/. Embryo/, exp. Morph. Vol. 50, pp. 47-55, 1979
Printed in Great Britain © Company of Biologists Limited 1979
47
Differentiation capacity of unincubated chick
blastoderm in culture
By NICHOLAS ZAGRIS 1
From the Tissue Culture Laboratory, School of Sciences,
University of Patras
SUMMARY
Unincubated (stage X) chick blastoderms cultured in plain Dulbecco's modified Eagle
medium in the absence of serum, synthesize haemoglobin and melanin 2-5-30 and 6 0 70 days, respectively, after the beginning of culture. The capacity of unincubated blastoderm
to form haemoglobin and melanin is a response to intrinsic genotypic instructions rather
than to the active intervention of substances continuously supplied by the yolk. 5-Bromodeoxyridine, applied during the first and second days of unincubated blastoderm in culture,
is incorporated into DNA and perturbs the formation of haemoglobin and melanin, perhaps,
by modifying gene expression.
INTRODUCTION
The blastoderm of the newly laid chicken egg contains about 60000 cells and
consists of the one- to two-cell thick area pellucida, the formation of which has
been just completed, and the peripheral thicker area opaca (Spratt & Haas,
1960; Eyal-Giladi, 1975). The morphology of the blastoderm from the beginning of cleavage to the appearance of the primitive streak is described in an
elegant paper by Eyal-Giladi & Kochav (1976).
Most of the experimental work with chick blastoderms intending to investigate early developmental processes such as haemoglobin (Hb) synthesis
has been performed on embryos from the primitive streak stage and older
(Ingram, 1974; Wainwright & Wainwright, 1974; Wilt, 1974; Pine & Tobin,
1976). The purpose of the present communication is to elucidate the differentiative capacity of unincubated chick blastoderm in a simple, chemically defined
medium independent of the influences operating in ovo. 5-Bromodeoxyuridine
(BUdR), the thymidine analog which is readily incorporated into DNA (Rutter,
Pictet & Morris, 1973), was used to manipulate the determination of cell type
and its subsequent expression.
1
Author's address: Tissue Culture Laboratory, School of Sciences, University of Patras,
Patras, Greece.
4-2
48
N. ZAGRIS
MATERIALS AND METHODS
Culture
Freshly laid fertilized chicken eggs of the White Leghorn breed were used the
day they were obtained from a local hatchery.
The explanted blastoderms were checked individually under a dissecting
microscope and staged accurately according to Eyal-Giladi & Kochav (1976).
Only blastoderms at stage X were used for experiments.
Blastoderms were explanted and washed free of the vitelline membrane and
any adhering yolk in chick saline under a dissecting microscope (15 x), were
placed ventral side down on 2 cm discs ('rafts': four embryos/raft) cut from
Whatman no. 4 filter paper, and were cultured in Petri dishes with plain
Dulbecco's modified Eagle medium (MEM) in the absence of serum, in a
moist air-CO 2 atmosphere adjusted to keep the culture medium neutral, at
38 °C. Blastoderms attached on filter rafts were placed in fresh medium every
day, and were examined under a dissecting microscope (10-25 x ) twice a day.
Unincubated blastoderms in chick saline were dissociated by pipetting in
and out of a 13-5 cm tipped Pasteur pipette until a fine cell suspension was
obtained. Slides were prepared from this suspension, and cells were stained
with Harris' haematoxylin overnight.
Embryos, media, and glassware were handled with sterile precautions.
5-bromodeoxyuridine
BUdR (Sigma) was dissolved in chick saline and was included in the culture
MEM at a final concentration of 1 - 6 2 X 1 0 ~ 4 M . TO determine the time of
sensitivity to BUdR, stage-X blastoderms were cultured in MEM containing
BUdR for their first day only, second day only, first and second days continuously, and their third day only in culture. At the end of incubation in
BUdR-containing MEM, blastoderms were rinsed five times by floating on
plain MEM (5-10 min each time), were transferred to plain MEM, and culture
was continued indefinitely. Parallel control blastoderms were cultured in plain
MEM for the same period of time.
Measurement of DNA
Groups of stage-X blastoderms which were cultured in MEM for 3 days,
2 days, 1 day, and freshly explanted were transferred to MEM containing either
6-6/tCi/ml [methyl-3H]thymidine 5'-triphosphate (30 Ci/mmol) or 13-3/*Ci/ml
5-bromo-2'-deoxyuridine-6-3H (728 mCi/mmol) and their culture continued for
one additional day.
At the end of culture, blastoderms of each group were rinsed with chick
saline and frozen ( - 4 0 °C) until use. The labelled blastoderms were thawed
and were homogenized in 0-1 ml ice-cold TKM buffer (0-01 M Tris-HCl,
Differentiation capacity of chick blastoderm
49
003 M-KC1, 002 M-MgCl2, pH 7-5) per embryo with a Dounce homogenizer
using 50 strokes of a tight pestle.
Whole homogenate (40 JLI\) from each group was placed in individual finger
tubes together with an equal volume of 0-6 N-KOH (final concentration 0-3 NKOH) to hydrolyse the RNA by incubation at 37 °C for 5 h.
The samples from the reaction mixture were collected on Whatman GF/C
glass fibre discs, 2-5 cm in diameter, which were suspended on pins mounted
on a styrofoam block. The discs were dried, were placed into 10 % trichloroacetic acid (TCA) at 4 °C for 10 min, into 5 % TCA (4 °C) for 10 min, rinsed
once with 5 % TCA, rinsed twice with ethanol, twice with ethanol-ether (3:1,
v/v) and twice with ether (Tobin et al. 1976). Radioactivity was determined in
10 ml of toluene containing 0-5 % PPO, 003 % POPOP in a liquid scintillation
spectrometer.
RESULTS
Figure 1 shows the cells of mechanically dissociated unincubated blastoderms
which seem to consist of at least two cell populations.
Within the first 2 days of culture in MEM which supplies their non-specific
needs, blastoderms raise a transparent bubble-like structure which becomes
cystic, and form outgrowths of whitish tissue. The following days the blastoderm
continues growing, but there is no axis formation nor apparent morphogenesis.
However, there are visible manifestations of cytodifferentiation.
The first visible differentiative event is the appearance of Hb, usually inside
the transparent bubble-like structure, 2-5-3-0 days after beginning of culture
(Fig. 2). Benzidine-peroxide staining revealed that the small red spots observed
(10-25 x) at 2-5 days of culture are groups of erythroblasts containing Hb.
These erythropoietic foci form isolated blood islands which are visible to the
naked eye under bright light by 30-3-5 days of culture. The blood islands
anastomose and form a network after day 5-0 of culture. Despite active mitotic
division of blood cells (Zagris, 1978), blood islands disappear after 10 days of
culture. These stream and are lost in the culture medium after disintegration of
the transparent bubble of which maintenance can, perhaps, no longer be
supported by the culture medium.
The second visible differentiative event is the appearance of melanin (M) in
the whitish tissue outgrowths, and in close, or, often, in intimate association
with the blood islands 6-0-7-0 days after the beginning of culture. Melanopoietic foci first appear (10-25 x) as small granules tracing two neighbouring
semi-circular pigmented areas, change from faint brown to solid black, readily
visible to the naked eye during the first day following their appearance, and
progressively increase in diameter. Occasionally melanin-forming tissue appears
around the edge of the blastoderm, as well. Figure 3 shows a typical embryo.
Although disintegration of the blastoderm sets in about 10 days after beginning
of culture, the melanin-forming tissue survives without difficulty and lends
50
w
Differentiation capacity of chick blastoderm
51
100
80
|
60.
o
o
*
40
20
1
2
1-2
Time (Day)
3
Fig. 4. Effect of BUdR (1-62 x 10~4 M) on haemoglobin and melanin synthesis in
stage-X blastoderms in culture. Groups of stage-X blastoderms were cultured in
MEM, and on the days indicated they were placed in MEM-containing BUdR.
Blastoderms were rinsed, their culture continued in plain MEM, and were scored
for the presence of haemoglobin (D) and of melanin ( • ) 4 days and 7 days,
respectively, after beginning of culture. The results are expressed as a comparison
(%) to blastoderms cultured continuously in plain MEM. The time scale refers to
the day blastoderms were cultured in MEM-containing BUdR.
itself to indefinite culture. We have kept, for example, melanin-forming tissue
in culture for more than 10 months now.
To define the time of acquisition of insensitivity to BUdR in the culture
system used here, stage-X blastoderms are transferred from control to BUdRcontaining MEM at various times of their culture. 1-62 x 10~4 M BUdR is used
because this concentration is known to suppress blood island formation
without introducing visible signs of toxicity (Miura & Wilt, 1970).
Fig. 1. Loosely packed cells of mechanically dissociated unincubated (stage-X)
chick blastoderms. The cells were stained with Harris' haematoxylin stain. Scale
bar = 10 /mi.
Fig. 2. Stage-X blastoderm, attached on Whatman no. 4 filter paper, was cultured
in liquid MEM for 2-7 days, 38 °C. Culture conditions as described in Materials and
Methods. The dark area shows haemoglobin stained with benzidine-peroxide stain.
Scale bar = 1 mm.
Fig. 3. Stage-X blastoderm, attached on Whatman no. 4 filter paper, was cultured
in liquid MEM for 12 days, 38 °C. Culture conditions as described in Materials
and Methods. The dark pigmented areas show formation of melanin. Scale bar =
1 mm.
52
N. ZAGRIS
14
12
10
Time (Day)
Fig. 5. Incorporation of [3H]thymidine (—•—) and of [3H]BUdR (— x—) into
total DNA by stage-X blastoderms. Stage-X blastoderms cultured in MEM were
pulsed for 1 day with 6-6/iCi/ml [methyl-3H]thymidine 5'-triphosphate (30 Ci/
mmol), or with 13-3/iCi/ml 5-bromo-2'-deoxyuridine-6-3H (728 mCi/mmol) on
the days indicated, and the culture was interrupted at the end of the pulse. Measurement of DNA as described in Materials and Methods. The time scale refers to the
day blastoderms were pulsed with [3H]thymidine or with [3H]BUdR. Values are
given as the means with S.E. as vertical bars. Each point is the average of three
experiments.
Figure 4 shows that only about 5 % and 22 % of stage-X blastoderms synthesize Hb and M, respectively, when their first day in culture is spent in BUdRcontaining MEM. In all cases the erythropoietic area is visible only under
stereoscopic observation (10-25 x) before and after staining with benzidineperoxide stain, while the melanophoretic area is 1/5 the size of controls and is
evident to the naked eye under very close inspection. Blastoderms appear
healthy on their second day of culture in plain MEM, then disintegration sets in.
When blastoderms are transferred to BUdR-containing MEM for only the
second day of their culture, 22 % of blastoderms form Hb, though the erythropoietic area is 1/4 the size of controls, while the melanopoietic area is indistinguishable from the control. Blastoderms remain apparently quite healthy.
The formation of both Hb and M is completely suppressed in blastoderms
Differentiation capacity of chick blastoderm
53
cultured continuously for their first and second days in BUdR medium, while
both Hb and M are at control levels in blastoderms placed in BUdR-containing
MEM for only the third day of their culture.
In all cases that Hb and M are synthesized, they are first observed 2-5-3-0
and 6-0-7-0 days, respectively, after the blastoderms were placed in culture at
38 °C.
Figure 5 shows DNA synthesis in blastoderms given 1 day pulses with
3
[ H]thymidine and [3H]BUdR for only the first, only the second, only the third,
or only the fourth day of culture. Culture was interrupted at the end of the
pulse rather than after a chase. There is extensive DNA synthesis on days 1-0
and 2-0 of culture, a decrease on day 3 0 and a slight increase on day 40 of
culture. [3H]BUdR reaches its highest level of incorporation into DNA the
second day in culture while [3H]thymidine reaches its highest level the first day
and then decreases somewhat on the second day of culture. The difference in
incorporation between [3H]BUdR and [3H]thymidine into DNA may reflect
preferential incorporation of BUdR into specific DNA sequences the first
2 days of culture, and in the following days its incorporation may become
non-specific. It is important to note that blastoderms incorporate [3H]BUdR
extensively even during the BUdR insensitive period.
DISCUSSION
It has been established that blood cells arise from cell condensations of the
splanchnopleuric mesoderm in close association with the underlying endoderm
(Wilt, 1967; Miura & Wilt, 1970), and that pigment cells arise from the neural
crest (Harrison, 1969; Weston, 1970). In the embryo in ovo, melanophores
begin to differentiate about the time blood circulation starts (Harrison, 1969).
Our results show that the in vitro chronological sequence of differentiative
events mimics that of differentiation in ovo in that synthesis of M follows Hb
synthesis. The survival and indefinite culture of the melanopoietic area under
our simple culture conditions indicate that melanophores are largely adaptive
to external conditions, and is an example of terminally differentiated cells
maintained under simple conditions in culture.
The nutritive culture medium in which unincubated blastoderms begin to
synthesize highly specific molecules seems to be adequate substitute for the
nutritive components of the yolk. This is an indication that the capacity of
unincubated blastoderm to form Hb and M is a response to intrinsic genotypic
instructions stamped upon it rather than to active intervention of substances
continuously supplied by the yolk.
BUdR action is associated with its incorporation into DNA. Several authors
have reported preferential incorporation of BUdR into moderately and highly
repetitive sequences of DNA rather than random incorporation over the whole
DNA as is the case with [3H]thymidine in various cell types (Schwartz & Kirsten,
54
N. ZAGRIS
1974; Strom & Dorfman, 1976; Durante et al. 1977; Seyer & Lescure, 1977).
Matthes et al. (1977) have shown that histones bind tighter to BUdR-containing
DNA in rat liver. This tight binding could, for instance, prevent the displacement of histones by certain regulatory proteins thus modifying their binding to
DNA and could block transcription at specific sites.
Our results show that the first and second days of stage-X blastoderm in
culture comprise a period of active DNA synthesis critical to the subsequent
formation of Hb and M. BUdR incorporated into DNA during this time
perturbs the formation of Hb and M, perhaps, by modifying gene expression.
It is likely that BUdR induces cellular identity changes and/or that BUdR
affects the development of important cellular 'homotypic' and/or 'heterotypic'
associations.
I wish to thank Professor Theony Valcana for reading the manuscript, and Ms Maria
Stefanopoulou for typing the manuscript.
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(Received 9 June 1978, revised 6 November 1978)