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Thyroid hormone (T3) is essential for development and organ metabolism in all vertebrates. T3 has both genomic and nongenomic effects on target cells. While much has been learnt on its genomic effects via T3 receptors (TRs) in vertebrate development, mostly through TR-knockout and knockin studies, little is known about the effects of T3 on gene expression in animals in the absence of TR. We have been studying Xenopus metamorphosis as a model for mammalian postembryonic development, a period around birth when plasma T3 level peaks and many organs/tissues mature into their adult forms. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals. This offers an opportunity to compare the effects of T3 on global gene expression in tadpole tissues in the presence or absence of TR.We analyzed the effects of T3 on gene expression in tadpole tail and intestine by using RNA-seq analysis on wild-type and TRDKO tadpoles with or without T3 treatment.We observed that removing TRs reduced the number of genes regulated by T3 in both organs. Gene Ontology (GO) and KEGG pathway analyses revealed that T3 affected distinct biological processes and pathways in wild-type and TRDKO tadpoles. Many GO terms and KEGG pathways were enriched among genes regulated in wild-type tissues are likely involved in mediating the effects of T3 on metamorphosis, e.g., those related to development, stem cells, apoptosis, and cell cycle/cell proliferation. However, such GO terms and pathways were not enriched among T3-regulated genes in TRDKO tadpoles. Instead, in TRDKO tadpoles, GO terms and pathways related to "metabolism" and "immune response" were highly enriched among T3-regulated genes. We further observed strong divergence in the TR-independent, nongenomic effects of T3 in the intestine and tail.Our data suggest that T3 have distinct and organ-dependent effects on gene expression in developing tadpoles. The TR-mediated effects are consistent with the metamorphic changes, in agreement with the fact that TR is necessary and sufficient to mediate the effects of T3 on metamorphosis. T3 appears to have a major effect on metabolism and immune response via TR-independent nongenomic processes.

Thyroid hormone (T3) is essential for normal development and metabolism, especially during postembryonic development, a period around birth in mammals when plasma T3 levels reach their peak. T3 functions through two T3 receptors, TRα and TRβ. However, little is known about the tissue-specific functions of TRs during postembryonic development because of maternal influence and difficulty in manipulation of mammalian models. We have studied Xenopus tropicalis metamorphosis as a model for human postembryonic development. By using TRα knockout (Xtr·thra

Thyroid hormone (T3) affects many diverse physiological processes such as metabolism, organogenesis, and growth. The two highly related frog species, diploid Xenopus tropicalis and pseudo tetraploid Xenopus laevis, have been used as models for analyzing the effects of T3 during vertebrate development. T3 regulates T3-inducible gene transcription through T3 receptor (TR)-binding to T3-response elements (TREs). We have previously identified sperm associated antigen 7 (spag7) as a candidate T3 target gene that is potentially involved in adult stem cell development and/or proliferation during intestinal metamorphosis. To investigate whether T3 regulates spag7 directly at the transcriptional level via TR, we first conducted qRT-PCR to analyze its expression during natural and T3-induced metamorphosis and found that spag7 was up-regulated during natural metamorphosis in the intestine, tail, brain and hindlimb, peaking at the climax of metamorphosis in all those organs, and upon T3 treatment of premetamorphic tadpoles. Next, we demonstrated that an intronic TRE in spag7, first identified through bioinformatic analysis, could bind to TR in vitro and in vivo during metamorphosis. A dual luciferase assay utilizing a reconstituted frog oocyte transcription system showed that the TRE could mediate promoter activation by liganded TR. These results indicate that spag7 expression is directly regulated by T3 through the TRE in the first intron during metamorphosis, implicating a role for spag7 early during T3-regulated tissue remodeling and resorption.

Background: Thyroid hormone (triiodothyronine [T3]) plays an important role in regulating vertebrate developmental, cellular, and metabolic processes via T3 receptor (TR). Liganded TR recruit coactivator complexes that include steroid receptor coactivators (SRC1, SRC2 or SRC3), which are histone acetyltransferases, to T3-responsive promoters. The functions of endogenous coactivators during T3-dependent mammalian adult organ development remain largely unclear, in part, due to the difficulty to access and manipulate late-stage embryos and neonates. We use Xenopus metamorphosis as a model for postembryonic development in vertebrates. This process is controlled by T3, involves drastic changes in every organ/tissue, and can be easily manipulated. We have previously found that SRC3 was upregulated in the intestine during amphibian metamorphosis. Methods: To determine the function of endogenous SRC3 during intestinal remodeling, we have generated Xenopus tropicalis animals lacking a functional SRC3 gene and analyzed the resulting phenotype. Results: Although removing SRC3 had no apparent effect on external development and animal gross morphology, the SRC3 (-/-) tadpoles displayed a reduction in the acetylation of histone H4 in the intestine compared with that in wild-type animals. Further, the expression of TR target genes was also reduced in SRC3 (-/-) tadpoles during intestinal remodeling. Importantly, SRC3 (-/-) tadpoles had inhibited/delayed intestinal remodeling during natural and T3-induced metamorphosis, including reduced adult intestinal stem cell proliferation and apoptosis of larval epithelial cells. Conclusion: Our results, thus, demonstrate that SRC3 is a critical component of the TR-signaling pathway in vivo during intestinal remodeling.

Background Thyroid hormone (T3) is critical for development in all vertebrates. The mechanism underlying T3 effect has been difficult to study due to the uterus-enclosed nature of mammalian embryos. Anuran metamorphosis, which is dependent on T3 but independent of maternal influence, is an excellent model to study the roles of T3 and its receptors (TRs) during vertebrate development. We and others have reported various effects of TR knockout (TRα and TRβ) during Xenopus tropicalis development. However, these studies were largely focused on external morphology. Results We have generated TRβ knockout animals containing an out-frame-mutation of 5 base deletion by using the CRISPR/Cas9 system and observed that TRβ knockout does not affect premetamorphic tadpole development. We have found that the basal expression of direct T3-inducible genes is increased but their upregulation by T3 is reduced in the intestine of premetamorphic homozygous TRβ knockout animals, accompanied by reduced target binding by TR. More importantly, we have observed reduced adult stem cell proliferation and larval epithelial apoptosis in the intestine during T3-induced metamorphosis. Conclusions Our data suggest that TRβ plays a critical role in intestinal remodeling during metamorphosis.

Thyroid hormone (T3) receptors (TRs) mediate T3 effects on vertebrate development. We have studied Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TRα knockout induces precocious hind limb development. To reveal the molecular pathways regulated by TRα during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on the hind limb of premetamorphic wild type and TRα knockout tadpoles, and identified over 700 TR-bound genes upregulated by T3 treatment in wild type but not TRα knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hind limb of premetamorphic TRα knockout tadpoles than stage-matched wild-type tadpoles, suggesting their derepression upon TRα knockout. Bioinformatic analyses revealed that these genes were highly enriched with cell cycle and Wingless/Integrated (Wnt) signaling-related genes. Furthermore, cell cycle and Wnt signaling pathways were also highly enriched among genes bound by TR in wild type but not TRα knockout hind limb. These findings suggest that direct binding of TRα to target genes related to cell cycle and Wnt pathways is important for limb development: first preventing precocious hind limb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hind limb development during metamorphosis by mediating T3 activation of these pathways.

Vertebrates organ development often takes place in two phases: initial formation and subsequent maturation into the adult form. This is exemplified by the intestine. In mouse, the intestine at birth has villus, where most differentiated epithelial cells are located, but lacks any crypts, where adult intestinal stem cells reside. The crypt is formed during the first 3 weeks after birth when plasma thyroid hormone (T3) levels are high. Similarly, in anurans, the intestine undergoes drastic remodeling into the adult form during metamorphosis in a process completely dependent on T3. Studies on Xenopus metamorphosis have revealed important clues on the formation of the adult intestine during metamorphosis. Here we will review our current understanding on how T3 induces the degeneration of larval epithelium and de novo formation of adult intestinal stem cells. We will also discuss the mechanistic conservations in intestinal development between anurans and mammals.

Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, TRα and TRβ, during postembryonic development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.

Background: Thyroid hormone (triiodothyronine [T3]) plays an important role in regulating vertebrate developmental, cellular, and metabolic processes via T3 receptor (TR). Liganded TR recruit coactivator complexes that include steroid receptor coactivators (SRC1, SRC2 or SRC3), which are histone acetyltransferases, to T3-responsive promoters. The functions of endogenous coactivators during T3-dependent mammalian adult organ development remain largely unclear, in part, due to the difficulty to access and manipulate late-stage embryos and neonates. We use Xenopus metamorphosis as a model for postembryonic development in vertebrates. This process is controlled by T3, involves drastic changes in every organ/tissue, and can be easily manipulated. We have previously found that SRC3 was upregulated in the intestine during amphibian metamorphosis. Methods: To determine the function of endogenous SRC3 during intestinal remodeling, we have generated Xenopus tropicalis animals lacking a functional SRC3 gene and analyzed the resulting phenotype. Results: Although removing SRC3 had no apparent effect on external development and animal gross morphology, the SRC3 (−/−) tadpoles displayed a reduction in the acetylation of histone H4 in the intestine compared with that in wild-type animals. Further, the expression of TR target genes was also reduced in SRC3 (−/−) tadpoles during intestinal remodeling. Importantly, SRC3 (−/−) tadpoles had inhibited/delayed intestinal remodeling during natural and T3-induced metamorphosis, including reduced adult intestinal stem cell proliferation and apoptosis of larval epithelial cells. Conclusion: Our results, thus, demonstrate that SRC3 is a critical component of the TR-signaling pathway in vivo during intestinal remodeling.

Background: There are two highly conserved thyroid hormone (triiodothyronine [T3]) receptor (TR) genes, TRα and TRβ, in all vertebrates, and the expression of TRα but not TRβ is activated earlier than T3 synthesis during development. In human, high levels of T3 are present during the several months around birth, and T3 deficiency during this period causes severe developmental abnormalities including skeletal and intestinal defects. It is, however, difficult to study this period in mammals as the embryos and neonates depend on maternal supply of nutrients for survival. However, Xenopus tropicalis undergoes a T3-dependent metamorphosis, which drastically changes essentially every organ in a tadpole. Of interest is intestinal remodeling, which involves near complete degeneration of the larval epithelium through apoptosis. Concurrently, adult intestinal stem cells are formed de novo and subsequently give rise to the self-renewing adult epithelial system, resembling intestinal maturation around birth in mammals. We have previously demonstrated that T3 signaling is essential for the formation of adult intestinal stem cells during metamorphosis. Methods: We studied the function of endogenous TRα in the tadpole intestine by using knockout animals and RNA-seq analysis. Results: We observed that removing endogenous TRα caused defects in intestinal remodeling, including drastically reduced larval epithelial cell death and adult intestinal stem cell proliferation. Using RNA-seq on intestinal RNA from premetamorphic wild-type and TRα-knockout tadpoles treated with or without T3 for one day, before any detectable T3-induced cell death and stem cell formation in the tadpole intestine, we identified more than 1500 genes, which were regulated by T3 treatment of the wild-type but not TRα-knockout tadpoles. Gene Ontology and biological pathway analyses revealed that surprisingly, these TRα-regulated genes were highly enriched with cell cycle-related genes, in addition to genes related to stem cells and apoptosis. Conclusions: Our findings suggest that TRα-mediated T3 activation of the cell cycle program is involved in larval epithelial cell death and adult epithelial stem cell development during intestinal remodeling.

Background Thyroid hormone (T3) is critical for development in all vertebrates. The mechanism underlying T3 effect has been difficult to study due to the uterus-enclosed nature of mammalian embryos. Anuran metamorphosis, which is dependent on T3 but independent of maternal influence, is an excellent model to study the roles of T3 and its receptors (TRs) during vertebrate development. We and others have reported various effects of TR knockout (TRα and TRβ) during Xenopus tropicalis development. However, these studies were largely focused on external morphology. Results We have generated TRβ knockout animals containing an out-frame-mutation of 5 base deletion by using the CRISPR/Cas9 system and observed that TRβ knockout does not affect premetamorphic tadpole development. We have found that the basal expression of direct T3-inducible genes is increased but their upregulation by T3 is reduced in the intestine of premetamorphic homozygous TRβ knockout animals, accompanied by reduced target binding by TR. More importantly, we have observed reduced adult stem cell proliferation and larval epithelial apoptosis in the intestine during T3-induced metamorphosis. Conclusions Our data suggest that TRβ plays a critical role in intestinal remodeling during metamorphosis.

Anuran metamorphosis is perhaps the most dramatic developmental process regulated by thyroid hormone (TH). One of the unique processes that occur during metamorphosis is the complete resorption of the tail, including the notochord. Interestingly, recent gene knockout studies have shown that of the two known vertebrate TH receptors, TRα and TRβ, TRβ appears to be critical for notochord regression during tail resorption in Xenopus tropicalis. To determine the mechanisms underlying notochord regression, we carried out a comprehensive gene expression analysis in the notochord during metamorphosis by using RNA-Seq analyses of whole tail at stage 60 before any noticeable tail length reduction, whole tail at stage 63 when the tail length is reduced by about one half, and the rest of the tail at stage 63 after removing the notochord. This allowed us to identify many notochord-enriched, metamorphosis-induced genes at stage 63. Future studies on these genes should help to determine if they are regulated by TRβ and play any roles in notochord regression.

Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.

Thyroid hormone (T3) is essential for vertebrate development, especially during the so-called postembryonic development, a period around birth in mammals when plasma T3 level peaks and many organs mature into their adult form. Compared to embryogenesis, postembryonic development is poorly studied in mammals largely because of the difficulty to manipulate the uterus-enclosed embryos and neonates. Amphibian metamorphosis is independent of maternal influence and can be easily manipulated for molecular and genetic studies, making it a valuable model to study postembryonic development in vertebrates. Studies on amphibian metamorphosis have been largely focused on the two highly related species Xenopus laevis and Xenopus tropicalis. However, adult X. laevis and X. tropicalis animals remain aquatic. This makes important to study metamorphosis in a species in which postmetamorphic frogs live on land. In this regard, the anuran Microhyla fissipes represents an alternative model for developmental and genetic studies. Here we have made use of the advances in sequencing technologies to investigate the gene expression profiles underlying the tail resorption program during metamorphosis in M. fissipes. We first used single molecule real-time sequencing to obtain 67, 939 expressed transcripts in M. fissipes. We next identified 4,555 differentially expressed transcripts during tail resorption by using Illumina sequencing on RNA samples from tails at different metamorphic stages. Bioinformatics analyses revealed that 11 up-regulated KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and 88 Gene Ontology (GO) terms as well as 21 down-regulated KEGG pathways and 499 GO terms were associated with tail resorption. Our findings suggest that tail resorption in M. fissipes and X. laevis shares many programs. Future investigations on function and regulation of these genes and pathways should help to reveal the mechanisms governing amphibian tail resorption and adaptive evolution from aquatic to terrestrial life. Furthermore, analysis of the M. fissipes model, especially, on the changes in other organs associated with the transition from aquatic to terrestrial living, should help to reveal important mechanistic insights governing mammalian postembryonic developments.

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.

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).

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.

Mammalian hematopoiesis begins in the yolk sac, and then shifts to the aorta-gonad-mesonephros, liver, spleen, and finally to the bone marrow. Hematopoietic tissue also shifts by environmental stress. In rats, the marrow contained hematopoietic progenitors at birth and contains no fat cells, and after birth, fat cells develop within the marrow cavity. However, hematopoiesis is retained in the tail vertebrae by transposing the tail into the warmer environment of the abdomen. To elucidate temperature-dependent regulation of hematopoiesis, we attempted to establish an African clawed frog (X. laevis) model which is systemically-resistant to low-temperature environment. Unlike mammals, X. laevis is filled with adipose cells, and blood cells are mainly produced in the liver. Moreover, cold stress (5°C) for 24 hour induced pancytopenia in X. laevis (Maekawa et al. J Exp Biol., 2012), demonstrating environmental regulation of hematopoiesis in animals has been largely conserved throughout evolution. However, dynamic behavior of hematopoietic stem progenitor cells (HSPCs) under the environmental stress, especially temperature stress, is unclear. We therefore attempted to identify the HSPCs in X. laevis to evaluate the ability of hematopoiesis under low-temperature. We have previously reported the function of Xenopus thrombopoietin (TPO) to stimulate proliferation of hematopoietic progenitor cell (Tanizaki Y et al. ASH. 2013, Tanizaki Y et al. Sci rep. 2015). To obtain hematopoietic progenitors with multipotent capacity, hepatic cells were cultured in serum-containing semisolid media with Xenopus TPO alone. Hepatic colonies stimulated by TPO could be cultured for more than 3 months, during which the cell number reached 1 × 106. These colonies expressed mRNAs of ESAM, c-kit, Oct-25, 60, 91 (Oct-4 orthologues) and Sox2, and have the potency to differentiate into myeloid cells. To test stemness of the cells, we resected the left lobe from the X. laevis,and cultured in the presence of TPO for 30 days. After that period, cells were labeled with PKH26 and transplanted autologously. After 30 days of transplantation, PKH26-positive cells were detected in the sinusoids of liver and spleen. Flow cytometric analysis showed that the PKH26-positive cells displayed low forward scatter (FSC) and side scatter, and had thin-layered cytoplasm and round nuclei, which are typical features of mammalian HSPCs. These results indicated that TPO stimulates proliferation of HSPCs, which can be engrafted and differentiated to multiple lineages. To enrich HSPCs from the liver, we generated anti-Xenopus MPl mouse monoclonal antibodies, and showed that anti-thrombocyte antibody (T12)-Mpl+FSClow population was enriched in high nuclear/cytoplasm (N/C) ratio-hematopoietic progenitors. The ratio of these cells to all hepatic cells was 0.28%. Surprisingly, T12-Mpl+FSClow cells were also identified (0.69±0.4%) in the fatty marrow. Furthermore, T12-Mpl+FSClow cells in the liver and the BM expressing mRNAs of ESAM, c-kit, GATA2, Oct-25, 60 and Sox2 showed the extremely high N/C ratio. These results demonstrated that HSPCs are enriched in T12-Mpl+FSClow population and localized in the both liver and the BM. We then focused on HSPCs as a marker of hematopoietic regulation under the low temperature condition. To explore the hematopoietic capacity in the BM, X. laevis were exposed to 5°C, which led to pancytopenia. After exposure to low temperature for 12 days, we observed a significant increase (approx. 2 fold) of the number of HSPCs only in the BM compared to controls. To reveal whether microenvironment of the BM was modified under the low-temperature stimulation, we analyzed the bone structure under the low-temperature stimulation by using micro-CT. Micro CT analysis showed that femoral bone density was higher than that before exposure to 5°C, indicating the modification of the BM microenvironment. Besides, TPO mRNA expression in the BM also increased to 15-fold by low-temperature stress, showing that microenvironment of the BM under low-temperature provided further signals for proliferation of HSPCs. These findings suggested that low-temperature stimulation induced proliferation of HSPCs. Hematopoietic switch from fatty to "active" bone marrow has the clue to make considerable progress in understanding the pathophysiology of hematological diseases, e.g. aplastic anemia. Disclosures No relevant conflicts of interest to declare.

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.

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.

P1116 PROPERTIES OF C-MPL EXPRESSION IN THROMBOPOIETINDERIVED HEPATIC HEMATOPOIETIC PROGENITORS OF XENOPUS LAEVIS Yoko Mochizuki, Yuta Tanizaki, Takato Otani, Kei Sato, Megumi Ichisugi, Ayaka Murase, Shunji Sakai, and Takashi Kato Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan; Department of Biology, School of Education, Waseda University, Tokyo, Tokyo, Japan

Hematopoiesis in animal models including non-mammalian zebrafish have demonstrated that the system of hematopoiesis is largely conserved throughout vertebrate evolution. We previously reported that hepatic cells of frog (X. laevis) differentiated into megakaryocytic cells and thrombocytic cells in response to X. laevis thrombopoietin (xlTPO). In this study, we identified the effects of xlTPO for immature hematopoietic progenitors such as hematopoietic stem cells (HSCs). The hepatic cell suspension was cultured in semi-solid culturewith xlTPO.After 24 days, multilineage colonies expressing various cell lineage markers appeared, and these colonies survived more than four months. These cells differentiated tomature blood cells by hepatic cell-conditioned-media stimulation, demonstrating that the ability of these cells were multipotent in the cell differentiation. Because inbred strains of X. laevis are not abundantly available, transplantation studies in X. laevis are limited. We therefore developed the cell transplantationmodel to identify theHSCs inX. laevis. A left liver lobeofX. laeviswas resected and cultured with xlTPO for 24 days. The cells were labeled by PKH26 and autologously transplanted by intracardiac injection. After 30 days, PKH26 cells detected in the sinusoid of the liver, and FACS analysis revealed that PKH26 cells divided at least one time. However it was hard to track the transplanted cells for long term by such fluorescent labeling, PKH26 labelled cellswere directly injected to the blastocele ofX. laevis embryos at stage 8 which have no immune system. After 3 days of injection, PKH26 cells were detected all over the embryos, and part of transplanted cells flowed into blood vessels. The cellular potency should be confirmed furthermore, but this unique transplantation model developed here should have clues to resolve the functional evolution of TPO system shared by hematopoietic progenitors.

Thrombopoietin (TPO) is required for megakaryopoiesis generating platelets via acting through its receptor c-Mpl, and supports hematopoietic stem cell maintenance. It is unknown whether there are evolutionary conserved signalling pathways and molecular functions governed by TPO/c-Mpl in non-vertebrates. We therefore attempt to develop a non-mammalian animal model in order to investigate thrombocyte development in adult African clawed frog (Xenopus laevis: X. laevis). We previously reported that Xenopus hepatic cells were differentiated to polyploid megakaryocyte-like cells and thrombocyte under X. laevis TPO (xlTPO) stimulation. The aim of this study is to determine the cellular characteristics of TPO-induced immature cells by establishing in vitro and in vivo Xenopus models. Hepatic cells were cultured in a 35mm diameter dish at 23°C under 5% CO2 with minimum essential medium containing 0.87% methylcellulose and 20% FCS in the presence of xlTPO (10 ng/ml). After 24 days of culture, the number of colonies reached 9.0±3.0 per dish. The size of colonies was heterogeneous, and a typical large colony was composed of more than 512 cells. Cells recovered from the large colonies at 24-day culture were then serially replated to a 96-well plate at a density of 1x103 cells/well with semi-solid medium in the presence of xlTPO (10 ng/ml) or X. laevis erythropoietin (xlEPO) (10 ng/ml). At day 8, a significant number of colonies (125±10/100μl culture well) were emerged in the xlTPO culture; but a few colonies in the xlEPO culture (3±1/100μl culture). The colony formation was inhibited by the addition of recombinant xlc-Mpl-FC fusion protein in the culture, indicating that the colony formation was directly stimulated by xlTPO. It was notable that xlTPO alone maintained and expanded the culture for over four months. The component cells in xlTPO-induced colonies resembled megakaryocytes or thrombocytic cells with spindle shapes, suggesting that xlTPO exerted the ability to support long-term maintenance and proliferation of thrombocytic progenitors. We then collected the cells after 24 days of liver cell culture in the presence of xlTPO. The RT-PCR revealed that the cells did not express hepatic markers of albumin and tpo; while they expressed not only mRNAs of thrombocytic markers such as c-mpl, fli-1, integrinαIIb, but also mRNAs of mpo, g-csfr, γ—globin , gata-1, esam and c-kit. The result proved that xlTPO expanded multilineage hematopoietic progenitors; not restricted to cells in the thrombocyte lineage. Subsequently we cultured cells collected from the day-24 colonies in conditioned medium of liver and spleen. After 8 days of culture with xlTPO, approximately 5% of the cells developed morphologies resembling mature eosinophils in the spleen-conditioned medium. As well, about 18% of the cells appeared to be macrophage-like cells, and they phagocytosed latex beads. Taken together, we concluded that xlTPO supported long-term maintenance of multipotent heamatopoietic cells in the Xenopus liver via TPO-c-Mpl signalling. Because inbred strains of X. laevis are not abundantly available, transplantation studies in X. laevis are limited. We therefore attempted to develop autologous cell transplantation of xlTPO-induced cells in X. laevis. Liver cells obtained by partial hepatic resection were cultured in the semi-solid culture system in the presence of xlTPO. After 24 days, the cells labeled with PKH26 were autologously transplanted by intracardiac injection. PKH26-positive cells were detected in the liver and the spleen of the auto-transplanted frogs after 12 days, though the cells were diminished from the body within 4 to 6 days in the allo-transplanted Xenopus. Flow cytometric analysis showed that PKH26-high cells contained much RNA and displayed lower forward scatter (FSC); while PKH 26-low cells were detected in the intermediate FSC. The findings suggested that a subpopulation of long-term maintained liver cells by xlTPO could barely proliferate and differentiate in the liver and the spleen, and the characteristics of these cells agrees with the features of hematopoietic stem cells or progenitors, as in mammals and zebrafish. This unique culture system developed in this study has clues to resolve the functional evolution of TPO-cMpl system shared by thrombocytic development and earlier hematopoietic progenitors. Disclosures: No relevant conflicts of interest to declare.

Abstract 4907 Immunohistochemical staining and flow cytometry are performed on the tissue to distinguish specific cells. The detection of diseases is made by the flow cytometry using monoclonal antibodies on peripheral blood (PB) or bone marrow cells. The diagnosis of leukemia is made with PB and marrow cells using antibodies to cluster of differentiation (CD) antigens specific to leukemic cells (e.g.,CD13, CD33). Therefore, conventional approaches to identify cellular phenotypes are being replaced by immunophenotyping using flow cytometry. However antibodies satisfying for needs are not always available. In this case, the generation of such antibody to each specific antigen also causes problems of producing the respective recombinant antigen for immunization. When attempting to construct new animal models using other than mice and humans, this issue becomes one of the serious limiting factors in developing research. In addition, the opportunity is increasing to classify unexperienced types of cells such as cells derived from iPS cells and others. We therefore consider that it is important to develop methods to classify and separate specific cells of interest without antibodies. As supravital cell staining with acridine orange (AO) is introduced in 1960's (Jacson JF, Blood, 1961; Lewis M. et al. Blood, 1962), this metachromatic fluorescent dye rapidly stains DNA and RNA independently. Morphological abnormalities of human erythrocytes such as red cell fragment and large platelets are detectable (Nagai Y et al., Int. J Lab Hematol., 2008). AO emits green fluorescence when it binds to the double-stranded DNA and also red fluorescence when it binds to the single-stranded RNA. Consequently flow cytometry of cells stained with AO is suitable for analyzing blood cells in four parameter, which are scattered light intensity (FSC and SSC) and fluorescence intensity (DNA content and RNA content). This analysis method has the possibility of developing the classifying and separating method for abnormalities. Xenopus laevis(X. laevis) has various nucleated blood cells, which are erythrocytes, leukocytes, and thrombocytes, and their progenitors that are not classified yet. As the first step to identify cells with increased RNA content, we chose X. laevis blood cells as a new model of AO staining for flow cytometry by FACS Aria II cell sorter based on the content of DNA and RNA. Since collected cells were stained by May-Griinwald-Giemsa on cytocentrifuge preparations. The population in lower content of DNA and RNA is composed of erythrocytes (95.7±1.3% of whole PB cells). The population in higher concentration of DNA and RNA was pure leukocytes fraction (0.7±0.6% of whole PB cells) expressing the mRNA of X. laevis myeloperoxidase. This population was then fractionated with the higher content of RNA containing eosinophils and basophils (48.7±29.9%), and lower content of RNA contained neutrophils (50.9±29.9%). The proportion of the peripheral thrombocytes in lower forward light scatter (FSC) and side light scatter (SSC) ranges was 0.8±0.8% of whole PB cells, and the expression of c-Mpl, CD41 and Fli-1 mRNA was detected in the sorted cells by RT-PCR. Furthermore, sorted cells were confirmed by immunohistochemical staining by anti X. laevis thrombocyte monoclonal antibody. To reveal the characteristics of the abnormal PB cells, we compared the normal PB with the PB of phenylhydrazine (PHZ) induced anemic X. laevis. At 8 days after treated with PHZ, immature hematopoietic cells were appeared in circulating blood. We isolated these cells with high content of DNA and RNA, and the population in lower FSC and higher SSC with crossover analysis of light emission by AO, suggesting that this method could identify abnormalities in whole PB. The character of these cells was morphologically small and high nuclear cytoplasmic ratio. We also applied this method to blood cells of Rana catesbeiana, American bull frog. Despite the differences in size of erythrocytes, thrombocytes and leukocytes, Rana catesbeiana PB was similarly sorted to X. laevis PB by cellular DNA/RNA content. These results suggest that this method can sort nucleated cells in various species by crossover analysis of light emission by AO. Our study showed that this method has the advantage to characterize of human leukemia cells, tumor cells, iPS-derived cells and nucleated blood cells. Disclosures: No relevant conflicts of interest to declare.