Extent and properties of the regeneration field in the larval legs of cockroaches (Leucophaea maderae)

Transcription

1 /. Embryol. exp. Morph. Vol. 32, 1, pp Printed in Great Britain Extent and properties of the regeneration field in the larval legs of cockroaches (Leucophaea maderae) III. Origin of the tissues and determination of symmetry properties in the regenerates ByHORST BOHN 1 From the Zoologisches Institut der Universitat, Munchen SUMMARY Previous papers dealing with the regeneration field of cockroach {Leucophaea maderae) legs have shown that two elements of the leg surroundings are indispensable for regeneration of a leg: the basal sclerites and the 'leg-inducing membrane' (LIM). The experiments of this paper partly clarify the way in which these two tissues interact; the origin of the regenerative tissues and the determination of polarity and symmetry in the regenerated legs were studied. By combination of sclerites and LIM of different species or segments it has been shown that each of the tissues makes up half of a leg regenerate - the sclerite tissues the anterior longitudinal half, the LIM the posterior longitudinal half. The trochantin was implanted in four different orientations (normal, turned 90 clockwise or anti-clockwise, turned 180 ) into a normally orientated field of LIM. The anteriorposterior and medio-lateral axes of the regenerates had the same orientation as those of the sclerites. Therefore, the sclerites alone determine symmetry of the regenerate; the LIM has no influence in this respect. Neither of the two axes is fixed irreversibly in the cells of the LTM; the cells seem able to accept any polarity which is forced upon them by the sclerites. The symmetry properties are more strongly fixed in the cells of the sclerites, but an inversion of polarity (through 180 ) is possible for both axes. Regeneration of a leg from the level of the trochantin seems to be initiated by or dependent on a small medial region of the trochantin. A tiny fragment of the medial wedge of the trochantin, when transplanted to a field of LIM, can form a complete leg regenerate; a lateral half of the trochantin never does. As long as this small medial part of the trochantin is prevented from making contact with LIM by a praecoxa fragment, no regeneration takes place at the anterior border of the trochantin, even when the greatest part of the border has contact with LTM. The regenerative capacities of the praecoxal sclerite are different at the anterior and posterior cut surfaces, irrespective of the level of the cut and the orientation of the sclerite. An anterior cut surface never forms a leg regenerate, but a posterior cut surface does. 1 Author's address: Zoologisches Institut der Universitat, 8000 Munchen 2, Luisenstr. 14, Germany. EM B 32

2 82 H. BOHN INTRODUCTION In the larvae of cockroaches, complete extirpation of a leg (including the coxa) is followed, as a rule, by regeneration of a new leg. The prerequisite of a successful regeneration is the presence of two kinds of tissue normally surrounding the leg-the basal sclerites, which lie anterior to the coxa, and the 'leginducing membrane' (LIM) adjoining the leg posteriorly. The interaction of these two tissues is necessary for leg regeneration (Bohn, 1914 a, b), but the nature of the interaction during regeneration is unknown. The goal of the experiments presented in this paper was to clarify at least part of this problem. One question was the origin of the tissues of the regenerates. The question could be answered by experiments (series H) in which sclerites and LIM from different segments or different species were combined. Another related question was whether the two co-operating tissues were of equal importance for the development of the regenerate; which of the two tissues, for instance, determines the symmetry properties of the new leg? Sclerites and LIM were combined in divergent orientations (series J); observation of the orientation and symmetry of the legs developing under such conflicting conditions permitted analysis of the respective influence of the two tissues on symmetry determination during regeneration. A third problem arose from the transplantation experiments described in Bohn (19746). It seemed that the capacities of different parts of the basal sclerites to regenerate legs upon contact with LIM were different; therefore, basal sclerites in various combinations were transplanted to a LIM to test their regenerative capacities (series G). MATERIALS AND METHODS The animals (larvae of Leucophaea maderae) and the experimental procedures were the same as in previous papers (Bohn, 1974 a, b). In one experiment (H 2 ) another species of cockroach (Gromphadorhina portentosa) was used in addition to Leucophaea to allow combination of sclerites and LIM from two different species. The three experimental series (G, H, J) of this paper continue the six series (A-F) of the preceding papers. EXPERIMENTS AND RESULTS Series G (Table 1) Experimental series G was undertaken to clarify some contradictory results of the preceding experimental series, concerning the regenerative capacity of the basal sclerites. When a trochantin was implanted into a field covered by LIM, two legs were regenerated; when a complete set of basal sclerites (trochantin plus praecoxa) was transplanted in a similar way, only one leg developed, always at the posterior margin of the trochantin. There were a few exceptions, in which a second leg was formed at the anterior margin of the transplant; it seemed that the medial part of the praecoxa had disappeared in these cases.

3 Tissue origin and symmetry in cockroach leg regenerates 83 Table 1. Experimental series G and H: types of operation and results The transplant (dotted area) in series G was taken from the basal sclerites of the right (Expts. G^) or left (Expts. G 7i8 ) midleg and was implanted immediately behind the basal sclerites of the left hindleg (clear area in third column) after removal of the distal parts of the leg. In Expts. G^6 the transplant was rotated through 180 before it was implanted. In Expt. H ± the basal sclerites of a left foreleg were transplanted in the same way as in series G. In Expt. H 2 the basal sclerites of Leucophaea were implanted at the site of the extirpated hindleg of Gromphadorhina (Gr. tissues indicated by cross-hatching). Expt. No. of operated animals Orientation of trans- plant Source of transplant Surviving No leg formed Normal leg regenerated Medial part of precoxa Missing Very small G " 9 * All legs mixed, the anterior half consisting of foreleg tissue, the posterior half of hindleg tissue. f Eight of the nine regenerated legs clearly mixed: anterior half Leucophaea tissue, posterior half Gromphadorhina tissue; character of one leg indeterminable. Therefore it was desirable to analyse the regenerative ability of a trochantin in combination with different parts of the praecoxa. For this purpose an experimental arrangement was used which gave a higher yield of successful regenerates as compared to series D and J. The left hindleg was removed at the base of the 6-2

4 84 H. BOHN

5 Tissue origin and symmetry in cockroach leg regenerates 85 coxa, and the sclerites to be tested were implanted into this wound area, immediately adjacent to the trochantin of the hindleg. Posteriorly they had contact with the LIM of this leg. When the capacities of anterior wound surfaces of the praecoxa and the trochantin were studied (Expts. G x _ & ), the sclerites were implanted with inverted anterior-posterior polarity to allow contact with the LIM. To avoid complications by divergent medio-lateral polarity of host and transplant tissues, the basal sclerites of right midlegs were used. When posterior wound surfaces were tested, basal sclerites of left midlegs were used and implanted in normal orientation (Expts. G 78 ). Experiments G^Q. The basal sclerites, from which different parts of the praecoxa had been removed, were implanted with reversed anterior-posterior polarity. Host and transplant sclerites grew together without any signs of regeneration (Fig. 1), but eventually a leg regenerated at the former anterior border of the implanted basal sclerites, now directed to the rear end of the animal. A leg with a normally orientated trochantin just behind the transplanted trochantin was regenerated in all cases when only a trochantin was transplanted (Expt. G 3 ; Fig. 3) or when at least the medial part of the trochantin had contact with the LIM (Expt. G 6 ). But no legs were regenerated when the LIM was combined with the following wound surfaces of the basal sclerites: anterior margin of the praecoxa (Expt. G 1 ; Fig. 1), intermediate level of the praecoxa (Expt. G 2 ), lateral half (Expt. G^) or lateral two-thirds of the posterior margin of the trochantin (Expt. G 5 ). When a regenerate formed in these experiments, at least part of the transplant had disappeared; either the medial half or third of the praecoxa was absent (Fig. 2; column 8 of Table 1) or, if present, it was reduced to a very thin sclerite (last column of Table 1). In all cases without leg regeneration, the sclerites were completed. Experiments G 7 _ 8. Part of the base of the left midleg (part of SIM + complete praecoxa in Expt. G 7 ; part of SIM + anterior half of praecoxa in Expt. G 8 ) were transplanted in normal orientation into the wound field. A third set of basal sclerites with inverted anterior-posterior polarity was regenerated between host and transplant sclerites (compare with Fig. 5 of Expt. H 4 ), except in two animals which had regenerated a normal leg at the posterior border of the host trochantin. In all experiments, legs were formed at the posterior wound borders of the transplants. FIGURES 1-3 Examples from experimental series G. When a complete set of basal sclerites of a right leg is combined in reverse anterior-posterior orientation with the basal sclerites of a left hind-leg, no leg regeneration takes place (Fig. 1). The arrow indicates the border between host (above) and transplant (below) tissues. But when the praecoxa of the transplant was obliterated after transplantation (Fig. 2) or had been removed before transplantation (Fig. 3) a leg was regenerated at the posterior border of the transplant. In these cases a third trochantin (/) formed between the transplanted trochantin and the coxa of the regenerated leg.

6 86 H. BOHN M.. FIGURES 4-6 Examples from experimental series H, dealing with the origin of regenerate tissues. Fig. 4. Ventral view of the first and second thoracic segments, showing the structural differences between the coxa of fore- and mid- (or hind-) legs. The left legs were removed at the trochanter-femur joint. The meron (m) is very narrow in the foreleg, but broad in the midleg and hindleg. There is a sharp linear edge on the coxa of the foreleg, which is bent and not very prominent in the midleg and hindleg (arrows). Fig. 5. Mixed leg which developed after the combination of basal sclerites of a foreleg with the LIM of a hindleg. Anteriorly (upper surface in the figure) there are foreleg structures (the arrow points to the sharp edge characteristic of the foreleg), posteriorly there are hindleg structures (broad meron (w) as in the hindleg). A third set of basal sclerites (t = trochantin, p = praecoxa) has regenerated between host and transplant sclerites. Fig. 6. Mixed leg which developed after the combination of basal sclerites of Leucophaea with LIM of Gromphadorhina. The anterior half of the leg consists of Leucophaea tissues (light), the posterior half of Gromphadorhina tissues (dark). The leg was cut between femur (/) and tibia (ti) and within the tarsus to allow better photography. The claw segment (c/) is shown from the dorsal surface, the other parts from the ventral surface.

7 Tissue origin and symmetry in cockroach leg regenerates 87 Series H (Table 1) In analysis of the respective roles of basal sclerites and LIM during regeneration it was desirable to clarify the origin of the cellular material composing the regenerates. Does it come from the LIM, from the sclerites, or from both tissues? The method for approaching this problem was to combine sclerites and LIM from different segments (Expt. Hj) or different species (Expt. H 2 ). Experiment H x. The experimental procedure was very similar to that of Expts. G 78. The complete set of basal sclerites of the left foreleg was implanted behind the basal sclerites of the left hindleg, which had been previously removed. Thus, the sclerites of a foreleg were combined with the LIM of a hindleg. All regenerates which formed at the posterior margin of the trochantin had a composite structure, with foreleg features at their anterior surface (straight at the anterior surface of the coxa, etc.) and hindleg features at the posterior surface (broad meron at the lateral border of the coxa, etc.) (Figs. 4, 5). The exact boundary between the two kinds of tissue was usually not visible, since the recognizably differing structures make up only a part of the leg, but it seemed that the anterior longitudinal half of the leg was formed by the sclerite (foreleg) tissues, and the posterior half by the LIM (hindleg) tissues. As in Expt. G 78, a third set of basal sclerites with inverted anterior-posterior polarity was regenerated between host and transplant sclerites (p and t in Fig. 5). Experiment H 2. The left hindleg, including its basal sclerites, was extirpated in larvae of Gromphadorhina portentosa and replaced by the basal sclerites of the left hindleg of Leucophaea maderae. Thus the Leucophaea basal sclerites came into contact with the Gromphadorhina LIM. All animals with successful transplants regenerated a leg. Host and transplant tissues were easily distinguished by their different pigmentation; transplanted Gromphadorhina tissues are nearly black, Leucophaea tissues have a light brownish colour. All legs were partly Gromphadorhina tissue (posterior half) and partly Leucophaea tissue (anterior half). The border between the two tissues ran fairly linearly at the mid-ventral and mid-dorsal surfaces over the whole length of the leg (Fig. 6). Series J (Table 2; Fig. 7) In the preceding series it was shown that each of the two interacting tissues, the sclerites and the LIM, contribute about equal amounts of material to the developing regenerate. This does not necessarily mean that both tissues are of equal importance in all respects and during all phases of regeneration. One possible means of separating the contributions of the two different tissues was to create a conflicting situation between sclerites and LIM, and see which of the two had more influence on the character of the developing regenerate; therefore a conflict of polarity and orientation between sclerites and LIM was produced (Expts. Ji_ 5 ). In the same series I attempted to settle another problem which arose from the results of series F. It seemed that the different parts of the

8 H. BOHN Table 2. Experimental series J: types of operation and results The figures in the second column represent the basal sclerites of the left (Expts. Ji-3 } e) or right (Expts. J 4> 5 ) midleg. Dotted area: part of the sclerite used as transplant. The third column shows orientation of the transplant just after operation: in Expts. J 2>3 the transplant was turned through 90 anti-clockwise or clockwise, respectively; in the other experiments there was no turning before implantation. The site of implantation was the membranous field, which is outlined by heavy dots in II of Fig. 7. Source of trans- Expt. plant Orientation of No. of trans- operated Surplant animals viving Animals with regenerated legs Orientation of leg regenerates Same Between as trans- Same trans- plant as plant and host host Fig. 7. Plan of the main experiments of series J. The experiment was performed in two steps. First (I) the left hindleg was removed completely. The figure at the left shows a situation where only the distal parts of the leg had been removed (crosshatched area: cut surface). In addition to this the basal sclerites and large parts of the anterior membranous area (SIM) had also been cut out (heavily dotted area). The midleg had not been cut, but is shown only up to the base of the coxa (broken line). After two moults (II), during which time no leg regeneration occurred, sclerites were implanted into the membranous field covering the former leg area. This area of implantation (encircled with heavily dotted outline in the right top figure) mostly consisted of LIM of the meso- and metathoracic segments. The lower part of the figure shows the origin of the transplant in the left column (transplant heavily dotted) and the orientation in which it was implanted, in the centre column. The thick black arrow indicates the medio-lateral polarity of the transplant and of the resulting regenerate (m, medial; 1, lateral). Two symmetrically arranged legs are formed, whose orientation mostly coincides with that of the transplant. Lightly dotted area: membranous parts; clear area: sclerotizedparts; longitudinally hatched area: wing anlagen.

9 Tissue origin and symmetry in cockroach leg regenerates Extirpation of left hind leg. II. Transplantation of basal sclerites Area of implantation Source of transplant Orientation of transplant Results Fig. 7. For legend see opposite. trochantin had different capacities for regeneration; therefore, either lateral or medial parts of the trochantin were implanted into an area covered by LIM (Expts. J 4 _ 6 ). The operation for preparing an implantation area was similar to that of series D. The left hindleg was removed, together with all basal sclerites. In order

10 FIGURES 8-11 Examples from experimental series J. Regeneration of differently orientated double legs after the implantation of a trochantin in various orientations into a field of LIM. Fig. 8. Normal orientation, knees (k) pointing laterally. Fig. 9. Trochantin turned 90 anti-clockwise, knees pointing anteriorly. Fig. 10. Trochantin turned 90 clockwise, knees pointing posteriorly. Fig. 11. Trochantin of a right leg implanted without turning (corresponds to a 180 turning), knees pointing medially. do O

11 Tissue origin and symmetry in cockroach leg regenerates 91 to have a large area covered only by LIM, the SIM in front of the hindleg was also removed. Thus the LIM of mid- and hindleg together formed the area to which sclerites could be transplanted (Fig. 7). Experiments J x _^. The trochantin of the left midleg was implanted in various orientations: normal orientation (Expt. Jj), turned 90 anti-clockwise (Expt. J 2 ) or clockwise (Expt. J 3 ). In experiment J 4 the medial half of the trochantin of the right midleg was implanted without turning it. This situation was nearly identical to a 180 turning of a left trochantin. In most cases, two legs were formed, as would be expected when a trochantin is used as transplant. Only the double legs were used for analysing the orientation of regenerates; orientation of the single legs was difficult to evaluate since they were often abnormally bent. The orientation of regenerated legs mostly coincided with that of the transplant (Figs. 7-11), but in some cases it seemed to lie between the orientation of the host tissues and that of the transplant's original position. Experiments / 5j6. A rectangular piece comprising about one-sixth of the whole area of the trochantin, cut out of the medial third of the trochantin of the right hindleg, was used in Expt. J 5. In experiment J 6 the lateral half of the trochantin of the left or right hindleg was transplanted. Implanted lateral halves of the trochantin were not able to elicit a leg regenerate, whereas medial halves did so (see experiment J 4 ); even pieces as small as those used in experiment J 6, if taken from the medial half, could initiate leg regeneration. Half of the regenerates of experiment J 5 had transplant orientation, the other half had an orientation which was somewhere between host and transplant orientation. DISCUSSION The regenerative capacity of praecoxa and trochantin It has been repeatedly shown that the regeneration field of the cockroach leg consists of two different types of tissue which are both necessary for initiating leg regeneration - the basal sclerites of the leg and the 'leg-inducing membrane' (LIM). Contact between the two tissues is sufficient to induce leg regeneration even if no leg has been removed (Expts. C 3 _ 6 ; E^). But the regenerative capacity of different parts of the basal sclerites appears to be different. When, for instance, a trochantin is transplanted to a LIM, two legs are usually regenerated, one at the posterior margin of the transplant and one at the anterior margin (Expts. D 2 ; Ji_ 4 ). The regenerate formed at the anterior margin has reversed anterior-posterior polarity. These two legs and their basal sclerites form exact mirror images of each other (Fig. 7). Therefore, after the implantation of a trochantin, the regenerate at the anterior margin also begins with a trochantin, symmetrical to the implanted one. In Expt. G 3, where only the anterior margin (which is facing posteriorly because of rotation of the trochantin) had contact with the LIM only one regenerate (with normal polarity) is formed. Again the regenerate begins with a trochantin, symmetrical to the implanted one. A

12 92 H. BOHN similar situation is found in Expts. C 4) 6, except that the anterior margin of the trochantin is pointing anteriorly and the regenerate therefore has inverted anterior-posterior polarity. The praecoxa never forms a leg regenerate at its anterior margin when combined with LIM, irrespective of whether the margin is facing anteriorly (Expt. E 3 ) or posteriorly (Expts. E l5 G x ). When the praecoxa is cut at an intermediate level, the success of regeneration after contact with LIM depends on which of the two cut surfaces is used. An anterior cut surface of the praecoxa never forms a leg regenerate, irrespective of the actual orientation of the sclerite (compare, for instance, Expts. C 7 8 with Expt. G 2 ), but a posterior cut surface does (Expts. A 3 _ 5 ; G 8 ). The posterior margin of the praecoxa upon contact with LIM forms a normal regenerate (Expts. A 2, G 7 ). It is not clear why anterior and posterior cut surfaces of the praecoxa, although of the same intermediate level, behave so differently. One could assume that the tissues of the praecoxa are not able to reverse their polarity, since, as in the case of the anterior margin of the trochantin, a leg with reverse anterior-posterior polarity should develop. But this cannot be true. For when a similar wound surface was combined with sclerites, a second set of basal sclerites with reverse polarity could be regenerated (Expt. B 4 ). So the cells of the praecoxa when exposed to an anterior cut surface are able to reverse their anterior-posterior polarity, but they are not able to form a leg regenerate upon contact with LIM. The developmental meaning of this is not clear; a possible biological meaning could be that the inability of an anterior cut surface of the praecoxa to elicit leg regenerates might reduce the chance of formation of a presumably hindering extra leg after accidental damage in the region between the sclerites and the LIM of the preceding leg (see experimental series C) without reducing the capacity for leg regeneration following the loss of a leg and large parts of its basal sclerites. But even within the trochantin the capacities for leg regeneration are not evenly distributed. Lateral halves of the trochantin were not able to elicit a leg regenerate, but pieces as small as one-sixth the size of the trochantin could initiate leg regeneration if taken from the medial part of the trochantin (Expts. J 4 _ 6 ). When a whole trochantin was transplanted, regeneration only occurred if this medial part of the trochantin came into direct contact with the LIM. The coverage of only the medial one-third of the trochantin with a praecoxal sclerite was sufficient to inhibit leg regeneration (Expts. G 45 ), whereas regeneration occurred in all cases when the medial half of the trochantin was free and the lateral half was protected by the praecoxa (Expt. G 6 ). These results seem to be in contrast to an earlier experiment in which it was possible to get complete leg regenerates from lateral halves of the basal sclerites (Expt. A 9 ); but in this case the missing parts of the basal sclerites, and thus also the important medial halves of the trochantin, had been regenerated. One could imagine that leg regeneration only started after the regeneration of the medial part of the trochantin. Such regeneration does not seem to be possible when the lateral

13 Tissue origin and symmetry in cockroach leg regenerates 93 halves of the trochantin are implanted into a field of LTM (Expt. J 6 ) and therefore no leg regenerate is formed. Restriction of the leg-promoting capacities of the trochantin to a small medial region seems to be responsible for the fact that the triangular trochantin only forms two leg regenerates when transplanted to a LIM, though there are three cut surfaces in contact with LIM. Leg regeneration is only possible when parts of the sclerites and part of the LIM are present and have the chance to interact. These two types of tissues seem to contain two different principles which are necessary for regeneration. It was shown in experiments H 12 that each of the two tissues make up half of the leg regenerate; the anterior half of the leg is formed by the sclerites, the posterior half by the LIM. The question was whether these two principles were still present in the respective tissues of more distal segments of the leg. This problem was touched by Expts. C 3j 5 in which anterior coxal tissues had contact with LIM; in these cases legs were regenerated. The results could be confirmed by transplantation experiments (Bohn, unpublished results); parts of the anterior and posterior surfaces of the coxa were cut out and transplanted to a LIM. Only the tissues of the anterior surface in combination with LIM were able to elicit leg regenerates. So it becomes clear that the tissues of the anterior surface of the coxa, when in contact with LIM, behave in the same way as the basal sclerites from which they are derived, and therefore they may have the same morphogenetic principles. In this connexion it is interesting to see whether there are correlations between the conditions of leg regeneration at the base and at more distal levels. It has often been observed that extra leg regenerates are formed when leg parts are combined in such a way that the polarities of the transverse axes do not harmonize in host and transplant tissues. The accessory regenerates normally sprout at the sites of the greatest incompatibility between host and transplant tissues. (Bohn 1965; Bart, 1971) but an anterior-posterior disharmony is as potent in creating additional regenerates as c dorso-ventral disharmony. Therefore it is not possible to directly correlate the results of experiments at the base and at more distal levels of the leg. On the other hand, Leucophaea and most other insects (Przibram, 1921; Bodenstein, 1937; Bulliere, 1970; Bohn, 1972; but see also Bart, 1971) never form more than two additional legs, even under conditions which should allow the formation of multiple legs. It seems that the development of regenerates at more distal levels of the leg is based on a dualistic principle similar to that observed in leg regeneration at the base. Nevertheless, one basic difference remains: when a leg is wounded on only one side, an incomplete distal regenerate may develop, containing only the side qualities of the wounded surface (Bohn, 1965; Bart, 1970); at the base, on the contrary, either a complete leg is formed or no distal parts are formed at all. Additional experiments are necessary to find a more satisfactory concept which eventually could explain both phenomena by a common principle.

14 94 H. BOHN Origin of the tissues and determination of polarity and symmetry of the regenerates The establishment of a leg regenerate is dependent on the interaction of sclerites and LIM; the nature of the interaction is still largely unknown. Experimental series H and J were designed to elucidate at least part of these problems, namely, the origin of the cellular material composing the regenerate, and the respective roles of sclerites and LIM in determining polarity of the regenerate. The first problem was studied by combining sclerites and LIM of different segments (Expt. H x ) or different species (Expt. H 2 ). All experiments showed the same results - both participants contributed equal amounts of cellular material to the regenerate. The anterior longitudinal half of the regenerate was formed by material derived from the sclerites, the posterior half by material proliferated by the LIM. In this connexion it should be emphasized once more that neither the LIM nor the sclerite is able to form its respective half of a leg without the other tissue. In series F, legs were formed on the abdomen after the implantation of basal sclerites. It would be interesting to see whether the abdominal tissues in this case had also provided half of the material for the regenerates; interspecific transplantation experiments are necessary to answer this question. The problem of polarity has already been touched upon in series C, D and E, in which eventually legs developed with inverted anterior-posterior polarity. Reversal of polarity in these series occurred when the anterior margin of the trochantin or coxa was contacted by LIM. The problem of polarity was more directly attacked by experimental series J. In Expts. J 2 3 the trochantin had been rotated through 90 clockwise or anti-clockwise before it was implanted into a field of normally orientated LIM. Thus, the orientation of the anterior-posterior axes of transplant and host tissues were at right angles. As a rule two legs were formed, one at the original posterior margin and one at the former anterior margin of the trochantin. The first had the same anterior-posterior polarity as the transplant, while the polarity of the latter was inverted through 180 with respect to the transplant's polarity, as is usual for regenerates developing at the anterior margin of the trochantin (see also Expts. G 36 ). The results allow the following conclusions: when a trochantin is implanted into a field of LIM, leg regeneration occurs. The trochantin determines the number and site of the developing regenerates. Irrespective of the shape of the trochantin, i.e. the number of cut surfaces and the orientation of the cut surfaces, only two regenerates are formed, anteriorly and posteriorly with respect to the original anterior-posterior axis of the sclerite. The trochantin also determines the anterior-posterior polarity of the regenerates. That the polarity of the anterior regenerate is inverted is a consequence of the reversal of the normal order: anterior sclerites, posterior LIM.

15 Tissue origin and symmetry in cockroach leg regenerates 95 The situation is very similar if we consider medio-lateral polarity. In Expts. J 2 _ 5 the orientation of the medio-lateral axis of the transplant was different from that of the LIM; the two axes formed an angle of 90 (Expts. J 2 3 ) or 180 (Expt. J 4 ). The developing regenerates had the same medio-lateral polarity as the sclerites. In this respect again the LIM had no influence on the polarity of the regenerates; the sclerite alone determined the medio-lateral polarity of the regenerates. The results of the experiments concerned with polarity of the regenerates allow some conclusions concerning the stability of the symmetry properties in the reacting tissues. It is assumed that as in normal regeneration (Expts. H 1) 2 ) each of the two participating tissues provides half of the tissue of the regenerate. (This assumption was confirmed by an experiment in which sclerites of the foreleg were implanted in incorrect medio-lateral orientation into a hindleg field of LIM; H. Bohn, unpublished observation.) From this it follows that neither the anterior-posterior nor the medio-lateral polarity is strongly fixed in the cells of the LIM; they may change their polarity and accept any other that is forced upon them by the sclerites. In the tissues of the sclerites, though the axes of symmetry are more stable, some change is possible. Though no change in the orientation of the two axes of symmetry was observed, at least the polarity of both could be reversed through 180. The regenerates which develop at the anterior margin of the trochantin have inverted anterior-posterior polarity (with respect to the trochantin's polarity). There was also one example of an inversion of the medio-lateral polarity. In Expt. A 9 all parts of a leg had been extirpated with the exception of the lateral half of the basal sclerites. Between these remaining sclerites and a newly formed complete set of basal sclerites another lateral half of basal sclerites developed; these latter had reversed mediolateral polarity (see Fig. 5, Bohn, 1974a). There are some cases in Expts. J 2 _ 5 in which the orientation of the regenerate is somewhat between that of the host tissues and that of the original position of the implanted sclerite. This could mean that both tissues have partly changed their polarity and that the regenerate represents this compromise, but there are other, more likely, possibilities. Firstly during an operation and the subsequent healing period, a transplant may well assume an orientation slightly different from the desired one. Secondly, an active re-rotation of the whole implant towards a condition where its medio-lateral polarity is in accordance with that of the surroundings must be taken into account. There is strong evidence for similar correcting movements of rotated pieces of integument on tergites of Leucophaea (Bohn, 1974 c). If such a re-rotation happened, it would indicate that LIM exerts some influence (if only indirectly) on the orientation of the regenerate.

16 96 H. BOHN Leg induction The ventral surface of the thoracic segment may be divided into four different zones (see Bohn, 1974a, fig. 1): most anteriorly the SIM, then the basal sclerites of the leg, the leg itself, and behind the leg the LIM. The anterior and posterior membranous areas are called sclerite-inducing membrane and leginducing membrane because additional sclerites or legs are formed when these membranes are combined with basal sclerites. These terms were at first used only for practical reasons. The question is whether we are justified in the use of the term 'inducing' in this connexion, in the sense that embryologists normally speak of induction. Unfortunately, it is not possible to make the decisive control experiment, i.e. isolation of the corresponding structures. The tissues under study always have contact with other tissues. Therefore, it is not possible to decide why a combination of basal sclerites and SIM cannot form leg regenerates, while a combination of the same sclerites and LIM can. Either the solerites need an inductive impulse, which can only be provided by the LIM, or the sclerites are prevented by the SIM from regenerating distal leg tissues, as they normally would do. The same holds true for the LIM itself, which in combination with the sclerites forms part of the leg: in combination with SIM (Expt. A 5 ) only membranous tissues are formed. There is still another possibility: the sclerites could have leg-inducing capacities, but SIM might not be competent to react to this inductive stimulus; only LIM is competent, and for its part induces the sclerites to form leg regenerates (without response from the LIM they could only form additional sclerites). Another phenomenon which must be taken into account has been previously discussed (Bohn, 1974a)-that is, the tendency in the segmented animals to avoid discontinuities of the pattern. It is not necessary that all given structures of a segment should be present, but it is indispensable to have an uninterrupted continuity between those structures which are present. At least some aspects of the regeneration process may be explicable in terms of this principle. In series G, for instance, there was no regeneration at the cut surface of host (or transplant) trochantin when it was combined with tissues of the same 'level' (Expts. Gi_ 6 ). In Expts. G 7 _ 8, H l9 where the same surface was combined with tissues of a more anterior level (SIM), the 'gap' was bridged by a third set of basal sclerites with reverse anterior-posterior polarity (Fig. 5). Similarly, a third, normally orientated trochantin developed behind the transplanted trochantin when leg regeneration occurred at the former anterior borderof the trochantin in experiments G^ (Figs. 2,3 ). But the principle is not very helpful for our main question, namely: why is there leg regeneration in one combination and no leg regeneration in the other combination? Though there is no exception to this principle in our experiments it cannot explain all phenomena of leg regeneration without additional assumptions. At this time there is no possibility of answering the question of whether

17 Tissue origin and symmetry in cockroach leg regenerates 97 'induction' really does take place. Therefore it would be better to avoid the term induction in this connexion. When I, despite this reservation, make use of the word, it is because I think the terms SIM and LIM are more informative than neutral expressions like 'anterior membrane' and 'posterior membrane'. Comparison of the regeneration field of Leucophaea and of other animals When features of the regeneration fields of other animals are compared with those of Leucophaea, one characteristic difference becomes very pronounced. In amphibia, for instance, one may elicit the regeneration of supernumerary legs by deviation of nerves to any point in the regeneration field. It seems that there are no qualitative differences in the different regions of the field; i.e. the cells lying dorsally or ventrally, anteriorly or posteriorly from the leg are equally qualified to build up a whole appendage, with its different surfaces. On the contrary, in Leucophaea there are the two principles represented by the sclerites and the LIM which only by a mutual interaction can produce leg regenerates. Neither of them alone is able to generate the structures formed after this interaction. The experiments of Noulin (1970, 1971) on Porcellio seem to indicate similar phenomena in Crustacea. Noulin transplanted pieces of the dorsal integument to the ventral surface of the abdomen. On two sides of the transplant, laterally and medially, supernumerary pleurepimers and pereiopods developed. Though the experiments are similar to ours, in so far as two different kinds of tissues are necessary for leg regeneration, they are not completely comparable. The incompatability of the combined ventral and dorsal tissues seems to initiate regeneration processes by which the missing tissues between host and implant are intercalated. Thus the pleural region is formed on both sides of the transplant and of course also the legs originating in this area. Therefore the experiments of Noulin concern the regeneration of the pleural region rather than the local conditions for leg regeneration. Some time ago Lender 8c Grobocopateli (1967) published a preliminary paper dealing with the regeneration field of the legs of Tenebrio molitor (Coleoptera) which unfortunately has not since been confirmed. According to the authors, the regeneration field includes major parts of the thoracic sternite. Even fragments of this regeneration field comprising only a quarter of the whole field are able to elicit a leg regenerate when transplanted to the second abdominal segment, irrespective of whether the pieces are taken from the anterior, posterior, medial or lateral region of the regeneration field. They also say that abdominal epidermis participates in leg regeneration when it is implanted into the regeneration field after extirpation of the leg, but this has not been proved. Recently, Bart & Browaeys-Poly (1973) reported experiments on the regeneration field of the wings of the orthopteran insect Sipyloidea. In this insect the regenerative capacity of the wing environment seems to be restricted to the latero-anterior region of the wing base. When this area is removed together with 7 E M B 32

18 98 H. BOHN the wing, no wing is regenerated; when the area is transplanted to an abdominal segment, a whole wing develops and, as it seems, without participation of the abdominal tissues. Contrary to the requirements for leg regeneration in Leiicophaea, this restricted area of the wing environment is sufficient for wing development; no interaction with another tissue seems to be necessary to allow the formation of the two surfaces of the wing. REFERENCES BART, A. (1970). Blessures et transformations trochanteriennes chez Carausius morosus Br. Annls. Embryol. Morph. 3, BART, A. (1971). Modalites de formation et de developpement d'un centre morphogenetique surnumeraire chez Carausius morosus Br. Wilhelm Roux Arch. EntwMech. Org. 168, BART, A., & BROWAEYS-POLY, E. (1973). Mise en evidence d'un territoire responsable de la regeneration del'aile metathoracique chez le phasme Sipyloidea sipylus Westwood (Insecte orthopteroide cheleutoptere). C. r. hebd. Seanc. Acad. Sci., Paris D 276, BODENSTEIN, D. (1937). Beintransplantationen an Lepidopterenraupen. IV. Zur Analyse experimentell erzeugter Bein-Mehrfachbildungen. Wilhelm Roux Arch. EntwMech. Org. 136, BOHN, H. (1965). Analyse der Regenerationsfahigkeit der Insektenextremitat durch Amputations- und Transplantationsversuche an Larven der Afrikanischen Schabe Leucophaea maderae Fabr. (Blattaria). II. Achsendetermination. Wilhelm Roux Arch. EntwMech. Org. 156, BOHN, H. (1972). The origin of the epidermis in the supernumerary regenerates of triple legs in cockroaches (Blattaria). /. Embryol. exp. Morph. 28, BOHN, H. (1974a). Extent and properties of the regeneration field in the larval legs of cockroaches {Leucophaea maderae). I. Extirpation experiments. /. Embryol. exp. Morph. 31, BOHN, H. (19746). Extent and properties of the regeneration field in the larval legs of cockroaches (Leucophaea maderae). II. Confirmation by transplantation experiments. /. Embryol. exp. Morph. 32, BOHN, H. (1974C). Pattern reconstitution in abdominal segment of Leucophaea madernae (Blattaria). Nature, Lond. 248, BULLIERE, D. (1970). Interpretation des regenerats multiples chez les Insectes. /. Embryol. exp. Morph. 23, LENDER, T. & GROBOCOPATELI, A. (1967). Etude du territoire de regeneration de la patte larvaire de Tenebrio molitor (Coleoptere). Bull. Soc. zool. Fr. 92, NOULIN, G. (1970). Conditions de la differentiation de pereiopodes surnumeraires uniques ou multiple chez l'oniscoide Porcellio dilatatus Brandt. C. r. hebd. Seanc. Acad. Sci., Paris D 270, NOULIN, G. (1971). Etude de la production de formations surnumeraires simples par greffe d'hypoderme et de la regeneration de formations surnumeraires doubles chez Porcellio dilatatus Brandt. C. r. hebd. Seanc. Acad. Sci., Paris D 272, PRZIBRAM, H. (1921). Die Bruchdreifachbildung im Tierreiche. Wilhelm Roux Arch. Entw- Mech. Org. 48, (Received 12 September 1973, revised 8 January 1974)