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/. Embryol. exp. Morph. 77, 167-182 (1983)
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Printed in Great Britain © The Company of Biologists Limited 1983
Morphological and molecular modifications induced
by heat shock in Drosophila melanogaster embryos
By GIORGIO GRAZIOSI1, FRANCO DE CRISTINI, ANGELO
DI MARCOTULLIO, ROBERTO MARZARI, FULVIO
MICALI AND ADRIANO SAVOINI
From the Istituto di Zoologia e Anatomia Comparata, Universitd di Trieste,
Italy
SUMMARY
The early embryo of Drosophila melanogaster did not survive treatment at 37 °C (heat
shock) for 25 min. The histological analysis of eggs treated in this way showed that the heat
shock caused disintegration of nuclei and of cytoplasmic islands, displacement and swelling of
nuclei and blocked mitoses. These effects were not observed in embryos treated after blastoderm formation. After this stage, we noticed that development was slowed down. The heat
shock proteins (hsp 83,70 and 68) were, under shock, synthesized at all developmental stages.
There was little or no synthesis of hsp 70 and 68 in unfertilized eggs, but synthesis increased
in proportion to the number of nuclei present. Most probably, hsp 70 synthesis was directed
by zygotic mRNA. DNA synthesis was not blocked by the heat shock though the overall
incorporation of pH]thymidine was substantially reduced, presumably because of the block
of mitoses. We did not find a direct relation between survival pattern and hsp synthesis. We
concluded that some, at least, of the heat shock genes can be activated at all developmental
stages and that heat shock could be used for synchronizing mitoses.
INTRODUCTION
The usual temperature for raising Drosophila melanogaster is around 24 °C
though this organism can also live at temperatures ranging from 15 to 34 °C
(Hedman & Krogstad, 1963; Powsner, 1935). Thisflycan also survive relatively
short exposure to more extreme temperatures such as 4°C or 40 °C. During the
exposure to high temperature, usually around 37 °C, Drosophila cells undergo
considerable molecular and physiological changes (Ashburner & Bonner, 1979,
review). Transcription and translation of the genes which were active before the
heat shock are halted (Bonner & Pardue, 1976; Lindquist, 1980,1981; McKenzie, Henikoff & Meselson, 1975; Spradling, Penman & Pardue, 1975), while a
newly synthesized set of RNAs is translated into at least seven different proteins
(Mirault et al. 1978; Tissieres, Mitchell & Tracy, 1974; Henikoff & Meselson,
1977; Spradling, Pardue & Penman, 1977).
1
Author's address: Istituto di Zoologia e Anatomia Comparata, Casella Universita, 34100
Trieste, Italy.
168
G. GRAZIOSI AND OTHERS
Such a response has been described for tissue culture cells and for various
organs at various developmental stages of Drosophila (Chomyn, Moller &
Mitchell, 1979; McKenzie et al. 1975; Lewis, Helmsing & Ashburner, 1975;
Peterson, Moller & Mitchell, 1979) and it has been found in all the eucariotic
organisms so far tested (Barnett, Altschuler, McDaniel & Mascarenhas, 1980;
Fink & Zuethen, 1970; Giudice, Roccheri & Di Bernardo, 1980; Kelley, Aliperti
& Schlesinger, 1980; McAlister & Finkelstein, 1980). Early embryos are the only
known possible exception to this response: the Drosophila embryo before blastoderm formation (Dura, 1981; Graziosi et al. 1980), and the sea urchin embryo
before gastrulation (Giudice et al. 1980) synthesize little or no heat shock
proteins. The fly embryo response to heat shock also differs from other
Drosophila systems in other respects. Hsp 83 has been found in large amounts
as a cold protein in eggs kept at normal temperature, although its synthesis can
be elicited by a heat shock of 30min (Graziosi et al. 1980; Savoini et al. 1981).
Furthermore, early embryos are extremely sensitive to the exposure to high
temperatures: 15min of treatment cause a very high mortality rate as well as
phenocopies (Mitchell & Lipps, 1978; Santamaria, 1979).
We undertook this study to investigate this high mortality rate in early embryos and, by quantitating the [35S]methionine-labelled hsps, to correlate the
survival pattern with hsp synthesis.
MATERIALS AND METHODS
Egg harvesting and fly maintenance
Eggs were collected from a population cage of roughly 5000 flies. The laying
population was kept under a fixed regime of food supply and of light/dark cycle
to ensure the deposition of a large amount of eggs between 10 am and 4 pm. The
light was switched on at 5 am and switched off at 5 pm. Fresh baker's yeast
deposited on Petri dishes of standard Drosophila food was offered every hour;
starting at 9 am until 5 pm. The feeding dishes were also used for harvesting eggs,
but they were left in the cage for lOmin only. An average age of 5min was
assumed when the eggs were withdrawn from the cage. The mass collections
were used for the survival curve and they contained 5-10 % of eggs in advanced
developmental stages. The eggs used for two-dimensional electrophoresis and
for the histological analysis were laid over a period of 5 min and were individually
checked for age.
A small cage containing a few hundred flies was prepared to collect eggs from
the cross of heterozygote flies Df (3R) 229, Df (3R) kar33 (Ish-Horowicz et al.
1979).
Egg labelling and heat shock
Upon removal of the chorion membrane with 20 % sodium hypochlorite and
several washes in Drosophila saline, the eggs were permeabilized according to
Modifications induced by heat shock in Drosophila embryos
169
the method of Limbourg & Zalokar (1973). Then the eggs were covered with
30 fi\ of incubation medium and transferred either to 24 °C (controls), or to 37 °C.
[35S]methionine or [3H]thymidine (final concentrations of 1 mCi/ml and 0-5 mCi/
ml respectively) were added 10 min after the transfer of the eggs to the desired
temperature. Similarly the controls had a preincubation of 10 min at 24 °C before
addition of the label. At the end of the incubation, the eggs were washed in cold
medium and either fixed for the histological analysis or sonicated for protein
extraction.
Electrophoresis and fluorography
Protein extracts underwent two-dimensional electrophoresis as reported by
O'Farrell (1975). After sonication, the samples were freeze-dried, resuspended
in 50 /il of lysis buffer, centrifuged for 30 min at 50 000 g and 3 x 1 //I aliquots of
the supernatant were used to count the total radioactivity in the TCA precipitable material. All first dimensions were loaded with 100 000 c.p.m. All the other
electrophoretic parameters were the same as reported by Savoini et al. (1981).
Since hsps 70 and 68 did not always appear as stainable spots, their foci were
identified matching the stained gels with autoradiographs run for other purposes
(Graziosi et al. 1980; Savoini et al. 1981). Hsp 83 and /3-tubulin were always
recognized as consistent stainable spots. The protein spots were cut out, placed
in 1 ml of 30 % H2O2 and evaporated at 60 °C until dry, resuspended in scintillation liquid and counted.
Single eggs were analysed on 9 % polyacrylamide slab gels using the discontinuous sodium dodecyl sulphate (SDS) method of Laemmli (1970). These gels
underwent fluorography as reported by Bonner & Laskey (1974) using Kodak
film RP X-Omat.
Histology and autoradiography
Both chorion and vitelline membranes were removed from the eggs for histological analysis. After fixation using the method of Zalokar & Erk (1977), the
eggs were embedded in paraffin and sectioned at 8 /im. The sections were stained
with haematoxilin and eosin for 10 and 2 min respectively and permanently
mounted in Canada balsam.
The autoradiographs were prepared by dipping the slides in the photographic
emulsion Ilford K5 diluted 1:2 with water at 37 °C. The excess of emulsion was
drained by leaving the slides vertical until completely dry. All slides were exposed for 7 days and developed by standard methods. The histological sections
used for the autoradiography were stained after exposure and development.
RESULTS
Embryonic viability
A total of 5452 eggs, subdivided into 43 experimental groups, was heat
170
G. G R A Z I O S I A N D
OTHERS
shocked for 25 min. Each egg collection was subdivided into four to eight batches
of eggs and each batch was aged at 24 °C until the embryos had reached the
desired developmental stage. Fig. 1 shows the percentage survival of heatshocked embryos of different ages and the equation describing this survival. A
third degree polynomial was the best fit which is statistically significant (F = 8-20;
D.F. = 39). We calculated the second derivative to find the point of inflection
which was around 130 min. Before this age the nuclei are engaged in a fast mitotic
cycle and the viability is very low. Soon after 130 min, when mitoses stop and the
embryos become cellularized, the survival rate was not dissimilar from that of the
controls.
A few hundred embryos were dechorionated and inspected under a dissecting
microscope 2-3 h after the treatment. The eggs treated before blastoderm
formation showed no sign of development while the eggs treated at later stages
showed a delayed development but normal morphology.
Heat-shock proteins
As reported previously (Graziosi et al. 1980) different amounts of hsps were
found at different developmental stages. To quantitate hsps we ran twodimensional gel electrophoresis of p5S]methionine labelled extracts of embryos
heat shocked for 30 min at seven different ages: from unfertilized eggs up to the
beginning of gastrulation (3 h). After localization of the proteins on stained gels,
90
y = 0-000012 x3 + 0-004971 x2 - 0-27988 x + 24-2136
100
80
f"(x)= 131
95
90
80
g
70
60 -
70 13
>
•>* 50 H
50 3
o
40 -
30 ^
30 -
20
20 -
o Controls
10
• 5
10 40
80
120
160
200
240
Age in min
Fig. 1. Survival of embryos following a heat shock of 25 min. The left-hand side
ordinate shows the survival percentages arc sinV^T transformed. This transformation was introduced to take into account the non-normal distribution of percentages
near 0 % and 100 % (Sokal & Rholf, 1969). The right-hand side ordinate shows the
survival rates in percentage but on an arc sin V p scale.
Modifications induced by heat shock in Drosophila embryos
111
the spots corresponding to hsp 83, 70 and 68 and /3-tubulin were cut out and
counted. Hsps 70 and 68 were counted together because they were not well
separated in this gel system.
The counts of hsp 83 increased from 150c.p.m. (out of 100000c.p.m. total
counts) in unfertilized eggs to about 350 at gastrulation. Hsps 70 and 68 were
virtually absent in very young eggs but, together, they showed 3000c.p.m. at
gastrulation (3 % of the total counts). Because of the consistent trend of the
curves, we considered the use of statistics unnecessary. The amount of synthesis
of hsps and the number of nuclei at each stage are shown in Fig. 2.
In order to investigate whether hsp 70 was translated from maternal messages,
we used the mutant Df(3R) 229, Df(3R) kar31 deficient for the loci coding for this
protein. We ran on SDS gels extracts of single eggs obtained from heterozygote
flies 90 min and 3 h after deposition. Hsp 83 was always present, hsp 68 was found
in all late embryos. Hsp 70 was recognized only in 27 late embryos, the remaining
8 hsp-deficient eggs were considered to be the hsp 70 null homozygotes (Fig. 3).
This proportion is not dissimilar from the expected Mendelian ratio.
640032001600 -
•
o
AA
• Nuclei
o hsp 70 + h s p 68
Ahsp83
ATubulin
-25000
10000
800
5000
512
2500
256/—\
$ 128-
-1000
-500
00
o
r
64-
250
I
oo
2
32-100
•§
1
16-1
50
8^
25
10
20
40
60
80 100 120
Age in min
140 160 180
Fig. 2. Counts of [35S]methionine-labelled spots of hsp 83, hsp 70 + 68 and /3-tubulin
cut out from two-dimensional gel electrophoresis of embryos heat shocked at various
developmental stages. We took j3-tubulin as a possible reference protein because its
synthesis was not suppressed by the heat shock. Allfirstdimensions were loaded with
100000 c.p.m. The embryonic ages refer to the mid-incorporation time. The
theoretical number of nuclei was taken from Zalokar & Erk (1977) tables.
172
G. GRAZIOSI AND OTHERS
C
D
E F
G
H
I
m
Fig. 3. Fluorography of SDS gel electrophoresis of 3h-old eggs (lanes A to H)
obtained from the cross between heterozygote flies deficient for the hsp 70 genes.
The eggs were incubated 30 min at 37 °C in medium containing 4mCi/ml [35S]methionine. We considered hsp 70 as missing in lanes B and F, we think that the faint
band at the level of hsp 70 is due to a protein of similar molecular weight but of
different isoelectric point as observed in two-dimensional gel electrophoresis. In lane
I a 90-min-old egg was run. Exposure two months.
Morphological modifications of heat-shock embryos
We prepared histological sections of control and heat-shocked eggs to identify
the possible cause of the early embryo mortality. Three experimental sets were
considered: freshly laid eggs (subjected to heat shock 25 min after deposition,
experiments A and B); advanced nuclear segmentation (50 min after deposition,
experiments C and D); and beginning of gastrulation (210 min after deposition,
experiments E and F). Each experimental set comprised three groups of about
ten eggs; the first one underwent 30min of heat shock (experiments A, C, E),
the second one was heated for 60min (experiments B, D, F) and the third one,
Modifications induced by heat shock in Drosophila embryos
173
D I A G R A M OF EXPERIMENTS
55'
25V
is,
25V
H240'
21O'i
50V
210V
H270'
80'
4110'
50V
.190'
50'
23O'l
= 270'
.270'
40
80
—I—
2C
160
120
Time after egg deposition (min)
240
1 heat shock; I
1 preincubation at 24°C; J* preincubation in heat shock;
o o o | thymidine at 24 °C; ! • • • • ! heat shock plus thymidine; —• time of fixation of controls.
Fig. 4
5A
B
Fig. 5. Histological sections of 85-min-old eggs. (A) Control showing nuclei in the
cytoplasmic islands; (B) heat-shocked egg from experiment B, showing no cytoplasmic islands and swollen nuclei at the surface of the egg. Bar = 100 fim.
the control, was kept at 24 °C until the end of the 60 min of heat shock and then
fixed. These experimental regimes are summarized in Fig. 4.
Nuclear segmentation stages
After sectioning, the three-dimensional structure of the eggs treated during
the nuclear segmentation stages was reconstructed to establish the exact number
of nuclei in each egg. The control embryos fixed at 85 min were at stages 8 to 9
174
G. GRAZIOSI AND OTHERS
Fig. 6. Enlargements to show the dimensions of nuclei. (A) Control; (B) heatshocked egg from experiment B. Bar = 10/im.
Fig. 7A. Nucleus found in one egg of experiment D. The arrows point where the
nuclear membrane appears disintegrated. B. The fibrous body shown above was
observed in many eggs of experiments A, B, C and D. We think that they are
disintegrated nuclei. Bar = 10/um.
(Fig. 5A, Zalokar & Erk, 1977, staging) and showed cytoplasmic islands, interkinetic nuclei or mitotic figures.
In many of the heat-shocked eggs we could not recognize morphological structures resembling normal nuclei, while in the others we counted a limited number
of nuclei (Table 1, Fig. 5B). We could not detect mitotic figures in the 40 heated
eggs. From Table 1 it is also apparent that the longer the heat shock, the fewer
the nuclei. In fact the average number of nuclei per egg in the experiments A and
B was 1-2 and 0-7 respectively, while in the experiments C and D it was 9-9 and
5-7 respectively.
Modifications induced by heat shock in Drosophila embryos
175
Table 1. Developmental stages reached by eggs after the heat shock
Experiments as reported in Fig. 4
A
f
A
Egg No
1
2
3
4
5
6
7
8
9
10
11
r
0
2
3
3
0
1
0
0
2
B
c
Number of nuclei
2
6
0
0
0
0
0
0
0
0
0
3
0
1
7
0
23
12
22
24
7
•\
D
E
•\
5
10
0
10
8
12
12
0
0
0
F
stage
15B
14/15
14/15
18
15B
15B
14/15
15/16
>
15A
15/16
15/16
15/16
15/16
15/16
16
15/16
15B
15B
15/16
Controls at 85 minutes (experiments A and B): 128/256 nuclei
at 110 minutes (experiments C and D): 256/512 nuclei
at 270 minutes (experiments E and F): Stage 16.
The diameter of the control egg nuclei was 6-8 jum (Fig. 6A) while all the
nuclei found in the treated eggs were considerably larger: 12-22/im (Fig. 6B).
These large nuclei were also located abnormally on the surface of the eggs. In one
egg, treated for 1 h, we found a swollen nucleus showing damage of the nuclear
membrane (Fig. 7A). In many cases we observed fibrillar structures which we
suppose were disrupted nuclei (Fig. 7B) while similar structures were never
observed in control eggs. We also noticed the absence or considerable reduction
in the number of the cytoplasmic islands.
Gastrulae
In gastrulating embryos the heat shock did not produce the dramatic changes
reported above. The eggs were able to continue development while under shock.
The two experimental groups E and F underwent heat shock at the same time,
but the group of eggs treated for a longer period reached a more advanced stage
of development. Nevertheless, this experimental group was less advanced than
the control eggs (Table 1) even if they were fixed at the same time. Thus, in late
embryos, the heat shock slowed development without blocking it.
During gastrulation we did not observe mitoses in either controls or heatshocked eggs but, because of the stage-dependent loss of mitotic synchrony and
the large number of cells, chromosomes could have escaped observation.
Thymidine incorporation
A preliminary pulse-chase experiment (Fig. 8) showed that, after lOmin of
176
G. GRAZIOSI AND OTHERS
CPM
H 24 °C
\ 37°C
5000-
4000-
3000-
2000I
A
I
1
1000 I
10
,
30
20
Time (min)
40
Fig. 8. Bars indicate the beginning and the end of a pulse of [3H]thymidine at 24 °C
and at 37 °C. The eggs were washed in TCA at the end of the pulse. At the beginning
of the experiment, the eggs were 210 min old. Ordinate: counts of 100 eggs. Abscissa:
time from the beginning of the heat shock or the incubation at 24 °C.
heat shock, the total incorporation of thymidine was substantially repressed.
Thus, in the following experiments, [3H]thymidine was added 10min after the
beginning of the heat shock to ensure that the label was incorporated under
shock conditions.
Segmentation stages
In control eggs (experiment G) we found no unlabelled nuclei. The observed
number of nuclei is indicated in Table 2. In many eggs there were some elongated
radioactive traces indicating active mitosis. Judging from the number of nuclei,
the control eggs (Fig. 9A) underwent two to four mitoses during the incubation
period. The difference between the expected and the observed number of nuclei
was due not only to the loss of some sections when coating the slides with the
photographic emulsion, but also to a possible inhibition of mitosis by the incubation medium. Permeabilization itself does not interfere with normal development (Roberts & Graziosi, 1977).
In the absence of nuclear disintegration, the heat-shocked eggs should show
a number of nuclei equal to, or higher than, that of controls. To test this, [3H]thymidine was added to the heat-shock egg incubation medium at the time of
fixation of the controls (see Fig. 4, experiments G and H). Labelled nuclei in the
Modifications induced by heat shock in Drosophila embryos
111
Table 2. Number of nuclei labelled by [3H]thymidine
Experiments as reported in Fig. 4
>
f
Egg No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Total
Average
G
H
109
146
168
105
51
247
59
227
95
241
36
49
60
33
46
85
54
28
48
54
62
121
29
35
33
33
1510
137-3/egg
744
49-6/egg
9A
Fig. 9. Autoradiographs of sections of eggs incubated in pH]thymidine. (A) Young
egg incubated at 24°C, experiment G. (B) Heat-shocked young egg, experiment H.
Arrows point at radioactive nuclei. (C) 270-min-old control egg, experiment I. (D)
270-min-old heat-shocked egg. Arrows point at radioactive vitellophages, experiment L. Bar = 100 |Um.
178
G. GRAZIOSI AND OTHERS
heat-shocked eggs (experiment H) were fewer than those counted in controls
(Table 2). In heat-shocked eggs we never observed elongated nuclear radioactive
traces. We found again large nuclei of 20 /im of diameter which were radioactive,
and generally located at the surface of the egg (Fig. 9B). Only a few unlabelled
nuclei were found.
Gastrulae
In the fifteen control eggs examined (experiment I), a large number of nuclei
were radioactive. The majority of the radioactive nuclei was clustered in specific
areas where the cells were invaginating: stomodeum, posterior midgut, germinal
band and cephalic furrow (Fig. 9C). Few or no vitellophages were labelled.
Out of the 20 heat-shocked embryos (experiment L) only a few showed
radioactive nuclei at the cephalic furrow and at the posterior midgut invagination. The majority of eggs had radioactive vitellophages (Fig. 9D).
DISCUSSION
In quantitating the amount of [35S]methionine incorporated into the hsps we
did not intend to establish the absolute rate of synthesis of these proteins.
Rather, our experimental procedure accounts only for the proportion of the label
which ended up in the heat-shock proteins. The synthesis of hsp 70 and 68 rose
steadily during development and did not show, on a logarithmic scale, any sharp
increase at blastoderm formation. Consequently it appears that these proteins
can be synthesized before blastoderm formation.
It has been reported that the bulk of the zygotic genome is activated at blastoderm formation (Anderson & Lengyel, 1979; Lamb & Laird, 1976; McKnight &
Miller, 1976; Zalokar, 1976). If this is also true for the heat-shock genes then hsp
synthesis during early stages should be supported by maternal mRNAs. At least
as far as hsp 70 and, possibly, hsp 68 are concerned, we do not favour this
hypothesis because: i) there was little or no synthesis in unfertilized or freshly
laid eggs, ii) their synthesis rose during the exponential phase of nuclear
multiplication and not at blastoderm formation and, iii) we did find mutant eggs
(229 hat33) unable to synthesize hsp 70.
If hsps 70 and 68 are indeed translated from zygotic messages, their low
synthesis or even absence in early stages is not surprising. If we compare the
number of nuclei in the zygote with that of the egg at blastoderm formation we
find a ratio 1:6000. Hence, assuming equal transcriptional and translational rates
at all stages, the zygote is expected to produce far less hsps than the following
stages. Furthermore many nuclei disintegrated and, presumably, disintegrated
nuclei cannot be transcribed normally. This interpretation cannot be applied to
hsp 83 whose synthesis was relatively high even in unfertilized eggs. Most probably its synthesis is sustained by maternal mRNAs, at least during the early
stages.
Modifications induced by heat shock in Drosophila embryos
179
The morphology of the heat-shocked eggs
In many eggs, heat shocked during the nuclear multiplication stages, we found
no nuclei or just a few. It is obvious that eggs without nuclei or with damaged
nuclei cannot undergo normal development and, on the basis of the mortality
curve, we think that such damage was caused only in eggs which did not reach
the stage of blastoderm formation. Our observations are very similar to those
reported by Zalokar & Erk (1976), who obtained swollen nuclei upon treatment
of eggs with dinitrophenol or anoxia. It is now known that both anoxia and
dinitrophenol induce a 'heat-shock-like' response (Ashburner, 1970; Ellgaard,
1972; Rensing, 1973).
Although our results can explain why the embryos did not survive the heat
shock, they cannot explain the degeneration of nuclei. The unique morphological and physiological characteristics of the early embryos, as the absence of
plasmalemma and the fast mitotic cycles (Sonnenblick, 1950; Zalokar & Erk,
1976), could account for the nuclear sensitivity to heat. But such characteristics
cannot fully explain why some nuclei survive the exposure to the high temperature and the subsequent reduction of nuclei number as reported in Table 1.
The unequal sensitivity of nuclei could be more convincingly explained by the
unequal distribution of some protective agents either the hsps or any other
component of the egg. We know that the heat-shock proteins migrate into the
nuclei (Arrigo, Fakan & Tissieres, 1980; Valazquez, Di Domenico & Lindquist,
1980) and it is possible that their concentration in the egg was too low to exert
a possible protective action on all nuclei. Furthermore, the early embryo is not
compartmentalized by plasmalemmas and substances could diffuse in nucleusfree areas.
Besides the disintegration of nuclei we found that the heat shock interfered
with the mitotic cycle of early embryos. Frequently in control eggs, we observed
mitotic figures which were never seen in sections of heat-shocked eggs. Thus the
heated nuclei were unable to enter mitoses. Furthermore, taking into account
the synchrony of mitoses and that almost all nuclei of all the labelled eggs were
radioactive, it is apparent that the nuclei reached the S phase. Consequently the
block should occur between S and M, presumably in G2. Alternatively it is
possible that the nuclei did not reach M because they died just after the S phase.
In any case DNA synthesis itself was not inhibited and the low incorporation of
thymidine during the heat shock was due to a low number of nuclei entering the
S phase.
As far as gastrulating heat-shocked embryos are concerned, we found only a
delay in development. Similar results were obtained in embryo by Dura (1981)
and in pupae by Lindsley & Poodry (1977). Delayed development could be
caused by a variety of reasons including the block of mitoses.
Finally, we must take into account the possibility that nuclear disintegration
as well as a slower development could be due to the perturbation of the normal
180
G. GRAZIOSI AND OTHERS
programme of embryonic gene expression and of coordinate maternal message
translation, as reported for tissue culture cells (McKenzie et al. 1975; Moran et
al. 1978; Tissieres, Mitchell & Tracy, 1974). This hypothesis could be tested by
an appropriate experimental design which could also show a way to use
hyperthermia to discriminate between the contribution of the embryonic
genome and that of the maternal genome to development. Further investigations
could also indicate if the heat shock may be used as a physiological method for
synchronizing mitoses, which might have practical applications in a variety of
biological systems.
We wish to thank Dr D. Ish-Horowicz for the kind gift of the mutant stock 229 kar31, and
Dr D. B. Roberts and C. R. Bebbington for critical reading and improvements to the
manuscript. This research has been supported by the CNR grant No. 81 00488, 85-115, 2143
and by the MPI grant 1981-82.
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{Accepted 27 April 1983)

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