J. Embryol. exp. Morph. 96, 245-266 (1986)
Printed in Great Britain © The Company of Biologists Limited 1986
245
Disruption of segmentation in a short germ insect
embryo
I. The location of abnormalities induced by heat shock
JANE E. MEE AND VERNON FRENCH
Zoology Department, University of Edinburgh, West Mains Road,
Edinburgh, EH9 3JT, UK
SUMMARY
The effect of heat shock (15 min at 48 °C) on segmentation has been investigated in the short
germ embryo of the locust (Schistocerca gregaria). Prior to formation of the germ anlage and at
the disc stage heat shock considerably reduced the survival of eggs but appeared to have little
effect upon segmentation. At later stages heat shock had no effect on survival but resulted in
disruptions of the segmental pattern. The location of abnormal segments depended upon the
stage at heat shock and the number affected depended on its severity. A constant number of
normal segments developed between the last segment visible at the time of heat shock and the
first abnormal segment. These results are similar to the disruptions observed in amphibian
somites following heat shock. However, different parts of the segment pattern varied in their
response; the head segments were very rarely affected, and disrupted regions rarely started in
the middle abdomen (segments A5 and A6).
The results are discussed in relation to two models (the clock and wavefront and progress zone
models) that have been proposed as an explanation for the specification of the somite pattern in
amphibians.
INTRODUCTION
The body of an insect consists of a constant number of similar, but not identical,
segmental units arranged in an anterior-to-posterior sequence. The mechanism by
which this segmented pattern is specified during early embryonic development is
not known.
At a descriptive level, insect embryos seem to form segments in two different
ways (reviewed Sander, 1976, 1981). In short germ insects (e.g. the locust,
Schistocerca gregaria) the embryo (or 'germ anlage') forms from a small region of
the blastoderm cell layer and segments become visible in a reliable sequence
during the gradual elongation of the posterior tip of the germ anlage. In contrast,
the germ anlage in long germ insects (e.g. Drosophila, Smittia) forms from a
large area of the blastoderm which is subdivided into segments. There are also
intermediate germ insects (e.g. Eucelis, Acheta) in which anterior segments form as
in a long germ embryo but the more posterior segments are added gradually as in a
short germ embryo.
Key words: Schistocerca gregaria, insect embryo, segmentation, locust, heat shock, models.
246
J. E. MEE AND V. FRENCH
In long germ insects, the results of a wide variety of experiments (such as
fragmentation and localized u.v.-irradiation) have suggested that the embryonic
segments form with reference to levels of a gradient (or gradients) established
between the anterior and posterior poles of the egg, prior to the formation of the
germ anlage (see Herth & Sander, 1973; Schubiger & Wood, 1977; Kalthoff,
1983). In short germ insects the response to experimental manipulation at similar
stages tends to be all-or-nothing; the embryo either develops normally or not at all
(Miya & Kobayashi, 1974). This suggests that the mechanism of segmentation may
differ from that in long germ embryos. The way in which segments appear during
development suggests that cell proliferation at the posterior tip of the embryo may
play an important role in the formation of the segment pattern (reviewed Sander,
1976).
Segmentation of insects appears to be rather similar to somitogenesis in anuran
amphibian embryos. Somites appear in anterior-to-posterior sequence, the posterior somites developing as the tail bud extends. The normal pattern of somites
is disrupted in particular ways following heat shock at specific stages during
development. A short heat shock early in development causes small disruptions
throughout the somite pattern. Heat shock given later (just prior to and during
visible somitogenesis) results in a small disruption at a predictable position,
posterior to the somites visible at the time of heat shock (Elsdale, Pearson &
Whitehead, 1976; Cooke, 1978; Pearson & Elsdale, 1979; Elsdale & Pearson,
1979). Thus, as heat shock is given at progressively later stages, the disrupted area
occurs in an increasingly posterior position, with a constant number of normal
somites forming between the most posterior somite visible at the time of heat
shock and the most anterior abnormal somite.
In the present experiments short germ locust embryos were given a short heat
shock at different times prior to and during visible segmentation and the location
and nature of the resulting segmental abnormalities were analysed (see Mee &
French, 1986).
MATERIALS AND METHODS
Populations of the locust, Schistocerca gregaria, were maintained at 31 ± 0-5 °C, with a 12h/l2h
light/dark cycle, on a diet of sprouted barley and bran. Mature adults were provided with honey
pots filled with damp sand for oviposition. Eggs are laid in a cluster (a 'pod') of between 25 and
90 eggs deposited over a period of 1-5 to 3h. When eggs of a known age were required,
ovipositing females were observed and pots removed when the female withdrew her abdomen
from the sand. At this time the age of all eggs in the pod was designated 0 h. Pods of eggs were
removed from the sand, the eggs carefully separated, washed and kept on moist cotton wool in
an incubator at 30 ± 0-5°C until required.
Embryos were examined after fixation. Eggs were punctured (in a region remote from the
embryo),fixedin acetic acid: formalin: ethanol: water (1:6:16:30) for 3 h at 60°C (see Lawrence,
1973), washed and then stored in 70 % ethanol. The chorion was removed by dissection, using
tungsten needles and watchmakers' forceps, or by treatment with 3 % sodium hypochlorite
solution. In older eggs the serosal cuticle, serosa and amnion were removed also.
The period of development from germ anlage to germ band was staged on the basis of the
morphological appearance of embryos. To determine the mean age at which each stage
occurred, 22 pods of varying ages were sampled at 2 h intervals over a period of 12 or 24 h. The
Locust segmentation after heat shock
247
size of the sample taken from each pod (two to seven eggs) was dictated by the number of eggs
per pod and the number of samples. The stage of each of the embryos within the sample (of
known age) was then determined.
Eggs were heat shocked at intervals between egg deposition and the completion of visible
segmentation. The eggs from one pod were treated together, as they develop fairly synchronously (Mee, 1984) while different pods develop at rather different rates (Tyrer, 1970; Bentley,
Keshishian, Shankland & Toroian-Raymond, 1979). Prior to the formation of the germ anlage,
eggs were heat shocked at a known age, subsequently at a recognizable morphological stage.
A pod was staged by examining a sample of 10 or 15 eggs fixed immediately before experimentation, and eggs from the pod were assumed to be at the median stage observed in the
sample. Eggs were heat shocked in water at 48°C for 15min and then returned to a 30°C
incubator until hatching. The consequences of altering the temperature or duration of heat
shock were investigated and the effect of handling was examined by giving a sham 'shock' of
30 °C.
The effect of heat shock on the segmental pattern was determined in first instar hoppers, fixed
shortly after hatching, usually after the development of pigmentation. Eggs that failed to hatch
were fixed, the embryos dissected out and scored.
RESULTS
(A) Stages of development
The development of the germ anlage into the fully segmented germ band can be
described by 14 morphological stages (see Fig. 1). During this period the segments
of the gnathos (3), thorax (3) and abdomen (11) become visible as the posterior tip
of the embryo elongates. The first four stages are the disc, heart shape (HS),
elongating protocorm (EP) and segmented thorax (sT) (see also Shulov & Pener,
1963; Bentley et al. 1979) and the remaining stages are based on the sequential
appearance of the eleven segments of the abdomen. These stages, named
according to the most posterior visible segment, are the segmented abdominal
segment 1 (sAl) stage, the segmented abdominal segment 2 (sA2) stage and so on to
the fully segmented stage (sA10/ll).
The fully segmented germ band is the presumptive ventral surface and appendages. The dorsal surface is derived from the flanks of the germ band which later
extend dorsally and finally fuse at the dorsal midline (Sander, 1976).
The mean age at which each stage of development was reached is shown in
Fig. 2. The disc stage germ anlage forms approximately 36 h after oviposition and
segmentation is completed at approximately 73 h. Comparison of the interval
between the mean ages for each stage shows that the duration of the first three
stages (disc, heart shape and elongating protocorm) is longer than that of
subsequent stages.
(B) The effects of heat shock
Following heat shock three categories of result were observed: (i) normal
animals; (ii) animals with segmental defects; (iii) eggs that failed to develop (most
were dead and discoloured, or had burst and extruded yolk).
The response, in terms of the proportion of animals falling into each of these
categories varied with the stage of development at which heat shock was given and
between different pods of eggs at a given stage.
248
J. E. MEE AND V. FRENCH
disc
sA1
sA2
1mm
A11
Fig. 1. Camera lucida drawings of embryonic stages of 5. gregaria. The embryo lies at
the posterior pole of the egg. Egg and embryo are viewed ventrally to show the first
four stages (disc, HS, EP and sT), two of the stages during segmentation of the
abdomen (sAl and sA2) and the fully segmented stage. Segments (of the gnathos and
thorax) first become visible in anterior regions of the embryo at the sT stage, and
subsequently the eleven segments of the abdomen appear in anterior-to-posterior
sequence. Gl, G2 and G3 are the mandibular, maxillary and labial segments; 77, 72
and T3 the pro-, meso- and metathoracic segments and Al, A2 and All the 1st, 2nd
and 11th abdominal segments.
Locust segmentation after heat shock
249
The location of defects in the head was based on the appearance of the eyes and
the appendages (labrum, antennae and mouthparts) as individual head segments
are not visibly delineated. In the abdomen and thorax defects were located in the
segments and their appendages. Abdominal segments A2 to A7 (and A8 in males)
are similar and therefore the location of heat shock defects was determined by
reference to unambiguously recognizable segments. This was a problem only when
one or more of the similar segments were absent. If there was an additional defect
it was assumed that the loss and the disruption had occurred in adjacent segments
(e.g. the absence of one segment and an abnormality in the 2nd and 3rd abdominal
segments present would be scored as disruption of abdominal segments 2,3 and 4).
Abdominal segments A10 and A l l are fused in the hopper and were treated as a
single segment (A10/11). Segments were scored without reference to the location
of the disruption within the segment circumference or its appendages.
In an affected animal, heat shock usually resulted in the disruption of two or
three consecutive segments. Two areas of disruption separated by one or more
normal segments occurred rarely (in only 15 % of animals with abnormal segmentation resulting from heat shock prior to germ anlage formation, and 6 % of
those resulting from heat shock given during the subsequent period).
(C) The response to heat shock before germ anlage formation
When comparing the results from heat- and sham-shocked eggs the variability
between eggs from different pods was taken into account by using an Analysis of
X1 Test. The results from eggs, heat and sham shocked at 3h, showed that heat
32
sAlO/ll
sA9
sA8
sA7
sA6
sA5
sA4
2
sA3
sA2
sAl
sT
EP
HS
disc
30
40
50
60
70
Age (h)
Fig. 2. Mean age (with standard deviation) at each morphological stage (for abbreviations see text). The total number of embryos observed at each stage is given.
250
J. E. MEE AND V. FRENCH
shock resulted in a significant increase in the proportion of eggs failing to develop,
but had no significant effect on the frequency of surviving animals with segmental
abnormalities.
The frequency of eggs failing to develop and of eggs developing into normal
animals fluctuated with age at heat shock (Table 1) and the proportion of animals
with abnormal segmentation was consistently less than 10 %. After heat shock at
all stages, disruptions were observed in the head, thorax and abdomen, and their
location within the segment file was apparently not related to age (Fig. 3).
(D) The response to heat shock after germ anlage formation
(1) Sensitivity to heat shock
At all stages after germ anlage formation (except disc and sA7) heat shock
resulted in an increase in the frequency of animals with segmental abnormalities
(with respect to that of the sham-shocked controls) with a peak in the frequency of
animals affected between the sT and sA2 stages. At disc and sA7 stages the
frequency of segmental defects was low and similar to that of the control (see
Table 2). The frequency of eggs failing to develop fluctuated following a sham or a
heat shock between heart shape and sA7 stages (see Table 2), but at any given
stage (apart from sA2 and sA3) values for sham and heat shock were not
significantly different. At the earlier disc stage, there was a significant increase in
the frequency of eggs failing to develop after a heat shock. At the disc stage,
therefore, the response to heat shock was similar to that observed following early
heat shock (failure to develop, no effect on segmentation) while at later stages it
differed (little effect on viability, disrupted segmentation).
(2) The segments affected by heat shock
At the disc stage the distribution of defects within the segment file resembled
that observed following early heat shock, and all segments (apart from T3) were
affected at similar low frequencies (Fig. 4).
In contrast, at the heart shape and subsequent stages heat shock induced defects
that were located in the thorax and abdomen at relatively high frequencies, while
the procephalon and gnathos were rarely disrupted. The location of the few
segmental defects observed following sham shocks at heart shape and later stages
(171 affected segments in .44 animals) showed a similar distribution.
The location of segmental defects depended upon the stage at which heat shock
was given (Fig. 4); heat shock at progressively later stages affected increasingly
posterior segments (whereas the location of the few defects occurring after a sham
shock was not stage specific). A latency was apparent between the heat shock and
the register of its effect upon the pattern. The first (i.e. most anterior) abnormal
segment was the third or fourth to become visible (Fig. 5); two or three normal
segments developing posterior to the last segment visible at the time of heat shock
(Table 3). When measured as the time between age at heat shock and age at which
8
370
42
7
44
6
no. of pods
no. of eggs
% failing to develop
% animals with defects
% normal animals
% unclassified
8
378
49
8
38
4
2 h 55 min
6
281
21
3
72
4
Control
3 h 5 min
9
428
59
5
32
3
6h
8
366
26
10
59
5
11 h 10 min
6
331
48
5
45
2
18 h 30 min
5
265
55
6
36
3
23 h 30 min
8
399
40
3
54
3
30 h 35 min
The times given are the median ages of pods in each group. Some animals, usually fixed prior to hatching, could not be classified; usually
because damage inflicted during dissection or deterioration of material prior to fixation made it difficult to ascertain whether the pattern was
abnormal. Embryos developing much more slowly than their heat shocked siblings were not classified. An analysis of x2 test comparing the
results from heat- and sham-shocked eggs (aged approximately 3 h) showed a significant increase in the proportion of eggs failing to develop
(percentage of total number of eggs) after heat shock (Fl/12 = 10-65, P < 0-01) but no significant difference between the frequencies of animals
with segmental abnormalities (percentage of total number surviving shock; F l / l l = 3-56, N.S.).
lh
Age at heat shock
Table 1. The response of eggs following early heat shock
&
usts
ion
252
J. E. MEE AND V. FRENCH
Age when
.
, . . .
lh
2h55min
3h5min
6h
llhlOmin
79
108
18h30min
23h30min
3Oh35min
nCtii snocKco
CONTROL
Total no. of
98
98
22
54
63
33
abnormal segments
lb
eye
ant
Gl
G2
G3
Tl
T2
T3
Al
A2
A3
A4
A5
A6
A7
A8
A9
A10/11
Fig. 3. The location of segmental abnormalities following heat shock prior to the
formation of the germ anlage, and following a sham shock (at 3h 5min). The kite
diagrams show the frequency with which each segment was abnormal, calculated as a
proportion (%) of the total number of abnormal segments in that age class. A frequency of 10 % is shown by the length of the scale bar. Segments are labelled, anterior
to posterior, gnathos, Gl to G3; thorax, 77 to 73; abdomen, Al to A10/11. The other
pattern elements scored for defects were the labrum (lb), eyes and antennae (ant).
the most anterior affected segment became visible (see Fig. 2), the latency was
12-9 h at early stages and 7 or 5 h during appearance of the abdomen.
A discontinuity was observed in the distribution of defects within the abdomen.
Although segments A5 and A6 were often affected by heat shock, they were
rarely found as the first abnormal segment in a disrupted sequence (Fig. 6). Since
large numbers of eggs were heat shocked at appropriate stages (i.e. sAl, sA2
and sA3), the absence of disruptions beginning with segments A5 and A6 was not
due to inadequate sampling. The effect of the discontinuity is clearly seen in the
distribution of defects following heat shock at sA2 (Fig. 5).
(3) The extent of defects
In any given animal the extent of a heat shock defect was usually two or three
segments (Fig. 5). However, at any given stage the range of segments affected
at relatively high frequencies was from two or three (at stages sA3, sA4 and sA5,
for example) to four, five or more (heart shape, sAl and sA2 stages - see Fig. 4).
The interpretation of these results is difficult as the overall extent of disruptions
was restricted; posteriorly, because A10/11 is the last segment of the animal;
anteriorly, because gnathal segments were rarely affected. The absence of defects
beginning with segments A5 and A6 also influenced the distribution.
4
125
7
0
90
2
(ii) Stage at sham shock
no. of pods
no. of eggs
% failing to develop
% animals with defects
% normal animals
% unclassified
9
273
17
2
81
0-4
19
780
24
38
31
7
HS
6
190
14
2
80
4
15
522
34
41
19
7
EP
7
231
11
2
85
2
7
231
11
70
8
11
sT
4
158
23
1
75
1
10
402
20
74
3
4
sAl
7
195
21
2
76
1
14
480
10
71
11
8
sA2
7
237
19
1
78
2
18
658
9
59
26
7
sA3
8
280
13
5
79
3
14
513
6
35
52
6
sA4
5
127
15
3
80
2
9
293
7
23
65
5
sA5
8
252
18
1
73
7
14
480
17
25
53
4
sA6
7
284
12
2
85
1
8
239
19
7
67
8
sA7
An analysis of x2 comparing the frequency of eggs failing to develop (percentage of total number shocked) showed no difference between
heat and sham shock at most stages (heart shape, Fl/26 = l-55; elongating protocorm, Fl/l9 = 3-29; segmented thorax, Fl/12 = 0; sAl,
Fl/12 = 0-10; sA4, Fl/20 = 1-75; sA5, Fl/12 = 3-44; sA6, Fl/20 = 0-03; sA7, Fl/13 = 1-00; N.S.) but a significant effect of heat shock at disc,
sA2 and sA3 stages (disc, Fl/15 = 4-76, P<0-05; sA2, Fl/19 = 8-32, P<0-01; sA3, Fl/23 = 6-69, P< 0-025). At the disc stage there was no
significant difference between the frequencies of animals with segmental abnormalities (percentage of total surviving shock) following a sham
or a heat shock (Fl/15 = 2-89, N.S.).
13
461
38
3
56
3
no. of pods
no. of eggs
% failing to develop
% animals with defects
% normal animals
% unclassified
(i) Stage at heat shock
Disc
Table 2. The results of (i) heat shock and (ii) control sham shock of eggs at stages between disc and sA7
a.
to
<"*"
3
©
Total no. of
abnormal segments
lb
eye
ant
Gl
G2
G3
Tl
T2
T3
Al
A2
A3
A4
A5
A6
A7
A8
A9
A10/11
Stage when
heat shocked
36
Disc
867
Heart shape
504
Elongating protocorm
416
Segmented thorax
976
sAl
sA2
O
W
to
A10/11
lb
eye
ant
Gl
G2
G3
Tl
T2
T3
Al
A2
A3
A4
A5
A6
A7
A8
A9
•j
sA4
385
sA3
901
132
sA5
294
sA6
41
sA7
10%
Fig. 4. The location of segments affected by heat shock at stages from disc to sA7. The data are presented as in Fig. 3. A frequency of
10 % is shown by the length of the scale bar. Frequencies of less than 1 % are represented by a line. (Defects in which the only
abnormality was the apparent loss of one (or more) of the middle abdominal segments could not be included - see Materials and
Methods.)
:
B
Stage when
heat shocked
Total no. of
abnormal segments
A
Stage when heat shocked
Heart shape
Total no. of affected animals
296 (1)
Mean no. of segments/disruption 2-9 ± 1-3
lb
eye
ant
Gl
G2
G3
Tl
T2
T3
Al
A2
A3
A4
A5
A6
A7
A8
A9
A10/11
Elongating protocorm
193 (19)
2-5 ±1-1
Segmented thorax
145 (17)
2-7 ±1-0
sAl
280 (17)
3-4 ±1-4
sA2
336 (6)
2-6±M
o
W
*
sA3
379 (8)
2-4 ±0-8
2-1 ±0-6
sA4
180 (0)
sA5
67(0)
l-9±0-8
sA6
122 (0)
2-4 + 1-2
Fig. 5. Identity of the first (most anterior) segment affected by heat shock at stages from heart shape to sA6. Kite diagrams show the
frequency with which a given segment was the most anterior in a series of affected segments as a proportion (%) of the total number of
animals with segmental defects (defects in which the only abnormality was the loss of one or two abdominal segments were not
included - the number of such defects is given in brackets). The mean number of segments/disruption (with standard deviation) is
also shown (defects in which the only abnormality was the loss of one or two abdominal segments were scored as affecting 1 and 2
segments respectively). Data were not plotted for disc and sA7 stages as at the former the pattern of response was unlike that
observed for the heart shape and subsequent stages (see text) and at the latter the frequency of animals with abnormal segments was
low, suggesting that the majority of eggs from pods staged sA7 had reached a stage at which heat shock no longer had any effect.
AlO/11
lb
eye
ant
Gl
G2
G3
Tl
T2
T3
Al
A2
A3
A4
A5
A6
A7
A8
A9
Stage when heat shocked
Total no. of affected animals
Mean no. of segments/disruption
10%
to
Cta
258
J. E. MEE AND V. FRENCH
At heart shape and sA2 stages it seems that the larger range of segments
affected was due, at least in part, to variability in the location of the disrupted
region within the segment file, as indicated by the identity of the most anterior
Table 3. The latency in the response to heat shock in terms of the number of normal
segments developed between the last segment visible at the time of heat shock and the
first (most anterior) abdominal segment affected (see Fig. 5), and the interval of time
between heat shock and the appearance of the first abnormal segment (see Fig. 2)
Stage at heat
shock (Median)
HS
EP
Lag (no. of hours)
First segment
before abnormal
Lag (no. of
affected (Median) normal segments) segments formed
—
T3
11
12
A2
—
sT
sAl
A3
A4
2
2
9
7
sA2
sA3
sA4
sA5
sA6
A6
A7
A8
A9
A8
3
3
3
3
1
5
5
7
2
5
400
800
700
600
300
500
400
200
300
200
100
100
I I I I II
lb
ant
eye
G2 Tl
T3
A2 A4
A6
AS
A10/11
Gl G3
T2
Al A3
A5
A7
A9
1 II I1 1
lb
ant
eye
Gl
G2 Tl
T3
A2 A4
A6
A8
A10/11
G3
T2
Al A3
A5
A7
A9
Fig. 6. The total number of times (i) each segment was affected by heat shock (total
number of affected segments was 5373); (ii) each segment occurred as the most
anterior in an affected region (total number of defects was 1998). Data taken from the
heart shape to sA6 stage. Again disruptions in which the only abnormality was the loss
of a complete segment(s) cannot be plotted.
Locust segmentation after heat shock
259
Table 4. The effect of varying the severity of heat shock at the elongating protocorm
stage
15min: temp. °C
Parameters of heat shock
no. of pods
no. of eggs
% failing to develop
% animals with defects
% normal animals
% unclassified
45
47
9
367
20
9
63
8
10
384
24
25
41
9
48 49
6
15
522 244
34 69
41 10
0
19
7 21
5
8
332
18
27
44
11
48°C: duration (min)
15
10
25
20
11
6
15
8
274 522 412 323
28
34
64
66
26
41
26
17
39
19
1
2
7
7
8
16
30
7
311
72
18
<1
10
abnormal segment (see Fig. 5). A greater number of segments would be affected
by heat shock within a stage if that stage was of longer duration than the others
(assuming the rate of segmentation is constant), as is the case at the heart shape
and other early stages (but not the sA2 stage - see Fig. 2).
At the sAl stage the location of the abnormal area was not particularly variable
but the mean number of segments per disruption was slightly higher than at other
stages (Fig. 5).
(4) The response to heat shock of different severities
Heat shocks of a different duration or temperature were delivered to embryos at
the elongating protocorm stage. As the heat shock became more extreme (i.e. the
temperature or duration of the heat shock increased) the frequency of normal
animals declined and the proportion failing to develop increased (Table 4). All
heat shocks raised the frequency of animals with abnormal segments above that
of the sham-shocked control (Table 2). The heat shock utilized in the main
experimental series (i.e. 48°C, 15min) gave the maximum frequency of animals
with segmental abnormalities.
The severity of the heat shock had little effect on the identity of the most
anterior segment affected (Fig. 7). The median position of the first abnormal
segment was A l or A2, regardless of the temperature or duration of heat shock.
However, as the heat shock became more extreme the mean number of segments
per defect increased as successively more posterior segments were affected.
DISCUSSION
The effect of heat shock early in development
Prior to germ anlage formation and at the disc stage heat shock has a
considerable effect on the survival of locust eggs. Eggs heat shocked at any time up
to and including the disc stage show a mortality of 40-60 %, compared to around
20 % in control sham-shocked eggs and in eggs heat shocked at the later stages
(Tables 1, 2). A similar result was observed in Drosophila following heat shock.
Bergh & Arking (1984) found that only a small proportion (6 %) of eggs survived a
heat shock given before blastoderm, but at gastrulation the frequency of eggs
lb
eye
ant
Gl
G2
G3
Tl
T2
T3
Al
A2
A3
A4
A5
A6
A7
A8
A9
A10/11
Temperature of 15 min heat shock
45 °C
Total no. of affected animals
33 (0)
Mean no. of segments/disruption 2-6 ± 2-5
47 °C
93(3)
2-l±0-7
48 °C
193 (19)
2-5 ± 1-1
T
T
49 °C
24(0)
7-1 ±2-3
lb
eye
ant
Gl
G2
G3
Tl
T2
T3
Al
A2
A3
A4
A5
A6
A7
A8
A9
A10/11
5min
87(3)
2-0 ±0-8
lOmin
70(2)
2-3 ±0-9
20min
106 (3)
3-8±2-l
25min
45(9)
3-6±l-7
30min
53(3)
51 ±2-1
Fig. 7. Identity of the first (most anterior) segment affected following heat shocks of varying severity delivered at the elongating
protocorm stage of development. Data plotted as in Fig. 6. Each kite diagram shows the result following a heat shock of temperature
and duration indicated. The mean number of segments per disruption is also shown.
Duration of 48°C heat shock
Total no. of affected animals
Mean no. of segments/disruption
262
J. E. MEE AND V. FRENCH
continuing to develop (85 %) was similar to that of control eggs (see also Graziosi
etal 1983).
Early heat shock, however, had no effect on the segmentation of locust
embryos; the frequency of animals with segmental defects was low and not
significantly different from that of the sham-shocked controls; the location of
defects within the segment file was not influenced by age at heat shock (see Figs 3,
4, for 3h and disc stages, respectively). However, the segment pattern of nondeveloping embryos was not investigated and some of these may have had segmental abnormalities. Berg & Arking (1984) observed morphological abnormalities,
following heat shock, in nonhatching Drosophila embryos but not in hatching
larvae.
These results are rather different to those described for anuran amphibians
following early heat shock (at stages during gastrulation, prior to somitogenesis).
In amphibians, mortality and the degree to which somitogenesis was disrupted
declined as heat shock was given at progressively later stages. The location of the
disruptions was also found to depend upon stage; heat shock at earlier stages
induced widespread somite abnormalities while at later stages they were restricted
to more posterior regions (Elsdale & Pearson, 1979).
The effect of heat shock late in development
From the heart shape stage onwards, the standard heat shock of 15 min at 48°C
had little effect on the survival of locust eggs but resulted in a high frequency of
animals with segmental abnormalities. The location of segmental defects was stage
specific and shifted posteriorly from thoracic to posterior abdominal segments as
the heat shock was delivered at progressively later stages (Fig. 4). A fairly
constant number of normal segments developed between the most posterior
segment visible at the time of heat shock and the most anterior segment affected,
regardless of the stage at which heat shock was delivered (Table 3) or the severity
of the heat shock (Fig. 7). The first abnormal segment was the third or fourth to
form after heat shock. A cellular sensitivity to heat shock therefore seems to
precede visible segmentation. The number of segments affected ranged from one
to four or more and was influenced by the severity of the heat shock; segments
posterior to the disrupted region were normal.
The effects of heat shock on the locust during this period of development are
similar in many ways to those described for amphibians heat shocked immediately
prior to and during visible somitogenesis (Elsdale et al. 1976; Pearson & Elsdale,
1979).
Several aspects of the results show, however, that all parts of the locust segment
pattern do not respond equally to heat shock.
(1) The frequency of heat-shock-induced abnormalities varied with stage,
reaching high levels at stages between sT and sA2 (Fig. 4). Low frequencies at
relatively early (e.g. heart shape) or late (e.g. sA6, sA7) stages may indicate that a
proportion of the eggs in these pods was outside the sensitive period. This is not a
complete explanation, however, since the frequency was also reduced at stages
Locust segmentation after heat shock
263
(e.g. EP and sA3) when the location of abnormalities was not at the anterior or
posterior end of the segment pattern.
(2) The range of segments affected by heat shock was greater at some stages
than at others and, as discussed above, this seems to reflect variability in location
and in one case, an increase in the size of defects.
(3) Defects involving gnathal segments were rare (Fig. 6). It seems unlikely that
this was due either to inadequate sampling (large numbers of eggs were shocked at
disc, HS and EP stages) or to the fact that only the appendages of the gnathos were
scored (since 84 % of thoracic disruptions involved the appendages). In short germ
embryos the development of gnathal segments is believed to be similar to that of
the thoracic and abdominal segments (for example, see Anderson, 1972; Sander,
1976), but the different response to heat shock suggests that this may not be the
case.
(4) It is also clear that, although segments A5 and A6 are frequently affected by
heat shock, areas of disruption do not often begin with these segments (Fig. 6),
and this suggests that the abdominal segments may not all be formed in the same
way.
Segmental abnormalities have also been described in the intermediate germ
cricket, Acheta, following irradiation with X-rays (Heinig, 1967, reviewed Sander,
1976). Defects induced at the time of germ anlage formation affected thoracic
and gnathal segments. At subsequent stages more anterior head segments were
affected and finally the segments of the abdomen. Within the abdomen, which
appears to form segments as in a short germ embryo, the location of disruptions
followed a different pattern to that observed in the locust. Initially defects
occurred throughout the abdomen but later they were restricted to increasingly
posterior segments. This difference in the location of defects in the cricket and the
locust may reflect different effects of heat shock and X-irradiation or a difference
in the process of segmentation in the intermediate and short germ insects.
Segmental disruptions have been induced by a severe heat shock (4 h at 35 °C)
to long germ Drosophila embryos but, in contrast to those in the locust, they
were distributed throughout the segment file irrespective of the stage at which
heat shock was given (Maas, 1949). The maximum frequencies of abnormalities
occurred following heat shock at about the blastoderm stage (when the segment
pattern is being formed) and then later, at about the time of dorsal closure.
Heat shock and models of segmentation
Pearson & Elsdale (1979) interpreted the effects of heat shock on amphibian
development in terms of the 'Clock and Wavefronf model (Cooke & Zeeman,
1976) in which somites are generated by the interaction of two components. These
are a gradient, set up early in development across the anterior-posterior length
of presumptive somite tissue, which specifies the time at which cells become
competent to undergo segmentation (the wavefront), and a cellular oscillation
with respect to which all cells are normally synchronized (the clock). During one
264
J. E. MEE AND V. FRENCH
short phase of oscillation all presumptive somite cells that have become competent
will co-operate and eventually form a visible somite.
In amphibians heat shock results in the chaotic subdivision of a region of the
presomite tissue. The disruption is most severe anteriorly, the pattern gradually
becoming normal posteriorly. It is proposed that heat shock disturbs the synchrony of cellular clocks (Pearson & Elsdale, 1979). The boundary between
anterior normal somites and the abnormal region would therefore locate the
position of the wavefront of competence at the time of heat shock. The increasingly normal appearance of posterior somites was interpreted in terms of
a recovery of synchrony within the unsegmented tissue; the severity of the
disruption at any position reflecting the time available for recovery before
competence was achieved (Pearson & Elsdale, 1979).
The clock and wavefront model was proposed for the formation of the somites
of the trunk, which appear to form in a similar way to segments in long germ
insects. However, the model can also be applied to somitogenesis in the elongating
tail bud, where the disruptions are similar to those of the trunk.
Variations in the timing and severity of heat shock had similar effects on the
location and extent of defects in anuran amphibians and locusts, but there are
problems in interpreting the locust results in terms of the clock and wavefront
model. The abnormality is not an irregular subdivision of the embryo (as in
amphibians), but a deletion of parts of the segmental pattern (see Mee & French,
1986). The disrupted patterns may not be directly comparable since amphibian
somite fissures were studied shortly after heat shock, while locust defects were
examined in the cuticular pattern formed after the further development of the
segments during embryogenesis. However, it is not clear how deletions could arise
in the clock and wavefront model and, although a period of sensitivity to heat
shock clearly precedes visible segmentation, the model cannot readily account for
the results observed in the locust.
The 'Progress Zone' model for sequential pattern formation during growth was
proposed for the specification of pattern elements in the developing chick limb
bud (Summerbell, Lewis & Wolpert, 1973), and has since been suggested for
somitogenesis in the amphibian tail (Cooke, 1975) and segmentation in the short
germ insect embryo (Sander, 1976). It is postulated that a 'progress zone' of
constant size is located at the tip of the chick limb bud and the positional value of
the cells within the zone is labile, becoming increasingly distal with time. As cell
division occurs within the progress zone, cells emerge from it proximally, retaining
their current positional value, so that the pattern is laid down in a proximal-todistal sequence. Following the X-irradiation of early limb buds proximal structures
were missing, and more distal structures were lost as irradiation was carried out at
successively later stages (Wolpert, Tickle & Sampford, 1979). It was suggested
that X-irradiation caused cell death and so depleted the cell population of the
progress zone. Consequently surviving cells remained within the zone for longer
than normal and thereby acquired more distal positional values. This would result
in the absence of cells possessing a particular range of positional values, according
Locust segmentation after heat shock
265
to the time of irradiation, and would lead to the formation of a limb lacking
corresponding structures.
In a short germ insect embryo, a progress zone may occur at the posterior tip of
the germ anlage, and cells emerging early in development would form thoracic
segments, while those leaving late would form posterior abdominal segments.
Heat shock could produce a stage-dependent loss of segmental structures if it
resulted in cell death or merely delayed cell division, cells remaining in the
progress zone and consequently acquiring a more distal positional value before
they emerged.
The applicability of the progress zone model to segmentation in short germ
insects will be reexamined in the light of results from fragmentation experiments
(Mee, 1986).
We thank David Wright and Neil Toussaint for stimulating discussion and comments, Linda
Partridge for help with the statistics and Bert Stewart for background noise. This work was
funded by an SERC studentship to JEM.
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