in Xenopus laevis to clarify the existence of colony-forming unit-erythroid cells, the functional erythroid progenitors identified in vitro. Using this assay, we showed that recombinant xlEPO induces erythroid colony formation in vitro and detected an increased level of erythropoietin activity in blood serum during acute anemic stress. In addition, our study demonstrated the possible presence of multiple, non-xlEPO, factors in anemic serum supportive of erythroid colony formation. These results indicate that erythropoiesis mediated by erythropoietin is present in amphibian species and, furthermore, that the regulatory mechanisms controlling peripheral erythrocyte number may vary among vertebrates.

Significant modulation of the hepatic proteome induced by exposure to low temperature in Xenopus laevis

Summary The African clawed frog, Xenopus laevis, is an ectothermic vertebrate that can survive at low environmental temperatures. To gain insight into the molecular events induced by low body temperature, liver proteins were evaluated at the standard laboratory rearing temperature (22°C, control) and a low environmental temperature (5°C, cold exposure). Using nano-flow liquid chromatography coupled with tandem mass spectrometry, we identified 58 proteins that differed in abundance. A subsequent Gene Ontology analysis revealed that the tyrosine and phenylalanine catabolic processes were modulated by cold exposure, which resulted in decreases in hepatic tyrosine and phenylalanine, respectively. Similarly, levels of pyruvate kinase and enolase, which are involved in glycolysis and glycogen synthesis, were also decreased, whereas levels of glycogen phosphorylase, which participates in glycogenolysis, were increased. Therefore, we measured metabolites in the respective pathways and found that levels of hepatic glycogen and glucose were decreased. Although the liver was under oxidative stress because of iron accumulation caused by hepatic erythrocyte destruction, the hepatic NADPH/NADP ratio was not changed. Thus, glycogen is probably utilized mainly for NADPH supply rather than for energy or glucose production. In conclusion, X. laevis responds to low body temperature by modulating its hepatic proteome, which results in altered carbohydrate metabolism.

Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies

Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog (Xenopus laevis), we needed to distinguish nucleated thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis thrombocytes. Whereas T5 recognized both thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped thrombocytes. The T12(+) thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl2, adenosine diphosphate, thrombin, or various collagens, T12(+) thrombocytes exhibited aggregation. These thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating thrombocytes exhibited lower DNA ploidy than hepatic thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating thrombocytes. Although differences between MKs and thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.

The influence of artificially introduced N-glycosylation sites on the in vitro activity of Xenopus laevis erythropoietin.

Erythropoietin (EPO), the primary regulator of erythropoiesis, is a heavily glycosylated protein found in humans and several other mammals. Intriguingly, we have previously found that EPO in Xenopus laevis (xlEPO) has no N-glycosylation sites, and cross-reacts with the human EPO (huEPO) receptor despite low homology with huEPO. In this study, we introduced N-glycosylation sites into wild-type xlEPO at the positions homologous to those in huEPO, and tested whether the glycosylated mutein retained its biological activity. Seven xlEPO muteins, containing 1–3 additional N-linked carbohydrates at positions 24, 38, and/or 83, were expressed in COS-1 cells. The muteins exhibited lower secretion efficiency, higher hydrophilicity, and stronger acidic properties than the wild type. All muteins stimulated the proliferation of both cell lines, xlEPO receptor-expressing xlEPOR-FDC/P2 cells and huEPO receptor-expressing UT-7/EPO cells, in a dose-dependent manner. Thus, the muteins retained their in vitro biological activities. The maximum effect on xlEPOR-FDC/P2 proliferation was decreased by the addition of N-linked carbohydrates, but that on UT-7/EPO proliferation was not changed, indicating that the muteins act as partial agonists to the xlEPO receptor, and near-full agonists to the huEPO receptor. Hence, the EPO-EPOR binding site in X. laevis locates the distal region of artificially introduced three N-glycosylation sites, demonstrating that the vital conformation to exert biological activity is conserved between humans and X. laevis, despite the low similarity in primary structures of EPO and EPOR.

Thrombopoietin induces production of nucleated thrombocytes from liver cells in Xenopus laevis

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). Hepatic and splenic cells were cultured in liquid suspension with recombinant xlTPO. These cells differentiated into large, round, polyploid CD41-expressing cells and were classified as X. laevis MKs, comparable to mammalian MKs. The subsequent culture of MKs after removal of xlTPO produced mature, spindle-shaped thrombocytes that were activated by thrombin, thereby altering their morphology. XlTPO induced MKs in cultured hepatic cells for at least three weeks; however, this was not observed in splenic cells; this result demonstrates the origin of early haematopoietic progenitors in the liver rather than the spleen. Additionally, xlTPO enhanced viability of peripheral thrombocytes, indicating the xlTPO-Mpl pathway stimulates anti-apoptotic in peripheral thrombocytes. The development of thrombocytes from MKs via the TPO-Mpl system in X. laevis plays a crucial role in their development from MKs, comparable to mammalian thrombopoiesis. Thus, our results offer insight into the cellular evolution of platelets/MKs in vertebrates. (200/200).

A comparative perspective on erythropoiesis

The acquisition of fundamental information by the use of recent technologies, including omics-based molecular analyses and total RNA sequencing, has opened the door to further advances in physiological studies on new animal models. Currently, we are endeavoring to develop a comparative hematology protocol in order to build a discovery platform. All vertebrates, with the exception of a few species, have universally peripheral erythrocytes and hemoglobin, suggesting erythropoiesis to be an evolutionary index.