/ . Embryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976
Printed in Great Britain
267
Leg regeneration in the cockroach,
Blatella germanica
II. Regeneration from a non-congruent tibial graft/host junction
By VERNON FRENCH 1
From the Developmental Biology Group,
University of Sussex, and Laboratoire de Zoologie,
Universite Scientifique et Medicate de Grenoble
SUMMARY
The interactions occurring between host and graft leg epidermis at a non-congruent
junction were studied in the cockroach, Blatella germanica. Graft and host tibia were cut
perpendicular to the proximal-distal axis and two heteropleural combinations were used
to reverse separately the two transverse axes of the graft relative to the host. Use of dark
and light cuticle colour mutants gave a good indication of the graft or host origin of
regenerated structures.
Graft/host junctions regenerated segmented structures in various spatial arrangements,
always comprising two copies of all structures distal to the level of the junction.
It is concluded that the categories - two separate laterals, double lateral, completely and
partially autonomous regeneration - reflect two processes.
(i) If the graft tarsus is removed, graft and host may not heal together and interact, but
form autonomous regenerates lying in mirror-image symmetry separating original graft
and host levels.
(ii) If interaction occurs between graft and host (or their developing autonomous regenerates) two laterals of dual origin are produced, one from each point of transverse axis
incongruity. These laterals may secondarily fuse together to form a double structure
originating from a point of congruity. The orientation and composition of the component
tarsi of the double structure depend on the site of origin and the extent to which the two
laterals fuse.
It is argued that the four 'faces' and two 'transverse axes' of the leg are merely descriptive
terms. A new model is developed whereby lateral regeneration arises directly from the
circumferential organisation of the leg epidermis. Previous work has shown that position
is specified continuously around the circumference, and that intercalary regeneration occurs
by the shortest route between confronted positions. After reversal of one 'transverse axis'
the shortest route between confronted graft and host positions is different on the two sides
of each of the two points of 'axis' incongruity, and at these points the two halves of a
complete circumference are formed. These lateral circumferences, like the terminal circumference exposed by amputation, cannot heal over by intercalary regeneration, and this leads
to regeneration of distal structures.
The model accounts for lateral regeneration after reversal of both 'transverse axes' by
180° rotation of a homopleural graft.
The possibility is discussed that there may be clonal restrictions on the circumferential
positions which the progeny of a cell may occupy.
1
Author's address: National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, U.K.
268
V. FRENCH
INTRODUCTION
The cuticle of insects is secreted by the underlying single layer of epidermal
cells, and a study of the cuticular patterns formed after surgical operations
which remove or spatially disturb part of the epidermis can give information
about the systems of cellular interaction within the epidermis which must form
some sort of 'map' from which the cells derive their 'positional information'
(Wolpert, 1969; Lawrence, 1970; Wolpert, 1971; Bryant, 1974).
Grafting together two different proximal-distal levels of a segment (e.g.
tibia) results in longitudinal intercalary regeneration of the intermediate levels
(Bohn, 1967 & 1970; Bulliere, 1971; French, 1976a). Similarly, after association
of different circumferential positions, there is transverse intercalary regeneration
of the intermediate positions (French & Bulliere, 1975a, b).
Lateral regeneration of supernumerary legs can also occur from a graft/host
junction. The leg has been considered to be organized along three mutually
perpendicular axes (Bohn, 1965): a longitudinal (proximal-distal) axis running
from the articulation with the body to the claws, and two transverse axes
(anterior-posterior and internal-external). Two complete lateral regenerates
have been shown to form from a non-congruent junction (i.e. host and graft
transverse axes not aligned) in several species of cockroach: Periplaneta
americana (Penzlin, 1965),Leucophaeamaderae (Bohn, 1965, \912a),Leucopheaej
Gomphadorhina portentosa combinations (Bohn, 1972a), and Blabera craniifer
(Bulliere, 19706). Similar laterals have also been produced in the stick insect,
Carausius morosus (Bart, 1971a), and in Lepidoptera (Bodenstein, 1937),
Hemiptera (Shaw & Bryant, 1975), Dermaptera (Furokawa, 1940) and in
Arachnida (Lheureux, 1970, 1971).
There has been general agreement about the number, position and orientation
of the laterals resulting from various graft combinations, but different conclusions have been reached concerning the tissue composition (graft or host
origin) of the laterals. Grafts have been made between different legs of animals
of the same species (Bart, Bodenstein, Bulliere, Lheureux, Penzlin) or between
the legs of animals of different species (Bohn). Laterals have been considered
to be of pure graft or pure host origin, forming because of a failure of the
two components to interact (Bodenstein, Bulliere, Lheureux, Penzlin), or it
has been concluded that the laterals are usually of dual graft and host origin,
produced by an interaction between misaligned regions of host and graft
(Bart, Bohn).
In the present study, non-congruent graft/host junctions were made at the
tibia level in Blatella germanica to confirm lateral regeneration in this species
of small cockroach, to provide more information about the range of possible
regenerated structures, and to determine the composition of the regenerated
structures by grafting between cuticle colour mutants (French, 1976a). The
results obtained from tibial junctions can also be used for comparison with
Leg regeneration in the cockroach
269
the regenerates produced from congruent and non-congruent junctions between
non-homologous leg segments (French, 19766).
The way in which the non-aligned graft and host tibial epidermis interacts
(or fails to interact) to produce the regenerated structures, is discussed. Various
earlier theories of the origin of lateral regenerates are found to'be unsatisfactory
and a new model is developed.
This model considers the leg epidermis to be effectively two-dimensional:
the surface of a cylinder upon which cellular interactions can occur longitudinally
and circumferentially. Lateral regeneration is shown to arise as a direct result
of this spatial organization of the epidermis.
MATERIALS AND METHODS
Laboratory colonies of Blatella germanica were maintained as described
previously (French, 1976 a), and the grafting operations were performed on
3rd and 4th instar larvae 1 or 2 days after moulting. In all experiments some
grafts were made on wild-type animals (usually between different legs of the
same animal), and some were made between the dark cuticle colour mutant,
Bl (Ross & Cochran, 1967) and the light cuticle colour mutant, br (French,
1976a). Grafting operations were performed under the dissecting microscope,
using fine forceps and small spring scissors. Animals were immobilized with
CO2. Graft and host tibiae were chosen to be of slightly different diameters
so the graft could be pushed slightly into the end of the host stump, where it
was secured by dried haemolymph. Experimental animals were kept until
a few days after the 1st or 2nd post-operative moult (p.o.m.) and then the
operated leg was removed fixed and cleared in Gum Chloral, and examined.
The structure of the leg of Blatella germanica and the labelling convention
to be used have both been described previously (French, 1976a). The leg will
be described in terms of four 'faces': anterior, posterior, internal and external
(but see Discussion). The position and orientation of structures produced
after the operations will be given with reference to the host axes. It should
be noted that a regenerated tarsus is 4-segmented instead of 5-segmented
(O'Farrel, Stock, Rae & Morgan, 1960).
The two basic operations involved reversing separately the two transverse
axes of the graft with respect to the host, and are illustrated in Fig. 1. The
right pro- or meso-thoracic donor leg was removed at proximal tibial level
and grafted on to the host left meta-thoracic leg which had been amputated
at mid-tibia level. The graft tarsus was either amputated (as in Fig. 1) or left
intact.
Anterior/posterior (A/P) reversal. The graft was rotated by 180° about its
longitudinal axis, leaving internal and external host faces adjacent to the
corresponding graft faces, but confronting anterior and posterior host with
posterior and anterior graft, respectively (Fig. 1A).
270
V. FRENCH
1E
(b4)
Tr
Fig. 1. Structures regenerated from the graft/host junction following reversal of one
transverse axis at the level of the tibia. (A) Reversal of the anterior-posterior axis;
(B) reversal of the internal-external axis. All views are anterior (with respect to
host axes) except A^ 1 ), A(64), B(62) and B(65) which are external, (a) Graft
situation. (6x-65) Classes of result after 1st or 2nd post-operative moult, (b1) Two
separate laterals; (b2) double lateral (one of the possible positions and orientations);
(63) completely autonomous regeneration; (64, bh) partially autonomous regeneration.
A, Anterior; /, internal; P, posterior; E, external.
Leg regeneration in the cockroach
271
InternalI external (I/E) reversal. The graft was not rotated, leaving anterior
and posterior host faces aligned with the corresponding graft faces, but confronting internal and external host with external and internal graft, respectively
(Fig. IB).
RESULTS
(A) Frequency of regeneration from the graft/host junction
As shown in Table 1, most operated legs which retained the graft regenerated
segmented structures from the junction by the 1st p.o.m. and all had done
so by the 2nd p.o.m.
(B) Classification of regenerates from the graft /host junction
Structures produced from the junction are classified in Table 2, and it will
be seen that the two experiments give rise to the same categories of structures
with comparable frequencies. In each experiment, some of the animals with
graft tarsus left intact at the time of operation subsequently lost the tarsus
(and regenerated four tarsal segments by the 1st p.o.m.). These are therefore
considered together with those animals which had the graft tarsus amputated
at the time of grafting. Table 3 relates the state of the graft tarsus to the
structure regenerated from the graft/host junction. Each category of regenerate
will now be considered.
(i) Two separate lateral regenerates
These structures could appear at the 1st or 2nd p.o.m. (Table 2), and at
the junction between the host and a graft which had retained its tarsus intact
or had lost and regenerated the tarsus (Table 3). The combination of graft
proximal tibia and host mid tibia often gave rise to reversed orientation
intercalary regeneration (French, 1976 a) and the lateral regenerates occurred
at its proximal limit (NB, all positions and orientations refer to the host
axes).
The two lateral structures each comprised a tibial apex (the coronet of
spines and articulation with the condyle on the external side of the proximal
tarsus), a 4-segmented tarsus and a set of two claws (Figs. 1 A^ 1 ), B^ 1 ),
2,3).
The position of origin of the laterals around the circumference of the tibia
depended on which transverse axis had been reversed by the operation. Following
reversal of the anterior/posterior (A/P) axis of the graft, the laterals were
usually (13/17 cases) positioned one anteriorly and one posteriorly (Figs. 1A (b1),
2A, B, 3 A); following reversal of the internal/external (I/E) axis, the laterals
were usually (21/29 cases) found one internally and one externally (Figs. 1B (b1),
2C, D, 3B). Thus, in both experiments, a separate lateral was formed at each
region of incongruity between the transverse axes of host and graft: at each
confrontation of opposite faces of the leg.
139
124
Axis reversed
Anterior-posterior
Internal-external
42
30
No
regeneration
48
44
Regeneration
91
80
p.o.m., post-operative moult.
19
19
No. with
surviving
graft
Completely
autonomous
regeneration
(21+0)21
(7+1) 8
Double
lateral
(31 + 12)43
(33 + 9) 42
2 separate
laterals
(13 + 4)17
(25 + 4)29
Total no.
of
regenerates
(91 + 19)110
(80+19) 99
Axis
reversed
Anterior-posterior
Internal-external
(22 + 3)25
(12 + 4)16
Partially
autonomous
regeneration
Structures regenerated from the graft/host junction
Parentheses denote number of structures appearing at the 1st post-operative moult (p.o.m.)
followed by number appearing at the 2nd p.o.m., added to give total.
19
19
(4 + 0)4
(3 + 1)4
Other
Graft/host
junction
regeneration
2nd p.o.m.
Table 2. Classification oj regenerates Jrom the tibial grajt /host junction Jollowing reversal oj one
oj the transverse axes oj the graft
No. with
surviving
graft
Graft/host junction
No. showing
no regeneration
at 1st p.o.m.
and kept to
2nd p.o.m.
1st p.o.m.
Table 1. Frequency oj regeneration Jrom the tibial grajtIhost junction Jollowing reversal oj
one transverse axis oj the grajt
o
m
to
to
273
Leg regeneration in the cockroach
Table 3. Relationship between state of the graft tarsus and regeneration from
the graft/host junction following reversal of one transverse axis of the graft
{combined data from the two experiments)
Structure regenerated from the graft/host junction
•
State of graft
tarsus
Intact (5 segments)
Regenerated
(4 segments)
Broken or unknown
Total
Completely
autonomous
regeneration
Partially
autonomous
regeneration
2 separate
laterals
Double
lateral
19
17
35
31
0
7
4
10
10
46
19
85
22
29
27
41
The A/P orientation of the laterals could not be determined, but their
I/E axis was almost always polarized in accordance with host and graft
following reversal of the graft A/P axis (15/17 cases), and in accordance with
the host following reversal of the graft I/E axis (27/29 cases).
(ii) Double lateral regenerates
Double lateral regenerates appeared at the 1st or 2nd p.o.m. (Table 2) on
legs which had retained the graft tarsus intact or had lost and regenerated it
(Table 3). As in the case of the separate laterals, the double lateral structure
was regenerated at the proximal limit of any reversed orientation intercalary
regenerate formed between host and graft.
In all cases there was a large tibial apex (probably two apices fused together
but this could not be determined), articulating with a large double tarsal
structure having two proximal condyles and two distal sets of two claws. The
tarsus was usually a double structure along its entire length (Fig. 4, 1A (62),
IB (b2)) but sometimes had two separate distal parts (Fig. 5D).
The position of origin of the double lateral around the circumference of
the tibia differed in the two experiments. Where the A/P axis of the graft had
been reversed, the lateral occurred in an external (25/43 cases, Figs. 4A, B,
C, 5A, B) or internal (17/43 cases, Figs. 4D, E) position; following reversal of
the I/E axis, the lateral usually originated posteriorly (30/34 cases, Figs. 4F,
5C). Thus, in both experiments, the double lateral was usually formed at a
region of congruity between the transverse axes of host and graft: at a position
midway between the two regions of incongruity where the laterals form when
they are separate.
Only the I/E orientation of the double tarsi could be determined and, as
shown in Fig. 6, the orientations were very variable. The tarsi were always
orientated in one plane, but this could be the plane of the graft and host
I/E axes (Fig. 5 A) or perpendicular to it (Fig. 5B). The component tarsi were
18
E MB 35
A
I
P
D
gc
hs
gc
Fig. 2. Separate lateral regenerates formed from the graft/host junction after
reversal of the anterior-posterior axis (A and B) or the internal-external axis
(C and D). A, I, P, E, Anterior, internal, posterior and external 'faces', gc, Claw of
graft origin; gh, tarsal hair of graft origin; gs, coronet spine of graft origin; he, claw
of host origin; hh, tarsal hair of host origin; hs, coronet spine of host origin.
275
Leg regeneration in the cockroach
B
p
\ i
Fig. 3. Camera lucida drawings of separate lateral regenerates formed from the
graft/host junction after reversal of the anterior-posterior axis (A) or the internalexternal axis (B). A, I, P, E, Anterior, internal, posterior and external 'faces'.
almost always oppositely orientated, with the two sets of claws facing directly
towards (Fig. 5 A) or away from each other (Fig. 5B). The range of variation
is shown in Fig. 6 as it is important in connexion with the composition and
modes of origin of the double structures.
(iii) Completely autonomous regeneration
Completely autonomous regeneration of the host and graft surfaces occurred
by the first p.o.m. but did not occur subsequently in animals which had not
regenerated from the junction at the first p.o.m. (Table 2). The distal parts
of the long and fragile structures were often broken off but, from the seven,
cases where this had not occurred, autonomous regeneration was seen to
have occurred only in association with loss of graft tarsus at or after the
operation (Table 3).
Following reversal of either axis, the autonomous structure consisted of
two regenerates each comprising distal tibia, four-segmented tarsus and two
claws, lying in mirror-image linear sequence separating the original graft and
host levels, and joined by their distal tips (Figs. 1A (bz), IB (b3), 7 A, B).
The I/E orientation of the more proximal regenerate conformed to that of
the host, while the more distal regenerate was orientated like the graft.
18-2
C 1,
hh
he1
Fig. 4. For legend see opposite.
Leg regeneration in the cockroach
hs
277
f •V
E \A I
Fig. 4. Double lateral structures regenerated from the graft/host junction after
reversal of the anterior-posterior axis (A and B which are anterior and posterior
views of the same specimen; C, D and E) or the internal external axis (F). A, I, P,
E, Anterior, internal, posterior and external 'faces'. A, B, C and F show a double
lateral with one component tarsus more or less host-derived and the other more
or less graft-derived. In D and E each component tarsus is of dual origin, c, c1,
Denote the two sets of claws; gc, gc1, claw of graft origin; gs, coronet spine of graft
origin; he, he1, claw of host origin; hh, tarsal hair of host origin; hs, coronet spine
of host origin.
(iv) Partially autonomous regeneration
Partially autonomous regeneration of the graft and host surfaces could
occur by the first or second p.o.m. (Table 2). As in the case of the completely
autonomous regenerates, distal parts of the long and fragile structures were
often broken off, but, from the 14 complete structures, it seems that partially
autonomous regeneration usually occurred in association with loss of the
graft tarsus at or after the operation (Table 3).
The structures consisted of two partial regenerates lying in mirror-image
linear sequence separating host and graft, and fused together at some level
proximal to the end of the tarsus. This level could be the tibia apex or in any
of the tarsal segments but was almost always the same for the two elements
concerned. From this level of fusion were formed either two separate laterals
or a double lateral branch, complete to two sets of claws (Figs. 1A (b4), (b5)
\B(b%(b%lC-E).
As in the case of the completely autonomous regenerates, the two axially
situated partial regenerates were orientated (in the I/E axis) like the host and
278
V. F R E N C H
Fig. 5. Camera lucida drawings of double laterals regenerated from the graft/host
junction after reversal of the anterior-posterior axis (A, B) or the internal-external
axis (C, D). A, I, P, E, Anterior, internal, posterior and external 'faces'.
FIGURE 6
Position, orientation and composition of double lateral regenerates developing
from the graft/host junction following reversal of one transverse axis. A, Results
of experiment reversing the anterior-posterior axis; B, results of experiment
reversing the internal-external axis. Figures denote number of cases.
'Position': schematic representation of the graft/host junction, distal view. The
outer circle shows the orientation of the host; the inner circle represents the graft;
the asterisk shows the position of the double lateral around the circumference.
A, Anterior; /, internal; P, posterior; E, external.
'Orientation'-'end-on' view of the double lateral structures, showing the
orientation with respect to the host axes, with position of origin of the lateral at
the top to facilitate comparison with Fig. 8. Tarsal claws curve from external to
internal 'faces' on the component tarsi which are shown separated by a dashed
line.
'Composition'-'end-on' view of the double laterals showing approximate
division into host-derived (stippled) and graft-derived parts. '?'-unknown
composition; '1 host/1 graft', one component tarsus of host origin and the other
of graft origin; 'both dual' - each component tarsus of dual graft and host origin.
279
Leg regeneration in the cockroach
Position
Composition
Orientation
1 host/ 1 graft
(E)
(E)
External - 25
Other - 5
(f)
Internal - 17
Other
4
Anterior - 1
(P)
B
vyy
13
(P)
(P)
Posterior - 30
Other
(A)
Other - 1 0
5
Both dual
Leg regeneration in the cockroach
281
graft respectively. The lateral elements were positioned and orientated just like
the laterals originating directly from the graft/host junction. Following A/P
reversal, the separate lateral elements were positioned one anteriorly and one
posteriorly, and were orientated like the graft and host; double lateral elements
were positioned externally or internally and had a range of orientations similar
to those in Fig. 6. After I/E reversal, separate laterals were positioned internally
and externally, and were both orientated like the host; double laterals were
usually positioned posteriorly, with various orientations.
(v) Other structures
In four cases only a single lateral was regenerated from the graft/host
junction and, in another four cases the leg bore a poorly segmented lateral
ending in two atypical claws. One of these animals was allowed to moult
again, and produced a normal double lateral.
Structures regenerated from the junction - Summary
In all cases where operated legs had retained the graft until the second p.o.m., the graft/host
junction developed segmented structures in various spatial arrangements but always comprising two copies of all structures normally lying distal to the level of the junction.
(C) Composition of regenerates from the graft I host junction
The cuticle colour difference between Bl and br Blatella proved to be of
definite, though limited, use in determining the origin of the epidermis of
regenerated structures (French, 1976 a). Although the colour difference between
the original host and graft tissues was usually clear, it was usually not possible
to draw a precise boundary between them or their derivatives. This may
just reflect an insufficient difference in colour and some cell mixing at the
boundaries, or there may be a local interaction affecting cellular phenotype
near the boundary (French, 1976a). In addition, lateral or autonomous
structures regenerated from the graft/host junction were often fragile and
poorly pigmented, reducing the number of analysable cases. Although only
FIGURE 7
Autonomous structures regenerated from the graft/host junction after reversal
of the anterior-posterior axis (B, C, D, E) or the internal-external axis (A). A, B,
Completely autonomous regeneration; C, D, E, partially autonomous regeneration
with a double lateral structure, with one component of host origin and the other
of graft origin (C, D), or both of dual origin (E).
A ,/, P, E, Anterior, internal, posterior and external 'faces', c, c1, the two sets
of claws, g, graft; gc, gc1, claw of graft origin; gh, tarsal hair of graft origin;
gr, autonomous regenerate from graft component of the junction; h, host; he, he1,
claw of host origin; hh, tarsal hair of host origin; hr, autonomous regenerate from
host component of the junction; //, double lateral structure; rt, tarsus regenerated
from the distal end of the graft.
282
V. FRENCH
an approximate boundary could be drawn over areas of bare cuticle, the
spines of the tibial apex, well-developed tarsal hairs and the tarsal claws
could usually be easily identified as Bl or br, and the origin of the epidermis
of the regenerates will be deduced from these criteria.
(i) Separate lateral regenerates
All cuticle on the axial member proximal to the bases of the laterals was
host-derived and all axial cuticle distal to them was graft-derived. Hence
the reversed orientation intercalary regenerate (if any) was of graft origin.
Grafts between Bl and br animals reversing the graft A/P axis gave eight
analysable legs with separate laterals anteriorly and posteriorly. In one case
one lateral appeared to be of dual origin and the other of host origin, but in
all other cases both laterals were clearly of dual origin. They had host-derived
spines in the tibial coronet and a host-derived claw on the side adjacent to
the host, and graft-derived spines and claw on the side adjacent to the graft
(Fig. 2A, B).
After reversal of the I/E axis there were 11 analysable legs with separate
laterals internally and externally (or approximately internally and externally).
In nine cases one lateral tarsus seemed host-derived with two claws of host
origin, and the other seemed graft-derived with both claws of graft origin.
However, many of these laterals were really of dual origin as indicated by the
presence of both host- and graft-derived spines in the lateral coronets (Fig. 2C).
In the other two cases, one of the laterals had one host-derived and one
graft-derived claw (Fig. 2D). These laterals developing after I/E axis reversal
are so orientated that a division into side-adjacent-to-host and side-adjacentto-graft (which separated the claws of the A/P axis reversal laterals) divides
them into an external half and an internal half, and hence does not separate
the claws. The two claws of a lateral tarsus might be expected to have approximately the same composition, and the dual origin of such a tarsus would not
be obvious.
(ii) Double lateral regenerates
The compositions of the double lateral structures are given in Fig. 6. In all
cases the double structure was composed of both host and graft-derived
tissue.
The double laterals regenerating from internal or external positions following
reversal of the A/P axis had one of the component tarsi more or less host
derived and the other more or less graft-derived when their I/E orientations
were in the same plane as those of the graft and host (Figs. 4A, B, C). When
the I/E orientations were perpendicular to that of the host and graft, each
component tarsus was composed of both host and graft-derived tissue (on the
sides adjacent to the host and graft respectively), as shown in Fig. 4D, E.
Following reversal of the I/E axis, the double laterals were usually composed
Leg regeneration in the cockroach
283
of one more or less host-derived member and one more or less graft derived
member, regardless of their point of origin on the circumference and their
orientation (Fig. 4F). In two cases where the I/E orientations were perpendicular
to those of the host and graft, component tarsi of the double lateral were
each composed of both graft and host tissue.
(iii) Completely autonomous regenerates
As implied by the nomenclature, the autonomous regenerates were derived
one from the host and the other from the graft (Fig. 7 A, B).
(iv) Partially autonomous regenerates
In the partially autonomous structure, the axial element lying between the
original host level and the point of fusion was host-derived, and that lying
between the point of fusion and the original graft level was graft-derived.
There were a number of analyzable partially autonomous regenerates developed
after reversal of the A/P axis and having a double lateral element. These were
composed exactly as the double laterals developing directly from the corresponding graft/host junction. When they were orientated (in the I/E axis) in.
the plane of the graft and host I/E axis, one of the components was more or
less graft-derived and the other more or less host-derived (Fig. 7C, D). When
they were orientated perpendicular to the graft and host, each component
was composed of both graft and host tissue (Fig. 7E).
Composition of structures regenerated from the junction: Summary
Although use of the Bl and br mutants did not produce precise boundaries between hostderived and graft-derived parts of the structures regenerated from the non-congruent
junction, it does allow certain conclusions to be made Lateral regeneration from the original
graft/host junction or from the junction between autonomous graft-derived and host-derived
partial regenerates involves both graft and host tissue.
When two separate laterals are produced they are usually of dual origin. This was
especially obvious following reversal of the A/P axis, where the side adjacent to the host
was host-derived and the other side was graft-derived. Double laterals formed after reversal
of the A/P axis had either one host-derived and one graft-derived component tarsus, or each
of dual origin depending on their orientation. When the I/E axis was reversed, one component tarsus was usually host-derived and the other graft-derived, regardless of orientation.
The composition of the regenerated structures is clearly of great importance in considering
their possible mode of origin, and this will be discussed below.
DISCUSSION
The results presented above will be compared with those of similar experiments performed on other insects by other workers. The available data will
then be compared with the predictions of three theories which have been
suggested to explain regeneration from a non-congruent junction. A new
model will be developed for the spatial organization of the leg epidermis, and
this will be used to explain regeneration from the non-congruent junction.
284
V. FRENCH
(A) Number, position and orientation of structures
regenerated from the junction
The results of reversing the A/P or I/E axis at the tibia level of the Blatella
leg agree very well with the results of the same experiment performed on
other insects, in that the operation typically provoked the formation of two
complete regenerates by the 1st or 2nd p.o.m. (Bohn (1965, 1972a) on Leucophaea and between Leucophaea and Gromphadorhina; Bulliere (19706) on
Blabera; and Lheureux (1970) on the spider Tegenaria). This is in sharp
contrast to the results from the congruent tibial graft/host junction which
typically does not regenerate any segmented structures (Bohn, 1965, 1967,
1970; Bulliere, 1970a, 1971; French, 1976a) although a congruent Blatella
junction often regenerated partial structures if the graft tarsus had been
amputated (French, 1976 a).
A/P and I/E axis reversal was also performed at the coxa level by Bohn
(1972a), Bulliere (19706) and by Bart (1971a) on the stick insect, with results
comparable with those from the tibia level.
There is general agreement that the two separate laterals originate from the
points of discontinuity between graft and host, and are orientated as shown
in Fig. l A ^ 1 ) , B (61). The results from Carausius (Bart, 1971a) have two
notable features: laterals appeared from exactly the points of discontinuity,
and there was a relatively high frequency (20/57 cases) of formation of only
a single lateral after reversal of either of the graft axes. Bulliere (19706) and
Bart (1971a) found fusion between one of the laterals and the regenerated
graft terminal tarsus (this was observed once in Blatella) but no cases of a
double lateral. Bohn (1965, 1972a) obtained double laterals from a point of
congruity (external from A/P reversal, and posterior from I/E reversal) as
was found in Blatella, but he did not give details of their orientations.
Bohn found cases of partial autonomous regeneration at the tibial level
(Bohn, 1965, fig. 7a) and Penzlin (1965) found these structures in Periplaneta
after reversal of both axes by 180° rotation of a homopleural graft. It is
noticeable, however, that Bulliere (from all of whose grafts the tarsus was
removed) found no complete or partially autonomous regenerates, which were
major categories of result in Blatella.
(B) Composition of separate lateral regenerates
Bulliere's grafts (19706) were done between the pro- and meta-thoracic legs
of Blabera. These legs can be distinguished by the relative sizes of their segments,
and the presence of spines on the anterior/internal ridge of the femur of the
pro-thoracic leg. Laterals regenerated from tibial level could not be identified
as pro- or meta-thoracic, but Bulliere concluded that laterals regenerated
from coxa level were one of pure graft origin and the other of pure host origin.
Lheureux (1971), grafting between pedipalps and hind legs of Tegenaria,
Leg regeneration in the cockroach
285
reached the same conclusion using the criterion of one or two claws at the
distal tip. In both of these experimental situations the criteria enable direct
identification of only parts of the laterals.
Bohn (1972a) has shown that laterals of dual origin are the usual result
of reversing one transverse axis at either tibia or coxa level. He grafted between
differently pigmented species (Leucophaea and Gromphadorhina) and was able
to draw boundaries between host- and graft-derived areas of a lateral. The
boundaries almost always ran longitudinally down the lateral. After reversal
of the A/P axis, Bohn found that nearly all lateral regenerates were of dual
origin with the boundaries usually on the internal and external faces of the
lateral. A lateral was divided into a host-derived half adjacent to the host,
and a graft-derived half adjacent to the graft. This agrees completely with
the composition of the corresponding Blatella laterals (Figs. 2 A, B). Separate
laterals developed from an I/E reversal were less uniform, however, and were
sometimes of pure graft or pure host origin (especially when produced at
coxa level). Boundaries on dual origin laterals tended to be external and
(in nearly every case) posterior, dividing the lateral into approximately threequarters and one-quarter. These results correlate well with the tendency of
the corresponding Blatella laterals to appear to be either host- or graft-derived
(according to the limited criteria).
(C) The formation of double lateral regenerates
In both experiments the double laterals were formed in a region of congruity
between the transverse axes of the graft and host or, in other words, midway
between the sites where separate laterals would form. The existence of laterals
which were double at the base but separated more distally (Fig. 5D) reinforces
the view that the double laterals result from the secondary fusion of the two
single laterals regenerating from the areas of incongruity.
Fig. 8 shows the various ways in which the two separate laterals could
fuse, and comparison with Fig. 6 shows that most of the categories of double
lateral can be explained.
Separate laterals formed after reversal of the A/P axis could fuse internally
or externally (accounting for 42/43 of the structures obtained). If the laterals
fuse incompletely in an external position (Fig. SAty1)), each will become one
of the component tarsi of the double structure and the claws will be orientated
away from each other in the plane of the host A/P axis (6/25 cases, e.g. Fig. 5B).
The component tarsi will each be of dual origin (5/5 of the analyzable results).
Complete fusion of the laterals in an external position will make a double
circumference (Fig. 8A(6 2 )). Claws curve from external towards internal so
each component tarsus of the double structure will be formed from half of
each of the original laterals. Hence the claws will be orientated towards each
other in the plane of the host I/E axis (14/25 cases, e.g. Fig. 5 A), with one
set of host origin and the other set of graft origin (8/8 cases).
286
V. FRENCH
I
(A) AjP axis reversal
P
E
A
(A)
(£)
(P)
(c1) Incomplete fusion
(/?') Incomplete fusion
/
(/)
p
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(c2) Complete fusion
(bz) Complete fusion
(B) IjE axis reversal
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(£)
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Fuse anteriorly
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{bx) Incomplete fusion
A
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(c1) Incomplete fusion
I
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(b2) Complete fusion
P
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(c2) Complete fusion
E
P
Leg regeneration in the cockroach
287
Similarly, incomplete or complete fusion of laterals in an internal position
(Fig. 8 A (c1), (c2)) will give claws orientated towards each other in the plane
of the host A/P axis (13/17 cases) and each of dual origin (9/9 cases). Fusion
of the separate laterals hence accounts for the position of 42/43, the orientations
of 33/42, and the composition of 22/22 of the double laterals developed after
A/P axis reversal.
Laterals developed after reversal of the I/E axis may fuse posteriorly or
anteriorly (32/42 of the structures obtained). Incomplete fusion of laterals in
a posterior position (Fig. 8B(^)) will give claws side-by-side orientated in
opposite directions in the plane of the host A/P axis (7/30 cases), as will
incomplete fusion of laterals in an anterior position (2/2 cases, Fig. 5D,
SB^ 1 )). Complete fusion of the laterals posteriorly (Fig. 8B(&2)) will give
claws orientated towards each other in a plane between the host A/P and
I/E axes (13/30 of the observed double laterals were classified as approximately
in the plane of the host A/P axis, e.g. Fig. 5C). Composition of these fused
double laterals will depend upon the composition of the separate laterals:
separate laterals developing after I/E reversal usually appear to be one hostand one graft-derived and this tendency is also seen in the components of the
double structure. Fusion of the separate laterals hence accounts for the position
of 32/42, and the orientations of 22/32 of the double laterals developed after
I/E axis reversal.
Because of the correspondence between prediction and observation with
respect to the position, orientation and (at least for the A/P reversal) the
composition of the double laterals they will be assumed to have resulted from
fusion of separate laterals.
FIGURE 8
Fusion of separate laterals regenerated from the graft/host junction after reversal
of one transverse axis. Schematic representation of graft and host tibiae split
internally (Aa, b1, b2), externally (Ac1, c2), posteriorly (Btf, b1, b2) or anteriorly
(B c\ c2) and opened out flat, with the graft/host junction shown by the dashed
line. A, I, P, E, Anterior, internal, posterior and external 'faces' of the host tibia;
(A), (I), (P), (E),' faces' of the graft tibia. Laterals are shown' end-on' developing out
from the junction with host-derived tissue stippled (in A).
(A) Results from reversal of the anterior-posterior axis, (a) Two separate
laterals; (b\ b2) fusion of the two laterals into a double lateral positioned externally.
(b1) Incomplete fusion of the external 'faces' of the laterals, (b2) Complete fusion
to give one double circumference with two sets of claws orientated from external
to internal positions on the double circumference, (c1, c2) Fusion of the two laterals
internally, (c1) Incomplete fusion of the internal 'faces' of the laterals, (c2) Complete
fusion.
(B) Results from reversal of the internal-external axis, (a) Two separate laterals,
(b1, b2) fusion of the two laterals into a double lateral positioned posteriorly.
(b1) Incomplete fusion of the posterior 'faces' of the laterals. (62) Complete fusion
to give one double circumference, (c1, c2) Fusion of the two laterals anteriorly.
(c1) Incomplete fusion of the anterior 'faces' of the laterals, (c2) Complete fusion.
288
V. FRENCH
(D) The formation of autonomous and partially autonomous regenerates
It was shown (French, 1976 a) that completely and partially autonomous
regeneration could occur from a congruent tibial junction (i.e. both transverse
axes of host and graft in alignment) when healing together of host and graft
was impeded by withdrawal of the graft epidermis from the cuticle, associated
with the regeneration of an amputated graft tarsus. It was also argued that
rotation of the graft further increased the chance that graft and host would
not heal but would regenerate independently (at least initially). It is interesting
that the autonomous categories of regeneration from a non-congruent junction
also usually occur in conjunction with loss and regeneration of the graft
tarsus (Table 3), suggesting that, in this situation also, they result from a
failure of graft and host to heal together and interact.
The junction between the two axial elements of a partially autonomous
regenerate resulting from a congruent graft regenerated no lateral or a single
(usually incomplete) lateral, just like the original graft/host junction (French,
1976a). In the present study the junction of a partially autonomous structure
developing after reversal of one transverse axis also behaved just like the
corresponding graft/host junction. It regenerated two separate or a double
fused lateral and, as described, these were orientated and probably composed
exactly like the laterals regenerating from the original graft/host junction.
Thus the categories of lateral and autonomous regeneration reflect two
different processes:
(i) If interaction occurs between host and graft (or between their developing
autonomous regenerates) two laterals of dual origin are produced from the
points of incongruity of the transverse axes. These laterals may subsequently
fuse together.
(ii) If the graft and host surfaces do not heal together and interact, each
regenerates independently. In Blatella these autonomous regenerates lie in
linear sequence but in Blabera perhaps they may 'slide' past each other and
project laterally as two 'laterals' of pure graft and pure host origin. This
may explain why Bulliere (19706) found 'pure origin' laterals and no autonomous regenerates.
(E) Previous theories of the formation of separate lateral regenerates
following the reversal of one transverse axis
There have been three major suggestions about the cause of lateral regeneration, and to be tenable they must be consistent with the number, position,
orientation and composition of the laterals formed after reversal of one
transverse axis and after similar operations.
(i) Lawrence (1970). Regeneration of distal elements of the pattern may be
inhibited at all levels by disto-proximal inhibition. This would obviously be
relieved after amputation of distal structures, allowing the disinhibited area
Leg regeneration in the cockroach
289
(the cut end) to regenerate. If information flow is not only polarized distoproximally but also can only occur between cells from similar positions on
the circumference, reversal of a transverse axis will disinhibit two areas of
the host cut surface, separated by two inhibited areas. This is shown in Fig. 9 A
and would give two laterals of host origin, orientated like the host.
(ii) Bulliere (1970b). Regeneration may be inhibited everywhere on the intact
leg by the normal disto-proximal and proximo-distal short-range cellular
interactions. Reversing one transverse axis effectively relieves the inhibition
on the adjacent but non-communicating host and graft surfaces, so that they
will behave independently and each will regenerate (Fig. 9B). The host- and
graft-derived regenerates will grow past each other, retaining contact at a
common region of the circumference and hence will project laterally from
the original points of axis incongruity. Both will be orientated like the host
(since the graft regenerates from a proximal-facing surface).
(iii) Bart (1971a, b). Bart considers regeneration not to be disinhibited but
actually to be induced by local confrontation of tissue from opposite faces
of the leg. This confrontation occurs when the cut end of an amputated stump
heals over before terminal regeneration, and it occurs at two positions following
reversal of a transverse axis (Fig. 9C). A lateral of dual origin will be induced
at each of the two regions of confrontation, and they will both be orientated
like the host.
All three theories account satisfactorily for the number, position and
orientation of the laterals, but only that of Bart accounts for the fact that they
are usually of dual origin.
After reversal of one transverse axis, there are two areas of confrontation,
two cut surfaces and ultimately two regenerates. One might hope to determine
the relevant correlation by producing four areas of confrontation. The result
of these experiments will now be given and discussed.
Reversal of both transverse axes
Both transverse axes can be reversed by 180° rotation of a homopleural
graft, and the results are very variable. At tibial and coxal levels, Bulliere
(19706) usually found no laterals or two laterals, and Bohn (1972a) usually
found two laterals, often distally incomplete. Blatella usually produces no
laterals or one incomplete lateral (French, 1976a). In all cockroaches there
is a strong tendency for a rotated graft to rotate back into alignment with
the host. Bart (1971a) grafting at coxa level in the stick insect, found almost
no tendency for grafts to rotate back. He obtained no laterals (1/63 cases), one
lateral (15/63), two laterals (38/63) or three laterals (9/63).
Despite the difference in maximum number of laterals obtained, Bart's
results share many features with Bulliere's and Bohn's. Laterals could form
on any opposite or adjacent faces of the leg and, if two laterals were formed,
one was a right and the other a left leg. The cases illustrated by Bulliere
19
EMB 35
290
V. FRENCH
A. Lawrence (1970)
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ft
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B. Bulliere (1970 b)
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C.Bart (1971)
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Leg regeneration in the cockroach
291
(19706, fig. 8) all conform to Bart's illustration (1971a, fig. 2) of the possible
positions and orientations of the laterals.
The theory of Lawrence (Fig. 9 A) predicts one lateral, as the entire host
cut surface would be disinhibited; that of Bulliere (Fig. 9B) predicts one
lateral from the host and one from the graft; only the theory of Bart (Fig. 9C)
predicts more than two laterals (a maximum of four). It is difficult to assess
the significance of the cases of three laterals in the stick insect. Bohn (1972#)
suggests that two is the maximum possible number directly caused by the
rotation, and that Bart's triple laterals resulted from secondary wounding.
However, Bart never found cases of three laterals developing from the very
similar operations reversing only one transverse axis. Bohn also points to the
failure to ever obtain four laterals, indicating that it argues against Bart's
hypothesis of the establishment of a 'morphogenetic centre' wherever opposite
faces are confronted.
The differences in results between Blabera, Leucophaea, Blatella and Carausius
could be the reflexion of relatively trivial factors (e.g. size, rate of wound
healing, extent of back rotation), and are unlikely to be due to major differences
of mechanism. It seems unwise to choose a model (Bulliere's) which restricts
possible laterals to two when, even in only one insect, one seventh of operated
legs bear three.
As Bart argues (1971a), it is likely that lateral regeneration occurs due to
some positive interaction between opposite faces confronted at the junction,
rather than just a lack of normal contacts. Grafts of sternite or tergite (Bart,
1966), coxo-pleural articulatory membrane, scape, or the same face of another
segment (Bart, 1971 b), or elimination of a part of a femur face (Bart, 1970), do
not result in regeneration of the leg tissue thus deprived of its normal neighbours. Also, an interaction theory accounts more satisfactorily for most laterals
being of dual origin. Such an interaction theory will now be developed and
will be shown to account in detail for many of the features of lateral regeneration.
FIGURE 9
Theories of the origin of lateral regenerates after reversal of one transverse axis
(the internal-external axis).
(a) Schematic representation with the tibia split posteriorly and opened out
fiat, with the graft/host junction shown by the dashed line. Hatching, area disinhibited (A and B) or induced (C) and able to regenerate, g, Graft; h, host.
A, I,P, E, 'faces' of the tibia.
(b) Regenerated structures, anterior view. Stippling, host-derived lateral structures.
(A) Regeneration from two areas of the host where 'tramlined' disto-proximal
inhibition cannot act.
(B) Regeneration from the host surface (isolated from normal disto-proximal
contacts) and graft surface (isolated from normal proximo-distal contacts).
(C) Regeneration from the two regions of confrontation of opposite (/ and E)
faces of the tibia.
19-2
292
V. FRENCH
(F) Theory for the initiation of lateral regeneration
from a non-congruent graft /host junction
So far, in describing the experiments and discussing the results, the leg has
been considered to have two 'transverse axes' and four 'faces' (anterior,
internal, posterior, external). Bart (1971 a, b) considers the four faces to be
distinct and qualitatively different from each other: opposite qualities can
interact in a unique way to induce a regenerate. After reversal of one 'transverse
axis' of the graft, two host 'faces' will be in contact with the corresponding
'faces' of the graft, and the other two host 'faces' will each confront the
opposite graft 'face': there will be interaction and induction of a regenerate
at these two positions.
Bohn (1972a) suggests that 'faces' are nothing more than a 'topographical
characterization', and that the cross-section of the limb could be arranged
in a two-dimensional co-ordinate system 'the axes of which coincide with the
antero-posterior and dorso-ventral (external-internal) axes'. In this coordinate
system every cell is different from any other. After reversal of one transverse
axis, only at two points on the circumference will the normal contacts be
made, and the laterals will develop from the two areas of maximum incongruity.
One might further suggest that the two 'transverse axes' are nothing more
than a topographical characterization. There is no evidence that the anteriorposterior and internal-external planes are of any particular significance to the
cockroach leg with respect to epidermal pattern or regeneration.
It is very likely that lateral regeneration is due solely to interactions occurring
within the epidermis, since it can be provoked by grafts of epidermis not
involving the translocation of any muscle or the sectioning of any major nerve
(Bart, 1966, 1971a; Bohn, 19726; French & Bulliere, 1975a, b). The leg
epidermis, being a single celled layer, is effectively two dimensional; the surface
of a cylinder upon which any position may be specified, not in relation to
three axes, but by the two co-ordinates of proximal-distal level and circumferential position (Bulliere, 1971; French & Bulliere, 1975a, b).
F I G U R E 10
Transverse intercalary regeneration between different circumferential positions
(adapted from French & Bulliere, 1975 a).
(A) Schematic cross-section of the left femur, distal view. A, I, P, E, Anterior,
internal, posterior and external 'faces'. Twelve positions are marked around the
circumference arbitrarily by numbers 0-12.
(B, C) Graft of internal face of the left femur into the anterior face of the left femur:
graft situation (a) and result after two moults (b): transverse section through the
graft (B) and anterior view (C). An internal face (r) is regenerated between positions
4 of the graft (g) and 8 of the host (h), and a part of the anterior face (r) is regenerated between positions 8 of the graft (g) and 9 of the host (/?). At the ends
of the graft, intercalary regenerates (r') are formed between graft and host, and
between regenerate (r) and host, to remove all discontinuities of position.
Leg regeneration in the cockroach
A
E
293
294
V. FRENCH
Bohn (19726) showed that circumferential position is not irreversibly determined since, in the course of lateral or terminal regeneration, a quarter of
the circumference of the tibia often gave rise to more than the corresponding
quarter of the tarsal circumference. French and Bulliere (1975a, b) showed
that circumferential position (la generatrice) is an aspect of position independent
of proximal-distal level and is specified in a continuous manner around the
leg epidermis, rather than along two perpendicular axes going through the
leg. By grafting a rectangular piece of cuticle plus epidermis to an abnormal
position around the circumference, we confronted non-homologous positions
along the lateral edges of the graft. In all such experiments (French, 1976 c)
there was an intercalary regeneration of the structures which normally separate
host and graft positions, as measured by the shortest route around the circumference (Fig. 10 A, B). At the proximal and distal ends of the graft, different
circumferential positions were placed adjacent to each other in a proximaldistal sense, and there was an identical intercalary regeneration of circumferential values to restore normal cellular contacts (Fig. IOC).
These experiments detected no 'boundary positions' having unique properties
which could correspond to a boundary between 'high' and 'low' values of
a circumferential 'gradient' analogous to the postulated proximal-distal segment 'gradient' (Bohn, 1967). The circumferential position 12/0 in Fig. 10
does not imply a boundary; it arises inevitably when labelling a circle (e.g.
a clock face) with numbers. Possible mechanisms for smoothly specifying
position around a closed circle will be considered elsewhere (French, Bryant
& Bryant, in preparation).
The position of an epidermal cell within a segment seems to be defined
by its level within a linear proximal-distal ordering, and its position within
a circular circumferential ordering. Intercalary regeneration occurs between
different proximal-distal levels and between different circumferential positions.
FIGURE 11
Initiation of lateral regeneration from non-congruent graft/host junctions.
(A) Reversal of anterior-posterior axis, giving laterals anteriorly and posteriorly.
(B) Reversal of internal-external axis, giving laterals internally and externally.
(C) Reversal of both axes by 180° rotation of a homopleural graft, giving no
laterals (C1), or two laterals on adjacent faces (C2, C3) or on opposite faces (C4).
Schematic cross-section of graft/host junction; outer circle, host circumference;
inner circle, graft circumference. A, I, P, E, Anterior, internal, posterior and
external 'faces'. Twelve positions are marked around the circumference by numbers
0-12 (as in Fig. 10) and numbers between the circles are the positions formed by
intercalary regeneration (by the shortest route) between the different confronted
positions of host and graft. Where the shortest route is different on the two sides
of a point, the two halves of a complete circumference are formed, confronting
each other; these are drawn spread apart to show the predicted orientation and
composition (in A and B) of the regenerate forming at that point. Claws curve from
external to internal positions on the laterally formed circumference, and stippling
denotes host-derived tissue.
Leg regeneration in the cockroach
A 9
A9
295
296
V. FRENCH
It will now be shown that lateral regeneration is initiated at a non-congruent
graft/host junction as a direct result of the spatial organization of the
epidermis.
Reversal of one 'transverse axis'
Consider the situation at the tibia graft/host junction following a graft
between left and right legs. Fig. 11A shows the A/P axis reversal and the
intercalary regeneration which will occur between the confronted graft and
host positions, always forming the intervening positions, as measured by the
shortest route. It will be seen that the shortest route between confronted graft
and host positions is different on the two sides of each point of maximum
incongruity (A and P), hence at these points the two halves of a complete
circumference are formed, confronting each other. Fig. 11B shows the I/E
axis reversal, forming the two halves of a circumference both internally and
externally.
Consider the confrontation of the two half-circumferences formed at each
point of maximum incongruity after the A/P axis (Fig. 11 A). The confrontations
10-8, 11-7, etc., will result in intercalary regeneration of the intervening
positions, as measured by the shortest route. The shortest route is different
on the two sides of the confrontation 12/0-6, hence at this point the two
halves of a complete circumference will again be formed, confronting each
other. Thus a round of intercalary regeneration produces new tissue (which
will bulge laterally from the junction) but just recreates the confrontation of
half circumferences.
After an amputation the epidermis from the circumference at the end of
the stump migrates under the clot of dried haemolymph to re-establish epidermal
continuity, resulting in the confrontation of cells from different circumferential
positions. This situation can be simplified by considering epidermal migration
to occur only in one plane, resulting in the confrontation of two half-circumferences which, exactly as in the lateral situation considered above, would
be perpetuated by intercalary regeneration. The two half circumferences
formed at each point of maximum incongruity after reversal of a transverse
axis, and the circumference left after an amputation, are both cases where
a complete circumference heals over because it is not sandwiched (as it is in
the intact leg) between other circumferences overlying it proximally and
distally. In these situations regeneration of distal structures occurs. Perhaps
the tissue of a regeneration blastema is produced by successive rounds of
intercalary regeneration between the different positions confronted by a circumference healing over at any level other than the distal tip of the tarsus. Clearly,
this is only one component of the regeneration process since, within the
blastema, the more distal levels of the leg must be specified. It is interesting
that a bilaterally symmetrical, partial circumference, which could heal over
and completely resolve confrontations between non-homologous positions by
Leg regeneration in the cockroach
297
intercalary regeneration, often does not regenerate distal structures or forms
distally incomplete partial regenerates (Bohn, 1965; French, 1976a).
Thus each of the two complete circumferences formed at the junction after
an A/P or I/E 'axis' reversal will be the origin of a lateral regenerate. Both
laterals will be orientated in the I/E axis like the host (I/E reversal) or like the
host and graft (A/P reversal). Assuming that both components contribute to
the intercalary regeneration which creates the complete circumference, the
laterals will each be composed of host-derived and graft-derived longitudinal
halves.
Reversal of both 'axes'1 by 180° rotation
After 180° rotation of a homopleural graft each host position around the
graft/host junction will be confronted by the opposite graft position. A confrontation between opposite positions may be resolved by intercalary regeneration of either of the half-circumferences which separate them (French,
1976 c). This can be visualized as going from the host position around a
circumference in a clockwise or anticlockwise 'direction' to the opposite graft
position. If intercalary regeneration occurs in the same 'direction' everywhere
on the junction, no lateral regenerates will be formed (Fig. 11C1). Any tendency
of the graft to rotate back into alignment will increase the probability that no
lateral will be formed by creating an unambiguous 'shortest route' in the
same 'direction' at all positions on the junction. If, by chance, one sector of
the junction forms the intercalary regenerate in one 'direction' while the
remainder of the circumference of the junction regenerates in the opposite
'direction', a complete circumference will be created at each boundary between
the two regions, and hence two laterals may be formed. This may occur anywhere on the circumference, forming two laterals situated opposite or adjacent
to each other. One left-handed and one right-handed lateral will be formed.
Consider the possible positions and orientations of these laterals. Considering
only the I/E axis, a lateral will be orientated like the host if it is formed
internally or externally (Fig. 11C2, C3), but either like the host or like the
graft it formed anteriorly or posteriorly (Fig. 11C2, C3, C4). If one lateral is
anterior and the other posterior, they will have opposite orientations (Fig. 11C4).
These predictions, concerning handedness, position and orientation, which
are not made by earlier models, are in good agreement with the data of
Bulliere, (1910b) Bart (1971a) and Bohn (1972a).
More than two laterals may be formed if the 'direction' of intercalary
regeneration in two sectors is opposed by that in the two sectors separating
them. A complete circumference will be created at the four boundaries, making
it possible for four laterals to form. Bart (1971a) found three laterals in 9/63
cases but no cases where four were formed. This is perhaps not surprising since
analysis of Bart's results for reversal of a single axis (Bart (1971a), table 4)
indicates that a point of incongruity has a probability of only 0-7 of leading
298
V. FRENCH
to the formation of a lateral. If cases of four changes of 'direction' around
a single junction occur fairly infrequently (two changes being the usual
situation) and if each circumference which forms at the site of such a change
has only a 0-7 probability of leading to regeneration of a lateral, two laterals
will be the most frequent result of the 180° rotation experiment and the
incidence of three laterals will be much higher than that of the maximum,
four.
It remains unclear why the laterals formed after 180° rotation were often
distally incomplete (Bart, 1971a; Bohn, 1972a). It was argued (French, 1976a)
that a distally incomplete lateral could arise from a sector of a congruent
junction if the graft and host did not initially heal together and interact.
Incomplete laterals may arise from a rotated junction if there is a failure of
healing at a position which is not a boundary between different 'directions' of
intercalary regeneration.
Composition of the lateral regenerates
It has been assumed so far that position is specified smoothly and continuously around the leg, with no positions having unique properties and that
all confrontations result in intercalary regeneration by the 'shortest route'.
This is consistent with the results of French & Bulliere (1975 a, b) and is the
basis of the model for initiation of lateral regeneration. The model is consistent
with the number, position and orientation of laterals formed after reversal of
one transverse axis and also explains the main features of regeneration after
180° rotation of a homopleural graft.
It has further been assumed in Figs. 6, 8 and 11 that the intercalary regeneration results from proliferation of both of the confronted regions creating
a complete circumference of dual origin, leading to a lateral regenerate composed of host-derived and graft-derived longitudinal halves. This assumes that
there are no restrictions with respect to the circumferential position which
the progeny of a cell can occupy. This assumption seems to be supported by
the results of A/P axis reversal, where the laterals were usually of dual origin
with borders between host- and graft-derived parts running approximately
mid-external and mid-internal, as predicted (see Results, and Bohn, 1972a).
After I/E reversal, however, the separate Blatella laterals were often largely
host-derived or graft-derived, and Bohn's interspecies grafts (1972 a) showed
that dual origin laterals had one border on the posterior side (predicted) but
the other border internal or external (not predicted). This suggests that an
anterior or posterior region can produce tissue more internal and more external,
but that an internal or external region may not always be able to produce
tissue both anterior and posterior to it.
In this connexion it is intriguing that, after grafting an external sector of
the tibia of Gromphadorhina to an internal position of the Leucophaea tibia,
Bohn (19726) found that the graft contributed more than one-quarter of the
Leg regeneration in the cockroach
299
circumference of the lateral tarsi which were formed. The four illustrations
(Bohn, 1912b, figs. 1, 3b-e) all show the graft forming anterior and even internal
portions of the tarsi, but not more posterior portions.
Clonal analysis has shown that the epidermal cells of the developing
appendages of Drosophila are restricted in the positions which they can occupy.
The imaginal wing disc becomes progressively subdivided into 'compartments'
in the course of normal development (Garcia-Bellido, Ripoll & Morata, 1973;
Garcia-Bellido, 1975). Clones initiated after a particular compartment boundary
has formed do not cross it in normal development. Even if they are able to
divide more rapidly than the rest of the tissue and nearly fill the compartment
in which they arise, cells of the clone will not cross the compartment boundary.
The disc seems to be divided into anterior and posterior compartments as
soon as it is segregated from the rest of the blastoderm, but thereafter
compartment boundaries form between dorsal and ventral, and wing and
notum (during the 1st larval instar), between regions of the notum (during
2nd instar) and between proximal and distal wing (during 3rd instar). During
regeneration from fragments of a disc, however, cells may cross the later
compartment boundaries although they may not be able to cross the anterior/
posterior boundary (Bryant, 1975, and personal communication).
If the cockroach leg were divided into anterior and posterior compartments
which were respected during regeneration, with the compartment boundaries
approximately mid-internal and mid-external, this might account for the
differences in composition between laterals resulting from A/P and I/E reversals.
Experiments are in progress to explore this possibility.
(F) Comparison between lateral regeneration
in insect and amphibian legs
When newt leg regeneration blastemas are grafted on to stumps in such
a way as to reverse the A/P or dorsal/ventral axis (D/V) or both, supernumerary
regenerates develop just as in cockroaches (Iten & Bryant, 1975; Bryant &
Iten, 1976). Reversal of A/P or D/V axis gives two laterals originating from
anterior and posterior, or from dorsal and ventral respectively, orientated like
the stump. After reversal of both axes, one right and one left-handed regenerate
are formed.
An interpretation of these newt laterals which is very similar to the present
model of lateral regeneration in cockroach legs has been developed by Bryant
& Iten (1976). It is suggested that position is continuously specified around
the circumference of the limb, that confrontation of non-homologous positions
results in intercalary regeneration by the shortest route, and that regeneration
occurs from the complete circumference generated wherever the 'direction' of
the shortest route changes over.
The similarities between insect and amphibian limbs will be developed
elsewhere (French, Bryant & Bryant, in preparation), but the occurrence of
300
V. FRENCH
similar lateral regeneration suggests that these anatomically quite different
systems may be organized in fundamentally the same way.
This work was financed by an S.R.C. Postgraduate Studentship and a Royal Society
European Exchange Research Fellowship. I thank Professor Sengel for the hospitality of
his laboratory in Grenoble; and Professor MacGregor of the Department of Zoology,
University of Leicester, for generously providing facilities for the preparation of this
manuscript.
I am deeply indebted to Gerry Webster for preserving the sanity and guiding the efforts
of his postgraduate student, and for advice on this manuscript.
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{Received 19 September 1975, revised 15 December 1975)