naturally occurring abnormalities (bruchdreifachbildungen) in the … · embryol. exp. morph. vol....

/. Embryol. exp. Morph. Vol. 63, pp. 285-304, 1981 285Printed in Great Britain © Company of Biologists Limited 1981

Naturally occurring abnormalities(Bruchdreifachbildungen) in the chelae of threespecies of Crustacea (Decapoda) and a possible

explanation

By P. M. J. SHELTON,1 P. R. TRUBY1 AND R. G. J. SHELTON2

From the Department of Zoology, University of Leicesterand the Marine Laboratory, Aberdeen

SUMMARYNaturally occurring abnormalities (Bruchdreifachbildungen) in decapod crustacean

appendages are described. They are similar to the range of structures experimentallyproduced by cutting notches in the sides of insect legs (Bohn, 1965). It is argued that theyresult from failure of wounds to heal. Regeneration from a free surface along the proximo-distal axis is always in a distal direction. Surfaces regenerating circumferentially can re-generate in either direction around the circumference. Regeneration will proceed until thetwo surfaces of the wound meet. Then, where the two surfaces on either side are non-congruent, intervening tissues will be intercalated. This explanation accounts for the rangeof structures observed after notching experiments (Bohn, 1965) and seen in crustaceanBruchdreifachbildungen. The explanation says that regeneration will occur wherever woundsfail to heal. This avoids the difficulties of the complete circle rule (French, Bryant & Bryant,1976) and explains why appendages with mirror-image symmetry are often capable ofregeneration.

INTRODUCTION

From time to time, fishermen capture lobsters and crabs with curious,mirror-image symmetrical lateral outgrowths from their chelae and occasionallyfrom other appendages. Such abnormalities occur in a variety of animals,including crustaceans, insects, amphibians, birds and mammals (Przibram,1921). They have been called' Bruchdreifachbildungen' because they are thoughtto result from damage to the original structure and they produce a triplicationdistal to the site of injury (Przibram, 1921). In this paper we describe a collectionof abnormal chelae which has been assembled over a period of at least 43years by the Marine Laboratory, Aberdeen, and provide a possible explanationfor the phenomenon. The deformities resemble the lateral outgrowths some-

1 Author's address: Department of Zoology, School of Biological Sciences, AdrianBuilding, University of Leicester, Leicester LEI 7RH, U.K.

2 Author's address: Marine Laboratory, P.O. Box 101, Victoria Road, Aberdeen AB9 8DB,U.K.

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I to oo

288 P. M. J. SHELTON, P. R. TRUBY AND R. G. J. SHELTON

times found in cockroach limbs after deep notches have been cut into theirsides during post-embryonic growth (Bonn, 1965). They take various formsfrom a small bump to pairs of extra segments. They often exhibit the pheno-menon of distal expansion, which has been noted previously in experimentallyformed double-posterior amphibian limbs (Slack, 1977, 1980a) and which isalso seen in some of the lateral regenerates of insect legs (Bohn, 1965). Thefact that crustacean limbs regenerate so readily after autotomy (see Bliss, 1960;Paul, 1914) and that they can respond locally to produce abnormal lateralsupernumeraries suggests that the crustacean limb might offer a valuable newsystem for studying pattern formation. Indeed preliminary reports suggestthat a gradient system, similar to that found in insects, may determine proximo-distal organisation of the crayfish leg (Mittenthal, 1978).

We have now examined 11 decapod crustacean limbs showing variousabnormalities. They include specimens from the lobster Homarus gammarus(L.), the edible crab Cancer pagurus L. and the Norway lobster Nephropsnorvegicus (L.). Together with some of the specimens originally described byBateson (1894) and Przibram (1921) there is now a sufficient range of examplesto permit a useful discussion of the cause of the phenomenon. The fact thatBruchdreifachbildungen occur in such a wide variety of animals suggests thatour findings may be of some general significance.

OBSERVATIONS

All the outgrowths described in this paper are on the terminal or thepenultimate segments of the chela (the dactylopodite and the propodite). How-ever, although this is the most common site for the abnormality, similar out-growths can occur on antennae (Przibram, 1921, specimen nos 13 a, 136), andwalking legs (Bateson, 1894, specimen no. 808). They can be derived from thecarpopodite (Przibram, 1921, specimen no. 16), the meropodite (Bateson, 1894,specimen no. 826; Przibram, 1921, specimen no. 37), the basipodite (Bateson,1894, specimen no. 808) and the coxopodite (Przibram, 1921, specimen no. 23).There is a natural dimorphism of chelae in decapods (Emmel, 1908) in whichone chela is adapted for 'cutting' and the other for 'crushing'. We foundexamples in both cutters and crushers. We adopted the following conventionto identify the origin of the lateral supernumeraries. The chela is flattenedlaterally and we call the surface which faces the contralateral chela the internalface. The outward-facing opposite side is called the external face. The propoditeextends ventrally as the index (or propopodite extension) to make the lowerjaw of the claw structure. The dactylopodite forms the moveable dorsal elementof the claw. Details of the various limbs and their abnormalities includingrelevant examples from Bateson (1894) and Przibram (1921) are tabulated(Table 1) and representative examples are illustrated diagrammatically (Fig. 1).Where appropriate, we have amplified this description with photographs.

Bruchdreifachbildungen in crustaceans 289

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Fig. 2. External (left) and internal (right) views of specimen no. 2 showing theorigin of the lateral outgrowth. Light coloured featureless cuticle at its baseprobably indicates the extent of the original damage. The mid-lateral ridges andadjacent cuticle dorsal to them are undisturbed. The knobs at the dactylopodite/propodite joint are typical of the dorsal part of the limb.

Although not all lateral outgrowths have the same structures, there areseveral features that most have in common:

(1) All but four of the outgrowths are symmetrical for at least part of theirlength (Fig. 1). Often the symmetry takes the form of a pair of mirror-imagestructures which may be fused (e.g. nos 1-4) or separate (no. 7) at the base.Exceptions to this are: no. 9, where the outgrowth is neither symmetrical nordivided and is in the form of a bump; no. 11, which is a curly spike lackingclear cuticular markers and thus of uncertain symmetry; no. 10, which hastwo lateral outgrowths, one narrow and featureless and one divided at theend to form a small pair of mirror image dactylopodite ends.

(2) Where features can be identified on the outgrowths they are alwaysones that lie distal to the level of the limb from which the outgrowth originates.This can include joints and more distal limb segments (nos. 2, 12, 15, Table 1,Fig. 1).

(3) Where an outgrowth is proximally fused, the base of the structure only


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Fig. 3. Ventral view of the dactylopodite of specimen no. 5. It shows severalfeatures typical of Bruchdreifachbildungen. The more proximal of the lateraloutgrowths (p) is longer than the other (d). Not all circumferential levels arerepresented at the base of the structure. Thus, teeth appear only just below thebifurcation level. On the distal side of the outgrowth there is a pattern discontinuityin the form of a bump (arrow), but on the proximal side the outgrowth emergessmoothly from the dactylopodite. Bar = 10 cm.Fig. 4. Ventral view of the distal side of the bump of specimen no. 9. It shows adistortion of the tooth row associated with the polarity reversal on the distal sideof the outgrowth, b, bump; d, dactylopodite. Bar = 50 mm.Figs. 5, 6. Outgrowth on specimen no. 9 at two moult stages. The same pattern ofteeth (arrows) is recognizable in the cast (Fig. 5) and in the living specimen (Fig. 6).The general appearance of the outgrowth is virtually unchanged from moult tomoult. Bars = 5-0 mm.


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Fig. 7. Regions of the limb cylinder can be arbitrarily defined in terms of circum-ferential and proximodistal coordinates (position values) (A) (see French et al.1976). Different shaped notches (B, C) can expose edges in which either one orboth sets of position values are arranged serially. A transverse cut exposes serialcircumferential values only (D). Such a surface will always regenerate distallyirrespective of stump polarity (D, E). A cut parallel to the proximodistal axis (F)exposes serial proximodistal values. It is proposed that regeneration of circum-ferential values may be in either direction around the circumference (G, H).

includes features from the same side of the circumference as that from whichit originates. This is particularly clear in no. 2, where the knobs around thepropodite/dactylopodite joint of the outgrowth are typical of the dorsal partof the limb (Fig. 2). In other cases, where there are no clear markers at thesite of origin, structures characteristic of other parts of the circumference arenever represented at the base of the outgrowth. For instance, teeth, which arefound on the side of the limb furthest from the outgrowth (e.g. nos. 3 and 4),are not present in the basal region of the structure. More distal regions ofsymmetrical outgrowths include features from further round the circumferenceso that at the bifurcation point where the outgrowth divides into two, twocomplete circumferences occur (Figs. 1, 3). We refer to this situation asdistal expansion after Slack (1980«).


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Fig. 8. A bump may be explained with reference to the proximodistal axis only.Proximodistal coordinates showing the position of the notch (A). The cut surfacesat the top of the notch are held apart (B). Distal regeneration occurs from bothfree cut surfaces (C). Regenerated tissues meet and fuse (D). When non-congruentvalues i and q meet, intercalation of intermediate values occurs to produce abump (E). Note that the values m to q are represented three times, the centre setbeing a mirror-image of each of the outer sets.

(4) Outgrowths can occur on many points of the chelae, including the carpo-podite (no. 12), the propodite (e.g. no. 2) and the dactylopodite (e.g. no. 4).Jn some instances the outgrowth arises from the joint at the base of thedactylopodite (nos. 6, 7). From these observations and the previous datashowing that similar supernumeraries have been recorded on walking legs andvirtually all limb segments (we have been unable to find an example of an out-growth on the ischiopodite) it appears that they can arise from any point on theproximo-distal axis of a limb including the intersegmental membranes of joints.

(5) Outgrowths can occur on the internal (nos. 1, 5), external (no. 10),dorsal (nos. 2, 4) and ventral (nos. 3, 8) faces of the limb.

(6) The bifurcation point can occur at different levels along the proximo-distal axis of the outgrowth. It may be at the base with little (no. 14) or no(no. 7) fusion, or it may be in the next segment up the series. Thus in onecase (no. 13) the origin of the structure is the meropodite, the carpopodite isfused and the two elements are separate only at the level of the carpopodite/propodite joint. Small outgrowths consist of bumps (no. 9) or spikes (no. 11)and are undivided.


Fig. 9. Production of two sets of circumferential values in the side of the limb aftera deep notch. The diagram shows the outline of the notch as seen from above withintercalation across the middle and free regeneration (arrows) to produce two setsof circumferential values. This will produce two complete lateral regenerateswithout fusion. Shaded area = open wound.

(7) The distal side of the base of the outgrowth often has a pattern dis-continuity where it meets the main part of the limb. If there is a clear patternat this point, it is symmetrical about this discontinuity. For instance, there isa ' V pattern in the row of teeth in no. 9 (Fig. 4). More often it is simplya bump or groove in the cuticle (nos. 4, 5, Figs. 1, 3).

(8) The proximal side of the outgrowth is often longer than the distal sideand this results in the outgrowth pointing distally (no. 3).

(9) Two specimens with lateral outgrowths moulted while in captivity. Thenew cuticle of no. 10 is a little damaged so that the tip of the internal lateralsupernumerary is broken and lost. This was the most interesting feature ofthe limb because it was formed into a pair of mirror-image dactylopodite tips(Fig. 1). Nevertheless, comparison of the structure before and after ecdysissuggests that other aspects of the pattern are the same from moult to moult.This is confirmed by no. 9 which is still alive and has moulted in captivity.Here the abnormality is in the form of a bump on the ventral edge of thedactylopodite (Figs. 1, 5). The shape of the structure is almost the same inthe cast and in the living specimens, and the tooth pattern remains unchanged

Fig. 10. A notch seen from above cutting the proximodistal axis at a shallowangle. Free regeneration (solid arrows) of values in the reverse direction aroundthe circumference proceeds to a point where, after fusion, intercalation (dottedarrows) produces two sets of circumferential values. The resulting structure willbe fused at the base and will bifurcate at a more distal level. Dashed lines in D joinpoints having the same circumferential values. (E) Plan view of the resultingstructure as it would appear from the side.


(Figs. 4, 5). From the fact that decapods can regenerate a complete limbwithin a moult cycle (Bliss, 1960) it seems likely that the lateral supernumerariesare formed within a single moult cycle. Our observations suggest that there-after the major pattern elements remain stable.

DISCUSSION

As the claws were all recovered from wild specimens the cause of thedeformities remains uncertain. They are unlikely to be due to a genetic abnor-mality in the pattern forming mechanism, because that would probably resultin much more widespread deformities. Damage during embryogenesis ordamage to a limb bud reforming after a limb had been autotomized wouldalso tend to produce more widespread effects. Much more likely is damageto the limb during fighting, possibly while the limb is still soft after moulting,followed by regeneration. Shortly after moulting the cuticle of H. gammarusis very brittle. Squeezing a limb with forceps at this stage causes splits in theexoskeleton and the damaged region can be pulled away easily (Truby, un-published observations). When two decapods fight they often grasp each otherby the chelae. It is easy to imagine how such behaviour could produce localizeddamage to the claws or their intersegmental membranes. Support for thishypothesis comes from the observation that the abnormalities have been foundfound mainly on the chelae and that most cases involve outgrowths on theclaws or preceding segment. It is, of course, possible that such abnormalitiesoccur with similar frequency on more proximal segments but that majordeformities there increase the likelihood of autotomy.

The idea that damage causes the outgrowths is supported by the views ofother workers. Huxley (1884) reported lateral outgrowths on crayfish limbsand suggested that they are due to regeneration following limb damage innewly moulted animals. Bodenstein (1953) concluded that naturally occurringtriple leg structures in insects are caused by a distal portion of the leg beingpartially broken off followed by distal regeneration from both the proximaland distal wound surfaces. Similarly, Przibram (1921) concluded that Bruch-dreifachbildungen, similar to the ones we have described, are caused by re-generation from both the proximal and distal sides of a wound in the side ofthe limb. There is now direct evidence from experiments with cockroaches tosupport the general principle that lateral regenerates are caused by wounding(Bohn, 1965). Bohn cut V-shaped notches in the ventral and dorsal faces oftibiae of Leucophaea maderae (Fabr.). Although the wound normally healedperfectly or left only a small bump, some of the ventral notches gave a rangeof Bruchdreifachbildungen of the same type that we have described forCrustacea. These may include raised bumps at the site of the injury, mirror-image lateral outgrowths of tibial tissue, similar structures but also includingthe tibia/tarsal joint and some tarsal structures terminally. In some cases


regeneration resulted in the formation of all proximodistal levels including thelimb tip. In such structures there is a range of types from those where there isa bifurcation at the tip to form two separate sets of tarsal claws to those wherethe bifurcation point is at the base of the structure before the first joint. Thedorsal notches never produced more than short outgrowths of tibial tissue.This difference between the effects of ventral and dorsal wounding may notreflect a physiological difference between cells at the two sites for the followingreasons. First, there are reported cases of naturally occurring dorsal outgrowthsfrom insect legs showing regeneration as far as the limb tip (Przibram, 1921,specimen nos. 173a, b). Second, in the crustaceans, which are probably builtaccording to similar rules, complete outgrowths can occur at any point onthe circumference (see above). It will be shown that our explanation for thephenomenon depends on failure of wounds to heal. Differences in the curvatureof the limb surface may well affect this process. We think that differences inregeneration behaviour at different points on the insect leg may be due tosome mechanical factor. It is important to establish this point because, accordingto a polar coordinate model (French et al. 1976), one would expect similartypes of cellular behaviour at all points on the circumference. If, however,cells are specified with reference to a Cartesian system of coordinates, onemight expect cells on opposite sides of the limb to exhibit different types ofbehaviour. For instance, they may be able to regenerate only in one directionalong a particular axis (see for instance Slack, 1980a, b).

Another special case of 'wounding' giving rise to pattern duplications ortriplications is known in Drosophila imaginal discs. Here, temperature-sensitivecell-lethal mutants can be used to produce localized cell death in the discs(Girton & Russell, 1980). Following such damage to the leg discs, structuresremarkably similar to those described by Bohn (1965) for the cockroach havebeen produced in Drosophila (see Girton & Bryant, 1980).

Regarding the general problem of the mechanism generating Bruchdreifach-bildungen, there has been no really satisfactory explanation. Why are theresults so variable and why does a notch cause the phenomenon infrequently?There have been numerous studies on the insect leg and from, them certainconsistent facts emerge. First, when tissues from different proximodistal levelsare recombined, tissues normally separating those levels are regenerated byintercalation. Cells forming the regenerate are derived from both proximal anddistal faces (Bohn, 1976). A similar pattern of intercalary regeneration followswhen tissues from different points around the circumference are confronted(French, 1978). Intercalation in that instance is by the shortest possible route(French, 1978) so that a slightly damaged limb repairs itself rather thanregenerates a mirror-image copy. These findings have led to the formulationof the 'clockface' or polar coordinate model for explaining distal regeneration(French et al. 1976). Complete distal regeneration occurs when a completecircumference is exposed or can be formed by intercalary regeneration from


sections of the circumference. According to the formal model (French et al.1976) distal transformation occurs only when these conditions are met. How-ever, distal regeneration can occur in other circumstances. In insects, experi-mentally created mirror-image symmetrical lateral limb outgrowths are capableof some regeneration (French, 1976a). In amphibians, mirror-image symmetricallimbs show varying abilities to regenerate. In the axolotl, double-posteriorlimbs give complete distal regeneration when amputated (Slack & Savage,1978). In the newt Notophthahnus viridescens, amputated double-half limbsshow partial regeneration. Here the distal regeneration is elicited by confront-ation of non-congruent circumferential values (Bryant & Baca, 1978). In thiscase only a few rounds of intercalation are necessary to resolve all patterndiscontinuities. However, Bryant & Baca (1978) still maintain that a completecircumference is necessary for total distal regeneration. In insects, after tele-scoping experiments where congruent proximodistal tibial levels are combined,lateral regenerates can form if there is partial failure of the host/graft junctionto heal (French, 1976a). Our interpretation of all these phenomena is thatregeneration follows wherever wounds fail to heal. Where a complete circleforms, healing is effectively prevented by intercalation across the clockface andthe production of an unresolvable point at the limb tip (French, 1976a).Healing is known to be inhibited when cells from different positions of anaxis are confronted in insect grafting experiments (Niibler-Jung, 1977). Thiscan lead to rounding up or even rejection of the graft. According to our hypo-thesis, amputated mirror-image symmetrical limbs regenerate because mech-anical factors prevent the exposed parts of the circumference from coming to-gether. To explain Bruchdreifachbildungen it is necessary to assume thatregeneration occurs when wounds fail to heal and that, where complete circlesare formed, distal regeneration will continue until terminal structures haveformed. The clockface model by itself fails to explain the range of structuresformed after notching (Bohn, 1965). It predicts only two outcomes. When thenotch is shallow the limb should repair itself, when it is deep two completelyseparate laterals should be produced with no fusion at the base. In the caseswe have described, there is only one instance (no. 7) where the two super-numeraries are separate. In the others, the bifurcation point is a considerabledistance from the base. This was also true of Bohn's (1965) examples in thecockroach. In addition, examination of some of our specimens shows that theoriginal damage was localized to much less than half of the circumference.A clear case is no. 2 (no. 1 is also derived from a small wound). This shows asymmetrical propodite/dactylopodite joint and a mirror-image pair of dactylo-podites arising from the dorsal side of the propodite. The base of the out-growth occupies no more than 20 % of the circumference of the main limb axis.In addition, the raised mid-lateral ridges which occur on the internal andexternal faces are undisturbed (Fig. 2). The base of the regenerate, whichshows mirror-image symmetry, consists of tissues normally occurring in the


dorsal third of the limb. This argues that notches extending much less thanhalf-way round the circumference can produce lateral regenerates. Accordingto the clockface model, damage to a small region of the circumference couldcause laterals to form if positional values are not evenly distributed aroundthe circumference. Local damage at a site where the values are clusteredcould expose more than half the circumferential levels. However, in that casethe supernumeraries should be separate at the base (see below). In all thecases but one (no. 7), that we have described, the laterals are fused at thebase. Finally, the clockface model does not explain how extra circumferentialpositional values are intercalated to cause the common phenomenon of distalexpansion.

Conditions necessary for the production of Bruchdreifachbildungen

Except in the case of the production of two completely separate mirror-image laterals it is necessary to assume that the wound remains open afternotching. Although the notch may be sealed with a clot of haemolymph, theedges of the wound may remain apart for a considerable period of time afterinjury. Where physical contact of the two sides of confronted tissues fails tooccur, each side may regenerate independently of the other. When proximaland distal levels of a cockroach limb are telescoped together, both faces usuallycontribute to the intercalary regenerate, with the distal face forming moreproximal levels and the proximal face forming more distal levels (Bohn, 1976;French, 1976a). However, if the two cut surfaces fail to establish cellularcontact, both surfaces behave like distally amputated limbs and, even withcongruent grafts, the proximal face completes the limb and the distal faceregenerates a mirror-image (along the proximodistal axis) of the original graft(Bohn, 1965; French, 1976a). Thus, although distal regions can give rise tomore proximal ones, when the surface is free, a cut surface always regeneratesmore distal parts of the proximodistal axis. There is no evidence concerning thedirection of regeneration around the circumference when a particular circumfer-ential level is exposed and prevented from joining the other side of the wound.However, we believe that it can regenerate and that it can regenerate in eitherdirection around the circumference. Only by making these assumptions canall Bruchdreifachbildungen be explained. According to the shape of the notchand position of cells on the cut edge, the exposed faces may behave like distallyamputated surfaces or exposed parts of the circumference. If the cells alongthe cut surface have serial circumferential values then regeneration will producemore distal structures. If they have serial proximodistal values then regenerationwill result in new circumferential values being formed (Fig. 7). In a situationwhere the surface is at an angle to both axes, they will both have serial values.Therefore regeneration will proceed along both axes. In the following account,for simplicity, we have considered only regeneration along one of the twoaxes. Thus for a steep-sided notch we have considered regeneration along the


proximodistal axis and for notches with a shallower angle we have consideredregeneration around the circumference. Later we will describe what happenswhen regeneration along both axes is considered at the same time.

The production of bumps or spikes undivided at the tip

Spikes or bumps are likely to arise where the two sides of the notch areat an acute angle to one another so that each surface behaves like a distallyamputated limb. In this case we will consider regeneration along the proximo-distal axis only. The proximal and distal faces of a notch may fail to fuse afternotching because of the mechanical factors involved. For instance the remainingintact side of the limb will sometimes hold the two edges apart. Following therule that a free cut end always regenerates distally (see above), both proximaland distal sides of the wound will regenerate in a distal direction. Since thetwo faces of the notch confront each other the regenerating faces will eventuallymeet (Figs 7 & 8). At the base of the notch the two faces will meet almostimmediately. Consequently there will be no great discrepancy of proximo-distal values at the fusion point. Further out along the sides of the notch,regeneration will proceed a considerable way until the two sides meet. Thisresults in the confrontation of tissues from significantly different proximo-distal levels. Intercalation of intervening values will follow to produce a stablebump (Fig. 8) or spike (specimen no. 11) which would persist unchanged frommoult to moult as observed in specimen no. 9 (Figs. 5, 6). Clearly the natureand size of the bump would depend on the geometry of the original notch.Observable features of our specimens are consistent with this explanation.Distal to the base of the outgrowth there is often a pattern discontinuity inthe form of a groove (no. 4) or a bump (Fig. 3) while on the proximal sidethe junction between the main limb and the sidegrowth is not visible. This isconsistent with distal regeneration from the two sides of the notch whichproduces a polarity reversal on the distal side but not on the proximal side(Fig. 4). We have noted that the lateral outgrowths often point distally (speci-men no. 11 and many of the mirror-image divided structures) (Fig. 1). Thisfollows from our explanation because the proximal side of the wound has toform more distal levels before reaching the tip than the distal side.

A feature of the theory is that it does not necessarily involve considerationof the circumferential positional values. Assuming that each side of the notchregenerates independently until fusion, we expect the newly regenerated cellsto derive their circumferential values from the free cut surface just as theydo when a distal amputation occurs. When the two sides of the wound meetduring regeneration the circumferential values should be approximately inregister.


The production of two complete lateral regenerates without fusion

The most complete mirror-image laterals, unjoined at the base and consistingof all levels distal to the wound site, require a different explanation. It is thatthe notch penetrated more than halfway through the appendage. In this casewe do not believe that opposite sides of the wound are necessarily held apart.Fusion of the two lateral faces, where they are closest together at the base ofthe notch, results in the intercalation of intervening circumferential values bythe shortest possible route across the middle of the damaged aiea. This createstwo sets of circumferential values on the side of the limb (Fig. 9). The woundwill effectively remain unhealed at the centre of each set because of theunresolvable points there. Distal regeneration will follow to completion.

Bruchdreifachbildungen fused at the base and showing distal expansion

Wherever the two lateral supernumeraries are completely separate at thebase, the inference must be that the notch extended more than halfway acrossthe circumference. If the notch extends less than halfway and the damage isfollowed by the two lateral edges of the wound coming together, then theshortest intercalation rule (French et al 1976) requires the missing circum-ferential values to be intercalated. The result is local repair without lateralsupernumeraries. This is probably the normal outcome of local damage to arestricted part of the circumference. Nevertheless in some of our specimensthe original damage was highly localized on one side of the limb and yetlaterals were produced. In these cases the Bruchdreifachbildungen are fusedat the base and distal expansion results in complete separation of the twolaterals at a more distal level. In this case we believe that the original notchwas shallow and/or had sections of the lateral wound surface approximatelyparallel to the proximodistal axis of the limb. It is assumed that the woundremains unhealed and that regeneration begins at the lateral edges. We alsoassume that such an exposed edge may regenerate in either direction aroundthe circumference. Each edge will regenerate without reference to the otheruntil the two sides finally meet (Fig. 10). After the lateral edges meet, we predictintercalation by the shortest route around the circumference if there is aconfrontation of non-congruent values. This could have three possible out-comes. First, the two edges can regenerate towards each other. In this casethere will be repair of the wound without lateral regenerates because, when thetwo sides meet, we expect near or complete congruences of values. Second, thetwo edges can regenerate values in the reverse direction (Fig. 10). If suchregeneration proceeds sufficiently far, values more than halfway round thecircumference from the centre of the original wound can be formed at theexposed edges. When they meet, intercalation at the interface will occur untiltwo complete sets of circumferential values have been created. Followingour previous reasoning, the inherent instability of this situation allows distal


regeneration to completion. Note that, at the base of such a structure, the twolaterals will each consist of less than half of the circumferential values andthey will be joined together in mirror image symmetry. The distal expansioncan be explained by the well established intercalation rule and does not requirespecial new rules (see for example Slack, 1980#, b). The third possibility isthat on one side of the wound, regeneration proceeds in one direction andthat on the other side, it is in the reverse direction. This would not result inlateral regenerates because, assuming equal rates of regeneration from eachedge, the relative difference in circumferential values will be approximatelythe same as the difference between the original exposed surfaces. However,intercalation after fusion will result in a bump at the site of the notch.

So far we have considered regeneration along each of the two axes separatelyand we have chosen ideal examples where the exposed surface is nearly parallelto one of the axes and cuts the other at an acute angle. However, in mostnotches the wound will be more or less U shaped when viewed from the side.So, in some places along the wound margin a given axis will be cut at a shallowangle and in others it will be cut at an acute one. For this reason the resultingoutgrowth will have features of bumps and of laterals with distal expansion.Thus, an outgrowth may have a polarity reversal (with respect to the proximo-distal axis) at the distal side of the lateral's base, but also show distal expansion(Fig. 3).

CONCLUSION

In this discussion we have attempted both to explain the phenomenon ofBruchdreifachbildungen and to provide an explanation for distal regenerationwhich does not depend upon the complete circle rule (French et al. 1976)alone. This is because the complete circle rule demonstrably fails in the caseof the distal regeneration of mirror-image symmetrical limbs (French, 1976;Slack & Savage, 1978) and because by itself the complete circle rule cannotexplain the range of structures found in the Bruchdreifachbildungen we havedescribed for crustaceans and Bohn (1965) has observed in insects. Afterdue consideration, we concluded that the main cause of regeneration in limbsis the failure of wounds to heal. Distal regeneration following the formationof complete circles is just a special case of wounds failing to heal. It may bebrought about because of the unresolvable point at the centre of a completecircumference or because cells on opposite sides of the circumference havesufficiently different surface properties that healing is inhibited. Our hypothesiscould explain why amputated mirror-image symmetrical limbs are capable ofdistal regeneration. All that is required is regeneration of all values distal tothe cut before the cells at the circumference come together at the tip. This isquite possible; most fields are small at the time of determination (Wolpert,1969; Crick, 1970) and subsequent cell divisions merely increase the size ofthe organ. In addition, just the geometry of an amputated cylinder is not


conducive to healing at the tip. An important point deriving from our argumentis that it is immaterial which circumferential values are represented in thewounded surface. For this reason we can explain the regeneration of anomalousmirror-image symmetrical lateral supernumeraries in amphibians after 180°rotations of the limb (Maden, 1980). In that case we propose that, after theoperation, the wound fails to heal over part of the circumference due to someobstruction (possibly a blood clot) or perhaps to a misalignment of host andgraft tissues. The partial set of values intercalated around the wound will havemirror-image symmetry and distal regeneration will result in these values beingperpetuated until either the most terminal levels are formed or the woundfinally heals. In conclusion, we can summarize our explanation as follows.A cut free surface will sequentially regenerate values along the axis orthogonalto the cut. Any exposed part of the circumference may begin to regenerate solong as the surface remains free. In the case of the proximodistal axis regenerationfrom a free surface will always proceed in a distal direction while in thecircumferential axis it can proceed in either direction around the circumference.Clearly our ideas are speculative and they are based on a rationalisation ofphenomena exhibited by a set of naturally occurring monstrosities. Nevertheless,they provide a possible way out of the complete circle impasse and they suggestthe need for further experiments and accurate observation of cell behaviourat wound surfaces.

We thank Angela Chorley for drawing the chelae and preparing Fig. 1. We are alsograteful to Dr P. J. Hogarth for helping with our literature search. P.R.T. was supported byan M.R.C. studentship.

REFERENCES

BATESON, W. (1894). Materials for the Study of Variation. London, New York: Macmillan.BLISS, D. E. (I960). Autotomy and regeneration. In Physiology of Crustacea, vol. 1 (ed.

T. H. Waterman), pp. 561-589. New York, London: Academic Press.BODENSTEIN, D. (1953). Regeneration. In Insect Physiology (ed. K. D. Roeder), pp. 886-

878. New York: Wiley.BOHN, H. (1965). Analyse der Regenerationsfahigkeit der Insekten-extremitat durch

Amputations- und Transplantationsversuche an Larven der afrikanischen Schabe Leuco-phaea maderae Fabr. (Blattaria). 1. Regenerationspotenzen. Wilhelm Roux Arch. Entw-Mech. Org. 156, 49-74.

BOHN, H. (1976). Regeneration of proximal tissue from a more distal amputation level inthe insect leg {Blaberus craniifer, Blattaria). Devi Biol. 53, 285-293.

BRYANT, S. V. & BACA, B. B, (1978). Regenerative ability of double-half and half upper armsin the newt, Notophthalmus viridescens. J. exp. Zool. 204, 307-324.

CRICK, F. H. C. (1970). Diffusion in embryogenesis. Nature, Lond. 225, 420-422.EMMEL, V. E. (1908). The experimental control of asymmetry at different stages in the

development of the lobster. / . exp. Zool. 189, 421-427.FRENCH, V. (1976a). Leg regeneration in the cockroach, Blatella germanica. I. Regeneration

from a congruent tibial graft/host junction. Wilhelm Roux' Arch, devl Biol. 179, 57-76.FRENCH, V. (19766). Leg regeneration in the cockroach, Blatella germanica. II. Regeneration

from a non-congruent tibial host/graft junction. / . Embryol. exp. Morph. 35, 267-301.FRENCH, V. (1978). Intercalary regeneration around the circumference of the cockroach

leg. J. Embryol. exp. Morph. 47, 53-84.


FRENCH, V., BRYANT, P. & BRYANT, S. (1976). Pattern regulation in epimorphic fields.Science, N.Y. 193, 969-981.

GIRTON, J. R. & BRYANT, P. J. (1980). The use of cell lethal mutations in the study ofDrosophila development. Devi Biol. 11, 233-243.

GIRTON, J. R. & RUSSELL, M. A. (1980). A clonal analysis of pattern duplication in atemperature-sensitive cell-lethal mutant of Drosophila melanogaster. Devi Biol. 77, 1-21.

HUXLEY, T. H. (1884). The Crayfish. London: Kegan Paul, Trench.MADEN, M. (1980). Structure of supernumerary limbs. Nature, Loud. 286, 803-805.MITTENTHAL, J. E. (1978). Intercalary regeneration in crayfish legs. Am. Zool. 18, 610.NUBLER-JUNG, K. (1977). Pattern stability in the insect segment. 1. Pattern reconstitution

by intercalary regeneration and cell sorting in Dysdercus intermedius Dist. Wilhelm RouxArch. devlBiol. 183, 17-40.

PAUL, J. H. (1914). Regeneration of the legs of decapod Crustacea from the preformedbreaking plane. Proc. R. Soc. Edinb. 35, 78-91.

PRZIBRAM, H. (1921). Die Bruchdreifachbildung im Tierreiche. Wilhelm Roux Arch. Entw-Mech. Org. 48, 205-444.

SLACK, J. M. W. (1977). Control of anteroposterior pattern in the axolotl forelimb by asmoothly graded signal. J. Embryol. exp. Morph. 39, 169-182.

SLACK, J. M. W. (1980a). Morphogenetic properties of skin in axolotl limbs. / . Embryol.exp. Morph. 58, 265-288.

SLACK, J. M. W. (19806). A serial threshold theory of regeneration. J. theor. Biol. 82,105-140.

SLACK, J. M. W. & SAVAGE, S. (1978). Regeneration of mirror symmetrical limbs in theaxolotl. Cell 14, 1-8.

WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation./ . theor. Biol. 25, 1-47.

(Received 15 October 1980, revised 27 January 1981)

naturally occurring abnormalities (bruchdreifachbildungen) in the … · embryol. exp. morph. vol....

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