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/. Embryol. exp. Morph. Vol. 60, pp. 389-404, 1980
Printed in Great Britain © Company of Biologists Limited 1980
389
The haemoglobins of developing duck
embryos
By C. CIROTTO,11. ARANGI AND F. PANARA
From the Institute of Histology and Embryology, Perugia University
SUMMARY
Three haemoglobins were isolated by ion-exchange chromatographyfrom the haemolysates
of embryonic duck erythrocytes up to 8 days of development. The component globins
were characterized both by electrophoresis in dissociating conditions and by finger-printing
analysis. The major haemoglobin fraction El appears to be an embryonic tetramer since
its constituent globins are different from all the others synthesized during embryonic and
adult life. The two minor fractions E2 and E3 show a-type subunits that are very similar
to those of the two adult haemoglobins Al and A2 respectively. They are present all through
embryonic life, as demonstrated by chromatographic analysis. For these reasons they have
been considered foetal.
The two haemoglobins typical of the adult animal are found in the red cells of the embryo
from 8 days of incubation. Their relative amounts change continuously during embryonic
development and reach the adult value after hatching.
INTRODUCTION
Erythroid cells have been considered very helpful models in investigating
mechanisms of differentiation. The main products of their biosynthetic activities
are haemoglobin molecules which can be easily isolated and characterized.
The correlation existing between haemoglobin types and red cell differentiation
has been widely demonstrated (Ingram, 1963; Manwell, Baker & Betz, 1966;
Paul, 1976). Haemoglobin types are also closely related to embryonic developmental stages (Kitchen & Brett, 1974; Bruns & Ingram, 1973). It is important
to obtain further evidence characterizing the haemoglobins synthesized by
the embryonic red cells at various stages of development.
Birds are suitable biological models because their embryos are completely
independent of the mother and a large number of embryos at the same
developmental stage can be obtained easily. Chick embryos are the most widely
investigated (Manwell et al 1966; Schalekamp, Schalekamp, Van Goor &
Slingerland, 1972; Bruns & Ingram, 1973; Brown & Ingram, 1974; Cirotto,
Scotto di Telia & Geraci, 1975; Schalekamp, Van Goor, Slingerland & Van
Noort, 1976; Cirotto & Geraci, 1977). Most authors agree that the red cells
of early chick embryos synthesize four haemoglobin types. Two of these are
1
Author's address: Istituto di Istologia, ed Embriologia, Via Elce di Sotto, 06100
Perugia, Italy.
390
C. CIROTTO, I. ARANGI AND F. PANARA
embryonic haemoglobins and the other two are foetal. At the seventh day of
incubation the two adult haemoglobins appear in the haemolysate. Moreover,
the relations between haemoglobin types and cell population are fairly clear:
the three haemoglobin couples are synthesized in three different cell lines
(Cirotto & Geraci, 1977; Shimizu, 1976; Cirotto, Panara & Geraci, 1977;
Mahoney, Hyer & Chan, 1977; Chapman & Tobin, 1979).
Unfortunately such experimental evidence is available only for the chick.
It is interesting however to extend similar investigations to other bird species.
The investigation of embryonic haemoglobins is helpful in the study of ontogenetic processes and phylogenetic problems. For this reason we have studied
the adult and embryonic haemoglobins of ducks, which are birds phylogenetically very different from chicks.
Since Peking ducks are widely used as laboratory animals, some reports are
found in the literature on the isolation and characterization of the adult
haemoglobins (Vandecasserie, Paul, Schnek & Leonis, 1973; Gander, Luppis,
Stewart & Scherrer, 1972). On the other hand, few studies have been carried
out on the embryonic haemoglobins (Borgese & Bertles, 1965; Stratil & Valenta,
1976;Borgese&Nagel, 1977).
In this work we report the characterization of the duck embryo haemoglobins
and the results obtained are discussed with reference to the data concerning
the chick.
MATERIALS AND METHODS
Haemoglobin preparation
Peking duck embryos at various stages of development were obtained from
different poultry farms. The blood was obtained from the embryos and collected
in buffered isotonic solution as described by D'Amelio & Costantino-Ceccarini
(1969). The erythrocytes were washed and lysed as described in a previous
paper (Cirotto et ah, 1975). The clear haemolysate was dialysed on a column
of Sephadex G 25 fine equilibrated with 10 mM potassium phosphate at pH 6-2
prior to the chromatographic analysis. Haemoglobins were isolated in a pure
form by chromatography on a CM cellulose column (Whatman CM 52), at
4°C, with the elution method previously described (Cirotto et al. 1975).
Quantitative evaluation of the chromatographic fractions was obtained by
cutting and weighing each peak of the tracings. Haemoglobin concentration
was determined spectrophotometrically using e = 11-lxl 03 at 540 nm for the
cyanmet derivative (Antonini, 1965).
Preparation and carboxymethylation of the globins
Haemoglobin molecules were depleted of the haems by the method of Rossi
Fanelli, Antonini & Caputo (1958). Sulphydryl groups were blocked by reaction
with a 10-fold molar excess of iodoacetamide in 8 M urea, 50 mM potassium
phosphate pH 7-5.
The haemoglobins of developing duck embryos
391
Fractionation of the globin chains
Analytical separation of the globins was obtained by electrophoresis on
polyacrylamide gels. The following solutions were prepared: (a) 45 g acrylamide, 0-3 g bisacrylamide in 100 ml of water; {b) 18 ml of 99 % formic acid,
3-93 ml of 0-6 % silver nitrate in 100 ml water; (c) 0-75 g ammonium persulfate
in 100 ml water. Equal volumes of these solutions were mixed and left standing
at 20 °C. The polymerization time was 2 h. The electrode solutions were 1 -4 M
formic acid. 60 jug of globins in 20 ju,\ of 1-4 M formic acid containing 0-5 M urea
were loaded on each gel. The gels were submitted to a pre-electrophoretic run at
4 mA/tube using methyl-green as tracing dye. Electrophoresis was carried out
at 4 mA/tube. Protein bands were stained with the direct method of Malik &
Berrie (1972) and the relative amounts of the globin bands were determined by
scanning the gels at 650 nm on a Gilford spectrophotometer. The percentages
of the globin types were obtained from the tracings as described for the
haemoglobin chromatographic patterns. A quantitative separation of the
globin chains was performed by chromatography on a CM cellulose column
according to Gander et al. (1972). The pooled chromatographic fractions were
lyophilized after dialysis against 1 % formic acid.
Fingerprint analysis
Tryptic digestion of the isolated and carboxymethylated globins was carried
out according to Hunt, Hunter & Munro (1969). Fingerprint analysis was
carried out by the method of Ingram (1958) for the electrophoresis and by the
method of Waley & Watson (1953) for the chromatography. A typical experiment has already been described (Cirotto et al. 1975). Tryptic maps were first
stained with the ninhydrin reagent (Clegg, Naughton & Weatherall, 1966)
and then with the specific stainings for histidine, tyrosine, arginine and
tryptophane as described by Lehmann & Huntsman (1974).
RESULTS
Haemoglobin separation
In Fig. 1 are shown the chromatographic patterns of total lysates from duck
embryos at 5 and 8 days of development, from hatched ducklings and from
the adult. Two distinct patterns of haemoglobins are observed: one, typical of
the early embryo is composed of the three fractions labelled El, E2 and E3
according to their order of elution from the column, the other, found in the
adult, is composed of two haemoglobins Al and A2. Only the haemoglobins
El, E2 and E3 are detected in the erythrocyte haemolysates up to 8 days of
incubation. The two adult haemoglobins Al and A2 appear from 8 days of
development on. It is evident from the patterns of Fig. 1 that the haemoglobin
E2 of the early embryo is chromatographically indistinguishable from the minor
adult haemoglobin Al. The two tetramers, however, appear to be different
by electrophoretic analysis of their globins (see Fig. 2). Haemoglobin E2
392
C. CIROTTO, I. ARANGI AND F. PANARA
Hatching
5 days
150
200
Fraction number
50
150
200
Fig. 1. Elution patterns from CM cellulose columns in phosphate buffer of the total
haemoglobins of 5- and 8-day-old duck embryos, of hatched ducklings and of the
adult.
A1 E2 E1
E3 A2
Fig. 2. Polyacrylamide disc gel electrophoresis in formic acid of the globins of the
individual haemoglobin fractions isolated by column chromatography as shown in
Fig. 1.
The haemoglobins of developing duck embryos
393
obtained from 5-day-old embryos differs at least in one globin from the
haemoglobin Al of the adult duck. Therefore, the inability of the chromatographic system to discriminate between these two haemoglobins makes the
second peak heterogeneous from 8 days of development, when the adult
haemoglobins appear, to hatching time. For this reason the globin compositions
of the embryonic haemoglobins have been determined on the haemolysates of
5-day-old embryos and the characterization of the adult haemoglobins on
the haemolysates of 1-year-old ducks.
In all the chromatographic profiles, in addition to the haemoglobin fractions
described above, some materials appear to be eluted with the void volume of
the column. Electrophoretic analysis, in denaturing conditions, of the molecules
contained in this peak, demonstrated the presence of the first haemoglobin
eluted from the column and other non-haem proteins.
Isolation of the globin chains
The electrophoretic analysis of the globins obtained from each chromatographic fraction shown in Fig. 1 is presented in Fig. 2. It is evident that each
tetramer differs from the others in at least one globin. The three haemoglobins
typical of the early embryo seem to share the fast migrating globin. Al and A2
tetramers also show identical cathodic chains. The cathodic subunits of the
haemoglobins E2 and Al, as well as those of the haemoglobins E3 and A2,
appear clearly similar by electrophoretic mobility. The presence of only two
protein bands in each gel confirms the purity of each chromatographic fraction.
Three globin bands were observed by electrophoresis of the proteins contained
in the second chromatographic peak from haemolysates of embryos older than
8 days. The globins correspond to those of the haemoglobins E2 and Al,
demonstrating that they were eluted at the same time. A more detailed
characterization of the structure of each globin was obtained by fingerprint
analysis of tryptic peptides. For this purpose, the globin chains were isolated
by chromatography on CM cellulose column in dissociating conditions as
described in 'Materials and Methods'. The chromatographic separation of the
chains of the haemoglobins El, E2 and E3 is shown in Fig. 3, while the patterns
of the globins from haemoglobins Al and A2 are shown in Fig. 4. In all cases
two peaks are eluted which differ in absorbance, but have almost equal dry
weights. The polypeptide chains present in each chromatographic fraction were
identified by gel electrophoresis in formic acid. A typical electrophoretic
pattern, shown in Fig. 4, reveals that each chromatographic peak is made of
only one type of chain. Possible cross contamination does not exceed 10 % of
the total proteins. The molecules eluted in the band corresponding to the void
volume do not give the colour reaction of the proteins.
Fingerprint analysis
The carboxymethylated globins were digested with trypsin and the peptides
analysed by fingerprint. In Fig. 5 are shown the peptide maps of the more
394
C. CIROTTO, I. ARANGI AND F. PANARA
El
0-8 -
04
AJ
LA
0-8
E2
04
E3
0-8
04
20
40
Fraction number
60
Fig. 3. Elution patterns from CM cellulose columns of the chains constituting the
haemoglobins El, E2 and E3.
cathodic subunits of Al and A2. The maps of the two chains show large
similarities in the relative position and in the specific staining of all the spots
with the exception of the dotted peptide in A2 map. A ninhydrin-stained spot
in an identical position appeared also in Al map, when the chain was pretreated
with performic acid. This finding suggests that the peptide typical of A2 is an
oxidized form.
Since the two maps of Fig. 5 are very similar to those of the /? globins of
adult chicken already reported (Cirotto, Petris, Panara & Manelli, 1974) it
may be concluded that the cathodic subunits of the adult duck haemoglobins
The haemoglobins of developing duck embryos
395
Al
Fraction number
Fig. 4. (A) Elution patterns from CM cellulose columns of the chains constituting
the haemoglobins Al and A2. (B) Disc gel electrophoresis in formic acid of globins
of A2 haemoglobin isolated by chromatography on CM cellulose column.
are /?-like chains. Therefore, the structural and functional differences between
the two adult duck haemoglobins arise essentially from the a chains.
Figure 6 shows the fingerprints of the anodic subunits of E3 and A2 tetramers.
The number, the relative position and the specific staining of the peptides are
identical in the two maps, thus suggesting that the identity of the electrophoretic
mobility (see Fig. 2) is due to a very similar primary structure. These two chains
appear to be of the cc type because their peptide maps are very similar to that
of the a chain of the major adult chicken haemoglobin A2 (Cirotto et al. 1974).
Very similar peptide maps were also obtained for the anodic globins of the
E2 and Al tetramers (Fig. 7). Comparison of these peptide maps with those
of the minor chicken haemoglobin chains, suggests that the former are of the
a type (Cirotto et al. 1974).
In addition to the characteristics described above, the electrophoretic patterns
of Fig. 2 show also identical mobilities for the /?-like chains of the haemoglobins
E2 and E3 and for the cathodic chain of the haemoglobin El. Fingerprints
of these globins show very similar patterns of trypdc peptides for the /?-like
396
C. CIROTTO, I. ARANGI AND F. PANARA
A
Fig. 5. (A) Fingerprint photographs and (B) fingerprint charts of the cathodic subunits of Al and A2 haemoglobins showing those peptides which have specific
staining reactions for histidine ([!), tyrosine ( • ) , arginine (@), tryptophan (HD).
The haemoglobins of developing duck embryos
397
B
0A1
o
oo
0
0
+•
Electrophoresis
0A2
0
oo© 0
•
Electrophoresis
globins of E2 and E3 (see Fig. 8). On the contrary, the /?-like chain of El differs
from the others in the relative position of at least two peptides (Fig. 9). The
difference is further emphasized by the distribution of spots positive for the
specific stainings. Above all it is interesting to note that tryptophan is absent
in the soluble peptides of the /Mike chains of E2 and E3, whereas it is present
in a nearly neutral peptide of the /?-like chain of El. The peptide map of the a-like
chain of El, shown in Fig. 9, confirms the electrophoretic data on the structural
difference of this chain from the other a-like globins of the duck embryo.
Globin synthesis during embryonic life
The results of chromatographic, electrophoretic and peptide mapping
analyses demonstrate the existence of an embryonic haemoglobin El, typical
of the early stages of development, which is different in both the constituent
chains from all the other embryonic and adult tetramers. This molecule is
detected in the haemolysates till the sixteenth day of development. The two
26
EMB 60
398
C. CIROTTO, I. ARANGI AND F. PANARA
0
o
r
a"E3
O 0
o
o
Electrophoresis
0
a" A2
0
o
0
Electrophoresis
Fig. 6. Tracings of the tryptic maps of the anodic subunits of E3 and A2 haemoglobins showing those peptides which have specific staining reactions for histidine
(ID), tyrosine ( • ) , arginine (j=j), tryptophan (ffl]).Tryptophan residues are absent in
both samples. Uncertainties in the specific staining are indicated by asterisks.
haemoglobins E2 and E3 found in the early embryo persist till hatching time.
Both E2 and E3 haemoglobins show a globin composition typical of mammalian
foetal haemoglobins, since their a-like subunits are quite similar, and possibly
identical, to the two a-chains of the adult haemoglobins Al and A2. At 8 days
of incubation the two adult haemoglobins Al and A2 appear in the haemolysate.
If the chains showing quite similar peptide maps are assumed to be identical
then six different globin chains appear to be synthesized in duck erythrocytes
during embryonic life. In accordance with the labelling adopted for mammalian
globins, the greek letters a and /? are used for the adult chains, the /?-like globin
of the foetal tetramers E2 and E3 is labelled y, the constituent globins of the
•embryonic haemoglobin El are labelled £ and e. On this basis globin composition
The haemoglobins of developing duck embryos
o
0
I
0
399
a'E2
0 *g?>
O
0
0
o,c?
0
- • Electrophoresis
03
t
E
2
6
0 *tf>l
? 8
o
a' Al
o
o.
Electrophoresis
Fig. 7. Tracings of the tryptic maps of the anodic subunits of E2 and Al haemoglobins showing those peptides which have specific staining reactions for histidine H,
tyrosine ( • ) , arginine (g) tryptophan (HD). Tryptophan residue are absent in both
samples. The dotted peptide of aE2 was absent in some experiments. Uncertainties
in the specific staining are indicated by asterisks.
of Al, A2, El, E2, E3 are in the order a'2/?2, a"2/?2, £2e2, a' 2 y 2 , a"2y2. The relative
amounts of each globin at different stages of development are shown in Fig. 10.
The percent values were obtained by quantitative evaluation of El, A2 and E3
haemoglobins and of the electrophoretic bands of chains constituting A1-E2
chromatographic fraction.
DISCUSSION
The electrophoretic isolation of haemoglobins present in the haemolysate
of early duck embryos was described some time ago by Borgese & Bertles
(1965) and more recently, by Stratil & Valenta (1976) and by Borgese &Nagel
26-2
400
C. CIROTTO, I. ARANGI AND F. PANARA
0
|
o
o
a <=>
o
0
Electrophoresis
o
7E3
0 o
o
Oo
-0
0
0
O
Electrophoresis
Fig. 8. Tracings of the tryptic maps of the cathodic subunits of E2 and E3 haemoglobins showing those peptides which have specific staining reactions for histidine
(H), tyrosine ( • ) , arginine (g), tryptophan (HI). Tryptophan residues are absent
in both samples.
(1977). All these authors agree in describing only one major haemoglobin
fraction, classified by Borgese & Bertles as embryonic on the basis of the time
over which it was present in the lysate of developing embryo and of its electrophoretic similarity to the chick embryonic haemoglobins. The chromatographic
isolation of its constituent chains reported in this paper confirms that it is
actually an embryonic haemoglobin since its globins differ from all the others
synthesized both in embryonic and in adult life.
As demonstrated by many authors, chick haemoglobins seem to occur in
pairs, each of them composed of a common /Mike globin chain and two
different a-like globin chains (Brown & Ingram, 1974; Cirotto & Geraci, 1977).
The probable genetic process underlying the formation of these two a-like
chain genes involves a duplication of an ancestor gene followed by independent
The haemoglobins of developing duck embryos
401
0
o
eg
E
o
_e
U
0
0
O°o
Electrophoresis
O
eEl
O
Electrophoresis
Fig. 9. Tracings of the tryptic maps of the two chains of El haemoglobin showing
those peptides which have specific staining reactions for histidine ( M), tyrosine ( • ) ,
arginine (g), tryptophan (OH). Uncertainties in the specific staining are indicated
by asterisks. Spots shared by e El, y E2 andy E3 are indicated by the arrows.
mutations. The duck foetal and adult haemoglobins may involve a similar
scheme. The exception seems to be the embryonic haemoglobin El. However,
it is likely that also in this case two distinct a-like globin structural genes are
present per haploid genome, but they do not undergo any diversification. Their
products are therefore chemically indistinguishable. There is substantial
evidence that also in man there are two structural genes for the a chains per
haploid genotype with a total of four a chains per diploid cell (Lang & Lorkin,
1976).
In duck embryo erythrocytes as in chick and in goose (Cirotto, Arangi &
Panara, 1979) there are two haemoglobins that can be defined as foetal
according to their globin composition and to their presence in the embryo and
in the newborn duckling. Stratil & Valenta (1976) described two minor electrophoretic haemoglobin fractions in the early embryo, whereas Borgese & Bertles
(1965) and Borgese & Nagel (1977) detected only one. In the case of chick
402
C. CIROTTO, I. ARANGI AND F. PANARA
60
40
X>
§
20
•A
10
15
20
Days of incubation
25
y
30
Fig. 10. Relative amounts of individual globins in developing duck embryos.
the problem concerning the identity of the foetal haemoglobins in the course
of embryonic life is not yet solved. Some electrophoretic evidence suggests
a sequential appearance in the embryo of different types of foetal haemoglobins
(Bruns & Ingram, 1973). Nevertheless, an analysis of the primary structure of
these molecules is lacking up to today. During our investigation of the
haemoglobins of the embryonic duck no variation of the chromatographic
elution properties of the two foetal fractions was found. For this reason we
have chosen to name with the same labels E2 and E3 these two haemoglobins,
independently of the developmental stage.
The persistence of foetal haemoglobins during the embryonic life seems to
be lacking in a plausible physiological meaning, since they constitute too small
a portion of the haemolysate. At the same time, their functional properties
in the presence of organic phosphates are very similar to those of adult
haemoglobins (Borgese & Nagel, 1977). It is likely that in duck, as in chick,
these two haemoglobins are synthesized by a foetal red cell population (Cirotto
et al. 1977).
The time of appearance of the two adult haemoglobins is delayed in ducks,
in comparison with chicks, in accordance with the longer duck incubation
time. Percent amounts of the adult haemoglobins change during embryonic
development and reach the typical adult values only after egg hatching. This
finding could be due to the different origins of the red cells of the definitive
line which produce the adult haemoglobins. Before hatching, the duck definitive
erythrocytes are markedly morphologically different from those of the adult,
as found in chicks by Lemez (1964). It is likely then, that they differ also in
the relative amounts of Al and A2.
The results reported in this paper demonstrate the existence of strong
similarities between the general picture of haemoglobins in the chick embryo
and that of the duck embryo. Further studies will demonstrate the existence
of a similar haemoglobin distribution in other bird species.
The haemoglobins of developing duck embryos
403
The authors are indebted to Professor G. Geraci for his helpful suggestions and to
Mr L. Barberini for his technical assistance.
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