THE ERYTHROID CELLS OF ANAEMIC XENOPUS LAEVIS. I. STUDIES ON CELLULAR MORPHOLOGY AND PROTEIN AND NUCLEIC ACID SYNTHESIS DURING DIFFERENTIATION

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1 y. Cell Sci. 19, (1975) 509 Printed in Great Britain THE ERYTHROID CELLS OF ANAEMIC XENOPUS LAEVIS. I. STUDIES ON CELLULAR MORPHOLOGY AND PROTEIN AND NUCLEIC ACID SYNTHESIS DURING DIFFERENTIATION NESTA THOMAS AND N. MACLEAN The Department of Biology, Southampton University, Medical and Biological Sciences Building, Southampton SOg 3 TU, England SUMMARY Phenylhydrazine has been used to induce anaemia in Xenopus laevis. The dosage used causes the complete destruction of all mature erythrocytes within twelve days. The anaemia results in the initiation of a wave of erythropoiesis and large numbers of immature erythroid cells are released into the circulation. The morphological and biosynthetic changes which these cells undergo as they differentiate in circulation are described. The origin of the circulating erythroid cells is also discussed. INTRODUCTION The process of erythropoiesis provides a convenient system for the study of differentiation in eukaryotes, since the maturing erythroid cell population passes through a series of recognizable morphological stages, and undergoes progressive changes in biosynthetic capacities (Maclean & Jurd, 1972). In adult vertebrates, the erythropoietic loci are well defined, residing in organs such as bone-marrow, liver and spleen, and in healthy adults it is generally only the mature erythrocytes which are released into the circulation. Studies of erythropoiesis are normally limited by the presence of non-erythropoietic cells in these organs, by the heterogeneity of the erythroid cells (since the erythropoietic loci contain representatives of many different stages of erythroid development), and, perhaps most of all, by the relative inaccessibility of these differentiating erythroid cells. Studies with adult newts, rendered anaemic by the administration of phenylhydrazine (Grasso, 1973), have shown that animals can recover from severe anaemia, even when splenectomized, although in the normal adult newt the spleen is the only recognized erythropoietic organ. These experiments further show that 'erythroid precursor cells' can divide and undergo subsequent differentiation into mature erythrocytes whilst in the peripheral circulation. The present study was undertaken in order to explore the possibility that the circulating blood cells of very anaemic Xenopus would provide an experimental system for studies on erythroid differentiation. This paper summarizes our findings on the characteristics of the erythroid cells in anaemic Xenopus, and is an extension of our earlier preliminary report on this interesting system (Thomas & Maclean, 1974).

2 510 N. Thomas and N. Maclean MATERIALS AND METHODS Animals Mature, adult Xenopus laevis were obtained from Harris' Biological Supplies, Weston-super- Mare, and maintained as previously described (Maclean & Jurd, 1971). Chemical reagents Tritiated reagents were obtained from the Radiochemical Centre, Amersham; all other reagents from British Drug Houses Ltd, Poole, except where stated in the text. Induction of anaemia and collection of blood Mature, female Xenopus, of body weight approximately 100 g, were made anaemic by 2 subcutaneous injections on 2 successive days of 0-5 ml of a 0-5 % solution of phenylhydrazine in Rugh's amphibian Ringer solution (Rugh, 1962). At varying times after the last injection of phenylhydrazine, the animals were anaesthetized in MS222 (Sandoz Products Ltd, London) and blood collected by ventricular puncture. An aliquot of the blood collected was used for a blood cell count and for preparing smears. The smears were subsequently stained with o-dianisidine (O'Brien, 1961) and haematoxylin or with May-Grunwald-Giemsa. In vitro labelling vrith radioisotopes The remainder of the blood was washed twice in twenty volumes of Ringer solution. The blood cells were resuspended in an incubation medium as previously described (Maclean, Hilder & Baynes, 1973) and agitated at 20 C. [s- 3 H]uridine (sp. act. 5 Ci/mM), [6-3 H]thymidine (sp. act. s Ci/mM) or L-[4,s- 3 H]leucine (sp. act. 50 Ci/mM) were added to a final concentration of 10/iCi/ml. Where appropriate, actinomycin D was added at io/tg/ml and cycloheximide at 100 /ig/ml. Scintillation counting and autoradiographic techniques were as previously described (Maclean et al. 1973). After developing, some of the autoradiographs were stained with Giemsa. Preparation of tissue touch imprints Liver, kidney and spleen from bled animals were cut with a sharp scalpel. The cut surfaces were blotted and then touched against the surface of a clean, dry slide. Bone-marrow was obtained by fracturing the femur. After air drying, the imprints were stained. Measurement of cell dimensions Cells stained with o-dianisidine and haematoxylin were photographed and measurements-'of cell and nuclear diameters taken from prints. The absolute dimensions of the cells were determined from a stage micrometer scale photographed and printed under the same conditions. For each cell, 2 readings of nuclear and cell diameters were taken at right angles to each other. We should emphasize, therefore, that measurements and comparisons are of fixed cells, not living ones. Alterations in shape during fixation may therefore affect the result. RESULTS The time course of recovery from phenylhydrazine-induced anaemia The number and types of cells present in the peripheral circulation at increasing times after phenylhydrazine administration have been summarized in Table 1. The values expressed are the results obtained from 2 animals at each time point. The total number of cells was measured by counting a known dilution of blood in a haemocyto-

3 Erythroid cells of anaemic Xenopus 511 meter; the relative proportions of the various cell types were determined from May- Grunwald-Giemsa stained blood smears. It can be seen that, at 5 days after the induction of anaemia, there is an elevated number of leucocytes present in the blood as compared to untreated animals. The leucocyte level reaches a peak at 20 days and then gradually declines to normal levels. Large numbers of new erythroid cells appear in the peripheral circulation only at about 15 days after the last phenylhydrazine injection. There is then a gradual increase in the number of circulating erythroid cells such that at 50 days after the induction of anaemia the erythroid cell count has almost reached the normal level. The phenylhydrazine-treated toads showed an approximately 90% survival rate, although there was a higher mortality during the summer months. No special conditions were employed for the maintenance of the anaemic animals. Table 1. The numbers and types of cells present in the peripheral circulation at various times after the administration of phenylhydrazine Days after last phenylhydrazine injection 5 10 IS Control The values expressed point. Total 17-9 io-6 9-S No. of cells present x io'/ml blood Damaged erythrocyte8, 0/ /o Other erythroid, 0/ /o are the mean results obtained from at least two animals at each time Leucocytes, % The homogeneity of erythroid cell populations The degree of homogeneity seen in circulating cells was assessed from histograms of average cell diameter and of nucleo-cytoplasmic ratios (Figs. 1, 2). At 15 days there is a relatively homogeneous population of cells of high nucleo-cytoplasmic ratio {njc) but small average cell diameter. At 20 days there are still cells present which are of similar size and njc to those at 15 days but the majority of cells present are larger with a smaller njc. At later times the cells become larger with smaller n/c and the range of variation of these parameters becomes narrower, suggesting that the cells have a relatively high degree of developmental synchrony. These conclusions are supported by microscopic examination of blood smears. The morphology of Xenopus erythroid cells The mature erythrocyte is an oval cell, of average dimensions 32 x 23 /tin, with a relatively small nucleus, 11x9 /tm, showing very condensed chromatin. The cell stains strongly with o-dianisidine, indicating a high concentration of haemoglobin;

4 512 N. Thomas and N. Maclean Romanowsky staining shows absence of cytoplasmic basophilia. At 15 days posttreatment the cells are small and rounded, 20 x 17 /*m, with large nuclei, 17x14 /tm. The cells stain slightly with o-dianisidine and are polychromatophilic. In phase contrast (Fig. 5) the cells have cytoplasmic inclusions, probably mitochondria, and a nucleus showing some chromatin clumping and a prominent nucleolus. Subsequent erythroid cells are charactemed by increased chromatin clumping, loss of cytoplasmic inclusions, loss of nucleoli, loss of cytoplasmic basophilia and an increasing concentration of haemoglobin. 20 o Mean cell diameter, //m l Fig. 1. Histograms of the mean diameters of cells present in the peripheral circulation at different times after phenylhydrazine treatment, A, control; B, 15 days; c, 20 days; D, 30 days; E, 40 days; and F, 50 days. The mean cell diameters were calculated from 2 diameters taken at right angles to each other from May-Grunwald-Giemsa stained smears of the blood cells. 100 cells were measured at each time point. Thus cells exhibiting a wide spectrum of morphological characteristics appear in the peripheral circulation at different times during recovery from anaemia. The sites of origin of circulating erythroid cells Tissue-touch imprints of the liver and, to a much lesser extent, the spleen, indicated the presence of large numbers of cells with a characteristic appearance suggestive of erythroid precursor cells (Figs. 6, 7), during the early stages of recovery from anaemia. Such cells cannot be detected in normal adult liver or spleen. The cells are large and round, of average dimensions, 30 x 27 /tm, with a large nucleus, 24 x 22 /tm. The cells are characterized by intense cytoplasmic basophilia but show either a little or no

5 Erythroid cells of anaemic Xenopus 513 staining with o-dianisidine. Because of their similarity to cells identified as basophilic erythroblasts in other systems (e.g. Lucas & Jamroz, 1961), the cells seen in tissuetouch imprints have been tentatively designated as such. A few similar basophilic erythroblasts are also present in the circulation at 10 days post-treatment. No such cells were seen in the kidney or bone-marrow, suggesting that in anaemia there is no shift in erythropoietic sites from that seen in healthy, adult Xenopus (Jurd, 1971). Although we cannot be certain, we suggest that these basophilic cells of the anaemic liver (and perhaps the spleen) are the source of the circulating erythroid cells Nucleo-cytoplasmlc ratio Fig. 2. Histograms of the nucleo-cytoplasmic ratios of cells present in the peripheral circulation at different times after phenylhydrazine treatment, A, control; B, 15 days; C, 20 days; D, 30 days; E, 40 days; and F, 50 days. The n/c values were calculated from mean nuclear and cell diameters measured on May-Grunwald-Giemsa stained smears of the blood cells. 100 cells were measured for each time point. Incorporation of \^H]thymidine The incorporation of [ 3 H]thymidine into TCA-precipitable material in cells from samples taken at different times after phenylhydrazine treatment and the number of cells involved in the incorporation are summarized in Table 2 C. At least 95 % of the [ 3 H]thymidine can be removed by DNase treatment, and in autoradiographs the grains are confined to the nucleus (Fig. 8). The rate of incorporation has therefore been taken as an index of the relative rates of DNA synthesis. The incorporation of ph]thymidine is linear for at least 12 h (Fig. 3 c). Polychromatophilic erythroblasts characteristic of 15 days post-treatment are very active in DNA synthesis, 50% of the cells becoming labelled during a 6-h incubation

6 514 N. Thomas and N. Maclean with the isotope. At subsequent stages in the recovery there is a gradual fall in the number of cells incorporating [ 3 H]thymidine, until there is very little, or no, incorporation into mature erythrocytes. The basophilic erythroblasts presumed to be present in the liver and spleen are also active in DNA synthesis as seen from autoradiographs of tissue-touch imprints from animals injected with [ 3 H]thymidine 2 h before bleeding (Fig. 7). Since the liver normally has a low mitotic index and very few cells in 5-phase, we interpret the thymidine incorporation as further evidence of their being erythroid precursor cells. Table 2. The incorporation of radioisotopes into TCA-precipitable material in cells at different stages during recovery from phenylhydrazine-induced anaemia Days after last phenylhydrazine injection % cells labelled cpm/10 7 cells Control 07 ±o-6 i3'6±o ±2-I O'2 ±0' ± ± ± ± ± 700 B Control 4-6± I-I 87-S ±2-3 oi-2± ± ± ± SSOO ± ± ± ± Control 1-2 ± 1-2 5O-2 ± o-6 ±o-i ! O ± I2IOO 8840± O ±160 Section A, [ H]uridine; B, [ 3 H]leucine; C, [ 3 H]thymidine. Figures represent means of two experiments. The cpm have been corrected for zero time and background values. Autoradiographs were developed for 15 days at 4 C, io 3 cells were counted for calculation of the percentage of cells labelled in each experiment. Autoradiography of normal erythrocytes labelled with [ 3 H]leucine has been extensively studied by Maclean et al. (1969). DNA-synthesizing activity is associated with mitosis in all the erythroid cells except the mature erythrocyte. Mitoticfiguresare most common among polychromatophilic erythroblasts; approximately 1 % of these cells are in division (Fig. 5), but mitoses are seen in fewer numbers in more mature erythroid cells (Fig. 9). From these observations it would appear that cells divide at least twice in the peripheral circulation, but further work is necessary to determine the number of divisions with certainty. Mitoses in cells which are well haemoglobinized have been reported in a number of other species, for example, chicken (Lucas & Jamroz, 1961), newt (Grasso & Woodard, 1967) and foetal mouse (de La Chapelle, Fantoni & Marks, 1969).

7 Erythroid cells of anaemic Xenopus 5*5 I" o V Mi 2 4 Incubation time, h 2 A Incubation time, h 20 x E CL C S- 10 y Incubation time, h Fig. 3. The time course of incorporation of (A) [ 3 H]uridine, (B) [ 3 H]leucine and (c) [ 3 H]thymidine into TCA-precipitable material in differentiating erythroid cells of Xenopus. Cells were removed from anaemic Xenopus 20 days after induction of anaemia and incubated in vitro. The results expressed are from one experiment; 4 such experiments were carried out, and cpm are per aliquot of cells; #, without actinomycin D; I, with actinomycin D at 10 fig [ml. [ 3 H]uridine incorporation The incorporation of [ 3 H]uridine into TCA-precipitable material has been taken as an index of the relative rates of RNA synthesis, since over 60 % of the activity can be removed from filter paper disks by RNase digestion and almost all can be removed by RNase if preceded by pronase treatment. Again, the incorporation is markedly inhibited by actinomycin D at a final concentration of 10/tg/ml (Table 3 and Fig. 3 A). Fig. 3A is the result of one experiment; 4 such experiments were performed. The incorporation of phjleucine is only slightly affected by the antibiotic (Fig. 3 B). The

8 516 N. Thomas and N. Maclean relative insensitivity of [^HJleucine incorporation to actinomycin D suggests that the inhibition of [ 3 H]uridine incorporation is a specific effect on cellular RNA synthesis, rather than a non-specific cytotoxic effect. The relative rates of RNA synthesis in cells at different stages of erythropoiesis have been summarized in Table 2 A. The RNA-synthesizing activity in all circulating cells is much lower than the DNA- or protein-synthesizing activity and the incorporation is more or less constant from the polychromatophilic to the late orthochromatic erythroblast, although more cells are involved in the incorporation at the polychromatophilic stage. Autoradiographs of liver and spleen tissue-touch imprints show that the basophilic erythroblasts are active in [ 3 H]uridine incorporation but much less active in protein synthesis. This finding corresponds to reports of similar behaviour in other species, for example, newt (Grasso & Woodard, 1966) and foetal rabbit (Grasso, Woodard & Swift, 1963) and has led to the hypothesis of the synthesis of long-lived messenger RNA in basophilic erythroblasts which supports, to a large extent, the synthesis of haemoglobin during subsequent differentiation of the erythroid cells. The insensitivity of [ 3 H]leucine incorporation to actinomycin D treatment further supports the concept of long-lived messenger RNA for globin synthesis in this species. [ 3 H]leucine incorporation The incorporation of ph]leucine into TCA-precipitable material has been taken as an index of protein synthesis, since cycloheximide (at 100 /tg/ml) reduces the incorporation by at least 90 % and pronase digestion on filter paper disks removes 85 % of the counts. From Table 2B it can be seen that the differentiating erythroid cells are initially very active in protein synthesis, although there is eventually a decrease to the levels found in mature erythrocytes from normal, untreated toads. The latter have been extensively studied by Maclean, Brooks & Jurd (1969). DISCUSSION The induction of anaemia in Xenopus with phenylhydrazine results in a dramatic erythropoiesis, with apparent premature release of erythroid precursor cells from the liver and their mitosis and differentiation in the peripheral circulation. Their differentiation has been summarized in Fig. 4. We would like to stress that studies such as ours, which compare physiological and biochemical parameters between individual animals, will show considerable variation, because of the uniqueness of the individual (see Maclean et al. 1969). It follows that it should not be expected that exact percentages of cells and cell types occurring in the blood at set times following phenylhydrazine injection will be matched exactly by other workers using our techniques. From the results presented it can be seen that the use of phenylhydrazine to induce anaemia in X. laevis results in the total destruction of the existing mature erythrocytes with subsequent initiation of a wave of erythropoiesis. Large numbers of immature

9 Erythroid cells of anaemic Xenopus Nomenclature.. Average njc 0-80 Response to o-dianisidine _ Cell division + Basophilic Polychromatophllic Early orthochromatic Late orthochromatic Mature eryth ro blast erythroblast erythroblast erythroblajt erythrocyte u-4x 4-4* 0-35 DNA synthesis + h RNA synthesis + Protein synthesis Fig. 4. A schematic representation of the morphological and synthetic characteristics of erythroid cells which occur in X. laevis during recovery from phenylhydrazineinduced anaemia. The shading of the cytoplasm and nucleus has been used as an indication of the degree of cytoplasmic basophilia and of nuclear condensation respectively. No attempt has been made to indicate the relative synthetic activities of the different cells; this has already been discussed in the text. has been used to indicate that in more extensive studies of the RNA and protein-synthetic activities of mature erythrocytes (Maclean et al. 1973; Maclean et al. 1969), not all the toads studied showed evidence of synthesis. Table 3. The effect of actinomycin D on [ 3 H]leucine and [ 3 H]uridine incorporation in cells from anaemic toads at days after phenylhydrazine treatment Without actinomycin D With actinomycin D (a) Incubation with ['H]uridine 2h 6h 2h 6h 477 ± ± ±540 I53 ±6io (b) Incubation with ['H]leucine The values are expressed as cpm for io 7 cells and those for ['FTJleucine have been rounded up. erythroid cells are released into the circulation where they undergo characteristic changes in morphology and biosynthetic activity. We believe that this system will prove useful for the further elucidation of the processes involved in differentiation. An accompanying paper (Hilder, Thomas & Maclean, 1975) reports on the changes in non-histone nuclear proteins which occur during the maturation of the erythroid cells. Other experiments are investigating the

10 518 N. Thomas and N. Maclean effect of bromouridine-deoxyribose (BUdR) administered in vivo on the subsequent protein synthesis of the circulating cells. The financial help of the Medical Research Council is gratefully acknowledged. REFERENCES DE LA CHAPELLE, A., FANTONI, A. & MARKS, P. A. (1969). Differentiation of mammalian somatic cells; DNA and haemoglobin synthesis in foetal mouse yolk sac erythroid cells. Proc. natn. Acad. Sci. U.S.A. 63, GRASSO, J. A. (1973). Erythropoiesis in the newt, Triturus cristatus Laur. I. Identification of the 'erythroid precursor cell'. J. Cell Sci. 12, GRASSO, J. A. & WOODARD, J. W. (1966). The relationship between RNA synthesis and haemoglobin synthesis in amphibian metamorphosis. Cytochemical evidence. J. Cell Biol. 31, GRASSO, J. A. & WOODARD, J. W. (1967). DNA synthesis and mitosis in erythropoietic cells. J. Cell Biol. 33, GRASSO, J. A., WOODARD, J. W. & SWIFT, H. (1963). Cytochemical studies of nucleic acids and proteins in erythrocytic development. Proc. natn. Acad. Sci. U.S.A. 50, HILDER, V. A., THOMAS, N. & MACLEAN, N. (1975). The erythroid cells of Xenopus laevis. II. Studies on nuclear non-histone proteins. J. Cell Sci. 19, JURD, R. D. (1971). The Haemoglobins of Xenopus laevis (Daudin): Their Characterization, Ontogeny and the Genetic Control of Their Syntliesis. University of Southampton, Ph.D. Thesis. LUCAS, A. M. & JAMROZ, C. (1961). An Atlas of Avian Haematology. Agriculture Monograph no. 25. Washington: United States Department of Agriculture. MACLEAN, N., BROOKS, G. T. & JURD, R. D. (1969). Haemoglobin synthesis in vitro by erythrocytes from Xenopus laevis Daudin. Comp. Biochem. Physiol. 30, MACLEAN, N., HILDER, V. A. & BAYNES, Y. A. (1973). RNA synthesis in Xenopus erythrocytes. Cell Differentiation 2, MACLEAN, N. & JURD, R. D. (1971). The haemoglobins of healthy and anaemic Xenopus laevis. J. Cell Sci. 9, MACLEAN, N. & JURD, R. D. (1972). The control of haemoglobin synthesis. Biol. Rev. 47, O'BRIEN, R. A. (1961). Identification of haemoglobin by its catalase reaction with peroxide and o-dianisidine. Stain Technol. 36, RUGH, R. (1962). Experimental Embryology. Minneapolis: Burgess. THOMAS, N. & MACLEAN, N. (1974). The blood as an erythropoietic organ in anaemic Xenopus. Experientia 30, {Received 3 April 1975) Fig. 5. Phase-contrast appearance of living cells present in the circulation at 15 days after phenylhydrazine treatment. There are 2 polychromatophilic erythroblasts adjacent to one another: one is exhibiting mitotic activity while the other shows cytoplasmic inclusions and a nucleus with some chromatin clumping and a prominent nucleolus. A granulocyte is also shown, x Fig. 6. Tissue-touch imprint of liver 15 days after the induction of anaemia; stained with May-Grunwald-Giemsa. A considerable number of large, round, basophilic cells are present. Some damaged erythrocytes can also be seen at times when they are no longer present in the blood, suggesting that the liver (and the spleen) are the sites of removal of these cells, x 200. Fig. 7. Autoradiograph of tissue-touch imprint of liver showing 3 basophilic erythroblasts labelled with [ 3 H]thymidine from a toad given an in vivo dose of the isotope 2 h before bleeding. Developed for 15 days at 4 C. Stained with May-Grunwald- Giesmsa. x 1200.

11 Erythroid cells of anaemic Xenopus 519

12 520 r N. Thomas and N. Maclean Fig. 8. Autoradiograph of [ 3 H]thymidine-labelled erythroid cells from anaemic Xenopus. 30 days post-treatment. Cells removed from the anaemic Xenopus and incubated in culture as described in Methods. Developed for 15 days at 4 C. Stained with Giemsa. x Fig. 9. Cell showing mitotic activity present in the circulation 30 days after phenylhydrazine treatment. Stained with o-dianisidine/haematoxylin; all cells are rich in haemoglobin, including the one in mitosis, x 1000.