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Figure 1. Characteristic changes in peripheral red blood cells (RBCs) during metamorphosis. (a) Changes of globin type in RBCs during Xenopus laevis and Rana ornativentris metamorphosis, shown using SDS-PAGE and AUT-PAGE. Larval and adult globins can be distinguished by SDS-PAGE based on their different mobilities: Two larval bands (L1 and L2, arrowheads) and a single adult band (A, arrow); however, L2 and A could not be separated clearly in R. ornativentris because of similar molecular weight. However, AUT-PAGE clearly separated the larval (L, arrowhead) and adult (A, arrow) globins of R. ornativentris. In X. laevis, a large amount of larval globin (arrowhead) remained in the peripheral RBCs even at the stage when morphological metamorphosis was completed (stage 66). In R. ornativentris, larval globin (arrowheads) disappeared simultaneously with the completion of morphological metamorphosis (stage XXV). Further, the band of L1 globin detected using SDS-PAGE in stage V (asterisk) shifted upward at stage VII and thereafter. (b) Western blotting using anti-PCNA antibody against proteins extracted from peripheral RBCs during metamorphosis. The PCNA signal was attenuated during premetamorphosis (from stage V to stage X) in R. ornativentris. In both X. laevis and R. ornativentris, the PCNA signal was drastically reduced during the metamorphic climax (stage 63 and XXII for X. laevis and R. ornativentris, respectively)
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Figure 2. Sorting of peripheral red blood cells (RBCs) by Percoll density gradient centrifugation during metamorphosis. Images of RBC separation using Percoll gradient centrifugation, western blotting of RBC extracts using an anti-PCNA antibody, and SDS-PAGE of hemolysate shown on the left, upper, and lower panels in (a–f), respectively. Images of AUT-PAGE, as well as SDS-PAGE are shown in (e) and (f). Larval and adult globins detected using PAGE are labeled using arrowheads and arrows, respectively, as in Figure 1. For Percoll gradient centrifugation, 80%, 70%, and 60% Percoll solutions were layered from the bottom up; thus, position 1 lies between the 60% and 70% Percoll solutions, while position 2 lies between the 70% and 80% Percoll solutions. The number of each lane indicates the migrating positions of RBCs in the Percoll density gradient and “whole” indicates RBCs before separation. (a) RBCs from Xenopus laevis at stage 59 could be separated into light (position 1) and intermediate (position 2) populations (left). The light RBCs were PCNA-positive (upper). Most globins were of the larval form both in the light and intermediate RBCs and the ratio of larval to adult globin was the same between the light and intermediate RBCs (lower). (b) RBCs in X. laevis at stage 62. This state was almost identical to stage 59. (c) RBCs in X. laevis at stage 63/64 could be separated into light (position 1), intermediate (position 2), and heavy (position 3) populations (left). The heavy RBCs were strongly PCNA-positive, while a faint signal was also detected in intermediate RBCs (upper). These heavy RBCs contained predominantly adult globin, whereas the light and intermediate RBCs possessed abundant larval globin (lower). (d) RBCs in X. laevis at 1 month after metamorphosis could be separated into intermediate (position 2) and heavy (position 3) populations (left). The intermediate RBCs were PCNA-positive, but the signal was totally weakened (upper). In three of four individuals, larval globin was faintly observed in the intermediate RBCs (lower, arrow). (e) RBCs in Rana ornativentris at stage XVII could be separated into intermediate (position 2) and heavy (position 3) RBCs (left). Intermediate RBCs were PCNA-positive (upper). Larval globin was hardly observed in the intermediate RBCs, while the heavy RBCs still contained larval globin (lower, SDS-PAGE and AUT-PAGE). (f) RBCs in R. ornativentris at stage XXII. This state was almost identical to stage XVII
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Figure 3. Proliferating red blood cells (RBCs)/erythroblasts in the metamorphosing liver of Xenopus laevis. Scale bar, 50 μm. (a) Liver sections stained with larval- (a, b, c) or adult- (d, e, f) globin probes. (a, d) the liver at stage 54. (b, e) the liver at stage 59. (c, f) the liver at stage 66. (b) Sections of the liver at stage 59 stained with larval or adult globin probes and anti-PCNA antibody. Cells labeled with both larval or adult globin and PCNA are indicated with arrowheads. (a) the liver stained with a larval globin probe. (d) the liver stained with an adult globin probe. (b, e) the liver stained with anti-PCNA antibody. (c, f) merged image with (a) and (b), and (d) and (e), respectively. (g) Ratio of the number of PCNA-positive larval RBCs/erythroblasts to the total number of larval RBCs/erythroblasts, and that of the number of PCNA-positive adult RBCs/erythroblasts to the total number of adult RBCs/erythroblasts. The percentage of PCNA-positive cells did not differ between larval and adult RBCs/erythroblasts (p > .05; Student's t-test). (c) Sections of the liver at stage 62 stained with larval or adult globin probes and anti-PCNA antibody. Cells labeled with both larval or adult globin and PCNA are indicated with arrowheads. (a) the liver stained with a larval globin probe. (d) the liver stained with an adult globin probe. (b, e) the liver stained with an anti-PCNA antibody. (c, f) merged image with (a) and (b), and (d) and (e), respectively. (g) Ratio of the number of PCNA-positive larval RBCs/erythroblasts to the total number of larval RBCs/erythroblasts and that of the number of PCNA-positive adult RBCs/erythroblasts to the total number of adult RBCs/erythroblasts. The percentage of PCNA-positive cells did not differ between larval and adult RBCs/erythroblasts (p > .05; Student's t-test)
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Figure 4. Comparison of globin switching in peripheral red blood cells (RBCs) and RBCs/erythroblasts in the liver. (a) Change of globin type in circulating RBCs and RBCs/erythroblasts in the liver during the metamorphosis of Xenopus laevis as determined using SDS-PAGE. Larval and adult globin bands are labeled with arrowheads and an arrow, respectively, as shown in Figure 1. The hemolysates of peripheral RBCs (B) and liver RBCs/erythroblasts (L) were prepared from the same individuals and electrophoresed. As typically observed in stage 64 and stage 65b, larval globin (arrowheads) began to decrease in liver erythroid cells during metamorphosis, whereas it remained in the peripheral RBCs. The degree of globin switching differed among individuals even if they were at the same developmental stage (e. g., stage 65a and stage 65b). (b) Change of globin type in peripheral blood and liver erythroid cells during the metamorphosis of Rana ornativentris as determined using SDS-PAGE. Larval and adult globin bands are labeled with arrowheads and an arrow, respectively as shown in Figure 1. The hemolysate of peripheral RBCs (B) and liver RBCs/erythroblasts (L) were prepared from the same individuals and electrophoresed. During the metamorphic climax, larval globin could be detected in both peripheral blood and liver erythroid cells but was less concentrated in the liver (arrowhead). It disappeared from both peripheral RBCs and liver RBCs/erythroblasts in the metamorphosed animal. (c) the percentage of the L1 band to total globin in peripheral RBCs and the livers of X. laevis (stage 65, n = 5) and R. ornativentris (stage XXII, n = 3). Asterisks indicate significantly different values between two groups (p < .05; Student's t-test). (d) Change of globin type in the peripheral blood and liver erythroid cells during metamorphosis of R. ornativentris as determined using northern blotting. Total RNAs from the RBCs and liver were extracted from the same individuals and hybridized with larval or adult globin-specific probes. During the metamorphic climax, larval globin could be detected in both peripheral blood and liver erythroid cells but the ratio of larval to adult globin was substantially lower in the liver
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Figure 5. (a) Hemolysates of tadpoles with no treatment (CON), treated only with phenylhydrazine (PHZ), only with methimazole (MMI), and with methimazole + phenylhydrazine (MMI + PHZ) were analyzed using SDS-PAGE. Xenopus laevis tadpoles at stage 57 were treated with or without MMI for 1 week, followed by anemia induction. Larval and adult globin bands are labeled with arrowheads and an arrow, respectively as shown in Figure 1. (b) Percentage of the L1 band to total globin in CON, PHZ, MMI, and MMI + PHZ. A significant difference was observed among different groups (ANOVA, p < .05). Asterisks indicate significantly different values compared to CON (p < .05; Dunnett's test). (c) Hemolysates of CON, PHZ, MMI, and MMI + PHZ analyzed using SDS-PAGE. Xenopus laevis tadpoles at stage 55 were treated with or without MMI for 1 week, followed by anemia induction. Larval and adult globin bands are labeled with arrowheads and an arrow, respectively as shown in Figure 1. (d, e) Expression of larval and adult globin genes in the peripheral RBCs of CON, PHZ, MMI, and MMI + PHZ, detected by northern blotting. Xenopus laevis tadpoles at stage 57 (d) or stage 55 (e) were treated with or without methimazole for 1 week, followed by anemia induction. (f) Expression of larval and adult globin genes in the livers of CON, PHZ, MMI, and MMI + PHZ detected by northern blotting. Xenopus laevis tadpoles at stage 55 were treated with or without methimazole for 1 week, followed by anemia induction
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Figure 6. (a) Hemolysates of tadpoles with no treatment (CON), treated only with phenylhydrazine (PHZ), only with 3,5,3′-triiodothyronine (T3), and with 3,5,3′-triiodothyronine + phenylhydrazine (T3 + PHZ) were analyzed using SDS-PAGE. Xenopus laevis tadpoles at stage 57 were treated with or without T3 for 3 days, followed by anemia induction. Larval and adult globin bands are labeled with arrowheads and an arrow, respectively as shown in Figure 1. (b) Percentage of L1 band to total globin in CON, PHZ, T3, and T3 + PHZ. A significant difference was observed among different groups (ANOVA, p < .05). Asterisks indicate significantly different values than those of CON (p < .05; Dunnett's test). (c) Hemolysates of CON, PHZ, T3, and T3 + PHZ analyzed using SDS-PAGE. Xenopus laevis tadpoles at stage 54 were treated with or without T3 for 3 days, followed by anemia induction. Larval globin bands are labeled with arrowheads as shown in Figure 1. (d) Expression of larval and adult globin genes in the peripheral RBCs of CON, PHZ, T3, and T3 + PHZ as determined by northern blotting. Xenopus laevis tadpoles at stage 57 were treated with or without T3 for 3 days, followed by anemia induction. (e) Expression of larval and adult globin genes in the peripheral RBCs of CON, PHZ, T3, and T3 + PHZ detected by northern blotting. Xenopus laevis tadpoles at stage 54 were treated with or without T3 for 3 days, followed by anemia induction
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Figure 7. Tentative model of red blood cell (RBC) transition in Xenopus laevis. Trace amount of T3 stimulates differentiation of adult RBC precursors in the liver during prometamorphosis. During early metamorphic climax, both larval and adult RBCs/erythroblasts proliferate in the liver and in circulation. At later climax, the decline in larval RBCs itself triggers expansion of adult RBCs; however, the possibility that peaked concentrations of TH can also facilitate adult RBC-specific proliferation cannot be ruled out. Pink triangles and orange squares represent larval and adult globins, respectively, and orange striped circle represent adult RBC precursor
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