y. Cell Sci. 19, 509-520 (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. MACLEANThe 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 causesthe complete destruction of all mature erythrocytes within twelve days. The anaemia resultsin the initiation of a wave of erythropoiesis and large numbers of immature erythroid cells arereleased into the circulation. The morphological and biosynthetic changes which these cellsundergo as they differentiate in circulation are described. The origin of the circulating erythroidcells is also discussed.

INTRODUCTION

The process of erythropoiesis provides a convenient system for the study of dif-ferentiation in eukaryotes, since the maturing erythroid cell population passes througha series of recognizable morphological stages, and undergoes progressive changes inbiosynthetic capacities (Maclean & Jurd, 1972). In adult vertebrates, the erythro-poietic 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 releasedinto the circulation. Studies of erythropoiesis are normally limited by the presence ofnon-erythropoietic cells in these organs, by the heterogeneity of the erythroid cells(since the erythropoietic loci contain representatives of many different stages oferythroid development), and, perhaps most of all, by the relative inaccessibility ofthese differentiating erythroid cells.

Studies with adult newts, rendered anaemic by the administration of phenyl-hydrazine (Grasso, 1973), have shown that animals can recover from severe anaemia,even when splenectomized, although in the normal adult newt the spleen is the onlyrecognized erythropoietic organ. These experiments further show that 'erythroidprecursor cells' can divide and undergo subsequent differentiation into matureerythrocytes whilst in the peripheral circulation. The present study was undertakenin order to explore the possibility that the circulating blood cells of very anaemicXenopus would provide an experimental system for studies on erythroid differentia-tion. This paper summarizes our findings on the characteristics of the erythroid cellsin anaemic Xenopus, and is an extension of our earlier preliminary report on thisinteresting system (Thomas & Maclean, 1974).

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 otherreagents 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 sub-cutaneous injections on 2 successive days of 0-5 ml of a 0-5 % solution of phenylhydrazine inRugh's amphibian Ringer solution (Rugh, 1962).

At varying times after the last injection of phenylhydrazine, the animals were anaesthetizedin 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 orwith May-Grunwald-Giemsa.

In vitro labelling vrith radioisotopes

The remainder of the blood was washed twice in twenty volumes of Ringer solution. Theblood cells were resuspended in an incubation medium as previously described (Maclean,Hilder & Baynes, 1973) and agitated at 20 °C. [s-3H]uridine (sp. act. 5 Ci/mM), [6-3H]thymidine(sp. act. s Ci/mM) or L-[4,s-3H]leucine (sp. act. 50 Ci/mM) were added to a final concentrationof 10/iCi/ml. Where appropriate, actinomycin D was added at io/tg/ml and cycloheximide at100 /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 surfaceswere blotted and then touched against the surface of a clean, dry slide. Bone-marrow wasobtained 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-'ofcell and nuclear diameters taken from prints. The absolute dimensions of the cells weredetermined 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, notliving 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 increasingtimes after phenylhydrazine administration have been summarized in Table 1. Thevalues expressed are the results obtained from 2 animals at each time point. The totalnumber of cells was measured by counting a known dilution of blood in a haemocyto-

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 elevatednumber of leucocytes present in the blood as compared to untreated animals. Theleucocyte 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 atabout 15 days after the last phenylhydrazine injection. There is then a gradual increasein the number of circulating erythroid cells such that at 50 days after the induction ofanaemia 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 con-ditions were employed for the maintenance of the anaemic animals.

Table 1. The numbers and types of cells present in the peripheral circulationat various times after the administration of phenylhydrazine

Days after lastphenylhydrazine

injection

51 0

IS2 0

3°5°

Control

The values expressedpoint.

Total

17-9io-69-S

1 9 0

2 9 763870-0

are the mean

No. of cells present x io'/ml blood

Damagederythrocyte8,

0//o

9177

2

0

0

0

0

Othererythroid,

0//o

0

1

5973899999

Leucocytes,%

922

39271 1

1

1

results obtained from at least two animals at each time

The homogeneity of erythroid cell populations

The degree of homogeneity seen in circulating cells was assessed from histogramsof average cell diameter and of nucleo-cytoplasmic ratios (Figs. 1, 2). At 15 days thereis 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 ofsimilar size and njc to those at 15 days but the majority of cells present are larger witha smaller njc. At later times the cells become larger with smaller n/c and the range ofvariation of these parameters becomes narrower, suggesting that the cells have arelatively high degree of developmental synchrony. These conclusions are supportedby 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 arelatively small nucleus, 11x9 /tm, showing very condensed chromatin. The cellstains strongly with o-dianisidine, indicating a high concentration of haemoglobin;

512 N. Thomas and N. Maclean

Romanowsky staining shows absence of cytoplasmic basophilia. At 15 days post-treatment 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 phasecontrast (Fig. 5) the cells have cytoplasmic inclusions, probably mitochondria, and anucleus showing some chromatin clumping and a prominent nucleolus. Subsequenterythroid cells are charactemed by increased chromatin clumping, loss of cytoplasmicinclusions, loss of nucleoli, loss of cytoplasmic basophilia and an increasing concentra-tion of haemoglobin.

20-

1 0 -

§ 20

o

1 0 -

20-

1 0 -

l20 30

Mean cell diameter, //m

Fig. 1. Histograms of the mean diameters of cells present in the peripheral circulationat 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 2diameters taken at right angles to each other from May-Grunwald-Giemsa stainedsmears of the blood cells. 100 cells were measured at each time point.

Thus cells exhibiting a wide spectrum of morphological characteristics appear inthe 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, indicatedthe presence of large numbers of cells with a characteristic appearance suggestive oferythroid 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 andround, of average dimensions, 30 x 27 /tm, with a large nucleus, 24 x 22 /tm. The cellsare characterized by intense cytoplasmic basophilia but show either a little or no

Erythroid cells of anaemic Xenopus 513

staining with o-dianisidine. Because of their similarity to cells identified as basophilicerythroblasts in other systems (e.g. Lucas & Jamroz, 1961), the cells seen in tissue-touch imprints have been tentatively designated as such. A few similar basophilicerythroblasts are also present in the circulation at 10 days post-treatment. No suchcells were seen in the kidney or bone-marrow, suggesting that in anaemia there is noshift 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 anaemicliver (and perhaps the spleen) are the source of the circulating erythroid cells.

2 0 -

1 0 -

£ 2 0 -

•5 10 -

2 0 -

10 -

03 0-5 07 0 9 0-3 0-5

Nucleo-cytoplasmlc ratio

Fig. 2. Histograms of the nucleo-cytoplasmic ratios of cells present in the peripheralcirculation 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 frommean nuclear and cell diameters measured on May-Grunwald-Giemsa stained smearsof the blood cells. 100 cells were measured for each time point.

Incorporation of \^H]thymidine

The incorporation of [3H]thymidine into TCA-precipitable material in cells fromsamples taken at different times after phenylhydrazine treatment and the number ofcells involved in the incorporation are summarized in Table 2 C. At least 95 % of the[3H]thymidine can be removed by DNase treatment, and in autoradiographs thegrains are confined to the nucleus (Fig. 8). The rate of incorporation has therefore beentaken as an index of the relative rates of DNA synthesis. The incorporation of pH]thy-midine is linear for at least 12 h (Fig. 3 c).

Polychromatophilic erythroblasts characteristic of 15 days post-treatment are veryactive in DNA synthesis, 50% of the cells becoming labelled during a 6-h incubation

514 N. Thomas and N. Maclean

with the isotope. At subsequent stages in the recovery there is a gradual fall in thenumber of cells incorporating [3H]thymidine, until there is very little, or no, incor-poration into mature erythrocytes. The basophilic erythroblasts presumed to bepresent in the liver and spleen are also active in DNA synthesis as seen from auto-radiographs of tissue-touch imprints from animals injected with [3H]thymidine 2 hbefore bleeding (Fig. 7). Since the liver normally has a low mitotic index and very fewcells in 5-phase, we interpret the thymidine incorporation as further evidence of theirbeing erythroid precursor cells.

Table 2. The incorporation of radioisotopes into TCA-precipitable material in cellsat different stages during recovery from phenylhydrazine-induced anaemia

Days after lastphenylhydrazine

injection % cells labelled cpm/107 cells

B

5152 0

3°Control

5152 0

3°• 5 0

•Control

5152 0

4 0

Control

07 ±o-6i3'6±o-6171075-2±2-IO'2 ±0'2

4-6± I-I

87-S ±2-3oi-2±3-593-1 ± 17

——

1-2 ± 1-25O-2 ±2-167117o-6 ±o-i

—

1840 ± 7008060 ± 950

10220 ±12008620±13501040 ± 700

5080 ±220071600 ±3700

27SSOO ±92500281110 ± 31200

980 ± 2201010 ±640

12701670727500! 22200

5883O ± I2IOO8840±1980

29O ±160

Section A, [°H]uridine; B, [3H]leucine; C, [3H]thymidine. Figures represent means of twoexperiments. The cpm have been corrected for zero time and background values. Autoradio-graphs were developed for 15 days at 4 °C, io3 cells were counted for calculation of the per-centage of cells labelled in each experiment.

• Autoradiography of normal erythrocytes labelled with [3H]leucine has been extensivelystudied by Maclean et al. (1969).

DNA-synthesizing activity is associated with mitosis in all the erythroid cellsexcept the mature erythrocyte. Mitotic figures are most common among polychromato-philic erythroblasts; approximately 1 % of these cells are in division (Fig. 5), butmitoses are seen in fewer numbers in more mature erythroid cells (Fig. 9). From theseobservations it would appear that cells divide at least twice in the peripheral circula-tion, but further work is necessary to determine the number of divisions with cer-tainty. Mitoses in cells which are well haemoglobinized have been reported in anumber of other species, for example, chicken (Lucas & Jamroz, 1961), newt (Grasso& Woodard, 1967) and foetal mouse (de La Chapelle, Fantoni & Marks, 1969).

Erythroid cells of anaemic Xenopus 5*5

I"

o

V

•Mi

2 4Incubation time, h

20

xECL

2 AIncubation time, h

CS- 10

y1

6 12 18Incubation time, h

24

Fig. 3. The time course of incorporation of (A) [3H]uridine, (B) [3H]leucine and (c)[3H]thymidine into TCA-precipitable material in differentiating erythroid cells ofXenopus. Cells were removed from anaemic Xenopus 20 days after induction ofanaemia and incubated in vitro. The results expressed are from one experiment; 4 suchexperiments were carried out, and cpm are per aliquot of cells; •—#—, withoutactinomycin D; — I — , with actinomycin D at 10 fig [ml.

[3H]uridine incorporation

The incorporation of [3H]uridine into TCA-precipitable material has been taken asan index of the relative rates of RNA synthesis, since over 60 % of the activity can beremoved from filter paper disks by RNase digestion and almost all can be removed byRNase if preceded by pronase treatment. Again, the incorporation is markedly in-hibited 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. Theincorporation of pHJleucine is only slightly affected by the antibiotic (Fig. 3 B). The

516 N. Thomas and N. Maclean

relative insensitivity of [^HJleucine incorporation to actinomycin D suggests that theinhibition of [3H]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 havebeen summarized in Table 2 A. The RNA-synthesizing activity in all circulating cellsis much lower than the DNA- or protein-synthesizing activity and the incorporationis more or less constant from the polychromatophilic to the late orthochromaticerythroblast, although more cells are involved in the incorporation at the poly-chromatophilic stage.

Autoradiographs of liver and spleen tissue-touch imprints show that the basophilicerythroblasts are active in [3H]uridine incorporation but much less active in proteinsynthesis. 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 messengerRNA in basophilic erythroblasts which supports, to a large extent, the synthesis ofhaemoglobin during subsequent differentiation of the erythroid cells. The insensi-tivity of [3H]leucine incorporation to actinomycin D treatment further supports theconcept of long-lived messenger RNA for globin synthesis in this species.

[3H]leucine incorporation

The incorporation of pH]leucine into TCA-precipitable material has been taken asan index of protein synthesis, since cycloheximide (at 100 /tg/ml) reduces the incor-poration by at least 90 % and pronase digestion on filter paper disks removes 85 % ofthe counts. From Table 2B it can be seen that the differentiating erythroid cells areinitially very active in protein synthesis, although there is eventually a decrease to thelevels found in mature erythrocytes from normal, untreated toads. The latter havebeen extensively studied by Maclean, Brooks & Jurd (1969).

DISCUSSION

The induction of anaemia in Xenopus with phenylhydrazine results in a dramaticerythropoiesis, with apparent premature release of erythroid precursor cells fromthe liver and their mitosis and differentiation in the peripheral circulation. Theirdifferentiation has been summarized in Fig. 4.

We would like to stress that studies such as ours, which compare physiological andbiochemical parameters between individual animals, will show considerable variation,because of the uniqueness of the individual (see Maclean et al. 1969). It follows thatit should not be expected that exact percentages of cells and cell types occurring in theblood at set times following phenylhydrazine injection will be matched exactly byother workers using our techniques.

From the results presented it can be seen that the use of phenylhydrazine to induceanaemia in X. laevis results in the total destruction of the existing mature erythrocyteswith subsequent initiation of a wave of erythropoiesis. Large numbers of immature

Erythroid cells of anaemic Xenopus

Nomenclature..

Average njc 0-80

Response to _o-dianisidine

Cell division +

DNA synthesis +

RNA synthesis +

Protein synthesis —

Basophilic Polychromatophllic Early orthochromatic Late orthochromatic Maturee r y th r oblast erythroblast erythroblast erythroblajt erythrocyte

072 0-56 u-4X

4-

4*

0-35

•h

Fig. 4. A schematic representation of the morphological and synthetic characteristicsof erythroid cells which occur in X. laevis during recovery from phenylhydrazine-induced anaemia. The shading of the cytoplasm and nucleus has been used as anindication of the degree of cytoplasmic basophilia and of nuclear condensation re-spectively. No attempt has been made to indicate the relative synthetic activities ofthe different cells; this has already been discussed in the text. • has been used toindicate that in more extensive studies of the RNA and protein-synthetic activities ofmature erythrocytes (Maclean et al. 1973; Maclean et al. 1969), not all the toadsstudied showed evidence of synthesis.

Table 3. The effect of actinomycin D on [3H]leucine and [3H]uridine incorporationin cells from anaemic toads at 12-20 days after phenylhydrazine treatment

Without actinomycin D With actinomycin D

2 h 6 h 2 h 6 h

(a) Incubation with['H]uridine

(b) Incubation with['H]leucine

477° ±2210 7370 ±2310 1290 ±540 I53°±6io

183000140000 2380001110000 1830001 32000 216000182000

The values are expressed as cpm for io7 cells and those for ['FTJleucine have been rounded up.

erythroid cells are released into the circulation where they undergo characteristicchanges in morphology and biosynthetic activity.

We believe that this system will prove useful for the further elucidation of theprocesses involved in differentiation. An accompanying paper (Hilder, Thomas &Maclean, 1975) reports on the changes in non-histone nuclear proteins which occurduring the maturation of the erythroid cells. Other experiments are investigating the

518 N. Thomas and N. Maclean

effect of bromouridine-deoxyribose (BUdR) administered in vivo on the subsequentprotein 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 mammaliansomatic cells; DNA and haemoglobin synthesis in foetal mouse yolk sac erythroid cells.Proc. natn. Acad. Sci. U.S.A. 63, 812-819.

GRASSO, J. A. (1973). Erythropoiesis in the newt, Triturus cristatus Laur. I. Identification of the'erythroid precursor cell'. J. Cell Sci. 12, 463-489.

GRASSO, J. A. & WOODARD, J. W. (1966). The relationship between RNA synthesis and haemo-globin synthesis in amphibian metamorphosis. Cytochemical evidence. J. Cell Biol. 31,279-294.

GRASSO, J. A. & WOODARD, J. W. (1967). DNA synthesis and mitosis in erythropoietic cells.J. Cell Biol. 33, 645-655.

GRASSO, J. A., WOODARD, J. W. & SWIFT, H. (1963). Cytochemical studies of nucleic acids andproteins in erythrocytic development. Proc. natn. Acad. Sci. U.S.A. 50, 134-140.

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, 521-529.

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 Monographno. 25. Washington: United States Department of Agriculture.

MACLEAN, N., BROOKS, G. T. & JURD, R. D. (1969). Haemoglobin synthesis in vitro by erythro-cytes from Xenopus laevis Daudin. Comp. Biochem. Physiol. 30, 825-834.

MACLEAN, N., HILDER, V. A. & BAYNES, Y. A. (1973). RNA synthesis in Xenopus erythrocytes.Cell Differentiation 2, 261-269.

MACLEAN, N. & JURD, R. D. (1971). The haemoglobins of healthy and anaemic Xenopus laevis.J. Cell Sci. 9, 509-528.

MACLEAN, N. & JURD, R. D. (1972). The control of haemoglobin synthesis. Biol. Rev. 47, 393-437-

O'BRIEN, R. A. (1961). Identification of haemoglobin by its catalase reaction with peroxide ando-dianisidine. Stain Technol. 36, 57-61.

RUGH, R. (1962). Experimental Embryology. Minneapolis: Burgess.THOMAS, N. & MACLEAN, N. (1974). The blood as an erythropoietic organ in anaemic Xenopus.

Experientia 30, 1083-1085.{Received 3 April 1975)

Fig. 5. Phase-contrast appearance of living cells present in the circulation at 15 daysafter phenylhydrazine treatment. There are 2 polychromatophilic erythroblasts ad-jacent to one another: one is exhibiting mitotic activity while the other shows cyto-plasmic inclusions and a nucleus with some chromatin clumping and a prominentnucleolus. A granulocyte is also shown, x 1500.Fig. 6. Tissue-touch imprint of liver 15 days after the induction of anaemia; stainedwith May-Grunwald-Giemsa. A considerable number of large, round, basophiliccells are present. Some damaged erythrocytes can also be seen at times when they areno longer present in the blood, suggesting that the liver (and the spleen) are the sitesof removal of these cells, x 200.Fig. 7. Autoradiograph of tissue-touch imprint of liver showing 3 basophilic erythro-blasts labelled with [3H]thymidine from a toad given an in vivo dose of the isotope 2 hbefore bleeding. Developed for 15 days at 4 °C. Stained with May-Grunwald-Giesmsa. x 1200.

Erythroid cells of anaemic Xenopus 519

520

rN. Thomas and N. Maclean

Fig. 8. Autoradiograph of [3H]thymidine-labelled erythroid cells from anaemicXenopus. 30 days post-treatment. Cells removed from the anaemic Xenopus andincubated in culture as described in Methods. Developed for 15 days at 4 °C. Stainedwith Giemsa. x 1000.

Fig. 9. Cell showing mitotic activity present in the circulation 30 days after phenyl-hydrazine treatment. Stained with o-dianisidine/haematoxylin; all cells are rich inhaemoglobin, including the one in mitosis, x 1000.