35
/. Embryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain 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 re- generates) 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 circum- ference 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.

Leg regeneration in the cockroach, Blatella germanicaEmbryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain Leg regeneration in the cockroach, Blatella germanica

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Page 1: Leg regeneration in the cockroach, Blatella germanicaEmbryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain Leg regeneration in the cockroach, Blatella germanica

/ . Embryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 2 6 7

Printed in Great Britain

Leg regeneration in the cockroach,Blatella germanica

II. Regeneration from a non-congruent tibial graft/host junction

By VERNON FRENCH1

From the Developmental Biology Group,University of Sussex, and Laboratoire de Zoologie,

Universite Scientifique et Medicate de Grenoble

SUMMARYThe 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 cutperpendicular to the proximal-distal axis and two heteropleural combinations were usedto reverse separately the two transverse axes of the graft relative to the host. Use of darkand light cuticle colour mutants gave a good indication of the graft or host origin ofregenerated 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 andpartially autonomous regeneration - reflect two processes.

(i) If the graft tarsus is removed, graft and host may not heal together and interact, butform autonomous regenerates lying in mirror-image symmetry separating original graftand host levels.

(ii) If interaction occurs between graft and host (or their developing autonomous re-generates) two laterals of dual origin are produced, one from each point of transverse axisincongruity. These laterals may secondarily fuse together to form a double structureoriginating from a point of congruity. The orientation and composition of the componenttarsi of the double structure depend on the site of origin and the extent to which the twolaterals fuse.

It is argued that the four 'faces' and two 'transverse axes' of the leg are merely descriptiveterms. A new model is developed whereby lateral regeneration arises directly from thecircumferential organisation of the leg epidermis. Previous work has shown that positionis specified continuously around the circumference, and that intercalary regeneration occursby 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 sidesof each of the two points of 'axis' incongruity, and at these points the two halves of acomplete circumference are formed. These lateral circumferences, like the terminal circum-ference exposed by amputation, cannot heal over by intercalary regeneration, and this leadsto regeneration of distal structures.

The model accounts for lateral regeneration after reversal of both 'transverse axes' by180° rotation of a homopleural graft.

The possibility is discussed that there may be clonal restrictions on the circumferentialpositions 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.

Page 2: Leg regeneration in the cockroach, Blatella germanicaEmbryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain Leg regeneration in the cockroach, Blatella germanica

268 V. FRENCH

INTRODUCTION

The cuticle of insects is secreted by the underlying single layer of epidermalcells, and a study of the cuticular patterns formed after surgical operationswhich remove or spatially disturb part of the epidermis can give informationabout the systems of cellular interaction within the epidermis which must formsome 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 associationof different circumferential positions, there is transverse intercalary regenerationof the intermediate positions (French & Bulliere, 1975a, b).

Lateral regeneration of supernumerary legs can also occur from a graft/hostjunction. The leg has been considered to be organized along three mutuallyperpendicular axes (Bohn, 1965): a longitudinal (proximal-distal) axis runningfrom the articulation with the body to the claws, and two transverse axes(anterior-posterior and internal-external). Two complete lateral regenerateshave been shown to form from a non-congruent junction (i.e. host and grafttransverse axes not aligned) in several species of cockroach: Periplanetaamericana (Penzlin, 1965),Leucophaeamaderae (Bohn, 1965, \912a),LeucopheaejGomphadorhina 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 inArachnida (Lheureux, 1970, 1971).

There has been general agreement about the number, position and orientationof the laterals resulting from various graft combinations, but different con-clusions have been reached concerning the tissue composition (graft or hostorigin) of the laterals. Grafts have been made between different legs of animalsof the same species (Bart, Bodenstein, Bulliere, Lheureux, Penzlin) or betweenthe legs of animals of different species (Bohn). Laterals have been consideredto be of pure graft or pure host origin, forming because of a failure of thetwo components to interact (Bodenstein, Bulliere, Lheureux, Penzlin), or ithas 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 thetibia level in Blatella germanica to confirm lateral regeneration in this speciesof small cockroach, to provide more information about the range of possibleregenerated structures, and to determine the composition of the regeneratedstructures by grafting between cuticle colour mutants (French, 1976a). Theresults obtained from tibial junctions can also be used for comparison with

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Leg regeneration in the cockroach 269

the regenerates produced from congruent and non-congruent junctions betweennon-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. Variousearlier theories of the origin of lateral regenerates are found to'be unsatisfactoryand 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 longitudinallyand circumferentially. Lateral regeneration is shown to arise as a direct resultof this spatial organization of the epidermis.

MATERIALS AND METHODS

Laboratory colonies of Blatella germanica were maintained as describedpreviously (French, 1976 a), and the grafting operations were performed on3rd and 4th instar larvae 1 or 2 days after moulting. In all experiments somegrafts were made on wild-type animals (usually between different legs of thesame 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 withCO2. Graft and host tibiae were chosen to be of slightly different diametersso the graft could be pushed slightly into the end of the host stump, where itwas secured by dried haemolymph. Experimental animals were kept untila few days after the 1st or 2nd post-operative moult (p.o.m.) and then theoperated leg was removed fixed and cleared in Gum Chloral, and examined.

The structure of the leg of Blatella germanica and the labelling conventionto be used have both been described previously (French, 1976a). The leg willbe described in terms of four 'faces': anterior, posterior, internal and external(but see Discussion). The position and orientation of structures producedafter the operations will be given with reference to the host axes. It shouldbe 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 transverseaxes of the graft with respect to the host, and are illustrated in Fig. 1. Theright pro- or meso-thoracic donor leg was removed at proximal tibial leveland grafted on to the host left meta-thoracic leg which had been amputatedat mid-tibia level. The graft tarsus was either amputated (as in Fig. 1) or leftintact.

Anterior/posterior (A/P) reversal. The graft was rotated by 180° about itslongitudinal axis, leaving internal and external host faces adjacent to thecorresponding graft faces, but confronting anterior and posterior host withposterior and anterior graft, respectively (Fig. 1A).

Page 4: Leg regeneration in the cockroach, Blatella germanicaEmbryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain Leg regeneration in the cockroach, Blatella germanica

270 V. FRENCH

(b4)

Tr

1 E

Fig. 1. Structures regenerated from the graft/host junction following reversal of onetransverse 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 tohost axes) except A^1), A(64), B(62) and B(65) which are external, (a) Graftsituation. (6x-65) Classes of result after 1st or 2nd post-operative moult, (b1) Twoseparate 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.

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Leg regeneration in the cockroach 271

Internal I external (I/E) reversal. The graft was not rotated, leaving anteriorand posterior host faces aligned with the corresponding graft faces, but con-fronting 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 regeneratedsegmented structures from the junction by the 1st p.o.m. and all had doneso 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 willbe seen that the two experiments give rise to the same categories of structureswith comparable frequencies. In each experiment, some of the animals withgraft 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 thereforeconsidered together with those animals which had the graft tarsus amputatedat the time of grafting. Table 3 relates the state of the graft tarsus to thestructure regenerated from the graft/host junction. Each category of regeneratewill now be considered.

(i) Two separate lateral regenerates

These structures could appear at the 1st or 2nd p.o.m. (Table 2), and atthe junction between the host and a graft which had retained its tarsus intactor had lost and regenerated the tarsus (Table 3). The combination of graftproximal tibia and host mid tibia often gave rise to reversed orientationintercalary regeneration (French, 1976 a) and the lateral regenerates occurredat its proximal limit (NB, all positions and orientations refer to the hostaxes).

The two lateral structures each comprised a tibial apex (the coronet ofspines and articulation with the condyle on the external side of the proximaltarsus), 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 tibiadepended on which transverse axis had been reversed by the operation. Followingreversal of the anterior/posterior (A/P) axis of the graft, the laterals wereusually (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 lateralswere 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 eachregion of incongruity between the transverse axes of host and graft: at eachconfrontation of opposite faces of the leg.

Page 6: Leg regeneration in the cockroach, Blatella germanicaEmbryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain Leg regeneration in the cockroach, Blatella germanica

Tab

le 1

. Fre

quen

cy o

j re

gene

rati

on J

rom

th

e ti

bial

gra

jtIh

ost

junc

tion

Jol

low

ing

reve

rsal

oj

one

tran

sver

se a

xis

oj

the

graj

t

to to

1st

p.o

.m.

Axi

s re

vers

ed

No.

w

ith

surv

ivin

ggr

aft

Gra

ft/h

ost

jun

ctio

n

No

Reg

ener

atio

n

rege

nera

tion

No.

sh

owin

gn

o r

egen

erat

ion

at 1

st p

.o.m

.an

d k

ept

to2n

d p

.o.m

.

2nd p

.o.m

.

No.

wit

hsu

rviv

ing

graf

t

Gra

ft/h

ost

junc

tion

rege

nera

tion

An

teri

or-

po

ster

ior

Inte

rnal

-ex

tern

al13

912

491 80

48 4442 30

19 1919 19

p.o

.m.,

pos

t-op

erat

ive

mo

ult

.

Tab

le 2

. C

lass

ific

atio

n o

j re

gene

rate

s Jr

om t

he

tibi

al g

rajt

/hos

t jun

ctio

n Jo

llow

ing

reve

rsal

oj o

neoj

th

e tr

ansv

erse

axe

s o

j th

e gr

aft

Par

enth

eses

den

ote

nu

mb

er o

f st

ruct

ures

app

eari

ng a

t th

e 1s

t p

ost

-op

erat

ive

mo

ult

(p.

o.m

.)fo

llow

ed b

y n

um

ber

app

eari

ng a

t th

e 2n

d p

.o.m

., a

dded

to

giv

e to

tal.

Str

uctu

res

rege

nera

ted

fro

m t

he g

raft

/hos

t ju

ncti

on

Axi

sre

vers

ed

To

tal

no

.of

rege

nera

tes

2 s

epar

ate

late

rals

Do

ub

lela

tera

l

Com

plet

ely

auto

no

mo

us

regen

erat

ion

Par

tial

lyau

tono

mou

sre

gene

rati

onO

ther

m o

Ant

erio

r-po

ster

ior

Inte

rnal

-ext

erna

l(9

1 +

19)

110

(80+

19)

99(1

3 +

4)1

7(2

5 +

4)2

9(3

1 +

12)

43(3

3 +

9)

42(2

1+0)

21(7

+1)

8

(22

+ 3

)25

(12

+ 4

)16

(4 +

0)4

(3 +

1)4

Page 7: Leg regeneration in the cockroach, Blatella germanicaEmbryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain Leg regeneration in the cockroach, Blatella germanica

Leg regeneration in the cockroach 273

Table 3. Relationship between state of the graft tarsus and regeneration fromthe graft/host junction following reversal of one transverse axis of the graft

{combined data from the two experiments)

State of grafttarsus

Intact (5 segments)Regenerated

(4 segments)Broken or unknown

Total

Structure regenerated

2 separatelaterals

1917

10

46

Doublelateral

3531

19

85

from the graft/host junction

Completelyautonomousregeneration

07

22

29

Partiallyautonomousregeneration

410

27

41

The A/P orientation of the laterals could not be determined, but theirI/E axis was almost always polarized in accordance with host and graftfollowing reversal of the graft A/P axis (15/17 cases), and in accordance withthe 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) onlegs 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 structurewas regenerated at the proximal limit of any reversed orientation intercalaryregenerate formed between host and graft.

In all cases there was a large tibial apex (probably two apices fused togetherbut this could not be determined), articulating with a large double tarsalstructure having two proximal condyles and two distal sets of two claws. Thetarsus 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 ofthe tibia differed in the two experiments. Where the A/P axis of the graft hadbeen 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 ofthe 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 aregion of congruity between the transverse axes of host and graft: at a positionmidway between the two regions of incongruity where the laterals form whenthey are separate.

Only the I/E orientation of the double tarsi could be determined and, asshown in Fig. 6, the orientations were very variable. The tarsi were alwaysorientated in one plane, but this could be the plane of the graft and hostI/E axes (Fig. 5 A) or perpendicular to it (Fig. 5B). The component tarsi were

18 E MB 35

Page 8: Leg regeneration in the cockroach, Blatella germanicaEmbryo!, exp. Morph. Vol. 35, 2, pp. 267-301, 1976 267 Printed in Great Britain Leg regeneration in the cockroach, Blatella germanica

A I P

gc

D

hs

gc

Fig. 2. Separate lateral regenerates formed from the graft/host junction afterreversal 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 ofgraft origin; gh, tarsal hair of graft origin; gs, coronet spine of graft origin; he, clawof host origin; hh, tarsal hair of host origin; hs, coronet spine of host origin.

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Leg regeneration in the cockroach 275

B

p \ i

Fig. 3. Camera lucida drawings of separate lateral regenerates formed from thegraft/host junction after reversal of the anterior-posterior axis (A) or the internal-external axis (B). A, I, P, E, Anterior, internal, posterior and external 'faces'.

almost always oppositely orientated, with the two sets of claws facing directlytowards (Fig. 5 A) or away from each other (Fig. 5B). The range of variationis shown in Fig. 6 as it is important in connexion with the composition andmodes of origin of the double structures.

(iii) Completely autonomous regeneration

Completely autonomous regeneration of the host and graft surfaces occurredby the first p.o.m. but did not occur subsequently in animals which had notregenerated from the junction at the first p.o.m. (Table 2). The distal partsof the long and fragile structures were often broken off but, from the seven,cases where this had not occurred, autonomous regeneration was seen tohave occurred only in association with loss of graft tarsus at or after theoperation (Table 3).

Following reversal of either axis, the autonomous structure consisted oftwo regenerates each comprising distal tibia, four-segmented tarsus and twoclaws, lying in mirror-image linear sequence separating the original graft andhost 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 ofthe host, while the more distal regenerate was orientated like the graft.

18-2

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C 1,

hh

he1

Fig. 4. For legend see opposite.

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Leg regeneration in the cockroach 277

hs f •V

E \A I

Fig. 4. Double lateral structures regenerated from the graft/host junction afterreversal of the anterior-posterior axis (A and B which are anterior and posteriorviews 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 doublelateral with one component tarsus more or less host-derived and the other moreor 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 graftorigin; he, he1, claw of host origin; hh, tarsal hair of host origin; hs, coronet spineof host origin.

(iv) Partially autonomous regeneration

Partially autonomous regeneration of the graft and host surfaces couldoccur by the first or second p.o.m. (Table 2). As in the case of the completelyautonomous regenerates, distal parts of the long and fragile structures wereoften broken off, but, from the 14 complete structures, it seems that partiallyautonomous regeneration usually occurred in association with loss of thegraft tarsus at or after the operation (Table 3).

The structures consisted of two partial regenerates lying in mirror-imagelinear sequence separating host and graft, and fused together at some levelproximal to the end of the tarsus. This level could be the tibia apex or in anyof the tarsal segments but was almost always the same for the two elementsconcerned. From this level of fusion were formed either two separate lateralsor 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 axiallysituated partial regenerates were orientated (in the I/E axis) like the host and

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278 V. FRENCH

Fig. 5. Camera lucida drawings of double laterals regenerated from the graft/hostjunction after reversal of the anterior-posterior axis (A, B) or the internal-externalaxis (C, D). A, I, P, E, Anterior, internal, posterior and external 'faces'.

FIGURE 6

Position, orientation and composition of double lateral regenerates developingfrom the graft/host junction following reversal of one transverse axis. A, Resultsof experiment reversing the anterior-posterior axis; B, results of experimentreversing the internal-external axis. Figures denote number of cases.

'Position': schematic representation of the graft/host junction, distal view. Theouter 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 theorientation with respect to the host axes, with position of origin of the lateral atthe top to facilitate comparison with Fig. 8. Tarsal claws curve from external tointernal 'faces' on the component tarsi which are shown separated by a dashedline.

'Composition'-'end-on' view of the double laterals showing approximatedivision into host-derived (stippled) and graft-derived parts. '? '-unknowncomposition; '1 host/1 graft', one component tarsus of host origin and the otherof graft origin; 'both dual' - each component tarsus of dual graft and host origin.

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Leg regeneration in the cockroach 279

Position Orientation Composition

1 host/ 1 graft Both dual

(E)

(E)

External - 25

Other - 5

(f)

Internal - 17 Other 4

Anterior - 1

B (P)

vyy13

(P)

(P)

Posterior - 30Other 5

(A)

Other -10

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Leg regeneration in the cockroach 281

graft respectively. The lateral elements were positioned and orientated just likethe laterals originating directly from the graft/host junction. Following A/Preversal, the separate lateral elements were positioned one anteriorly and oneposteriorly, and were orientated like the graft and host; double lateral elementswere positioned externally or internally and had a range of orientations similarto those in Fig. 6. After I/E reversal, separate laterals were positioned internallyand externally, and were both orientated like the host; double laterals wereusually positioned posteriorly, with various orientations.

(v) Other structures

In four cases only a single lateral was regenerated from the graft/hostjunction and, in another four cases the leg bore a poorly segmented lateralending in two atypical claws. One of these animals was allowed to moultagain, 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/hostjunction developed segmented structures in various spatial arrangements but always com-prising 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 ofdefinite, though limited, use in determining the origin of the epidermis ofregenerated structures (French, 1976 a). Although the colour difference betweenthe original host and graft tissues was usually clear, it was usually not possibleto draw a precise boundary between them or their derivatives. This mayjust reflect an insufficient difference in colour and some cell mixing at theboundaries, or there may be a local interaction affecting cellular phenotypenear the boundary (French, 1976a). In addition, lateral or autonomousstructures regenerated from the graft/host junction were often fragile andpoorly pigmented, reducing the number of analysable cases. Although only

FIGURE 7

Autonomous structures regenerated from the graft/host junction after reversalof the anterior-posterior axis (B, C, D, E) or the internal-external axis (A). A, B,Completely autonomous regeneration; C, D, E, partially autonomous regenerationwith a double lateral structure, with one component of host origin and the otherof graft origin (C, D), or both of dual origin (E).

A ,/, P, E, Anterior, internal, posterior and external 'faces', c, c1, the two setsof 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 fromhost component of the junction; //, double lateral structure; rt, tarsus regeneratedfrom the distal end of the graft.

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an approximate boundary could be drawn over areas of bare cuticle, thespines of the tibial apex, well-developed tarsal hairs and the tarsal clawscould usually be easily identified as Bl or br, and the origin of the epidermisof 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 washost-derived and all axial cuticle distal to them was graft-derived. Hencethe reversed orientation intercalary regenerate (if any) was of graft origin.

Grafts between Bl and br animals reversing the graft A/P axis gave eightanalysable legs with separate laterals anteriorly and posteriorly. In one caseone lateral appeared to be of dual origin and the other of host origin, but inall other cases both laterals were clearly of dual origin. They had host-derivedspines in the tibial coronet and a host-derived claw on the side adjacent tothe 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 separatelaterals internally and externally (or approximately internally and externally).In nine cases one lateral tarsus seemed host-derived with two claws of hostorigin, 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 thepresence 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 onegraft-derived claw (Fig. 2D). These laterals developing after I/E axis reversalare so orientated that a division into side-adjacent-to-host and side-adjacent-to-graft (which separated the claws of the A/P axis reversal laterals) dividesthem into an external half and an internal half, and hence does not separatethe claws. The two claws of a lateral tarsus might be expected to have approxi-mately the same composition, and the dual origin of such a tarsus would notbe obvious.

(ii) Double lateral regenerates

The compositions of the double lateral structures are given in Fig. 6. In allcases the double structure was composed of both host and graft-derivedtissue.

The double laterals regenerating from internal or external positions followingreversal of the A/P axis had one of the component tarsi more or less hostderived and the other more or less graft-derived when their I/E orientationswere in the same plane as those of the graft and host (Figs. 4A, B, C). Whenthe I/E orientations were perpendicular to that of the host and graft, eachcomponent tarsus was composed of both host and graft-derived tissue (on thesides 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

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of one more or less host-derived member and one more or less graft derivedmember, regardless of their point of origin on the circumference and theirorientation (Fig. 4F). In two cases where the I/E orientations were perpendicularto those of the host and graft, component tarsi of the double lateral wereeach composed of both graft and host tissue.

(iii) Completely autonomous regenerates

As implied by the nomenclature, the autonomous regenerates were derivedone 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 theoriginal host level and the point of fusion was host-derived, and that lyingbetween the point of fusion and the original graft level was graft-derived.There were a number of analyzable partially autonomous regenerates developedafter reversal of the A/P axis and having a double lateral element. These werecomposed exactly as the double laterals developing directly from the corre-sponding 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 orless graft-derived and the other more or less host-derived (Fig. 7C, D). Whenthey were orientated perpendicular to the graft and host, each componentwas composed of both graft and host tissue (Fig. 7E).

Composition of structures regenerated from the junction: SummaryAlthough use of the Bl and br mutants did not produce precise boundaries between host-

derived and graft-derived parts of the structures regenerated from the non-congruentjunction, it does allow certain conclusions to be made Lateral regeneration from the originalgraft/host junction or from the junction between autonomous graft-derived and host-derivedpartial regenerates involves both graft and host tissue.

When two separate laterals are produced they are usually of dual origin. This wasespecially obvious following reversal of the A/P axis, where the side adjacent to the hostwas host-derived and the other side was graft-derived. Double laterals formed after reversalof the A/P axis had either one host-derived and one graft-derived component tarsus, or eachof dual origin depending on their orientation. When the I/E axis was reversed, one com-ponent 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 consideringtheir possible mode of origin, and this will be discussed below.

DISCUSSION

The results presented above will be compared with those of similar experi-ments performed on other insects by other workers. The available data willthen be compared with the predictions of three theories which have beensuggested to explain regeneration from a non-congruent junction. A newmodel will be developed for the spatial organization of the leg epidermis, andthis will be used to explain regeneration from the non-congruent junction.

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(A) Number, position and orientation of structuresregenerated from the junction

The results of reversing the A/P or I/E axis at the tibia level of the Blatellaleg agree very well with the results of the same experiment performed onother insects, in that the operation typically provoked the formation of twocomplete regenerates by the 1st or 2nd p.o.m. (Bohn (1965, 1972a) on Leuco-phaea and between Leucophaea and Gromphadorhina; Bulliere (19706) onBlabera; and Lheureux (1970) on the spider Tegenaria). This is in sharpcontrast to the results from the congruent tibial graft/host junction whichtypically does not regenerate any segmented structures (Bohn, 1965, 1967,1970; Bulliere, 1970a, 1971; French, 1976a) although a congruent Blatellajunction often regenerated partial structures if the graft tarsus had beenamputated (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 resultscomparable with those from the tibia level.

There is general agreement that the two separate laterals originate from thepoints of discontinuity between graft and host, and are orientated as shownin Fig. l A ^ 1 ) , B (61). The results from Carausius (Bart, 1971a) have twonotable features: laterals appeared from exactly the points of discontinuity,and there was a relatively high frequency (20/57 cases) of formation of onlya single lateral after reversal of either of the graft axes. Bulliere (19706) andBart (1971a) found fusion between one of the laterals and the regeneratedgraft terminal tarsus (this was observed once in Blatella) but no cases of adouble lateral. Bohn (1965, 1972a) obtained double laterals from a point ofcongruity (external from A/P reversal, and posterior from I/E reversal) aswas 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 Periplanetaafter reversal of both axes by 180° rotation of a homopleural graft. It isnoticeable, however, that Bulliere (from all of whose grafts the tarsus wasremoved) found no complete or partially autonomous regenerates, which weremajor categories of result in Blatella.

(B) Composition of separate lateral regenerates

Bulliere's grafts (19706) were done between the pro- and meta-thoracic legsof 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 thepro-thoracic leg. Laterals regenerated from tibial level could not be identifiedas pro- or meta-thoracic, but Bulliere concluded that laterals regeneratedfrom 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,

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reached the same conclusion using the criterion of one or two claws at thedistal tip. In both of these experimental situations the criteria enable directidentification of only parts of the laterals.

Bohn (1972a) has shown that laterals of dual origin are the usual resultof reversing one transverse axis at either tibia or coxa level. He grafted betweendifferently pigmented species (Leucophaea and Gromphadorhina) and was ableto draw boundaries between host- and graft-derived areas of a lateral. Theboundaries almost always ran longitudinally down the lateral. After reversalof the A/P axis, Bohn found that nearly all lateral regenerates were of dualorigin with the boundaries usually on the internal and external faces of thelateral. 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 withthe composition of the corresponding Blatella laterals (Figs. 2 A, B). Separatelaterals developed from an I/E reversal were less uniform, however, and weresometimes of pure graft or pure host origin (especially when produced atcoxa level). Boundaries on dual origin laterals tended to be external and(in nearly every case) posterior, dividing the lateral into approximately three-quarters and one-quarter. These results correlate well with the tendency ofthe 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 congruitybetween the transverse axes of the graft and host or, in other words, midwaybetween the sites where separate laterals would form. The existence of lateralswhich were double at the base but separated more distally (Fig. 5D) reinforcesthe view that the double laterals result from the secondary fusion of the twosingle laterals regenerating from the areas of incongruity.

Fig. 8 shows the various ways in which the two separate laterals couldfuse, and comparison with Fig. 6 shows that most of the categories of doublelateral can be explained.

Separate laterals formed after reversal of the A/P axis could fuse internallyor externally (accounting for 42/43 of the structures obtained). If the lateralsfuse incompletely in an external position (Fig. SAty1)), each will become oneof the component tarsi of the double structure and the claws will be orientatedaway 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 doublecircumference (Fig. 8A(62)). Claws curve from external towards internal soeach component tarsus of the double structure will be formed from half ofeach of the original laterals. Hence the claws will be orientated towards eachother in the plane of the host I/E axis (14/25 cases, e.g. Fig. 5 A), with oneset of host origin and the other set of graft origin (8/8 cases).

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(A) AjP axis reversal

V. FRENCH

I P E A

(A) (£) (P) (/)

(/?') Incomplete fusion

/ p A I

(c1) Incomplete fusion

E A I

(bz) Complete fusion (c2) Complete fusion

(B) IjE axis reversal P I A E P

(a)

{P) (£) (A) (/) (P)

Fuse posteriorly

E P I A

/ \

{bx) Incomplete fusion

A E P I A

Fuse anteriorly

PI A E P

(c1) Incomplete fusion

P I E P

A

(b2) Complete fusion (c2) Complete fusion

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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 planeof the host A/P axis (13/17 cases) and each of dual origin (9/9 cases). Fusionof the separate laterals hence accounts for the position of 42/43, the orientationsof 33/42, and the composition of 22/22 of the double laterals developed afterA/P axis reversal.

Laterals developed after reversal of the I/E axis may fuse posteriorly oranteriorly (32/42 of the structures obtained). Incomplete fusion of laterals ina posterior position (Fig. 8B(^)) will give claws side-by-side orientated inopposite directions in the plane of the host A/P axis (7/30 cases), as willincomplete fusion of laterals in an anterior position (2/2 cases, Fig. 5D,SB^1)). Complete fusion of the laterals posteriorly (Fig. 8B(&2)) will giveclaws orientated towards each other in a plane between the host A/P andI/E axes (13/30 of the observed double laterals were classified as approximatelyin the plane of the host A/P axis, e.g. Fig. 5C). Composition of these fuseddouble laterals will depend upon the composition of the separate laterals:separate laterals developing after I/E reversal usually appear to be one host-and one graft-derived and this tendency is also seen in the components of thedouble structure. Fusion of the separate laterals hence accounts for the positionof 32/42, and the orientations of 22/32 of the double laterals developed afterI/E axis reversal.

Because of the correspondence between prediction and observation withrespect to the position, orientation and (at least for the A/P reversal) thecomposition of the double laterals they will be assumed to have resulted fromfusion of separate laterals.

FIGURE 8

Fusion of separate laterals regenerated from the graft/host junction after reversalof one transverse axis. Schematic representation of graft and host tibiae splitinternally (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 dashedline. 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 outfrom the junction with host-derived tissue stippled (in A).

(A) Results from reversal of the anterior-posterior axis, (a) Two separatelaterals; (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 fusionto give one double circumference with two sets of claws orientated from externalto internal positions on the double circumference, (c1, c2) Fusion of the two lateralsinternally, (c1) Incomplete fusion of the internal 'faces' of the laterals, (c2) Completefusion.(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 fusionto 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.

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(D) The formation of autonomous and partially autonomous regenerates

It was shown (French, 1976 a) that completely and partially autonomousregeneration could occur from a congruent tibial junction (i.e. both transverseaxes of host and graft in alignment) when healing together of host and graftwas impeded by withdrawal of the graft epidermis from the cuticle, associatedwith the regeneration of an amputated graft tarsus. It was also argued thatrotation of the graft further increased the chance that graft and host wouldnot heal but would regenerate independently (at least initially). It is interestingthat the autonomous categories of regeneration from a non-congruent junctionalso usually occur in conjunction with loss and regeneration of the grafttarsus (Table 3), suggesting that, in this situation also, they result from afailure of graft and host to heal together and interact.

The junction between the two axial elements of a partially autonomousregenerate 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 structuredeveloping after reversal of one transverse axis also behaved just like thecorresponding graft/host junction. It regenerated two separate or a doublefused lateral and, as described, these were orientated and probably composedexactly like the laterals regenerating from the original graft/host junction.

Thus the categories of lateral and autonomous regeneration reflect twodifferent processes:

(i) If interaction occurs between host and graft (or between their developingautonomous regenerates) two laterals of dual origin are produced from thepoints of incongruity of the transverse axes. These laterals may subsequentlyfuse together.

(ii) If the graft and host surfaces do not heal together and interact, eachregenerates independently. In Blatella these autonomous regenerates lie inlinear sequence but in Blabera perhaps they may 'slide' past each other andproject laterally as two 'laterals' of pure graft and pure host origin. Thismay explain why Bulliere (19706) found 'pure origin' laterals and no autono-mous regenerates.

(E) Previous theories of the formation of separate lateral regeneratesfollowing the reversal of one transverse axis

There have been three major suggestions about the cause of lateral re-generation, and to be tenable they must be consistent with the number, position,orientation and composition of the laterals formed after reversal of onetransverse axis and after similar operations.

(i) Lawrence (1970). Regeneration of distal elements of the pattern may beinhibited at all levels by disto-proximal inhibition. This would obviously berelieved after amputation of distal structures, allowing the disinhibited area

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(the cut end) to regenerate. If information flow is not only polarized disto-proximally but also can only occur between cells from similar positions onthe circumference, reversal of a transverse axis will disinhibit two areas ofthe host cut surface, separated by two inhibited areas. This is shown in Fig. 9 Aand would give two laterals of host origin, orientated like the host.

(ii) Bulliere (1970b). Regeneration may be inhibited everywhere on the intactleg by the normal disto-proximal and proximo-distal short-range cellularinteractions. Reversing one transverse axis effectively relieves the inhibitionon the adjacent but non-communicating host and graft surfaces, so that theywill behave independently and each will regenerate (Fig. 9B). The host- andgraft-derived regenerates will grow past each other, retaining contact at acommon region of the circumference and hence will project laterally fromthe 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 butactually to be induced by local confrontation of tissue from opposite facesof the leg. This confrontation occurs when the cut end of an amputated stumpheals over before terminal regeneration, and it occurs at two positions followingreversal of a transverse axis (Fig. 9C). A lateral of dual origin will be inducedat each of the two regions of confrontation, and they will both be orientatedlike the host.

All three theories account satisfactorily for the number, position andorientation of the laterals, but only that of Bart accounts for the fact that theyare 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 determinethe relevant correlation by producing four areas of confrontation. The resultof these experiments will now be given and discussed.

Reversal of both transverse axes

Both transverse axes can be reversed by 180° rotation of a homopleuralgraft, and the results are very variable. At tibial and coxal levels, Bulliere(19706) usually found no laterals or two laterals, and Bohn (1972a) usuallyfound two laterals, often distally incomplete. Blatella usually produces nolaterals or one incomplete lateral (French, 1976a). In all cockroaches thereis a strong tendency for a rotated graft to rotate back into alignment withthe host. Bart (1971a) grafting at coxa level in the stick insect, found almostno tendency for grafts to rotate back. He obtained no laterals (1/63 cases), onelateral (15/63), two laterals (38/63) or three laterals (9/63).

Despite the difference in maximum number of laterals obtained, Bart'sresults share many features with Bulliere's and Bohn's. Laterals could formon 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

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290 V. FRENCH

A. Lawrence (1970)

P\ I : A

ft

P '• E A

(o)

B. Bulliere (1970 b)

P , I , A E . P

P ' E

C.Bart (1971)

E \P

P ' E ' A ' I P

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(19706, fig. 8) all conform to Bart's illustration (1971a, fig. 2) of the possiblepositions and orientations of the laterals.

The theory of Lawrence (Fig. 9 A) predicts one lateral, as the entire hostcut surface would be disinhibited; that of Bulliere (Fig. 9B) predicts onelateral 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 assessthe significance of the cases of three laterals in the stick insect. Bohn (1972#)suggests that two is the maximum possible number directly caused by therotation, and that Bart's triple laterals resulted from secondary wounding.However, Bart never found cases of three laterals developing from the verysimilar operations reversing only one transverse axis. Bohn also points to thefailure to ever obtain four laterals, indicating that it argues against Bart'shypothesis of the establishment of a 'morphogenetic centre' wherever oppositefaces are confronted.

The differences in results between Blabera, Leucophaea, Blatella and Carausiuscould be the reflexion of relatively trivial factors (e.g. size, rate of woundhealing, extent of back rotation), and are unlikely to be due to major differencesof mechanism. It seems unwise to choose a model (Bulliere's) which restrictspossible laterals to two when, even in only one insect, one seventh of operatedlegs bear three.

As Bart argues (1971a), it is likely that lateral regeneration occurs due tosome 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 anothersegment (Bart, 1971 b), or elimination of a part of a femur face (Bart, 1970), donot result in regeneration of the leg tissue thus deprived of its normal neigh-bours. Also, an interaction theory accounts more satisfactorily for most lateralsbeing of dual origin. Such an interaction theory will now be developed andwill 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 outfiat, with the graft/host junction shown by the dashed line. Hatching, area dis-inhibited (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-proximalinhibition cannot act.(B) Regeneration from the host surface (isolated from normal disto-proximalcontacts) 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

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(F) Theory for the initiation of lateral regenerationfrom a non-congruent graft /host junction

So far, in describing the experiments and discussing the results, the leg hasbeen considered to have two 'transverse axes' and four 'faces' (anterior,internal, posterior, external). Bart (1971 a, b) considers the four faces to bedistinct and qualitatively different from each other: opposite qualities caninteract in a unique way to induce a regenerate. After reversal of one 'transverseaxis' 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 theopposite graft 'face': there will be interaction and induction of a regenerateat these two positions.

Bohn (1972a) suggests that 'faces' are nothing more than a 'topographicalcharacterization', and that the cross-section of the limb could be arrangedin a two-dimensional co-ordinate system 'the axes of which coincide with theantero-posterior and dorso-ventral (external-internal) axes'. In this coordinatesystem every cell is different from any other. After reversal of one transverseaxis, only at two points on the circumference will the normal contacts bemade, and the laterals will develop from the two areas of maximum incongruity.

One might further suggest that the two 'transverse axes' are nothing morethan a topographical characterization. There is no evidence that the anterior-posterior and internal-external planes are of any particular significance to thecockroach leg with respect to epidermal pattern or regeneration.

It is very likely that lateral regeneration is due solely to interactions occurringwithin the epidermis, since it can be provoked by grafts of epidermis notinvolving the translocation of any muscle or the sectioning of any major nerve(Bart, 1966, 1971a; Bohn, 19726; French & Bulliere, 1975a, b). The legepidermis, being a single celled layer, is effectively two dimensional; the surfaceof a cylinder upon which any position may be specified, not in relation tothree axes, but by the two co-ordinates of proximal-distal level and circum-ferential position (Bulliere, 1971; French & Bulliere, 1975a, b).

FIGURE 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 thecircumference 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 thegraft (B) and anterior view (C). An internal face (r) is regenerated between positions4 of the graft (g) and 8 of the host (h), and a part of the anterior face (r) is re-generated between positions 8 of the graft (g) and 9 of the host (/?). At the endsof the graft, intercalary regenerates (r') are formed between graft and host, andbetween regenerate (r) and host, to remove all discontinuities of position.

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293

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Bohn (19726) showed that circumferential position is not irreversibly deter-mined since, in the course of lateral or terminal regeneration, a quarter ofthe circumference of the tibia often gave rise to more than the correspondingquarter of the tarsal circumference. French and Bulliere (1975a, b) showedthat circumferential position (la generatrice) is an aspect of position independentof proximal-distal level and is specified in a continuous manner around theleg epidermis, rather than along two perpendicular axes going through theleg. By grafting a rectangular piece of cuticle plus epidermis to an abnormalposition around the circumference, we confronted non-homologous positionsalong the lateral edges of the graft. In all such experiments (French, 1976 c)there was an intercalary regeneration of the structures which normally separatehost and graft positions, as measured by the shortest route around the circum-ference (Fig. 10 A, B). At the proximal and distal ends of the graft, differentcircumferential positions were placed adjacent to each other in a proximal-distal sense, and there was an identical intercalary regeneration of circum-ferential values to restore normal cellular contacts (Fig. IOC).

These experiments detected no 'boundary positions' having unique propertieswhich could correspond to a boundary between 'high' and 'low' values ofa circumferential 'gradient' analogous to the postulated proximal-distal seg-ment 'gradient' (Bohn, 1967). The circumferential position 12/0 in Fig. 10does not imply a boundary; it arises inevitably when labelling a circle (e.g.a clock face) with numbers. Possible mechanisms for smoothly specifyingposition 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 definedby its level within a linear proximal-distal ordering, and its position withina circular circumferential ordering. Intercalary regeneration occurs betweendifferent 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 nolaterals (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 andexternal 'faces'. Twelve positions are marked around the circumference by numbers0-12 (as in Fig. 10) and numbers between the circles are the positions formed byintercalary regeneration (by the shortest route) between the different confrontedpositions of host and graft. Where the shortest route is different on the two sidesof a point, the two halves of a complete circumference are formed, confrontingeach other; these are drawn spread apart to show the predicted orientation andcomposition (in A and B) of the regenerate forming at that point. Claws curve fromexternal to internal positions on the laterally formed circumference, and stipplingdenotes host-derived tissue.

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A 9

A9

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It will now be shown that lateral regeneration is initiated at a non-congruentgraft/host junction as a direct result of the spatial organization of theepidermis.

Reversal of one 'transverse axis'

Consider the situation at the tibia graft/host junction following a graftbetween left and right legs. Fig. 11A shows the A/P axis reversal and theintercalary regeneration which will occur between the confronted graft andhost positions, always forming the intervening positions, as measured by theshortest route. It will be seen that the shortest route between confronted graftand host positions is different on the two sides of each point of maximumincongruity (A and P), hence at these points the two halves of a completecircumference are formed, confronting each other. Fig. 11B shows the I/Eaxis reversal, forming the two halves of a circumference both internally andexternally.

Consider the confrontation of the two half-circumferences formed at eachpoint of maximum incongruity after the A/P axis (Fig. 11 A). The confrontations10-8, 11-7, etc., will result in intercalary regeneration of the interveningpositions, as measured by the shortest route. The shortest route is differenton the two sides of the confrontation 12/0-6, hence at this point the twohalves of a complete circumference will again be formed, confronting eachother. Thus a round of intercalary regeneration produces new tissue (whichwill bulge laterally from the junction) but just recreates the confrontation ofhalf circumferences.

After an amputation the epidermis from the circumference at the end ofthe stump migrates under the clot of dried haemolymph to re-establish epidermalcontinuity, resulting in the confrontation of cells from different circumferentialpositions. This situation can be simplified by considering epidermal migrationto occur only in one plane, resulting in the confrontation of two half-circum-ferences which, exactly as in the lateral situation considered above, wouldbe perpetuated by intercalary regeneration. The two half circumferencesformed at each point of maximum incongruity after reversal of a transverseaxis, and the circumference left after an amputation, are both cases wherea complete circumference heals over because it is not sandwiched (as it is inthe intact leg) between other circumferences overlying it proximally anddistally. In these situations regeneration of distal structures occurs. Perhapsthe tissue of a regeneration blastema is produced by successive rounds ofintercalary regeneration between the different positions confronted by a circum-ference 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 theblastema, the more distal levels of the leg must be specified. It is interestingthat a bilaterally symmetrical, partial circumference, which could heal overand completely resolve confrontations between non-homologous positions by

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intercalary regeneration, often does not regenerate distal structures or formsdistally incomplete partial regenerates (Bohn, 1965; French, 1976a).

Thus each of the two complete circumferences formed at the junction afteran A/P or I/E 'axis' reversal will be the origin of a lateral regenerate. Bothlaterals will be orientated in the I/E axis like the host (I/E reversal) or like thehost and graft (A/P reversal). Assuming that both components contribute tothe intercalary regeneration which creates the complete circumference, thelaterals will each be composed of host-derived and graft-derived longitudinalhalves.

Reversal of both 'axes'1 by 180° rotation

After 180° rotation of a homopleural graft each host position around thegraft/host junction will be confronted by the opposite graft position. A con-frontation between opposite positions may be resolved by intercalary re-generation of either of the half-circumferences which separate them (French,1976 c). This can be visualized as going from the host position around acircumference in a clockwise or anticlockwise 'direction' to the opposite graftposition. If intercalary regeneration occurs in the same 'direction' everywhereon the junction, no lateral regenerates will be formed (Fig. 11C1). Any tendencyof the graft to rotate back into alignment will increase the probability that nolateral will be formed by creating an unambiguous 'shortest route' in thesame 'direction' at all positions on the junction. If, by chance, one sector ofthe junction forms the intercalary regenerate in one 'direction' while theremainder of the circumference of the junction regenerates in the opposite'direction', a complete circumference will be created at each boundary betweenthe two regions, and hence two laterals may be formed. This may occur any-where on the circumference, forming two laterals situated opposite or adjacentto each other. One left-handed and one right-handed lateral will be formed.Consider the possible positions and orientations of these laterals. Consideringonly the I/E axis, a lateral will be orientated like the host if it is formedinternally or externally (Fig. 11C2, C3), but either like the host or like thegraft it formed anteriorly or posteriorly (Fig. 11C2, C3, C4). If one lateral isanterior and the other posterior, they will have opposite orientations (Fig. 11C4).These predictions, concerning handedness, position and orientation, whichare not made by earlier models, are in good agreement with the data ofBulliere, (1910b) Bart (1971a) and Bohn (1972a).

More than two laterals may be formed if the 'direction' of intercalaryregeneration in two sectors is opposed by that in the two sectors separatingthem. A complete circumference will be created at the four boundaries, makingit possible for four laterals to form. Bart (1971a) found three laterals in 9/63cases but no cases where four were formed. This is perhaps not surprising sinceanalysis 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

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to the formation of a lateral. If cases of four changes of 'direction' arounda single junction occur fairly infrequently (two changes being the usualsituation) and if each circumference which forms at the site of such a changehas only a 0-7 probability of leading to regeneration of a lateral, two lateralswill be the most frequent result of the 180° rotation experiment and theincidence of three laterals will be much higher than that of the maximum,four.

It remains unclear why the laterals formed after 180° rotation were oftendistally incomplete (Bart, 1971a; Bohn, 1972a). It was argued (French, 1976a)that a distally incomplete lateral could arise from a sector of a congruentjunction 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 ofhealing at a position which is not a boundary between different 'directions' ofintercalary regeneration.

Composition of the lateral regenerates

It has been assumed so far that position is specified smoothly and con-tinuously around the leg, with no positions having unique properties and thatall confrontations result in intercalary regeneration by the 'shortest route'.This is consistent with the results of French & Bulliere (1975 a, b) and is thebasis of the model for initiation of lateral regeneration. The model is consistentwith the number, position and orientation of laterals formed after reversal ofone transverse axis and also explains the main features of regeneration after180° rotation of a homopleural graft.

It has further been assumed in Figs. 6, 8 and 11 that the intercalary re-generation results from proliferation of both of the confronted regions creatinga complete circumference of dual origin, leading to a lateral regenerate com-posed of host-derived and graft-derived longitudinal halves. This assumes thatthere are no restrictions with respect to the circumferential position whichthe progeny of a cell can occupy. This assumption seems to be supported bythe results of A/P axis reversal, where the laterals were usually of dual originwith borders between host- and graft-derived parts running approximatelymid-external and mid-internal, as predicted (see Results, and Bohn, 1972a).After I/E reversal, however, the separate Blatella laterals were often largelyhost-derived or graft-derived, and Bohn's interspecies grafts (1972 a) showedthat dual origin laterals had one border on the posterior side (predicted) butthe other border internal or external (not predicted). This suggests that ananterior or posterior region can produce tissue more internal and more external,but that an internal or external region may not always be able to producetissue both anterior and posterior to it.

In this connexion it is intriguing that, after grafting an external sector ofthe tibia of Gromphadorhina to an internal position of the Leucophaea tibia,Bohn (19726) found that the graft contributed more than one-quarter of the

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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 internalportions of the tarsi, but not more posterior portions.

Clonal analysis has shown that the epidermal cells of the developingappendages 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 boundaryhas formed do not cross it in normal development. Even if they are able todivide more rapidly than the rest of the tissue and nearly fill the compartmentin which they arise, cells of the clone will not cross the compartment boundary.The disc seems to be divided into anterior and posterior compartments assoon as it is segregated from the rest of the blastoderm, but thereaftercompartment boundaries form between dorsal and ventral, and wing andnotum (during the 1st larval instar), between regions of the notum (during2nd instar) and between proximal and distal wing (during 3rd instar). Duringregeneration from fragments of a disc, however, cells may cross the latercompartment 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 compartmentswhich were respected during regeneration, with the compartment boundariesapproximately mid-internal and mid-external, this might account for thedifferences in composition between laterals resulting from A/P and I/E reversals.Experiments are in progress to explore this possibility.

(F) Comparison between lateral regenerationin insect and amphibian legs

When newt leg regeneration blastemas are grafted on to stumps in sucha way as to reverse the A/P or dorsal/ventral axis (D/V) or both, supernumeraryregenerates develop just as in cockroaches (Iten & Bryant, 1975; Bryant &Iten, 1976). Reversal of A/P or D/V axis gives two laterals originating fromanterior and posterior, or from dorsal and ventral respectively, orientated likethe stump. After reversal of both axes, one right and one left-handed regenerateare formed.

An interpretation of these newt laterals which is very similar to the presentmodel of lateral regeneration in cockroach legs has been developed by Bryant& Iten (1976). It is suggested that position is continuously specified aroundthe circumference of the limb, that confrontation of non-homologous positionsresults in intercalary regeneration by the shortest route, and that regenerationoccurs from the complete circumference generated wherever the 'direction' ofthe shortest route changes over.

The similarities between insect and amphibian limbs will be developedelsewhere (French, Bryant & Bryant, in preparation), but the occurrence of

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similar lateral regeneration suggests that these anatomically quite differentsystems may be organized in fundamentally the same way.

This work was financed by an S.R.C. Postgraduate Studentship and a Royal SocietyEuropean Exchange Research Fellowship. I thank Professor Sengel for the hospitality ofhis laboratory in Grenoble; and Professor MacGregor of the Department of Zoology,University of Leicester, for generously providing facilities for the preparation of thismanuscript.

I am deeply indebted to Gerry Webster for preserving the sanity and guiding the effortsof his postgraduate student, and for advice on this manuscript.

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{Received 19 September 1975, revised 15 December 1975)