/. Embryol. exp. Morph. Vol. 25, 3, pp. 385-403, 1971
385
Printed in Great Britain
The effects of actinomycin D on the early
development of quail and chick embryos
By F. S. BILLETT 1 , PATRICIA BOWMAN2
AND DOREEN PUGH 1
From the Department of Zoology, University of Southampton
SUMMARY
In agreement with previous work it has been found that treatment of chick embryos at an
early stage of development with small concentrations of actinomycin D (20-40 x 10~8 M)
produces abnormalities of the brain, neural tube and somites but allows near normal development of heart and blood islands. Similar effects are produced on quail embryos by even smaller
concentrations of actinomycin D (0-8-10 x 10~8 M).
In addition to the effect on the embryo, actinomycin D severely restricts the outgrowth of the
explanted blastoderm; an effect which becomes obvious after about 8 h culture and coincides
with the detachment of the blastoderm edge from the supporting vitelline membrane. Histological examination of the edge of the blastoderm shows that many cells have been killed by the
actinomycin D. The pronounced effect of actinomycin D on the growth of the blastoderm
cannot be ignored as a factor which causes the embryos to develop abnormally and it is
suggested that it may even be the primary cause of the abnormalities. Under these circumstances any interpretation of the effects produced in the embryos in terms of the selective
inhibition, or lack of inhibition, of specific messenger RNAs by actinomycin D seems to be
unwarranted.
Using [3H]actinomycin D an estimate has been made of the concentration of the compound
in the blastoderm tissues at the time when the embryonic abnormalities are produced. The
total DNA content of the blastoderms has also been determined. These measurements indicate that the ratio between actinomycin D molecules and guanosine residues in the treated
tissue is very low (of the order of 1:20000) and would probably not allow an effective
inhibition of DNA-dependent RNA synthesis.
INTRODUCTION
The ability of actinomycin D to bind specifically to the guanosine residues of
DNA and thus inhibit DNA-dependent RNA synthesis (Reich, Goldberg &
Rabinowitz, 1962; Shatkin, 1962; Reich, 1964) has led to its widespread use
as a chemical means of blocking the transcription process and thus depriving
cells of genetic information. In many cases the results obtained can be explained
so convincingly in terms of its known mode of action that the assumption is
increasingly made that, provided a low enough concentration of the compound
is used, the prime site of action of actinomycin D in living cells is DNA and its
1
Authors' address: Department of Zoology, The University, Southampton, SO9 5NH,
U.K.
2
Author's address: Institute of Animal Genetics, West Mains Road, Edinburgh, 9, U.K.
386
F. S. BILLETT, P. BOWMAN AND D. PUGH
effects on a multitude of biological systems can be interpreted as a direct consequence of this action. Although our own work is not in direct conflict with the
prevailing view we feel that the effects of actinomycin D on explanted avian
embryos are not entirely consistent with it as the evidence suggests that the
compound acts indirectly, rather than directly, on the embryos. Thus an interpretation in terms of the specific inhibition of mRNA in particular tissues, or
lack of inhibition due to preformed mRNA in others, may not be necessary to
explain the teratological effects of actinomycin D on explanted chick embryos
(Collini & Ranzi, 1967).
The first recorded observations of the effect of actinomycin D on chick
embryos are those of Pierro (1961 a, b) who found that the injection of 006250-125/tg of actinomycin D into the yolk sac of 48-h chicks resulted in the
formation of rumpless and trunkless embryos. This was followed by work on
explanted embryos (Klein & Pierro, 1963) using a modified Spratt technique
(Britt & Hermann, 1962). The application of similar small concentrations of
actinomycin D to later somite stages was found to inhibit the growth and
development of the posterior regions of the embryos.
Further work on explanted embryos by Heilporn-Pohl (1964), using another
modification of the Spratt technique (Spratt & Haas, 1960), directed attention to
the deleterious effect of actinomycin D on the development of neural tissue. This
confirmed observations previously made on amphibian embryos (Brachet &
Denis, 1963; Denis, 1963). The experiments on chick embryos demonstrated
that the application of 0-1-0-5 /tg/ml (8-40 x 10~8 M) actinomycin D to primitivestreak stages resulted in an almost complete inhibition of the development of
neural tissue. Treatment at a later stage (Hamburger & Hamilton (1951) stage 7,
about 30 h incubation) allowed normal development of the anterior part of the
nervous system but inhibited its development posteriorly. Further experiments
on the chick system include those of Hell (1964), and Wilt (1965), who showed
that haemoglobin synthesis in the early blastoderm was only partially inhibited
by small concentrations of actinomycin D, and, more recently, Gallera (1970)
who has shown that actinomycin D interferes with the inductive interaction
between Hensen's node and the ectoderm.
The observations reported in this paper concern the effects of actinomycin D
on both chick and quail embryos explanted according to modifications of the
New technique (New, 1955). The observations confirm the observations of
previous authors that whereas the developing neural tissue is very sensitive to
actinomycin D the blood islands and heart are apparently not (Collini & Ranzi,
1967). However, our own work leads us to conclude that the most sensitive part
of the system is not the embryo itself but the leading edge of the extra-embryonic
blastoderm. Damage to this tissue leads to a lack of growth of the entire blastoderm and it is impossible to ignore this fact either in interpreting the abnormalities produced or their significance in terms of actinomycin D action.
Apart from the difficulty of interpreting the morphological effects of actino-
Actinomycin D and avion development
387
mycin D in terms of the inhibition of DNA-dependent RNA synthesis in the
case of avian embryos it is apparent that the deleterious concentration of
actinomycin D would be sufficient to combine only with a very small proportion
of the DNA present in the explanted blastoderms. This has been confirmed by
estimations of the total DNA present in early chick blastoderms and measurements of the uptake of radioactive actinomycin D during the period of culture
when the abnormalities arise.
MATERIALS AND METHODS
The chick eggs were from a White Leghorn-Rhode Island cross and were
obtained from a local breeder. We are indebted to Vitamins Ltd for their
kindness in supplying quail eggs.
The actinomycin D was a gift from Merck, Sharpe and Dohme. The tritiated
compound was prepared from the non-radioactive sample at the Radiochemical
centre (Amersham) by a modification of Wilzbach's method; it was of
high activity (2 mCi/ml) and was supplied at a concentration of 200 /^g/ml in
ethanol. The final concentration applied to the embryo was within the range
used for the non-radioactive compound, i.e. 003/tg/ml. This concentration
of radioactive actinomycin D is sufficient for radioactive assay by scintillation
counting but not for autoradiography.
Both chick and quail embryos were cultured by modifications of the New
technique (1955). Embryos were removed from the eggs at early stages ranging
from the fully formed streak to six somites. Actinomycin D was added to both
the Pannet-Compton solution which covers the upper surface of the embryo and
to the albumen which lies beneath it. Before incubation at 38-5 °C embryos
were kept at 15 °C for about 18 h to allow time for the passive diffusion of the
culture media containing the actinomycin D into the embryonic tissues. Delaying the start of the incubation in this way does not seem to harm the embryos and appears to be a necessary precaution when teratogens are used in the
New technique (Billett, Collini & Hamilton, 1965). If the cultures are incubated
immediately after the addition of the teratogen it seems likely that its concentration, unless it diffuses very quickly in the embryonic tissues, will not correspond
to that in the external medium and one would be treating the selected stage with
an unknown concentration of the added compound.
Quail eggs are much smaller than those of chicks and consequently the amount
of vitelline membrane available for suspending the blastoderm is very much
reduced. Simple modifications were necessary to adapt New's method for these
smaller embryos. The essential features of the modification are illustrated in
Fig. 1. The glass ring of New's method is replaced by a ring of approximately
18 mm diameter made of stainless steel wire (cross-sectional diameter ca. 1 mm).
The ring is mounted on a circular ledge cut into a glass block approximately
6 mm thick. The central hole of the block is slightly less than the diameter of the
388
F. S. BILLETT, P. BOWMAN AND D. PUGH
wire ring. The hole is sealed off at the base by a glass plate held in position by
silicon grease. This arrangement forms a chamber which holds about 1 ml of
albumen. The quail blastoderm attached to the vitelline membrane is mounted
in the assembly in a similar manner to the chick embryo in the New culture. As in
the original method a Petri dish serves as a culture chamber to house the
assembly in an incubator.
Complete culture chamber
Base plate
Glass block
Stainless steel ring
40 mm
Fig. 1. Modified New culture chamber used for explanted quail embryos.
Both the chick and quail embryos were observed from time to time during
the period of culture which lasted from 8 to 24 h. At the end of the culture
period the blastoderms were fixed and stained according to the method of
Mahoney (1963). Standard histological procedures were used to examine the
blastoderm tissues. The areas of both fresh and fixed blastoderms were measured
according to a method already described (Billett, Collini & Hamilton, 1965).
The uptake of radioactive actinomycin D into the chick embryos was measured
in the following way. After incubation for a known period in the presence of the
radioactive compound the blastoderms were removed from the vitelline membrane, weighed, and homogenized in 1 or 2 ml of ice-cold water. The samples
Actinomycin D and avion development
389
were then frozen until required. For assay the homogenate was thawed and 0-2 ml
aliquots pipetted on to paper discs, 2-2 cm diameter, cut from Whatman No. 3
filter paper, and dried. The discs were then treated according to the procedure
of Mans & Novelli (1961). The samples were counted on a model 574 Tricarb
liquid scintillation counter (Packard Instrument Co.).
The DNA content of both area opaca and area pellucida was measured by a
procedure based on established techniques (Solomon, 1957). After removal from
the eggs the chick blastoderms were separated into the two areas using fine
scissors and the pieces pooled and deep frozen until required. After storage
appropriate numbers of blastoderm pieces were thawed and washed twice, by
centrifugation at 5000 g with ice-cold 5 % trichloracetic acid. The material was
then homogenized in ice-cold 80 % ethanol and centrifuged as before. Similar
successive treatments were made with ethanol/ether and ether to remove lipid
and the residue left to dry overnight. Soluble nucleotides were removed using
ice-cold 5 % perchloric acid and 1 M-hydrochloric acid. Finally the DNA was
extracted from the residue by three successive treatments with 1 M-HCI at
about 90 °C. After cooling, the DNA was measured in the pooled extracts by the
colorimetric procedure of Ceriotti (1952). At the end of the treatment the
extracted residue contained no detectable DNA.
Table 1. Development of explanted quail embryos {controls)
Embryos explanted and kept at ca. 15 °C for 18-20 h before incubation
at 38-5 °C for 24 h.
Stage
No. of
explanted* embryos
ES-MS
FS
HP
HF
5
24
9
20
,
Normal development
*
» Blood
Brain Neural tube Heart
islandsf
3
15
9
18
3
15
8
19
4
19
9
20
0
9
4
14
Av. no.
somites
9-4
155
220
19-6
* ES-MS = early to mid streak; FS = full streak; HP = head process; HF = head fold.
f Blood islands occupy more than two-thirds of the area vasculosa.
~
.,
,
RESULTS
Quad embryos
Using the modified New technique, and in the absence of actinomycin D, the
majority of the explanted quail embryos developed normally (Fig. 2 A; Table 1).
However, the streak stages survived less well, ca. 60 % normal development,
than the head-process and head-fold stages, over 90 % normal development. In
nearly all cases the hearts and somites developed normally. The most obvious
abnormalities in the controls, chiefly occurring in embryos explanted at the
streak stage, were seen in the brain and neural tube. Blood islands only fully
26
E M B 25
390
F. S. BILLETT, P. BOWMAN AND D. PUGH
occupied the area vasculosa after explanted head-fold embryos had been cultured
for 18-20 h. In embryos explanted earlier the blood islands although normal in
appearance were mostly reduced in number.
Table 2 and Fig. 2B show that a very small concentration of actinomycin D,
1-6 x 10~8 M, may completely inhibit normal development and an even smaller
concentration, 0-8 x 10~8 M, allows only 50 % of the embryos to develop normally. As with the controls abnormalities were seen chiefly in the nervous
system, especially the neural tube which in some cases was almost absent at the
Table 2. Effect of actinomycin D on the early development of the quail
Embryos explanted and kept at ca. 15 °C for 18-20 h before incubation
at 38-5 °C for 24 h.
Concn
actinomycin D
Stage
individual
Development f
embryos
t
explanted at* Brain Neural tube Heart
/MS
1-6X10- 8 M
(0-02/ig/ml)
MS
FS
FS
FS
HP
HP
IHF
0-8 x 10- 8 M
(0-01 /tg/ml)
/MS
FS
FS
FS
FS
FS
FS
HP
HF
HF
HF
HF
HF
Blood
islands
Somite
no.
ND
ND
A
A
N
ND
A
N
ND
ND
A
A
N
ND
A
N
ND
ND
N
A
N
ND
A
N
ND
ND
N
A
N
ND
A
N$
ND
ND
io§
8§
19
ND
0
20
N
N
N
N*
10
A
N
N
N
N
N
N
Abs
A
A
N
N
A
N
N
N
N
N
N
Abs
A
A
N
N
N
A
A
N
N
N
N
A
N
N
N
N
Nt
10§
9§
18
18
18
18
22
0
A
N
A
N
N
N
NJ
NJ
N
N
N
io§
12§
15§
22
* MS = mid streak; FS = full streak; HP = head process; HF = head fold;
t N = normal development; A = abnormal development; Abs = absent; ND = no
development.
\ Blood islands normal but reduced in number.
§ Somites poorly developed.
end of the culture period (Fig. 2D). Although the number of somites reached
normality in many cases the somites themselves were frequently badly developed;
this effect being associated with a generally poor formation of the postaxial
structure of the embryo (Fig. 2B, D). Apart from those cases in which treated
Actinomycin D and avion development
391
D
5 mm
Fig. 2. All embryos are shown at the same magnification.
(A) Quail embryo explanted at full streak stage, control, 24 h incubation.
(B) Quail embryo explanted at full streak stage, 002/tg/ml actinomycin D, 24h
incubation. Anterior part of embryo is fairly normal, but there is almost complete
failure of axial development beyond the 6th somite.
(C) Quail embryo explanted at full streak stage, 0-05 /^g/ml actinomycin D. There
is marked shrinkage of blastoderm and the embryo region is a small nob of tissue
after 24 h incubation.
(D) Quail embryo explanted at head-process stage, 005/tg/ml actinomycin D,
24 h incubation. There are about 14 pairs of somites; postaxial development is poor
and there is restricted outgrowth of blastoderm beyond the area vasculosa.
(E) Chick embryo explanted at head-fold stage, control, 24 h incubation.
(F) Chick embryo explanted at head-process stage, 005 /«g/ml actinomycin D, 25 h
incubation. Notice absence of somites, trace of neural tube, well-developed blood
islands and heart.
26-2
392
F. S. BILLETT, P. BOWMAN AND D. PUGH
100//m
Actinomycin D and avian development
393
blastoderms failed to develop an embryo there appeared to be little difference
between the development of the heart and blood islands in treated and control
embryos.
Chick embryos
The results of incubating chick embryos at
ment in 2-4 x 10~8 M actinomycin D for 12 and
and 4 respectively. These results indicate that
to the compound than quail embryos. Some
various early stages of develop24 h are summarized in Tables 3
chick embryos are less sensitive
two to three times the effective
Table 3. Effect of 2-4 x 10~8 M actinomycin D on the early
development of chick embryos cultured in vitro for 12 h
Embryos explanted and kept at ca. 15 °C for 15-20 h before incubation at 38-5 °C
Percentage of embryos showing
No. of abnormalities in particular regions
embryos
treated Neural tube Somites Blood islands
A
Stage treated
Full length streak
Head process
Head fold
1-3 Somites
4-6 Somites
Treatment*
AD
C
AD
C
AD
C
AD
C
AD
C
6
8
6
10
6
14
8
9
9
6
100
0
50
10
67
22
63
0
33
0
67
13
33
0
0
7
13
0
0
0
33
0
33
20
17
14
13
11
0
0
* AD = actinomycin D; C = control.
Fig. 3. All photographs are of chick embryos, A and B at the same magnification,
C-F at the same magnification.
(A) Embryo explanted at head-fold stage, control, 12 h incubation; there are
1.1 pairs of somites.
(B) Embryo explanted at head-fold stage, 004/ig/ml actinomycin D, 12h incubation. There are 12 pairs of somites; the neural tube is a streak of tissue beyond the
2nd somite.
(C) Transverse section through somite region of control embryo, explanted at headfold stage and incubated for 24 h.
(D) Transverse section through somite region of embryo explanted at head-fold
stage and treated with 003/<g/ml actinomycin D, and incubated for 24h. Neural
tube and somites are poorly developed and contain many dead cells.
(E) Transverse section through blastoderm edge of control embryo, explanted at
full streak stage and incubated for 24 h.
(F) Transverse section through blastoderm edge of embryo explanted at full streak
stage, treated with 003/*g/ml actinomycin D and incubated for 24h.
394
F. S. BILLETT, P. BOWMAN AND D. PUGH
teratogenic concentration for quails produces a similar degree of damage in the
chicks. At higher concentrations, 4-0 x 10~8 M actinomycin D, severe damage is
caused. After 24 h in this concentration, embryos explanted at the full-streak
and head-process stage frequently lack both neural structures and somites
although exhibiting fairly normal development of heart and blood islands
(Fig. 2F).
Tables 3 and 4 show that, as in the quail, the neural tube and somites are the
most sensitive structures (Fig. 3 A, B). Histological examination confirms that as
far as the embryo itself is concerned cell death and damage are largely confined to
the neural and somite tissue (Fig. 3 C, D) and reveals that in addition to the heart
and blood islands the notochordal tissue is comparately normal. The results also
show that as with other teratogens the early somite stages are less sensitive than
the presomite stages and that there appears to be a straightforward relation
between stage and the degree of abnormality.
Table 4. Effect of 2-4 x 10~8 M actinomycin D on the early
development of chick embryos cultured in vitro for 24 h
Embryos explanted and kept at 15 °C for 16-20 h before incubation at 38-5 °C
Treatment*
ON ON
Stage treated
Percentage of embryos showing
No. of abnormalities in particular regions
A
embryos
treated Neural tube Somites Blood islands
AD
C
AD
C
AD
C
AD
C
AD
C
Full length streak
Head process
Head fold
1-3 Somites
4-6 Somites
12
11
12
11
9
12
7
10
100
0
100
18
91
9
67
33
57
20
94
0
33
0
58
9
33
8
29
0
25
0
8
0
18
9
0
0
14
0
* AD = actinomycin D; C = control.
Table 5. Effect of actinomycin D on formation of somites after 8 and 24 h treatment of blastoderms explanted at head-process and head-fold stages and incubated
for a total of 24 h at 38-5 °C
Control
24 h
No. of observations
I Mean ± S.E.
Somite no.
1 Range
13
21-5± 17-30
Actinomycin D
24 h
11
9-4 ±2-4
0-20
Actinomycin D 8 h
+ 16 h under
control conditions
13
14-2 ±2-7
0-26
Actinomycin D and avion development
395
Fig. 4 shows that in actinomycin D-treated embryos the number of somites
formed during the first 12 h of culture matches that of the control. After 12 h
there appears to be a slight drop in the average number of somites in the treated
embryos which now have significantly less than the controls. This represents a
real situation, for although in some cases the number of somites reaches control
values, in others (about 30 % of the treated embryos) the somites have disappeared at the end of the 24 h incubation period (Table 5). The observations
show that actinomycin D treatment can result in the degeneration of previously
formed somites after about 12 h. This may be indicative of a general postaxial
degeneration as the disappearance of the somites is often associated with a
degeneration of the neural tube which sometimes assumes a beaded appearance
during the course of the culture.
30 r
Control
S 20
Z 10
Actinomycin D
10
20
Time of incubation (h)
30
Fig. 4. Comparison of number of somites developed in control and actinomycin
D-treated embryos. Blastoderms were treated with 2-4 x 10~8 M actinomycin D at
head-process to head-fold stage and incubated for 24 h at 38-5 °C. Each point is a
mean value derived from 5 to 16 embryos.
Effect of removing actinomycin D after 8 h incubation
Observations on somite numbers and on the growth of the blastoderm during
the course of 24 h treatments of chick blastoderms with low concentrations of
actinomycin D indicate an interruption of normal growth and development
about 8 h after the start of treatment. This suggests that the initial period of
treatment may be the most damaging. A limited number of experiments were
carried out in which actinomycin D (2-4 x 10~8 M) was removed after 8 h
incubation and replaced by normal culture media. The results are summarized
in Table 6 and show that as far as the embryo itself is concerned continuous
396
F. S. BILLETT, P. BOWMAN AND D. PUGH
treatment with actinomycin D is more damaging. However, the number of
somites formed is similar in both 8 and 24 h treatments (Table 5) and furthermore complete somite loss, after an initial period of formation, is also encountered in the 8-h treatments. The interruption of blastoderm growth also occurs
at about the same time whether the blastoderms are treated continuously for
24 h or merely for an initial period of 8 h; this is discussed in more detail later.
Table 6. Effect of removing actinomycin D after 8 h incubation in
2-4 x 10~8 M actinomycin D
Percentage of embryos showing
abnormalities in particular regions
A
Duration of No. of ,
,
treatment embryos Neural tube Somites Blood islands
Controls
Actinomycin D
8h
24 h
8h
24 h
9
11
14
12
11
9
50
91
11
9
27
58
0
9
7
18
50
Abnormal
40
30
o 20
10
Control
0-8X10"8M
1-6X10"8M
40X10"8M
Concentration of actinomycin D
Fig. 5. Comparison of outgrowth of individual blastoderms in control and actinomycin D-treated quail embryos which were explanted at full-streak to head-fold stages
and incubated for 24 h.
The effect of actinomycin D on the outgrowth of the blastoderm
The outgrowth of the blastoderm in both explanted chicks and quails is
drastically reduced by small amounts of actinomycin D. This is shown in
Fig. 5 (quail) and Fig. 6 (chick) and clearly illustrated in Fig. 2C, D, F. After
Actinomycin D and avian development
397
24 h treatment the areas attained by the blastoderms are in fact usually little
more than the areas they had when first explanted, indicating that little or no
growth occurs in the presence of actinomycin D. Outgrowth is restricted in all
cases in which the embryo exhibits abnormality and in many cases in which it
does not. The growth of individual blastoderms is shown in Fig. 7 and indicates
400
Controls
Actinomycin Dtreated
200
Som
150
Control
Actinomycin
D-treated
Fig. 6
10
20
Time of incubation (h)
Fig. 7
Fig. 6. Comparison of outgrowth of control chick blastoderms and those treated
with 2-4 x 10~8 M actinomycin D. Columns represent mean values and the number of
measurements, each corresponding to an individual blastoderm, are in parentheses.
FS = full-streak stage; HP = head-process stage; HF = head-fold stage; Som —
somite stage.
Fig. 7. Comparison of growth of individual control, and actinomycin D-treated, blastoderms. Embryos explanted at full-streak and head-process stages.
that the growth of treated and control blastoderms diverges sharply after about
8 h in culture. Fig. 8 shows the effect of removing actinomycin D after 8 h
culture: blastoderms do not resume their outgrowth when returned to normal
culture conditions and this suggests that the blastoderms are damaged irreversibly at this stage.
The lack of growth, and in some cases actual shrinkage, of the blastoderm in
the presence of actinomycin D is clearly associated with damage to the leading
edge of the blastoderm. In both quails and chicks this edge quickly becomes
398
F. S. BILLETT, P. BOWMAN AND D. PUGH
300
Controls
o
Actinomycin D- #
treated
o
#
200
10
20
Time of incubation (h)
Fig. 8. Effect on growth of blastoderm of removing actinomycin D after 8 h incubation and then culturing under control conditions. Embryos explanted at full
streak to head-fold stages.
S 100
•Activity of actinomycin D
in medium
•*=
50 <
Incubated at 38-5 °C
o Left at 15°C
5
10
Time in culture (h)
15
Fig. 9. Uptake of [3H]actinomycin D, 2-4 x 10~8 M, into explanted chick blastoderms
during the first 12 h of culture.
opaque, thickened and detached during the initial stages of treatment. Histological examination reveals what observation of the living cultures suggests,
namely, extensive areas of cell death in that part of the blastoderm which would
normally be attached to the vitelline membrane (Fig. 3E, F).
Actinomycin D and avian development
399
Uptake of [sH]actinomycin D by chick blastoderms
A number of determinations were made of the amount of [3H]actinomycin D
in chick blastoderms at the start of the culture and during the first 12 h of
incubation. Twelve hours cover the period when the actinomycin D appears to
exert its effective action. After 12 h the blastoderms are usually detached and the
embryos appear to be degenerating; under these circumstances further measurement would be exceedingly difficult to interpret. The concentration of actinomycin D was 2-4 x 10~8 M. The observations are summarized in Fig. 9; they
show that at the start of the incubation the tissues are more or less in equilibrium
with the surrounding medium and that there is a slight rise in the actinomycin D
content of the blastoderm tissues as the incubation proceeds at 38-5 CC. This rise
corresponds to a small increase in the concentration of actinomycin D in the
blastoderm tissues, possibly rising from 2-4 to about 3-0 x 10~8 M.
The DNA content of chick blastoderms
Table 7 summarizes the measurements made using Ceriotti's reaction to
determine the DNA content of chick blastoderms. These measurements cover
the initial and effective period of action of actinomycin D. Although no great
stress can be placed on the accuracy of the determinations they indicate that the
embryos contain approximately 1 fig and the extra embryonic tissue from
5-30 /tg DNA, the latter obviously increasing as the blastoderms expand.
Table 7. DNA content of chick blastoderms
[ig DNA /embryo*
Area pellucida
Stage
Full streak-head
process
Head fold 2 somites
3-6 Somites
7-9 Somites
No. of
determinations Mean
Area opaca
Range
No. of
determinations Mean
Range
0-9
0-7-1-1
6-6
3-8-10-7
1-1
0-7-1-6
9-1
6-4-150
1-3
3-8
1-1-1-5
17-6
32-6
10-4-240
23-8-38-0
* No. of embryos/determination: 4-29 (area pellucida), 1-12 (area opaca)
DISCUSSION
The observations presented in the previous section suggest that the teratogenic
effects of actinomycin D on explanted chick and quail embryos are not obviously
related to a selective inhibition of DNA-dependent RNA synthesis (Collini &
Ranzi, 1967) and that the results can be interpreted in a simpler, but less topical,
400
F. S. BILLETT, P. BOWMAN AND D. PUGH
fashion. This interpretation rests on a consideration first of the nature of the
abnormalities and the way in which they are produced and secondly of the very
small concentration of actinomycin D required to produce the deleterious
effects on the embryos.
After 24 h culture in the presence of concentrations of actinomycin D ranging
from approximately 1 to 4 x 10~8 M both chick and quail embryos show more or
less extensive damage to neural structures and somites but in contrast the heart
and blood islands are almost normal. This particular pattern of abnormalities
cannot, however, be regarded as specific to actinomycin D. Several teratogens
produce a similar picture, for example, 2,4-dinitrophenol (Bowman, 1967), and it
must be remembered that the developing neural and somite structures appear to
be generally sensitive to chemical insults whereas the heart and blood islands
frequently survive similar conditions. Apart from its lack of specificity the
pattern of damage produced by 24 h culture in actinomycin D is, at least partly,
caused by the degeneration of previously formed somite and neural tissue
which particularly affects the posterior axis of the developing embryo. This
is seen especially in the case of the somites where observations on the chick
embryo revealed that in some cases previously formed somites disappeared
during the latter part of the culture. In other words the absence of neural
structures and somites after 24 h culture in actinomycin D is not due to a primary inhibition of these structures by the compound. Under such circumstances
extensive examination of dead and dying cells in the affected areas is unrewarding and was not pursued in the present investigation.
If the embryonic abnormalities are produced indirectly, what is the primary
effect of the treatment which could lead to the degeneration of previously
formed structures in the embryo ? A sufficient answer to this question appears to
be given by the observed effect of actinomycin D on the growth of the extraembryonic blastoderm. The treatment not only completely inhibits growth in
many cases but causes extensive cell death in the leading edge of the tissue. The
presence of dead and dying cells at the blastoderm edge often leads to its complete detachment from the vitelline membrane after about 8 h culture. Under
such conditions the embryo will be deprived both of adequate nutrition and of
the simple mechanical requirements for normal development in the New culture
(New, 1959; Bellairs, Bromham & Wylie, 1967). It is a common observation of
workers using the technique that if the blastoderm fails to attach to the vitelline
membrane during an early stage of the culture then embryonic abnormalities
are produced and in our own experience this can result in gross abnormalities
in which only the heart and a small number of blood islands survive as recognizable parts of the embryo. The occurrence of early and extensive cell death in the
edge of the blastoderm in the presence of actinomycin suggests some kind of
selective action, possibly based on the characteristic phagocytic activity of the
cells of that region (Bellairs & New, 1962).
The other consideration which leads one to suspect that the effects produced
Actinomycin D and avian development
401
by actinomycin D are not simply related to its ability to bind with the guanosine
residues of DNA in vitro is the very small concentration of the compound
required to upset development in the case of avian embryos. In this connexion it
is worth recalling that for sea-urchin embryos about 3 x 10~6 M actinomycin D
is required for the inhibition of mRNA production (Gross & Cousineau, 1964).
This concentration is over 100 times that used in the present study. The only
conceivable target, in terms of the inhibition of DNA-dependent RNA synthesis, in the case of the chick system might be the ribosomal cistron. According
to Perry (1963), working with tissue culture cells, nucleolar RNA synthesis is
some 10-100 times more sensitive than that which produces mRNA. However,
even an inhibition of ribosomal RNA does not seem likely in the case of the
chick system if the ratio between the amount of actinomycin D in the blastoderm and its DNA content is considered at the time when the damage is caused.
It is of course possible that the actinomycin D is not uniformly distributed
throughout the blastoderm tissues and that sufficiently high local concentrations
of the compound are reached in particular cells to inhibit RNA synthesis. For
instance a high concentration of actinomycin D might be expected in the cells
of the blastoderm edge because of their phagocytic activity (Bellairs & New,
1962). Preliminary autoradiographic studies using [3H]actinomycin D have,
however, failed to reveal any local accumulations of actinomycin D either in
the embryo or in the extra-embryonic blastoderm (F. S. Billett & D. Pugh,
unpublished observations). The calculation of the ratio between actinomycin D
molecules and guanosine residues, which is given below, is based on the assumption of a uniform distribution of actinomycin D in the blastoderm tissues.
The observations on the uptake of [3H]actinomycin D indicate that when the
blastoderms are incubated at 38-5 °C the level in the tissues rises slightly above
that of the surrounding medium and reaches a concentration of approximately
3-5 x 10~8 /tg/mg tissue. Estimations of the DNA content of the blastoderms
indicates that the tissue contains approximately 1 fig DNA/mg tissue. This
finding agrees with that of Solomon (1957) and is in general agreement with the
amount of DNA found in a variety of animal tissues (Long, 1961). Assuming the
extreme case in which all the DNA in the tissue combines with all the available
actinomycin D, and calculating on a molar basis, the ratio between actinomycin
D molecules and guanosine residues would be of the order of 1:20000. This does
not seem sufficient for an effective inhibition of DNA-dependent RNA synthesis.
Although the DNA measurements may lack accuracy in view of the small
amounts of tissue used and the technique employed the calculated ratio between
actinomycin D molecules and their presumed targets deserves serious consideration, not only in the present study, but in any attempt to explain the effects
of actinomycin D on complex biological systems.
This work was supported by a grant from the Medical Research Council. We are grateful to
John Baynham for taking the photographs.
402
F. S. BILLETT, P. BOWMAN AND D. PUGH
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{Manuscript received 15 October 1970)