Cell behaviour during postembryonic pattern regulation in the insect abdomen

15 Pages·2005·9.74 MB·English

Development 101, 221-235 (1987) 221
Printed in Great Britain © The Company of Biologists Limited 1987
Cell behaviour during postembryonic pattern regulation in the insect
abdomen (Oncopeltus fasciatus)
I. Regeneration of segment borders
GERARD L. CAMPBELL* and PETER M. J. SHELTON
Department of Zoology, University of Leicester, Adrian Building, University Road, Leicester LEI 7RH, UK
* Present address: Department of Zoology, University of Western Ontario, London, Ontario N6A 5B7, Canada
Summary
The confrontation of cells from the anterior region of result in any cell division or elongation. The results
an abdominal segment of Oncopeltus with those from are consistent with the view that there is a disconti-
the posterior region of the same or the adjacent nuity in positional values at the segment border. The
segment results in the generation of a segment border. stability of such a discontinuity and the regeneration
The behaviour of epidermal cells during this regu- of segment borders are discussed in terms of there
lation is described. It consists primarily of cell div- being a special population of cells at the segment
ision and transverse elongation of cells at the site of border that have the property of isolating other cells
confrontation. This behaviour can be separated from with the maximum difference in positional values.
any associated purely with wound healing because a
similar-sized wound to that used to ablate the segment Key words: cell behaviour, segment border, cell division,
border, performed within the segment, does not insect epidermis, pattern regulation, Oncopeltus fasciatus.
Introduction continuous series (French, Bryant & Bryant, 1976).
In that case confrontation of cells with different
The confrontation of cells from the anterior region of positional values leads to intercalation of the missing
an abdominal segment of the milkweed bug, Oncopel- values via the shortest route. Similar arguments can
tus fasciatus, with those from the posterior region be used to explain regeneration of segment borders.
results in the generation of a segment border at the This model contrasts with the more traditional view
site of confrontation (Wright & Lawrence, 1981). of the insect abdomen in which there is a repeating
Such a confrontation can be created by ablating the series of gradient systems; each gradient corresponds
segment border itself, resulting in its regeneration, or to a segment so that each segment is considered to be
by the removal of most of the central portion of the an independent developmental field (Locke, 1959,
segment, resulting in a supernumerary border in the 1960; Stumpf, 1966; Lawrence, 1966,1973). Here, the
centre of the segment. This regulation has been so-called 'intersegmental membranes', the boundary
explained in terms of a model in which the abdominal between adjacent segments, are assumed to form a
pattern is specified by a repeating series of positional barrier between adjacent segmental gradients. The
values, each series being equivalent to a segment gradients can be represented as series of positional
length. The segment borders do not constitute discon- values along the anteroposterior axis (Lawrence,
tinuities in the series so that the difference in pos- Crick & Munro, 1972) so that there is a discontinuity
itional values between two anteroposterior levels can in positional values at the segment border. According
be expressed as an angle (Wright & Lawrence, 1981; to this model, the discontinuity produced by ablation
Russell, 1985). This model follows the rules of the of the segment border could be removed by the
polar coordinate model for limb regeneration where intercalation of a series of intrasegmental levels with
circumferential values are similarly arranged in a reversed polarity. However, except very rarely, that
222 G. L. Campbell and P. M. J. Shelton
does not occur and a segment border reforms (Law- preparations fixed after the time at which cell division was
rence, 1966; Locke, 1960; Nubler-Jung, 1979; Wright known to have ceased. The total number of cells in each of
& Lawrence, 1981). This was explained by immi- these areas was counted and the mean cell density was
gration of preexisting cells of the intersegmental calculated for that preparation. Ten preparations were
membrane over the ablated area to restore their analysed to give the final mean for the centre of that class of
scrape.
continuity and isolate the separate gradient fields
All means are given with their standard errors. To
(Lawrence, 1966). However, this explanation is in-
determine whether the amount of cell division or the final
valid because segment borders can reform from
cell density after any particular scrape was significantly
normal segmental cells (Wright & Lawrence, 1981).
different from that following another scrape, the results
Therefore, in its original form, the gradient model is
were treated with a Student Mest.
unable to account for the regeneration of segment
borders.
(B) The operations
This paper describes the behaviour of epidermal
cells during the regeneration of segment borders. Cells from different anteroposterior levels were confronted
by an indirect method. Strips of epidermis between two
This consists largely of localized cell division and cell
levels were scraped from the cuticle with a needle and the
elongation. The results are discussed in terms of the
cells on either side were allowed to migrate across the
two models described above and the latter gradient
wound and meet in the centre. The area from which cells
model is favoured. However, although this model will
have been removed is easily discerned because the epider-
explain cell behaviour, it has to be modified to
mal cells of Oncopeltus are orange in colour (due to the
account for the regeneration of segment borders and
presence of pigment granules) and the cuticle is trans-
formation of supernumerary borders.
parent.
Cell behaviour, especially the amount of cell division,
was observed in the period following the operation by
Materials and methods
preparing stained, whole mounts of integument at different
times after the operation. For this study 4th instars (Onco-
(A) General
peltus has five larval instars) have been used that were
Stocks of Oncopeltus fasciatus (Lygaeidae, Hemiptera) were starved following a moult. This eliminates any modification
kept at 24°C in a D: L cycle of 12:12 and fed sunflower seeds
of behaviour by hormones secreted during the moult cycle,
and water. Wounds were performed on animals that had
because the moult cycle of Oncopeltus is normally initiated
been starved soon (at most 14 h) after moulting. They were
by feeding (Nijhout, 1979). No epidermal cell divisions are
starved for about 48 h before any operation. Animals were
found in unwounded, starved larvae (Campbell, 1987a).
anaesthetized either with ice (for operations lasting under
Identical confrontations were produced in 3rd instars but
one minute) or CO (for experiments requiring more time).
2 were scaled down because of the smaller size of this instar.
The integument was cut with slivers of razor blade and cells
The 3rd instars were fed and the pattern generated follow-
were scraped from the underside of the cuticle by inserting
ing the operation was recorded two moults later at the
a blunted tungsten needle through a cut. To help plot the
beginning of the 5th instar.
positions of mitotic figures, 4th instars were injected
through the base of a metathoracic leg with 0-5 ml of a The area of integument used was the 3rd tergite
1-Omgml"1 solution of colchidne (about 5-Omgg"1 body (Fig. 1A), together with the 4th when the scrapes extended
weight) 12 h before fixation. For whole mounts, integument across a segment border. The scrapes extended transversely
was fixed-for about l-0h in Carnoy's fixative. Hydration across the segment from an anteroposterior incision
through an alcohol series was augmented with 880 ammonia (required to intro'duce the needle) level with the muscle
at the 70 % and 50 % stages to depigment the epidermal insertions on one side to past the muscle insertions on the
cells. The whole mounts were stained in Hansen's trioxy- other side of the segment. This distance is about 1-7 mm in
haematin (Pantin, 1969). Integument for scanning electron 4th instars. The anteroposterior locations and widths of the
microscopy was fixed in acetone, critical-point dried with scrapes are given below and in Fig. 2. In a rectangular
CO , coated with gold and viewed and photographed on a
2 scrape, the length is the mediolateral distance. The exact
Cambridge S100 SEM.
dimensions of these scrapes are for 4th instars. When
For a quantitative measure of cell division, the total repeated on 3rd instars these dimensions have to be scaled
number of mitotic figures was counted in preparations down. The length of the segment in 4th instars is just under
(treated with colchicine) over a length (mediolateral dis- 0-6 mm.
tance) of 1-0 mm in the centre of the wound. At the times
The scrapes are as follows.
that measurements were made, virtually all of the mitotic
figures occurred within an anteroposterior width of 0-1 mm.
12 preparations were analysed to give the final mean. The (1) 0-1 mid and 0-1 bord
final cell density in the centre of a scrape was determined by These operations remove a 0-1 mm wide strip of cells in the
the following procedure. Three sample areas, each measur- middle of the segment (0-1 mid) and across the border
ing 0-1x0-1 mm, were randomly chosen from within the between segments 3 and 4 (0-1 bord). The width of these
centre of the scrape (at different mediolateral positions) in scrapes corresponds to about a sixth of a segment length.
Regeneration of segment borders in insects 223
(2) 0-5 mid this scrape is about 0-5 mm so that it leaves 0-05 mm strips
of cells behind the anterior border and in front of the
This operation removes most of the epidermal cells be-
posterior border.
tween the segment borders. The anteroposterior width of
• ••_\
Fig. 1. (A) A recently moulted fourth instar of Oncopeltus fasciatus, dorsal side. The abdomen is divided clearly into
segments. Segment 3 is marked. The sites of the lateral muscle insertions (m) that occur approximately midway between
the segment borders indicate the mediolateral extent of the scrapes shown in Fig. 2. (B) Unstained, whole mount of the
segment border between 2nd and 3rd tergites in a 4th instar. The cells at the anterior of the 3rd segment are clearly more
darkly pigmented than surrounding cells. (C,D) The cuticular pattern in and around the segment border between the 2nd
and 3rd tergites of a 5th instar. (C) Scanning electron micrograph which shows that there is a groove in the surface of the
cuticle at the segment border. (D) Whole mount, depigmented and photographed under phase contrast. At the top and
bottom, this photograph shows the normal cuticular pattern found over most of the segment. At the segment border, the
pattern of polygons is obscured. Here it appears that adjacent polygons fuse laterally and their posterior margins are aligned
and continuous over long distances. Anterior at top. Bars: (A) 0-5 mm, (B) 01 mm, (C) 005 mm, (D) 0-02 mm.
224 G. L. Campbell and P. M. J. Shelton
(3) 0-6 bord ments are characterized by a groove in the surface of
In this case, cells are removed from the middle of the 3rd the cuticle and by transverse elongation of the cells
segment to the middle of the 4th. The sciape is about (Lawrence & Green, 1975; Wright & Lawrence, 1981;
0-6 mm wide. Fig. 1C). The cells situated at the segment border are
At the outset it was assumed that the cells migrating over usually more darkly pigmented than surrounding
a scrape maintain their original positional values. The cells, especially in 4th instars (Fig. IB). The surface
validity of this assumption is discussed below.
of the cuticle over most of the segment is sculptured
into a pattern of irregularly packed polygons each
Results representing the area of cuticle secreted by a single
epidermal cell (Hinton & Gibbs, 1971; Fig. ID). This
(A) The Oncopeltus abdomen pattern is polarized because the posterior margin of
The abdomen of Oncopeltus is clearly divided into each polygon is raised slightly and represents the light
segments (Fig. 1A). The borders between the seg- areas visible in phase-contrast microscopy.
OPERATION CELL BEHAVIOUR FINAL PATTERN
2/3 Cell division Cell elongation
segment
01 mid \\\\\\V*\\\\\\\\\Vfc\\\.t border No No
3/4
segment
border
Regenerated
2/3
border
Yes, Yes
01 bord often extensive
3/4
Band of
" • • • • •- reversed
polarity
2/3
Yes, Yes
0-5 mid often extensive Supernumerary
border
3/4
Fused
~ • • • • "
segments
Usually little, Usually no,
0-6 bord occasionally but occasionally
extensive
Region of
-?_?_?-?_?_?_•}_ distorted
pattern
Fig. 2. The location and widths of scrapes designed to confront cells originally found at different levels in the
anteroposterior axis of the 3rd and 4th tergites and a summary of the results (cell behaviour and final pattern). Each scrape
extends laterally from the incision on one side (required to introduce the needle used to remove the cells) to beyond the
muscle insertions (Fig. 1A) on the opposite side. The 3rd tergite appears normal two moults after 0 1 mid scrapes. The
segment border usually regenerates after 01 bord scrapes, but occasionally it does not and a narrow region of reversed
polarity is found instead. Supernumerary segment borders are usually found after 05 mid scrapes. The polarity of the
central region which contains this border is reversed. Following 0-6 bord scrapes, in half of the cases the 3rd and 4th
segments become fused; in the remaining half there is cell elongation and distortion of the cuticular sculpturing pattern
where the 3/4 border was originally located.
Regeneration of segment borders in insects 225
(B) Cell behaviour in fourth instars cell division (four mitotic figures in 43 preparations
(1) Cell behaviour after 0-1 scrapes fixed 2, 3 and 4 days after wounding) and no visible
The cells spread over the wound and cover it within build up of cell numbers in the centre of the wound
(Fig. 3A). Cell behaviour after an identically sized
24 h. Following 01 mid scrapes there is virtually no
wound in other locations within the segment is very
similar (data not given).
The behaviour following the same-sized wound but
removing the cells located at the segment border
between segments 3 and 4 (0-1 bord) is very clearly
different from that described above (Figs 3,4). Often
there is quite extensive cell division in the centre of
the wound, leading to a local increase in cell density
(Fig. 3B,C). Here the cells also show a change in
morphology from the usual polygonal shape with
round nuclei to a spindle-shaped form with elongated
nuclei orientated transversely. This is an exaggeration
of the morphology normally found at the segment
border (Lawrence & Green, 1975). The number of
divisions found increases from day 3 to peak 4 days
after wounding (Fig. 4). All this division is confined
to a band usually no wider than 0-05 mm found in the
centre of the wound and there is no spread of cell
division from the site of confrontation. The high
division rate is reflected in a final cell density in the
centre that is significantly larger (P < 0-001) than that
at the centre of a 01 mid scrape.
However, it should be noted that the increased cell
division following a 0-1 bord scrape is variable and
sometimes does not occur. Then the behaviour is very
similar to that following other 01 mm scrapes within
the segment. Thus, in a minority of cases (2/12) no
mitotic figures were found in 01 bord preparations
from animals treated with colchicine and fixed after 4
A
50' >,
Qa
40' 6000
1*1
a 9.
30'
4000
20
r-j
>-.
a. 2000
B 10' rQ
3
Fig. 3. 0-/ scrapes. Stained whole mounts of dorsal, 0-1 mid 01 bord 01 mid 01 bord
abdominal integument from 4th instars fixed 4 days after
wounding and injected with colchicine 12 h before fixation. Fig. 4. Cell division and final cell density after 0-1 mm
(A) 0-1 mid. An area of low cell density has been created scrapes in 4th instars. (A) The number of mitotic figures
by the wound. No mitotic figures can be found among the per sample area per preparation 3 and 4 days after the
activated cells that have migrated over the region originally operation. Animals were treated with colchicine 12h before
scraped free of cells (s). (B,C) 01 bord. There is an area of fixation. No mitotic figures are found after 01 mid scrapes
higher cell density in the centre of the wound (z). At in contrast to 01 bord scrapes which ablate the segment
higher magnification, (C), numerous mitotic figures (m) border. (B) Final cell density in the centre of the scrapes
can be seen here, and many of the nuclei are transversely after the cessation of cell division. The cell density in the
elongated. Anterior at top. Bars: (A,B) 0-1 mm, (C) centre of the scrapes is greater after 01 bord scrapes than
002 mm. after 01 mid scrapes.
226 G. L. Campbell and P. M. J. Shelton
days. In all other cases, there was evidence of cell zone Olmm-0-3mm wide. After 4 days, this band
division and elongation. has narrowed significantly to a strip usually no more
than 0-07 mm wide located in the centre of the scrape
(2) Cell behaviour after 0-5 mid and 0-6 bord (Fig. 5). Again, like the pattern of mitosis following
scrapes 0-1 bord scrapes, mitotic figures are confined within
A 0-5 mid scrape confronts cells originally located at this narrow band. The final cell density in the centre is
the same levels in the segment as confronted by a 0-1 much higher than in the sparse zone immediately
bord scrape, but differs from the latter scrape in that adjacent to the zone of higher cell density (Fig. 6).
the cells originate from the same segment rather than In preparations not treated with colchicine, the
adjacent segments and in the wound being much spindles of the mitotic figures are orientated predomi-
larger. After a 0-5 mid scrape, two processes, wound nantly perpendicular to the anteroposterior axis
healing and pattern regulation, take place. The com- (Fig. 5C) as judged from alignment of the metaphase
ponent of cell behaviour due to wound healing alone plates. Daughter cells will therefore separate to lie
is detectable in scrapes of a comparable size where alongside each other laterally. Cells in the vicinity of
the confronted cells have the same positional value. the band of high mitotic activity are transversely
The 0-6 bord scrape is of this type; elongated. All 0-5 mid preparations show evidence of
0-5 mid scrapes are rapidly repaired and in most cell division and elongation in the centre.
cases have been covered with cells within 48 h of the The pattern of cell behaviour during the first 72 h
operation. By 72h numerous mitotic figures are after 0-6 bord scrapes is similar to that after 0-5 mid
present in all preparations. Injection of colchicine scrapes. Many are repaired within 48 h and after 72 h
reveals a central band of extensive mitotic activity in a mitotic figures are often found in a broad band in the
Jl
:;::vf •..'*••; •.'. C\V **:.-,
Fig. 5. Stained whole mounts of dorsal, abdominal integument after 05 mid scrapes in 4th instars. In (A) and (B), the
animal was fixed 4 days after the operation and was treated with colchicine. There is a zone of higher cell density (z) in the
centre of the area of low cell density created by the scrape (s). Numerous mitotic figures (m) are found in this zone, as
shown in B. (C) 7 days after the operation (no colchicine), at the site of confrontation in the centre of the scrape. Some
nuclei are transversely elongated and a mitotic figure is present in which the orientation of the metaphase plate is clearly
parallel to the anteroposterior axis. Some cell death (d), revealed by small darkly staining nuclei that gradually become
more and more diffuse, is also present. Anterior at top. Bars: (A) 005 mm, (B) 0-02 mm, (C) 002 mm.
Regeneration of segment borders in insects 227
2 60
D
Centre
40 6000'
Sparse zone
4000-
20
2000'
3
0-5 mid 0-6 bord 0-5 mid 0-6 bord
Fig. 6. Cell division and final cell density after OS mid and 0-6 bord scrapes in 4th instars. (A) The number of mitotic
figures per sample area/preparation 4 days after the operation. Animals were treated with colchicine 12 h before fixation.
The number of mitotic figures after a 0-5 bord scrape is considerably greater than the number found after a 0-6 bord scrape.
(B) Final cell density in the central region of the scrapes and in the sparse zone to either side of it after the cessation of cell
division. The cell density in the centre of a 0-5 mid scrape is greater than that in the sparse zone surrounding it. The
central cell density is also greater than that following the 0-6 bord scrape. The density in the centre of 0-6 bord scrapes is
not much greater than that in the area surrounding it.
Fig. 7. Stained whole mounts of dorsal abdominal integument from animals fixed 4 days after 0-6 bord scrapes in 4th
instars treated with colchicine 12 h before fixation. The most common result is shown here. (A) The area originally scraped
free of cells (s) has relatively uniform, low cell density with no increase in the centre. (B) The cells in the centre are
obviously activated with large round (no elongated) nuclei. An, anterior; Po, posterior. Bars: (A) 0-05 mm, (B) 0-01 mm.
228 G. L. Campbell and P. M. jf. Shelton
centre of the scrape zone. However, 4 days after Pattern two moults after 0-5 mid scrapes
wounding, in contrast to 0-5 mid scrapes, a region of When 0-5 mid scrapes are made on segment 3 of 3rd
high mitotic activity in the centre of the wound is not instars and the animals are examined as 5ths, a
usually found (Fig. 7). The number of mitotic figures supernumerary segment border (characterized by a
found is significantly smaller (P < 0-001) than found 4 cuticular groove and characteristic sculpturing pat-
days after a 0-5 mid scrape (Fig. 6). tern, Fig. 1C,D) is present in 80% of cases (32/40)
If the increased cell density, division and transverse (Fig. 9). The polarity pattern is also altered so that
elongation at the site of confrontation is termed a the polarities of bristles and cuticular sculpturing are
'reaction', then 5 days after the 0-6 bord operation, normal immediately posterior to the original 2/3
the majority (8/12) shows no reaction while the border and immediately anterior to the original 3/4
minority (4/12) shows a varying amount. Where no border, but the central region containing the super-
reaction occurs there is an almost uniform cell density numerary border has reversed polarity (Fig. 9B). The
over all the wounded area with no increase in the pattern appears to consist of a mirror-image dupli-
centre (Figs 6, 7A). The nuclei of the cells in the cation of the anterior-most part of the segment in
centre of these preparations are round (Fig. 7B). The front of the supernumerary border and a mirror-
cell density in the centre is significantly smaller image duplication of the posterior-most part of the
(P< 0-001) than that in the centre of 0-5 mid scrapes segment behind the new border (Fig. 9B). The final
(Fig. 6). pattern of the remaining 20 % is variable and difficult
to interpret.
(C) Cell behaviour and pattern one and two moults
Apart from the polarity patterns, the pigmentation
after the operation
of the segment also helps interpretation. The pos-
Cell behaviour at later moult stages terior region of the tergites in 5th instars is often less
When 0-1 bord scrapes are fixed as 4th instars soon heavily pigmented than more anterior regions so that
(1—3 days) after the postoperative moult, the cells in it appears lighter in colour. Thus, at normal segment
the regenerating border region are often activated borders there is a discontinuity in the level of pigmen-
(having large round nuclei and prominent nucleoli tation (in addition to the heavily pigmented cells that
(Wigglesworth, 1937)), and on a minority of prep- are usually found at the segment border (Fig. IB)).
arations (5/16, not treated with colchicine) some Where a supernumerary border forms, the region
mitotic figures are present (Fig. 8A). These animals posterior to it often has the typical pigmentation of
were also starved following the postoperative moult the posterior segment. The region anterior to the
to prevent initiation of the next moult cycle (during supernumerary border has a higher level of pigmen-
which all the epidermal cells would become acti- tation characteristic of anterior levels of the segment,
vated). The extent of this activation is variable and so that there is also a discontinuity in the level of
sometimes it is absent. At this stage, activation and pigmentation at this border (Fig. 9A).
cell division are never observed following 01 mid
scrapes. Pattern two moults after 0-6 bord scrapes
In theory, 0-6 bord scrapes confront cells with the
Pattern two moults after 0-1 scrapes same positional value from adjacent segments, so that
Following 0-1 mid scrapes in the 3rd instar virtually no these operations should result in a segment extending
alteration can be found in the tergite pattern of the from the 2/3 segment border to the 4/5 border. Out
5th instar. The cuticular pattern of 5th instars, two of 37 0-6 mid scrapes performed on 3rd instars and
moults after a 0-1 bord scrape, is variable, but analysed in 5ths, approximately half (19) have pat-
generally preparations can be separated into two terns resembling that expected, i.e. a large segment
classes. The majority (20/25) appears to have regen- extending from the 2/3 border to the 4/5 border
erated the segment border, which is often indis- (Fig. 10). No grooves in the cuticle are found where
tinguishable from that at an unwounded segment the 3/4 segment border would normally occur and the
border (Fig. 1) and polarity is normal. However, cuticular pattern resembles that found over most of
occasionally it is imperfect and more than one cuticu- an unoperated segment (Fig. 10B). There is also no
lar groove is found (Fig. 8B). Preparations in the sign of transverse elongation of cells at this site.
second class (5/25) have a band of reversed polarity The pattern in the remaining 50% of scrapes
in the region where the 3/4 border used to be. (18/37) is very variable and difficult to interpret
This extends anteroposteriorly for about 0-1-0-2 mm (Fig. 11). Here, in contrast, the cells do show trans-
(Fig. 8C). In this region, a groove is absent and the verse elongation in the centre of the original wound at
cuticular sculpturing more closely corresponds to that the level where the ablated 3/4 segment border was
over most of the normal segment rather than at the located. However, its extent may vary from a single
segment border (Fig. 8C). line of very modestly elongated cells to a 0-4 mm wide
Regeneration of segment borders in insects 229
Fig. 8. Whole mounts of O'l bord scrapes fixed after the first or second postoperative moult when the animal was wounded
as a 3rd instar. (A) Fixed 3 days after the first postoperative moult and stained with trioxyhaematin (animal not treated
with colchicine). The animal has been starved since the moult. The cells in the regenerating border region are activated
(ac) and mitotic figures (m) are found here. (B-D) Fixed soon after the second postoperative moult, illustrate the two
possible outcomes of this operation. (B) Scanning electron micrograph of the regenerated border region showing the usual
outcome. There is a groove in the cuticle in the centre of the field of view (arrowed), which probably corresponds to the
regenerated border. However, there are also additional grooves running more or less parallel and anterior to this one.
Regenerated borders are therefore often somewhat abnormal. (C) Whole mount, under phase contrast showing the minority
outcome. There is a band of polarity reversal of about 01 mm in width over the region where the 3/4 border was originally
situated (pr). The polarity change is apparent in the locally reversed orientation of the hairs and in the reversed pattern of
the cuticular sculpturing. The sculpturing does not resemble that found at a segment border (compare with Fig. ID).
(D) The epidermis of the regenerated region (pr) in C, shown stained with trioxyhaematin, revealing the transverse
elongation of the cells in this region. This change in cell shape may be responsible for the slightly distorted cuticular
pattern shown in C. Anterior at top. uc, unactivated cells. Bars: (A) 005 mm, (B) 005 mm, (C) 0-03 mm, (D) 002 mm.
230 G. L. Campbell and P. M. J. Shelton
9A
Fig. 9. 0-5 mid scrapes. Recently moulted 5th instars two moults after the operation. The resulting pattern has a
supernumerary border (su.b) in the 3rd segment. The polarity of hairs and cuticular sculpturing is reversed in the vicinity
of this new border. Within the 3rd segment, the area anterior to the supernumerary border is always larger than that
posterior to it. (A) In situ. The area posterior to the supernumerary border has a lower level of pigmentation than the area
anterior to it. (B) Unstained, depigmented whole mount, showing the polarity reversal. The points at which polarity
reversal begins and ends, anterior and posterior to the supernumerary border, is approximately half-way between this
border and the normal (2/3 and 3/4) borders. The polarity reversal is clearer anterior to the supernumerary border than
posterior because the former region is larger and more hairs are present here. (C) Scanning electron micrograph. All three
borders are represented by a groove in the cuticle, p, pigment spot around stink gland situated at the 4/5 border. Bars: (A)
0-5 mm, (B) 02mm, (C) O2mm.
band of extremely elongated cells (Fig. 11B). The observations show that this behaviour cannot be
cuticular sculpturing pattern associated with this cell attributed simply to wound healing. Firstly, when an
elongation is often very modified and the polarity in identically sized wound to that used to remove the
this region can be abnormal (Fig. 11A). segment border is performed in the centre of the
segment (0-1 mid) no cell division is found (Fig. 4).
Secondly, 0-6 bord scrapes, which confront cells from
Discussion the same position in adjacent segments, result in
much less cell division than after 0-5 mid scrapes even
(A) Cell behaviour during the generation of segment though the wounds are similar in size (Fig. 6).
borders Two possible explanations for the cell behaviour
Cells located in the anterior region of a segment can pattern after 0-1 bord and 0-5 mid scrapes are that it
be confronted with those found in the posterior may result from the interaction of cells with large
region either by removing the epidermis at a segment differences in positional values or that it may be
border (01 bord scrape), confronting cells from directly associated with features peculiar to regener-
adjacent segments, or by ablating most of the seg- ation of the segment border. The second possibility is
ment (0-5 mid scrape), confronting cells from the ruled out because similar cell behaviour patterns can
same segment. Both these operations normally result be found after confrontations that do not result in
in localized cell division and transverse elongation of segment border formation (0-3 post scrapes in Camp-
the cells at the confrontation site (Figs 3, 5). Two bell, 1987/?). Therefore, it seems likely that the cell