The regulation of hematopoiesis in non-mammalian vertebrates is poorly understood. This is partly because the structures and effects of most hematopoietic regulators have not been identified. As a first step towards studies on the key mechanism of hematopoietic regulation among phyla as well as the diversity of organisms, we have focused on amphibian hematopoiesis. A cDNA sharing the highest degree of homology with mammalian erythropoietin (EPO) receptors, tentatively named xlEPOR, was cloned from a cDNA library of Xenopus laevis immature erythrocytes. The comparative identities of the deduced entire amino acid sequence to mammalian EPO receptors were quite low, although functional domains indispensable for erythropoietic activities were found in the molecule. Northern analysis revealed that xlEPOR were expressed in peripheral blood cells. In the peripheral blood of phenylhydrazine-treated adult Xenopus, immature erythrocytes expressing xlEPOR were identified by in situ hybridization and immunostaining with polyclonal antibodies to xlEPOR. To confirm the biological functions of this molecule, the extracellular domain of xlEPOR (i.e., soluble xlEPOR) was administered to adult Xenopus by consecutive intracardiac injection. The peripheral erythrocyte counts were decreased gradually; meanwhile, immature erythrocytes appeared in the circulation, demonstrating that xlEPOR plays a significant physiological role in erythropoiesis in Xenopus laevis.
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.
Erythropoietin (EPO) is a main regulator of erythropoiesis ensuring oxygen supply in mammalian species. However the functions of EPO in nonmammalian vertebrates remain unclear. In this study, EPO was identified in Xenopus laevis (X. laevis), and its contribution to definitive erythropoiesis was studied. The X. laevis EPO (xlEpo) cDNA revealed that the deduced amino acid sequence had only 38% identity to human EPO (hEPO), while all four cysteine residues were conserved. xlEPO mRNA was expressed predominantly in the liver and lung. In order to assess the biological activity, recombinant xlEPO was produced by transfecting COS-1 with CMV promotor-driven vector. A mouse FDC/P2 cells stably expressing xlEPOR cDNA, that is a putative EPO receptor, showed proliferation in response to recombinant xlEPO in a dose dependent manner. This confirmed the ligand-receptor relationship of nonmammalian xlEPO and xlEPOR. To our surprise, xlEPO stimulated proliferation of EPO-dependent human cell line UT-7/EPO as well as murine EPOR expressing FDC/P2 cell lines. The cross-reactivity suggests the tertially structure is conserved through xlEPO to mammalian EPOs. In addition, the amino acid residues that are essential for hEPO binding to hEPOR are highly conserved in xlEPO. Since potent N-glycosylation site is absent in xlEPO, the glycosylation characteristics of recombinant xlEPO was studied by fractionation using wheat germ aggulutinin (WGA) and concanavalin A (ConA) lectin affinity chromatography. XlEPO activity was seen in flow-through fractions indicating the absence of O-glycosylation as well as N-glycosylation in xlEPO molecule. The absence of glycosylation suggests the high affinity of xlEPO to xlEPOR, and the shorter blood half-life. In order to investigate the biological function of xlEPO, in vitro colony forming assay of X. laevis erythroid progenitors was developed. Magnetic cell sorting analysis showed that xlEPOR-positive cells reside in the liver possessing typical erythroblastic morphology with high nucleus-to-cytoplasm ratio containing hemoglobin. The formation of erythroblast colonies from liver cells on addition of recombinant xlEPO was observed. The colonies formed were erythroblast colonies composed of hemoglobin-synthesising erythroblasts, confirming the erythropoietic function of xlEPO in X. laevis erythropoiesis. These results and the detection of xlEPO mRNA in liver hypothesized the paracrine regulation of xlEPO. In the colony assay, erythropoietic activity was observed in the serum of phenylhydrazine (PHZ) induced anemic X. laevis. The highest erythropoietic activity was observed 4 days after PHZ-administration, prior to the peripheral erythrocyte number reaches a nadir at day 8. These results proved that xlEPO is a functional ortholog of mammalian EPO and its role in vertebrate hematopoietic system, providing new insights into the basis of erythropoietic regulations.
Mature microRNA (miRNA) originated from primary miRNA (pri-miRNA) is a new group of potential regulator for cell differentiation, apoptosis, proliferation and oncogenesis. Some miRNAs were recently identified in hematopoietic cells, while the roles of miRNAs in erythrocytic and megakaryocytic cells had not been well examined. As a first step to explore for miRNAs specific for hematopoietic lineage, the expressions of several known primary microRNAs in erythrocytic and megakaryocytic cell lines, such as TF-1, HL-60, HEK293 and UT-7 leukemia cells, were examined by RT-PCR. We consequently focused on the pri-miR-10a, a primary transcript of miR-10a located within Hox gene clusters, and found the significant expression in TF-1 cells and UT-7/EPO cells. The UT-7/EPO cells were a subline established from the original UT-7 cells, as well as UT-7/GM and UT-7/TPO cells; therefore it was suitable for the further comparative analysis. Interestingly, in UT-7/EPO cells, the expression of pri-miR-10a increased under stimulation of erythropoietin (EPO; 1U/mL and 10U/mL). Based on these observations, it was postulated that pri-miR-10a might involve in modulating erythrocyte differentiation or proliferation. To clarify the role of pri-miR-10a in UT-7/EPO, we have established clonal cell lines by transfecting UT-7/EPO cells with either the control vector or the pri-miR-10a expression vector pCMV-pri-miR10a. Overexpression of pri-miR-10a in the UT-7/EPO cell line (miR10a-UT-7/EPO) was confirmed by RT-PCR. MiR10a-UT-7/EPO showed higher proliferation rate even at low concentration of EPO (0.1 mU/mL). Overexpression of pri-miR-10a did not appear to affect HOXB4 and HOXA1 expression, as similar mRNA levels were seen in both cell lines. It was notable that the cellular size of miR10a-UT-7/EPO became larger than its parental cells. Morphological studies of miR10a-UT-7/EPO were performed in detail. It is possible that miR-10a was capable to modulate morphological features particularly in cellular size relating to cell cycle regulation. For instance, loss of the E2F family members result in marked macrocytic anemia with megaloblastic features in adult mice (Mol Cell. 2000 Aug;6(2):281–91., Mol Cell Biol. 2003 May;23(10):3607–22., Blood. 2006 Aug 1;108(3):886–95.). Data presented here hypothesized that the roles of miR-10a in erythroid cells are tightly associated with cell cycle.
Cross-species comparisons of hematopoietic systems will elucidate the conservation and diversity among species such as zebrafish, Xenopus, chick and mouse, which are not only of interest but different approaches would contribute to general hematology. To begin to understand their hematopoietic systems, particularly the whole animal-physiology, across non-mammalian vertebrates, we have focused on amphibian hematopoiesis. We tried to clarify the localization of definitive hematopoietic progenitors in adult Xenopus laevis, which is still to be determined. When Xenopus was induced acute hemolytic anemia by intraperitoneal phenylhydrazine (PHZ) administration, immature erythroblasts emerging in the circulation and notable increase in erythropoiesis within the liver were observed. We first screened putative hematopoietic tissues, liver, spleen, bone marrow and kidney, for erythroid progenitors using polyclonal antibodies to putative Xenopus erythropoietin receptor (xlEPOR) that we recently identified. MACS and FACS sorting and analysis revealed the existence of xlEPOR expressing cells in both liver and anemic peripheral blood. These xlEPOR positive cells were hemoglobin-positive with o-dianisidine staining, and had typical blastic morphology with high nucleus-to-cytoplasm ratio. We next developed and established an in vitro colony assay system to identify and score the hematopoietic progenitors retrospectively. The method enabled the identification and quantification of erythroid progenitors. Briefly, cells were prepared from liver, spleen, bone marrow and kidney followed by placing in semi-solid culture medium (α-MEM containing 0.8% methylcellulose, 20% FCS with appropriate hematopoietic stimulators), and cultured at 23°C with 5% CO2. The anemic serum exhibited the apparent erythropoietic stimulating activity toward the formation of remarkable number of colonies derived from anemic peripheral blood cells, resembling typical mammalian hematopoietic colony formation. Most of the colonies consisted of hemoglobin-expressing erythroids after two days culture, indicating that colony-forming units-erythroid (CFU-e) appeared in anemic blood. The normal and anemic liver also contained CFU-e, resulting in the formation of mixed and pure hematopoietic colonies. This also proved to be a useful in vitro assay system for identifying and quantifying various hematopoietic progenitors and activities of related cytokines. Figure shows the number of erythroid colonies derived from PHZ-induced anemic peripheral blood and liver stimulated with anemic serum. We furthermore examined spleen and bone marrow side-by-side, since amphibian hematopoietic system is known to unique as erythropoiesis, granulopoiesis, and thrombopoiesis occur at distinct organs. The results demonstrated the direct evidences of predominant contribution of adult liver to erythropoiesis rather than bone marrow or spleen. A new animal model developed here should provide new insights into the basis of hematopoietic regulations. Figure Figure
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