Experimental Hematology 2015;43:125–136

Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies Yuta Tanizakia,b, Takako Ishida-Iwatac, Miyako Obuchi-Shimojic, and Takashi Katoa,c a

Integrative Bioscience and Biomedical Engineering, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan; Research Fellow of the Japan Society for the Promotion of Science, Tokyo, Japan; cDepartment of Biology, Education and Integrated Arts and Sciences, Waseda University, Tokyo, Japan

b

(Received 18 February 2014; revised 3 October 2014; accepted 7 October 2014)

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. Copyright Ó 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.

Platelets in mammals originate from the bone marrow or lung megakaryocytes (MKs) through a unique differentiation process [1,2]. After MKs complete repetitive cycles of nuclear duplication without cell division and undergo cytoplasmic maturation, they expand the plasma membrane and form elongated proplatelets, which are tubelike extensions of the MK cytoplasm. Megakaryocytes separate the proplatelet fragments from the cell body, then release individual platelets into circulation [1,3,4]. Our understanding of megakaryopoiesis and thrombopoiesis in humans and rodents has improved since the discovery of the thrombopoietin (TPO)–c-Mpl cellular signaling pathway [5–7]. An ultrastructural analysis of murine and

YT and TI contributed equally to this work. Offprint requests to: Dr. Takashi Kato, Waseda University, Graduate School of Advanced Science and Engineering, 2-2 Wakamatsu, Shinjuku, Tokyo 162-8480, Japan; E-mail: [email protected]

human platelets revealed that TPO drives the MKs to full maturation by generating the surface-connected canalicular system, alpha granules and dense granules, and through cytoplasmic fragmentation [8,9]. Typical platelet agonists, such as thrombin, collagen, arachidonic acid, adenosine diphosphate (ADP), and Ca2þ [10–12], activate the platelets. A priming effect of recombinant TPO in human platelet aggregation induced by multiple agonists has also been observed [13]. We previously reported that erythropoiesis in adult African clawed frog (Xenopus laevis) occurred predominantly in the liver following paracrine stimulation of erythropoietin (EPO) receptor (xlEPOR) by erythropoietin (xlEPO) [14–16]. The EPOR-expressing erythroid progenitors were observed to localize on the inner wall of the hepatic sinusoids [17]. In addition, we found that X. laevis exposed to low environmental temperature developed acute pancytopenia, including thrombocytopenia [18] with modulation of the hepatic proteome [19]. Thus, X. laevis is a valuable

0301-472X/Copyright Ó 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.exphem.2014.10.005

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animal model for analysis of hematopoiesis and environmental response. As a first step, we attempted to characterize circulating thrombocytes and their progenitors in adult X. laevis. Most previous studies on X. laevis thrombocytes have been based only on morphologic classification [20,21]. After the identification of human TPO/c-Mpl, Kakeda et al. [22] reported the cross-reactivity of rat TPO in the early developmental stage of X. laevis, suggesting that their hematopoiesis is likely to be regulated by TPO–cMpl signaling. In association with these observations in erythropoiesis, the biology of thrombopoiesis could be interpreted in a similar way; however, our knowledge of X. laevis thrombocytes and their progenitors is limited owing to the lack of tools other than microscopic methods available for cellular identification. Because MKs in the mammalian bone marrow form a rare subpopulation (approximately 0.1%), specific staining with chemical reagents or antibodies to MKs have been essential for the direct visualization and isolation of MKs from other cells. Acetylcholinesterase (AchE) staining has been applied to chemical cell staining for mature MKs and platelets of rodents [23,24]; however, this staining technique is not applicable in other species, including humans, because of specificity. In such cases, specific megakaryocytic molecular markers are used in situ, such as glycoprotein IIb/ IIIa (CD41), c-Fos, c-Myb, and Fli-1 [25]. In rats, colonyforming-unit MKs (CFU-MKs) were purified directly by immunoadsorption (panning) using monoclonal antibodies (mAbs) against CD41 [26]. In the course of our TPO identification [27], this antibody enabled us to specifically assess TPO activity toward rat CFU-MKs [28] and to develop an immunothrombocytopenic rat model. The generation of specific antibodies to thrombocytes is important to explore, not only the features of thrombocytes, but also the physiology of thrombopoiesis in X. laevis. In X. laevis, because of the lack of breadth of assortment of specific antibodies against particular cell subpopulations of interest, it has been one of the major issues to develop cellular and biochemical analyses, as described in Horb et al [29]. In this study, we initially generated mouse mAbs T5 and T12 by immunizing adult X. laevis peripheral blood cells in mice. T5 reacted to both peripheral thrombocytes and leukocytes, whereas T12 reacted specifically to peripheral thrombocytes. We then characterized T12þ thrombocytes from the various cellular aspects of activation by platelet agonists, DNA content, and tissue localization. Furthermore, we demonstrate an immunothrombocytopenic X. laevis model by administration of T12 to examine the probable presence of humoral factors involved in thrombopoiesis. Results of these studies with X. laevis should provide new insights into the diversity and universality of the vertebrate biology of thrombocytes.

Material and methods Animals and preparation of peripheral blood Wild-type African clawed frogs, X. laevis (10–30 g body weight), were purchased from Aquatic Animal Supply (Misato, Saitama, Japan). Frogs were maintained in plastic tanks at ambient room temperature in a controlled light cycle environment (12-h light/ dark; 22 C), fed with dry carp food, and provided with constant running water. As previously reported [14,15], blood samples were collected after anesthetizing the frogs in ice-cold water by cardiac puncture with a 27-gauge needle attached to a capillary tube (Drummond Scientific, Broomall, PA) coated with the anticoagulant sodium citrate or ethylenediaminetetraacetate (EDTA)2Na. We used 0.8 Dulbecco’s modified phosphate-buffered saline, treated to remove Mg2þ and Ca2þ ions (dDPBS), to prevent coagulation during dilution of whole blood, when necessary. Standard saline solution was diluted in this study to adjust for amphibian osmotic balance. Male BALB/c mice were purchased from Charles River Japan (Kanagawa, Japan). Peripheral blood (20–30 mL) was extracted from the eye orbitals of the mice. All animal experiments in this study were conducted according to the Regulations for Animal Experimentation at Waseda University. Cytological analysis Cytospin preparations of X. laevis blood cells collected by heart puncture were stained using May-Gr€ unwald Giemsa solution (MGG; Wako, Osaka, Japan) as previously described [15], or methyl green pyronin (MGPY; Muto Pure Chemicals, Tokyo, Japan). As described by Karnovsky [30], AchE staining was modified by the inclusion of 4% formalin-acetone solution (1.4 mmol/L Na2HPO4, 7.3 mmol/L KH2PO4, 45% [v/v] acetone, 25% [v/v] formalin) as a fixing solution. For immunostaining, cells were fixed with 4% formalin-acetone solution for 60 min at room temperature (RT). Nonspecific antibody binding was blocked with 4% BlockAce (Snow Brand, Tokyo, Japan) for 60 min at RT, and washed with 0.8 diluted Dulbecco’s modified Tris-buffered saline (dTBS). Sheep polyclonal antibody to human platelet glycoprotein CD41 (Affinity Biologicals, Ancaster, Canada), which Jagadeeswaran et al. reportedly crossreacted with zebrafish thrombocytes [31], was diluted 1:1,000 in 0.4% BlockAce/dTBS. The glass slides were incubated biotin-antisheep immunoglobulin G (IgG; American Qualex, San Clemente, CA) diluted 1:100 in 0.4% BlockAce/dTBS for 60 min at RT. Then, glass slides were incubated in Strep-Tactin–conjugated alkaline phosphatase (AP; BioRad, Hercules, CA) for 60 min at RT. Color development was performed using a solution of 5-bromo-4-chloro-3-indoyl phosphate p-toluidine salt (BCIP)/Nitro-BT (NBT; Dojindo, Kumamoto, Japan) containing 5 mmol/L levamisole. Generation of monoclonal antibodies to Xenopus laevis thrombocytes Peripheral blood (0.4 mL) was overlaid onto 2.5 mL of discontinuous Percoll (Amersham Biosciences, Freiburg, Germany, or GE Healthcare, Piscataway, NJ) gradients (100%, 60%, 50%, and 45% in dDPBS), centrifuged at 800 g for 15 min at 22 C, and then cells containing mature thrombocytes without erythrocytes were recovered from a layer of 50% Percoll. The thrombocyte fraction (1  107 cells in 100 mL dDPBS) was immunized into BALB/ c mice by intraperitoneal injection three times, at days 0, 14, and 25. The immunization was boosted during the final injection

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with recombinant human interleukin 6, expressed in Escherichia coli and purified to homogeneity after the refolding process. Three days after the final immunization, spleen cells were fused with SP2/o-Ag14 murine myeloma cells (provided by the RIKEN BioResource Center [Ibaraki, Japan] through the national BioResource Project of the MEXT, Japan), by using polyethylene glycol 1500 (Roche Diagnostics, Mannheim, Germany). The fused cells were distributed into the wells of a 96-well cell culture plate (Corning, Corning, NY), cultured in 10% heat-inactivated bovine serum optimized for hybridomas (Hyclone, Logan, UT) in Dulbecco’s modified Eagle medium (Nissui, Tokyo, Japan), and fed with the same culture medium containing hypoxanthine-aminopterinthymidine (Sigma, St. Louis, MO). Applying the limited dilution method, we screened IgG-producing hybridomas by assessing culture supernatants using a sandwich enzyme-linked immunosorbent assay (ELISA) [32]. Out of 288 IgG-producing hybridomas, we selected 19 stable hybridomas for further screening based on specific abilities associated with immunostaining against thrombocytes. Smears and cytospin preparations of peripheral blood cells were fixed with 4% formalin-acetone solution for 30 min. Glass slides were then incubated for 60 min with 1 mg/mL of biotinsp-conjugated goat-antimouse IgG (HþL), followed by incubation with Strep-Tactin–conjugated AP for 60 min. After washing three times again, color development was performed with BCIP/NBT. Finally, the new mAbs, named T5 and T12, were selected. The subclasses of T5 and T12 were determined by sequencing. Purification of antibodies Each hybridoma-producing T5 and T12 was cultured in lowprotein medium (CHO-S-SFM II DPM; Gibco, Grand Island, NY). The pH of culture supernatants was adjusted to 5.0 with acetic acid and applied onto an SP Sepharose Fast Flow (Amersham Biosciences or GE Healthcare) column pre-equilibrated with 20 mmol/L citrate buffer (pH 5.0). Each antibody was then purified by optimized elution with 20 mmol/L citrate buffer (pH 5.0) containing NaCl. Flow-cytometric analysis Cells obtained from the spleen, the liver and the kidney of adult X. laevis were passed through a 100-mm nylon mesh and washed three times with dDPBS containing 2% fetal calf serum and 2 mmol/L EDTA (dDPBS-FCS-EDTA). Peripheral blood cells were incubated with T5 or T12 for 30 min and washed with dDPBS-FCS-EDTA, followed by incubation with goat-antimouse IgG-phycoerythrin conjugate (Beckman Coulter, Brea, CA) for 30 min, then analyzed and sorted by fluorescence-activated cell sorting (FACS) with FACSAria II (BD Biosciences, Franklin Lakes, NJ). As respective isotype controls for T5 and T12, mouse IgG2b and mouse IgG1 (DAKO, Glostrup, Denmark) were used. DNA ploidy analysis was performed with flow cytometry using Hoechst33342 (Dojin, Kumamoto, Japan). Gene expression of T12þ cells Total RNA of T12þ cells isolated by FACS was extracted and converted into cDNA using ReverTra Ace (Toyobo, Osaka, Japan), as previously described [15]. Expression levels of the X. laevis CD41 gene (cd41), erythropoietin receptor (epor), and glyceraldehyde-3phosphate dehydrogenase (gapdh) as a control gene were measured by polymerase chain reaction (PCR) using a thermal cycler (Biometra, G€ottingen, Germany and MJ Research, Waltham, MA). The

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primers used were as follows: cd41-Forward (Fw), 50 -AGCACCGT CACCTTCC-30 ; cd41-Reverse (Re), 50 -TCCTCTCTTGGTTCC CAG-30 ; fli-1-Fw, 50 -GGGCTCTGCTCAACTCACA-30 ; fli-1-Re, 50 -GGGCTGACCATAATCGGG-30 ; epor-Fw, 50 -AAACTACAGC AATCCTGGGAAGATCC-30 ; epor-Re, 50 -GTAAACTCCAACTC CAGCGATTAAGG-30 ; gapdh-Fw, 50 -ATGGTGAAGGTTGGAAT TAACGG-30 ; and gapdh-Re, 50 -GACAGGTGACAGTGCTTAT TCC- 30 . Measurement of thrombocyte activation Whole peripheral blood (PB) samples were collected from X. laevis. Five mL of blood cells were incubated with 1 mL of 10 mmol/L CaCl2 (Nakalai Tesque, Kyoto, Japan), 10 mg/mL porcine skin collagen solution containing type I and III collagen (Nippon Meat Packers, Osaka, Japan), 10 mg/mL chicken cartilage collagen solution type II (Nippon Meat Packers), 5 mg/mL bovine dermis collagen (Koken, Tokyo, Japan), 2 mL of 1 mol/L ADP (Sigma), and 10 units/mL thrombin (Amersham Bioscience or GE Healthcare) in the presence or absence of 1 mL of 2.8 mmol/L of prostaglandin E1 (Sigma). After 20 min, samples were smeared and immunostained by T12, and the ratio of spindle-shaped T12þ cells was determined. Immunohistology and immunocytochemistry For immunohistochemistry, fresh frozen sections (7 mm) obtained from the liver and the spleen were fixed in 4% paraformaldehyde for 5 min at 4 C and washed three times with 12 mmol/L Tris-HCl buffered saline (pH 7.5). After permeabilizing in 0.1% Triton (Sigma) for 10 min at RT, sections were blocked with 4% BlockAce, then reacted with 1 mg/mL of T12, followed by secondary goatantimouse IgG-AP conjugate (BioRad) and subsequent visualization with NBT/BCIP solution containing 5 mmol/L levamisole. Immune thrombocytopenic Xenopus laevis model T12 (1 mg/kg body weight) in dDPBS was administered into male X. laevis (n 5 16) twice, at days 0 and 1, by intracardiac injection. As the control, IgG1 was injected (n 5 10). Peripheral blood cells were collected by heart puncture at days 0, 1, 3, 5, 8, and 14. The blood cells were stained with crystal violet (Sigma) solution, and blood cell types (erythrocytes, leukocytes, and thrombocytes) were clarified and counted under a microscope with an improved Neubauer counting chamber. We counted spindle-shaped or awkward-shaped cells as thrombocytes and validated them by immunostaining with T12, when necessary. Peripheral blood cells were collected, stained by MGPY according to the manufacturer’s instructions, and observed under a light microscope. Statistical analysis The results are given as means 6 SEM. Statistical analysis was conducted by using the analysis of variance with the Student’s t test. A p value of 0.05 or less was considered to be statistically significant.

Results Morphologic determination of Xenopus laevis thrombocytes by conventional methods In X. laevis, typical thrombocytes are spindle-shaped, nucleated cells, as described by Hadjiazimi et al. [22]

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(Fig. 1A). Acetylcholinesterase staining, which detects MKs in mice, rats, and cats [23,33], was used to differentially stain spindle and mature thrombocytes among other blood cells in X. laevis (Fig. 1B). We further observed that rabbit polyclonal antibody to human CD41 recognized X. laevis thrombocytes (Fig. 1C), which has also been noted with zebrafish thrombocytes [30], suggesting that the structure and expression of CD41 molecules are widely conserved across the species. Characterization of monoclonal antibodies T5 and T12 We screened two independent hybridomas that produced mAbs T5 and T12. To determine the subclass distributions of T5 and T12 antibodies, we performed direct sequence analysis of IgG cDNA and immunoblotting with specific antibodies to each IgG subclass. Results of both analyses identified the respective subclasses of T5 and T12 as IgG2b and IgG1. Using flow cytometry, the established antibodies, labeled with appropriate fluorescence in combination with forward-scattered (FSC) light and side-scattered

(SSC) light, enabled us to classify and analyze X. laevis blood cells. We initially examined T5þ cell morphology in peripheral blood immunocytochemically. T5 reacted with, not only thrombocytes, but also subpopulations of lymphocytes and granulocytes (Fig. 1D). We next assessed whether CD41 colocalized with antigens of either T5 or T12 on thrombocytes. Immunofluorescence staining and flow-cytometric analysis revealed that the cells binding T12 also reacted to antihuman CD41 polyclonal antibodies (Fig. 2A and 2B). Collectively, these results indicate that T12 recognizes a specific antigen in X. laevis thrombocytes. In contrast, T5þ cells consisted of two subpopulations: T5þ/CD41þ cells that displayed thrombocyte morphologies, and spherical T5þ/CD41 cells similar in appearance to lymphocytic cells (Fig. 2B). Flow-cytometric analysis demonstrated that there were two subpopulations of T5þ cells, and T5lowþ cells that coexpressed CD41 (Fig. 2A). Collectively, we concluded that T5lowþ cells were thrombocytes. Flow-cytometric analysis also revealed that T5þ cells could be resolved further by FSC and SSC (Fig. 2C). Based

Figure 1. Morphology of peripheral thrombocytes in X. laevis. (A) Cytocentrifuge smears were made as described in Materials and Methods and then stained with MGG. (B) X. laevis thrombocytes are rich in AchE. (C) Immunofluorescence analysis of CD41 expression in X. laevis thrombocytes was performed as previously described. (D) Immunostaining of whole blood cells with T5 and T12. T5 antibodies recognized both thrombocytes and leukocytes, based on morphologic observation (left panel), whereas the T12 antibody recognized only thrombocytes (right panel). White arrow point indicates leukocytes, and black arrow points indicate thrombocytes. Bars represent 20 mm.

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on morphology, we classified T5highþ/FSClow/SSClow cells as lymphocytes and T5highþ/FSChigh/SSChigh cells as granulocytes (Fig. 2C and 2E). This provided evidence of an unknown T5 antigen on the cell surface that was common to lymphocytes, granulocytes, and, to a lesser extent, thrombocytes. Conversely, immunohistochemical analysis demonstrated that T12 specifically recognized typical thrombocytes (Fig. 1D), but not erythrocytes or leukocytes, in peripheral blood cells. Morphologically, the T12þ cells were oblong, and flow-cytometric analysis showed that T12þ cells were classified with the FSClow/SSClow population (Fig. 2D). These cellular features were in agreement with previous reports of thrombocyte morphology (Fig. 1D) [21]. The proportion of T12þ cells was only 1.5% 6 0.3% of the entire population of peripheral blood cells (n 5 29; Table 1). To further confirm whether the flow-cytometric cell sorting with T12 could enrich the thrombocyte population, T12þ and T12 cells were sorted from the population of peripheral blood cells. We then examined expressions of specific molecules such as CD41 and Fli-1 in the sorted cells by reverse transcription PCR (RT-PCR) and found that they were highly expressed only in T12þ cells, whereas EPOR mRNA was not (Fig. 2F). Since T5þ cells and T12þ cells were readily distinguished by immunohistochemistry, we then explored the distribution of T5þ and T12þ cells in hematopoietic organs. Immunostaining of cytospin samples and flow-cytometric analysis were performed with peripheral blood, the spleen, and the liver in adult X. laevis, following which the numbers of antibody-positive cells were measured (Table 1). The proportions of these cell numbers in the tissues were distinctly different from those of the peripheral blood. In the spleen, 12.9% of the cells detected were T12þ cells, whereas a much smaller percentage of T12þ cells were observed in the liver. Likewise, T5þ cells (thrombocytes, leukocytes, and lymphocytes) comprised 75.7% of the cells in the spleen and 21.0% of those in the liver. Morphologically, the T12þ hepatic cells localized in the sinusoid in the liver (Fig. 3B) were spherical with large nuclei (Fig. 3C), whereas the T12þ splenic cells surrounding the white pulp in the spleen (Fig. 3A and 3C) exhibited spindle-shaped features resembling peripheral thrombocytes and round cells (Fig. 1A). Although T12þ cells in the spleen and the liver hardly expressed epor, these cells expressed cd41 (Fig. 3D). Activation of Xenopus laevis thrombocytes by CaCl2, adenosine diphosphate, thrombin, and collagen When thrombocytes were activated by various stimuli, such as temperature, shear stress, and contact with a synthetic surface, the shape of the thrombocytes became spherical. Therefore, we collected whole blood cells very carefully for the analysis. After immunostaining with T12, 86.0% 6 7.1% of the T12þ cells on the glass slides were spindle-shaped. In contrast, after treatments with CaCl2,

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ADP, thrombin, or various types of collagens, the proportion of spindle-shaped thrombocytes decreased to approximately 10%–30% (Fig. 4A and 4B). In the presence of prostaglandin E1, an inhibitor of platelet activation, the number of spindle-shaped thrombocytes increased to levels comparable with the control slide, indicating that X. laevis thrombocytes are activated in a manner similar to mammalian thrombocytes (Fig. 4A). DNA ploidy analysis of T12þ cells The T12þ cells exhibited typical ploidy histograms (Fig. 5A); therefore the DNA contents of peripheral thrombocytes were much higher than those of peripheral erythrocytes whose ploidy class was 4N (Fig. 5A). In comparison with peripheral thrombocytes, T12þ cells in the liver or the spleen exhibited twofold higher DNA contents than T12 cells (Fig. 5B and 5C). By comparing the ploidy of T12þ cells in the liver, spleen, and PB, we observed that high ploidy cells were localized mainly in the liver (Fig. 5D). This finding should be of significant interest when considering the development and maturation of thrombocytes in X. laevis. Immunothrombocytopenia induced by T12 antibody When T12 was injected into male X. laevis by intracardiac injections on day 0 and day 1, the number of peripheral thrombocytes began to decrease immediately, and thrombocytopenia was significantly induced, reaching a nadir at day 3. Erythrocyte counts were observed to decrease slightly, whereas white-blood-cell counts were not changed (Fig. 6A). However, this was not seen in control IgG-injected X. laevis (Fig. 6B). The thrombocyte counts recovered to normal levels between days 10 and 15. During the recovery, younger, RNA-rich blood cells stained by methyl green-pyronin solution emerged (Fig. 6C). Although these present studies did not completely elucidate the in vivo mechanism of thrombocyte depletion, coincubation of mature thrombocytes with T12 did not increase the number of apoptotic cells or reduce the number of thrombocytes in the in vitro culture, thereby suggesting that an antibody-related immunoreactive process might be involved. In this model, the ELISA-detectable levels of T12 in the circulation lasted for more than 150 days.

Discussion In this study, we attempted to determine whether X. laevis possessed cells equivalent to mammalian MKs, which are thrombocyte progenitors. Most nonmammalian vertebrates possess nucleated, spindle-shaped thrombocytes instead of platelets. To date, numerous studies have reported nucleated thrombocytes in avian, reptilian, amphibian, and teleost models. Although several challenging issues in platelet biology remain, new

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Figure 2. Characterization of monoclonal antibody to X. laevis thrombocytes. (A) Flow-cytometric analysis of peripheral blood cells stained with mouse IgG and rabbit IgG as controls (left panel). Flow-cytometric analysis of peripheral blood cells stained with T5 or T12 and CD41 (center and right panels). (B) PB was smeared and immnostained by CD41 and T5 or T12. (C) Flow-cytometric analysis of buffy coat cells with T5 revealed three populations. Populations in blue, green, and pink represent T5highþ/FSClow, T5highþ/FSChigh, and T5lowþ, respectively (left panel). Right panels showed FSC and SSC biparametric dot plot. (D) Flow-cytometric analysis of whole blood cells was conducted using T12 antibody. Subpopulations of T12þ cells are shown in the low FSC/SSC area. The population in pink represents T12-labeled cells, compared with negative cells in gray (left panel). Right panels showed FSC and SSC biparametric dot plot. (E) Cells from each gate were cytocentrifuged on glass slides and stained by MGG. (F) RT-PCR analysis for the expression of cd41, fli-1, and epor in the T12 and T12þ cells. (Color version of figure is available online.)

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Table 1. Distributions of cells in peripheral blood and hematopoietic organs Antibody positive cells (%) Immunostaining

PB Spleen Liver

FACS

T12þ

T5þ

T12þ

T5þ

T5lowþ

T5highþ

2.4 6 1.0 12.9 6 0.6 3.3 6 0.2

3.4 6 0.6 75.7 6 4.9 21.0 6 2.4

1.5 6 0.3 10.3 6 1.1 2.0 6 1.2

3.6 6 0.6 83.8 6 0.3 19.1 6 6.7

2.7 6 0.4 NT NT

0.9 6 0.2 NT NT

T5 or T12 cell counts were obtained by both immunostaining and flow-cytometric analysis, as described above. NT 5 Not tested.

perspectives would be gained by comparative studies of platelets and nucleated thrombocytes in animals [31,34]. For example, it would be of interest to examine whether nucleated thrombocytes have the same ability to separate

in the blood stream as is observed in circulating anuclear platelet bodies [35,36]. Additionally, it remains unclear whether MKs can be defined as mature thrombocyte progenitors in species with

Figure 3. Localization of thrombocytes and morphology of T12þ cells in the spleen and the liver. (A,B) Immunohistochemistry using T12 of spleen and liver tissue, as indicated. Spleen and liver were collected from normal X. laevis and cut into 7 mm-thick cryosections. High-power magnifications are shown in the right panels. Arrows point to pigment cells and melanomacrophage. Magnification is 20. Bars indicate 100 mm. No staining is seen in IgG-negative control (left panels). (C) Dissociated cells obtained from the X. laevis spleen and liver were immunostained by T12. Arrows points indicate T12þ cells. (D) RT-PCR analysis for the expression of cd41, epor, and gapdh in the T12þ cells of the spleen and the liver. CV 5 Central vein; RP 5 red pulp; WP 5 white pulp.

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Figure 4. Activation of thrombocytes and morphology of T12þ cells. (A) Activation of X. laevis thrombocytes in whole blood cells with different agonists. Whole blood cells were incubated with activators; after 20 min, cells were smeared on glass slides and stained by immunostaining with T12. The ratios of spindle thrombocytes were calculated (black bar). Whole blood cells were incubated with activator plus prostaglandin E1, and the ratios of spindle thrombocytes were calculated (open bar). *p ! 0.05 for prostaglandin E1–treated thrombocytes vs. prostaglandin E1–untreatead thrombocytes. (B) The cell morphologies of peripheral thrombocytes after treated with pig collagen, calcium, thrombin, and ADP. Smear samples of blood were immunostained by T12. The left panel indicates the negative control as described in Materials and Methods. Black arrow points indicate T12þ cells.

nucleated thrombocytes in their circulatory systems. The cellular identification of MKs/thrombocytes should serve as an important step toward understanding the physiology of hematopoiesis in vertebrates. In fact, we previously developed a unique pancytopenic model of X. laevis by exposure to reduced temperatures. In that model, thrombocyte count gradually decreased at 5 C to approximately 60% of the original values [18,19]. However, it was difficult to analyze thrombocyte metabolism owing to lack of specific methods to trace thrombocytes. Therefore, it was a logical extension of our previous work to explore specific methods to identify thrombocytes in X. laevis.

Thrombocyte-specific antibodies have been prepared in the rainbow trout, carp, and chicken [37–39]. CD41 is the most abundant glycoprotein on the human platelet membrane and a receptor for fibrinogen. CD41 on platelets/ thrombocytes is structurally conserved among humans, chicken, catfish, and zebrafish [40,41]. Although overall amino acid sequence identity between human and X. laevis CD41 is only 44% [42], polyclonal antibodies to human CD41 will bind to X. laevis thrombocytes. However, CD41 antibody lacks affinity to thrombocytes in X. laevis and gave a high background signal in the tissue of X. laevis. We demonstrated the cellular characterization of

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Figure 5. DNA content analysis of T12þ cells in the peripheral blood, spleen, and liver. (A–C) Peripheral blood, splenic cells, and hepatic cells were prepared for DNA content analysis as described in Materials and Methods. Each panel shows the peripheral blood cells labeled with T12 and hoechst33342; overlay of T12þ (black line) and T12 (gray fill) DNA content histograms. (D) White, black, and gray areas of the histogram indicate peripheral, liver, and spleen T12þ cells, respectively.

thrombocytes in X. laevis using T5 or T12 antibodies, which are high-affinity and high-specificity antibodies to X. laevis antigens. When X. laevis thrombocytes were perfused with the platelet-activating factors, the shapes of the thrombocytes changed from a spindle to a spherical form (Fig. 4B). Thrombocytes of birds, amphibians, reptiles, and fish have surface-connected canalicular systems, which are features of mammalian MKs characterized by dense granules that contain nucleotide ADP, adenosine triphosphate, serotonin and Ca2þ [43–45]. In the activation assay, thrombocytes were activated with CaCl2, ADP, thrombin, and various types of collagens (Fig. 4A). Although some vertebrates do not exhibit ADP activity, it is observed in X. laevis, as it is in zebrafish and rainbow trout [31,46]. We next analyzed the distribution of T5þ and T12þ cells in hematopoietic tissues. It is not clear where thrombopoiesis occurs, although we have recently identified erythropoietic progenitors in the liver and the spleen [16,17]. As indicated in Table 1, we detected higher percentages of

T12þ cells in the spleen than in the liver. However, the organ weight of the spleen is approximately 1/47 of the liver [18], and the number of total T12þ cells in liver and spleen were nearly equivalent. Thrombocytes in the spleen were predominantly spindle-shaped, whereas the liver contained significant numbers of larger, spherical T12þ cells, and these cells expressed the cd41 gene (Fig. 3C and 3D). Our results therefore suggest that both the spleen and the liver are thrombocytic organs. Xenopus laevis frogs are identified as pseudo-tetraploids, and ploidy analysis revealed that peripheral thrombocytes displayed twofold higher DNA contents than 4N peripheral erythrocytes (Fig. 5A). Additionally, hepatic thrombocytes contained about twice the DNA content of peripheral thrombocytes (Fig. 5D). In the zebrafish kidney, CD41low cells, which are putative thrombocyte precursors, have large nuclei [47], and thrombocyte progenitors in chicken were also large [48]. If the hepatic thrombocytes in X. laevis were the major source of circulating lower polyploid thrombocytes, hepatic thrombocytes should undergo cell division

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Figure 6. Effect of T12 on peripheral thrombocyte counts in X. laevis. We injected (A) 1 mg T12/g body weight at day 0 and day 1 (n 5 16) and (B) 1 mg IgG1/g body weight at day 0 and day 1 as controls (n 5 10). RNA-rich cells appeared in the circulation in T12 injected X. laevis. The open square, solid dot, and open triangle indicate the counts of the erythrocytes, thrombocytes, and leukocytes, respectively. *p ! 0.05 for peripheral thrombocyte count at day 3 vs. day 0. (C) Cytocentrifuge smears were made and then stained with MGPY.

without polyploidization as they progress toward final maturation. Because there are no platelets in X. laevis, thrombocyte progenitors were not the same as MKs. However, from these findings, we hypothesized that the features of thrombocyte progenitors are similar to MKs. To confirm whether T12þ cells in the spleen were at the termi-

nal development stage of thrombocyte lineage or retained the potential to differentiate, we examined the content of mRNA by thiazole orange staining, according to cell staining methods for reticulated platelets in mammals [49,50] and zebrafish thrombocytes [51]. However, we could not stain young X. laevis thrombocytes differently, because

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the entire population of peripheral thrombocytes and some leukocytes were positive. Throbopioetin, NFE2, or these counterparts in X. laevis might have potential roles in MK maturation and platelet production, but, to our knowledge, they have not yet been characterized. To identify the thrombocyte progenitors, further investigation is needed to demonstrate whether differentiation and maturation of X. laevis thrombocytes is stimulated by TPO. These findings will be important to consider cellular evolution of MKs and platelet production in vertebrates. Immunohistochemistry with T12 demonstrated that T12þ cells were located near xlEPOR-expressing cells in the sinusoid in the liver, but each T12þ cell was not identical to an xlEPOR expressing cell [18]. Although we did not examine directly whether thrombocyte progenitors resided in the liver, in conjunction with an ultrastructural observation of thrombocytic cells in the hepatic endothelial lining, as shown by Spornitz et al. [52], we hypothesize that the niche for thrombopoiesis in X. laevis is in the liver sinusoid. Likewise, T12þ cells in the spleen were observed to occupy the red pulp that surrounded white pulp (Fig. 3A and 3B). Considering the effect of DNA contents and the morphology of T12þ cells in the spleen and the liver, we demonstrated that thrombocyte progenitors at different development stages resided in the liver and the spleen. When T12 was injected into X. laevis, thrombocytopenia was induced, and thrombocyte counts recovered slowly (Fig. 6A). The mean ploidy of T12þ cells was equal to that of peripheral thrombocytes (Fig. 5), and the T12þ cells also resembled the peripheral thrombocytes morphologically (Fig. 3C). These observations suggested that peripheral thrombocytes that emerged at 4 days after injection of T12 were derived from the spleen. At the same time, the circulating number of erythrocytes decreased slightly in response to IgG (Fig. 6A and 6B). In mammals, erythrocytes were captured and ingested by macrophages [53], and macrophages were activated by IgG stimulation via CD16 [54]. Since macrophages in X. laevis were located in hepatic sinusoids and in the spleen [17], erythrocytopenia in IgG- and T12-injected frogs might relate the functions of phagocytic macrophages that ingested ectopic IgG. To our knowledge, this is the first reported example of a nonmammalian thrombocytopenic model, and represents a powerful model for thrombocyte progenitor analysis. The cellularity of thrombocytes in peripheral blood cells was significantly lower in X. laevis (2%) than in the mouse (Table 1). Because erythrocyte progenitors localized in the X. laevis liver represented approximately 25% of all cells, these thrombocyte progenitors represented a minority population [17]. T12þ cells comprise only 3.3% 6 0.2% of liver cells; consequently, it was difficult to analyze the thrombocyte progenitors. Both the circulating number of erythrocytes and that of other blood cells decreased in either a Phenylhydrazine-injected model and a low-temperature exposure model of X. laevis. By contrast, only thrombocyte counts significantly

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decreased in the T12-injected model, though we expected enhanced thrombocyte production in this model. Collectively, the thrombocytopenic animal model induced by T12 should become one of the valuable animal models to analyze vertebrate megakaryopoiesis/thrombopoiesis in vivo. Although the cell-surface antigens recognized by T5 and T12 remain to be identified, we were able to characterize the X. laevis thrombocytes and their tissue localization. In addition, the possibility of systemic thrombopoietic regulation was demonstrated through the immunethrombocytemic X. laevis model. These findings are the first step toward understanding nucleated thrombocyte production. Although we do not elucidate the process of the ploidy alteration and cell division in the course of generating peripheral thrombocytes, future studies of thrombocytes using T12 mAbs will offer new understandings in evolution of megakaryopoiesis/thrombopoiesis. Acknowledgments This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 244479), KAKENHI Grants-in-Aid for Scientific Research (No. 20570063 and 26440171), and from Waseda University grants for special research projects (No. 2005B-077, 2007B-061, 2011B-047, 2012B-045 and 2013B-056). Part of this study was performed as a component of a Private University High-Tech Research Center project supported by the Japanese Ministry of Education, Culture, Sports, Science, and Technology (07H016). Part of this study was performed as a MEXT-Supported Program for the Strategic Research Foundation at Private Universities.

Conflict of interest disclosure No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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