/ . Embryol. exp. Morph. Vol. 58, pp. 209-216,1980
Printed in Great Britain © Company of Biologists Limited 1980
209
Erythroid cell differentiation in unincubated
chick blastoderm in culture
By NIKOLAS ZAGRIS 1
From the Tissue Culture Laboratory, University of Patras, Greece
SUMMARY
Morphologically distinct erythroid cell types characteristic of the primitive and the definitive
erythroid cell lines, and embryonic and adult haemoglobins are produced when the unincubated chick blastoderm is cultured ventral side down on afilterraft to inhibit morphogenetic
movements and subsequent primitive-streak formation mechanically in serum-free minimal
essential medium. The primitive and definitive erythroid cell populations appear consecutively
in culture even though there is no axis formation nor apparent morphogenesis. The information to produce both the early and late haemoglobins and erythroid cell types is independent
of axis formation and of specific extra-embryonic influences, such as progressive induction
exerted by the yolk mass.
INTRODUCTION
Erythropoiesis in the chick is far from simple and the sequential appearance
in early chick embryos of morphologically distinct erythrocytes and their
haemoglobins (Hb) has been thoroughly described (Sabin, 1920; Fraser, 1961;
Lucas & Jamroz. 1961; Wilt, 1967; Schalekamp, Schalekamp, Van Goor &
Slingerland, 1972; Bruns & Ingram, 1973; Brown & Ingram, 1974; Wainwright,
& Wainwright, 1974; Cirotto, Scotto Di Telia & Geraci, 1975; Zagris & Melton,
1978; Chapman & Tobin, 1979).
The presumptive erythropoietic area of primitive streak and older chick
embryos has been maintained successfully in tissue culture (Murray, 1932;
Settle, 1954; O'Brien, 1960; Spratt & Haas, 1960; Hell, 1964; Levere & Granick,
1967; Wilt, 1967; Hagopian & Ingram, 1971; Wenk, 1971) and in suspension
culture (Hagopian, Lippke & Ingram, 1972; Chan & Ingram, 1973; Pine &
Tobin, 1976). In all cases potential erythropoietic cells continue to differentiate
into mature erythrocytes during the culture period.
Whereas previous work on erythroid blood cells and Hb biosynthesis in chick
embryos concentrated generally on embryos from the primitive streak stage and
older, we showed recently (Zagris, 1979) that unincubated blastoderm can be
maintained in a simple, serum-free chemically-defined medium in culture for a
number of days during which it forms haemoglobinized blood islands which
1
Author's address: Tissue Culture Laboratory, School of Sciences, University of Patras,
Patras, Greece.
210
N. ZAGRIS
anastomose to form a network. Even though there is no primitive streak formation and the unincubated blastoderm is deprived of the usual flow of materials
from the yolk, it forms Hb and this is inhibited when 5-bromodeoxyuridine is
applied during the first two days of culture (Zagris, 1979). It seemed of interest
to see whether the unincubated blastoderm, cultured ventral side down on filter
rafts, to inhibit morphogenetic movements and subsequent primitive-streak
formation mechanically, in a protein-free medium, contained also instructions
to produce the early and late blood cells and Hbs without participation of the
embryo proper, and deprived of possible extra-embryonic influences exerted by
the yolk mass.
MATERIALS AND METHODS
Culture
Freshly-laid fertilized eggs of White Leghorn chickens were used on the day
that they were obtained from a local hatchery.
The explanted unincubated blastoderms were checked individually under a
dissecting microscope and staged accurately (Eyal-Giladi & Kochav, 1976) so
that only blastoderms at stage X were used for experiments.
Blastoderms, washed free of the vitelline membrane and any adhering yolk,
were attached on filter rafts, and were cultured in Petri dishes with plain
Dulbecco's modified Eagle Medium (MEM) in the absence of serum as described elsewhere (Zagris, 1979).
In experiments involving labelling of haemoglobins, [U-14C]protein hydrolysate (56 mCi/mAtom, Amersham) mixture was added to the culture medium
to a final concentration of 6-25 /tCi/ml.
Blastoderms, media and glassware were handled with sterile precautions.
Collection of blood cells and preparation of Hb
Erythroid cells were harvested from blastoderms under a dissecting microscope using a specially prepared fine-tipped Pasteur micropipette.
By exercising gentle pressure in one direction with a fine-tipped Pasteur
pipette, the anastomosed blood islands were directed to form a single large
blood pool. The transparent tissue surrounding the blood pool (Zagris, 1979) was
punctured gently and blood cell clusters started streaming into the micropipette opening. This method gave very good yields of red cells in that the area
that the blood pool occupied originally appeared colourless after harvesting.
Blood cells from five blastoderms were suspended and washed three times in a
tenfold volume of ice-cold chick Ringer's solution. The packed cells, combined
with 20 /A carrier Hb from 8-day-old chick embryo blood cells (packed cells:
H 2 O, 1:2), were lysed in 30-50 /<! of H 2 O, then mixed vigorously with 10 /A
toluene-20 /A carbon tetrachloride and centrifuged (Hagopian & Ingram, 1971;
Tobin et al. 1976). The red aqueous layer was used for electrophoresis within
Erythroid cell differentiation in unincubated chick blastoderm 211
24 h after preparation, and after conversion of Hbs to the cyanomethemoglobin form with ferricyanide.
To study cell morphology, collected blood cells were placed in one drop of
chick Ringer's solution on a slide. After drying, cells were stained with Harris'
haematoxylin overnight.
Analysis of haemoglobin
Electrophoresis was performed in 10% polyacrylamide gels according to
Barker (1968) as modified by Zagris & Melton (1978). At the end of the run, the
gels were fixed in 7 % (v/v) acetic acid for 5 min, then stained with benzidineperoxide solution for 1 h. Hydrogen peroxide (30 %) at 0-2 % (v/v) in benzidine
solution [0-2% (w/v) benzidine in 0-5 M acetic acid] was added immediately
before use. Colour developed within the first few minutes after application of
the staining solution, and gels were scanned at 510 nm in a Gilford 240 spectrophotometer.
After scanning, gels were cut in 1-5 cm slices which were digested overnight in
0-2 ml of 30 % H 2 O 2 each in scintillation vials at 37 °C. The sample in each vial
was counted for radioactivity in a toluenefluor [0-5 % PPO, 0-03 % POPOP in
toluene (w/v)]-Triton X-100 (4:1, v/v) scintillation cocktail.
RESULTS
Unincubated blastoderms produce blood islands which appear after 2-5-3-0
days in culture as dense bright red clusters visible to the naked eye, and on subsequent days as a tissue which is heavily haemoglobinized. The first erythroblasts appear in similar structures in ovo, but with the blood island networks
being much more reticulate. After pricking the haemoglobinized tissue with a
glass microneedle, the blood cells stream in groups (clusters) indicating that the
blood island is constructed by tight grouping of erythroid cells. This observation
is in accord with the view of Miura & Wilt (1970) on the blood island cytoarchitecture.
Microscopic examination of erythroblasts showed morphological differences
in the erythroid cells of the blastoderms. There seem to be at least four distinct
erythroid cell populations appearing during the 10-0-day culture.
On day 3-8 of culture the erythroid cell population that appears to develop
from precursor cells as a cohort is uniform, roughly circular in shape, and
granulate in appearance, with most cells in mitosis. These cells, which, according
to the terminology of Lucas & Jamroz (1961), correspond to early primary
('primitive') erythrocytes, constitute the only cell type (Fig. 1 A).
On day 6-0 of culture there are three erythroid cell types present. The cells
described previously as early primary erythrocytes seem to be at a resting phase
with only an occasional cell in mitosis. These cells display delicate extensions of
cytoplasm as if to facilitate their anchoring within the blood island, or to begin a
212
N. ZAGRIS
Fig. 1. Blood cells collected from unincubated blastoderms which were cultured for
(A) 3-8, (B) 60, (C) 80 and (D) 100 days in serum-free MEM. Cells stained with
Harris' haematoxylin. Scale bar = 10 /tm. ep, Early primary erythrocyte; Id, late
polychromatic definitive erythrocyte; md, mid-polychromatic definitive erythrocyte;
mp, mid-polychromatic primary erythrocyte; pc, pycnotic cell.
degenerative pycnotic process. The most numerous population consists of small,
pycnotic cells with fragmented nuclei. These could be early primary erythrocytes
in the process of pycnosis and degeneration. The larger cells with the irregular
shape and sharper nuclear contour could be mid-polychromatic primary erythrocytes. An occasional semi-elliptical cell with a well-defined nucleus is also
observed (Fig. I B ) . .
On day 8-0 of culture, blood consists primarily of cells semi-elliptical in form
with a well-defined roughly oval nucleus. These cells resemble morphologically
the mid-polychromatic definitive erythrocytes characteristic of 6-5- to 8-5-day
in ovo developing chick embryos. A few pycnotic cells are still present. An
occasional erythroid cell, ellipsoid in general and nuclear shape, is evident.
There is absence of cell divisions in the blood cell population (Fig. 1C).
By day 10 of culture, blood consists primarily of mid-polychromatic definitive
erythrocytes, most of which seem to be in the process of degeneration. The rest of
the blood population consists of erythrocytes, elliptical in general and nuclear
shape, with a low nuclear-to-cytoplasmic ratio. These cells resemble morphologically late polychromatic definitive erythrocytes characteristic of older than
9-0-day embryos in ovo, and of the adult chicken (Fig. 1D).
Erythroid cell differentiation in unincubated chick blastoderm 213
100
80
60
40
1
20
3-8
60
80
Time in culture (days)
100
Fig. 2. Alterations in the blood cell population of unincubated blastoderm during
100-day culture in terms of percentage of the cells morphologically classified as early
primary erythrocytes (| 1), pycnotic cells (H^>mid-polychromatic primary erythrocytes ([JTflD, mid-polychromatic definitive erythrocytes (1—|) and late-polychromatic
definitive erythrocytes ( ^ | ) . The time scale refers to the day blood cells were collected.
Slides with the fixed blood cells were scanned microscopically, and percentages of the various cell populations were calculated from about 500 cells per
culture point. The results were reproducible in three separate experiments.
Figure 2 shows histograms of the frequencies of erythroid cell populations at
3-8, 6-0, 8-0 and 10-0 days of unincubated blastoderm in culture. The entire
erythroid cell population consists of early primary erythrocytes on day 3-8 of
culture. On day 6-0 the proportion of early primary erythrocytes has dropped to
20% with the pycnotic cells being the predominant cell type, while the midpolychromatic primitive erythrocytes comprise 22 % of the total erythroid cell
population. On day 8-0 of culture the proportion of mid-polychromatic definitive erythrocytes has risen to about 97 % of the entire cell population with
2 % mid-polychromatic primitive erythrocytes and 1 % pycnotic cells present.
By day 10-0 of culture the cells defined as mid-polychromatic definitive erythrocytes constitute about 65% of the total blood cell population, while the late
polychromatic definitive erythrocytes comprise the rest of the erythroid cell
population.
Electrophoresis on polyacrylamide gels of the Hbs produced by the unincubated chick blastoderm after 9 days of culture shows the presence of all four
Hbs (Fig. 3). The electrophoretic profile of Hb was reproducible in two separate
experiments (one electrophoretic run per experiment) performed. In keeping
with the nomenclature advocated by Bruns & Ingram (1973), these are Hbs E,
A, P, D, the embryonic type being E and P, with A and D being the adult Hbs.
According to Hagopian & Ingram (1973), the peak of radioactivity which is
present between Hbs P and D is a breakdown product from Hb A as a result of
overnight storage in the refrigerator.
214
N. ZAGRIS
-0-4
-0-3
-0-2
-01
00
10
Gel sections
Fig. 3. Polyacrylamide gel electrophoretic profile of Hbs extracted from unincubated
blastoderms cultured in serum-free MEM for 90 days. On the last 2 days of culture,
the medium contained in addition 6-25 /*Ci/ml [U-14C]protein hydrolysate (56 mCi/
mAtom). Haemoglobin preparation for electrophoresis and counting of radioactivity
as described in Materials and Methods. Direction of migration from left to right.
, Absorbance at 510 nm; # — # , radioactivity of [14C]amino acid-labelled
products.
DISCUSSION
The results show that unincubated chick blastoderm which, cultured in a
serum-free medium, supports differentiation of precursor cells to the erythroid
cell line (Zagris, 1979), contained also the information to produce the early and
late Hbs and erythroid cell types. This shows that the formation of morphologically distinct cell populations and Hbs is independent of specific extra-embryonic
influences, such as progressive induction exerted by the yolk mass.
The numerous cells in mitosis give evidence that blood cells are the haemopoietic tissue around day 3-5 of culture. Later in culture, there is an occasional
blood cell observed in mitosis, and by day 8-0 of culture there is a conspicuous
absence of mitotic figures.
The small pyenotic cells which comprise the largest cell population on day
6-0 of culture seem to be early primary erythrocytes in the process of degeneration, fragments of degenerated cells, and/or these could be the innermost
blood island cells which according to Sabin (1920) disintegrate to form plasma.
In a thorough study of chick haemopoiesis in ovo, Lucas & Jamroz (1961)
consider similar elements to be embryonic thrombocytes that clump accompanied by degeneration as shown by loss of cytoplasm and by pycnosis of the
nucleus.
Erythroid cell differentiation in unincubated chick blastoderm 215
The electrophoretic pattern of Hb from the blood of unincubated blastoderm
after 9 days in culture is comparable to that of the 8-day chick embryo in ovo
(Bruns & Ingram, 1973; Brown & Ingram, 1974). The electrophoretic profile of
Hb is also comparable to that observed with primitive streak and older blastoderms in tissue culture (Hagopian & Ingram, 1971), and in suspension culture
(Hagopian et al. 1972; Chan & Ingram, 1973; Pine & Tobin, 1976) in a serumenriched defined medium.
Perhaps the most important aspect of our results is that, even though there is
neither axis formation nor apparent morphogenesis (Zargis, 1979), the morphologically distinct erythroid cells of the primitive and the definitive series appear
consecutively in culture as is the case with in ovo development.
Because blastoderms are cultured ventral side down on filter rafts, morphogenetic cell movements are inhibited mechanically, and the primitive streak is
not formed. This shows that the interacting components for the erythroid cell
formation in this system need not invaginate through a primitive streak. It is
possible that the endodermal component is the primary hypoblast which is
formed from the epiblast by a process of polyinvagination (Vakaet, 1962), while
the mesodermal component is the dispersed cells which are found between the
epiblast and the hypoblast even in the absence of a primitive streak (Azar &
Eyal-Giladi, 1979). The appearance of morphologically distinct erythrocytes
consecutively in culture shows that the embryo proper is not essential for the
formation of the early and late erythroid eelte. However, k is possible that some
of the erythroid cell populations, such as those of the definitive series, originate
inside the embryo in normal development.
I wish to thank Professor Hefzibah Eyal-Giladi (Hebrew Univeristy of Jerusalem, Israel)
for the critical reading of the manuscript.
REFERENCES
Y. & EYAL-GILADI, H. (1979). Marginal zone cells-the primitive streak-inducing component of the primary hypoblast in the chick. /. Embryol. exp. Morph. 52, 79-88.
BARKER, J. (1968). Development of the mouse haematopoietic system. I. Types of hemoglobin
produced in embryonic yolk sac and liver. Devi Biol. 18, 14-29.
BROWN, J. L. & INGRAM, V. M. (1974). Structural studies on chick embryonic hemoglobins.
/. biol. Chem. 249, 3960-3972.
BRUNS, G. A. P. & INGRAM, V. M. (1973). The erythroid cells and haemoglobins of the chick
embryo. Proc. Trans. R. Soc. B 265, 225-305.
CHAN, L. L. & INGRAM, V. M. (1973). Culture of erythroid cells from chick blastoderms.
/. Cell Biol. 56, 861-865.
CHAPMAN, B. S. & TOBIN, A. J. (1979). Distribution of developmentally regulated hemoglobins
in embryonic erythroid populations. Devi Biol. 69, 375-387.
CIROTTO, C., SCOTTO, Di TELLA A. & GERACI, jG. (1975). The hemoglobins of the developing
chicken embryos. Fractionation and globin composition of the individual component of
total erythrocytes and of a single erythrocyte type. Cell Differ. 4, 87-99.
EYAL-GILADI, H. & KOCHAV, S. (1976). From cleavage to primitive streak formation: a
complementary normal table and a new look at the first stages of the development of the
chick. I. General morphology. Devi Biol. 49, 321-337.
AZAR,
216
N. ZAGRIS
R. C. (1961). Haemoglobin formation in the chick embryo. Expl Cell Res. 25,
418-427.
HAGOPIAN, H. K. & INGRAM, V. M. (1971). Developmental changes of erythropoiesis in cultured chick blastoderms. / . Cell Biol. 51, 440-451.
HAGOPIAN, H. K., LIPPKE, J. A. & INGRAM, V. M. (1972). Erythropoietic cell cultures from
chick embryos. / . Cell Biol. 54, 98-106.
HELL, A. (1964). The initial synthesis of haemoglobin in de-embryonated chick blastoderms.
I. Metabolism of the blastodisc cultured in vitro. J. Embryol. exp. Morph. 12, 609-619.
LEVERE, R. D. & GRANICK, S. (1967). Control of haemoglobin synthesis in the cultured chick
blastoderm. / . biol. Chem. 242, 1903-1911.
LUCAS, A. B. & JAMROZ, C. (1961). Atlas of Avian Haematology. Washington, D.C.: United
States Department of Agriculture.
MIURA, T. & WILT, F. H. (1970). The formations of blood islands in dissociated-reaggregated
chick embryo yolk sac cells. Expl Cell Res. 59, 217-226.
MURRAY, P. D. F. (1932). The development in vitro of the blood of the early chick embryo.
Proc. R. Soc. Lond. B 111, 497-521.
O'BRIEN, B. R. A. (1960). The presence of haemoglobin within the nucleus of the embryonic
chick erythroblast. Expl Cell Res. 21, 226-228.
PINE, K. S. & TOBIN, A. J. (1976). Hemoglobin synthesis in isolated erythroid colonies from
the chick embryo. Devi Biol. 49, 556-562.
SABIN, F. R. (1920). Studies on the origin of blood-vessels and of red corpuscles as seen in the
living blastoderm of chicks during the second day of incubation. Contr. Embryol. 9,
215-262.
FRASER,
SCHALEKAMP, M., SCHALEKAMP, M., VAN GOOR, D. & SLINGERLAND, R. (1972). Re-evalu-
ation of the presence of multiple haemoglobins during the ontogenesis of the chicken:
electrophoretic and chromatographic characterization, polypeptide composition and
immunochemical properties. /. Embryol. exp. Morph. 28, 681-713.
SETTLE, G. W. (1954). Localization of the erythrocyte-forming areas in the early chick blastoderm cultivated in vitro. Contr. Embryol. 241, 223-237.
SPRATT, N. T. & HAAS, H. (1960). Morphogenetic movements in the lower surface of the
unincubated and early chick blastoderm. / . exp. Zool. 144, 139-157.
TOBIN, A. J., COLOT, H. V., KAO, J., PINE, S. K., PORTNOFF, S., ZAGRIS, N. & ZARIN,N. (1976).
Analysis of erythroid development. In Eukaryotes at the Subcellular Level: Development and
Differentiation (ed. J. Last), pp. 211-255. New York, Basel: Marcel Dekker.
VAKAET, L. (1962). Some new data concerning the formation of the definitive endoblast in the
chick embryo. / . Embryol. exp. Morph. 10, 38-57.
WAINWRIGHT, S. D. & WAINWRIGHT, L. K. (1974). Isolation of two erythrocyte cell populations from the early chick blastodisc and the further resolution of one into two essential
sub-populations. Expl Cell Res. 88, 143-152.
WENK, M. (1971). Effect of BUdR on initiation of hemoglobin synthesis by erythroid precursor cell. Anat. Rec. 169, 453.
WILT, F. H. (1967). The control of embryonic hemoglobin synthesis. In Advances in Morphogenesis (ed. M. Abercrombie & J. Brachet), pp. 89-125. New York, London: Academic
Press.
ZAGRIS, N. & MELTON, C. G. (1978). Hemoglobins in single chick erythrocytes as determined
by a differential elution procedure Z. Naturf. 33 c, 330-336.
ZAGRIS, N. (1979). Differentiation capacity of unincubated chick blastoderm in culture.
/. Embryol. exp. Morph. 50, 47-55.
{Received 26 November 1979, revised 16 January 1980)