Erythroid progenitors that respond to erythropoietin (Epo) are present in the liver of adult Xenopus laevis. However, cells responding to Epo in the larval liver and through the metamorphosis period under hepatic remodeling have not been characterized. In this study, tadpoles were staged using the tables of Nieuwkoop and Faber (NF). Liver cells from pre- (NF56) or post- (NF66) metamorphic stage were cultured in the presence of Epo. β2-globin mRNA expression peaked at day 7 after the start of culture. Larval β2-globin was highly expressed in NF56-derived cells, while adult β2-globinwas detected in those of NF66. In both NF56- and NF66-derived cells, mRNA expression of eporand gata2 peaked at day 5 and days 3–4, respectively. In contrast, gata1 expression peaked at day 6 in NF56 cells and at day 5 in NF66 cells. Half maximal proliferation of erythrocytic blast cells derived from the liver at NF66 was observed at day 3, which was earlier than that of NF56. These results indicate that erythroid progenitors that respond to Xenopus laevis Epo are maintained in pre- and post-metamorphic liver, although the tissue architecture changes dramatically during metamorphosis. Additionally, the globin switching occurred, and/or the erythroid progenitors for larval erythrocytes were replaced by those for adult erythrocytes in the metamorphic liver.
Erythropoietin (Epo) is a glycoprotein that is the major physiological regulator of erythropoiesis. Epo supports the survival of late erythroid progenitors and enhances their proliferation and differentiation by binding to Epo receptor (Epor) (Krantz, 1991; Jelkmann, 2010; D'Andrea and Zon, 1990). Epo stimulates burst-forming unit-erythroid cells (BFU-e) expressing Epor on the surface, and successively colony-forming unit-erythroid cells (CFU-e), at sites of erythropoiesis (Broudy et al., 1991; Gregory and Eaves, 1978; Sawada et al., 1987). Further, mature erythrocytes are produced through proerythroblasts, erythroblasts, and reticulocytes. The Epo dependency of erythroid progenitors varies with developmental stages (Boussios et al., 1989; Masuda et al., 1992; Emerson et al., 1989). Erythrocytes convert from primitive to definitive type in the fetal liver, a major site for the development of definitive erythrocytes (Koury et al., 2002; Isern et al., 2008).
During mammalian development, the hematopoietic system, including the embryonic yolk sac, liver, thymus, spleen, and bone marrow, is established in a continuous manner. As the hematopoietic organ changes during ontogeny, the nature of the hematopoietic microenvironment must also change (Orkin and Zon, 2008). The mechanism(s) involved in the transition of hematopoietic organs, however, are not well understood. The embryonic liver acts as a hematopoietic organ, unlike the adult liver, which performs various metabolic functions, such as serum protein synthesis, lipogenesis, detoxification, and urea synthesis (Oliver et al., 1983; Perry et al., 1983; Shelly et al., 1989; Orkin et al., 1996). As it acquires these metabolic functions, the embryonic liver loses its hematopoietic activity during late fetal development (Miyajima et al., 2000). Finally, hematopoietic cells move into the bone marrow or spleen around the perinatal stage to constitute the adult-type hematopoietic system.
In the African clawed frog, Xenopus laevis (X. laevis), hematopoiesis occurs first in the analog of yolk sac called the ventral blood island (VBI), and subsequently in the equivalent of the aorta-gonad-mesonephros (AGM) called the dorsal lateral plate (DLP) (Kau and Turpen, 1983; Flajnik et al., 1984; Maeno et al., 1985; Ciau-Uitz et al., 2000). Next, hematopoietic stem cells, which are derived from a part of VBI and DLP, migrate to the liver and maintain their hematopoietic activity until post-metamorphosis (Chen and Turpen, 1995; Turpen et al., 1997). During metamorphosis, hemoglobin isoform switching changes the oxygen affinity in the liver (Mukhi et al., 2010); this process is physiologically important for the adaptation from an aquatic to a terrestrial habitat (Riggs, 1951). After metamorphosis, X. laevis grows into adulthood; however, unlike in mammals, in adult X. laevis, hematopoiesis does not occur in the bone marrow. Hepatic properties change during metamorphosis. For example, fetuin B and alcohol dehydrogenase are expressed in the pre-metamorphic stage, but their expression decreases in the post-metamorphic stage. Conversely, the expression of albumin, cytochrome p450, and carbomyl phosphate synthetase increase in the post-metamorphic stage (Mukhi et al., 2010). We previously identified cDNA sequences of X. laevis Epo (Nogawa-Kosaka et al., 2010) and Epor (Aizawa et al., 2005), and it has been shown that Epor-expressing cells localized to the inner wall of the sinusoid in the adult liver and Epo was mainly expressed in hepatocytes (Nogawa-Kosaka et al., 2011; Okui et al., 2013). Epo does not have an N-linked sugar chain, but exerts biological activity in vivo (Nogawa-Kosaka et al., 2010; Nagasawa et al., 2015), suggesting a paracrine action through the hepatic Epo-Epor pathway, similar to that in the fetal liver in mammals. Thus, hepatic erythropoiesis induced by the Epo-Epor pathway and the proliferation and differentiation of erythrocytes in adult X. laevis became clear. Likewise, thrombocyte progenitors reside in the liver and the spleen in X. laevis(Tanizaki et al., 2015). Therefore, the liver is one of the predominant multilineage hematopoietic organs. However, larval hematopoiesis remains to be elucidated, especially that at stages around the metamorphic climax approaching the adult stage.
In the present study, we examined erythroid progenitors that respond to Epo in the liver around metamorphic period at NF56 (pre-metamorphosis) and NF66 (post-metamorphosis). Developmental stages were classified based on the report of Nieuwkoop and Faber (1956). Epo stimulated the hepatic erythroid development toward hemoglobinized cells at NF56 and NF66. We further analyzed the differences in proliferation, morphology, and the expression of larval erythroid progenitors between NF56 and NF66 to understand the dynamics of Xenopus erythropoiesis from the metamorphic to the adult stage.
MATERIALS AND METHODS
Sexually mature adult X. laevis were purchased from an Aquatic Animal Supply (Misato, Japan). Tadpoles were obtained by the induction of breeding. For breeding, chorionic gonadotropin (hCG-Mochida, Mochida Pharmaceutical, Tokyo, Japan) was injected subcutaneously into adult X. laevis. Tadpoles were raised in a conventional culture room under light- (12-h/12-h light/dark cycle) and temperature- (22°C) controlled conditions. Green juice, including kale and green barley, was fed to the tadpoles at the pre-metamorphic stages, and fish food pellets were added after metamorphosis. All animal experiments in this study were conducted in accordance with the Regulations for Animal Experimentation of Waseda University, Tokyo, Japan. Larvae were staged based on the definition of Nieuwkoop and Faber (Nieuwkoop and Faber, 1967).
Tadpoles were anesthetized with 1 mg/L of ethyl 3-aminobenzoate methanesulfonate (MS-222; Sigma-Aldrich, St Louis, MO, USA) in ice-cold water. The livers were immediately collected from anesthetized tadpoles. Tissue samples were fixed for 24 hours at 4°C in Bouin's solution (Sigma-Aldrich, St Louis, MO, USA), dehydrated through a graded series of ethanol, embedded in Paraplast (Kendall-Tyco Healthcare, Mansfield, MA, USA) and cut into 4-µm thick sections. The sections were stained with hematoxylin-eosin solution (Wako Pure Chemical Industries, Osaka, Japan) and mounted with Bioleit (Okenshoji, Tokyo, Japan).
Dulbecco's modified phosphate-buffered saline without Mg2+ and Ca2+ ions (dDPBS (-)) was used to avoid blood coagulation. Cytocentrifuged preparations were made with 2 × 105 cells diluted in 0.8 × dDPBS (-), containing 50% fetal calf serum (FCS) (AusGeneX, Brisbane, Australia). The cell suspension was centrifuged at 1,100 × g for four minutes onto glass slides. After air drying, preparations were fixed for 5 minutes in methanol, incubated in 0.1% o-dianisidine (Wako Pure Chemical Industries, Osaka, Japan) in methanol, incubated 30% hydrogen peroxide for 1.5 minutes, washed in running water, and counterstained with Giemsa solution (Okui et al., 2013).
The isolation of hematopoietic progenitor cells from the liver was performed as previously described (Nogawa-Kosaka et al., 2011). Briefly, livers were dissected and placed in 0.8 × dDPBS (-) or 0.8 × alpha minimum essential medium (α-MEM) (Invitrogen, Carlsbad, CA, USA). Organs were washed, minced, and drawn in and out of a syringe through a 27 gauge needle, and treated with a hemolysis solution (150 mM NH4Cl, 14 mM NaHCO3, 0.12 mM EDTA-2Na) to remove most erythrocytes. To prepare a single-cell suspension, the cell suspension was further filtered through a 40-µm nylon cell strainer (BD Biosciences, San Jose, CA, USA). Cells (1.5 × 104) were cultured in 0.8 × α-MEM including 10% heat-inactivated FCS, streptomycin (Invitrogen, Carlsbad, CA, USA), penicillin (Invitrogen, Carlsbad, CA, USA), and 1% adult X. laevis serum in 96-well plates. Epo produced by Escherichia coli was added to culture medium. The assay for cell proliferation was conducted in the presence of 100 ng/ml Epo in accordance with the protocol of a previous report (Nogawa-Kosaka et al., 2011). Cells on each day were stained with Trypan Blue solution and counted with a hemocytometer.
Sequences of primers for RT-PCR.
Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted from cells on each culturing day and whole liver using TriPure Isolation Reagent (Roche Applied Science, Indianapolis, IN, USA) according to manufacturer's instructions. Total RNA was reverse-transcribed into cDNA using ReverTra Ace (Toyobo, Osaka, Japan). Fragments of ribosomal protein l13a (rpl13a) and glyceraldehyde-3-phosphate dehydrogenase (gapdh) were amplified as an internal standard by reference to a previous study (Nogawa-Kosaka et al., 2010). The primer sequences were listed in Table 1. For genes and proteins of X. laevis, we followed the notation method declared in Xenbase ( http://www.xenbase.org/gene/static/geneNomenclature.jsp).
Liver structure at the metamorphic stage
At NF56, the length of the body reached approximately 30 mm under our breeding conditions. Forelimb and hind limb were formed clearly, and elimination of the caudal portion started at NF60 (middle metamorphic stage). At the climax of metamorphosis (NF66), caudal elimination was complete, and the body morphology transformed to that of adult frogs (Fig. 1A). In the metamorphic stages, from NF56 to NF66, changes occurred in both the external morphology and the internal structure. The lung could be observed at NF56, and tissue arrangement of the intestine, which was in a spiral configuration at NF56, changed to a more complicated structure at NF66. Sternum formation began at NF60. From NF56 to NF60, larval liver increased in size and turned red (Fig. 1B).
The structure of the liver changed greatly during metamorphosis. The number of lobes varied from three at NF56 to two at NF66. HE-stained liver sections at NF49, 56, 60, and 66 clearly demonstrated the morphological changes in hepatocytes and the increase in sinusoid diameter (Fig. 1C). At NF49, the structure of the hepatic cell cord was unclear, and the diameter of the sinusoid was wider than that at NF56, 60, and 66. It is known that the pigment cells, melanomacrophages, appear in adult liver (Wiechmann et al., 2003). Melanomacrophages were not observed in the liver at NF49 and NF56, and they began to appear at NF60 (Fig. 1C). The staining property also changed during the development, and the area of sinusoid decreased from NF56 (Fig. 1D). The size of hepatocytes increased significantly at NF66 (Fig. 1E). We then examined erythroid progenitors in the liver during the metamorphic period and evaluated their response to Epo.
Reactivity of pre- and post-metamorphic liver cells to Epo
When whole liver cells of NF56 larvae were cultured in the presence of Epo, the survival rates of the cells increased from that of the control, but the cell counts did not increase (Fig. 2A). Since it was possible that mature erythrocytes express Epor to avoid apoptosis due to Epo-Epor signaling (Aizawa et al., 2005; Tamura et al., 2015), we removed mature erythrocytes by hemolysis prior to liquid culture. This also suppressed the absorption of unbound Epo to Epor in the culture. After removal of mature erythrocytes by hemolysis, the number of proliferating cells increased (Fig. 2B).
Previous studies have shown that erythropoiesis takes place in the liver of tadpoles; therefore, we examined whether erythroid progenitors that responded to Epo resided in the larval liver. When larval hepatic cells at NF56 or NF66 were cultured in the presence of Epo, the cells proliferated, and several types of erythrocytic cells emerged in the respective cultures at day 8 (Fig. 3A). To characterize the types of erythrocytic cells induced by stimulation by Epo, we stained the cultured cells on each day with o-dianisidine to classify them based on hemoglobinization. A number of blast cells and pale brown cells that appeared on day 5 were not validated as erythrocytic cells. Subsequently, mature erythrocytes emerged abundantly at day 8 from liver cells at both NF56 and NF66 (Fig. 3B and 3C). Next, viable blast cells and mature erythrocytes from liver cells at NF56 or NF66 were counted on each culture day. We classified cultured cells into three cell subpopulations based on the size of nuclei and the results of o-dianisidine and Giemsa staining: (1) mature erythrocytes robustly stained with o-dianisidine (brown cytoplasm), (2) blast cells with large nuclei (blue or very pale brown cytoplasm stained with Giemsa and o-dianisidine), and (3) other cells with lobulated nuclei (negative to o-dianisidine-staining) (Fig. 3D).
The viable cells increased in the cultures of the liver cells of NF56 and NF66 (Fig. 4A, B). In the culture of the cells derived from the NF56 liver, half maximal of the number of viable cells was attained at 5 days and the maximal was attained at day 7 after the start of culture (Fig. 4A). At NF66, the cell number did not reach the maximum plateau until day 8 (Fig. 4B). The difference in the pattern of cell proliferation between NF56 and NF66 was that the NF56 cells increased in a sigmoid pattern, while NF66 cells expanded in a linear pattern. Dead cell counts for NF56 and NF66 were similar to those for the control (data not shown).
Blast cell numbers at both NF56 and NF66 started to increase at day 3, peaked at day 5 (NF56) and day 4 (NF66), and then decreased at day 7 (Fig. 4C, D). The number of o-dianisidine+ cells decreased by day 5 for NF56, while a slight rise was observed at day 2 for NF66 (Fig. 4C, D). However, a rapid increase was observed at day 7 (NF56) and day 6 (NF66). Additionally, at day 8, the number of o-dianisidine+ cells continued to increase for NF66, while it decreased for NF56. In the light of cell proportion in the culture of both NF56- and NF66-derived cells, blast cells increased at first, and o-dianisidine+ cells increased thereafter, then blast cells decreased (Fig. 4E, F).
Gene expression in pre- and post-metamorphic tissues
In adult X. laevis, the liver is the predominant hematopoietic organ (Hadji-Azimi et al., 1987; Nogawa-Kosaka et al., 2011). The whole liver at NF56 and NF66 expressed epo, epor, gata2, and gata1(Fig. 5A). To characterize the cells responding to Epo, the expression of various genes at each day was analyzed by RT-PCR (Fig. 5B, C). We evaluated the mRNA expression of Epor and Gata, which participate in erythropoiesis in mammals and fish. We first examined the Gata-binding regions on the X. laevisglobin gene (data not shown). In the NF56 and NF66 liver cells cultured in the presence of Epo, the expression of both larval and adult globin were up-regulated at days 6–8, consistent with the growth pattern of o-dianisidine+ cells. Interestingly, the expression of larval or adult globin gene increased predominantly in NF56- and NF66-derived cells, respectively. The wavy change of globin expression was detected in the culture (Fig. 5B, C). Both larval and adult globin were expressed initially in the cultures NF56 and NF66 liver cells, and their expression levels gradually declined to the nadir at around day 4, and then the expression levels increased again at day 6. The expression of epor reached a peak in both liver cultures (NF56 and NF66) as erythrocytic blast cells expressing eporemerged. Similar to globin expression, the expression of epor was detected at day 0, decreased to the nadir at days 1–2, and then increased to reach a peak at around day 3. The expression of gata2 was detected at days 3–4 (NF56 and NF66). Theexpression of gata1 peaked at day 6 in the NF56 liver cells, while that in NF66 liver cells peaked at day 5. The peak of gata1 expression was reached about 2 days after that of gata2. Albumin expression was observed in the early culture days, and it showed a sequential decline, indicating that exogenous Epo could not maintain hepatocyte culture under this condition. The changes in gene expression over the course of erythroid development are shown in Fig. 6.
The liver of X. laevis has been thought to function as a hematopoietic organ in both larva and adult. However, it has been shown that the properties of the liver change throughout the metamorphic period (Mukhi et al., 2010), suggesting that the hematopoietic environment differs in larval and adult tissues. The key findings of the present study are: (1) erythroid progenitors remain in the liver, despite dramatic changes in the environment and tissue organization during the metamorphosis period and (2) larval hepatic erythroid progenitors at pre- and post-metamorphic stages respond differently to Epo. It is known that hematopoiesis is closely associated with the hematopoietic “niche” in the tissue microenvironment (Suda et al., 2005; Miyajima et al., 2000). During metamorphosis, the size and shape of hepatocytes, the diameter of sinusoid, staining properties, and the number of erythrocytes in the liver varied greatly (Fig. 1C-E). In this study, we found that erythroid progenitors exist in the larval liver. The structure and function of the liver undergoes significant changes during the pre- and post-metamorphosis stages (Fig. 1B) (Brown and Cai, 2007).
We also investigated whether the properties of hepatic erythroid progenitors changed in response to the transmutation of the niche. Previous studies have shown that erythropoiesis occurs through the Epo-Epor system in adult X. laevis (Nogawa-Kosaka et al., 2011); however, the mechanism in X. laevis larvae has remained unclear. We revealed in the present study that the erythroid progenitors in the larval liver proliferated and matured under recombinant Epo stimulation. The number of blast cells with large nucleus and blue-stained cytoplasm (Fig. 3) increased in the presence of Epo, which was consistent with the epor expression pattern, demonstrating that these blast cells were erythroblasts. The number of o-dianisidine+cells was higher than that of blast cells at respective peak days, indicating that blast cells had the capacity to mature in the presence of Epo. The transcription factors GATA2 and GATA1 play key roles in erythroid development in mammals (Doré et al., 2011). Their expressions are strictly regulated in the erythroid lineage. We observed the expression period of gata1 and gata2changed sequentially, suggesting that their expression was controlled at stages of erythroid development and differentiation in the analogous way of mammals. The crossover in the expression of larval and adult globin mRNAs between NF56 and NF66 under stimulation with Epo indicated the difference in the properties of erythroid progenitors undergoing differentiation. The timing of the initiation of blast cell proliferation was also different for NF56 and NF66. This suggested that the progenitor cells residing in NF56 liver may be more immature than those at NF66; therefore, the NF66 liver cells responded to Epo sooner than NF56 liver cells.
The gene expression profiles of larval liver and cultured hepatic cells were also analyzed in this study. The larval whole liver expressed epo at pre- (NF56) and post- (NF66) metamorphosis (Fig. 5A) similar to that in adult X. laevis (Nogawa-Kosaka et al., 2010). This finding is consistent with epo expression in the liver of NF57, 60, 62, and 64 reported previously (Tamura et al., 2015). This expression is probably derived from hepatocytes like adult X. laevis (Nogawa-Kosaka et al., 2010). In addition, epor, gata2, and gata1were also detected in the whole liver of NF56 and NF66 (Fig. 5A). In adult frogs, Epo has been suggested to bind to Epor through a paracrine system in the liver (Nogawa-Kosaka et al., 2010). The same erythropoietic system likely exists in larvae, as suggested by the expression of Epo mRNA in the larval liver. The expression of globin and eporwas observed early in culture, after which their expression levels declined gradually (Fig. 5B, C). It is possible that the globin and epor mRNAs detected at the early stages were produced by the originally developed hepatic erythrocytes or progenitors, although we removed erythrocytes by hemolyzing before seeding cells. In the culture of NF56 liver cells, gata2 expression peaked at day 4, and gata1 expression reached a peak at day 6. This suggests that the erythroid progenitors expressing gata2 differentiated into erythroblasts expressing gata1 (Fig. 6). In the culture of NF66 liver cells, gata2 expression peaked at day 3, and gata1 expression reached a peak at day 5. The difference in the time of reaching maximal expression for gata2 and gata1 between NF56 and NF66 indicated that the hematopoietic cells residing in the liver of NF56 and NF66 were at different stages of development. The life span of larval developing erythrocytes compared to that of adult ones remains to be addressed, as larval type erythrocytes are replaced by adult type erythrocytes. The life span of erythrocytes was 20 to 40 days in mice (Burwell et al., 1953; Van Putten and Fineke, 1958), while it is approximately 220 days in adult X. laevis(Maekawa et al., 2012). In contrast, the life span of the erythrocytes derived at various developmental stages from larval livers is unknown.
In fish, the kidney is a major hematopoietic organ, as has been reported in zebrafish (Davidson and Zon, 2004). Furthermore, a variety of hematopoietic organs have been reported among animals. In addition, a transition of the hematopoietic organs in accordance to ontogenic stage has also been observed in many animals. Xenopus laevis may thus represent a useful animal model for elucidating the functions/characteristics of the hematopoietic niche, as hematopoiesis occurs consistently in its liver throughout its lifetime from tadpole to adult frog. In this regard, the culture system for larval liver cells used in this study is well-suited to analyses of the changes that occur during metamorphosis with respect to erythrocyte development.
In conclusion, erythroid progenitors that respond to Epo were maintained in pre- and post-metamorphic liver, although the tissue architecture changed dramatically during metamorphosis. The erythroid progenitors for larval erythrocytes were replaced by adult erythrocytes in the post-metamorphic liver.
We thank Mr. Kazuo Ouchi for advice on breeding technique. We also thank Dr. Yusuke Yamamoto for constructive advice. This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (No. 20570063 and 26440171), Grants for Special Research Project from Waseda University (No. 2007B-061, 2011B-047, 2012B-045 and 2013B-056) and by “High-Tech Research Center” Project for Private Universities: matching fund subsidy from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (No. 07H016). A part of this study was performed as MEXT-Supported Program for the Strategic Research Foundation at Private Universities.