/. Embryol exp. Morph. Vol. 39, pp. 253-259, 1977
253
Printed in Great Britain
Developmental effects of X-irradiation of
early Drosophila embryos
By M. BOWNES 1 AND L. A. SUNNELL 2
From the Center for Pathobiology, University of California at Irvine
and The Department of Biology, University of Essex
SUMMARY
Drosophila embryos were treated at specific stages during early embryogenesis with
various doses of X-irradiation. The lethality at various times during development was
established and pattern defects in the adults noted. It was observed that the most sensitive
stages of embryogenesis to X-ray-induced lethality were also the stages where most morphological defects were found in the adults which emerged. This suggests that presumptive
larval and adult cells are sensitive to X-rays at the same stages of embryogenesis.
INTRODUCTION
X-irradiation of embryos causes imaginal and abdominal defects in some of
the adults which hatch (Ulrich, 1951; Postlethwait & Schneiderman, 1973;
Wieschaus, 1974,1975). These defects were induced after 7 ± 1 h of development,
yet in other techniques such as microcautery or puncturing, imaginal defects
were induced at the blastoderm stage (Bownes & Sang, 1974 a, b; Bownes,
1915a, 1976). These experiments were designed to see when, precisely, the
embryo is sensitive to X-rays. There are two possible points of sensitivity; the
first is in the formation of the larva and may lead to death of the egg or embryo
(Wurgler & Ulrich, 1976) and the second is the formation of the adult and may
lead to a defective morphology. It would be of interest to know if these two
systems were most sensitive at the same or different times during embryogenesis, and to know when the prospective disc cells are most susceptible to
the induction of defects in the adult using X-rays. Eggs were X-rayed at three
different doses for the first ten stages (9 h) of embryogenesis (Bownes, 1975 b)
and the embryonic, larval and pupal mortality and the proportion of defective
adults were calculated for each stage.
1
Author's address: Department of Biology, University of Essex, Wivenhoe Park, Colchester,
CO4 3SQ, Essex, England.
2
Author's address: School of Biological Sciences, University of California, Irvine, California
92717, U.S.A.
17
EMB
39
254
M. BOWNES AND L. A. SUNNELL
MATERIALS AND METHODS
Eggs were collected from Oregon R females of DrosophiJa melanogaster on
yeasted agar plates and left at 25 °C, until they reached an appropriate age.
They were then dechorionated in 3 % sodium hypochlorite. Eggs were then
selected at precise developmental stages using a Wild dissecting microscope.
Stage
Stage
Stage
Stage
Stage
Stage
2,
3,
4,
5,
6,
7,
nuclear multiplication
(1/4-1 h)
pole cells present
(1-2 h)
early syncytial blastoderm
(2-2^ h)
late syncytial blastoderm
(2|-3 h)
cellular blastoderm
(3-3^ h)
ventral furrow formed, cephalic furrow formed, beginning
posterior midgut invagination
(3|-4i h)
( 4 | - 5 | h)
Stage 8, posterior midgut pocket invaginates into the embryo
Stage 9, fore and hind guts continue to invaginate; segmentation
begins to form
(5^—8 h)
Stage 10, head segment invaginated; segmentation becomes distinct
ventrally. A dark central yolk patch is present which spreads
to the dorsal edge half-way along the anterior-posterior
axis. At this point there is a gap between the yolk and the
vitelline membrane
(8-9 h)
The selected eggs were placed on agar plates and were X-rayed at 500 rpm.
(using a general electric X-ray machine; 250 kV; 3mA). Eggs at each stage were
placed in the machine for 1, 2 or 4 min. The plates were then left at 25 °C for
24 h. Control eggs were selected and treated in every way like experimental
eggs but not exposed to X-irradiation. At some developmental stages, especially
the stages during syncytial blastoderm formation, the eggs were particularly
sensitive to the large degree of handling necessary for staging accurately. For
this reason staged control eggs were taken from every egg collection and the
experimental results of X-irradiation were always corrected for lethality in the
appropriate controls. Experiments at each stage and X-ray dose were repeated
on a minimum of six different collections of eggs. Often many more experiments
were done in order to obtain sufficient adults. The number of eggs irradiated
at each stage and dose ranged from 200 to 500. The variability between individual
runs ranged from standard errors of 0 % through most samples with standard
errors around 3-4% to one stage with large variability between runs and a
standard error of 8 %.
After 24 h the eggs were classified into (1) hatched larvae; (2) dead eggs with
no differentiated structures, the eggs had either a mottled white appearance or
a yolk mass shrunk at the centre of the egg, (3) abnormal embryos which developed but failed to hatch (some of these have a brown decaying appearance). For
a detailed explanation of how these different kinds of lethality are thought to
X-irradiation of Drosophila embryos
255
arise from X-irradiation see Wurgler & Ulrich (1976). After this yeast paste
was added to the plates. Any pupae which formed were transferred to vials.
Hatched flies were checked for any morphological defects. Pupae failing to
hatch were also dissected and any pharate adults were also analysed for
morphological defects. Adults were mounted in Gurr's water mounting medium
for a more detailed analysis of the defects.
RESULTS
Figure 1 is a graph showing the percentage larval hatch after irradiation at
500 R, 1,000 R and 2,000 R, corrected for the control hatch at each stage.
Clearly, young stages of embryogenesis at stages of nuclear migration and early
syncytial blastoderm formation are most sensitive to X-rays. However, the
youngest embryos in the early stages of nuclear division survive better than the
later stages of pole cell formation and the syncytial blastoderm. This was previously observed by other authors and is reviewed by Wurgler & Ulrich (1976).
This observation is probably the result of there being fewer nuclei which could
be hit by X-irradiation at the earlier stages of embryogenesis. Survival remains
above 70 % after 500 R and 1000 R, yet irradiation at 2000 R shows another
sensitive period at stage 8 when posterior midgut invagination is in progress.
Figure 2, which plots the percentage of treated eggs which survive to form
pupae, reflects the extra sensitivity to X-irradiation at stages 3, 4 and 8 even
more dramatically. Here there is a decrease in the number of treated, stage 8
eggs which pupate after all doses of irradiation. This graph also shows that at
2000 R very few eggs eventually form a puparium except at the stages around
blastoderm formation when the egg is especially resistant to X-rays.
Table 1 summarizes the number of pharate adults which either emerged
or were dissected from the puparium with defective imaginal disc or histoblast
derivatives. These defects included deletions of complete or partial imaginal
disc or histoblast derivatives, and pattern duplications of parts of disc or
histoblast derivatives. Some morphological defects were observed after irradiation at 2000 R but too few adults survived the treatment for them to be statistically valid. Figure 3 demonstrates the percentage of the puparia which were
formed which led to defective pharate adults. (Again the data are corrected for
any defects in the controls at each stage). Too few adults hatched before stage 5
after any dose of irradiation to give valid percentages. There were some defective
adults after irradiation of all stages at 1000 R and all stages from blastoderm
formation onwards at 500 R. At 500 R the percentage of defective flies remained
around 10 % for all the stages of development studied. At 1000 R, however,
there is a large peak in the number of defective adults at stage 8.
It appears then, that stage 8, which is one of the most sensitive periods
during embryogenesis and leads to the death of many embryos and larvae, is
also the stage when the prospective imaginal disc cells are most sensitive to
X-irradiation.
17-2
256
M. BOWNES AND L. A. SUNNELL
100
90
80
70
60
50
40
30
20
10
-O
4
5
6
7
8
Stage of development
I
.|-1
1-2
l
I
l
10
I
2-2} 21-3 3-31-31-41 41-51 51 8 8 9
Equivalent age of standard embryo at 25 ' C (h)
Fig. 1. Percentage larval hatch (corrected for control hatch into larvae at each
stage) plotted against developmental stage at time of irradiation. (x — x), 500 R;
(O—O), 1000 R; (A
A), 2000 R.
^
^
100
90
_x
i
i 80
I 70 -
^x
i
i
o
I
50 -
|
40
30
</oo>
\
it
/
/ A NJ
g? 20
"8 io
2
3
1
l
|-1
1-2
5
6
7
8
Stage of development
4
l
I
i
i
/
A
A
9
10
i
l
2-21- 21-3 3-31 31-4141-5i5i 8
i
8-9
Equivalent age of standard embryo at 25'C (h)
Fig. 2. Percentage treated eggs forming puparia (corrected for control pupation at
that stage) plotted against developmental stage at time of irradiation. ( x — x ), 500
R; (O
O), 1000 R; (A
A), 2000 R.
50
— 40
30
|
20
10
10
Stage of development
I
l
i
l
21-3 3-3131-4141-5151-8 8-9
Equivalent age of standard embryo at 25 °C (h)
Fig. 3. Percentage pupae which produced abnormal pharate adults (corrected for
number of abnormal controls at each stage) plotted against developmental stage at
time of irradiation. (x — x), 500 R; (O
O), 1000 R.
2
3
4
5
6
7
8
9
10
2
3
4
5
6
7
8
9
10
2
3
4
5
6
7
8
9
10
Stage of
embryos
117
94
100
112
189
138
273
179
163
18
0
5
121
108
129
231
111
130
20
0
2
213
114
106
88
74
83
0
0
0
0
0
0
0
0
0
500
500
500
500
500
500
500
500
500
1000
1000
1000
1000
1000
1000
1000
1000
1000
Total
pupae
formed
Dose
X-rays
(Roetgen)
4
0
4
8
5
2
6
4
2
4
0
1
6
13
12
39
11
18
2
0
0
12
15
10
43
30
24
Total
abnormal
4
0
4
8
5
2
5
4
2
3
0
1
6
12
10
31
7
17
2
0
0
12
10
6
20
15
19
0
0
0
0
0
0
1
0
0
1
0
0
0
1
3
11
5
2
0
0
0
0
8
4
30
16
5
Adults with Adults with
abnormal abnormal disc
Heads
abdomens
derivatives
Mouth
parts
Table 1. Distribution of defects in pharate adults
Wing
thorax
5
2
18
10
1
Halteres
Legs
3
1
1
2 7
3
1 1
2 1 1
1
12
Imaginal disc affected
Genitalia
o
o
d
a
a
258
M. BOWNES AND L. A. SUNNELL
DISCUSSION
The results presented here show that eggs at different developmental stages
have a different sensitivity to X-rays. Generally the results agree well with those
of Ulrich (1951); Wieschaus (1974, 1975) and Wiirgler & Ulrich (1976). They
also show that X-rays can induce morphological defects in the adults from
blastoderm formation to stage 9 when they were first observed by Wieschaus.
In fact, the peak of their production occurs at stage 8; however, these would
not have been detected by Wieschaus since they did not obtain sufficient
numbers of adults to analyse them from 3-5-6 h of development which covers
approximately stage 7 and stage 8.
One of the problems of analysing the results of X-irradiation studies is that
we understand very little about the way in which cell death arises. For example,
it is not known whether a mitosis is needed in order for cell death to result
from irradiation. However, since both larval cells, which are dividing endomitotically at the later developmental stages, and the presumptive imaginal
disc cells are both sensitive to X-irradiation it seems likely that cell death can
result without an actual cell cleavage. Also we cannot assume that the regulative
processes undertaken by cells to produce pattern duplications are necessarily
initiated at the time of irradiation. The damage to the embryo may not be
recognized by neighbouring cells until later in development.
The most interesting finding from these experiments is that all the cells of
the embryo are sensitive to X-rays at the same stages of development and the
peak of production of imaginal defects occurs at the same stage as the highest
frequency of embryonic lethality. This suggests that the development of the
larval and prospective adult cells is not occurring independently since there is
no differential sensitivity to X-rays between the two cell types.
We would like to thank Ms Susanne Glenn for technical assistance and Dr S. Long for
his assistance with the statistical analysis.
This study was supported by grants from the National Science Foundation and the
National Institutes of Health to H. A. Schneiderman and P. J. Bryant and from the Science
Research Council to M. Bownes.
REFERENCES
M. (1975a). Adult deficiencies and duplications of head and thoracic structures
resulting from microcautery of blastoderm stage Drosophila embryos. /. Embryol. exp.
Morph. 34, 33-54.
BOWNES, M. (19756). A photographic study of development in the living embryo of Drosophila melanogaster. J. Embryol. exp. Morph. 33, 789-801.
BOWNES, M. (1976). Larval and adult abdominal defects resulting from microcautery of
blastoderm staged Drosophila embryos. /. exp. Zool. 195, 369-392.
BOWNES, M. & SANG, J. H. (1974 a). Experimental manipulations of early Drosophila embryos.
I. Adult and embryonic defects resulting from microcautery at nuclear multiplication and
blastoderm stages. /. Embryol. exp. Morph. 32, 253-272.
BOWNES, M. & SANG, J. H. (19746). Experimental manipulations of early Drosophila embryos.
IT. Adult and embryonic defects resulting from the removal of blastoderm cells of
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BOWNES,
X-irradiation 0/Drosophila embryos
259
J. H. & SCHNEIDERMAN, H. A. (1973). Pattern formation in imaginal discs
of Drosophila melanogaster after irradiation of embryos and young larvae. Devi Biol. 32,
345-360.
ULRICH, H. (1951). Sensitive periods and egg-regions in production of the modification
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WIESCHAUS, E. (1974). X-ray induction of pattern abnormalities in early embryos. Dros.
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WIESCHAUS, E. (1975). Clonal analysis of early development in Drosophila melanogaster.
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POSTLETHWAIT,
(Received 17 December 1976, revised 3 February 1977)