/. Embryol. exp. Morph. 90, 57-78 (1985)
57
Printed in Great Britain © The Company of Biologists Limited 1985
Cell division during intercalary regeneration in the
cockroach leg
HILARY ANDERSON*
European Molecular Biology Laboratory, Heidelberg, F.R.G.
AND VERNON FRENCH
Department of Zoology, University of Edinburgh, The King's Buildings, West Mains
Road, Edinburgh, U.K.
SUMMARY
In a series of grafting operations on cockroach legs, epidermal cells from different positions or
from the same position on the circumference of the femur were placed together. Where cells
from different positions were confronted, new cuticular structures corresponding to the positions which would normally have lain between them were formed during the following moults.
At the control junctions, where cells from the same positions were placed together, no new
structures were formed.
Grafted legs were examined histologically at various times after the operation. The events
following grafting fell into four phases: wound healing - when epidermal cells migrated over the
wound to re-establish epidermal continuity and cells adjacent to the wound divided to compensate for cell emigration; intercalation - when cell divisions took place at the host-graft
borders where there was a positional discrepancy; proliferation - when the general growth of the
epidermis occurred by widespread cell division; cuticle secretion - when apolysis occurred, cell
division ceased, and cuticle secretion began.
The results show that intercalary regeneration is associated with local cell division at the
graft-host borders, and that these divisions are not confined to the normal proliferative phase of
the moult cycle, but begin much earlier in the cycle, as soon as wound healing is complete.
These results support epimorphic models (such as the Polar Coordinate Model) of pattern
regulation, where change of positional value is tied to cell division, but they do not discount the
possibility of a limited initial morphallactic phase.
INTRODUCTION
In many developing animals cellular interactions have been shown to be
fundamental in determining the fate of individual cells and the extent to which
they divide. In the single-layered insect epidermis, for example, grafting and other
surgical operations which juxtapose cells which are not normally neighbours lead
to a modification in the pattern of cuticular structures formed and an increase in
growth of the tissue. This has been demonstrated in the epidermis of the leg (e.g.
Bohn, 1970; French, 1978), and abdominal segment (e.g. Niibler-Jung, 1977;
* Present address: Department of Zoology, University of California, Davis, California 95616,
U.S.A.
Key words: intercalary regeneration, cockroach leg, cell division.
58
H. ANDERSON AND V. FRENCH
Wright & Lawrence, 1981), and in the imaginal discs of Drosophila (e.g. Haynie &
Bryant, 1976; Abbott, Karpen & Schubiger, 1981).
In the cockroach leg the single layer of surface epidermal cells forms precise
patterns of structures (e.g. bristles, spines, pigmented regions) in the overlying
cuticle. If different proximal-distal levels of a leg segment are grafted together,
growth occurs in the region of the junction and the intervening levels are formed
by intercalary regeneration (Bohn, 1970; Bulliere, 1971). Similarly, if cells from
different circumferential positions on a segment are grafted together, the
intervening circumferential positions are produced by intercalary regeneration
(French, 1978). These results (and others) lead to the formulation of the Polar
Coordinate Model (French, Bryant & Bryant, 1976; Bryant, French & Bryant,
1981). This proposes that the limb epidermis has a two-dimensional map of
positional values arranged along its longitudinal and circumferential axes, and that
pattern regulation occurs by epimorphosis (Morgan, 1901). Interaction between
cells with different values provokes division of those cells and the new daughter
cells adopt intermediate values, producing an intercalary regenerate.
The Polar Coordinate Model (PCM) was derived almost entirely from
observation of the final pattern of cuticular structures formed after various
experimental perturbations. To evaluate the model, and more generally to explore
the relationship between pattern formation and cell division, it is vital to also
observe cell behaviour during the processes of wound healing and regeneration.
Some such studies have been made on imaginal disc fragments (e.g. Reinhardt,
Hodgkin & Bryant, 1977; Reinhardt & Bryant, 1981; Dale & Bownes, 1980), and
on cockroach limbs undergoing distal regeneration (Truby, 1983). In the
experiments reported here we have grafted together epidermal cells from different
circumferential positions on the cockroach femur and have looked directly at
healing and cell division during subsequent intercalary regeneration. We
demonstrate that intercalary regeneration involves local cell divisions which start
around the time of healing, well before the normal cell divisions associated with
growth during the moult cycle.
MATERIALS AND METHODS
Larvae of the cockroach, Blabera craniifer were kept at 28-5°C (±1°C) in constant darkness,
and were provided with laboratory rat pellets and moist cotton wool. Grafting operations were
performed on CO2-anaesthetized 5th instar animals, 2-3 days after moulting. Using fine forceps
and a razor-blade knife, a longitudinal strip of cuticle plus epidermis was removed from the
femur of the donor mesothoracic leg (after the leg had been removed from the animal) and
grafted into a site prepared on the metathoracic femur of the same animal. In this way the same
confrontation of graft and host cells (from different positions or from the same circumferential
position) was created all along each longitudinal edge of the graft. Grafts were secured by dried
haemolymph and the operated cockroaches were then kept in small groups in plastic sandwich
boxes.
Some animals were kept until after the 1st or 2nd postoperative moult when their operated
legs were removed, fixed in 70% ethanol and examined for cuticular features. Other animals
were selected for histological examination and were taken at progressively later times after the
Cell division and intercalary regeneration in cockroach leg
59
operation, anaesthetized, injected with colchicine (10 jul of a 0-25 % solution made up in distilled
water) and left for 11 h. The grafted femurs were then removed,fixedfor 2 h in alcoholic Bouin,
stored for several weeks in 70 % isopropyl alcohol to soften the cuticle, embedded in paraffin
wax and sectioned at 10 fjm in the transverse plane. Sections were stained in haemalum and
eosin and, in the region of the centre of the graft, 20 sections taken atfive-section(i.e. 50jum)
intervals were examined in detail and the positions of any arrested mitoses were plotted on
standard diagrams of the femur circumference. The thick cuticle was sometimes difficult to
section and in cases where a chosen section was incomplete or damaged, an adjacent section was
scored instead.
RESULTS
(A) Structure of the femur
The metathoracic (host) or mesothoracic (donor) femur of Blabera is an
anteroposteriorly flattened cylinder of cuticle bearing clearly recognizable rows of
bristles and bands of pigmentation at different circumferential positions (mainly
on the medial and lateral faces), as shown in Fig. 1. The femur also contains
apodemes which are cuticular invaginations from the femur/tibia joint and from
the distal tip of the tarsus. The surface cuticle and internal apodemes are secreted
every moult by an underlying single-layered epidermis. The femur contains large
muscles which attach to the apodeme epidermis and to particular regions of
lateral, midanterior, and midposterior surface epidermis (see Fig. 1).
(B) Graft I: left medial face grafted to left anterior face
A strip between circumferential positions 8 and 4 (see labelling of femur
circumference in Fig. 1) was removed from the left mesothoracic femur and
grafted to a site between positions 9 and 8 on the left metathoracic femur, as shown
in Fig. 2. On the same animal, a control graft was performed on the right
metathoracic femur: a strip between positions 9 and 8 was removed from the leg
and then replaced in the same site. Both grafts involved freeing some muscle
attachments in the region of position 9.
Cuticular features of graft I legs
A series of 29 operated animals moulted after 18-24 days (27/29 moulting in
20 ± 1 days); 13 were fixed and the rest left to the 2nd postoperative moult.
After the 1st moult the experimental (left) femur had a considerably increased
overall circumference in the grafted region. The bristle rows and well-defined
pigmentation bands of the original host femur and the grafted medial face were
unmodified (except for the growth normally occurring between 5th and 6th instars)
but along both graft-host junctions intercalary regeneration had produced new
tissue. At the junction between graft position 8 and host position 9 (the 8-9
junction) this tissue had no recognizable cuticular features. At the junction
between graft position 4 and host position 8 (the 4-8 junction) the new tissue
60
H. ANDERSON AND V. FRENCH
M
*
1A
B
Fig. 1. Structure of the left metathoracic femur. (A) Anterior/medial view with the
host site for Grafts I and II shown by a dashed line. A, P, M, L, - anterior, posterior,
medial and lateral faces of the femur; p, proximal; d, distal. (B) Schematic transverse
section showing the cuticle (cu) and the underlying epidermis (e) with sites of muscle
insertion (m). Twelve positions (1-12) are marked around the circumference and
characteristic cuticular features are shown: rows of bristles (positions 12-1,5,7 and 10),
medial band of light-coloured cuticle (positions 5-7) shown by dots, and the lateral
band of dark cuticle (positions 12-1) shown by thick line.
had either no recognizable features, occasional short bristles and some light
pigmentation (Fig. 2Aiii), or had two irregular rows of short bristles separated by a
poorly defined lightly pigmented band.
After the second moult, all parts of the femur circumference appeared to have
grown fairly evenly, the 8-9 intercalary regenerate still bore no recognizable
features, and the 4-8 intercalary regenerate was in all cases a clear medial face
(i.e. positions 5,6 and 7 - Fig. 2Aiv).
61
Cell division and intercalary regeneration in cockroach leg
-2
-1
-12
-10
-9
8
•7
-6
-6
• 5
•4
-3
/-/•/•
77-;
Fig. 2. Graft I (A) and the matched Control Graft (B). Schematic cross-sections of the
femur show the grafting operation (i) and the result after one moult (ii). (iii) and (iv)
are camera lucida drawings of short lengths of the grafted femur after one (iii) or two
(iv) moults, cut along the posterior side (approximately position 3), opened out, and
mounted flat. G, graft; H, host; R, intercalary regenerate. (A) Grafting a left medial
face into the anterior face of the host left femur creates positional discontinuities at
both graft-host junctions (i). Intercalary regeneration ensues and, after two moults
(iv) the new tissue is clearly the portion of circumference normally separating graft and
host (e.g. a new medial face, stippled, positions 7,6,5 forms at the 8-4 junction). (B)
Removing and replacing part of the anterior face produces no positional discontinuities
(i) and no intercalary regeneration (ii—iv).
62
H. ANDERSON AND V. FRENCH
After the 1st moult, the control (right) femur had the circumference normal for
the 6th instar, and the control graft had healed in, provoking no extra growth or
modification of cuticular structures (Fig. 2Biii).
Cellular events and cell division
On each alternate day following the grafting operations (Days 1,3,5,7,9,
11,13,15,17), some operated animals were treated with colchicine, and their
experimental and control legs fixed, sectioned and examined for the appearance of
the epidermis and the occurrence and distribution of mitotic cells.
The length of the normal moult cycle varies between animals within the same
instar. Therefore experimental and control operations were performed on the
same animals so that the numbers of mitotic cells could be compared statistically as
pairs by analysis of variance. We compared the total numbers of mitotic cells, the
numbers of mitotic cells at the graft-host borders, and the numbers of mitotic cells
elsewhere, in the experimental and control legs for each day. The numbers of
mitotic cells in each class on each day are shown in Fig. 7.
The graft-host junction was indicated by the obvious break in the leg cuticle.
On either side of this break was a region where epidermis had been damaged or
removed, to a greater or lesser extent, during the grafting operation, and which
was filled initially by clotted haemolymph and haemocytes. Subsequently epidermal cells adjacent to this region detached from their overlying cuticle and
migrated to form a continuous sheet under the wound region. In experimental legs
these epidermal cells were considered to be 'the graft-host border' but in control
legs, where the degree of wounding was less, an equivalent band of 20-30 cells was
considered to be the 'graft-host border'.
Day 1. The first event after grafting is closure of the wound by clotted
haemolymph and accumulation of one or more layers of haemocytes beneath the
clot. These are distinguishable from epidermal cells by their small darkly staining
nuclei and flattened shape. Sections through experimental grafts showed that at
neither the 8-9 nor the 4-8 borders were the host and graft epidermis in contact
(Fig. 3A,B). The control grafts fitted better than the experimental grafts (Fig. 3D)
so that the host and graft epidermis were nearly in contact (Fig. 3E). At all
host-graft junctions haemocytes had accumulated under the cut edges of epidermis, so that a continuous cell layer was present under the wound (Fig. 3B,E).
Very few mitotic cells were observed at this stage (Fig. 7).
Day 3. By Day 3 there had been some migration of epidermal cells through the
plug of haemocytes so that in experimental legs host and graft epidermis were in
contact or in close proximity (Fig. 3C) and in control legs they were usually in
contact (Fig. 3F). The epidermal cells remain as a coherent sheet during
migration.
There were few dividing cells in regions away from the graft-host borders on the
experimental and control legs, and the difference was not significant. There were
more divisions at the graft-host borders and there were significantly (P = 0-05)
more at those of experimental legs than those of control legs (Fig. 7).
Cell division and intercalary regeneration in cockroach leg
3A
Fig. 3. Sections of Graft I legs on Days 1 and 3. (A) Experimental (left) leg on Day 1.
Scale, 0-5 mm. (B) Experimental 4-8 junction on Day 1. Host (h) and graft (g)
epidermis are separated by a plug of haemocytes beneath clotted haemolymph (c).
Scale, 80jian. (C) Experimental 8-9 junction on Day 3. Epidermis from the host (h)
and graft (g) are almost in contact through migration of epidermal cells under the clot
(c) of haemolymph and haemocytes. Scale, 100 jum. (D) Control (right) leg on Day 1.
Scale, 0-5 mm. (E) Control 9-9 junction on Day 1. Host (h) and graft (g) epidermis are
separated by haemocytes. The host and graft cuticles are held together by clotted
haemolymph (c). Scale, 80^m. (F) Control 8-8 junction on Day 3. Migrating
epidermal cells have connected the host (h) and graft (g) epidermis in the region of the
graft-host border beneath the clot (c) of haemolymph. Scale, 100 ^m.
63
64
H. ANDERSON AND V. FRENCH
Day 5. On Day 5 the host and graft epidermis had formed a continuous sheet
(Fig. 4A,B,E,F) a n ( j j i a ( j p r o c j u c e d a continuous layer of endocuticle (Fig. 4B,F).
There were still relatively few dividing cells at regions other than the graft-host
borders- and these did not differ significantly between experimental and control
legs. There were however consistently more mitotic cells at the experimental
graft-host borders than at the control graft-host borders (Fig. 7), although this
was not statistically significant at the 5 % level.
Day 7. The epidermis along the anterior face had begun to thicken slightly and
the chromatin within the epidermal cells to condense. Some degenerating cells
were observed, either as pycnotic nuclei or as clusters of darkly stained droplets,
but these were always confined to the graft-host border.
Experimental legs showed significantly (P = 0-05) more dividing cells than
control legs especially at the graft-host borders (Figs 4C,G and 7), but also
elsewhere around the circumference.
Day 9. The appearance of the epidermis was much the same as on Day 7.
The numbers of mitotic cells had increased greatly throughout the legs (Figs
4D,H and 7). At the graft-host borders experimental legs showed a significantly
(P = 0-05) higher number of mitotic cells than did control legs, but elsewhere there
was no significant difference.
Day 11. At this stage all of the epidermis around the circumference of the leg
was thickened, including that of the posterior face.
Mitotic cells were observed in large numbers all over both control and
experimental legs (Figs 5A,B and 7). At the graft-host border experimental legs
showed significantly (P = 0-05) more mitotic cells than did controls. At other
positions, however, control legs had significantly more mitoses.
Day 13. Apolysis, the withdrawal of the epidermis from the overlying cuticle,
had started along the host medial face in a few animals. There were numerous cell
divisions throughout the epidermis of experimental and control legs (Fig. 5C,D)
and the difference in numbers was not significant, either at the graft-host borders
or elsewhere (Fig. 7).
Day 15. The epidermis had completely withdrawn from the cuticle and
expanded to form extensive folds (Fig. 6A,B) over which the cells had begun to
secrete a new cuticle. The folding was more extensive in the region of the
experimental graft than in the region of the control graft (compare Fig. 6A,B). No
dividing cells were observed at this stage.
Day 17. The overall appearance of the epidermis was similar to that of Day 15
except that the newly secreted cuticle was considerably thicker (Fig. 6C,D). No
mitotic cells were observed.
These results suggest that events after grafting fall into the following four
phases:
1. Wound healing which takes place during Days 1-3 and results in the
production of a continuous epidermis. By Day 3 mitotic cells are observed at the
graft-host borders and are more numerous in experimental legs.
Cell division and intercalary regeneration in cockroach leg
65
H
Fig. 4. Sections of Graft I legs on days 5,7 and 9. (A) Experimental leg on Day 5.
Scale, 0-5 mm. (B) Experimental 4-8 junction on Day 5. The host (h) and graft (g)
epidermis are joined together by migrated epidermal cells which have secreted a new
endocuticle (airow) at the graft-host border. Scale, 160 ptm. (C) Experimental 4-8
junction on Day 7. A dividing cell (arrow) is shown within the graft-host border.
Scale, 160 jwm. (D) Experimental leg on Day 9. A dividing cell (arrow) is shown at
approximately position 1. Scale, 80/an. (E) Control leg on Day 5. Scale, 0-5mm. (F)
Control 8-8 junction on Day 5. The graft (g) and host (h) epidermis are joined
together by epidermal cells which have secreted a new endocuticle (arrow). Scale,
80 pan. (G) Control 9-9 junction on Day 7. A dividing cells (arrow) is shown within the
graft-host border. Scale, 160 jLtm. (H) Control leg on Day 9. A dividing cell (arrow) is
shown in position 5. Scale, 160 jwm.
66
H . A N D E R S O N AND V. F R E N C H
D
t
Fig. 5. Camera-lucida drawings of sections of Graft I legs on Days 11 and 13. (A)
Experimental leg on Day 11. (B) Control leg on Day 11. (C) Experimental leg on Day
13. (D) Control leg on Day 13. Asterisks indicate location of dividing cells. Scale,
0-5 mm in all cases.
2. Intercalation which takes place on Days 5,7 and 9, during which there are
low levels of mitosis at the control graft-host borders, and much higher levels at
experimental graft-host borders, with mitosis initially low elsewhere but becoming frequent by Day 9.
3. Proliferative cell divisions which occur throughout the epidermis on Days 11
and 13 with mitosis still considerably higher at the graft-host border in the
experimental legs on Day 11 but not significantly different on Day 13.
4. Cuticle secretion which takes place during Days 15-17 when no mitoses are
observed.
Thus this experiment has shown a considerable difference in behaviour at the
graft-host borders, with cell divisions much higher at the experimental borders
from the end of healing through the intercalation and into the proliferation phase.
The experimental borders have a positional disparity between the graft and host
epidermis, which the control borders do not have. However, the experimental
borders also differ from control borders in that, because of poorer fit, more cell
migration is necessary to attain epidermal confluence in the healing phase and this
may be accompanied by cell divisions in the surrounding area of low cell density
(Wigglesworth, 1937). Hence it could be that the observed difference in the
67
Cell division and intercalary regeneration in cockroach leg
number of dividing cells in our experiments resulted from the differing extent of
repair in control and experimental legs rather than the positional disparity.
However, the enhanced cell division at experimental borders lasted long after a
new cuticle was secreted (c.f. Wiggles worth, 1937), so that it is likely to indicate
intercalation rather than healing. However, to confirm this we performed another
set of grafting operations where the degree of mechanical disruption of the
epidermis at control and experimental junctions was well matched.
(C) Graft II: right medial face grafted to left anterior face
A strip between circumferential positions 4 and 8 was removed from the right
mesothoracic femur and grafted to a site between positions 9 and 8 on the left
metathoracic femur, as shown in Fig. 8. As in Graft I, muscle attachments in the
region of position 9 had to be broken in preparing the host site.
OC
>*£?
?:.',)
Fig. 6. Sections of Graft I legs on Days 15 and 17. (A) Experimental leg on Day 15.
Scale, 400 fjm. (B) Control leg on Day 15. Scale, 400 fjm. (C) Experimental graft region
on Day 17. Scale, 130/mi. (D) Control graft region on Day 17. Scale, 130/zm. The
epidermis (e) has withdrawn from the old cuticle (oc) and begun to secrete a new
cuticle (nc). The epidermis has expanded more in the experimental (A,C) than in the
control legs (B, D).
68
H. ANDERSON AND V. FRENCH
200-1 B
I 100
100
-
n= 3 n= 3 n= 3 n=4
D3
D5
D7
Dl
n=4 n=4 n=3 n=3 n=3
D9
D l l D13 D15 D17
n=3 n=3
Dl
D3
n= 3 n=4 n=4 n=4 n= 3 n= 3 n=3
D5
D7
D9 D l l
D13 D15 D17
Fig. 7. Location of dividing cells after Graft I (A) and the matched Control Graft (B).
Histograms show the means and standard errors for the total number of dividing cells
found on 20 sections through the grafted femurs on alternate days after the operation.
Dividing cells found at the graft-host junctions are shown by the solid black (A wounding plus positional discontinuity) or the stippled (B - wounding) portions of the
histograms, while the blank portions show the mitoses occurring elsewhere around the
circumference.
Cuticular features of graft II legs
A series of 25 operated animals moulted in 18-28 days (20/25 moulting in 20-25
days): 14 were fixed and the rest left to the 2nd postoperative moult.
After the 1st moult the original graft and host tissues had grown as normal and
their cuticular structures remained unchanged. At the control 8-8 junction, the
graft had healed in with no production of extra tissue, but at the experimental 4-9
junction there was a large intercalary regenerate with either no recognizable
structures, scattered short bristles and some light pigmentation (Fig. 8iii), or two
irregular rows of short bristles separated by a lightly pigmented region.
After the 2nd postoperative moult there was still no extra tissue at the 8-8
junction and, in all cases, the large intercalary regenerate at the 4-9 junction
included a clear medial face (positions 5,6 and 7, Fig. 8iv).
Cellular events and cell division
On Days 3,5 and 12 some animals were treated with colchicine and their
operated legs fixed, sectioned and examined. These days were chosen to give a
representative picture of the stages of wound healing, intercalary regeneration,
and general growth as observed for graft I.
At one junction, graft epidermis from position 8 was confronted with host
epidermis from position 8 (the 8-8 junction, Fig. 8) and at the other, graft
69
Cell division and intercalary regeneration in cockroach leg
epidermis from position 4 was confronted with host epidermis from position 9 (the
4-9 junction, Fig. 8) providing control (non-intercalating) and experimental
(intercalating) regions within the same leg. We compared the numbers of mitotic
cells at the 8-8 and 4-9 graft-host borders (the graft-host border denned as for
graft I) for each of the 3 days using the same statistical methods as before. These
results are shown in Fig. 10.
The general appearance of the epidermis was the same as for Graft I on the
corresponding day and a description will not be repeated here. As anticipated, the
8-8 and 4-9 junctions looked very similar in configuration (Fig. 9A,B) and
presumably required a similar amount of wound healing and cell migration to
produce, in all cases, a continuous sheet of epidermis with its overlying cuticle by
Day 5.
On Day 3 there were consistently more dividing cells at the 4-9 border than at
the 8-8 border (Fig. 10), although the difference was not statistically significant.
On Day 5 there were significantly more mitotic cells at the 4-9 border than at the
8-8 border which now showed fewer than on Day 3. On Day 12 there were again
significantly more dividing cells at the 4-9 border than at the 8-8 border. The
latter again showed fewer dividing cells than on Day 3.
-9
Fig. 8. Graft II - right medial face grafted to left anterior face. Notations and
abbreviations as in Fig. 2. The graft creates a 9-4 positional discontinuity at one
graft-host junction (i) and here intercalary regeneration produces new tissue which,
after two moults (iv), can be identified as the intermediate portion of circumference
(i.e. a new medial face - positions 7,6,5 - is formed). At the other junction there is no
discontinuity (8-8) and no intercalary regeneration.
70
H. ANDERSON AND V. FRENCH
9A
Fig. 9. Sections of a Graft II leg on Day 5. At the experimental 4-9 border (A) and the
control 8-8 border (B) the host (h) and graft (g) epidermis are joined by epidermal
cells which have migrated from the host and graft tissues and which have secreted a
new cuticle (arrow). The size and arrangement of this epidermis is similar to the two
(A,B) borders. Scales, 100 jiim.
Since the amount of wound healing is similar at the two borders, the far greater
number of dividing cells found at the 4-9 border on Days 5 and 12 is caused by
the positional discrepancy created in this region by the grafting operation. This
result therefore strongly supports the conclusion reached from Graft I that cell
division occurring at graft-host borders on and after Day 5 reflects intercalary
regeneration.
DISCUSSION
(A) Cuticular features
Grafts which disturb normal neighbourhood relationships within the insect leg
epidermis result in intercalary regeneration whereby growth is stimulated and new
pattern elements are formed. In the present experiments a strip graft is moved
around the circumference, creating circumferential positional disparities at both
(Graft I) or one (Graft II) graft-host junctions. The total limb circumference
increases and the new structures formed at each graft-host junction correspond,
Cell division and intercalary regeneration in cockroach leg
71
by the shortest route, to the missing part of the circumference (French, 1978,
1980).
Examination of the cuticular structures formed at the first and subsequent
postoperative moults suggests that the extra growth occurs locally at the junction
and that the new structures form within the new tissue. Hence the graft and host
retain their differentiated structures (e.g. bristle rows some 10 cell diameters away
from the cut edges at the time of grafting) but are separated by an intercalary
regenerate which initially bears few cuticular structures (e.g. Figs 2Aiii, 8iii) but
which subsequently develops them (Figs 2Aiv, 8iv).
The present histological study was undertaken to show directly that a positional
discontinuity does provoke cell divisions in addition to those resulting from
wounding inevitably accompanying grafting and those normally occurring in the
moult cycle, and to investigate when these divisions occur and whether they are
indeed localized at the site of the discontinuity.
(B) The timing of cell divisions within the moult cycle
During each instar the epidermis grows by proliferative cell divisions and new
bristles, hairs and chemosensilla are added to the increased body surface by
differentiative cell divisions. Proliferative divisions generally occur during the
middle third of the moult cycle (Lawrence, 1966ft; Bulliere, 1972; Kunkel, 1975;
200
100
n=4n=4n=4
D3 D5 D12
Fig. 10. Location of dividing cells after Graft II. Histograms show the means and
standard errors for the number of dividing cells found on 20 sections through grafted
femurs on days 3,5 and 12 following the operation. Dividing cells found at the
experimental (9-4) graft-host junction are shown in solid black, those found at the
control (8-8) graft-host border are shown by stippling, and those found elsewhere on
the circumference are shown by the blank portion of the histograms.
72
H. ANDERSON AND V. FRENCH
Truby, 1983), while differentialve divisions occur either before proliferative
divisions, in the case of chemosensilla or bristles (Lawrence, 19666; Kunkel, 1975),
or after, in the case of hairs (Lawrence, 19666). In all cases, cell divisions have
ceased at the time of cuticle secretion.
After a wound, cell divisions occur at other times. Following a cut, epidermal
cells adjacent to the wound migrate across it to re-establish a continuous epidermis
(Wigglesworth, 1937). Nearby regions of reduced cell density undergo a period of
local cell division which ceases when epidermal continuity has been restored and
the cells in the wound area have secreted a new cuticular covering (Wigglesworth,
1937). After amputation of the distal part of the leg, cell divisions also appear
much earlier in the moult cycle than normal. Truby (1983) observed DNA
synthesis and mitosis at 2 days (in a 16-day moult cycle), while Bulliere (1972)
detected DNA synthesis on Day 7 (in a 40-day moult cycle) but, presumably
because he did not harvest mitoses with colchicine, he did not detect cell divisions
until Day 18. Following amputation late in the instar, wound healing and some cell
division occurs prior to the moult, but regeneration only starts after the moult and,
again, cell divisions are found as early as Day 2 of the following moult cycle
(Truby, 1985). However, unlike cell divisions occurring after a simple incision
(which cease shortly after the wound has healed), the additional cell divisions
following amputation continue into the proliferative stage of the moult cycle
(Bulliere, 1972; Truby, 1983). The additional cell divisions result not only from the
extensive migration required for healing over the amputation site, but also from
the production from stump tissue of a regenerated leg.
In the experiments described here we were able to perform experimental and
control grafts and could therefore distinguish cell divisions involved in healing,
intercalation, and growth. We observed the following pattern of cell divisions after
grafting operations:
(a) In regions away from the graft-host borders, cell divisions in both experimental and control legs were negligible on Day 1, were present in low numbers on
Days 3 and 5, increasing on Days 7 and 9 to give very high levels on Days 11 and
13. On Days 15 and 17 cuticle secretion had begun and mitosis had ceased.
The few early divisions (Days 3 and 5) could be differentiative divisions giving
rise to new hairs and bristles. Most were too far away from the graft-host borders
to represent the wound healing response, although some adjacent to the borders
may be involved in this. The later divisions correspond to the proliferative
divisions observed in the absence of colchicine in non-regenerating epidermis of
Blabera craniifer from Day 22 to Day 34 in a cycle of 40 days (Bulliere, 1972).
We therefore interpret mitosis away from the borders as corresponding to the
normal events of the moult cycle.
(b) At the graft-host border, the number of cell divisions was significantly
higher on Day 3 in the poorly fitting Graft I experimental grafts than in the control
grafts. However, in Graft II where there was no difference in the degree of
wounding at the control and experimental borders, there was no significant
difference on Day 3 in cell division at the experimental and control graft-host
Cell division and intercalary regeneration in cockroach leg
borders. Cell divisions at the graft-host borders on Day 3 therefore represent a
wound healing response.
(c) At the graft-host borders on Day 5, epidermal continuity had been restored
and a new endocuticle had been secreted by cells under the wound. Any mitotic
cells observed at, and after this stage are therefore probably associated with
intercalary regeneration of new tissue. This view is confirmed by the very different
levels of cell division at the poorly fitting Graft II experimental and control
junctions. We interpret the low levels seen at control junctions on Day 5 and
subsequently, as representing a small degree of intercalation provoked by
inexactly fitting grafts, or as a residual wound response. Much higher levels of cell
division were observed at experimental graft-host borders than at control
graft-host borders on all days from 5 to 13 following Graft I and on Days 5 and 12
following Graft II. We interpret this local cell division as representing intercalary
regeneration of tissue at the region of positional discrepancy.
Intercalary regeneration therefore does not result from an augmentation of cell
division during the normal proliferative phase of the moult cycle, nor from an
extension of this phase later into the cycle, but results from cell divisions beginning
much earlier in the moult cycle and extending into the proliferative phase (Fig.
11).
(C) The location of cell divisions
We now have shown directly that intercalation is associated with division of cells
at the 'graft-host border', defined as a band of 20-30 cells disrupted by damage
and subsequent healing. Because of the mechanical disturbance involved in the
grafting operation, it is not possible at present to be more accurate about the
localization of the initial mitoses of intercalation. As intercalation proceeds, a
region of folded and thickened epidermis is formed, bulging a little away from the
old cuticle, and again it is difficult to be precise but it seems that mitoses can occur
anywhere within it, rather than just at a narrow front corresponding to the
confrontation of host- and graft-derived cells. From the histological observations,
it is not possible to say whether both host and graft contribute to the intercalary
regenerate, but this is known to be the case in similar grafts between leg segments
with different cuticular structures (French, 1980).
Intercalary regeneration following grafting in cockroach legs is very similar to
the pattern regulation observed when fragments of Drosophila imaginal disc are
cultured for some time in vivo before metamorphosis (Haynie & Bryant, 1976;
French et at. 1976). In general, small fragments of the wing disc duplicate, forming
a mirror-symmetrical partial pattern, while complementary large fragments
regenerate, completing the pattern (Bryant, 1975). Many fragments have been
shown to heal their cut edges together in such a way that shortest route
intercalation would generate the observed pattern (Reinhardt et al. 1977;
Reinhardt & Bryant, 1981), and other fragments which show variable patterns of
regeneration show corresponding variability in their mode of healing (Dale &
Bownes, 1985).
73
H. ANDERSON AND V. FRENCH
/ Intercalation .'
Healing
Growth
Operation
0
1
2_J_# 4 5
Healing
6 7
Moult
8 9 10 11 12 13 14 15 16 17 18 19 20 Days
Growth
Fig. 11. Summary of the timing and location of cell division following grafting
operations. (A) Experimental graft I involving positional disparities 9-8 and 8-4 (see
Fig. 2A); (B) Control graft (see Fig. 2B). Legs are shown in diagrammatic cross section
through the grafted region and the distribution of colchicine-arrested epidermal
mitoses (dots) is given at various times between the operation and the first moult.
Thick horizontal bars indicate the duration of the localized cell divisions of healing and
intercalation (far more numerous in A) and the widespread divisions of growth and
bristle differentiation.
Fig. 12. Cell division and the change of positional values during intercalation. Each
row of circles represents epidermal cells near the junction (arrow) between positional
value 4 and 8, as in Graft I (see Fig. 2A). Only thefirstthree stages of intercalation are
shown. Cells stimulated to divide are marked with a dot and those which have divided
are stippled. A-C show different versions of epimorphosis and D shows a model with
an initial morphallactic phase. (A) Cells at the positional discontinuity divide giving
rise to one daughter cell with unchanged positional value and the other with a value
slightly nearer that of the inappropriate neighbour. Intercalation will progressively
reduce the discontinuity and cell division will occur only at the confrontation of graftand host-derived cells. (B) Cell division gives rise to one daughter with unchanged
value and the other with a value much nearer that of the inappropriate neighbour.
Intercalation will reduce the discontinuities within the new tissue and cell division will
occur throughout the intercalary regenerate. (C) After cell division both daughter cells
adopt values nearer to that of the inappropriate neighbour. In contrast with A and B,
cell division can spread back to cells not at the original junction, but extreme positional
values (4-0 and 8-0) are lost. (D) There is an initial respecification of cells to remove the
large discontinuity at the junction followed by cell division to restore normal
relationships between neighbouring cells. In this case cell division will not necessarily
start at the junction, it need not be localized, and extreme values (4-0 and 8-0) will be
lost. The greater the initial morphallactic phase, the fewer the subsequent cell divisions
and the greater the loss of positional values.
Cell division and intercalary regeneration in cockroach leg
Q
75
76
.
H. ANDERSON AND V. FRENCH
Cell division is necessary for regeneration of disc fragments and, by wholemount autoradiography after thymidine pulse labelling (Dale & Bownes, 1980)
and mitotic counts of sectioned discs (O'Brochta & Bryant, submitted) it has been
shown that the dividing cells are localized in the region of wound healing. The
estimated width of the zone of cell division is 4-13 cells (Dale & Bownes, 1980) or
15 cells (O'Brochta & Bryant, submitted) although, using rather different techniques of long-term labelling with thymidine, Adler (1984) suggests that an
increase in cell division spreads much further back from the wound surface (see
also Adler, 1981).
In Drosophila, partial duplication and triplication of legs can arise after heat
treatment during larval development of temperature-sensitive cell-lethal mutants.
These structures have been interpreted as resulting from localized cell death,
healing, and intercalary regeneration within the developing disc (Girton, 1981).
From clonal analysis, Girton & Russell (1980) estimated that, in a duplicated
pattern, the extra leg was derived from only 10-20 cells within the damaged disc,
again suggesting that intercalary regeneration involves only very local cell division
at a site of positional discontinuity. Abbott, Karpen & Schubiger (1981) used
clonal analysis similarly to demonstrate that the regenerate or duplicate produced
by an imaginal leg disc fragment derives from cells originating at or near the edge
(in this case, only one edge) of the fragment, but since they used the Minute
technique they could not estimate the number of cells involved.
An epimorphic model (such as the PCM) involves stable cellular positional
values changing only during cell division provoked by a discontinuity between
adjacent cells, and it therefore predicts that regeneration will involve only local
cell division at a graft-host junction or at an amputation surface. Clearly,
circumferential intercalary regeneration in grafted cockroach legs or in imaginal
disc fragments involves localized cell division, and is thus broadly compatible with
epimorphosis rather than, at the other extreme, a morphallactic model based
on widespread respecification of cells. There are several possible forms of
epimorphosis (Fig. 12A-C) and all predict that the first cell divisions will be
precisely at the confrontation of disparate cells and that all cells of the intercalary
regenerate will have divided. The forms differ in predictions concerning subsequent spread of cell division back from the junction, loss of extreme values at the
junction, and the location of cell division within the developing intercalary
regenerate (Fig. 12). In practice, however, with the complication of damage and
healing, with the limited cuticular markers for cellular positional value, and with
the difficulty of precisely locating divisions relative to the confrontation of grafted
cells, and determining whether all cells of the regenerate have indeed divided, it is
difficult to distinguish between some of these possibilities (e.g. Fig. 12B,C) or even
to rule out a rather different model involving an initial morphallactic phase of
resetting cellular positional values (Fig. 12D).
In a recent histological study of distal regeneration after autotomy of the
cockroach leg at the femur-trochanter level, Truby (1983) found that the initial
cell divisions occurred during wound healing, just proximal to the amputation site.
Cell division and intercalary regeneration in cockroach leg
77
Cell division then spreads back into the stump to the level of the distal coxa (50-80
cell diameters in 2nd instar animals). After an autotomy late in the instar, wound
healing and associated cell divisions occurred bui: regeneration only commenced
after the next moult. Cell divisions then started at the distal tip of the stump and
spread back, again to distal coxa level (Truby, 1985). Cell division continued at all
levels within the blastema, visible segmentation occurred, and the regenerate
continued to grow.
The PCM proposes that distal regeneration occurs because healing across the
end of the stump provokes intercalation between cells with different values and,
because of the presence of adjacent cells with unaltered values, the new cells take
up more distal positional values (Bryant etal 1981). Thus distal regeneration, like
intercalation after a graft, should involve only local cell division at the site of
discontinuity. Some versions of the PCM would allow cell division to spread back
into stump tissue (as in Fig. 12C) but this effect would not be expected to extend
very far back from the amputation site (Lewis, 1981) and most cell divisions would
still occur at the tip of the developing blastema. The difference between Truby's
results and our own may indicate that distal regeneration is different from
intercalation or that they are similar processes both involving a degree of cellular
respecification before cell division (Truby, 1983). Because much more new tissue is
formed after a proximal amputation than after a strip graft, respecification and
subsequent cell division would be much more extensive in distal regeneration than
in intercalation.
This work has been supported by the British Science Research Council, the European
Molecular Biology Organization, and the Royal Society.
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(Accepted 1 May 1985)