/. Embryol. exp. Morph. 96, 267-294 (1986)
267
Printed in Great Britain © The Company of Biologists Limited 1986
Disruption of segmentation in a short germ insect
embryo
II. The structure of segmental abnormalities induced by heat shock
JANE E. MEE AND VERNON FRENCH
Zoology Department, University of Edinburgh, West Mains Road,
Edinburgh EH9 3JT, UK
SUMMARY
A heat shock (of 15min at 48°C) given to early embryos of the locust, Schistocerca gregaria,
results in localized abnormalities in the segment pattern subsequently formed. Most defects
involve two consecutive segments of the thorax or abdomen, and these are analysed in detail.
The abdominal defects fall into three main classes each of which involves the absence of a
particular region of the segment pair and, in one class, duplication of the region which remains.
The thoracic defects similarly involve absence of parts of the segments and the formation of a
single limb base from which one, two, or three limbs develop.
Heat shock may result in the absence of parts of segments in two distinct ways. It may
interfere with the process of segmentation or it may delete parts of already formed segment
primordia. These possibilities are discussed although, at present, neither can be excluded.
The duplication observed in some abdominal disruptions and the formation of triple limbs
indicates that the absence of parts of embryonic segments is followed by pattern regulation
similar to that occurring in regeneration studies on larval segments and appendages of other
insects. Two out of the three classes of abnormality can be explained in terms of intercalary
regeneration restoring pattern continuity, but it is possible that discontinuities persist in the
remaining class.
INTRODUCTION
Early in insect development the ventral part of the blastoderm cell layer, the
'germ anlage', becomes divided into the head, thoracic and abdominal segments of
the embryo. This process of segmentation has been most thoroughly studied in
Drosophila, a 'long germ' insect. The results of ligation (e.g. Schubiger & Wood,
1977), cell transplantation (e.g. Simcox & Sang, 1983) and clonal analysis (e.g.
Wieschaus & Gehring, 1976) show that the segment primordia become determined
around cellular blastoderm stage (at approximately 3|h). This process may be
related to the further restriction of cells to anterior or posterior compartments
which occurs at about the same time. Segment primordia seem to be evenly spaced
on the blastoderm, each about three to four cells in anterior-posterior length
(Lohs-Schardin, Cremer & Nusslein-Volhar^, 1979; Technau & Campos-Ortega,
1985). The segmental organization of the embryo becomes visible later, at
approximately 5 h (see Martinez-Arias & Lawrence, 1985).
Key words: Schistocerca gregaria, locust, segmentation, heat shock, insect.
268
J. E. MEE AND V. FRENCH
In 'short germ' insects such as the locust, the embryo forms from a small region
of the blastoderm, and the segments become visible sequentially during the
elongation of the posterior part of the germ anlage. At present there are no
estimates of the size of the segment primordia and it is not clear when they become
determined. The results of ligation studies indicate that segmentation may occur in
an anterior-to-posterior sequence just in advance of the visible appearance of
segments, although it is also possible that segmentation could occur much earlier
(Mee, 1986). It seems likely that the mechanism of segmentation in short germ
insects depends on growth of the germ anlage, and therefore differs fundamentally
from that in long germ insects, but there is no unequivocal evidence that this is the
case (see Sander, 1976).
Although segments are determined early in embryogenesis, the pattern of
cuticular structures subsequently formed depends upon cellular interaction within
the segment. These interactions have been studied by damage experiments on
embryos and, particularly, by excision and grafting experiments on the abdominal
segments of larval insects (reviewed Lawrence, 1981). Many grafting experiments
show that the epidermal cells have stable 'positional values' (Wolpert, 1971)
related to their position on the anterior-posterior axis of the segment. Positional
values form a regular sequence (or a gradient) which is repeated in each segment
and if cells with different values are confronted they will interact to restore pattern
continuity.
Following the excision of a strip of epidermis from a segment, Wright &
Lawrence (1981) showed that the pattern regenerated is precisely related to the
position of the cells confronted by the operation. The original pattern was
reformed following the removal of a half-segment length or less, but replaced by
other structures in reversed orientation when more than half a segment length was
excised. When a piece equivalent to a segment length was excised across a border,
so confronting cells of equivalent position from the two segments, the remaining
regions healed forming a chimaeric segment. These patterns of regeneration were
explained by intercalary regeneration via the 'shortest route' (see French, Bryant
& Bryant, 1976) whereby discontinuities are removed by inserting cells with the
shortest possible sequence of intervening positional values.
The segment border was regenerated when excised as part of a narrow strip, and
an extra segment border was formed ectopically when a broad strip was removed
from the middle of a segment. Hence, the segment border can be regarded as part
of the repeating pattern, rather than a special boundary responsible for maintaining the gradient within the larval segment.
As a means of studying segmentation in short germ insects we have heatshocked locust embryos at different stages. In the preceding paper (Mee &
French, 1986) we show that segmental abnormalities are induced in a predictable
location, two or three segments posterior to the segments visible at the time of
heat shock. Here the structure of the abnormal segments is analysed and discussed
in relation to the mechanism of segmentation and to pattern regulation within
embryonic segments.
Segmental abnormalities in heat-shocked locusts
269
MATERIALS AND METHODS
Disruptions of the segment pattern were generated following a 15min heat shock of 48 °C
delivered at stages prior to and during visible segmentation of the embryo of the locust,
Schistocerca gregaria (see Mee & French, 1986). Usually the segmental abnormalities were
examined in pigmented first instar hoppers. However, some animals were fixed as hatchlings
(prior to shedding the embryonic cuticle) or as late embryos which were dissected out of the egg
after fixation. Abnormal segments were examined in the animal or as whole-mount preparations
of cuticle cut from fixed animals digested with 10 % KOH (at 60 °C for 2 to 3 h), dehydrated and
mounted in Euparol.
RESULTS
(A) Disruptions of the abdomen
(1) The normal abdomen
The cuticular pattern characteristic of most abdominal segments of the first
instar hopper is shown in Fig. 1. Some segments differ slightly in structure; the first
abdominal segment (Al) is not distinct from the thorax ventrally and bears a pair
of tympanal organs and a pair of pleuropodia; A8 (in females only) and A9 bear
rudimentary genitalia and A10 and A l l are fused and bear the cerci. The features
of the segment pattern examined in heat-shocked animals were the extent of the
pigmented regions of the tergite and sternite, the extent of the 'intersegmental
membrane' (ism), and the polarity and location of bristles. The pleura was not
examined for abnormalities as it is relatively featureless and highly folded.
(2) The location of defects
The abdominal disruptions resulting from heat shock most frequently involved a
pair of consecutive segments (Mee & French, 1986) and affected all or only part of
their circumference. For convenience, the segment circumference was divided into
quadrants, consisting of the two hemisternites and two hemitergites, and twosegment defects are denned as those in which one or more quadrants are affected
in both segments. Many (31 %) of the disruptions involved all four quadrants of
both segments. Slightly less common were defects affecting one lateral half of the
animal (23%) or the whole ventral surface (23%). Defects affecting the whole
dorsal surface, one hemitergite, one hemisternite, a hemitergite and the contralateral hemisternite, or three quadrants of the circumference were rare (5 % or
less).
(3) The structure of two-segment defects
The abdominal defects analysed in detail resulted from heat shock between
formation of the germ anlage and the appearance of the 7th abdominal segment
(sA7 stage) and all involve two consecutive segments. Defects involving segments
A10 and A l l were excluded as these segments are fused and difficult to score
accurately. The defects are described with reference to a pair of consecutive
270
J. E. MEE AND V. FRENCH
hemitergites or hemisternites (and the intervening ism) and can be grouped into
four classes.
Class 1 (loss of a segment). This abnormality appeared to involve the absence of
one of the hemitergites or hemisternites of the pair; the remaining structures were
normal (Fig. 2). However, it was not clear whether (i) one of the hemitergites or
hemisternites and the ism were absent or (ii) the posterior of one hemitergite or
hemisternite, the ism, and the anterior of the next hemisegment were missing,
with the remaining parts fused to form a single, chimaeric, segment (see thoracic
defects below).
Class 2 (loss of the ism). A dorsal class 2 defect was characterized by the absence
of ism for some distance along the transverse axis and the fusion of consecutive
hemitergites. The total anterior-posterior length of the hemitergites, at the site of
ster
anterior
1mm
Fig. 1. The cuticular pattern of abdominal segments. Camera lucida drawing of the
circumference of two segments, cut along the pleura on the left side, opened out and
mounted flat. The dorsal sclerotized tergite is mostly darkly pigmented (stippling) with
a narrow unpigmented stripe at the dorsal midline (D) and also two irregular
unpigmented bands (bl and bli) laterally. Bristles are absent from the anterior margin
of the tergite (ter) and elsewhere they are sparsely distributed and directed posteriorly.
The ventral sclerotized sternite (ster) consists of two lightly pigmented patches
separated by a wide upigmented band at the ventral midline (V). Bristles occur in
pigmented areas (except at the anterior margin) and point posteriorly. The lateral
unsclerotized pleura and the 'intersegmental membrane' (ism) are unpigmented and
the pleura contains the spiracles (sp). The border between ism and sternite is indistinct.
In this preparation the anterior-posterior extent of the ism is reduced slightly by folds
and creases. In the intact animal the posterior region of each segment overlaps the
anterior region of the next (posterior) segment and the ism is folded away.
Segmental abnormalities in heat-shocked locusts
271
fusion, was usually less than twice the normal tergite length, and all bristles
appeared to be normally orientated (Fig. 3). The defect was usually restricted to
the midline region, but occasionally the ism was absent in lateral regions or across
the whole hemisegment. In a ventral class 2 defect there was a disturbance in the
normal overlap between consecutive hemisternites in the vicinity of the midline,
suggesting loss of ism in this region (Fig. 3). It was usually not clear whether the
ism was completely absent because the border between ism and sternite is not
distinct and consecutive pigmented regions were not fused. In a few cases
however, the ism was clearly missing between the hemisternites, and there was a
single enlarged pigmented region with bristles in normal orientation (Fig. 3B).
Class 3 (loss of part of the ism, polarity reversal). In class 3 defects the
anterior-posterior extent of the ism and the two hemitergites or hemisternites
(particularly that posterior to the ism) was reduced. In the posterior segment,
bristles of reversed or abnormal orientation were located near the anterior margin,
in a region normally devoid of bristles (Fig. 4). Dorsal class 3 disruptions usually
had all of these characteristics, while ventral class 3 defects often had only a
reduction in the size of the pigmented region of the hemisternites. As in class 2
defects, it was difficult to assess the extent to which the ism was reduced in length
ventrally but occasionally it was completely absent and there was a single large
patch of pigment, with a marked transverse ridge bearing bristles in reversed
orientation (Fig. 4B).
Class 4 (defects at the dorsal midline). In most class 4 disruptions right and left
hemitergites were incorrectly aligned so that noncorresponding segments were
fused at the dorsal midline (Fig. 5). In a few cases the hemitergites of a single
segment did not extend to the midline to form a normal tergite.
Although most abdominal segment abnormalities can be classified in this way,
the classes may not be distinct. For example, in class 1 defects the single
ter
Fig. 2. Abdominal segment disruption - class 1. Camera lucida drawings of dorsal
(i) and ventral (ii) surfaces of an animal with a segment missing on the left side.
Dorsally, the single segment gradually increases in anterior-posterior length towards
the midline.
272
J. E. MEE AND V. FRENCH
'abnormal' sclerite may be of greater than normal length at the midline where it is
fused with the two normal sclerites from the opposite side of the animal (Fig. 2),
while class 2 defects may have a fused sclerite of less than twice the normal length
(Fig. 3B). Also, some class 3 defects resemble class 1 or 2 in that the ism remaining
occurs intermittently and is absent in some regions (Fig. 4B).
Table 1 shows the frequency of each of these classes of disruption dorsally and
ventrally. Frequently hemisegments have defects ventrally and dorsally, although
an abnormal ventral pattern is often associated with a normal dorsal pattern. In
ism
ism
Bii
V
Fig. 3. Abdominal segment disruptions - class 2.
(A) Dorsally (i), the ism is absent and the hemitergites are fused in the vicinity of
the midline (see arrows). Bristle polarity is normal and the total length of the fused
tergites is similar to that of two tergites. Ventrally (ii), the sternites do not overlap
normally at the midline and appear to be fused (arrow), although the pigment patches
are separate.
(B) An animal with class 2 defects on the right. Dorsally (i), the ism is absent and
the hemitergites fused from bii to the midline (arrows). The total length of the
hemitergites is reduced and bristle orientation is normal. Ventrally (ii), there is a single
enlarged patch of pigmentation bearing bristles with normal orientation (arrow).
Segmental abnormalities in heat-shocked locusts
273
ism
ism
Fig. 4. Abdominal segment disruptions - class 3.
(A) Dorsally (i), the ism is reduced in length in some places and totally missing
elsewhere. The hemitergites of the posterior segment are reduced in length and bear a
reversed bristle (arrow). Ventrally (ii), there is no overlap between sternites and the
posterior hemisternites are small and bear a bristle with abnormal orientation (arrow).
(B) An animal with a class 3 defect on the left side. The dorsal pattern (i) is normal
at the midline but laterally there is no overlap between the segments, and bristles with
reversed or abnormal polarity (arrows) occur along the anterior margin of the posterior
hemitergite. On the ventral surface (ii) the ism is absent (except laterally) and the two
hemisternites are fused to give a single large patch of pigmentation with a marked ridge
bearing bristles with reversed polarity (arrows).
general, class 2 defects are associated with normal patterns, and class 1 and 3
defects with a similar defect.
(4) Disruptions involving more than two segments
Extensive defects usually involved a number of consecutive segments (see Mee
& French, 1986) but sometimes there were two regions of disruption separated by
274
J. E . M E E A N D V . F R E N C H
Fig. 5. Abdominal segment disruption - class 4. In this example, showing dorsal
mismatch, both hemitergites on the left are fused with a single hemitergite on the right
(arrowed). The posterior right hemitergite is isolated.
a normal segment border (Fig. 6) or one or more normal segments. Within a
disrupted area, defects between adjacent segments could be classified in the same
way as the two segment defects (Fig. 6).
(B) Disruptions of the thorax
(1) The normal thorax
The structure of the thoracic segments and their appendages is shown in Fig. 7.
Dorsally, the cuticular pattern is similar to that of the abdomen but ventrally the
segments are not clearly delineated. The metathoracic leg has a double row of
spines on the lateral face of the tibia but, on the pro- and mesothoracic legs, the
spines are smaller and located on the medial face of the tibia. The four spurs
present on the distal tibia were also larger on the metathoracic leg.
Table 1. The occurrence of abdominal segment defects (classes 1-3) on each
hemisegment of 660 out of 678 experimental animals with two-segment defects
Normal
Class 2
Class 1
Class 3
Total ventral
Normal*
Normal or
class 2f
Class 2%
Class 1
Class 3
Total
dorsal
29
63
0
8
190
20
2
7
318
46
1
12
20
7
247
146
38
13
35
118
595
149
285
291
100
219
377
420
204
The ventral patterns are plotted against the accompanying dorsal patterns and total values give
the number of times each class of hemitergite or hemisternite pattern occurred.
* A number of hemisternite, hemitergite and lateral half patterns were normal while other
regions of the segment circumference were abnormal.
t Where one hemisternite was clearly abnormal it was not possible to distinguish between the
normal pattern and a class 2 defect at the midline on the other hemisternite.
X Most ventral class 2 defects were restricted to the region of the midline.
15 animals had class 4 disruptions at the dorsal midline (11 with normal ventral patterns and 4
with ventral class 2 defects) and 3 other animals were unclassified.
Segmental abnormalities in heat-shocked locusts
275
IV
V
Fig. 6. Disruptions affecting more than two segments. Dorsal (i) and ventral
(ii) surfaces of an animal with a disruption affecting five segments (/to V). There are
two regions of disruption, of two and three segments, separated by a normal segment
border. Defects affecting pairs of consecutive segments are labelled class 1 to class 3
(1-3) and reversed bristles are indicated by arrows.
In heat-shocked animals the dorsal cuticular pattern was examined and scored in
the same way as that of the abdomen. Limb structures were scored for the
presence of the leg segments and the cuticular markers illustrated in Fig. 7B. The
segmental composition of limbs associated with meso- and metathoracic segment
defects could often be determined from the location of the tibial spines.
(2) Thoracic defects
The thoracic disruptions analysed in this section resulted from heat shock prior
to the sA7 stage and involve pairs of segments and their appendages. There were
151 scoreable disruptions (right and left sides scored separately); 4 involving the
labium (G3) and Tl; 61, segments Tl and T2; 44, segments T2 and T3 and 42,
segments T3 and A l .
In 34 other animals thoracic disruptions involved the dorsal or ventral surface
but not the limbs. Disruptions involving the limb of a single segment (these
included minor pattern defects and also occasional cases of partial duplication or
truncation of the limb) and disruptions involving three or more segments are not
described here.
(3) Abnormal limb patterns
In disruptions affecting a pair of thoracic segments there was a single leg base,
usually associated with two pleural plates (but occasionally three or four) and loss
of the metathoracic spiracle in defects involving segments T2 and T3. These
276
\
J. E. MEE AND V. FRENCH
T1
\T2\T3
V ^ y
ti
Bii
tl
I
CO
X^-
s
/.SP
/
c\/
M.
S9D
tao^
ci^K
sp ^JL
1
Segmental abnormalities in heat-shocked locusts
277
Table 2. Abnormal limb patterns following fusion of segments Tl with T2, and T2
with T3
T1/T2
defects
T2/T3
Total
Sets of limb structures
10
Class 1
1 complete
2
15
31
1 complete + spike(s)
4
3
1
incomplete
1
complete
+
1
Class 2
2
1 complete + 1 incomplete + spike
2
12
2 complete
12
7
2 complete + spike
4
43
7
2 complete + 1 incomplete
13
Class 3
4
30
3 complete
6
1
1
0
3 complete + 1 incomplete
Other
61
44
105
Total
Limbs are classified according to the number of sets of structures present at the distal tip(s)
(see text). 'Spikes' are unsegmented branches bearing no markers.
defects therefore involved loss of at least the regions of the thorax normally
separating successive leg bases. The limbs had between one and three complete
sets of limb structures and usually separated into a corresponding number of
branches.
The number of sets of pattern elements could only be scored in the tibia (two
rows of spines, four spurs per set), the tarsus (two rows of tarsal pads per set) and
at the distal tip (two claws). The total number of sets present on all branches often
remained constant from the proximal to the distal end of the limb (Figs 8, 9), but
sometimes the number increased (the pattern diverged - see Fig. 10) and
occasionally it decreased (the pattern converged).
Limbs were classified according to the number of sets present at the distal tip of
the limb (all branches). If two claws were present the set was scored as 'distally
complete', while sets with a single claw, no claws, or missing distal segments were
scored as 'incomplete'. The segmentation of distally complete limbs was usually
normal, but the tibia was sometimes absent or much reduced in length.
The limbs fell into three classes (Table 2).
Class LI. Class LI limbs often appeared to be completely normal (Fig. 8), with
one complete set of pattern elements, but in other cases had an enlarged
Fig. 7. The normal thorax of thefirstinstar hopper. (A) Lateral view of the thorax.
The prothorax is covered by the saddle-like pronotum (77) which extends laterally and
partly covers the mesonotum (72). The lower posterior angles of meso- and
metanotum (T3) give rise to the wing buds. The pleura of each segment bears an
anterior and a posterior sclerotized plate (1 and 2). T3s and Als are the spiracles of the
metathoracic andfirstabdominal segments. (B) Anterior views of (i) prothoracic and
(ii) metathoracic left legs, each with an enlarged medial view of the tarsus, illustrating
the segments of the limb - coxa (co), trochanter (tr), femur (/<?), tibia (ft) and tarsus
(ta); and the circumferential markers - the two rows of tibial spines (s), four tibial spurs
(sp), pairs of tarsal pads (tap) and the two claws (cl). The mesothoracic limb is similar
to the prothoracic. The faces of the limb are labelled medial (M), anterior (A), lateral
(L) and posterior (P).
278
J. E. MEE AND V. FRENCH
circumference or bore a spike on the appendage or pleural plates. The limb was
orientated normally with respect to the body.
Class L2 and L3. As shown in Table 2, these limbs had, at most, two (class L2) or
three (class L3) distally complete sets of pattern elements. Of these 73 limbs, 47
branched proximally, at a level between the leg base and the distal end of the
femur (Fig. 9) and the majority of the remaining limbs branched within the tarsus
(Fig. 10). The class L3 limbs usually branched proximally into one normal limb
and a second branch, which often branched again at a more distal level. A few
class L2 and L3 limbs did not branch (Fig. 11 A) and some class L3 limbs only
branched once (Fig. 10) so that the tip of a limb could bear up to six claws. Many
of the limb branches were fused or twisted so that it was difficult to determine their
orientation, but in scoreable cases the medial-lateral axes of all limb branches
were approximately parallel and orientated normally with respect to the body.
T1/T2
A1
8
Fig. 8. A class LI limb resulting from a disruption of segments Tl and T2. The leg has
a slightly enlarged coxa (co) but is otherwise normal. Dorsally, there is a single tergite
(72/77) similar to the pronotum.
Segmental abnormalities in heat-shocked locusts
B
Fig. 9. Class L2 and class L3 limbs, both involving segments Tl and T2. (A) shows a
limb which branches at the proximal femur {fe), to give two normal distally complete
branches (class L2). The common limb base, coxa (co) and trochanter, are enlarged.
(B) shows a class L3 limb with three distally complete branches each with a set of two
claws (c/). The limb branches at the femur/tibia joint and the anterior branch divides
again at the proximal tibia to give a normal anterior limb and a middle branch which is
distally complete but lacks the tibia. The common leg base, coxa, trochanter and femur
are considerably enlarged.
279
280
J. E . M E E AND V.
FRENCH
Cl
Fig. 10. A class L3 limb, involving segments Tl and T2, which was fused for most of its
proximal-distal length. The coxa, femur and tibia are enlarged and the limb branches
in the tarsus (ta) to give two complete branches and a branch which is probably
damaged. The pattern diverges; the tibia has two sets of pattern elements - four rows
of medial spines (s) and eight spurs (sp - seven shown); proximally, the tarsus has two
and a half sets - five rows of tarsal pads (tap), and one branch with three rows of pads
terminates with two complete sets of claws (cl).
(4) The segmental origin of limbs
The criteria for distinguishing between limbs are confined to the tibia. In proand mesothoracic limbs the double row of tibial spines occurs on the medial face; in
the metathoracic limb on the lateral face. Metathoracic tibial spines and spurs are
also larger than those of the pro- and mesothorax (Fig. 7). Thus, the segmental
origin of abnormal limbs could be investigated only where the disruption involved
segments T2 and T3 (see Table 2). The following patterns were found.
(i) A class LI limb with characteristics of either segment T2 or T3 (5 cases).
(ii) Class L2 or L3 limbs with characteristics of either segment T2 or T3 (4
cases). These limbs branched in the tarsus and had only one or two rows of tibial
spines and four or five spurs (Fig. 11 A).
(iii) Class L2 limbs separating proximally into one T2 and one T3 branch (10
cases).
Segmental abnormalities in heat-shocked locusts
281
(iv) Class L3 limbs separating proximally into a normal T2 or T3 branch and a
second branch bearing more than one set of pattern elements but markers of only
T3 or T2 respectively (Fig. 11B; 5 cases).
(v) Class LI limb with one row of T2 tibial spines and one row of J 3 tibial spines
(1 case).
(vi) Class L2 or L3 limbs with composite tibiae bearing rows of spines and
spurs characteristic in location and size, of both segments T2 and T3 (7 cases;
Fig. 11C,D).
In a further 12 class L2 or L3 limbs only one set of cuticular structures could be
identified and these were usually T2 in origin.
Thus most (23/32) of the scoreable T2/T3 limbs had structures characteristic of
both the T2 and the T3 limbs, although they were present on the same branch of
the limb in only 8 cases.
(5) Disruptions of the dorsal thorax
In order to relate the abnormal limbs to the disrupted patterns of segments
observed in the abdomen, the accompanying dorsal thorax patterns were
examined. Of the 105 abnormal limbs, however, only 26 occurred in pigmented
animals in which the dorsal pattern could be scored in detail. The dorsal
disruptions were similar to dorsal abdominal defects of class 1 (16 cases) and class
3 (10 cases). Both classes of dorsal defect were found with all types of abnormal
limbs, from class LI limbs to class L3 limbs with three distally complete branches.
(6) Disruptions involving segments T3 and Al
Disruptions involving segments T3 and Al were also often accompanied by
abnormal appendages (Fig. 12A). However, the pleuropodia of A l lack specific
markers and are normally lost on hatching, so the composition of abnormal
appendages was difficult to assess. They were usually deformed and swollen
proximally but often had two or four tibial spurs and a tarsus ending with one or
two claws.
(7) Disruptions involving segments G3 and Tl
In four animals the posterior head segment (labium) and the prothorax were
disrupted and formed two or three appendages fused at the base. The three
appendages (2/4 cases) were an anterior labium, a posterior prothoracic leg and,
between them, a short swollen appendage with two or three tibial spurs and one
tarsal claw (Fig. 12B). When only two appendages were formed they were either
the swollen appendage (with one claw) and the leg, or the labium plus a
malformed leg (with no spurs and a single claw).
(C) Disruptions of the head
Individual head segments are not clearly defined (Fig. 13A) and in heat-shocked
animals only the labrum, eyes and appendages could be scored. Disruptions of the
282
J. E. MEE AND V. FRENCH
spT3
ta
cl
Segmental abnormalities in heat-shocked locusts
283
ST3
spT3
Fig. 11. Segmental origin of limbs in T2/T3 defects. (A) A class L3 limb with a single
row of tibial spines (s) and four spurs (sp) typical of T3. The limb terminates with three
pairs of claws (c/). (B) A class L3 limb which branches proximally to give a normal,
distally complete, T2 branch and posteriorly, a branch with an enlarged tibia (ft')
bearing three distinct rows of T3 spines and eight T3 spurs (two views of the tibia and
tarsi are shown). A further short row of smaller spines is present (arrow) and these may
be T2 structures. (C) A class L2 limb with a composite tibia (ft) bearing two rows of
spines (s) and three spurs (sp) typical of T3, and two rows of spines (s) and three spurs
(sp) typical of T2. (D) A class L2 limb branching in the proximal tibia to give one
complete branch, with spines and spurs characteristic of T3, and another complete, but
composite, branch with a lateral row of T3 spines and a medial row of T2 spines.
eyes, antennae and labrum often involved either duplication or fusion and loss of
structures (Fig. 13C,D). In the gnathos, disruptions included absence of appendages and, or, fusion of the appendages of adjacent segments (Fig. 13B) and
sometimes duplication of appendages was observed.
Dorsally, the head and thorax (and sometimes anterior abdomen) were
occasionally fused in the vicinity of the midline, but it was not possible to
determine which head segments were involved.
DISCUSSION
A brief heat shock to the locust embryo at stages just before and during the
anterior-to-posterior progress of visible segmentation frequently produces a
discrete disruption in the segment pattern of the hatching larva. The defect usually
affects two consecutive segments and is located two or three segments posterior to
the last segment visible on the embryo at the time of heat shock (Mee & French,
1986). The abnormal patterns are most readily analysed in the abdomen where the
segments are fairly simple in structure and clearly delineated dorsally and ventrally. In the thorax, the presence of more numerous cuticular markers and the
morphological differences between T2 and T3 legs allows the segmental
composition of the disrupted area to be assessed. Head defects were infrequent
(Mee & French, 1986) and only the appendages, labrum and eyes could be readily
scored.
284
J. E. MEE AND V. FRENCH
T1
12A
B
Segmental abnormalities in heat-shocked locusts
285
eye
\J
par
\G3
G1
Fig. 13. Disruptions of the head. Heads of late embryos are shown in anterior view.
Only the eyes and the teeth of the mandibles, and laciniae were pigmented (indicated
by stippling). (A) Normal head showing the eyes, labrum (lab), antennae (ant) and
mouthparts: mandibles (Gl), the lacinia (la), galea (ga) and palp (pa) of the maxillary
segment (G2) and the paraglossa (par) and palp (pa) of the labial segment (G3).
(B) Fusion of appendages. On the left of the animal the appendages of segments Gl
and G2 were fused proximally (bracket) and the antenna was absent. (C) Duplication
of pattern elements. This animal bore one extra eye dorsally (not visible) and one extra
antenna. (D) Fusion and loss of pattern elements. The eyes and antennae were fused
and the labrum absent.
Fig. 12. (A) Fusion of segments T3 and Al. The example shown was dissected out of
the egg as a late embryo and is only partially pigmented. The femur (fe) and tibia (ft) of
the abnormal appendage are grossly enlarged and swollen. Distally the limb is normal
in size and bears four spurs (sp - two visible here) and a single claw (ct). The T2 leg has
been removed. (B) Fusion of segments G3 and Tl. The segmental appendages are
fused proximally. The palp (pa) and paraglossa (par) of G3 and the Tl leg are present.
Between them there is a supernumerary appendage, limb-like in shape and size, with
limb segments corresponding to the trochanter, femur and tibia. The tibia bears three
spurs (not visible here) and the limb terminates with a single claw.
286
J. E. MEE AND V. FRENCH
Segmental abnormalities
Heat shock results in three main classes of pattern abnormality in the abdomen,
all of which appear to involve the absence of regions of the segment;
Class 1 - loss of a segment (Fig. 2). This may correspond to either absence of a
single segment (including the ism) or absence of the posterior of one segment, the
ism and the anterior of the following segment (see below).
Class 2 - absence of the ism (Fig. 3).
Class 3 - absence of most of the ism plus the anterior part of the following
segment (Fig. 4). In class 3 patterns the presence of bristles with reversed
orientation suggests that deletion may be followed by pattern regulation (see
below).
The remaining defects (class 4) appear to involve abnormalities of the late
process of dorsal closure, during which the two edges of the germ band extend
around the remains of the yolk and fuse at the dorsal midline.
The dorsal surface of the thoracic segments is very similar to that of the
abdomen, and the defects found in the heat-shocked animals are also similar, and
involve loss of a segment (class 1) or loss of part of a segment with disturbed
polarity (class 3). Both classes of dorsal defect were found with all classes of
abnormal limbs.
Differences in leg structure enable the segmental composition of limbs formed
in ventral meso-/metathoracic segmental disruptions to be determined. Assuming
that thoracic and abdominal segments respond similarly to heat shock, as
suggested by the similarities in dorsal defects, the leg structures also indicate the
probable composition of the abdominal defects. Conclusions about composition
must be tentative, however, since markers are restricted to the tibia and consist
only of the size of structures and their lateral or medial position. The majority
(23/32, 72 %) of the scoreable appendages formed in meso-/metathoracic defects
bore structures characteristic of both segments, suggesting that segmental disruptions usually result from loss of parts of both segments and fusion of the
remnants. Of the six scoreable single (class LI) limbs however, only one had
structures from both segments, implying that these (and perhaps abdominal class
1) defects often result from the deletion of an entire segment.
The classes of abnormalities induced in the short germ locust embryo following
heat shock appear to be similar to the segmental defects induced in long germ
dipteran larvae by X-irradiation (Pearson, 1974) or localized microcautery
(Bownes, 1976) at approximately blastoderm stage, and by severe heat shock at
blastoderm and later stages (Maas, 1949). Polarity cannot readily be assessed in
these larvae but Bownes (1976) examined the resulting adults and found deletions
associated with mirror-image duplications of the posterior or, occasionally, the
anterior parts of the segment. She also found duplicated leg structures (Bownes,
1975). Similar abdominal and limb defects were also found after X-irradiation of
intermediate germ cricket embryos at stages before and during visible segmentation (Heinig, 1967). Higher X-ray doses resulted in some animals lacking all
Segmental abnormalities in heat-shocked locusts
287
or most of the abdomen. Locust embryos completely lacking the anterior or
posterior part of the segment pattern were occasionally found after heat shock
(Mee, 1984).
Pattern regulation
The abnormal cuticular patterns observed in the locust abdomen following heat
shock to the early embryo can be explained in the same way as the results of
excision experiments performed on larval segments of other insects (e.g. see
Wright & Lawrence, 1981). However, it is not necessarily the case that heat shock
alters a pattern which is already laid down (e.g. removing tissue by causing cell
death). It is possible that the process of segmentation may be affected directly
(e.g. by inappropriate specification of some positional values), resulting in the
formation of segments which lack some positional values and consequently undergo intercalary regeneration to form the observed patterns.
Class 1 abdominal defects could result from the absence of one segment length
of tissue, either corresponding exactly to one morphological segment or lying
across the segment border. Cells of equivalent position would be confronted and
the pattern would be stable. Absence of one to one and a half segments would
confront cells with slightly different positional values and the resulting intercalary
regeneration would give rise to a class 1 defect (Fig. 14B). The pattern would be
restored if less than half a segment were absent.
Class 3 defects may result if half to one segment length of tissue is missing,
confronting cells from very different positions. For example, in the absence of
most of the ism and anterior margin of the next segment, intercalary regeneration
would lead to the duplication of the posterior part of the segment (Fig. 14C). This
corresponds to the most frequent form of class 3 defect (Fig. 4A). Absence of this
length of tissue from other positions could result in the complete loss of the ism
and an enlarged sclerite with a midregion of bristle reversal (Fig. 14D), or in the
absence of most or all of the ism and a mirror-image duplication of the anterior
region of the sclerite (Fig. 14E). The first of these patterns, but not the second,
was found among the heat-shocked animals (Fig. 4B). However, since bristles are
found mainly on the posterior part of the sclerite, the pattern shown in Fig. 14E
may have been scored as class 2 (with no obvious polarity reversal). Class 2 defects
could also result if the loss of the ism is not followed by intercalary regeneration to
restore pattern continuity.
The abnormal limbs induced by heat shock can be interpreted in a similar way.
In some insects, extirpation of most of the ventral thorax between successive
larval legs leads to the formation of an extra leg in reversed anterior-posterior
orientation (Bohn, 1974; French & Rowlands, in preparation). These results
suggest that the larval leg and the surrounding ventral thorax bear a polar coordinate map of positional values (see French etal. 1976; Bryant, French
& Bryant, 1981). If the embryonic limb primordium contains some of these
J. E. MEE AND V. FRENCH
0/10 987654
321
09 87 6 54
3210/iQ
Normal
ter
ism
ter
ism
B
543
mm
Classi
23456
-mm
34
Class 3
567
Class 3
Segmental abnormalities in heat-shocked locusts
0/10
289
6 7 8 90/1Q
Class 2?
Fig. 14. Interpretation of the formation of cuticular pattern in two abdominal segments in normal (A) and heat shocked (B-E) animals. (A) The positional values (pv)
of each segment are represented as a graded series, 10 to 0, with the segment border at
0/10. The dorsal cuticular pattern corresponding to the positional values is given below
and bristle orientation shown by arrows. (B) In the absence of slightly more than one
segment length of tissue, cell with values 5 and 3 are confronted and intercalary
regeneration inserts value 4, restoring continuity and producing a normal-sized
composite segment, as in a class 1 defect. (C-E) If rather more than half a segment is
missing, intercalary regeneration inserts the shorter set of intervening values in
reversed order. The precise pattern formed depends on the location of the defect.
Confrontation of cells with values 2 and 6 (C) leads to a mirror-image duplication of
posterior tergite (5, 4) and ism (3), as in a class 3 defect. Confrontation of positions 3
and 7 leads to a similar duplication, but complete absence of ism, and confrontation of
6 and 10 leads to a duplication of the anterior tergite (7, 8, 9) which bears few bristles
and might be scored as class 2 (absence of ism, no polarity reversal).
positional values, the absence of thoracic tissue may stimulate intercalary regeneration, resulting in the formation of an abnormal limb base and multiple limbs
(Fig. 15).
Intercalary regeneration provoked by the absence of one to one and a half
segments would lead to the formation of a normal limb base and a single normal
limb (Class LI), as shown in Fig. 15B. The absence of half to one segment would
result in duplications of remaining structures, including parts of the limb bases.
Such a loss extending across the segment border into both limb primordia would
lead to formation of a large fused limb base within which interactions could
complete three sets of circumferential positional values forming a triple branched
leg, as in class L3 (Fig. 15C). The class L2 limbs are less readily explained.
Absence of half to one segment across the segment border, but affecting only one
limb primordium, could lead to the formation of a symmetrical double-half base.
This could converge to form a truncated limb or diverge to form two distal parts
(Fig. 15D). A few limbs like these were observed but were not scored as 'twosegment' defects as they only affected one limb base. The absence of one and a
half to two segments, however, could result in the complete absence of one limb
base plus a diverging symmetrical limb, which would be scored as class L2. This
requires the absence of a large and precisely positioned region and therefore class
L2 limbs would be expected to occur infrequently. However, class L2 limbs were
as frequent as the other classes. An alternative possibility is that they were formed
by the fusion of two consecutive limb bases, without intercalary regeneration.
290
J. E. MEE AND V. FRENCH
t
CO
•
Y—
CM
med
CO
V. CM
"£:
CO
CO
N.
00
O)
O
r—
CM
t:
CO
00
Y-~
r
"~^
CO
IV
00
CD
o
\12'
*
Fig. 15. Interpretation of the formation of abnormal limbs after heat shock. (A) The anterior-posterior values (10 to 0, as in Fig. 14)
are shown on two thoracic segments (Tl and 72). Each segment has a leg primordium (//?) with proximal circumferential positional
values (1' to 12'), as in the polar coordinate model (French etal. 1976). During development distalization will complete the
primordium, forming proximal-to-distal levels A to E. (B) If more than one segment is absent, intercalary regeneration will form
value 4, restoring continuity and completing one leg base which will grow out to form a single class LI leg. (C,D) In the absence of half
to one segment, intercalary regeneration will insert the alternative shorter set of values (as in Fig. 14C-E). If the deletion extends into
two limb bases (C), values 4, 5, 6 are formed, leading to an enlarged leg base (with three copies of values 12' and 6') within which
intercalary regeneration will lead to divergence into three distal tips (class L3). A similar absence involving part of only one of the
primordia (D) will lead to regeneration of values 2,3,4 and the formation of a mirror-image double-half limb base. In the course of
distal outgrowth this may lose or gain values at the plane of symmetry, forming either (a) a duplicated limb or (b) an incomplete
truncated limb (see Bryant etal. 1981).
72345
-—>fi^.
" - • " ^ • ^
—-oa
2 34
i
i ift.
to
a.
292
J. E. MEE AND V. FRENCH
This interpretation of the abnormal limbs induced by heat shock is consistent
with the relative positions and mediolateral orientation of the limbs and limb
branches. It predicts that the middle branch of a class L3 leg (and perhaps one
branch of a class L2 leg) is reversed in the anterior-posterior axis, but this cannot
be resolved due to the absence of suitable markers. No strict predictions are made
about the composition of the limbs. If intercalary regeneration occurs equally from
both sides of the deletion, class LI limbs and the middle branch of class L3 limbs
would be expected to be of dual origin. The composition of class L2 legs would
depend on their mode of origin. The limbs in T2/T3 defects show, however, that
(i) most LI and occasional L2 and L3 limbs have markers of only one segment and
(ii) some L3 legs have middle branches with markers of only one segment type.
However, as explained above, the available markers can only give an approximate
indication of limb origin.
The analysis suggests that pattern regulation is similar in classes 1 and LI and
classes 3 and L3. In class 2 and L2 defects it seems that intercalary regeneration
may not have occurred and pattern discontinuities persist. It is perhaps relevant that limb buds do not regenerate when amputated at slightly later stages
(Whitington & Seifert, 1982) and intercalary regeneration does not occur in limbs
or abdominal segments of locust larvae (unpublished results).
Segmentation and heat shock abnormalities
Heat shock given at early embryonic stages results in an absence of regions of
the cuticular pattern formed in the late embryo and scored in the first instar
hopper. It is not clear by what mechanism heat shock produces the segmental
disruptions or whether it acts upon the process of segmentation or by altering an
existing normal segment pattern.
In the short germ locust embryo segmentation may occur sequentially, just
posterior to the visible segments (see Mee, 1986; Mee & French, 1986) and may be
directly affected by the heat shock. In Drosophila, and many other organisms, heat
shock evokes a characteristic temporary cellular response, consisting of the
transcription and translation of a small number of 'heat-shock' genes and the
repression of the normal pattern of RNA and protein synthesis (see Mitchell &
Lipps, 1978; Ashburner & Bonner, 1979). This response develops in Drosophila
only after the blastoderm stage and is correlated with ability to survive the heat
shock (Bergh & Arking, 1984). Characteristic bristle abnormalities are induced by
heat shocks given at particular times during pupation and these may result from
the temporary failure of protein synthesis occurring at critical times (Mitchell &
Lipps, 1978). In the locust embryo, heat shock temporarily stops cell division
(M. Bate, personal communication) and, if segmentation depends on cell division
(as in the Progress Zone Model - see Mee & French, 1986), then heat shock could
directly interrupt the formation of segments. If this is the case, the formation of
partial segments (as in class 2 and 3 defects) implies that segmentation involves
more than the specification of a series of segment borders, between which
segmental positional values are subsequently formed. Similarly, because variable
amounts of the segments appear to be formed, segmentation cannot just consist
Segmental abnormalities in heat-shocked locusts
293
of establishing an alternating pattern of anterior and posterior compartments
(Kornberg, 1981).
It is possible, however, that segmentation occurs much earlier in the locust
embryo (see Mee, 1986) and that heat shock produces defects by removing parts of
already formed segment primordia. Segmental abnormalities similar to the locust
heat-shock defects follow the localized cell death caused by microcautery of
Drosophila embryos at around the time of segmentation (Bownes, 1975,1976), and
defects following severe X-irradiation at later stages have been interpreted in the
same way (Postlethwaite & Schneiderman, 1973). The heat-shock-induced disruptions are also similar to the phenotypes of many of the Drosophila 'segmentation
mutants' (Nusslein-Volhard & Wieschaus, 1980). In the 'pair-rule' mutants a
segment length is missing from within each pair of segments and the remaining
tissue is fused to form a single composite segment, as in locust class 1 and LI
defects. In the 'segment polarity' mutants each segment has part of the pattern
missing and the remainder duplicated, as in locust class 3 and L3 defects. In several
of these mutants pattern deletions are associated with cell death occurring in
approximately the corresponding regions, well after segmentation (MartinezArias, 1985; Ingham, Howard & Ish-Horowicz, 1985). It seems that duplications in
the segment polarity mutants may arise through pattern regulation after cell death
(Martinez-Arias, 1985).
If the locust heat shock defects are due to cell death within segment primordia,
the localization of defects implies that only segments at a particular developmental
stage are vulnerable. Class 2 and L2 defects may then result from cell death
delayed until a stage when pattern regulation is no longer possible, so discontinuities persist. There is, however, no indication that heat shock does cause
cell death in locust embryos (M. Bate, personal communication).
To establish whether heat shock affects the process of segmentation or the
already formed segment primordia, more information is required about the time
of segmentation in short germ insects and about the effects of heat shock on cell
proliferation and cell death.
This work was supported by an SERC studentship to J.E.M. We thank David Wright, Mike
Bate and Neil Toussaint for useful comments over the years, and especially we thank Klaus
Sander for advice, encouragement, and inspiration.
REFERENCES
M. & BONNER, J. J. (1979). The induction of gene activity in Drosophila by heat
shock. Cell 17, 241-254.
BERGH, S. & ARKING, R. (1984). Developmental profile of the heat shock response in early
embryos of Drosophila. J. exp. Zool. 231, 379-391.
BOHN, H. (1974). Extent and properties of the regenerationfieldin the larval legs of cockroaches
(Leucophaea maderae). I. Extirpation experiments. /. Embryol. exp. Morph. 31, 557-572.
BOWNES, M. (1975). Adult deficiencies and duplications of head and thoracic structures resulting
from microcautery of blastoderm stage Drosophila embryos. /. Embryol.-exp. Morph. 34,
33-54.
ASHBURNER,
294
J. E . M E E AND V.
FRENCH
M. (1976). Larval and adult abdominal defects resulting from microcautery of
blastoderm staged Drosophila embryos. J. exp. Zool. 195, 369-392.
BRYANT, S. V., FRENCH, V. & BRYANT, P. J. (1981). Distal regeneration and symmetry. Science
212, 993-1002.
FRENCH, V., BRYANT, P. J. & BRYANT, S. V. (1976). Pattern regulation in epimorphicfields.Science
193, 969-981.
HEINIG, S. (1967). Die Abanderung embryonaler Differenzierungsprozesse durch totale
Rontgenbestrahlung im Ei von Gryllus domesticus. Zool. Jb. (Anat.) 84, 425-492.
KORNBERG, T. (1981). Compartments in the abdomen of Drosophila and the role of the engrailed
locus. Devi Biol. 86, 363-372.
LAWRENCE, P. A. (1981). The cellular basis of segmentation in insects. Cell 26, 3-10.
INGHAM, P. W., HOWARD, K. R. & ISH-HOROWICZ, D. (1985). Transcription pattern of the
Drosophila segmentation gene hairy. Nature, Lond. 318, 439-445.
LOHS-SCHARDIN, M., CREMER, C. & NUSSLEIN-VOLHARD, C. (1979). A fate map for the larval
epidermis of Drosophila melanogaster: Localized cuticle defects following irradiation of the
blastoderm with an ultraviolet laser microbeam. Devi Biol. 73, 239-255.
MAAS, H. (1949). Uber die Auslosbarkeit von Temperaturmodifikationen wahrend der
Embryonalentwicklung von Drosophila melanogaster. Wilhelm Roux Arch. EntwMech. Org.
143,515-572.
MARTINEZ-ARIAS, A. (1985). The development of fused embryos of Drosophila melanogaster.
J. Embryol. exp. Morph. 87, 99-114.
MARTINEZ-ARIAS, A. & LAWRENCE, P. (1985). Parasegments and compartments in the Drosophila
embryo. Nature, Lond. 313, 639-642.
MEE, J. E. (1984). The development of segments in locust embryos. Ph.D. thesis, University of
Edinburgh.
MEE, J. E. (1986). Pattern formation in fragmented eggs of the short germ insect, Schistocerca
gregaria. Wilhelm Roux Arch. devlBiol. (submitted).
MEE, J. E. & FRENCH, V. (1986). Disruption of segmentation in a short germ insect embryo. I.
The location of abnormalities induced by heat shock. /. Embryol. exp. Morph. 96, 000-000.
MITCHELL, H. K. & LIPPS, L. S. (1978). Heat shock and phenocopy induction in Drosophila. Cell
15, 907-918.
NUSSLEIN- VOLHARD, C. &WIESCHAUS,E. (1980). Segmentation in Drosophila: Mutations affecting
segment number and polarity. Nature, Lond. 287, 795-801.
PEARSON, M. J. (1974). The relation between larval and adult abnormalities in the abdominal
segmentation of Calliphora erythrocephala (Diptera). /. Embryol. exp. Morph. 32, 533-555.
POSTLETHWAITE, J. & SCHNEIDERMAN, H. (1973). Pattern formation in imaginal discs of Drosophila
melanogaster after irradiation of embryos and young larvae. Devi Biol. 32, 345-360.
SANDER, K. (1976). Specification of the basic body pattern in insect embryogenesis. Adv. Insect
Physiol. 12, 125-238.
SCHUBIGER, G. & WOOD, W. J. (1977). Determination during early embryogenesis in Drosophila
melanogaster. Am. Zool. 17, 565-576.
SIMCOX, A. & SANG, J. (1983). When does determination occur in Drosophila embryos? Devi
Biol. 97, 212-221.
TECHNAU, G. M. & CAMPOS-ORTEGA, J. A. (1985). Fate-mapping in wild-type Drosophila
melanogaster. II. Injections of horse-radish peroxidase in cells of the early gastrula stage.
Wilhelm Roux Arch, devl Biol. 194,196-212.
WHITINGTON, P. M. & SEIFERT, E. (1982). Axon growth from limb motor neurons in the locust
embryos: the effect of target limb removal on the path taken out of the central nervous
system. DevlBiol. 93, 206-215.
WIESCHAUS, E. & GEHRING, W. (1976). Clonal analysis of primordial disc cells in the early embryo
of Drosophila melanogaster. DevlBiol. 50, 249-263.
WOLPERT, L. (1971). Positional information and pattern formation. Curr. Top. devl Biol. 6,
183-224.
WRIGHT, D. A. & LAWRENCE, P. A. (1981). Regeneration of the segment boundary in Oncopeltus.
DevlBiol. 85,317-327.
BOWNES,
(Accepted 23 April 1986)