J. Embryol. exp. Morph. 77, 153-165 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
153
Synthesis of globin chains in the erythropoietic sites
of the early chick embryo
ByL. FUCCI 1 , C. CIROTTO 2 , L. TOMEI AND G. GERACI 1
From the Institute of General Biology and Genetics, Faculty of Sciences,
University of Naples, and the Institute of Cell Biology, University of Perugia
SUMMARY
The synthesis of globins in the chick embryo before the onset of circulation has been studied
in situ by specific immunofluorescence labelling of embryonic sections and by labelling newly
synthesized proteins in ovo and in vitro in embryonic explants with pHjleucine.
The presence of major primitive haemoglobins is observed by 28 h of incubation. The minor
primitive haemoglobins become detectable by immunofluorescence after 40 h of development, shortly before the onset of circulation. 3H-labelling shows that one definitive a chain is
synthesized, though in low concentration, from the initial globin detection. The other
definitive a chain is observed in embryos of at least 40 h of development. The relative concentration of the two definitive a chains changes rapidly with development indicating a specific
mechanism of regulation.
An erythropoietic site is observed in the wall of the dorsal aorta in embryos of about 45-50 h
of development. From the initial detection, those cells contain all four primitive embryonic
haemoglobins, in contrast to what is observed for the cells of the blood islands.
INTRODUCTION
The knowledge of the timing of haemoglobin synthesis in the erythropoietic
sites and the possible sequence of haemoglobin types is of interest in defining an
erythropoietic model that would take into consideration the initial formation of
the stem cell(s) and its maturation through the series of precursor cells that
finally lead to the formation of the erythrocyte. Many studies have been made
to define the erythroid cells present in the embryo at different stages of development (Bruns & Ingram, 1973a), their different haemoglobin types (Schalekamp,
Schalekamp, Van Goor & Slingerland, 1972; Shimizu & Hagiwara, 1973; Brown
& Ingram, 1974; Cirotto, Scotto di Telia & Geraci, 1975) and their distribution
in the erythroid cells (Bruns & Ingram, 19736; Wilt, 1974; Shimizu, 1976). There
are different opinions concerning the presence of the stem cell in the unincubated
chick blastoderm (Samarut, Jurdic & Nigon, 1979; Zagris, 1979; DieterlenLievre & Martin, 1981). It has been shown that erythroid cell differentiation
1
Author's address: Institute of General Biology and Genetics, Faculty of Sciences, University of Naples, Via Mezzocannone 8, 80134, Naples, Italy.
^Author's address: Institute of Cell Biology, University of Perugia, Via Elce di Sotto, 06100
Perugia, Italy.
154
L. FUCCI, C. CIROTTO, L. TOMEI AND G. GERACI
occurs in unincubated chick blastoderm in culture on filter rafts that prevent cell
movements (Zagris, 1980). Both primitive and definitive cell lines are found after
appropriate incubation time in the cell culture indicating that the unincubated
blastoderm has all the necessary information to develop in due time the required
erythrocyte lines. Keane, Lindblad, Pierik & Ingram (1979) have also shown that
cultures of mesenchymal cells are able to synthesize both embryonic and adult
haemoglobin chains. Recently the haemoglobin switch has been studied in erythroid nuclei in vitro. The data concern primarily events occurring from 5 days
of development onwards (Landes & Martinson, 1982; Landes, Villeponteau,
Pribyl & Martinson, 1982) and the structure and activity of the globin genes in
precursor cells obtained from 20-23 h embryo and individually cultivated in vitro
for several days (Groudine & Weintraub, 1981).
We decided to investigate the timing of the various embryonic haemoglobins
in the single cells directly in the embryos from the initial time of haemoglobin
detection until the onset of circulation at 45-50 h of development. Immunocytochemical methods provide a convenient tool to detect with high sensitivity and
specificity the protein contents of individual cell (Shimizu, 1976; Cirotto, Panara
& Geraci, 1977; Chapman & Tobin, 1979). By using this approach we attempted
to obtain a more exact estimate of the initial time of globin synthesis in the early
chick embryo and information concerning the haemoglobin types in the erythropoietic territories at different times of development. Direct evidences on the
synthesis of the individual globins were obtained by labelling the newly
synthesized proteins with [3H]leucine both in ovo and in vitro.
MATERIALS AND METHODS
Fertilized chicken eggs of several strains were obtained from a local poultry
farm and were grown in the laboratory at 38 °C in a humid incubator. Embryonic
stages were classified as described by Lillie (1952) on the basis of somite
number.
Preparation of antibodies
Circulating erythrocytes were isolated from 4- and 15-day-old embryos and
major embryonic and adult haemoglobins were prepared by fractionation on
CM-cellulose columns with a phosphate gradient as previously described (Cirotto & Geraci, 1974). Electrophoretically pure haemoglobins were used for antibody preparation in rabbit by lymphonode injection. Antibodies were isolated
from the immune sera by standard procedures using ammonium sulphate
precipitation and DEAE- and CM-cellulose column chromatography (Barrett,
1970). The IgG fractions were stored at - 2 0 °C. Antibody specificity was tested
using the Outcherlony double diffusion method. Antibodies against adult
haemoglobins did not react with major primitive embryonic haemoglobins, HbP
Globins in the early chick embryo
155
(1)
and HbP' , but reacted with minor primitive haemoglobins, HbM and HbE, as
already reported by Cirotto et al. (1977), and were used as specific for these
haemoglobins. Antibodies prepared against the mixture of all four embryonic
haemoglobins were used to obtain the highest efficiency of detection of traces of
primitive haemoglobins in embryonic sections. This antibody preparation
showed almost the same titre against the individual haemoglobin species.
Immunofluorescence method
Blastoderms dissected from embryos between one and three days of incubation were rinsed in PBS, fixed in Carnoy and embedded in paraffin. The presence
of haemoglobin molecules in the different embryonic areas was tested on 5 jumthick sections by using the indirect immunofluorescence method of Brahma &
Van Doorenmaalen (1971).
The best results were obtained by fixing the blastoderms in Carnoy at 4 °C for
2 h; use of formaldehyde caused alteration of cell morphology and an artifactual
condensation of fluorescent precipitate around the cell membrane. Fluoresceineisothiocyanate-labelled goat anti-rabbit antibodies (Nordic Pharmaceuticals and
Diagnostics, The Nederlands) were used to detect the presence of antigenantibody complexes. A Zeiss fluorescence microscope was used for examination
with 09 filter combination under magnification xl60. Photographs were taken
using Ilford PANF films.
Benzidine staining of blastoderm
Blastoderms, isolated from yolk mass, were washed several times in cold PBS
and fixed in neutral-buffered formaldehyde (Bancroft & Stevens, 1977) for 24 h
at room temperature. After rinsing in PBS and in methanol, the blastoderms
were dipped for 5min in a solution containing benzidine 0-2 g, methanol 30 ml
and glacial acetic acid 0-8 ml and then in a solution containing H2O2 (3%),
methanol and ethyl ether, 1:2:1 (v/v/v). The reaction was carried out for 10 min
at room temperature, then the blastoderms were rinsed in water for about 5 min,
dehydrated in absolute alcohol, cleared in xylene and mounted in Canada
balsam.
Labelling of proteins in ovo
Groups of 10 embryos at 24 h of incubation were exposed by opening a window
on a lateral position on the egg and 25|Ul of [3H]leucine Amersham, 150 Ci/
mmol, 1 /iCi/zil) equivalent to 170pmolesof total label were injected per embryo
through the area opaca using a tuberculin needle at an angle of about 45 ° to
(1)
Abbrevations. HbP and HbP': major primitive embryonic haemoglobins; respective
chain composition: KI QI ,n'iQi.
HbM and HbE: minor primitive embryonic haemoglobins; respective chain composition:
$ £2, <h £2.
PBS: 40mM-NaCl in 30mM-sodium phosphate, pH7-2.
156
L. FUCCI, C. CIROTTO, L. TOMEI AND G. GERACI
deposit the solution underneath the embryo at a depth of about 2 mm. The
window was sealed and the incubation resumed.
The low amount of amino acid injected did not affect the growth rate of the
embryos because after an additional 16 h of incubation at 38 °C, the embryos
were usually at the stage 11 as expected, showing 13 somites (Lillie, 1952).
Embryos at more advanced stages were discarded. The embryonic areas were
dissected from the yolk, washed several times with 0-9 % NaCl solution, gently
blotted with filter paper and homogenized in a Dounce L Homogenizer in 2
volumes of distilled water. The supernatant after 30min of centrifugation at
30-000 g was brought to 8M-urea, 5 % acetic acid and 5 % ^-mercaptoethanol.
The solution was analysed electrophoretically on 7 % acrylamide slab gels in 8 Murea, 5 % acetic acid, in the presence of 0-6 % Triton X-100. In this condition
there is a better separation of the adult types of a chains (Keane et al. 1979). The
slab gels were stained with Coomassie and fluorographed (Laskey & Mills, 1975).
Labelling of proteins in vitro
Explants of blood islands obtained from a minimum of 15 embryos at stage 10
(8 somites, 32 h of development) and at stage 12 (16 somites, 45 h of development) were incubated for 1 h at 38 °C in 500 jul of incubation buffer containing
150mM-NaCl, 15mM-sodium phosphate pH7-4 and 15/^Ci of [3H]leucine
equivalent to 102pmoles of label.
Circulating erythrocytes coming out of umbilical veins cut near embryos of 3-5
days of development were used to prepare 3H-labelled globins. Erythrocytes
were obtained also from 7-day-old embyos. 15-20 //I of red cells were suspended
in 500 jUl of incubation buffer, as for the explants of blood islands, and incubated
for 1 h at 38 °C. At the end of the incubation time soluble proteins were prepared
by lysing the cells with 2vols of distilled water, incubating at 4°C for 15min,
centrifuging at 30-000 g for 30min and precipitating the globins from the clear
supernatant solutions by addition of 20 vol of acid acetone at - 2 0 °C. The suspensions were left at - 2 0 °C overnight, the precipitates collected by centrifugation and dissolved in 50 [A of water. Each sample was analysed for radioactive
incorporation in order to introduce in each slot of acrylamide slab gel a minimum
of 30000 c.p.m. of labelled proteins to be electrophoretically fractionated and
analysed by fluorography. Densitometric analyses of the fluorographic patterns
were performed on a Varian 634 gel scanner at A = 540 nm.
RESULTS
Fig. 1 shows the positive reaction of the cells of the area opaca of a 28 h, 6somite embryo stained with benzidine for haem detection. It should be noted
that if o-dianisidine (O'Brien, 1960) is used instead of benzidine, the reaction is
positive only on embryos at 7 somites corresponding to 30-32 h of incubation.
Globins in the early chick embryo
157
Fig. 1. Benzidine reaction of the blood islands of an embryo at the 6-somite stage
corresponding to about 28 h of incubation. The formation of a blue colour, that is
indicated by the darker zones, shows the presence of the haem. Two black arrows
point to a region of positive reaction. Magnification X60.
Fig. 2 shows embryonic sections at two stages of development reacted with
antibodies against all primitive embryonic haemoglobins. The immunofluorescence reaction is observed starting from 28-30 h of incubation, 6-somite embryos
as shown in Fig. 2A. The fluorescence intensity of the cells reacted with
antibodies against all primitive haemoglobins increases rapidly on embryos of
increasing incubation time. At 35-38 h, 10-somite embryos, it is as shown in Fig.
2B. In this picture two types of erythroid cells are clearly distinguishable: clusters
and isolated cells. Reaction with antibodies against the adult haemoglobins on
embryos up to about 38 h of incubation (10 somites) does not cause any
fluorescence above background level indicating the absence, or very low concentration, of the minor primitive embryonic haemoglobins. The difference
between background fluorescence level and fluorescence of erythroid cells can
be seen in Fig. 3. This compares the results on two sections through the area
opaca of a 38 h 10-somite embryo. The embryonic section treated with the antibody preparation that does not react with the major embryonic haemoglobins,
HbP and HbP', and that reacts with the minor embryonic haemoglobins, HbM
and HbE, does not show any fluorescence above background level in the erythroid cells (Fig. 3A). In contrast, the cells are highly fluorescent when treated
EMB77
158
L. FUCCI, C. CIROTTO, L. TOMEI AND G. GERACI
with the antibody preparation that reacts with all chicken haemoglobins including the major primitive embryonic haemoglobins, HbP and HbP' (Fig. 3B). Only
from 45 h of development (stage 12) is the immunofluorescence reaction observed also with the antibody preparation that reacts only with the minor primitive
haemoglobins. At this time circulation begins and immunofluorescent cells are
observed also in the embryo. At 50-55 h of development, a cluster of cells is
observed in the wall of dorsal aorta that is reported to be, at this stage, the only
intraembryonic territory that produces erythroid cells to be put in circulation
(Lillie, 1952; Dieterlen-Lievre & Martin, 1981). The circulating cells, as well as
those in the wall of dorsal aorta, react both with antibodies against major
primitive embryonic haemoglobins and with antibodies against minor primitive
embryonic haemoglobins as clearly shown in Fig. 4A-D. One field (Fig. 4B) is
shown at higher magnification for a better definition of the cell morphology. Two
B
Fig. 2. Immunofluorescence reaction on frontal sections through the blood islands
region of an embryo at the 6-somite stage, corresponding to 28-30 h of incubation
(A) and at the 10-somite stage, corresponding to 35—38 h of incubation (B). Sections
reacted with rabbit antibodies against all primitive embryonic haemoglobins were
madefluorescentby indirect labelling, as described in Materials and Methods. Magnification X160.
Globins in the early chick embryo
159
types of erythroid cells can be distinguished: those with a round shape and welldefined individual boundaries and those in the erythropoietic site, tightly packed
with individual cell boundaries not clearly defined. However, in both cases, the
nucleus shows up as a dark region over uniform fluorescence background of
cytoplasm containing haemoglobin.
In order to obtain evidence of the individual globin synthesis, embryos at 24 h
of incubation were injected with [3H]leucine and incubated for additional 16 h.
Total proteins were extracted from embryos showing 12-13 somites and were
analysed by acrylamide gel electrophoresis. The fluorographs of the
electrophoretic separations in the presence of the acetic acid-urea-Triton X^lOO
are reported in Fig. 5. No attempts to purify the globins in the homogenate of
the in ovo labelling has been made to avoid possible selective losses of proteins.
Protein synthesis was studied also in explants of blood islands of embryos at stage
_ —
B
Fig. 3. Immunofluorescence reaction on frontal sections through the blood island
region of a 10-somite embryo, 38 h of incubation, reacted with rabbit anti-adult
chicken haemoglobin antibodies (A) and with rabbit antibodies against all primitive
embryonic haemoglobins (B). Labelling as in Materials and Methods. Magnification
X160.
160
L. FUCCI, C. CIROTTO, L. TOMEI AND G. GERACI
\
•p
4A
i
B
l#
«t*
Fig. 4
Globins in the early chick embryo
5A
B
R
C
R
161
D
R
Fig. 5. Globin chains synthesized in ovo between 24 and 40 h of development (stage
7 to stage 11). The results are shown in the lanes R. Lanes L show reference globin
chains prepared in vitro in 1 h pulse in erythrocytes from 3-5-day-old embryos (stage
18). A, B, C are fluorographs of the same slab gel at increasing exposure times to
show minor components. In D, globins synthesized in vitro in 1 h pulse on explants
of blood islands of embryos at 45 h of development (stage 11-12,15 somites). Labelling with pH]leucine. Electrophoresis in acetic acid-urea polyacrylamide slab gels
containing 0-6 % Triton X-100. The arrowheads indicate the positions of the globin
chains as marked in panel A. Note that no protein band is apparent at the position
corresponding to globin aA after a long exposure time, when a° globin band appears
overexposed.
Fig. 4. Immunofluorescence' reaction on frontal sections through the area of the
aortic arches of chicken embryos. (A) 24-somite embryo, corresponding to 50-55 h
of incubation. An erythropoietic site is apparent in the wall of the dorsal aorta, in
agreement with previous reports (Lillie, 1952; Dieterlen-Lievre, 1981). (B) The
erythropoietic site of panel A is shown at higher magnification (x 500). (C) 30-somite
embryo, corresponding to 62 h of incubation. (D) 37-somite embryo corresponding
to 76 h of development. The arrows in the panels indicate the caudal-to-cephalic
directions. Sections were treated with antibodies that react only with minor primitive
and adult haemoglobins. Note that all cells arefluorescentindicating that all contain,
in addition to the initial major primitive haemoglobins, also the minor primitive
haemoglobins. Magnification X160.
162
L. FUCCI, C. CIROTTO, L. TOMEI AND G. GERACI
-20
-10
Hours
Somites
32
8
45
16
84
168
Fig. 6. Relative amounts of the definitive a chains in the developing chick embryo.
Newly synthesized proteins were labelled in vitro with pHjleucine in 1 h pulses on
explants of erythroid cells. The definitive a chains were identified by urea-Triton
X-100 gel electrophoresis, as shown in Fig. 5, and the fiuorographs of the gels were
scanned in a densitometer. The area of each definitive a chain band was measured
for the determination of the relative amounts of each a globin.
10 and stage 12 in l h pulses to reduce possible variations in the types and
amounts of mRNA molecules existing in the cells at the time of the explants. The
globins in the circulating erythrocytes of 3-5-day-old embryos (stage 18) and 7day-old embryos were analysed with the same method. The adult types of a
chains, of^ and oP, that are specific also for the minor primitive embryonic
haemoglobins, HbE and HbM respectively (Brown & Ingram, 1974; Cirotto et
al. 1975), migrate in the presence of Triton X-100 much faster than the bulk of
the protein bands and are easy to identify (Keane et al. 1979). In Fig. 5 it is
evident that of" chains are not synthesized in the embryo before stage 10-11
since there is no trace of aminoacid incorporation in proteins in the gel at a
position corresponding to that globin band. Some radioactivity is observed at a
position corresponding to aD chains though the amount is very small compared
to the three globin bands of the major primitive embryonic haemoglobins. At
stage 11-12, between 40 and 45 h of development, both aA and aD are clearly
synthesized in the explants of blood islands. The ratio aD/ aA changes regularly
during development, as shown in Fig. 6. The ratio has been estimated as at least
Globins in the early chick embryo
163
20 for the embryos at stage 10-11 but this is a limit value because no protein band
corresponding to a* is actually detectable in the fluorography. For embryos at
stage 11-12 the ratio is six to eight. In circulating erythroid cells of 3-5-day-old
embryos the ratio becomes about two to three and it is about one in the circulating cells of the 7-day-old embryo. The globin values for the 3-5-day-old and
7-day-old embryos are as expected from the relative amounts of haemoglobins
in the circulating red cells as determined by chromatography (Cirotto et al.
1975).
DISCUSSION
The initial presence of haem, as well as haemoglobins, is clearly apparent in
chick embryos at 28-30 h of incubation. Thefindingof haemoglobins at this time
of development shows that the megaloblast (Romanoff, 1960) or other precursor
cells, supposed to be present at this stage, already synthesize some haemoglobin
molecules. The concentration of haemoglobin does not appear to increase substantially in the cells until 35 h of incubation when the intensity of the
fluorescence labelling with antibodies against all primitive embryonic haemoglobins begins to increase (Figs 2, 3). Antibodies that react with the minor
primitive haemoglobins, HbM and HbE, and that do not react with the major
primitive haemoglobins, HbP and HbP' do not show any reaction above background level with erythroid cells of the embryo up to about 38-40 h of development (stage 10-11). In agreement with these findings, analyses on explants of
stage-10 embryos and of the in ovo synthesis of globin chains between 24 and 40 h
of development show no synthesis of a A , the a chain of HbE, and very little
synthesis of aD, the a chain of HbM (Fig. 5A-C, lanes R). Soon after, starting
at stage 11 at about 40-45 h of development, the immunoreaction against minor
primitive haemoglobins becomes positive and rapidly increases in intensity with
the time of development. Analysis of globin chains synthesized at this time in the
blood islands shows now the presence of both aA and oP chains. When circulation begins, erythroid cells are found also in the embryo, but they all show the
presence of all four primitive haemoglobins.
It is interesting to note that at stage 12 the relative amounts of o^ and cP are
very much in favour of a? and the ratio changes with increase in time of development to become that typical of the circulating red cells of the 3-5-day-old embryo
as reported in Fig. 6. At 7 days of development the ratio is one and eventually
it continues to change becoming in favour of aA as demonstrated by the higher
percent of HbA over HbD observed at hatching time (Cirotto et al. 1975).
The results reported here show that initially, the erythroid cells of the area
opaca of the chick embryo synthesize the major primitive haemoglobins HbP and
HbP' and a small amount of HbM. After 40 h of development, from about stage
11-12 on, the minor primitive haemoglobin HbE is also synthesized and both
types of minor primitive haemoglobins increase rapidly in concentration. When
164
L. FUCCI, C. CIROTTO, L. TOMEI AND G. GERACI
circulation begins all cells appear to contain all four primitive haemoglobins as
revealed by immunofluorescence labelling. The rapid appearance at stage 11, or
immediately after, of the minor primitive haemoglobins in all the erythroid cells
indicates that those cells not only develop as a cohort (Bruns & Ingram, 1973a)
but also express synchronously in ovo the sequential synthesis of the major and
then of the minor primitive a globin chains. Interestingly the erythroid cells of
the erythropoietic site in the wall of the dorsal aorta show the presence of all four
primitive haemoglobin types from the very initial detection by immunofluorescence analysis. This suggests an origin of those cells (Beaupain, Martin &
Diterlen-Lievre, 1980; Dieterlen-Lievre & Martin, 1981). Their detection in the
embryo is almost coincident with the very initial time of embryonic circulation
suggesting that they were there before the actual conjunction of the network of
vessels outside and inside the embryo. Since the relative amounts of haemoglobin types in the erythoid cells of the blood islands change during the initial
development, as demonstrated here, the finding that the erythoid cells in the
intraembryonic site contain the same haemoglobin types observed in the cells
outside the embryo speaks in favour of a sort of coordinated ontogeny between
the two cellular populations. It appears difficult to discard the possibility that the
progeny of an initial stem-cell line populated both the yolk-sac sites and the area
of formation of the embryonic body undergoing similar stages of maturation.
Those in the blood island expressing the globins from the very beginning while
those in the embryo later on, possibly depending on a coordination with haem
synthesis. As soon as the restraint of globin synthesis is removed, e.g. by turning
on haem synthesis, the cells in the embryo would express the same globin chains
as those of the cells outside the embryo, since the internal development clock
would be the same for all of them.
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(Accepted 26 May 1983)