J. Cell Sci. 9, 509-528 (1971)

Printed in Great Britain

THE HAEMOGLOBINS OF HEALTHY AND

ANAEMIC XENOPUS LAEVIS

N. MACLEAN AND R. D. JURD

Department of Zoology, Tlie University, Southampton, SO9 $NH, England

SUMMARY

The haemoglobins of Xenopus laevis have been studied by carboxymethyl-cellulose columnchromatography and by polyacrylamide gel disk electrophoresis. In Xenopus tadpoles, 2 haemo-globins are found (Xenopus-HbFx and Xenopus-HbF^). Both haemoglobins persist throughouttadpole life: Xenopus-HbF\ is the main tadpole haemoglobin; Xenopus-HbFt is present inrather smaller amounts. The proportion of Xenopus-HbF\ to Xenopus-HbF3 increases as thetadpoles age. Almost all the tadpole haemoglobin disappears soon after metamorphosis, althoughtraces persist throughout adult life. Two adult haemoglobins occur in Xenopus: they first appearduring the later tadpole stages. Xenopus-HbAx comprises the majority of the adult haemo-globin. Between one-twentieth and one-tenth of the adult haemoglobin consists of Xenopus-HbAt, which is not a polymer of Xenopus-HbA1.

Immunoglobulins respectively specific to pooled Xenopus-HbF^ and Xenopus-HbFt, andto pooled Xenopus-HbA1 and Xenopus-HbA2 were raised. Samples of these antibodies wereconjugated with fluorescein isothiocyanate.

Adult Xenopus were made anaemic by bleeding or by injection of phenylhydrazine. Fourteendays after the induction of anaemia it was found, using both chromatographic and electro-phoretic techniques, that most anaemic toads had started to resynthesize Xenopus-HbF^ Insome animals up to 11 % of the haemoglobin present was found to be Xenopus-HbF1; andprecipitin lines were obtained when the pooled haemoglobins from these animals were testedwith a specific anti-_Xeno/WM-HbF immunoglobulin. During recovery from anaemia greatlyenhanced amounts of Xenopus-HbA^ (up to 58 % of the total haemoglobin) were present in theblood. The amounts of tadpole haemoglobins present in such recovering toads were notnoticeably greater than in healthy animals. Between 42 and 120 days after the induction ofanaemia the haemoglobin profile of the toads regained normality.

Blood cell smears were made from anaemic toads which were known to possess abnormalamounts of Xenopus-HbF x 14 days after the induction of anaemia. The smears were treatedwith specific anti--Xeno/>Ui-HbF immunoglobulin conjugated with fluorescein isothiocyanate.Although up to 11 % of the haemoglobin present in the animals was Xenopus-HbF\, the numberof fluorescing cells was no greater than in similarly treated smears from healthy control adultspossessing less than 1 % Xenopus-HbF y.

It is concluded that during anaemia Xenopus starts to resynthesize one of its tadpole haemo-globins. This resynthesis occurs in many or most of the circulating red cells, and is not confinedto a small population of cells. During recovery from anaemia enhanced quantities of Xenopus-HbA2 are made.

INTRODUCTION

The African Clawed Toad, Xenopus laevis (Daudin), like most other tetrapods,makes different haemoglobins during larval (tadpole) and adult life respectively.Moreover, both in larval and adult life, more than one haemoglobin is manufacturedat a time. It is now well authenticated that, under conditions of anaemia, some adultanimals alter the pattern of haemoglobins produced. In man enhanced synthesis of the

510 N. Maclean and R. D. Jurd

foetal haemoglobin, HbF, occurs in various anaemias (Beaven, Ellis & White, i960;Shahadi, Gerald & Diamond, 1962). Some breeds of sheep and goats, when renderedanaemic by injection of phenylhydra2ine, synthesize abnormally large amounts of a'Haemoglobin C which, in healthy adults, is present in only small amounts (Boyer,Hathaway, Pascasio, Orton& Bordley, 1966; Beale, Lehmann, Drury & Tucker, 1966;Huisman, Adams, Dimmock, Edwards & Wilson, 1967).

We have artificially induced anaemia in adult Xenopus laevis and find that manysuch anaemic individuals possess substantial amounts of one of their tadpole haemo-globins which, in healthy adults, is present only in trace amounts. Moreover, duringrecovery from anaemia these experimental animals yield adult haemoglobins in whichthe normal ratio between the different types is altered.

We have already demonstrated, by immunofluorescent techniques, that during themetamorphic changeover from tadpole to adult haemoglobins in Xenopus, some redblood cells contain haemoglobin of both the adult and the tadpole types (Jurd &Maclean, 1970). By applying the same immunofluorescent techniques to red blood cellsfrom anaemic adult Xenopus, we conclude that whilst every red cell contains adulthaemoglobins, the tadpole haemoglobin produced during anaemia is distributedthroughout a large proportion of the cell population.

MATERIALS AND METHODS

Animals

Mature adult male and female Xenopus laevis were obtained from Harris' Biological Supplies,Weston-super-Mare, England. Animals from selected adult pairs were each injected, over a2-day period, with 350 i.u. of Chorionic Gonadotrophin, B.P. (Organon Labs., Ltd., Morden,England) to induce amplexus. Tadpoles reared from the resulting eggs were used in our ex-periments. Tadpoles were kept in tap water which had been 'aged' by standing overnightto eliminate chlorine and to equilibrate it with the room temperature of 20 °C. Adult toadswere fed twice weekly on chopped ox heart and monthly on chopped ox liver; tadpoles werefed daily with a thin suspension of Complan (Glaxo, England) in water.

Chemical reagents

Except where otherwise specified, all reagents were supplied by British Drug Houses Ltd.,Poole, England.

Induction of anaemia

Two methods of induction were used. (1) Mature adult Xenopus, between 60 and 100 mmlong from mouth to anus, were anaesthetized by immersion in 0-2 % methane tricaine sulphonate(MS 222, Sandoz Products Ltd., London). The body cavity was opened by making a smallventral incision in the skin and body wall just posterior to the xiphisternal cartilage, and between2 and 11 ml of blood, depending upon the size of the toad, were extracted from the heart byventricular puncture. The ventral incision was then sutured and the toad was allowed to recoverfrom anaesthesia. A volume of Rugh's Amphibian Ringer Solution (Rugh, 1962) equal to thevolume of blood extracted was injected into the dorsal lymph sac to restore the salt balance ofthe animals. A count of red blood cells per ml of blood before the induction of anaemia wasmade. (2) Other adult Xenopus, of similar size, were made anaemic by 2 subcutaneous injections,each of 0-05 ml of a 1 % solution of phenylhydrazine in 001 N HC1, on day 1 and on day 5.A red blood cell count of peripheral blood extracted from a severed vein in the interdigital footweb was made before the first injection.

Haemoglobins in Xenopus 511

Fourteen days after the toads were initially bled, or 14 days after the first injection of phenyl-hydrazine hydrochloride, the animals were anaesthetized in MS 222, and 0-5 ml of blood wascollected by ventricular puncture. A red blood cell count was again made. In some animalsfurther samples of blood were taken 28 and 42 days after the induction of anaemia.

In vitro labelling of haemoglobin with pH] leucine

The haemoglobin from O'S ml of blood extracted from adult toads before or after the inductionof anaemia was labelled by incubating the blood cells in vitro with ['Hjleucine, as previouslydescribed (Maclean, Brooks & Jurd, 1969). After incubation the cells were lysed in 5-0 ml ofdistilled water to which 03 ml of toluene had been added. The mixture was shaken thoroughlyand stood for 10 min to ensure complete cell lysis; after centrifuging for 60 min at 8000g,the clear red supernatant haemolysate was collected.

The haemolysate to be used for carboxymethyl-cellulose cation exchange chromatographywas dialysed overnight at 2 °C against 200 vol. of stirred o-oi M sodium phosphate at pH 610,containing 001 % dithiothreitol (DTT - Calbiochem Ltd., Los Angeles, U.S.A.) to reducedisulphide bridges between adjacent globin chains, and so prevent polymerization of thehaemoglobin (Cleland, 1964; Sullivan & Riggs, 1967). Haemolysates to be used for polyacryl-amide gel disk electrophoresis were similarly dialysed against a tris-(hydroxymethyl)-methylamine (Tris)/diamino-ethane-tetra-acetic acid (EDTA)/boric acid buffer at pH 8-6, alsocontaining 001 % DTT. Haemolysates were oxygenated by shaking in air or by bubbling oxygenthrough them for 10 min at 20 °C prior to electrophoresis and chromatography.

Control haemolysates were similarly prepared from tadpole red blood cells (extracted byincising the hearts of anaesthetized tadpoles whilst they were immersed in Ringer solution),and from artificially mixed tadpole and adult cells. All cells were incubated with tritiated leucineprior to lysis.

Separation of haemoglobins

Carboxymethyl-cellulose column chromatography. Haemolysates from the incubated red bloodcells were subjected to cation-exchange column chromatography on Whatman Chromediagrade CM52 preswollen carboxymethyl cellulose (sold by H. Reeve Angel & Co. Ltd., London)in a sodium phosphate pH gradient, as previously described (Maclean et al. 1969). The pHvalues of fractions eluting from the chromatography column were measured; the fractions werethen assayed for optical density at 410 nm, and, by liquid scintillation counting, for tritiumactivity.

Polyacrylamide-gel disk electrophoresis. Polyacrylamide-gel disk electrophoresis was carriedout in a Shandon SAE 2731 disk electrophoresis apparatus using a continuous TEB buffersystem. The buffer used was an aqueous solution containing 10-9 g/1. Tris, o-6 g/1. EDTA, and3-1 g/1. boric acid. The pH was corrected to 860 with o-i N NaOH; o-oi % DTT was addedas a thiol agent. Gels containing 10% acrylamide were used: it was found that the addition of10% glycerol to the gels greatly reduced diffusion of the protein bands.

The system was pre-run for 2 h at 25 mA per gel tube to effect equilibration and to removeexcess persulphate ions (added to facilitate the polymerization of the acrylamide) which couldcause an artificial heterogeneity of the proteins (Mitchell, 1967).

Oxygenated haemolysates from red cells from anaemic and healthy control Xenopus adults,dialysed against the TEB electrophoresis buffer as described above, were diluted with the bufferto bring their haemoglobin concentration to 15 mg/ml; 100 mg of sucrose was added to eachml of haemolysate to increase density, then 002 ml of the haemolysate was layered on to thetop of the polyacrylamide gel and electrophoresis of the proteins was effected at 2-5 mA pergel tube for between 2 and 4 h. Haemoglobin migrated towards the anode. The apparatus wasmaintained at 2 °C in a refrigerator throughout electrophoresis.

On completion of electrophoresis the gels were photographed against transmitted whitelight, and were then fixed and stained for 24 h in a solution consisting of 50 ml methanol, 50 mlglycerol, 10 ml glacial acetic acid and o-8 g naphthalene black 12B. Stain not bound to proteinwas removed electrophoretically by placing the gel at 90° to an applied electric field in a Petridish containining 7% acetic acid, as described by Shaw (1969); unbound dye migrated towards

33 C E L 9

512 N. Maclean and R. D. Jurd

the cathode. Gels were subsequently stored in 5 % acetic acid. Some gels were stained specificallyfor haemoglobin with benzidine immediately after electrophoresis, as described by Moss &Ingram (1968 a).

Identification of liaemoglobins by agar-gel immunodiffusion

Antisera specific to the pooled adult haemoglobins ('Xenopus-HbA') and to the pooledtadpole haemoglobins ('Xenopus-HbF') of Xenopus were raised in rabbits and guinea-pigsrespectively as previously described (Jurd & Maclean, 1969, 1970). The y-globulin fractionwas separated on diethylaminoethyl cellulose at pH 7-5 according to the method of Stanworth(i960). The anti-Xenopus-HbA immunoglobulin was treated with acetone-dried pig-liverpowder (Burroughs Wellcome & Co. Ltd., London) and with Xenopus-HbF to remove non-specific activity; similarly the anti-Xenopus-HbF was treated with pig-liver powder andXenopus-HbA.

Ouchterlony (1949) agar-gel immunodiffusion plates were prepared using a 1% Bacto-Agar(Difco Labs., Detroit, U.S.A.) gel containing 0-15% sodium azide as a bacteriostatic agent.

Haemoglobin from anaemic animals was tested on the plates against anti-Xenopus-HbA andanti-Xenopus-HbF antisera, and the appearance of any precipitin lines between the immuno-globulin and the haemoglobin was noted. Control wells on the same immunodiffusion plateswere set up using haemoglobin taken from the same adult toad before induction of anaemia,and Xenopus-HbF from young tadpoles. All haemolysates contained 001 % DTT.

Permanent preparations of the plates, stained with 0 1 % azocarmine, were subsequentlymade following the method of Uriel (1964).

Preparation and use of fluorescent antisera

Anti-Xeno/wi-HbA and anti-Xenopus-HbF immunoglobulins, prepared and purified asdescribed above, were conjugated with fluorescein isothiocyanate (FITC - Calbiochem Ltd.,Los Angeles, U.S.A.) after the method of Nairn (1969). The anti-adult haemoglobin and anti-tadpole haemoglobin conjugates, known as anti-Xenopus-HbA-FITC and anti-Xenopus-HbF-FITC respectively, were further purified by a second absorption with pig-liver powder to removenon-specific fluorescence (Curtain, 1958).

Red blood cells from anaemic Xenopus were washed in Ringer solution and smeared on toslides which were then air-dried and fixed for 2 min in 95% ethanol - a method modified fromSainte-Marie (1962) and Wild (1970). The smears were treated with the fluorescent immuno-globulin as previously described (Jurd & Maclean, 1970). The proportion of cells fluorescingwhen the smear was viewed by ultraviolet illumination at 360 nm was noted.

Red blood cell smears from anaemic animals were treated with anti-Xenopus-HbA-FITC andanti-^Xeno^iu-HbF-FITC. Both fluorescent antisera were also used to test the blood cells fromthe same animals prior to the induction of anaemia, and cells from other control animals, fromtadpoles, and from artificial mixtures of adult and tadpole cells.

RESULTS

Chromatography of haemoglobins from healthy adult Xenopus and from Xenopus tadpoles

The elution pattern obtained when adult Xenopus red blood cells were incubatedwith tritiated leucine and the haemoglobin chromatographed on carboxymethylcellulose in a sodium phosphate pH gradient has already been described (Macleanet al. 1969). We now feel that this pattern must be slightly modified in the light offurther experimentation. Fig. 1 illustrates an elution pattern typical of the haemo-globins of healthy adult toads. Optical absorption at 410 nm indicates 2 significanthaemoglobin peaks. The first is eluted from the column at pH 7-38 and contains 92%of the total haemoglobin eluted (as measured by optical absorption), and 92 % of thetritium activity; the second peak is much smaller, eluting at pH 7-85 and containing

Haemoglobins in Xenopus 513

5 % of the haemoglobin as measured by optical absorption, and 5 % of the radioactivity.Similar elution patterns were obtained from the haemoglobins of all other healthyadult toads tested: chromatography data from 8 such toads are summarized in the toppart of Table 1. It will be noted that most of the toads show very small optical absorp-tion and radioactivity peaks in the fraction eluting from the column at pH 6-5 (± o-i)and pH 7-15 (±o-i): the amount of haemoglobin in these peaks is always less than2% of the total.

1-5 r pH 7-38- -1 300

- 200

Ea.

Fig. 1. Elution pattern obtained when 15 mg of haemoglobin from one femaleXenopus adult, length 85 mm mouth to anus, were chromatographed on carboxy-methyl cellulose in a sodium phosphate pH gradient. The red cells were incubated intritiated leucine prior to lysis. Eluate fractions were assayed for optical density at410 nm, and for tritium activity. O, Optical density; • , 3H.

The main adult haemoglobin peak, which elutes at pH 7-35 (±0-07), we designate' Xenopus-HbA1': this haemoglobin corresponds to the combined 'H1 ' and 'H2'haemoglobins described by Maclean et al. (1969): our recent studies show that the' H2' peak closely associated with the ' Hj ' peak is an artifact caused by varying degreesof oxygenation of the haemoglobin. The minor adult haemoglobin eluting at pH 7-85(±0-05) is designated 'Xenopus-HbA2', and corresponds to the previously described'H3' haemoglobin (Maclean et al. 1969).

Fig. 2 shows the elution pattern obtained when the pooled red blood cells from10 stage-50 (Nieuwkoop & Faber, 1967) tadpoles were incubated with tritiated leuclneand the haemoglobin was subsequently chromatographed on carboxymethyl cellulose.Three elution peaks are seen. The first elutes at pH 6-53 and contains 56% of thehaemoglobin as measured by optical absorption at 410 nm, and 57% of the tritiumactivity; the second elutes at pH 7-13, contains 20% of the haemoglobin and 17% ofthe radioactivity; the final peak is eluted from the column at pH 7-36 and comprises

33-2

N. Maclean and R. D. Jurd

Table i. Elution data for haemoglobin from 8 healthy adult Xenopus, and for 6 samples

of pooled tadpole haemoglobin, chromatographed on carboxymethyl cellulose in a o-oi M

sodium phosphate pH gradient; i$mg of oxygenated haemoglobin, from red blood cells

previously incubated with tritiated leucine, were applied to the column

(Figures in parentheses indicate the pH values at which the haemoglobin peaks eluted from thecolumn. The upper figure is the percentage of the haemoglobin which was eluted in each peak,measured by optical absorption at 410 nm; the lower figure is the percentage of tritium activityin each peak.)

Toad

i

2

3

4

5

6

7

8

Tadpoles

i

2

3

4

5

6

Sex

M

M

M

M

F

F

F

F

No.pooled

13

16

I I

15

IO

8

Size, mmmouthto anus

6o

6o

7°

7 0

85

85

95

1 0 0

Stage

45

45

47/8

47/8

5°

53

Xenopus-HbFi

—

(6-55) < 1< i

(649) < 1< 1

—

( 6 S I ) < I< 1

(642) < 1< i

(6-50) < 1< i

(660) < 1< 1

Xenopus-HbF

(6-49)

(646)

(6-5°)

(658)

(6-53)

(6-44)

51

556 0

6 2

6 0

58

535°565761

64

Xenopus-HbFj

—

(7-25)<1< i

( 7 - I I ) < I

< i

—

(7-06) < 1< i

(7-17) < 1< i

—

( 7 i S ) < i< 1

Xenopus-HbA

(7-32)

(7-42)

(7-39)

(736)

(7-38)

(7-32)

(7-3O)

(7-29)

Xenopus-HbF

(7-12)

(7-05)

(711)

(709)

(7-i3)

(7-10)

434 0

3433

3429

4 0

39

2 0

17

1 1

4

'i

9693

9 0

93

899 i

9496

9292

9693

9593929 2

Xenopus-HbA,

(7'8i) 36

(789) 64

(7-80) 87

(778) 42

(7-85) 55

(7'84) 25

(7-90) 46

(7-89) 66

Xenopus-HbAx

(7-34) 55

(7-35) 24

(738) 61 2

(732) 61 0

(7-36) 2223

(7-33) 2832

Haemoglobins in Xenopus 515

22 % of the haemoglobin and 23 % of the radioactivity. That this pattern is repeatableis shown from the data in the lower part of Table 1. It will be noticed that, as thetadpoles get older the proportion of haemoglobin eluting at pH y io (± 0-05) decreasesslightly and the proportion eluting at pH 6-50 ( ± 0-08) increases.

We find that the haemoglobin from tadpoles eluting from the column at pH 7-35( + 0-05) gives a positive precipitin reaction with specific anti-Xenopus-HbA antiserum:the haemoglobins eluting at pH 6-5 ( + 0-08) and pHy-io (±0-05) give no suchreaction. We therefore conclude that the pH 7-35 haemoglobin is identical to Xenopus-

06

0-4

qd

0-2

-pH 653

pH 7-36

12

8 „

xEQ.

6-5 70 75 80pH

Fig. 2. Elution pattern obtained when 15 mg of haemoglobin from 10 stage-50Xenopus tadpoles were chromatographed on carboxymethyl cellulose in a sodiumphosphate pH gradient. The red cells were incubated in tritiated leucine prior tolysis. Eluate fractions were assayed for optical density at 410 ran, and for tritiumactivity. O, Optical density; • , 3H.

^ This conclusion is further justified by the fact that as tadpoles get older therelative amount of this haemoglobin progressively increases until, about 10 weeks aftermetamorphosis, it comprises over 80% of the haemoglobin present and the overallelution pattern resembles that of a mature adult toad.

The pH 6-50 and pH7-io eluate fractions are designated 'Xenopus-HbF^ and'Xenopus-HbF2' respectively. After metamorphosis these haemoglobins disappearalmost completely: the existence of very small absorption peaks at pH 6-50 and pH 7-15in haemolysates from mature adult toads may be explained by the persistence ofminute amounts of the foetal haemoglobins into post-metamorphic life.

When haemoglobins from artificially mixed tadpole and adult red blood cells arechromatographed, 4 optical absorption peaks are observed which elute at pH valuescorresponding to the 2 foetal and the 2 adult haemoglobin peaks respectively.

516 N. Maclean and R. D. Jurd

Polyacrylamide-gel disk electrophoresis of Xenopus adult and tadpole haemoglobin

Haemoglobin from the red blood cells of healthy adult Xenopus, when electro-phoresed on polyacrylamide gel in a continuous TEB buffer system at pH 8-6o,migrates towards the anode in 2 visible bands. Most of the haemoglobin migrates inone fast band (a1 in Fig. 5), but a faint slower band (a2) is also detectable. Benzidinestaining shows that both bands are haemoglobins. Naphthalene black staining alsoreveals these 2 bands, together with 2 fainter, faster bands which are not detectable bybenzidine staining. These faster bands probably represent non-haem proteins suchas carbonic anhydrase present in the cell lysate.

When haemolysates from Xenopus tadpole red blood cells are similarly electro-phoresed, 4 visible bands, which also stain with benzidine, are present in the gel. Astrong band (£3 in Fig. 5) migrates towards the anode somewhat faster than the mainadult band. Another band, tv migrates more slowly than the main adult, but fasterthan the slow adult band. A third band (t2) migrates at a speed intermediate to t1 andt3, and at the same speed as the main adult band. A faint, very fast fourth band (24)is also detectable. If the bands are excised from the gel and the haemoglobins areeluted into 0-3 ml of 0-85 % saline, eluates from the fast (£3), very fast (f4), and slow(ti) tadpole bands give a positive precipitin ring reaction with specific anti-Xenopus-HbF antiserum, but not with anti-Xenopus-HbA antiserum; the t% eluate, and theeluates from both the adult bands give a positive precipitin ring reaction with anti-Xenopus-HbA, but not with anti-Xenopus-HbF antiserum.

We therefore conclude that the tadpole haemoglobin comprising electrophoresisband t2 is identical to that which comprises the main adult haemoglobin band. Thisview is reinforced by the observation that in haemolysates from young, pre-stage 50,tadpoles the t2 band is almost or completely absent, but as the tadpoles get older theproportion of this band increases. The remaining 3 tadpole bands, tlt t^, t4, are genuinetadpole haemoglobins. Artificial mixtures of adult and tadpole haemoglobins showthe presence of both adult bands and all 3 true tadpole bands.

When the Xenopus-HbA1 (pH 7-35) eluate fractions from carboxymethyl-cellulosechromatography were dialysed and concentrated by ultrafiltration against TEB bufferat pH 8-6o and subsequently electrophoresed on polyacrylamide it was found thatthe haemoglobin migrated at the same speed as the main adult (oj) and the t2 tadpoleband: it is thus concluded that the main adult band (which is also identical to the t2

tadpole band) represents Xenopus-HbAl haemoglobin. Using similar techniques itcan be shown that the pH 7-85 Xenopus-HbA2 chromatography eluate corresponds tothe slow adult electrophoresis band a?, that the pH 6-50 Xenopus-HbF1 eluate corre-sponds to the fast tadpole band t^, and that the pH7>i5 Xenopus-HbF2 eluatecorresponds with the slow tadpole band t±. It has not proved possible to correlate thevery fast tadpole band £4 with any of the carboxymethyl cellulose fractions.

Detection of altered haemoglobin profiles in anaemic adults

Following the induction of anaemia in adult Xenopus by bleeding, the blood cellcount 14 days later was reduced by between 28% and 63%. It was noted that the

Haemoglobins in Xenopus

Table 2. Percentages of haemoglobin and tritium eluting in each carboxymethyl-cellulosechromatography elutionpeak when haemoglobin from adult Xenopus was chromatographedbefore, and 14 days after induction of anaemia by bleeding (Bl) or phenylhydrazine (Ph);red cells were incubated with tritiated leucine before lysis

(Upper row of figures for each toad shows percentages on day 1; lower row percentages onday 14. Figures in parentheses indicate percentages of tritium; figures not in parentheses showpercentages of haemoglobin measured by optical absorption.)

Toad

i

2

3

4

5 '

6

7

8

9

1 0

I I

1 2

13

14

15

16

17

18

Methodof

induction

Bl

Bl

Bl

Bl

Bl

Bl

Bl

Bl

Ph

Ph

Ph

Ph

Ph

Ph

Ph

Ph

Ph

Ph

Reduction incell count

after 14 daysas % of count

on day 1

40

33

28

31

4 2

63

36

31

7 0

7 1

63

54

9 0

72

9 2

98

55

61

Xenopiis-HbF,

< j

I

< I

2

< I

< I

< I

I

I

< I

I

< I

< I

< I

I

< I 1I I

< I

2

< I

9< i

1

< i

1

< i

8< i

2

< i

1

< i

1

< i

2

* No data available

( < j )

(5)( < i )

(12)

( < i )

( < ! )

( < l )

( 2 )

(14)

( < l )

(36)( < l )

( 2 )

( < l )(25)

( < l )

(14)

( < l )

(31)

( < l )

(16)( < l )

(19)

( < l )

(39)( < l )

(14)

( < l )

(48)( < l )

(39)( < l )

(28)( < l )

(31)

on day 1

Xenopus-HbF,

<i (<i )< i (<i )

< i (<i )

< i (2)

< i (<i )<i (<i)

< i (<i )< i (<i)

—

1 (1)

< i ( < i )

< i (2)

<i (<i)<i (<i)

<i (<i)

<i (2)

<i (<i)

1 (3)

< i ( < i )

1 (2)

< i ( < i )

1 (3)

< i ( < i )

1 (1)

< i ( < i )

< i (2)

< i ( < i )

1 (3)

< i ( < i )

1 (1)

<i (<i)

1 (2)

<i (<i)

1 (1)

< i ( < i )

1 (1)

for toad No.

Xenopus-HbAi

90 (93)91 (89)

89 (91)87 (82)

92 (93)94(94)

94 (95)93 (95)

—89 (79)92 (92)

9i (57)93 (92)91 (92)

92 (93)92 (68)

96 (93)85 (78)92 (92)91 (62)

93 (94)85 (73)

95 (94)95 (74)96 (93)94 (55)

93 (94)88 (79)

92 (91)92 (46)

89 (90)9i (52)

93 (92)92 (65)

95 (94)93 (64)

5-

Xenopus-HbAj

6(4)5(3)8(7)8(4)7(7)5(4)5(3)5 ( 2 )

—

9(6)6(5)7(5)5(6)7(5)6(5)4(5)2 ( 5 )

3(5)6(6)6(5)5(4)4(6)3(4)3(6)2(4)

4(4)5(4)3(4)6(7)5(5)9(7)7(7)5(6)6(6)3(4)4(4)

518 N. Maclean andR.D. Jurd

peripheral blood cells contained abnormally large numbers of early and mid-poly-chromatic erythrocytes in addition to the usual mature erythrocytes - the nomen-clature used is taken from Lucas & Jamroz (1961). Fig. 6 shows a smear made fromblood cells from an adult toad before it was made anaemic and its red blood cell countwas 731 x io6 red cells/ml; Fig. 7 shows blood cells from the same animal 14 days after6-3 ml of blood had been extracted from the ventricle and the cell count had beenreduced to 281 x io8 cells/ml.

1-5 1—

1 0

rid

OS

65

Fig. 3. Elution patterns obtained when 15 mg of haemoglobin from toad 9 (Table 2)were chromatographed on day 1 (O O) and 14 days after induction 'of anaemiawith phenylhydrazine (• • ) .

Injection of phenylhydrazine hydrochloride reduced the blood cell count of adultXenopus by between 54% and 98% after 14 days. In extreme anaemia the majority ofcirculating cells are seen to be blast cells and erythroblasts (Fig. 8). Four out of 10animals made anaemic in this way died between 14 and 20 days after the initial phenyl-hydrazine injection; the others recovered. The count of circulating cells started toincrease again after about 21 days, but it was between 6 and 12 weeks before the normalpopulation of mature erythrocytes was restored.

Table 2 shows the results of chromatography of haemolysates from red blood cellsof 18 adult Xenopus before and 14 days after they were made anaemic. Prior to chromato-graphy the cells were incubated with tritiated leucine: as might be expected, in anaemiathere is an overall increase in the synthetic activity of the red blood cells measured interms of tritiated leucine incorporation. It will be noted that in 15 out of the 18 animals(the exceptions are animals 3, 4 and 7) there is a significant increase in the proportionof tritium activity in the pH 6-5 Xenopus-HbF1 elution peak. Before anaemia there

Haemoglobins in Xenopus 519

was never more than 1 % of the total radioactivity in the fractions eluted from thecolumn in the pH 6-5 peak: in 14 anaemic animals the proportion of the total radio-activity in the pH 6-5 peak varies between 12% and 48%. The proportion ofradioactivity in the pH 7-15 Xenopus-HbF2 peak increases by up to 3 %, but the per-centage of radioactivity in the pH 7-85 Xenopus-HbA2 peak does not increase. In three

Table 3. Percentages of haemoglobin and tritium eluting in each carboxymethyl-cellulosechromatography elutionpeak when haemoglobin from adult Xenopus was chromatographedbefore, and 14, 28, and 42 days after induction of anaemia; red cells were incubated withtritiated leucine before lysis

(Top row of figures for each toad shows percentages on day 1; second row percentages on day14; third row percentages on day 28 and bottom row percentages on day 42. Figures inparentheses indicate percentages of tritium; figures not in parentheses show percentages ofhaemoglobin measured in terms of optical absorption.)

Toad(Nos. refer to Table 2)

11

1 2

14

15

17

18

Xenopus-H

< !

91

< i

< i

1

< 1

< i

< i

81

< i

< i

2

1

< i

< i

1

1

< i

< i

2

1

( < i )(16)

(3)( < i )

( < i )

(19)( < 1)

( < i )

( < i )

(14)( 2 )

( < i )

( < i )

(48)(')

( < ! )( < l )

(28)( 2 )

( l )

( < l )

(31)( 2 )

( < l )

Xenopus-HbF2

< i ( < i )

1 (3)< 1 ( < 1)< i ( < i )

< i ( < i )

1 (1)< i< i

<

<

< 1

<

<

<

( < 1)

( < i )

( < i )

(3)(< ')( < l )

( < I )

( I )( < i)

( < l )

< i ( < i )

1 (1)< i ( < i )< i ( < i )

< i ( < i )

1 (1)< 1 ( < 1)< i ( < i )

Xenopiu-HbAt

93 (94)85 (73)72 (71)75 (78)

95 (94)95 (74)75 (74)77 (80)

93 (94)88 (79)40 (42)

75 (78)

92 (91)92 (46)68 (74)72 (78)

93 (92)92 (65)93 (9i)94(9i)

95 (94)93 (64)73 (73)77 (82)

Xenopus-HbA,

5 (4)4 (6)

26 (27)23 (20)

3 (4)3 (6)

23 (24)21 (18)

5 (4)3 (4)

58 (56)23 (20)

6 (7)5 (5)

3° (24)26 (20)

5 (6)6 (6)5 (6)4 (7)

3 (4)4 (4)

25 (24)21 (16)

of the animals made anaemic (Nos. 9, 11 and 14) there is a markedly enhanced opticalabsorption peak, measured at 410 nm, in the pH 6-5 fraction. It is noticeable that theproportion of tritium activity in the pH 6-5 fractions of these 3 animals is not as highas in some toads which do not show increased optical absorption, demonstrating thatafter an appreciable amount of the new haemoglobin has been made, its synthesistends to slow down. The optical absorption data from animal 9 are representedgraphically in Fig. 3.

520 N. Maclean and R. D. Jurd

When the haemolysate from anaemic toad 9 was submitted to polyacrylamide-geldisk electrophoresis a faint haemoglobin band was seen migrating at the same speedas Xenopus-HbF1: the usual Xenopus-HbA1 and Xenopus-HbA2 bands were alsopresent (Fig. 9).

1 0

Q

d0-5

6-2I

6-5I

70 751

80PH

Fig. 4. Elution patterns obtained when 15 mg of haemoglobin from toad 11 (Table 2)were chromatographed on day 1 (O O), and 42 days after induction of anaemiawith phenylhydrazine ( • • ) .

Six toads which had been made anaemic with phenylhydrazine recovered. On the28th and 42nd days after the initial phenylhydrazine injection blood cells extractedfrom the animals were chromatographed in the usual way. The results are shown inTable 3. In 5 of the toads, after both 28 and 42 days, an abnormally high proportionof the haemoglobin (between 21 % and 58%), measured in terms both of radioactivityand optical absorption, eluted from the chromatography column in the pH 7-85Xenopus-HbA2 elution peak. The amount of haemoglobin eluting from the columnin the pH 6-5 Xenopus-HbF1 and pHy-i5 Xenopus-HbF^ fractions was not signifi-cantly greater than in healthy adult toads. The optical absorption data for animal 11,bled 42 days after the induction of anaemia, are represented in Fig. 4.

One toad, No. 15, was bled and its haemoglobin chromatographed 120 days afterthe induction of anaemia. It was found that the haemoglobin profile had regainednormality. Optical absorption measurements showed the haemoglobin to be presentin the following proportions: Xenopus-HbF1 <o-5%; Xenopns-HbF2 <o-5%;Xenopus-HbA^ 93%; Xenopus-HbA^, 4%.

Polyacrylamide-gel disk electrophoresis of the 28-day haemolysate from toad No. 14is shown in Fig. 10. Two bands can be seen, both of approximately equal strength;

Haemoglobins in Xenopus 521

one migrates at the same speed as Xenopus-HbA1 and the other at the same speed asXenopus-HbA2.

Identification of haemoglobins by agar-gel immunodiffusion

Fig. 11 shows an Ouchterlony agar-gel immunodiffusion plate on which haemoglobintaken from toad 9 (Table 2) 14 days after the induction of anaemia was tested againstspecific anU-Xenopus-HbF antiserum. Precipitin lines are visible between the anti-serum well and the wells containing the haemoglobin from the anaemic toad. No lineis detectable between the antiserum and a control well containing haemoglobinremoved from the animal before the induction of anaemia.

Fig. 12 shows a plate on which haemoglobin from toad 11 (Table 2) 42 days afterthe induction of anaemia was tested against specific anti-Xenopus-HbA antiserum.Two precipitin lines between the antiserum and the haemoglobin from the recoveringtoad are visible; only one line is detectable between the antiserum and the control wellcontaining haemoglobin removed from the toad before the induction of anaemia.

From these results it is concluded that a greatly enhanced amount of Xenopus-WbF x

was present in the blood of toad 9, 14 days after the induction of anaemia: similarpositive results for the presence of tadpole haemoglobin were obtained from toads11 and 14 (Table 2).

The 2 precipitin lines between the anti-Xenopus-HbA immunoglobulin and thehaemolysate taken from toad 11, 42 days after the induction of anaemia, suggest thepresence of a haemoglobin which normally occurs in adult toads in very small amounts.This haemoglobin cannot normally be detected on immunodiffusion plates but isdetectable by the host animal when it is injected so that antibody is raised against it.On recovery from anaemia adult Xenopus have sufficient of this haemoglobin for it tobe detectable by immunodiffusion. The haemoglobin is not a polymer because it isunaffected by the presence or absence of DTT. The chromatographic and electro-phoretic evidence strongly implies that the enhanced amount of haemoglobin producedduring recovery from anaemia is Xenopus-HbA2.

Use of fluorescent antisera

When blood smears from healthy Xenopus adults are treated with anti-Xenopus-HbA-FITC the percentage of cells fluorescing is 100%. Treatment of tadpole cellswith the same conjugate results in the number of cells fluorescing never exceeding 2%of the total. If tadpole cells are treated with anti-Xenopus-HbF-FlTC the proportionof fluorescing cells always exceeds 98%; no more than i -5% of the cells fluorescewhen adult blood cells are treated with the same conjugate.

When artificial mixtures containing known proportions of adult and tadpole redblood cells were treated with anti-Xenopi^-HbA-FITC or anti-Xenopus-HbF-FlTC,the proportion of cells fluorescing corresponded to the known proportions of adult ortadpole cells present (Fig. 13).

Blood smears from anaemic Xenopus 9 (Table 2) taken 14 days after the inductionof anaemia, and treated with anti-Xenopus-HbA-FITC showed that 100% of the cellsfluoresce, and thus 100% of the cells contain adult haemoglobins. Treatment with

522 N. Maclean and R. D. Jurd

anti-Xenopus-HbF-FITC (Fig. 14) shows that only 1-5% of the cells fluoresce, ournormal background error for the technique, despite the fact that the amount ofXenopus-HbF x eluting from the chromatography column comprises 11 % of the total.It is therefore concluded that the tadpole haemoglobin is spread throughout a largeproportion of the blood cells. Similar results were obtained from other anaemic toads.

DISCUSSION

An altered pattern of haemoglobin synthesis during anaemia is already known tooccur in man (Beaven et al. i960; Shahadi et al. 1962), sheep (Beale et al. 1966;Gabuzda, Schuman, Silver & Lewis, 1968), and goats (Huisman et al. 1967). We havereported 2 distinct changes in the haemoglobin pattern of anaemic Xenopus. Oneresembles the phenomenon in man in which a foetal haemoglobin, normally presentin the adult as less than 1 % of the total haemoglobin, appears in increased amountsfollowing blood loss. The other change observed in the blood picture of anaemicXenopus is the synthesis of increased amounts of Xenopus-HbAit a haemoglobinnormally present only in the adult, but in very small amounts. This parallels thephenomenon observed in some breeds of sheep and goats, in which the ratios of theadult haemoglobins are changed during anaemia (Beale et al. 1966; Huisman et al.1967). In Xenopus, therefore, the evidence is that 2 haemoglobins normally presentonly in very small quantities in the adult appear in increased amounts after blood loss;one of these haemoglobins is a major haemoglobin in the tadpole, the other is exclu-sively adult. Moreover, there is complete asynchrony between the 2 changes, oneappearing early and the other late after the induction of anaemia.

We have discussed elsewhere (Jurd & Maclean, 1970) the evidence for more thanone haemoglobin co-existing in the same blood cell. It seems clear that in man, bothadult and foetal haemoglobins occur together in some cells of the new-born (Hosoi,1965; Dan & Hagiwara, 1967; Schneider & Haggard, 1955; Rosenburg, 1970). Wepredict that a similar situation occurs in induced types of anaemia in man, that thefoetal haemoglobin will be found widely distributed in many blood cells together withadult haemoglobin. Our evidence from the use of fluorescent antisera with anaemicXenopus strongly suggests that the tadpole haemoglobin is present, in low concentra-tion, in many red blood cells.

Two conclusions emerge from this work. The first is that there is considerableflexibility inherent in the machinery responsible for haemoglobin synthesis. Webelieve that this should not necessarily be regarded as flexibUity operated at the geneticlevel. On the one hand, the altered pattern of synthesis is distributed throughoutmany cells, and is not achieved by a total' switch' in a few cells. On the other hand, inno case is it apparent that the synthesis of a previously absent protein is involved. Theproduction of haemoglobin C in some breeds of sheep (Beale et al. 1966) and in goats(Huisman et al. 1967) during anaemia appears to involve a change in proportions ofthe different haemoglobins, but not the appearance of a totally novel protein. So, inXenopus, we find greatly increased production after blood loss of 2 haemoglobinspreviously present only in very small amounts. It follows that the phenomenon of

Haemoglobins in Xenopus 523

altered haemoglobin synthesis may involve only a rate control mechanism; rate controlof the synthetic product at a translational level is already established as a mechanismin the synthesis of haemoglobin (Baglioni & Colombo, 1964).

The second conclusion is that the experience of a cell during erythropoiesis probablydetermines its eventual synthetic products. Since the tadpole haemoglobin seems tobe widely distributed amongst many cells during anaemia, its reappearance cannot beattributed to the entry into circulation of the descendants of stem cells committedsolely to its production. Instead it appears that the protein-synthetic pattern of manycells is altered by their exposure during erythropoiesis to plasma factors releasedduring anaemia. Evidence for this is particularly clear from the work of Gabuzda onanaemic sheep (Gabuzda et al. 1968) and the work of Thurmon et al. (1970) providesstrong evidence for identifying one plasma factor as an erythropoietin. What shouldnot be overlooked is that the process of increased rate of cell division in the erythro-poietic tissue induced by anaemia may itself lead to the altered pattern of globinsynthesis, an idea originally proposed by Baglioni (1963).

The changing pattern of haemoglobin synthesized during the metamorphosis ofamphibians (Moss & Ingram, 1968 a, b; Jurd & Maclean, 1970) also provides informa-tion on the regulation of protein synthesis. It is noteworthy that the adult type haemo-globin which appears in the blood of thyroxin-treated bullfrog tadpoles (Moss &Ingram, 19686) is located predominantly within apparently immature erythrocytes.

We are currently investigating these problems further by assaying the haemoglobinsynthesized by erythropoietic tissues cultured in vitro in the presence and absence ofvarious plasma factors.

This work was supported by grants from the Wellcome Trust and the Medical ResearchCouncil. The technical assistance of Mrs B. Streets and Mrs Y. Baynes is gratefullyacknowledged.

REFERENCES

BAGLIONI, C. (1963). Genetics and human hemoglobin chemistry. In Molecular Genetics, Part I(ed. I. H. Taylor), pp. 405-475. New York and London: Academic Press.

BAGLIONI, C. & COLOMBO, B. (1964). Control of hemoglobin synthesis. Cold Spring Harb.Symp. quant. Biol. 29, 347-356.

BEALE, D., LEHMANN, H., DRURY, A. & TUCKER, E. M. (1966). Haemoglobins of sheep. Nature,Lond. 209, 1099-1102.

BEAVEN, G. H., ELLIS, M. J. & WHITE, J. C. (i960). Studies on human foetal haemoglobin. II.Foetal haemoglobin levels in healthy children and adults, and in certain haematologicaldisorders. Br.jf. Haemat. 6, 201-222.

BOYER, S. H., HATHAWAY, P., PASCASIO, F., ORTON, C. & BORDLEY, J. (1966). Hemoglobins insheep: multiple differences in amino-acid sequences of three beta-chains and possible origins.Science, N.Y. 153, I539-I543-

CLELAND, W. W. (1964). Dithiothreitol, a new protective reagent for SH groups. Biochemistry,N. Y. 3, 480-482.

CURTAIN, C. C. (1958). Electrophoresis of fluorescent antibody. Nature, Lond. 182, 1305-1306.DAN, M. & HAGIWARA, A. (1967). Detection of two types of hemoglobin (HbA and HbF) in

single erythrocytes by fluorescent antibody technique. Expl Cell Res. 46, 596-598.GABUZDA, T. G., SCHUMAN, M. A., SILVER, R. K. & LEWIS, H. B. (1968). Erythropoietic

kinetics in sheep studied by means of induced changes in haemoglobin phenotype. J. clin.Invest. 47, 1895-1904.

524 N. Maclean and R. D. Jurd

Hosoi, T. (1965). Studies on haemoglobin F within single erythrocyte by fluorescent antibodytechnique. Expl Cell Res. 37, 680-683.

HUISMAN, T. H. J., ADAMS, H. R., DIMMOCK, M. O., EDWARDS, W. E. & WILSON, J. B. (1967).

The structure of goat hemoglobins. I. Structural studies of the beta-chains of the hemoglobinsof normal and anemic goats. J. biol. Chem. 242, 2534-2541.

JURD, R. D. & MACLEAN, N. (1969). The investigation of Xenopus laevis hemoglobins duringdevelopment by a fluorescent antibody. Experientia 25, 626-628.

JURD, R. D. & MACLEAN, N. (1970). An immunofluorescent study of the haemoglobins inmetamorphosing Xenopus laevis. J. Embryol. exp. Morph. 23, 299-309.

LUCAS, M. & JAMROZ, C. (1961). In Atlas of Avian Hematology (Agriculture Monograph 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. Comp. Biochem. Physiol. 30, 825-834.

MITCHELL, W. M. (1967). A potential source of electrophoretic artifacts in polyacrylamide gels.Biochim. biophys. Acta 147, 171-174.

Moss, B. & INGRAM, V. M. (1968a). Haemoglobin synthesis during amphibian metamorphosis.I. Chemical studies on the haemoglobins from the larval and adult stages of Rana catesbeiana.J. molec. Biol. 32, 481-492.

Moss, B. & INGRAM, V. M. (19686). Haemoglobin synthesis during amphibian metamorphosis.II. Synthesis of adult haemoglobin following thyroxine administration. J. molec. Biol. 32,493-504.

NAIRN, R. C. (ed.) (1969). In Fluorescent Protein Tracing, 3rd Edition. Edinburgh and London:Livingstone.

NIEUWKOOP, P. D. & FABER, J. (1967). Normal Table of Xenopus laevis (Daudin). Utrecht,Netherlands: Hubrecht Laboratory.

OUCHTERLONY, O. (1949). Antigen-antibody reactions in gels. Acta path, microbiol. scand.26, 507.

ROSENBURG, M. (1970). Electrophoretic analysis of hemoglobin and isozymes in individualvertebrate cells. Proc. natn. Acad. Sci. U.S.A. 67, 32-36.

RUGH, R. (1962). In Experimental Embryology, p. 50. Minneapolis, U.S.A.: Minnesota Pub-lishing Co.

SAINTE-MARIE, G. (1962). A paraffin embedding technique for studies employing immuno-fluorescence. J. Histochem. Cytochem. 10, 250-256.

SCHNEIDER, R. G. & HAGGARD, M. E. (1955). Sickling, a quantitatively delayed genetic character.Proc. Soc. exp. Biol. Med. 89, 196-199.

SHAHADI, N. T., GERALD, P. S. & DIAMOND, L. K. (1962). Alkali-resistant hemoglobin in

aplastic anemia of both acquired and congenital types. New Engl.jf. Med. 266, 117-120.SHAW, A. R. E. (1969). Developmental Aspects of Rodent Haemoglobin Syntliesis. Ph.D. Thesis:

University of Southampton.STANWORTH, D. R. (i960). A rapid method for preparing pure serum gamma-globulin. Nature,

Lond. 188, 156-157.SULLIVAN, B. & RIGGS, A. (1967). Structure, function and evolution of turtle haemoglobins.

Comp. Biochem. Physiol. 23, 437-474.THURMAN, T. F., BOYER, S. H., CROSBY, E. F., SHEPARD, M. K., NOYES, A. N. & STOHLMAN, F.

(1970). Hemoglobin switching in nonanemic sheep. III. Evidence for presumptive identitybetween the A-C factor and erythropoietin. Blood 36, 598-606.

URIEL, J. (1964). Quantitative estimation of proteins. In Immuno-Electrophoretic Analysis (ed.P. Grabar & P. Burtin), pp. 58—81. Amsterdam, London and New York: Elsevier.

WILD, A. E. (1970). Protein transmission across the rabbit foetal membranes. ,7. Embryol. exp.Morph. 24, 313-33°-

(Received 15 February 1971)

Haemoglobins in Xenopus 525

4

•fit 4

ftFigs. 5-8. For legend see next page.

526 N. Maclean and R. D. Jurd

Fig. 5. Electrophoresis in polyacrylamide gels of stage-47 tadpole (left) and 60-mmmouth-to-anus adult (right) Xenopus haemoglobin. The haemoglobin was electro-phoresed at 2-5 mA in Tris/EDTA/borate buffer at pH 8-6o for 3 h at 2 °C; arrowindicates origin. Photographed unstained.Figs. 6—8. Blood smears stained with Wright's Stain, x 1250 approx.

Fig. 6. Healthy adult Xenopus; red cell count, 731 x io6 cells/ml.Fig. 7. The same animal 14 days after the induction of anaemia. Cell count,

281 x io6 cells/ml.Fig. 8. Anaemic Xenopus with a red blood cell count of 78 x 10' cells/ml.

Figs. 9, 10. Samples electrophoresed at 2-5 mA per gel in Tris/EDTA/borate buffer atpH 8-6o for 3 h at 2 °C; arrows indicate origin. Photographed unstained.

Fig. 9. Electrophoresis in polyacrylamide gels of haemoglobins of healthy adult(left), stage-52 tadpole (centre) and anaemic toad 9 (Table 2) 14 days after the inductionof anaemia (right).

Fig. 10. Electrophoresis in polyacrylamide gels of haemoglobins of healthy adult(left), and anaemic toad 14, 28 days after the induction of anaemia.

Figs. 11, 12. Ouchterlony agar-gel diffusion plates, stained with azo-carmine by themethod of Uriel (1964) before photography.

Fig. 11. Specific anti-Xenopus-HbF immunoglobulin tested against haemoglobinsof healthy adult, stage-45 tadpole, and anaemic toad 9 (Table 2) 14 days after inductionof anaemia.

Fig. 12. Specific anti-.Xeno/>us-HbA immunoglobulin tested against haemoglobinsof healthy adult, stage-45 tadpole, and toad 11 (Tables 2, 3) 42 days after inductionof anaemia.

Haemoglobins in Xenopus 527

Tadpole

Anti-Anaemic Hbp Anaemic Anti-

anaemic f H b A Anaemil

Adult Tadpole

11 12

CE L 9

N. Maclean and R. D. Jurd

Fig. 13. An artificial mixture of adult and tadpole red cells treated with anti-Xenopus-HbF-FITC. x 1000 approx.

Fig. 14. Red cells from toad 14 (Tables 2, 3) taken 14 days after the induction ofanaemia and treated with ^nti-Xenopus-llbF-FITC. x 1000 approx.