/. Embryol. exp. Morpli. Vol. 49, pp. 27-46, 1979
Printed in Great Britain © Company of Biologists Limited 1979
27
Post-embryonic determination of
the ecdysial line in the cockroach: evidence for
pattern regulation in the medio-lateral axis
By P. M. J. SHELTON 1
From the Department of Zoology, University of Leicester
SUMMARY
By removing or transplanting sections of the ecdysial line in the cockroach Gromphadorhinaportentosa, pattern regulation in medio-lateral axis of an insect has been demonstrated.
The line can reform after sections have been removed and when epidermal grafts from either
side of the midline are grafted with confronting medial edges. The results are explicable in
terms of known mechanisms for pattern regulation in insects.
INTRODUCTION
Although there have been many studies on the regulation and determination
of spatial patterns of cellular differentiation in the antero-posterior (a-p) axis of
the insect segment during post-embryonic development (see Lawrence, 1973 a),
little is known about determination in the medio-lateral (m-1) axis. This is in
spite of the fact that there is well documented evidence for m-1 pattern regulation
during embryonic stages (see Sander, 1971, 1976). The absence of studies on
post-embryonic stages is due to the lack of suitable markers in. the m-1 axis.
However, one such marker does exist and this is the ecdysial line. Using the
cockroach Gromphadorhina portentosa, I have investigated the formation of this
structure by ablation and grafting experiments.
The ecdysial line defines the dorsal thoracic midline and is a narrow zone of
untanned cuticle which splits to allow the emergence of the next larval instar.
The line is produced by a strip of underlying epidermis only a few cells wide.
Since there is epidermal cell growth and proliferation between moults there
must be some mechanism which ensures that the line remains patent over
many cell cycles.
In pattern regulation a structure adapts to interference or loss of parts by
reconstructing missing regions from the remaining cells or their division products. Three types of transplant were performed and all provide good evidence
for pattern regulation in the m-1 axis. In the first type of experiment, pieces of
1
Author's address: Department of Zoology, School of Biological Sciences, Adrian Building, University of Leicester, University Road, Leicester LEI 7RH, U.K.
28
P. M. J. SHELTON
segment containing the ecdysial line were removed. At the following moult a
perfect ecdysial line reformed. In a second type of operation, squares of cuticle
containing the ecdysial line were moved laterally away from the midline. When
the distance was no greater than 1 mm the ends of the transplanted line reconnected with the host line. When the distance was increased to 2-5 mm the line
did not reconnect one moult later. In the third type of experiment, two pieces of
epidermis from either side of the line were transplanted into dorsal abdominal
segments so that their most medial edges were apposed. When the grafts were
taken from close to the midline a new ecdysial line formed medially in a high
proportion of cases. These and other experiments are described in detail below.
A number of factors must be considered when interpreting the results. There
may be local cell migration after damage to the epidermis (Wigglesworth,
1940). The missing structure could reform by morphallaxis where there is a local
reassignment of positional information to make a properly proportioned
structure without the production of new cells (Wolpert, 1969). There could be
epimorphic regeneration so that the intervening structures are formed by
localized cell division and differentiation from the cut surfaces wherever noncongruent levels are apposed (French, Bryant & Bryant, 1976). Finally rotation
experiments may be complicated by derotation of the grafts (Bohn, 1974;
Lawrence, 1974; Nubler-Jung, 1974). For a logical exposition the Results and
Discussion sections are combined.
MATERIALS AND METHODS
Cultures of Gromphadorhina portentosa were maintained in large flbreglass
tanks at a constant temperature of 21 °C. They were fed on rat pellets and water.
All operations were performed on recently moulted animals which were
identified by the rounded shape of their abdomens. Most animals used were
about half the adult size. They were first anaesthetized with CO2 for 1 min,
then they were held for about 5 min under distilled water that had been flushed
previously with CO2. They were carefully dried with tissue and restrained with
plasticine strips. Pieces of graft cuticle with attached epidermis were cut out
using fine scalpels made from razor-blade chips. The host site was prepared in
the same way and was trimmed, if necessary, using iridectomy scissors. Once in
position the grafts were sealed in place using insect wax (Krogh & Weis-Fogh,
1951). Experimental animals were regularly inspected and removed after the
following moult which was normally about one month after grafting. For
photography, insects were immersed in water and photographed from above
after initial fixation in Duboscq-Brasil fixative (Pantin, 1969). Semi-thin
Araldite sections of the ecdysial line were prepared using standard. EM preparative methods after an initial fixation in a paraformaldehyde/glutaraldehyde
mixture (Karnovsky, 1965). To confirm that regenerated lines had a normal
appearance three experiment-(i) and two experiment-(vi) animals were
Ecdysial line determination
29
Fig. 1
Fig. .1. A line diagram of the three thoracic segments to show the ecdysial line and
features such as tubercles and underlying muscle insertions.
Fig. 2. A photograph of the ecdysial line in the metathoracic segment.
Fig. 3. A semi-thin section of the ecdysial line stained with toluidine blue to show
the characteristic appearance of the line in cross-section.
sectioned using wax-embedded material. This material was then fixed in.
Duboscq-Brasil fixative and the sections were stained with haemalum and eosin
(Pantin, 1969).
RESULTS AND DISCUSSION
Nature and extent of the ecdysial line
The ecdysial line defines the dorsal midline of the pro-, meso- and metathoracic tergites (Figs. 1, 2). Anteriorly it extends over the dorsal head region
and here it divides symmetrically to form two branches, each terminating at one
of the two lateral ocelli; there is no ecdysial line on the abdominal segments.
The line is continuous from segment to segment and is recognizable as a narrow
strip of untanned cuticle (up to 100 /.cm wide) which is produced by the epidermal cells of the midline. In sections of the newly moulted cuticle the ecdysial
line forms a depression on the surface of the cuticle. The line of weakness does
not stain with toluidine blue and it can be visualized in 1 /an Araldite sections
(Fig. 3). The ecdysial line cuticle is twice as thick as the surrounding stained
3
EMB
49
30
P. M. J. SHELTON
r: rr\
i.
•
i-
•
• -1
•
•
I1
1
L 1 I'J
Control
(b)
T
Kxpt (i)
•1
(c)
\
Expt (ii)
Fig. 4. Diagrams to show the operation, gradient of distance configuration and the
result of various experiments, (a) After control removal and replacement the line is
perfectly normal in the majority of cases, (b) When a narrow strip containing the line
is removed and discarded, the missing section is regenerated by the following
moult, (c) Cuts across the line produce only very small disturbances in the region of
the cut (see Fig. 9).
cuticle and it is underlain by densely packed epidermal cells. The line is wider in
some regions than others and is from 6 to 20 cells wide.
Diagrammatic representation of operations and results
The operations and results for each graft configuration are described by
means of plan diagrams (Figs. 4, 5, 6 and 7) and where possible the polarities of
the grafts and their origin within the m-1 axis have been represented with
reference to a bilaterally symmetrical gradient of distance from the midline.
The convention adopted is that the high point of the gradient is at the centre of
the segment. The gradient convention is purely a means of displaying the
Ecdysial line determination
r.-.-.
31
„
:
L:
L:: J
f
-T
1V
•
/
•
'I
n::•):
K x p l (iii)
«r
1
L
.1:. J-.
^ i
1
1
Hxpt(iv)
(<•)
l:xpt (v)
Fig. 5. (a) This exchange of squares between the meso- and metathoracic segments
produces small lateral shifts of the ecdysial line. At the following moult it has
reconnected, (b) Exchanges between left and right sides of the same segment can
produce large lateral displacements of the line. The displaced line fails to rejoin but
a supernumerary line forms where in terms of the gradient convention there is a
ridge, (c) Squares of cuticle from more lateral levels can be exchanged without the
formation of a line at the next moult.
3-2
32
P. M. J. SHELTON
1
1 J
fc
i-
-i-
-i
•
^
....
(a)
i:.x P t
Lli-.J
^
Kxpt (vii)
Fig. 6. (a) Thoracic cuticle from close to the ecdysial line can be transplanted into
similar sites in the abdomen. An ecdysial line reforms where the medial edges of
thoracic grafts are apposed in the abdomen but not where similar abdominal grafts
are apposed in the thorax, (b) When grafts of thoracic epidermis come from more
lateral levels no line forms when they are similarly apposed in the abdomen.
maximum information about each graft situation. It is not intended to imply
that the epidermal cells have graded physiological properties
Control operations
As control grafts, 2-5 mm squares of cuticle and attached epidermis from the
middle of the meso- (five animals) and metathoracic (five animals) segments
were replaced and sealed in their original locations (Fig. 4a). The majority
result of the control operations was a perfectly normal ecdysial line at the
following moult (six animals). In two cases the line was slightly wider at the
points where the anterior and posterior margins of the graft intersected the
Ecdysial line determination
n
j
r.-.-.-i
V
/i
i
(a)
Hxpt (viii)
r
(h)
v! ...
ITi
( • • . • • : • • _ - - •
T.xpt (ix)
F •
n
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Expt (x)
Expt (xi)
(e)
p
1
1
Hxpt (xii)
Fig. 7. (a) By exchanging between meso- and metathoracic segments, epidermis
from close to the midline can be confronted so that the confronted edges are situated
in the metathoracic segment at the bottom of a valley according to the gradient
convention. After such grafts there is no formation of a line at the following moult.
In the mesothoracic segment the line does reform as might be predicted from preceding experiments, (b) Epidermis from graded distances away from midline can
be confronted by another graft configuration resulting from exchanges between
meso- and metathoracic segments. In the mesothoracic segment an ecdysial line
forms only anteriorly between the graft squares where the apposed tissues are from
close to the midline. More posteriorly the line fails to form. In the metathorax small
sections of line may form at the medial corners. Careful consideration of the
gradient situation could provide an explanation. There is a precipice at this corner
and the host and graft tissues are from close to the midline. Along the anteriormedial border the height of the precipice decreases. Along the posterior-medial
border the height of the precipice increases but the graft is confronted with host
cells of progressively more lateral levels. Consequently the most favourable position
for forming ecdysial line is at the medial corner, (c) After 180° rotations the line is
undisturbed at the following moult, (d) When large grafts are rotated by 90° the
line fails to reconnect. The cut ends of the line are deflected terminally in the opposite direction to the direction of rotation of the graft, (e) After small 90° rotations
the line may rejoin along any of the graft edges
33
34
P. M. J. SHELTON
ecdysial line. In the two remaining animals the segmental organisation was
disrupted, due to damage to the intersegmental membranes at the time of the
operation. It is concluded that removal and replacement of the line does not
disrupt the normal m-1 pattern.
Cuts in the line and removal of narrow strips containing the ecdysial line
The simplest operations consisted of either (i) removing a very narrow strip
of epidermis including that which forms the ecdysial line (Expt. i; Fig. 4b) or
(ii) making simple cuts with a razor-blade fragment through the epidermis of the
midline with a direction of cut of 45° to the a-p axis (Expt. ii; Fig. 4c). Five
animals were used for these experiments. In each a section of line was removed
from the metathoracic segment and a single cut made in the mesothoracic
segment. The former experiment tested the ability of the line to reform after
removal. The cuts were made because simple cuts are known to cause the reorientation of epidermal cells in Rhodnius (Lawrence, Crick & Munro, 1972).
After removal of the line, four animals had a perfect and continuous line at the
following moult (Fig. 8) but one animal failed to reform the line and there was a
break at the site of the operation. Almost certainly this animal had been
wrongly staged and was in the second half of the moult cycle at the time of the
operation. This animal also showed no evidence of the cut in the mesothoracic
segment. The other four animals did show evidence of the cut. In one case there
was a small lateral spur at the site of the cut; in the other three cases there
appeared to be a slight reorientation of the ecdysial line along the direction of
the cut (Fig. 9). From these results it is concluded that a new line can regenerate
after a section has been removed and that cuts may produce a small but detectable reorientation of the ecdysial-line-forming cells.
Lateral shifts of the ecdysial line
In two series of experiments sections of ecdysial line were shifted laterally
across the segment. In the first series similar sized 2-5 mm squares from the
meso- and metathoracic segments were exchanged. The ecdysial line in the
square from the metathoracic segments was located in the centre of the graft
while in the mesothoracic segment the square was cut so that the line was 1 mm
closer to one lateral edge than to the other (Expt. iii; Fig. 5 a). When exchanged
between segments this results in a 1 mm lateral displacement of the transplanted
sections of the line in each of the two segments. Although laterally displaced by
1 mm the line normally rejoined the host line at the following moult (Fig. 10).
Six animals were used with two transplants per animal. In eight cases the lines
rejoined at both ends. In one case the line rejoined at only one end of the
transplant and in another case there was no reconnexion at either end. The two
remaining grafts were unsuccessful.
Successful rejoining could be explained if there is local migration of cells
along the borders of the graft so that host cells could meet with graft cells from
Ecdysial line determination
9.
Fig. 8. Expt. (i). After a narrow strip containing the ecdysial line is removed, the
ecdysial line reforms at the following moult. Dotted lines indicate the approximate
region where epidermis has been removed.
Fig. 9. Expt. (ii). After simple cuts there may be some reorientation of the line.
Dotted line indicates the extent and direction of the cut.
Fig. 10. Expt. (iii). After small lateral shifts of the line continuity is re-established
at the following moult.
Fig. 11. Expt. (iv). Exchange of squares from either side results in large lateral
shifts. The ecdysial line fails to rejoin and a supernumerary may develop (see
Fig. 5 b).
35
36
P. M. J. SHELTON
a corresponding level in the m-1 axis. An alternative explanation is that the new
ecdysial line forms by intercalary regeneration along the host/graft border
where confronting cells are from regions immediately next to the midline. Such
intercalary regeneration is known to occur in the regeneration of insect leg
structures (see Bohn, 1970; French et al. 1976). It is also possible that pattern
regulation is occurring by morphallaxis with local reassignment of positional
information. The present experiments do not allow me to distinguish between
these possibilities.
In the second series of lateral shift experiments, adjacent 2-5 mm squares from
the left- and right-hand sides on the metathoracic segment were exchanged with
each other laterally. The squares were cut so that the ecdysial line was included
just within the left-hand edge of the right-hand square. Thus there was no
ecdysial line in the left-hand square. The two squares were then exchanged so
that the midline was displaced to the left by about 2-5 mm (Expt. iv; Fig. 5 b).
At the following moult the transplanted line was visible on the left-hand side.
In addition, a second line formed on the right-hand side (five out of eight
operations) along the right-hand edge of the transplanted left-hand square
(Figs. 5 b, 11). Invariably this additional line was shorter than the transplanted
one. In the three unsuccessful cases the segment was extensively damaged and
these animals were discarded. In the five successful cases neither the transplanted nor the supernumerary lines joined at their ends with the host line.
The most significant finding from this series of experiments is the formation
of a supernumerary line. The supernumerary line could not form by migration
of cells from the host line because the two are disconnected. Thus the supernumerary line must have reformed by a pattern regulating mechanism such as
epimorphosis or morphallaxis. The failure of the original or supernumerary
lines to reconnect along the anterior and posterior edges of the grafts could be
explained by the fact that at the host/graft borders cells from very different
levels on the m-1 axis are confronted. This would not favour the total restoration
of pattern within one moult by either of the suggested pattern forming mechanisms. Since the distances are relatively great the possibility of cell migration to
reconnect the lines is also greatly reduced.
Exchanges of left and right squares distant from the ecdysial line
The previous experiments do not exclude the possibility that supernumerary
ecdysial lines merely form when there is a confrontation between tissues of left
and right sides. It was decided to test the ability of apposed squares from more
lateral levels in the m-1 axis to form midline. Left and right squares from
similar m-1 levels midway between the ecdysial line and the lateral margins of
the segments were exchanged in ten animals (Expt. v; Fig. 5c). Of the 20
transplants, 16 were clearly visible at the following moult. In no case did an
ecdysial line structure form. This experiment demonstrates that the ecdysial
line does not form whenever left and right squares are confronted with opposite
Ecdysial line determination
37
polarity and shows that the ability to reform the midline does not exist at more
lateral levels.
Left and right thoracic squares grafted into abdominal segments
In some experiments displaced or excised sections of ecdysial line could form
by inward migration of line forming cells from the adjacent host epidermis. The
following series of experiments was designed to exclude cell migration as a
means for reconstituting the line. Operations were performed in which left and
right grafts from various levels in the m-1 axis of the metathorax were recombined adjacent to one another in the centre of the second or third abdominal
segment at a corresponding level in the a-p axis. Abdominal segments do not
possess an ecdysial line so that an ecdysial line forming after this type of graft
must have done so by a pattern regulating mechanism. Abdominal tissue
removed to provide locations for the thoracic implants was relocated at the
thoracic sites (Fig. 5 a, b). Two series of operations of this type were carried out.
In the first series, a narrow strip of epidermis containing the ecdysial line was
removed from the metathoracic segment and discarded. Grafts of an oblong
shape (1 x 2-5 mm) were taken from either side of this strip and they were
apposed in the third abdominal tergite with their short medial edges in contact
in the centre of the segment. The abdominal site was prepared by making a
single cut along the midline and then removing appropriately sized oblongs
from either side of the cut. These two pieces of tissue were then relocated in the
thorax with their medial edges in contact (Expt. vi; Fig. 6a). At the following
moult no ecdysial line formed between confronted abdominal tissues located in
the thorax (Fig. 12). Where thoracic grafts were confronted in the abdomen
ecdysial lines formed in 11 of the 21 cases at the junction of left and right grafts
(Figs. 13-15). In five cases no line formed although the grafts appeared normal.
In the five remaining cases there was extensive wound damage at the sites of the
operations. These results show that (i) a new line can form when two epidermal
grafts with opposing m-1 polarities from either side of the midline are confronted, (ii) the regenerated line does not form by inward migration of host line
cells, (iii) between abdominal grafts which were similarly apposed and located in
the thorax, where such migration would be possible, no ecdysial line formed.
In a second series of operations the procedure was the same except that the
thoracic tissue was taken from more lateral positions midway between the
ecdysial line and the lateral segment margins (Expt. vii; Fig. 6b). When
examined at the following moult no reformation of the line occurred at the confronted edges of thoracic tissue in any of the ten successful operations. It is
concluded that after a single moult apposed thoracic grafts from regions
distant from the midline cannot form midline.
38
12
P. M. J. SHELTON
13
Ecdysial line determination
39
The importance of graft polarities at the confronting edge
In the previous experiments wherever a supernumerary ecdysial line demonstrably forms by pattern regulation rather than by cell migration there are
certain common features. At least one of the edges is from close to the midline
and in all cases it is the medial borders of the confronted tissues which are
apposed. According to the gradient convention used to describe each graft
situation, the ecdysial line is forming at a ridge. An experiment was devised to
establish whether an ecdysial line would form at a valley when grafts from close
to the midline are apposed. Grafts were exchanged between meso- and metathoracic segments. A square containing a section of ecdysial line was removed
from the mesothoracic segment for exchange with a graft from the left of the
ecdysial line in the metathoracic segment. The right-hand border of this site
was approximately 1 mm from the ecdysial line (Expt. viii; Fig. la). The
results of 11 operations were consistent at the following moult. As expected
from previous confrontation experiments a new line formed at the junction
between left and right squares in the majority of grafts to the meso thorax (6 out
of 11; Fig. 16). In the metathorax the transplanted section of line was visible
orientated parallel to the host line and in no case did a supernumerary line form
at the junction of host and graft tissues (10 out of 11 grafts successful; Fig. 17).
It is concluded that the m-1 polarities of the apposed grafts are a decisive
influence in determining whether or not a line will be formed. Apparently even
though the apposed edges are from close to the midline a supernumerary line
will not form if the graft configuration forms a valley.
FIGURES
12-19
Fig. 12. Expt. (vi). When abdominal grafts from close to the midline are confronted in the middle of the thorax no line forms and the host line remains interrupted.
Fig. 13. Expt. (vii). Thoracic grafts from either side of the midline do reform an
ecdysial line when confronted in the abdomen.
Figs. 14, 15. Two other examples of ecdysial line formation in an abdominal site
where thoracic grafts have been confronted.
Figs. 16, 17. Expt. (viii). Grafts have been exchanged between the meso- and
metathoracic segments, so that a section of the ecdysial line lies parallel to the host
line in the metathorax (Fig. 17). There is no formation of an additional line between
the graft and host lines. In the mesothorax (Fig. 16) the line rejoins as might be
expected from earlier results (see Fig. la).
Figs. 18, 19. Expt. (ix). Grafts were exchanged between meso- and metathoracic
segments according to the scheme as defined in Fig. l(b). The line reforms at the
anterior end of the confrontation in the mesothorax (Fig. 18) but not where the
graft borders are from more lateral levels. In the metathorax (Fig. 19) small regions
of line may form at the medial corners of the grafts.
40
P. M. J. SHELTON
A test for a gradient of ability to make ecdysial line
The preceding experiments have established that a supernumerary line can
form when epidermis from either side of the midline is recombined in a graft
situation. It is established that the ecdysial line will form only if at the site of
apposition at least one of the two edges is from close to the midline and if, in
terms of the gradient of distance from the midline, the graft configuration forms
a ridge. In order to test for any graded ability in the m-1 axis to form ecdysial
line an operation involving two pairs of graft squares was carried out (Expt. ix;
Fig. 7 b). A section of ecdysial line from the mesothoracic segment was removed
and squares from either side were used to graft into the metathoracic segment.
Here two similar sized squares were cut so that their sides were inclined at 45°
to the a-p and m-1 axes. The most medial corner of each of these squares was
approximately 1 mm from the ecdysial line which was left in place. The two metathoracic grafts were then inserted into the mesothoracic site with apposed medial
corners and with their inner posterior edges confronted (Fig. 7 b). Thus at the confronting edge the most anterior part was from close to the midline and the most
posterior part was from more lateral regions. The mesothoracic squares were
implanted into the metathoracic segment with their most medial sides along the
posterior-medial edge of the prepared site. At this junction a graft edge from close
to the midline confronts host epidermis from a continuous range of m-1 levels.
At the following moult ecdysial line structures formed in both segments. In
the mesothorax the ecdysial line was interrupted (12 successful grafts from 18
operations). At the posterior edge of the grafts the host ecdysial line was
expanded and did not enter the graft tissue (Fig. 18). Anteriorly, however, the
ecdysial line did extend into the graft tissue and this was particularly clear in six
animals where host and graft tissues were distinguishable by the colour of the
cuticle. In the metathoracic segment ecdysial line structures were visible as small
patches at the most medial corners of the grafts in 13 cases (Fig. 19). In five
cases the line extended from this patch about halfway along the posteriormedial edge of the graft.
The interpretation of these results is as follows. In the mesothoracic segment
an ecdysial line forms only where the apposed edges are from the most medial
levels. Hence the ecdysial line can form anteriorly between the apposed edges
but in more posterior regions of the confrontation the apposed edges are from
sites too far removed from the midline. The failure of the line to reform posteriorly shows that in this case migration of ecdysial line forming cells along the
medial junction of the grafts did not take place.
In the metathoracic segment the medial corner of the square was transplanted
to a more lateral level. The new gradient situation shows there to be a precipice
at this corner (Fig. 7 b). Further along the posterior-medial border the height of
the precipice increases because the host border is from increasingly more
lateral levels. In contrast as one proceeds along the anterior-medial border of
Ecdysial line determination
41
the graft the height of the precipice decreases. According to the gradient convention a pronounced ridge is found only along the posterior-medial border.
Nearest to the medial corner of the graft, tissues from both host and graft are
from close to the midline. It is here that ecdysial lines most commonly appear.
180° and 90° rotations
For comparison with experiments on the a-p axis where medio-laterally
orientated lines have been rotated by 180° and 90° (see Lawrence et al, 1972;
Bohn, 1974) similar rotations were performed on squares bisected by the
ecdysial line. At the outset it was realised that difficulties in interpretation of
results might arise since these operations involved moving tissues with respect
to both a-p and m-1 axes.
After 180° rotations of 3-5 mm squares (Expt. x) the ecdysial line remained
patent at the following moult with no disturbance of pattern (five operations)
(Fig. 7 c). This result was expected since it did not result in movement of tissues
with respect to the m-1 axis, merely an inversion of the a-p axis.
After rotation of 3-5 mm squares by 90° (Expt. xi) the host line consistently
failed to rejoin with the rotated section (three clockwise and two anti-clockwise
rotations) (Fig. Id). A constant feature of these grafts was a deflexion of the
cut ends of the rotated section and the end of the host line in the opposite
direction to the direction of rotation of the grafts. In consequence the line in the
vicinity of the graft often had the appearance of an incomplete S (Fig. 20). The
failure of the line to rejoin is consistent with the results of the lateral shift
grafts where sections of the line were displaced from the midline by 2-5 mm and
there was a failure of the transplanted section to join with the host line. Only
when the line was moved by 1 mm in the lateral shift operations did the line
rejoin. In that case the reconnexion may have occurred by cell migration or by
pattern, regulation. After these relatively large 90° operations the ends of the
rotated section are separated from the host line by a distance around the edge
of the graft of 3-5 mm. This would be a very great distance for cells to migrate.
The graft configuration may be unfavourable for intercalary regeneration
because along no border of a large square rotated by 90° is there a gradient
ridge and although one side of the confrontation may be from close to the
midline, the tissue on the other side is from much further away.
The direction of deflexion of the ends of the rotated section of the host line is
contrary to what one might intuitively expect. In experiments on the a-p axis
where sections of mediolaterally oiientated lines have been rotated by 90°
(Bohn, 1974), an S-shaped regulated pattern is produced which respects the
handedness of the graft and which restores the continuity of the line. In that
case the ends of the rotated section reconnect with the end of the host line to
which they were formerly connected (Bohn, 1974). Although in the case of the
ecdysial line the connexion is incomplete, the resulting broken S is of the
opposite handedness to what might be expected. An explanation for this result
42
P. M. J. SHELTON
Fig. 20. Expt. (xi). After large grafts have been rotated by 90° the line fails to rejoin.
The cut ends of the line are deflected in the opposite direction to what might have
been expected. Clockwise rotation shown.
Fig. 21. Expt. (xii). When the 90° rotations are small reconnexion is possible along
some or all of the edges.
could be that the graft physically derotates because of the 90° shift with respect
to the a-p axis. Because of this the ends of the host and graft lines are deflected
by the turning of the whole graft. This is a possibility because such derotation
is known to occur in a number of insects after rotation of squares by 90°
(Bohn, 1974; Lawrence, 1974; Nubler-Jung, 1974).
Since sections of the line shifted laterally by small distances reconnect, 90°
rotations of smaller squares were carried out. After eight successful rotations of
2 mm squares, seven gave similar results to those described previously. In the
remaining case the host line connected anteriorly with the left hand end of the
graft line which had been rotated clockwise. A further series of experiments
using 1-5 mm squares (Expt. xii) gave six successful grafts. In three cases there
was no reconnexjon of the line but in the other three cases various patterns of
reconnexion resulted (Figs, le and 21). In two of these the line joined anteriorly
and posteriorly from the left-hand end of the graft line after a clockwise rotation. In the other case the line joined anteriorly with the right-hand end of the
graft after an anticlockwise rotation. Such reconnexion after small grafts is
compatible with either limited cell migration or pattern regulation at the
Ecdysial line determination
43
graft/host border. The distance around the circumference of the graft is sufficiently small to allow the former and the latter is possible because at the graft
border all edges are from close to the midline, although once again there would
be no gradient ridge. According to either mechanism a symmetrical pattern of
reconnection might be expected. However, asymmetries may arise from damage
during the operation and the graft itself may be asymmetrical.
CONCLUSIONS
Since only the ecdysial line provides a clearly definable pattern discontinuity
in the m-1 axis of the thorax, the statements which can be made concerning
general pattern forming mechanisms in that axis are limited. However, with
regards to the mechanism for determination of the ecdysial line itself certain
possibilities are excluded. Firstly, the ecdysial line occurs at the junction between
right- and left-hand sides of the segment and in other insects the two halves are
derived from separate polyclones (Lawrence, 19736; Lawrence, Green &
Johnston, 1978). The hypothesis that the ecdysial line forms along the shortest
possible line separating the two polyclones is excluded by experiments where
squares from each side of the segment are exchanged without the formation of
an ecdysial line surrounding the grafts. Many of the other experiments also
confront tissues from the two sides without ecdysial line formation.
Cell migration along graft borders is one mechanism which has not been
entirely excluded for some experiments. In the case of small lateral shifts and
small 90° rotations it could be important but is not necessarily the only mechanism in those cases. In several experiments the possibility of cell migration was
excluded. The most convincing of these is where medial thoracic tissues from
either side of the line are confronted in the abdomen and form the line. In that
case pattern regulation must have occurred.
Pattern regulation in insects is well established for a variety of systems. There
is demonstrable regulation of pattern in the a-p axis of the abdominal segment
(Lawrence, 1973 a), in the proximo-distal axis of the leg (Bohn, 1976) and in its
circumferential axis (French & Bulliere, 1975 a, b). When tissues from different
a-p gradient levels are confronted in the abdomen it has been assumed that
there is local reassignment of positional information to provide a regulated
pattern (Lawrence et ah 1972). On the other hand, in the leg, confrontation of
tissues from different levels in either of the two axes results in local cell proliferation and the intervening values are restored by epimorphic growth and
differentiation. However, there is now evidence to suggest that epimorphic
pattern regulation also occurs in the abdomen (Nubler-Jung, 1977). When cells
are transplanted to new a-p gradient positions there is some local cell proliferation which produces conspicuous folds in the epidermis.
Lawrence (1970) has suggested that both the length of the segment and the
spatial pattern of cellular differentiation are determined by different features of
44
P. M. J. SHELTON
the same gradient system. The length is determined by the slope and the pattern
of spatial differentiation by the distribution of local positional values. It has
been speculated that the basis of the gradient of differentiation is a gradient of
a diffusible morphogen (see Lawrence et al. 1972). Although probably a great
oversimplification and it is difficult to apply the model to the circumferential
axis of the leg which has no ends, similar reasoning could be used for much
more complicated models. For instance, each epidermal cell could provide its
own unique spectrum of diffusible substances. In graft situations, when cells
from different levels on the gradient are apposed, the new distribution of
diffusible substances could specify the regulated pattern. An important finding
is that the epidermal cells resist the effects of transplantation to new gradient
levels and have a homeostatic ability (Lawrence et al. 1972). In the abdomen
the cells of the segment borders have high homeostatic ability while the intervening cells of the segment have lower homeostatic ability. Thus the ends of the
system act as organizers in the classical sense. Transplants within the segment
not involving the segment borders were thought to result in respecification of
pattern by a mechanism similar to morphallaxis (Lawrence et al. 1972). When
non-congruent levels are combined in either of the proximo-distal or circumferential axes of the leg the missing regions are restored by epimorphic regeneration from the cut surfaces (see French et al. 1976). This behaviour is in
accord with the Lawrence, Crick & Munro model if it is assumed that all cells
in the leg have high homeostatic ability and that local steepnesses of the epigenetic landscape cause cell proliferation. From this reasoning it is clear that
any difference in the behaviour of abdomen and leg cells are due to differences in
homeostatic ability and not to fundamentally different mechanisms of pattern
regulation. According to this interpretation, there should be some epimorphic
growth in the abdomen at the anterior and posterior borders of a graft moved
relative to the a-p axis. The recent demonstration that this is the case in the
abdomen of Dysdercus intermedius helps to unify our understanding of the
mechanism of pattern regulation in different parts of the insect (Nubler-Jung,
1977). Any difference in behaviour of leg and abdomen almost certainly arises
because of the enormous selective advantage derived from the ability to regenerate the full length of the leg after damage.
An interesting result from work on the circumferential axis of the leg is that
after confrontation of non-congruent levels the intercalary regenerate restores
the missing values by the shortest possible route (French et al. 1976). The circumference of the leg can be numbered like a clock face and if regions 7 and 11
are confronted, values 8, 9 and 10 regenerate rather than 6 to 12 (French &
Bulliere, 1975 b). In addition, the polarity of the regenerated tissues is not
dependent upon the polarity of the epidermis to either side of the graft (French
& Bulliere, 1975a). The regulative behaviour of imaginal discs in Drosophila
melanogaster after experimental manipulation can also be explained in terms of
the clock face model and epimorphic regeneration (Bryant, 1975). In the
Ecdysial line determination
45
bilaterally symmetrical genital disc, confronted identical values from either side
of the axis of symmetry fail to form an intercalary regenerate (French et ah
1976). Intercalary regeneration occurs only if different values are apposed.
From the present study ecdysial line regeneration appears to differ in two
ways from the cockroach limb and D. melanogaster genital disc cases. Firstly,
regeneration does occur when identical values from either side of the ecdysial
line are apposed and secondly, the polarity of the confronted tissues determines
whether ecdysial line regeneration will occur; a new line will form at a ridge but
not at a valley even though the confronted values are the same in each case.
At the outset I emphasized that the use of a gradient convention to explain
the various experimental confrontations was not intended to imply that a m-1
gradient of differentiation exists. Nevertheless many of the results would be
consistent with the presence of a bilaterally symmetrical gradient for determination of pattern in the m-1 axis. However, in order to demonstrate the presence or
absence of such a system more cuticular markers in the m-1 axis would be
required. Whatever the mechanism it is not likely to differ in a fundamental
way from the mechanisms operating in other parts of the insect.
Although a detailed explanation for the preceding results is not yet possible,
the observation that pattern regulation takes places in the m-1 axis is important.
In experiments on the a-p axis, squares of cuticle have been shifted to new
positions on the m-1 axis (Locke, 1959; Lawrence et ah 1972; Bohn, 1974). The
possibility of pattern regulation in the m-1 axis is not considered in these
experiments. If pattern formation in the m-1 axis is determined by a generalized
mechanism as in an epimorphic field, that would complicate the interpretation
of experiments where tissues are moved with respect to the two axes simultaneously.
I am indebted to Mark Nowel for reading the manuscript and to the technical staff of the
department for maintaining the animals. 1 am grateful to Frances Barker for typing the manuscript, and to referees for valuable comments.
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{Received 12 June 1978, revised 8 September 1978)