THE TRANSITION FROM TADPOLE TO FROG HAEMOGLOBIN DURING NATURAL AMPHIBIAN METAMORPHOSIS
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J. Cell Sci. 16, 143-156 (i974) 143
Printed in Great Britain
THE TRANSITION FROM TADPOLE TO FROG
HAEMOGLOBIN DURING NATURAL
AMPHIBIAN METAMORPHOSIS
II. IMMUNOFLUORESCENCE STUDIES
J. BENBASSAT
Department of Medicine A, Hadassah University Hospital,
Jerusalem, Israel
SUMMARY
Rabbits were immunized with frog or tadpole haemoglobin purified either by chromato-
graphy or by polyacrylamide gel electrophoresis. The obtained rabbit antisera were shown to be
specific for frog or tadpole haemolysates by double diffusion, immunoprecipitation and
immunonuorescence. Indirect immunofluorescent staining of peripheral blood smears of Rana
catesbeiana tadpoles at the metamorphic climax revealed that 16 % of the red cells were stained
with antibodies against frog haemolysates, while almost all of them (98 %) were stained with
antibodies against tadpole haemolysates. These results are compatible with the possibility that
some of the circulating red cells in metamorphosing tadpoles contain both tadpole and frog
haemoglobins.
INTRODUCTION
During amphibian metamorphosis, the transition from tadpole to frog haemoglobin
is associated with the appearance of a poorly haemoglobinized, morphologically
immature microcyte in the circulation (Moss & Ingram, 1968; Vankin, Brandt &
DeWitt, 1970), which incorporates actively precursors of protein and nucleic acid
synthesis (Moss & Ingram, 1968; Benbassat, 1970; Thiel, 1970), and which contains
frog haemoglobin (DeWitt, 1968). These findings indicate that the switch from tadpole
to frog haemoglobin involves a replacement of the tadpole erythrocytes by a population
of cells containing frog haemoglobin. The question, whether these 2 populations
belong to different clones or to the same cell line is subject to controversy. Human
embryonic and foetal haemoglobins (Kleihauer, Tang & Betke, 1967) as well as
human foetal and adult haemoglobins (Betke & Kleihauer, 1958; Dan & Hagiwara,
1967; Gitlin, Sasaki & Vuopio, 1968) may coexist in the same cell. There is also
evidence that frog and tadpole haemoglobins are present simultaneously in some of
the erythrocytes of metamorphosing and adult anaemic Xenopus laevis (Jurd &
Maclean, 1970; Maclean & Jurd, 1971). On the other hand, however, ithasbeen found
that red cells of metamorphosing Rana catesbeiana tadpoles contain either tadpole or
frog haemoglobins but not both (Rosenberg, 1970; Maniatis & Ingram, 19716).
This paper reports the results of immunofluorescence studies of the content of
individual erythrocytes of Rana catesbeiana tadpoles and frogs by means of specific
rabbit antibodies against tadpole and frog haemolysates. It is shown that during144 J- Benbassat
metamorphosis some of the circulating red cells are stained by both types of antisera.
This rinding is consistent with the possibility that tadpole and frog haemoglobins are
produced by erythroid cells belonging to the same red cell line.
METHODS
The source of the animals and the methods for blood collection, preparation of labelled
haemolysates, polyacrylamide gel electrophoresis, reduction and alkylation of red cell proteins
and electrophoresis in 8 M urea have been described previously (Benbassat, 1974).
Preparation of the immunogen
Red cells oiRana catesbeiana adult frogs and premetamorphic tadpoles (total length 55-70 mm)
were washed 3 times with amphibian Ringer solution (Rugh, 1962). The visible buffy coat
was removed after each centrifugation, and the washed cells were lysed in 10 volumes of
0-0015 M MgClj. The stroma, nuclei and the ribosomes were removed by centrifugation at
105000g for 90 min at 5 °C. About 2 ml of the ribosome-free supernatant were applied to a
column of CM-cellulose (Serva) and washed through with o-oi M sodium phosphate buffer
(pH 70) until the eluate was clear and without absorbance at 280 nm. Then the haemoglobin
was eluted with 0-5 M Na2HPO4 (pH 95), and used as immunogen.
Immunization
One ml of the eluted haemoglobin solution containing about 1 mg of the immunogen was
mixed with complete Freund's adjuvant (Difco Laboratories, Detroit, Michigan), thoroughly
emulsified and injected at different sites in the footpads and skin of adult New Zealand white
rabbits. In some experiments the immunogen consisted of the major tadpole or frog haemo-
globin fractions obtained by polyacrylamide gel electrophoresis (Benbassat, 1974). The haemo-
globin bands were cutout, thoroughly homogenized as described by Maniatis& Ingram (1971a),
and injected into the rabbits; 4 injections were given at 2- to 3-week intervals. One week after
the last injection, the rabbits were bled by cardiac puncture. The sera were separated and kept
frozen until used.
Immunoprecipitation of labelled haemolysates
14
C-labelled haemolysates, prepared as detailed previously (Benbassat, 1974) from tadpole
or frog erythrocytes, were incubated at 4 °C for 48 h with antisera prepared as described in
Immunization (above). After incubation the mixtures were spun for 10 min at 5000J* in a
refrigerated centrifuge. The obtained precipitate was washed 3 times in cold Ringer's
solution, dissolved in formic acid and mounted on planchets for determination of radioactivity.
In other experiments, the immunoprecipitates were dissolved in deionized 8 M urea, reduced,
alkylated and electrophoresed on polyacrylamide gels prepared in alkaline 8 M urea. All the
immune precipitations were carried out at antibody excess as judged by standard immune
precipitation curves (Fig. 6).
Staining of tadpole and frog blood cells with fluorescent antibodies
The indirect method of Weller & Coons (1954) was used. Air-dried smears were prepared
from washed blood cells of early premetamorphic tadpoles, animals at the metamorphic climax,
and adult frogs, and left at —20 °C until used. The smears were covered with antisera against
tadpole or frog haemoglobins and incubated for 30 min at 37 °C with continuous shaking. Serum
of non-immunized rabbits was used in control experiments. The coated smears were then
washed 3 times with amphibian Ringer's solution and covered for 30 min with Fluorescein
Conjugated Goat anti (Rabbit IgG) globulin (Microbiological Associates, Inc., Bethesda)
diluted 1:60 in amphibian Ringer's solution. They were then washed again, dried and examined
in u.v. illumination under glycerol buffered to pH 70 with sodium phosphate. Cell counts were
performed from photographs of the smears.Synthesis of red cell protein during metamorphosis. II 145
RESULTS
Determination of the specificity of the antisera
The characterization of the antisera was performed by double diffusion on agar,
immunoprecipitation and characterization of the precipitated material by polyacryl-
amide gel electrophoresis in 8 M urea, and immunofluorescence.
Double diffusion on agar. Agar-gel diffusion plates were used. The reagents con-
sisted of undiluted antisera and of tadpole or frog haemoglobin solutions. The haemo-
globin solutions consisted of either unfractionated haemolysates or purified major
haemoglobin fractions after separation by polyacrylamide gel electrophoresis.
Sera of rabbits immunized with tadpole haemolysates purified by chromatography
produced one precipitin line when tested against haemolysates of prometamorphic
tadpoles, animals at the metamorphic climax or against the purified major tadpole
haemoglobin (T-J; the same antisera did not react when tested against frog or mouse
haemolysates (Fig. 4). Sera of rabbits immunized with the purified major tadpole
haemoglobin fraction (Tj), produced one precipitation line when tested against
unfractionated tadpole haemolysates, purified major tadpole haemoglobin (Tx) and
the purified minor tadpole haemoglobin fraction (T2). The type of reaction obtained
suggested an identity between the antigens (Fig. 5).
Rabbit antisera against frog haemoglobin reacted with unpurified haemolysates of
animals during the metamorphic climax and of adult frogs, as well as with the purified
frog haemoglobin fractions (Fx and F3). One precipitation line was observed in all
cases. The same antisera did not react when tested against tadpole or mouse haemo-
globins (Figs. 6, 7).
These results confirm the findings by Maniatis & Ingram (1971a) and by Jurd &
Maclean (1970), that frog and tadpole haemoglobins are relatively strong immunogens
for the rabbit. The absence of any detectable cross-reaction between these haemo-
globins is consistent with the findings by Baglioni & Sparks (1963), Moss & Ingram
(1968) and Aggarwal & Riggs (1969), that the transition from tadpole to frog haemo-
globin involves the beginning of synthesis of a completely different set of polypeptide
chains. The major tadpole haemoglobin (Tj) cross-reacted with the minor tadpole
haemoglobin (T2), while the major frog haemoglobin (F3) cross-reacted with the minor
one (Fj); this could indicate that these haemoglobin fractions share one or more
common subunits. Haemolysates of animals at the metamorphic climax reacted with
antisera against either tadpole or frog haemoglobin; this is in agreement with the
finding that tadpoles at this stage of metamorphosis contain both types of haemoglobin
(Fig. 8).
Immunoprecipitation. The amount of antiserum needed to provide an excess of
antibodies in the reaction mixtures was determined by standard immune precipitation
curves. In these experiments a constant amount (3-4 /tg) of 14C-labelled haemoglobin
was titrated with increasing volumes (5-500 /A) of antiserum. The relative amount of
antiserum which precipitated the maximal amount of radioactivity was used in further
experiments (Fig. 1).
In three different experiments, 76, 83 and 89% of the total radioactivity of
IO C EL 16146 J. Benbassat
100 -
20
200 300 400 500 900 1000
it\ antiserum added
Fig. 1. Immunoprecipitation of tadpole (solid line) and frog (dashed line) 14C-
labelled haemolysates by rabbit anti-tadpole hb and anti-frog hb antisera, respectively.
The indicated amount of antiserum was incubated for 48 h at 4 CC with a constant
amount of 14C-labelled haemolysate containing 3 fig haemoglobin. After incubation
the immunoprecipitate was washed, dissolved in formic acid and the radioactivity
determined in a Nuclear Chicago Gas Flow Counter.
Table 1. Immunoprecipitation of tadpole and frog labelled haemolysates
cpm precipitated by
TT
TCA-
globin, precipitable Control
Antigen /*g cpm Anti-T* Anti-F* serum*
Unfractionated 32 640 (100%) 356(56%) 14 (3 %) 10(2%)
tadpole haemo-
lysate
Purified major 17 137(100%) 82(60%) 6(4%) 7 (5 %)
tadpole haemo-
globin (Tj)
Unfractionated 4i 318(100%) 21(6%) 240 (76 %) 24(7%)
frog haemo-
lysate
Purified major 2-O 112(100%) 5(4%) 79(7O%) 3 (3 %)
frog haemo-
globin (F3)
* Anti-T and anri-F refer to non-absorbed rabbit antisera against tadpole or frog haemo-
lysates purified by chromatography on CM-cellulose. The control consisted of the serum of
a non-immunized rabbit.Synthesis of red cell protein during metamorphosis. II 147 unfractionated frog haemolysates could be precipitated by antisera against frog haemo- globin. The highest amounts of radioactivity, which could be precipitated by antisera against tadpole haemoglobin from unfractionated haemolysates were 56, 53 and 38%. Similar results were obtained when the purified major haemoglobin fractions were used as antigens in the reaction mixture. The amounts of tadpole haemoglobin pre- cipitated by antibodies against frog haemoglobin and vice versa did not exceed those precipitated by sera of non-immunized rabbits. The results of a representative experiment are given in Table 1. In order to confirm that the antisera reacted with haemoglobin and not with any other red cell protein, which might have contaminated the immunogen, the immuno- precipitated material was characterized by polyacrylamide gel electrophoresis. For this purpose, immunoprecipitated haemolysates were dissolved in 8 M urea, in order to dissociate between the antibody and the labelled antigen. Possible S—S bonds were reduced by the addition of mercaptoethanol and blocked by amidation. Then the dissociated protein subunits were resolved by electrophoresis on polyacrylamide gels prepared in alkaline 8 M urea. After electrophoresis the gels were sliced and the radio- activity was determined in each of the gel fractions. The radioactive profiles of the immunoprecipitated material were compared to those of the reduced and alkylated purified major haemoglobin components. As shown in Figs. 2 and 3, the purified major haemoglobin components of both tadpoles and frogs were resolved into one major and several smaller peaks of radioactivity (Figs. 2C and 3 c). The major peak consisted of both globin chains, which could not be separated electrophoretically under these conditions. The smaller peaks represented probably small amounts of con- taminating red cell (haemoglobin or non-haemoglobin) protein. The pattern of resolution of the immunoprecipitated protein of frog haemolysates was almost identical with that of reduced and alkylated globin prepared from the purified major frog haemo- globin fraction (Fig. 2). The immunoprecipitated protein of tadpole haemolysates resolved into several peaks of radioactivity, one of which coelectrophoresed with the major tadpole reduced and alkylated globin (Fig. 3). The nature of the remaining peaks of immunoprecipitated radioactivity remains unclear: they could be globin subunits of the minor tadpole haemoglobins, or non-haemoglobin red cell proteins. Similar results were obtained whether the immunogen employed in the preparation of the antisera was purified by chromatography or by polyacrylamide gel electro- phoresis. Immunofiuorescence. The antisera against frog or tadpole haemoglobins character- ized in the two preceding sections, were used for immunofluorescent staining after cross-absorption with tadpole and frog lysed red cells, respectively. The degree of specificity of the antisera employed is shown in Figs. 9-12 and in Table 2. When peri- pheral blood smears of premetamorphic tadpoles were stained with antisera against tadpole haemoglobin, 98-7 % of the cells showed a brightfluorescence,which was evenly distributed throughout the cytoplasm; the nucleus was not stained (Fig. 9). Only occasional cells (less than 2 %) were stained when tadpole blood smears were treated with antisera against frog haemoglobin (Fig. 10), normal rabbit serum or antiserum against tadpole haemoglobin, which had been absorbed with tadpole haemoglobin.
148 J. Benbassat
When frog peripheral blood cells were stained with antisera against frog haemo-
globin, 99-2 % of the cell showed a bright fluorescence, which was most prominent
around the nucleus; the remaining parts of the cytoplasm and the nucleus appeared
unevenly stained (Fig. 11). Less than 3 % of the cells were stained when frog blood
300 400
200 200
100
10 20 30 40
0 10 20 30
150
600 |- k
100
400 -
E
-A
Q.
50
200
1 i i i
10 20 30 40
) 10 20 30 40
300
c
200 - 200
li
100 -
/
ft I
100
10 20 30 40 10 20 30 40
Fraction no. Fraction no.
Fig. 2 Fig. 3
I4
Figs. 2, 3. Electrophoresis of C-labelled reduced and alkylated globin in alkaline 8 M
urea. Immunoprecipitation, reduction and alkylation of the immune precipitates,
electrophoresis and fractionation were carried out as previously described (Benbassat,
)
Fig. 2. A, untreated frog haemolysates; B, immune precipitates of frog haemolysates
treated with antibodies against frog haemoglobin; c, purified major frog haemo-
globin (F3). Origin to the left, migration towards the anode.
Fig. 3. A, untreated tadpole haemolysates; B, immune precipitates of tadpole haemo-
lysates treated with antibodies against tadpole haemoglobin; c, purified major tadpole
haemoglobin (T^). Origin to the left, migration towards the anode.
smears were treated with antisera against tadpole haemoglobin (Fig. 12) or with
control sera.
Imtnunofluorescent staining of peripheral blood cells of animals at the metamorphic climax
The results of the immunofluorescent staining of the peripheral blood cells of animalsSynthesis of red cell protein during metamorphosis. II 149
at the metamorphic climax are given in Table 2 and Figs. 13 and 14. Of these cells,
98'1 % exhibited a bright fluorescence after treatment with antisera against tadpole
haemoglobin. The pattern offluorescenceof the cells was identical with that of tadpole
red cells stained with antisera against tadpole haemoglobin (Fig. 13). In contrast to
the homogeneity of the smears stained with antisera against tadpole haemoglobin,
smears treated with antibodies against frog haemoglobin exhibited a mosaic-like
pattern of 16% positively stained cells (Fig. 14).
Table 2. Staining of frog tadpole peripheral blood smears with
fluorescent antibodies
Total no. of % cells showing
Stage Antiserum used* cells counted fluorescence
Premetamorphosis Anti-tadpole Hb 304 987
Premetamorphosis Anti-frog Hb 228 17
Metamorphic climax Anti-tadpole Hb 527 98-1
Metamorphic climax Anti-frog Hb 231 160
Adult Anti-tadpole Hb 287 24
Adult Anti-frog Hb 240 99-2
• The antisera were prepared by immunizing rabbits with frog or tadpole haemolysates
purified by chromatography on CM-cellulose.
DISCUSSION
The degree of specificity of the antisera employed was determined by double
diffusion on agar, immunoprecipitation of labelled haemolysates and fluorescent
staining of peripheral blood smears. The amounts of labelled tadpole and frog haemo-
globin, precipitated by antisera against frog and tadpole haemoglobin, respectively,
did not exceed those precipitated by control sera of non-immunized rabbits. Less than
3 % of the tadpole or frog blood cells were stained with antisera against frog or tad-
pole haemoglobin, respectively, while more than 98 % of the cells in tadpole and frog
peripheral blood smears were stained with antibodies against the haemoglobin of the
same developmental stage. Therefore, the finding that 16% of the peripheral blood
cells of metamorphosing animals were stained with antisera against frog haemoglobin,
while almost all of them were stained with antisera against tadpole haemolysates,
suggests the coexistence of both types of haemoglobin in some of the erythrocytes.
These results conform with the evidence presented by Shukuya (1966) and by Jurd
& Maclean (1970) that some of the erythroid cells of metamorphosing tadpoles con-
tain both tadpole and frog haemoglobins. The presented observations are consistent
also with the coexistence of human embryonic and foetal haemoglobins (Kleihauer
et al. 1967) and of human foetal and adult haemoglobins (Dan & Hagiwara, 1967)
within the same erythrocyte. They are at variance, however, with the conclusions of
Rosenberg (1970) and Maniatis & Ingram (19716), that during metamorphosis the
erythroid cells carry either frog or tadpole haemoglobin but not both.
The reason for this inconsistency is uncertain. One possibility could be, that the150 J.Benbassat antisera against tadpole and frog haemoglobins employed by Maniatis & Ingram (1971 a) may have had a higher degree of monospecificity than those used in the present investigation. As shown in Fig. 3, treatment of tadpole haemolysates with antisera against tadpole haemoglobin resulted in the precipitation of several protein subunits in addition to the main tadpole globin; this additional immunoprecipitate could have consisted either of minor tadpole globin fractions or of non-haemoglobin red cell proteins. It is possible, therefore, that the antisera against tadpole haemoglobin employed in the present study were not monospecific. Thus, the antigen involved in the immunofluorescent staining of the red cells with antisera against tadpole haemo- globin, could have been a non-haemoglobin protein, present in the red cells of pre- metamorphic and metamorphosing tadpoles, but not in mature frog erythrocytes. Alternatively, the discrepancy in results could be explained by differences in the sensitivity of the methods employed for immunofluorescent staining. The indirect 'sandwich' method for immunofluorescent staining employed in this paper is more sensitive than the direct 'double label' method used by Maniatis & Ingram (1971). Conceivably, small amounts of tadpole haemoglobin in a cell containing principally frog haemoglobin may have escaped detection by direct immunofluorescent staining, particularly when applied to cells already pretreated with fluorescent antibodies against frog haemoglobin. I am grateful to Dr I. M. London for advice and encouragement and to Dr S. Baum for help in the staining with fluorescent antibodies. This investigation was supported by a Public Health Service International Research Fellowship (FOTW5-1224) while the author was a post- doctoral fellow in the Department of Medicine, Albert Einstein College of Medicine, New York, and by a grant from the Joint Research Fund of the Hebrew University and Hadassah. REFERENCES AGGARWAL, S. J. & RIGGS, A. (1969). The hemoglobins of the bullfrog, Rana catesbeiana. I. Purification, amino acid composition and oxygen equilibria.^, biol. Chem. 244, 2372-2383. BAGLIONI, C. & SPARKS, C. E. (1963). A study of hemoglobin differentiation in Rana catesbeiana. Devi Biol. 8, 272-285. BENBASSAT, J. (1970). Erythroid cell development during natural amphibian metamorphosis. Devi Biol. 21, 557-583. BENBASSAT, J. (1974). The transition from tadpole to frog haemoglobin during natural amphibian metamorphosis. I. Protein synthesis by peripheral blood cells in vitro. J. Cell Sci. 15,347-357. BETKE, K. & KLEIHAUER, E. (1958). Fetaler und blebender Blutfarbstoff in Erythrozyten und Erythroblasten. Blut 4, 241. 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. DEWITT, W. (1968). Microcytes response to thyroxine administration. J. molec. Biol. 32, 502-504. GITLIN, D., SASAKI, T . & VUOPIO, P. (1968). Immunochemical quantitation of proteins in single cells. Blood 32, 796. JURD, R. D. & MACLEAN, N. (1970). An immuno-fluorescent study of the haemoglobins in metamorphosing Xenopus laevis. J. Embryol. exp. Morph. 23, 299—309. KLEIHAUER, E., TANG, T . E. & BETKE, K. (1967). Die intrazellnare Verteilung von embryo- nalem Hamoglobin in roten Blutzellen menschlicher Embryonen. Ada haemat. 38, 264. MACLEAN, N. & JURD, R. D. (1971). The haemoglobins of healthy and anaemic Xenopus laevis. J. CeU Sci. 9, 509-528.
Synthesis of red cell protein during metamorphosis. II 151
MANIATIS, G. M. & INGRAM, V. M. (1971a). Erythropoiesis during amphibian metamorphosis.
II. Immunochemical study of larval and adult hemoglobins of Rana catesbeiana. J. Cell Biol.
49- 380-389.
MANIATIS, G. M. & INGRAM, V. M. (19716). Erythropoiesis during amphibian metamorphosis.
III. Immunochemical detection of tadpole and frog hemoglobins in single erythrocytes.
J. Cell Biol. 49, 390-404.
Moss, B. & INGRAM, V. M. (1968). Hemoglobin synthesis during amphibian metamorphosis.
I. Chemical studies on the hemoglobins from larval and adult stages of R. catesbeiana.
J. molec. Biol. 32, 481-492.
ROSENBERG, M. (1970). Electrophoretic analysis of hemoglobin and isozymes in individual
vertebrate cells. Proc. natn. Acad. Set. U.S.A. 67, 32-36.
RUGH, R. (1962). Experimental Embryology. Minneapolis: Burgess.
SHUKUVA, R. (1966). As cited by Frieden, E. in Metamorphosis (ed. W. Etkin & L. I. Gilbert).
New York: Appleton-Cenrury-Crofte.
THIEL, E. (1970). Red blood cell replacement during the transition from tadpole to frog
hemoglobin in Rana catesbeiana. Comp. Biochem. Physiol. 33, 717-720.
VANKIN, G. L., BRANDT, E. M. & DEWITT, W. (1970). Fine structure of erythroid cells during
thyroxjn-induced metamorphosis of bullfrog tadpoles. Am. Zool. 10, 321.
WELLER, T. H. & COONS, A. H. (1954). Fluorescent antibody studies with agents of Varicella
and Herpes Zoster propagated in vitro. Proc. Soc. exp. Biol. Med. 86, 789-794.
{Received 15 February 1974)is* J. Benbassat
Figs. 4-7. Double diffusion in 1 % agarose. The reagents consisted of undiluted rabbit
antisera (central wells) and haemoglobin solutions in concentrations varying between
O-O2-O-3 mg/ml (peripheral wells).
Central wells: Figs. 4 and 5, rabbit anti-tadpole hb serum; Figs. 6 and 7, rabbit anti-
frog hb serum; the antisera in Figs. 4 and 6 were prepared by immunization of rabbits
with haemolysates purified by chromatography on CM-cellulose; the immunogen used
for the preparation of the antisera in central wells Figs. 5 and 7 consisted of the purified
major hb fraction.
Peripheral wells: T, unfractionated haemolysate of a prometamorphic tadpole;
Tj, purified major tadpole hb fraction; T2, purified minor tadpole hb fraction (see
Fig. 8); E, haemolysate of an animal at emergence of the front legs (beginning of
metamorphic climax); C, haemolysate of an animal at the end of the metamorphic
climax; F, haemolysate of an adult frog; Flt purified minor frog hb fraction; F3, purified
major frog hb fraction (see Fig. 8); M, mouse hb.Synthesis of red cell protein during metamorphosis. II
8
A B E C F
Fig. 8. Polyacrylamide gel electrophoresis (discontinuous Tris-glycine/Tris-HCl alka-
line system) of haemolysates of R. catesbeiana tadpoles and frogs. Unstained gels.
A, early premetamorphic tadpole (total length, 35mm, estimated age 2-3 weeks);
B, prometamorphic tadpole (total length, 85 mm, hind legs 2 mm, estimated age, at
least 1 year); E, tadpole at the beginning of the metamorphic climax (total length,
98 mm, hind legs 45 mm, 2 days after emergence of front legs); C, animal at the end
of metamorphic climax (tail almost completely reabsorbed, 12 days after emergence
of front legs); F, adult frog.154 J. Benbassat
Figs. 9-12. R. catesbeiana tadpoles and frogs. Peripheral blood smears treated with
fluorescent antibodies, x 750.
Fig. 9. Tadpole blood cells stained with antibodies against tadpole haemoglobin.
Fig. 10. Tadpole blood cells stained with antibodies against frog haemoglobin. The
fluorescent round body is probably either a white cell or an artifact.Synthesis of red cell protein during metamorphosis. II Fig. I I . Frog blood cells stained with antibodies against frog haemoglobin. Fig. 12. Frog blood cells stained with antibodies against tadpole haemoglobin.
156 J. Benbassat
Figs. 13, 14. R. catesbeiana metamorphosing tadpoles. Peripheral blood smears
treated with fluorescent antibodies, x 750.
Fig. 13. Stained with antisera against tadpole haemoglobin.
Fig. 14. Stained with antisera against frog haemoglobin.You can also read
