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Regeneration and duplication following operations in situ on the imaginal discs of Drosophila melanogaster
DEVELOPMENTAL
26, 606-615 (1971)
BIOLOGY
Regeneration
and Duplication
on the lmaginal
Following
Discs of Drosophila
Operations
in Situ
melanogaster’
PETERJ. BRYANT Developmental
Biology
Laboratory
and Center for Pathobiology, Irvine, California 92664
University
of California,
Irvine
Accepted August 13, 1971 A technique is described which allows defects to be made in situ in the imaginal discs of immature Drosophila larvae. Bisection of the second leg disc across the upper-lower axis results in regeneration of the remainder of the disc from the upper portion, and mirror-image duplication of the anlagen in the lower half (Figs. 2-5, Table 1). The upper half, retaining its connection to the larval epidermis, is able to evert; the lower half metamorphoses as an uneverted implant. Partial bisection of the disc often results in the production of branched legs in which one branch is complete but the other is a double half (Figs. 7-9). These cases can be interpreted as resulting from regeneration from one cut surface and duplication from the other. Pattern triplications have been obtained by partial bisections of the wing disc (Fig. 10). and these can be interpreted in a similar manner. It is suggested that regeneration and duplication are identical phenomena, resulting from the properties of of the anlagen at the cut edge. Cases of regeneration and duplication in other insect and vertebrate systems are discussed, and interpreted on the basis of gradients of developmental capacity (Fig. 11). INTRODUCTION
sophilu, suggested to Bodenstein (1941) that this stage was developmentally equivalent to the last larval instar of Lepidoptera, insofar as the determination of imaginal anlagen was concerned. More recently, several experiments have shown that mature imaginal discs of Drosophila can give rise to structures other than those for which they were determined, provided extra time is available before metamorphosis (Ursprung, 1959; Liiiind, 1961; Gehring, 1966). In the most thorough study of this question Schubiger (1971) has demonstrated the capacity for regeneration, duplication, transdetermination, and multiplication of units in imaginal leg disc fragments which are cultured in the adult for a few days prior to inducing metamorphosis by transplantation to a larval host. Furthermore, by several techniques it has been shown that the cells of the imaginal discs of Drosophila are not firmly committed to a particular pathway of differentiation in the early third instar (Vogt, 1946a,b; Bryant and Schneiderman, 1969), although quite a specific determination can be demonstrated in the ready-to-pupate larva (Ursprung, 1959; Schubiger, 1968).
In a classic series of experiments Bodenstein (1935, 1937, 1941) amassed a considerable amount of evidence to show that the imaginal leg anlagen of last instar lepidopteran larvae are not finally determined for adult structures; appropriate operative techniques could produce a variety of regenerative and duplicative phenomena in the resulting adult structures. On the other hand, the imaginal discs of last instar Drosophila larvae, when transplanted to host larvae, were able to produce only a fixed set of structures and appeared neither to duplicate nor to regenerate anlagen (Bodenstein, 1941). To reconcile these differences, Bodenstein (1941) suggested that perhaps the state of determination of the imaginal anlagen of larval Lepidoptera was passed through much earlier in the imaginal discs of Drosophila. The fact that Geigy (1931) could produce duplications of adult structures by UV-irradiating the embryo of Dro‘This research was HE-13194-01 from the and by Grant E-600 from awarded to Dr. Howard
supported in part by Grant National Institute of Health the American Cancer Society A. Schneiderman. 637
638
DEVELOPMENTAL
BIOLOGY
In view of the evidence for lability of determination in imaginal discs, it seemed worthwhile to reconsider Bodenstein’s (1941) conclusion that there is a substantial difference in the timing of determination of imaginal anlagen in Diptera as compared with that in Lepidoptera. Accordingly, we developed a technique for producing defects in the imaginal discs of Drosophila larvae without extirpation. Some previous studies have employed a similar technique. For instance, Braun (1940) and Lees (1941) were able to produce defects in wing development by puncturing the developing wing in the pupa. Zalokar (1943) and Pantelouris and Waddington (1955) were able to extirpate various imaginal discs from the larva, and they noted the absence of the corresponding structures from the resulting adult. No regeneration or duplication was detected in these earlier studies, except that Pantelouris and Waddington (1955) observed a few cases where mesonotal structures developed on the side from which a wing disc had been removed. Since the mesonotum is a derivative of the wing disc, Pantelouris and Waddington (1955) concluded that the effects of extirpation could sometimes be compensated for by regulation by the contralateral wing disc. This interpretation was later disproved by Murphy (1967), who showed that thorax material developed on the operated side only in cases of partial extirpation. Hence, none of the previous studies of operations on mature imaginal discs in situ have provided convincing evidence for regeneration or duplication of anlagen. In this report it will be shown that operations in situ on the imaginal discs of Drosophila result in both regeneration and duplication of anlagen. The experiments differ from those of previous workers in that they were performed on young larvae (approximately one day before puparium formation) and that the operations were simple bisections rather than extirpations.
VOLUME26, 1971 MATERIALS
AND
METHODS
We used larvae of the Oregon R-C stock grown at 25’C on cornmeal-yeast-agar medium. The operations were performed at 96 f 2 hr after oviposition; this corresponds to approximately one day before puparium formation under our culture conditions, although the operation itself possibly causes some delay in puparium formation. A few experiments performed at 72 f 2 hr after oviposition gave similar results, but with a lower success rate. The larvae were etherized for 1.5-2 min, placed in Ringer’s solution, and viewed with transmitted light. Most of the internal organs including all the major imaginal discs are visible through the transparent cuticle. The experiments reported here are restricted to the second leg disc and the wing disc, since these are most accessible. The operations were performed with a tungsten needle sharpened by erosion in hot sodium nitrite. One side of the larva was compressed, in the region of an imaginal disc, between the needle and the floor of the dish. The needle was held perpendicular to the anterior-posterior axis of the larva. In many cases the operation could be performed without injury to the larval cuticle, but in other cases the cuticle was punctured. Only in favorable cases was it possible to determine, by direct observation, whether the disc had been bisected. The data presented in this paper are based on a total of 804 operations at 96 hr, and some results from operations at 72 hr have been included. The larvae were placed in yeasted vials and allowed to proceed with development and metamorphosis. All the resulting adults were dissected, and whole mount preparations were made, using Gurr’s water mounting medium, of any structures of interest. These were then examined at 1000 x magnification. RESULTS
Second Leg Discs The results are best understood by refer-
BRYANT
Regeneration and Duplication in Drosophila
ence to the anlage plan of the leg disc (Fig. 1) constructed by Schubiger (1968). Schubiger’s map refers to the male first leg, from which the second leg differs in the following ways: (1) The second leg lacks sensilla trichodea (GSt) on the thorax segment; has only one, instead of two rows of sensilla trichodea (RSt) on the coxa; and has no transverse bristle rows (TR) on the tibia or basitarsus, or sex combs (GK) on the basitarsus. (2) Some additional useful markers are present on the derivatives of the second leg disc: the large sternopleural bristles on the side of the thorax, and the preapical (PA) and apical (A) bristles on the distal
639
part of the tibia. Extrapolation from the morphology of the leg indicates that the preapical bristle is located in the anlage plan near the femur Scl, and the apical bristle near the femur Sell (see Fig. 1). These two bristles can be distinguished by the fact that the apical bristle is more distal than the preapical, and is associated with spur bristles (see Figs. 4, 7, and 8). The survival rate in these experiments was approximately 50%. In about 20% of the survivors the operation was successful: either the appendage was abnormal in appearance, or there was extra second leg material, uneverted, inside the thorax or abdo-
FIG. 1. Anlageplan of the male first leg disc, according to Schubiger (1968). See Schubiger (1968) for description of markers. PT, thorax; Co, coxa; Tr, trochanter; Fe, femur; Ti, tibia; TG, tarsus; Scl, single sensillum campaniforme; Stl, single sensillum trichodeum; Sc+5, group of 5 sensilla campaniformia on hairy cuticle; StE, group of 8 sensilla trichodea; GSt, group of sensilla trichodea; TR, transverse bristle rows; GK, sex comb; GTr, joint from coxa to trochanter; Sell, group of 11 sensilla campaniformia; St5, group of 5 sensilla trichodea; BH, bristle on hairy island; Sc3, group of 3 sensilla campaniformia; grB, large bristles; Kl, claw organ; SC-~, group of 8 sensilla campaniformia on naked cuticle; EB, edge bristle; ZRSt, 2 rows of sensilla trichodea; GTh, joint from coxa to thorax. Sc+5 and SC-8 are here considered together as Sc13. TR, GK, thoracic GSt and one of the coxa RSt are absent from the second leg.
640
DEVELOPMENTAL
BIOLOGY
men of the animal. These cases can be divided into six classes: 1. No second leg derivatives on one side of the animal (15 cases). In most of these cases an uneverted second leg, complete with sternopleura was found inside the animal. These cases are interpreted as resulting from severance of the stalk to the epidermis without damage to the disc itself. They were found frequently in another series of experiments where the operations were performed on mature (120 hr) larvae; in those cases it was possible to observe that the disc frequently slipped from under the needle, making severance of the stalk extremely likely. 2. The second leg on one side was represented by a small amount of material on the outside of the fly, with a large amount of material present as an uneverted “implant” (13 cases). In these cases, the “implant” was usually a complete leg including coxa and sternopleura, and the structures present on the outside included a recognizable sternopleura, sometimes with a reduced number of bristles. In five cases coxal structures were present on both outside and inside. However, it was sometimes difficult to ascertain whether coxal structures were present on the outside or whether the sternopleura was duplicated. Our interpretation of these cases is that the disc was cut through the presumptive sternopleural region, and that this was followed by a limited regeneration from one or both of the resulting fragments. 3. The most interesting cases are those in which the second leg was complete, or fairly complete, on the outside of the animal, but in which a rather large amount of material derived from the second leg disc was also present as an uneverted “implant” (14 cases). In these cases the implant invariably showed mirror-image duplication of some or all of the structures depicted in the lower half of the anlage plan. Figure 2 shows part of such an implant, in which the markers Sc3, St5, and BH- are all clearly duplicated. In this fragment as with other
VOLUME
26, 1971
favorable cases, there is also clear evidence of mirror-image duplication of bristle patterns which are not included in the inventory of specific markers. Table 1 catalogs in detail the occurrence of specific markers in the animals showing this class of result. This table includes some results from operations performed at 72 hr, which gave essentially similar results to those performed at 96 hr. It can be seen from this table that duplication of the Sc3 and St5 of the trochanter is especially common in the implant, as is that of the BH- and RSt of the coxa, of the Sell of the femur (Fig. 3) and of the apical bristle of the tibia (Fig. 4). All of these markers are to be found in the lower half of the anlage plan with the exception of the RSt of the coxa. This exception could be due to a difference of the position of this marker in the anlage plan of the second leg disc, compared to the first, or to a limited regenerative capacity of the lower half of the disc. In two cases (Fig. 5) the tarsal segments were duplicated and sepaxated in the implant. In the case of the GSt of the trochanter, St8 of the coxa, and the tarsus, it was often difficult to classify the structures as either single or duplicated. This was because in many cases, the size of the marker was increased to less than double the normal size; for example Fig. 6 shows a group of 13 sensilla trichodea on the coxa, which correspond to the normal St6 Our interpretation of these cases is that they result from duplication of a part of the anlage for the markers. All three of the structures in which this phenomenon was encountered, lie approximately half way along the upper-lower axis of the disc; hence a bisection of the disc would be expected frequently to bisect these anlagen. In many cases of class 3, the second leg on the operated side was abnormal, incompletely everted, or lacking the distal segments. However in two cases, the operated leg was completely normal, as far as could be ascertained. In nearly all cases some markers could be found on the everted leg which were present or duplicated in the
BRYANT
FIG. 2. Mirror-image
Regeneration
pattern duplication
FIG. 3. Two Sell
and Duplication
641
in Drosophila
of part of the coxa and part of the trochanter.
Markers
as in Fig. 1.
markers in the femur from the same implant.
implant. We therefore conclude that the half of the disc which remains attached to the epidermal stalk (the upper half in the anlage plan) is capable of regenerating the remainder of the disc. Duplication of markers was found in only one case in the everted part, and is evidently exceptional (Table 1). 4. There were four cases where all the everted legs were normal, but in which there was a small amount of material, uneverted inside the animal. In these cases it was dif-
ficult to analyze the uneverted portion due to lack of markers, so they will not be discussed further. They are interpreted as having been cut in the lower part of the disc. 5. Nine cases were obtained where the operated leg was partially or completely missing. These are probably due to extensive damage of the disc during the operation. 6. Fifteen cases of branched, everted legs were obtained. In five cases the branch occurred in the sternopleura-coxa region;
642
hVF&OPMENTAL
BIOLOGY
VOLUME
FIG. 4. Duplicated apical bristle apical bristle; S, spur bristle.
FIG.
5. Duplicated
26, 1971
of the tibia.
A,
tarsus.
FIG. 6. Part of a duplicated
coxa, showing enlarged St8 marker.
in four cases in the femur region; in five cases in the tibia region; and in one case in the tarsal region. In eight of these cases one of the branches was small and sometimes poorly differentiated. However, the other seven cases showed two branches of roughly equal size (Fig. 7). In three cases it was pos-
sible to ascertain that one of the branches of the leg consisted not of complete segments, but of two fused longitudinal halves of the segments. These fused halves correspond to the lower halves of the segments in the anlage plan, as shown by the duplication in these cases of the tibia1 apical bristle,
Regeneration
BRYANT
and Duplication TABLE
ABSENCE, PRESENCE, AND DUPLICATION
643
in Drosophila
1
OF MARKERS
BISECTION OF THE SECOND LEG Dac~
FOLLOWING
Everted part Case No.
RSt
St8
SC13
St1
EB
Scl
PA
lb 2 3b 4 5 6 7 8 96 10 11 12 13 14
+ + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + +
+ +
+ +
+
+ + +
+ + + +
t + + + + + + + + ‘2 +
5 9
2 12
Totals Absent Single Duplicated
+ + +
+ +
2
11 1
+ + + + + +
+ + +
+
+ + +
+ + + +
3 11
1 13
+
+ +
8 6
Noneverted lb 2 36 4 5 6 7 8 96 10 11 12 13 14
1 12 1 part
+ +
+ + 2 2 2
TG
A
SC11
SC3
+ + + + + + + + + +
+ + + + + + + + +
+ + +
+ + + +
+ +
+
+
+
+
+ +
+ +
+
+
+ +
+
+ +
+
2 12
3 11
6 8
6 8
5 9
5 9
10 4
2 (+I (f) + ($1
2 2 2 + 2
C-t) 2 2 2 2
+ 2 + 2 2
+ 2 2 2 2
2 2 2 2
2
+
+ ($1 +
2 + + ‘+
($)
+
+
+
+
+
(+I (+I t-t) (f)
+
+
2
+ +
+ +
+
2
2 2
2 2 2 2 2
2 2 2 2 2
2 2 2 2 2 2
+ 2
Totals Absent 13 3 3 1 7 14 14 14 14 4 Single 1 11 12 4 3 7 3 Duplicated 7 2 7 10 u See Fig. 1 and Schubiger (1968) for nomenclature of markers. PA, tibia1 preapical bristle; bristle; f, present; (+), enlarged or partially duplicated; 2, duplicated. b Operation performed at 72 * 2 hr after oviposition. The remainder were performed at oviposition.
with absence of the preapical bristle (Fig. 8). We interpret these cases as being due to incomplete cutting of the disc. This would then be followed by regeneration from one of the surfaces bared by the cut, and mirrorimage duplication from the other surface. The arrangement of tibia1 apical and preapical bristles indicates that it is the upper
+ + + + +
+
+
-t
2
+ + t + +
BH
+
+ + + + +
St5
+ +
+ (1,
2 2
2
GSt
+
+ 2 2 2 2 + 2 2
2 2 2 3 2 9 11 10 A, tibia1 apical 96 i
2 hr after
surface which regenerates and the lower surface which duplicates. In one case the two distal parts in the duplicated branch have separated, thus giving an appendage with three extremities (Fig. 9). The wing Disc The results from 100 operations in situ on
644
DEVELOPMENTAL
FIG.
7. Branched
leg resulting
BIOLOGY
from operation
FIG. 8. Distal parts of the tibias from the preparation
the wing disc at 96 hr. indicate that it behaves in a similar fashion to the leg disc. Eleven of the cases where the entire operated disc everted showed clear evidence of duplication or triplication of parts of the pattern. This could be seen most clearly in five cases where six dorsocentral bristles were present on the operated side, instead of the normal two. Figure 10 shows one
VOLUME
26, 1971
in situ on the imaginal disc. Fe, femur; Ti, tibia.
of Fig. 7. PA, preapical bristle; A, apical bristle.
such case, in which the half-scutellum on the operated side also appears to be triplicated. This response is analogous to the production of branched legs (Fig. 7), which also show triplication of parts of the structure (e.g., apical tibia1 bristle). DISCUSSION
The results described here can be sum-
BRYANT
Regeneration
/
/
/
/
/
and Duplication
645
in Drosophila
/-
,
O.lmm
FIG. 9. Camera lucida drawing of a branched leg in which the two halves of the “double separated, giving a triple extremity.
half’
branch are
646
DEVELOPMENTAL BIOLOGY
VOLUME 26, 1971
0.1mm
FIG. 10. Camera lucida drawing of a thorax with partial triplication resulting from an operation performed at 96 hr on the wing disc in situ (microchaetes omitted). DC, pairs of dorsocentral bristles; S, scutellum; W, wing.
marized by the statement that bisection of the leg disc (in the horizontal direction in the anlage plan) is followed by regeneration of the remainder of the disc from the upper part and duplication of structures, with mirror-image symmetry, from the lower part. The regenerated portion is able to ever& since it retains a connection to the larval epidermis. The latter connection is crucial to the process of eversion, which involves a sequential replacement of larval epidermal cells by the contiguous cells from the imaginal disc (Poodry and Schneiderman, 1970). During this process the integrity of the single-layered epithelium is retained. The duplicated portion, lacking a connection to the epidermis, does not evert but forms an “implant,” which is a vesicle with bristles and hairs differentiated on the inside, just as is formed by transplanted imaginal discs.
Partial bisection of the leg disc appears also to be followed by regeneration from the upper surface of the cut and duplication from the lower cut surface. This results in triplication of certain structures, producing legs with two, or sometimes three, distal parts. In an exactly analogous manner, partial bisection of the wing disc results in triplication of certain of its derivatives. These experiments establish that “multiple regeneration” can be induced in Drosophila, by surgical operations on the imaginal discs of immature larvae. The multiple regenerates obtained are exactly analogous to those obtained by disturbing the integrity of leg anlagen in the last larval instar of Lepidoptera, such as F’hryganidiu (Bodenstein, 1941). Hence, there is no longer any reason to assume that the determinative events occurring in the imaginal discs of the last larval instar of Drosophila are in any
BRYANT
Regeneration
and Duplication
fundamental way different from the events occurring in the leg anlagen of the last larval instar of Lepidoptera (cf. Bodenstein, 1941). The availability of the two alternatives of regeneration or mirror-image duplication is a common observation in developing insect systems subjected to surgical intervention. Using transplantation techniques, Schubiger (1971) has obtained results closely similar to those reported here; the upper half of the mature leg disc can regenerate the remainder of the disc, but the lower half usually duplicates its anlagen. Furthermore he was able to show that bisection of the disc into medial and lateral parts gives similar results; the medial half regenerates and the lateral half duplicates. A similar result was also obtained for the antenna1 disc by Gehring (1966), who showed that the posterior region was able to regenerate the anterior, palpus region, but that the palpus region showed a high tendency to duplicate. This type of behavior is well known from studies of leg regeneration in other insects, which show that proximal parts can regenerate the remainder of the leg, but that distal parts are only able to duplicate. For example, when the developing leg of Periplaneta is amputated, rotated axially through 180 degrees, and attached back to the stump in the new orientation, two wound surfaces are created. The surface of the stump regenerates the remainder of the leg, while the surface of the distal portion produces a mirror-image duplication of the amputated part (Bodenstein, 1962). Similarly, transplantations of limbs with reversed antero-posterior or dorsoventral (interior-exterior) axes in Phryganidia (Bodenstein, 1937); Caruusius (Bart, 1965a, 1971); Leucophaea (Bohn, 1965b) ; Periplaneta (Penzlin, 1965); or Blabera (Bulliere, 1970) results in triplicated distal portions. Many of these cases can also be interpreted as resulting from regeneration from part of the proximal wound surface, with duplication from part of the distal wound surface (Przi-
in Drosophila
647
bram, 1931; Bodenstein, 1962; Bull&e, 1970). The supernumerary regenerates are often formed by a fusion of tissue from both sources (Bohn, 1965b). When a piece of the coxa in Curausius is transplanted on to the opposite surface of the coxa of the host, it causes regeneration of a supernumerary limb. However, if a piece of femur is so grafted, the resulting regenerate contains only those segments distal to the femur (Bart, 1965b; see also Bohn, 1965b). In Culex (Spinner, 1969), Drosophila (Schubiger, 1971), and Tenebrio (Hadom et al., 1969), parts of the leg anlagen corresponding to presumptive proximal parts can regenerate presumptive distal parts, but the reverse is not the case. In some cases, duplication of distal parts occurs (Hadom et al., 1969). The regenerating limbs of amphibians appear to be subject to similar limitations. When the distal part of a limb is amputated and grafted back on to the animal with its proximodistal axis reversed, regeneration proceeds from the free surface. The regenerate forms a new distal structure which is a mirror-image of the transplanted piece (Przibram, 1931; Dent, 1954; Butler, 1955). If a limb is cut part way across, thus producing a proximal and a distal wound surface, both surfaces regenerate distal parts (Przibram, 1931). Regeneration of proximal parts from more distal levels is not observed in these cases. All these findings can be interpreted in terms of a gradient of developmental capacity, the basic feature of which is that cells at any one level can, when given the opportunity for growth and cell division, produce only those structures further down the gradient (Fig. 11). Following bisection of the system, the cells at both cut surfaces produce those structures which are at lower levels in the gradient. In the representation of Fig. 11, cells at a level C have the developmental capacity to construct structures D and E but not A and B. The cells at each side of the cut surface are at roughly the
DEVELOPMENTAL BIOLOGY
VOLUME26, 1971
POSITION
I +
A
BISSECTION
B C
C
D b
E
b GROWTH
\
REGENERATION FIG. 11. The postulated
DUPLICATION
gradient of developmental
same point in the gradient, so they are able to produce the same structures, D and E. With the cut surface of the ABC fragment, the addition of DE corresponds to regeneration but with the CDE fragment it represents mirror-image duplication. Hence, with this interpretation regeneration and duplication are considered as identical phenomena, the final results differing only in the topological relationships of the new
capacity and its response to bisection.
parts to the old. Bohn (1965a,b) proposes a similar gradient of developmental potency to account for various aspects of leg regeneration in Leucophaea. In the case of the proximodistal axis of limbs, this is Rose’s (1962) “rule of distal transformation.” Wolpert (1971) reasserts this rule in suggesting that cells can change their positional values only in the distal direction. We suggest, however, that this behavior is not merely a
BRYANT
Regenemtion
and. Duplication
property of the proximodistal axis but, in the case of imaginal discs at least, reflects a more general feature of their developmental organization. The credibility of this model would be enhanced if it could be shown that regeneration or duplication of insect zppendages or imaginal disc fragments results from the activity of cells at the cut edge. Bohn’s (1965a) transplantation experiments and Bulliere’s (1971) X-irradiation experiments indicate that this is the case for leg regeneration in Orthoptera. As far as imaginal discs are concerned, evidence from autoradiography (Wildermuth, 1968); genetic cell marking (Postlethwait et al., 1971; Ulrich, 1971); direction observations of developing fragments (Ursprung, 1962); and mitotic counts (in Ephestia wing discs; Kroeger, 1958) are all consistent with, but do not give direct proof for this suggestion. However, the assumption is not crucial for the model presented; it can also be discussed in terms of the regeneration or duplication of anlagen without any regard for the exact clonal origin of the cells. The response to bisection would then be a function of the anlagen which are left at the cut surface; C produces D and E whether it is a part of the ABC or CDE fragment. This in fact, is what is indicated by the results presented in this paper. However, an experiment by Niithiger and Schubiger (1966) is rather difficult to explain on this basis. These authors showed that if a UV burn was made in a half-genital disc which was subsequently allowed to duplicate, the defect was found in both copies of the pattern. This somewhat surprising result cannot be accommodated into our model except by assuming that the genital disc has a complex anlage structure. For instance, if it were an assemblage of separately regulating and duplicating anlagen, one of which is completely removed by the UV burn, then the difficulty of explaining the experiment is reduced. Hadorn et al (1949) and Liiijnd (1961) have in fact shown that the genital disc is a complex of separately regulating
in Drosophila
649
morphogenetic fields. The gradients of developmental capacity which we have discussed reflect graded differences in the prospective potencies of the parts of the developing system. Growth and cell division occur during, and are presumably indispensable for, regeneration and duplication in many of the cases we have discussed (Kroeger, 1958; Niithiger and Schubiger, 1966; Wildermuth, 1968). Hence, the postulated gradient refers, strictly, to the developmental capacity of the progency of the cells involved. Underlying gradients of morphogenetic influences have been postulated to account for the control of growth, polarity, and pattern in insect appendages (see Lawrence, 1970 for review). Whether all these concepts can be referred to a single underlying gradient is not clear from the reported experiments, but such a unified control mechanism has, of course, the advantage of simplicity. The results of fragmentation experiments on imaginal discs can alternatively be explained, not on the basis of graded, quantitative differences in developmental capacity, but by assuming that the discs are composed of two types of qualitatively different cells (Wildermuth, 1968; Schubiger, 1971). One cell type would have the capacity to regenerate missing parts of the disc, whereas the other type would have only the ability to duplicate structures. A gradient in the concentration of regenerative cells could, given certain assumptions, mimic the behavior of the gradient of developmental capacity postulated here. The author would like to express his sincere appreciation to Dr. Howard A. Schneiderman for his interest, enthusiasm, and criticism throughout the course of this work. REFERENCES BART, A. (1965h) Induction experimentale dune numeraires au tours de la regeneration des pattes chez Carau.&.s m~r~su.s Br. C. R. Acad. Sci. 261, 190-1903. BART, A. (1965b) Induction exljerimentale d’une morphogenese accompagnant ou non la regeneration de la patte de Camusius morosus Br. C. R.
650
DEVELOPMENTAL BIOLOGSY
Acad. Sci. 261, 5619-5622. BART, A. (1971). Morphogenese sumumeraire au niveau de la patte du phasme Camusius morosas Br. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 166, 331-364. BODENSTEIN D. (1935). Beintransplantationen an Lepidopterenraupen. III. Zur Analyse der Entwicklungspotenzen der Schmetterhngsbeine. Wilhelm Roux’ Arch. Entwicklnngsmech. Organismen 133, 156-192. BODENSTEIN, D. (1937). Beintransplantationen an Lepidopterenraupen. IV. Zur Analyse Experimentell-erzeugter Bein-Mehrfachbildungen. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 136, 745-785. BODENSTEIN, D. (1941). Investigations on the problem of metamorphosis. VIII. Studies on leg determination in insects. J. Exp. Zool. 87, 31-53. BODENSTEIN, D. (1962). Regeneration in insects. Symp Genet. Biol. Ital. 9, l-19. BOHN, H. (1965a). Analyse der Regenerationsmigkeit der InsektenextremiEt durch Amputations-und Transplantationsversuche an Larven der afrikanischen Schabe Leucophaea maderue Fabr. I. Mitt. Regenerationspotenzen. Wilhelm Roax’ Arch. Entwicklungsmech. Organismen 156, 49-74. BOHN, H. (1965b). Analyse der Regenerationsflihigkeit der Insektenextremitst durch Amputations und Transplantationsversuche an Larven der afrikanischen Schabe Leuocophaea maderae Fabr. II. Mitt. Achsendetermination. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 156, 449-503. BRAUN, W. (1940) The effect of punctures on the developing wings of several mutants of Drosophila melanogaster. J. Exp. Zool. 84, 325-349. BRYANT, P. J., and SCHNEIDERMAN, H. A. (1969). Cell lineage, growth, and determination in the imaginal leg discs of Drosophila melanogaster. Deuelop. Biol. 20, 263-290. BULL&RE, D. (1970). Interpretation des regenerats multiples chez les Insectes. J. Embryol. Exp. Morphol. 23, 337-357. BULLI~RE, D. (1971). Les mecanismes de la regeneration Studies chez B!abera craniifer (Insecte Dictyopt&e) a l’aide d’irradiations localisees aux rayons X. Deuelop. Viol. 24, 443-463. BUTLER, E. G. (1955). Regeneration of the urodele forelimb after reversal of its proximo-distal axis. J. Morphol. 96, 265-282. DENT, J. N. (1954). A study of regeneration emanating from limb transplants with reversed proximo-distal polarity in the adult newt. Anat Rec. 118, 841-856. GEHRING, W. (1966). ijbertragung und bderung der Determinationsqualitiiten in AntennenscheibenKulturen von Drosophila metanogaster. J. Embryol. Exp. Morphol. 15, 777111. GEIGY, R. (1931). Erzeugung rein imaginaler Defekte durch ultraviolette Eibestrahlung bei Drosophila
VOLUME 26, 1971
melnnogaster. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen. 125, 406-447. HADORN, E., BERTANI, G., and GALLERA, J. (1949). Regulationsfahigkeit und Feldorganisation der mtinlichen Genital-Imaginalscheibe von Drosophila melanogaster. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 144, 31-70. HADORN, E., HAURI, H., MUELLER,P., and STEINER, E. (1969). Transplantationsexperimente mit Imaginalanlagen von Tenebrio molitor L. Rev. Suisse 2001. 76, 642-649. KROEGER, H. (1958). l?ber Doppelbildungen in die Liebeshiihle verpflanzter Fliigelimaginalschieben von Ephestia ktihnielia 2. Wilhelm Roux’ Arch. Entwicklnngsmech. Organismen 150, 401-424. LAWRENCE, P. A. (1970). Polarity and patterns in the postembryonic development of insects. Aduan. Insect Physiol. 7, 197-226. LEES, A. D. (1941). Operations on the pupal wing of Drosophila melanogaster. J. Genet. 42, 115-142. MIND, H. (1961). Untersuchungen zur Mustergliederung in fragmentierten Primordien des m&mlichen Geschlectsapparates von Drosophila seguyi Deuelop. Biol. 3, 615-656. MURPHY, C. (1967). Determination of the dorsal mesothoracic disc in Drosophila. Deuelop. Biol. 15, 368-394. NGTHIGER, R., and SCHUBIGER, G. (1966). Developmental behaviour of fragments of symmetrical and asymmetrical imaginal discs of Drosophila melanogaster (Diptera). J. Embryol. Exp. Morphol. 16, 355-368. PANTELOURIS, E. M., and WADDINGTON, C. H. (1955). Regulation capacities of the wing and haltere discs of wild type and bithorax Drosophila. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 147, 539-546. PENZLIN, H. (1965). Experimentelle Erzeugung von Dreifachbildungen bei einem heterometabolen Insekt. Naturwissenschaften 52, 217. POODRY, C. A., and SCHNEIDERMAN, H. A. (1970). The ultrastructure of the developing leg of Drosophila melanogaster. Wilhelm Roux’ Arch. Entwicklangsmech. Organismen 166, l-44. POSTLETHWAIT, J., POODRY, C. A., and SCHNEIDERMAN, H. A. (1971). Cellular dynamics of pattern duplication in imaginal discs of Drosophila melanogaster. Deuelop. Biol. in press. Laws in Animal PRZIBRAM, H. (1931). “Connecting Morphology.” Univ. of London Press, London. BOSE, S. M. (1962). Tissue-arc control of regeneration in the amphibian limb. Symp. Sot. Study Deuelop. Growth 20. SCHUBIGER, G. (1968). Anlageplan, Determinationsz&and und Transdeterminationsleistungen der mannlichen Vorderbeinscheibe von Drosophila mekmogaster. Wilhelm Roax’ Arch. Entwicklungsmech. Organismen 160, 9-40.
BRYANT SCHUBIGER, G. (1971). Regeneration,
Regeneration
and Duplication
duplication, and transdetermination in fragments of the leg disc of Drosophila melnnogaster. Develop. Biol. in press SPINNER, W. (1969). Transplantationsversuche zur Blastemgliederung, Regenerationsund Differenzierungsleistung der Beinanlagen von Culex pipiens (L.) Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 163, 259-286. ULRICH, E. (1971). Cell lineage, Determination und Regulation in der weiblichen Genitalimaginalscheibe von Drosophila melanogaster. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 167, 64-82. URSPRUNG, H. (1959). Fragmentierungsund Bestrahhmgsversuche zur Bestimmung von Determinationszustand und Anlageplan der Genitalscheiben von Drosophila melanogaster. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 151, 504-558. URSPRUNG, H. (1962). Einfluss des Wirtsalters auf die Entwickhmgsleistung von Sagittalhalften marmlicher Genitalschieben von Drosophila melanogaster.
in Drosophila
653
Develop. Biol. 4, 22-39. VOGT, M. (1946a). Zur labilen Determination der Imaginalscheiben von Drosophila. I. Verhalten verschiedenaltriger hnaginalanlagen bei operativer Defektsetzung. Biol. Zent. 65, 223-238. VOGT, M. (194613). Zur labilen Determination der Imaginalscheiben von Drosophila. II. Die Umwandlung prasumptiven Ftihlergewebes in Beingewebe. Biol. Zentralbl. 65, 238-254. WILDERMUTH, H. (1968). Autoradiographische Untersuchungen zum Vermehrungsmuster der Zellen in proliferierenden Riisselprimordien von Drosophila melanogaster. Develop. Biol. 18, 1-13. WOLPERT, L. (1971). Positional information and pattern formation. Current Topics in Developmental Biology 6 (in press). ZALOKAR, M. (1943). L’ablation des disques imaginaux chez la larve de Drosophile. Rev. S&se Zool. 50, 232-237.
26, 606-615 (1971)
BIOLOGY
Regeneration
and Duplication
on the lmaginal
Following
Discs of Drosophila
Operations
in Situ
melanogaster’
PETERJ. BRYANT Developmental
Biology
Laboratory
and Center for Pathobiology, Irvine, California 92664
University
of California,
Irvine
Accepted August 13, 1971 A technique is described which allows defects to be made in situ in the imaginal discs of immature Drosophila larvae. Bisection of the second leg disc across the upper-lower axis results in regeneration of the remainder of the disc from the upper portion, and mirror-image duplication of the anlagen in the lower half (Figs. 2-5, Table 1). The upper half, retaining its connection to the larval epidermis, is able to evert; the lower half metamorphoses as an uneverted implant. Partial bisection of the disc often results in the production of branched legs in which one branch is complete but the other is a double half (Figs. 7-9). These cases can be interpreted as resulting from regeneration from one cut surface and duplication from the other. Pattern triplications have been obtained by partial bisections of the wing disc (Fig. 10). and these can be interpreted in a similar manner. It is suggested that regeneration and duplication are identical phenomena, resulting from the properties of of the anlagen at the cut edge. Cases of regeneration and duplication in other insect and vertebrate systems are discussed, and interpreted on the basis of gradients of developmental capacity (Fig. 11). INTRODUCTION
sophilu, suggested to Bodenstein (1941) that this stage was developmentally equivalent to the last larval instar of Lepidoptera, insofar as the determination of imaginal anlagen was concerned. More recently, several experiments have shown that mature imaginal discs of Drosophila can give rise to structures other than those for which they were determined, provided extra time is available before metamorphosis (Ursprung, 1959; Liiiind, 1961; Gehring, 1966). In the most thorough study of this question Schubiger (1971) has demonstrated the capacity for regeneration, duplication, transdetermination, and multiplication of units in imaginal leg disc fragments which are cultured in the adult for a few days prior to inducing metamorphosis by transplantation to a larval host. Furthermore, by several techniques it has been shown that the cells of the imaginal discs of Drosophila are not firmly committed to a particular pathway of differentiation in the early third instar (Vogt, 1946a,b; Bryant and Schneiderman, 1969), although quite a specific determination can be demonstrated in the ready-to-pupate larva (Ursprung, 1959; Schubiger, 1968).
In a classic series of experiments Bodenstein (1935, 1937, 1941) amassed a considerable amount of evidence to show that the imaginal leg anlagen of last instar lepidopteran larvae are not finally determined for adult structures; appropriate operative techniques could produce a variety of regenerative and duplicative phenomena in the resulting adult structures. On the other hand, the imaginal discs of last instar Drosophila larvae, when transplanted to host larvae, were able to produce only a fixed set of structures and appeared neither to duplicate nor to regenerate anlagen (Bodenstein, 1941). To reconcile these differences, Bodenstein (1941) suggested that perhaps the state of determination of the imaginal anlagen of larval Lepidoptera was passed through much earlier in the imaginal discs of Drosophila. The fact that Geigy (1931) could produce duplications of adult structures by UV-irradiating the embryo of Dro‘This research was HE-13194-01 from the and by Grant E-600 from awarded to Dr. Howard
supported in part by Grant National Institute of Health the American Cancer Society A. Schneiderman. 637
638
DEVELOPMENTAL
BIOLOGY
In view of the evidence for lability of determination in imaginal discs, it seemed worthwhile to reconsider Bodenstein’s (1941) conclusion that there is a substantial difference in the timing of determination of imaginal anlagen in Diptera as compared with that in Lepidoptera. Accordingly, we developed a technique for producing defects in the imaginal discs of Drosophila larvae without extirpation. Some previous studies have employed a similar technique. For instance, Braun (1940) and Lees (1941) were able to produce defects in wing development by puncturing the developing wing in the pupa. Zalokar (1943) and Pantelouris and Waddington (1955) were able to extirpate various imaginal discs from the larva, and they noted the absence of the corresponding structures from the resulting adult. No regeneration or duplication was detected in these earlier studies, except that Pantelouris and Waddington (1955) observed a few cases where mesonotal structures developed on the side from which a wing disc had been removed. Since the mesonotum is a derivative of the wing disc, Pantelouris and Waddington (1955) concluded that the effects of extirpation could sometimes be compensated for by regulation by the contralateral wing disc. This interpretation was later disproved by Murphy (1967), who showed that thorax material developed on the operated side only in cases of partial extirpation. Hence, none of the previous studies of operations on mature imaginal discs in situ have provided convincing evidence for regeneration or duplication of anlagen. In this report it will be shown that operations in situ on the imaginal discs of Drosophila result in both regeneration and duplication of anlagen. The experiments differ from those of previous workers in that they were performed on young larvae (approximately one day before puparium formation) and that the operations were simple bisections rather than extirpations.
VOLUME26, 1971 MATERIALS
AND
METHODS
We used larvae of the Oregon R-C stock grown at 25’C on cornmeal-yeast-agar medium. The operations were performed at 96 f 2 hr after oviposition; this corresponds to approximately one day before puparium formation under our culture conditions, although the operation itself possibly causes some delay in puparium formation. A few experiments performed at 72 f 2 hr after oviposition gave similar results, but with a lower success rate. The larvae were etherized for 1.5-2 min, placed in Ringer’s solution, and viewed with transmitted light. Most of the internal organs including all the major imaginal discs are visible through the transparent cuticle. The experiments reported here are restricted to the second leg disc and the wing disc, since these are most accessible. The operations were performed with a tungsten needle sharpened by erosion in hot sodium nitrite. One side of the larva was compressed, in the region of an imaginal disc, between the needle and the floor of the dish. The needle was held perpendicular to the anterior-posterior axis of the larva. In many cases the operation could be performed without injury to the larval cuticle, but in other cases the cuticle was punctured. Only in favorable cases was it possible to determine, by direct observation, whether the disc had been bisected. The data presented in this paper are based on a total of 804 operations at 96 hr, and some results from operations at 72 hr have been included. The larvae were placed in yeasted vials and allowed to proceed with development and metamorphosis. All the resulting adults were dissected, and whole mount preparations were made, using Gurr’s water mounting medium, of any structures of interest. These were then examined at 1000 x magnification. RESULTS
Second Leg Discs The results are best understood by refer-
BRYANT
Regeneration and Duplication in Drosophila
ence to the anlage plan of the leg disc (Fig. 1) constructed by Schubiger (1968). Schubiger’s map refers to the male first leg, from which the second leg differs in the following ways: (1) The second leg lacks sensilla trichodea (GSt) on the thorax segment; has only one, instead of two rows of sensilla trichodea (RSt) on the coxa; and has no transverse bristle rows (TR) on the tibia or basitarsus, or sex combs (GK) on the basitarsus. (2) Some additional useful markers are present on the derivatives of the second leg disc: the large sternopleural bristles on the side of the thorax, and the preapical (PA) and apical (A) bristles on the distal
639
part of the tibia. Extrapolation from the morphology of the leg indicates that the preapical bristle is located in the anlage plan near the femur Scl, and the apical bristle near the femur Sell (see Fig. 1). These two bristles can be distinguished by the fact that the apical bristle is more distal than the preapical, and is associated with spur bristles (see Figs. 4, 7, and 8). The survival rate in these experiments was approximately 50%. In about 20% of the survivors the operation was successful: either the appendage was abnormal in appearance, or there was extra second leg material, uneverted, inside the thorax or abdo-
FIG. 1. Anlageplan of the male first leg disc, according to Schubiger (1968). See Schubiger (1968) for description of markers. PT, thorax; Co, coxa; Tr, trochanter; Fe, femur; Ti, tibia; TG, tarsus; Scl, single sensillum campaniforme; Stl, single sensillum trichodeum; Sc+5, group of 5 sensilla campaniformia on hairy cuticle; StE, group of 8 sensilla trichodea; GSt, group of sensilla trichodea; TR, transverse bristle rows; GK, sex comb; GTr, joint from coxa to trochanter; Sell, group of 11 sensilla campaniformia; St5, group of 5 sensilla trichodea; BH, bristle on hairy island; Sc3, group of 3 sensilla campaniformia; grB, large bristles; Kl, claw organ; SC-~, group of 8 sensilla campaniformia on naked cuticle; EB, edge bristle; ZRSt, 2 rows of sensilla trichodea; GTh, joint from coxa to thorax. Sc+5 and SC-8 are here considered together as Sc13. TR, GK, thoracic GSt and one of the coxa RSt are absent from the second leg.
640
DEVELOPMENTAL
BIOLOGY
men of the animal. These cases can be divided into six classes: 1. No second leg derivatives on one side of the animal (15 cases). In most of these cases an uneverted second leg, complete with sternopleura was found inside the animal. These cases are interpreted as resulting from severance of the stalk to the epidermis without damage to the disc itself. They were found frequently in another series of experiments where the operations were performed on mature (120 hr) larvae; in those cases it was possible to observe that the disc frequently slipped from under the needle, making severance of the stalk extremely likely. 2. The second leg on one side was represented by a small amount of material on the outside of the fly, with a large amount of material present as an uneverted “implant” (13 cases). In these cases, the “implant” was usually a complete leg including coxa and sternopleura, and the structures present on the outside included a recognizable sternopleura, sometimes with a reduced number of bristles. In five cases coxal structures were present on both outside and inside. However, it was sometimes difficult to ascertain whether coxal structures were present on the outside or whether the sternopleura was duplicated. Our interpretation of these cases is that the disc was cut through the presumptive sternopleural region, and that this was followed by a limited regeneration from one or both of the resulting fragments. 3. The most interesting cases are those in which the second leg was complete, or fairly complete, on the outside of the animal, but in which a rather large amount of material derived from the second leg disc was also present as an uneverted “implant” (14 cases). In these cases the implant invariably showed mirror-image duplication of some or all of the structures depicted in the lower half of the anlage plan. Figure 2 shows part of such an implant, in which the markers Sc3, St5, and BH- are all clearly duplicated. In this fragment as with other
VOLUME
26, 1971
favorable cases, there is also clear evidence of mirror-image duplication of bristle patterns which are not included in the inventory of specific markers. Table 1 catalogs in detail the occurrence of specific markers in the animals showing this class of result. This table includes some results from operations performed at 72 hr, which gave essentially similar results to those performed at 96 hr. It can be seen from this table that duplication of the Sc3 and St5 of the trochanter is especially common in the implant, as is that of the BH- and RSt of the coxa, of the Sell of the femur (Fig. 3) and of the apical bristle of the tibia (Fig. 4). All of these markers are to be found in the lower half of the anlage plan with the exception of the RSt of the coxa. This exception could be due to a difference of the position of this marker in the anlage plan of the second leg disc, compared to the first, or to a limited regenerative capacity of the lower half of the disc. In two cases (Fig. 5) the tarsal segments were duplicated and sepaxated in the implant. In the case of the GSt of the trochanter, St8 of the coxa, and the tarsus, it was often difficult to classify the structures as either single or duplicated. This was because in many cases, the size of the marker was increased to less than double the normal size; for example Fig. 6 shows a group of 13 sensilla trichodea on the coxa, which correspond to the normal St6 Our interpretation of these cases is that they result from duplication of a part of the anlage for the markers. All three of the structures in which this phenomenon was encountered, lie approximately half way along the upper-lower axis of the disc; hence a bisection of the disc would be expected frequently to bisect these anlagen. In many cases of class 3, the second leg on the operated side was abnormal, incompletely everted, or lacking the distal segments. However in two cases, the operated leg was completely normal, as far as could be ascertained. In nearly all cases some markers could be found on the everted leg which were present or duplicated in the
BRYANT
FIG. 2. Mirror-image
Regeneration
pattern duplication
FIG. 3. Two Sell
and Duplication
641
in Drosophila
of part of the coxa and part of the trochanter.
Markers
as in Fig. 1.
markers in the femur from the same implant.
implant. We therefore conclude that the half of the disc which remains attached to the epidermal stalk (the upper half in the anlage plan) is capable of regenerating the remainder of the disc. Duplication of markers was found in only one case in the everted part, and is evidently exceptional (Table 1). 4. There were four cases where all the everted legs were normal, but in which there was a small amount of material, uneverted inside the animal. In these cases it was dif-
ficult to analyze the uneverted portion due to lack of markers, so they will not be discussed further. They are interpreted as having been cut in the lower part of the disc. 5. Nine cases were obtained where the operated leg was partially or completely missing. These are probably due to extensive damage of the disc during the operation. 6. Fifteen cases of branched, everted legs were obtained. In five cases the branch occurred in the sternopleura-coxa region;
642
hVF&OPMENTAL
BIOLOGY
VOLUME
FIG. 4. Duplicated apical bristle apical bristle; S, spur bristle.
FIG.
5. Duplicated
26, 1971
of the tibia.
A,
tarsus.
FIG. 6. Part of a duplicated
coxa, showing enlarged St8 marker.
in four cases in the femur region; in five cases in the tibia region; and in one case in the tarsal region. In eight of these cases one of the branches was small and sometimes poorly differentiated. However, the other seven cases showed two branches of roughly equal size (Fig. 7). In three cases it was pos-
sible to ascertain that one of the branches of the leg consisted not of complete segments, but of two fused longitudinal halves of the segments. These fused halves correspond to the lower halves of the segments in the anlage plan, as shown by the duplication in these cases of the tibia1 apical bristle,
Regeneration
BRYANT
and Duplication TABLE
ABSENCE, PRESENCE, AND DUPLICATION
643
in Drosophila
1
OF MARKERS
BISECTION OF THE SECOND LEG Dac~
FOLLOWING
Everted part Case No.
RSt
St8
SC13
St1
EB
Scl
PA
lb 2 3b 4 5 6 7 8 96 10 11 12 13 14
+ + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + +
+ +
+ +
+
+ + +
+ + + +
t + + + + + + + + ‘2 +
5 9
2 12
Totals Absent Single Duplicated
+ + +
+ +
2
11 1
+ + + + + +
+ + +
+
+ + +
+ + + +
3 11
1 13
+
+ +
8 6
Noneverted lb 2 36 4 5 6 7 8 96 10 11 12 13 14
1 12 1 part
+ +
+ + 2 2 2
TG
A
SC11
SC3
+ + + + + + + + + +
+ + + + + + + + +
+ + +
+ + + +
+ +
+
+
+
+
+ +
+ +
+
+
+ +
+
+ +
+
2 12
3 11
6 8
6 8
5 9
5 9
10 4
2 (+I (f) + ($1
2 2 2 + 2
C-t) 2 2 2 2
+ 2 + 2 2
+ 2 2 2 2
2 2 2 2
2
+
+ ($1 +
2 + + ‘+
($)
+
+
+
+
+
(+I (+I t-t) (f)
+
+
2
+ +
+ +
+
2
2 2
2 2 2 2 2
2 2 2 2 2
2 2 2 2 2 2
+ 2
Totals Absent 13 3 3 1 7 14 14 14 14 4 Single 1 11 12 4 3 7 3 Duplicated 7 2 7 10 u See Fig. 1 and Schubiger (1968) for nomenclature of markers. PA, tibia1 preapical bristle; bristle; f, present; (+), enlarged or partially duplicated; 2, duplicated. b Operation performed at 72 * 2 hr after oviposition. The remainder were performed at oviposition.
with absence of the preapical bristle (Fig. 8). We interpret these cases as being due to incomplete cutting of the disc. This would then be followed by regeneration from one of the surfaces bared by the cut, and mirrorimage duplication from the other surface. The arrangement of tibia1 apical and preapical bristles indicates that it is the upper
+ + + + +
+
+
-t
2
+ + t + +
BH
+
+ + + + +
St5
+ +
+ (1,
2 2
2
GSt
+
+ 2 2 2 2 + 2 2
2 2 2 3 2 9 11 10 A, tibia1 apical 96 i
2 hr after
surface which regenerates and the lower surface which duplicates. In one case the two distal parts in the duplicated branch have separated, thus giving an appendage with three extremities (Fig. 9). The wing Disc The results from 100 operations in situ on
644
DEVELOPMENTAL
FIG.
7. Branched
leg resulting
BIOLOGY
from operation
FIG. 8. Distal parts of the tibias from the preparation
the wing disc at 96 hr. indicate that it behaves in a similar fashion to the leg disc. Eleven of the cases where the entire operated disc everted showed clear evidence of duplication or triplication of parts of the pattern. This could be seen most clearly in five cases where six dorsocentral bristles were present on the operated side, instead of the normal two. Figure 10 shows one
VOLUME
26, 1971
in situ on the imaginal disc. Fe, femur; Ti, tibia.
of Fig. 7. PA, preapical bristle; A, apical bristle.
such case, in which the half-scutellum on the operated side also appears to be triplicated. This response is analogous to the production of branched legs (Fig. 7), which also show triplication of parts of the structure (e.g., apical tibia1 bristle). DISCUSSION
The results described here can be sum-
BRYANT
Regeneration
/
/
/
/
/
and Duplication
645
in Drosophila
/-
,
O.lmm
FIG. 9. Camera lucida drawing of a branched leg in which the two halves of the “double separated, giving a triple extremity.
half’
branch are
646
DEVELOPMENTAL BIOLOGY
VOLUME 26, 1971
0.1mm
FIG. 10. Camera lucida drawing of a thorax with partial triplication resulting from an operation performed at 96 hr on the wing disc in situ (microchaetes omitted). DC, pairs of dorsocentral bristles; S, scutellum; W, wing.
marized by the statement that bisection of the leg disc (in the horizontal direction in the anlage plan) is followed by regeneration of the remainder of the disc from the upper part and duplication of structures, with mirror-image symmetry, from the lower part. The regenerated portion is able to ever& since it retains a connection to the larval epidermis. The latter connection is crucial to the process of eversion, which involves a sequential replacement of larval epidermal cells by the contiguous cells from the imaginal disc (Poodry and Schneiderman, 1970). During this process the integrity of the single-layered epithelium is retained. The duplicated portion, lacking a connection to the epidermis, does not evert but forms an “implant,” which is a vesicle with bristles and hairs differentiated on the inside, just as is formed by transplanted imaginal discs.
Partial bisection of the leg disc appears also to be followed by regeneration from the upper surface of the cut and duplication from the lower cut surface. This results in triplication of certain structures, producing legs with two, or sometimes three, distal parts. In an exactly analogous manner, partial bisection of the wing disc results in triplication of certain of its derivatives. These experiments establish that “multiple regeneration” can be induced in Drosophila, by surgical operations on the imaginal discs of immature larvae. The multiple regenerates obtained are exactly analogous to those obtained by disturbing the integrity of leg anlagen in the last larval instar of Lepidoptera, such as F’hryganidiu (Bodenstein, 1941). Hence, there is no longer any reason to assume that the determinative events occurring in the imaginal discs of the last larval instar of Drosophila are in any
BRYANT
Regeneration
and Duplication
fundamental way different from the events occurring in the leg anlagen of the last larval instar of Lepidoptera (cf. Bodenstein, 1941). The availability of the two alternatives of regeneration or mirror-image duplication is a common observation in developing insect systems subjected to surgical intervention. Using transplantation techniques, Schubiger (1971) has obtained results closely similar to those reported here; the upper half of the mature leg disc can regenerate the remainder of the disc, but the lower half usually duplicates its anlagen. Furthermore he was able to show that bisection of the disc into medial and lateral parts gives similar results; the medial half regenerates and the lateral half duplicates. A similar result was also obtained for the antenna1 disc by Gehring (1966), who showed that the posterior region was able to regenerate the anterior, palpus region, but that the palpus region showed a high tendency to duplicate. This type of behavior is well known from studies of leg regeneration in other insects, which show that proximal parts can regenerate the remainder of the leg, but that distal parts are only able to duplicate. For example, when the developing leg of Periplaneta is amputated, rotated axially through 180 degrees, and attached back to the stump in the new orientation, two wound surfaces are created. The surface of the stump regenerates the remainder of the leg, while the surface of the distal portion produces a mirror-image duplication of the amputated part (Bodenstein, 1962). Similarly, transplantations of limbs with reversed antero-posterior or dorsoventral (interior-exterior) axes in Phryganidia (Bodenstein, 1937); Caruusius (Bart, 1965a, 1971); Leucophaea (Bohn, 1965b) ; Periplaneta (Penzlin, 1965); or Blabera (Bulliere, 1970) results in triplicated distal portions. Many of these cases can also be interpreted as resulting from regeneration from part of the proximal wound surface, with duplication from part of the distal wound surface (Przi-
in Drosophila
647
bram, 1931; Bodenstein, 1962; Bull&e, 1970). The supernumerary regenerates are often formed by a fusion of tissue from both sources (Bohn, 1965b). When a piece of the coxa in Curausius is transplanted on to the opposite surface of the coxa of the host, it causes regeneration of a supernumerary limb. However, if a piece of femur is so grafted, the resulting regenerate contains only those segments distal to the femur (Bart, 1965b; see also Bohn, 1965b). In Culex (Spinner, 1969), Drosophila (Schubiger, 1971), and Tenebrio (Hadom et al., 1969), parts of the leg anlagen corresponding to presumptive proximal parts can regenerate presumptive distal parts, but the reverse is not the case. In some cases, duplication of distal parts occurs (Hadom et al., 1969). The regenerating limbs of amphibians appear to be subject to similar limitations. When the distal part of a limb is amputated and grafted back on to the animal with its proximodistal axis reversed, regeneration proceeds from the free surface. The regenerate forms a new distal structure which is a mirror-image of the transplanted piece (Przibram, 1931; Dent, 1954; Butler, 1955). If a limb is cut part way across, thus producing a proximal and a distal wound surface, both surfaces regenerate distal parts (Przibram, 1931). Regeneration of proximal parts from more distal levels is not observed in these cases. All these findings can be interpreted in terms of a gradient of developmental capacity, the basic feature of which is that cells at any one level can, when given the opportunity for growth and cell division, produce only those structures further down the gradient (Fig. 11). Following bisection of the system, the cells at both cut surfaces produce those structures which are at lower levels in the gradient. In the representation of Fig. 11, cells at a level C have the developmental capacity to construct structures D and E but not A and B. The cells at each side of the cut surface are at roughly the
DEVELOPMENTAL BIOLOGY
VOLUME26, 1971
POSITION
I +
A
BISSECTION
B C
C
D b
E
b GROWTH
\
REGENERATION FIG. 11. The postulated
DUPLICATION
gradient of developmental
same point in the gradient, so they are able to produce the same structures, D and E. With the cut surface of the ABC fragment, the addition of DE corresponds to regeneration but with the CDE fragment it represents mirror-image duplication. Hence, with this interpretation regeneration and duplication are considered as identical phenomena, the final results differing only in the topological relationships of the new
capacity and its response to bisection.
parts to the old. Bohn (1965a,b) proposes a similar gradient of developmental potency to account for various aspects of leg regeneration in Leucophaea. In the case of the proximodistal axis of limbs, this is Rose’s (1962) “rule of distal transformation.” Wolpert (1971) reasserts this rule in suggesting that cells can change their positional values only in the distal direction. We suggest, however, that this behavior is not merely a
BRYANT
Regenemtion
and. Duplication
property of the proximodistal axis but, in the case of imaginal discs at least, reflects a more general feature of their developmental organization. The credibility of this model would be enhanced if it could be shown that regeneration or duplication of insect zppendages or imaginal disc fragments results from the activity of cells at the cut edge. Bohn’s (1965a) transplantation experiments and Bulliere’s (1971) X-irradiation experiments indicate that this is the case for leg regeneration in Orthoptera. As far as imaginal discs are concerned, evidence from autoradiography (Wildermuth, 1968); genetic cell marking (Postlethwait et al., 1971; Ulrich, 1971); direction observations of developing fragments (Ursprung, 1962); and mitotic counts (in Ephestia wing discs; Kroeger, 1958) are all consistent with, but do not give direct proof for this suggestion. However, the assumption is not crucial for the model presented; it can also be discussed in terms of the regeneration or duplication of anlagen without any regard for the exact clonal origin of the cells. The response to bisection would then be a function of the anlagen which are left at the cut surface; C produces D and E whether it is a part of the ABC or CDE fragment. This in fact, is what is indicated by the results presented in this paper. However, an experiment by Niithiger and Schubiger (1966) is rather difficult to explain on this basis. These authors showed that if a UV burn was made in a half-genital disc which was subsequently allowed to duplicate, the defect was found in both copies of the pattern. This somewhat surprising result cannot be accommodated into our model except by assuming that the genital disc has a complex anlage structure. For instance, if it were an assemblage of separately regulating and duplicating anlagen, one of which is completely removed by the UV burn, then the difficulty of explaining the experiment is reduced. Hadorn et al (1949) and Liiijnd (1961) have in fact shown that the genital disc is a complex of separately regulating
in Drosophila
649
morphogenetic fields. The gradients of developmental capacity which we have discussed reflect graded differences in the prospective potencies of the parts of the developing system. Growth and cell division occur during, and are presumably indispensable for, regeneration and duplication in many of the cases we have discussed (Kroeger, 1958; Niithiger and Schubiger, 1966; Wildermuth, 1968). Hence, the postulated gradient refers, strictly, to the developmental capacity of the progency of the cells involved. Underlying gradients of morphogenetic influences have been postulated to account for the control of growth, polarity, and pattern in insect appendages (see Lawrence, 1970 for review). Whether all these concepts can be referred to a single underlying gradient is not clear from the reported experiments, but such a unified control mechanism has, of course, the advantage of simplicity. The results of fragmentation experiments on imaginal discs can alternatively be explained, not on the basis of graded, quantitative differences in developmental capacity, but by assuming that the discs are composed of two types of qualitatively different cells (Wildermuth, 1968; Schubiger, 1971). One cell type would have the capacity to regenerate missing parts of the disc, whereas the other type would have only the ability to duplicate structures. A gradient in the concentration of regenerative cells could, given certain assumptions, mimic the behavior of the gradient of developmental capacity postulated here. The author would like to express his sincere appreciation to Dr. Howard A. Schneiderman for his interest, enthusiasm, and criticism throughout the course of this work. REFERENCES BART, A. (1965h) Induction experimentale dune numeraires au tours de la regeneration des pattes chez Carau.&.s m~r~su.s Br. C. R. Acad. Sci. 261, 190-1903. BART, A. (1965b) Induction exljerimentale d’une morphogenese accompagnant ou non la regeneration de la patte de Camusius morosus Br. C. R.
650
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Acad. Sci. 261, 5619-5622. BART, A. (1971). Morphogenese sumumeraire au niveau de la patte du phasme Camusius morosas Br. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 166, 331-364. BODENSTEIN D. (1935). Beintransplantationen an Lepidopterenraupen. III. Zur Analyse der Entwicklungspotenzen der Schmetterhngsbeine. Wilhelm Roux’ Arch. Entwicklnngsmech. Organismen 133, 156-192. BODENSTEIN, D. (1937). Beintransplantationen an Lepidopterenraupen. IV. Zur Analyse Experimentell-erzeugter Bein-Mehrfachbildungen. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 136, 745-785. BODENSTEIN, D. (1941). Investigations on the problem of metamorphosis. VIII. Studies on leg determination in insects. J. Exp. Zool. 87, 31-53. BODENSTEIN, D. (1962). Regeneration in insects. Symp Genet. Biol. Ital. 9, l-19. BOHN, H. (1965a). Analyse der Regenerationsmigkeit der InsektenextremiEt durch Amputations-und Transplantationsversuche an Larven der afrikanischen Schabe Leucophaea maderue Fabr. I. Mitt. Regenerationspotenzen. Wilhelm Roax’ Arch. Entwicklungsmech. Organismen 156, 49-74. BOHN, H. (1965b). Analyse der Regenerationsflihigkeit der Insektenextremitst durch Amputations und Transplantationsversuche an Larven der afrikanischen Schabe Leuocophaea maderae Fabr. II. Mitt. Achsendetermination. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 156, 449-503. BRAUN, W. (1940) The effect of punctures on the developing wings of several mutants of Drosophila melanogaster. J. Exp. Zool. 84, 325-349. BRYANT, P. J., and SCHNEIDERMAN, H. A. (1969). Cell lineage, growth, and determination in the imaginal leg discs of Drosophila melanogaster. Deuelop. Biol. 20, 263-290. BULL&RE, D. (1970). Interpretation des regenerats multiples chez les Insectes. J. Embryol. Exp. Morphol. 23, 337-357. BULLI~RE, D. (1971). Les mecanismes de la regeneration Studies chez B!abera craniifer (Insecte Dictyopt&e) a l’aide d’irradiations localisees aux rayons X. Deuelop. Viol. 24, 443-463. BUTLER, E. G. (1955). Regeneration of the urodele forelimb after reversal of its proximo-distal axis. J. Morphol. 96, 265-282. DENT, J. N. (1954). A study of regeneration emanating from limb transplants with reversed proximo-distal polarity in the adult newt. Anat Rec. 118, 841-856. GEHRING, W. (1966). ijbertragung und bderung der Determinationsqualitiiten in AntennenscheibenKulturen von Drosophila metanogaster. J. Embryol. Exp. Morphol. 15, 777111. GEIGY, R. (1931). Erzeugung rein imaginaler Defekte durch ultraviolette Eibestrahlung bei Drosophila
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melnnogaster. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen. 125, 406-447. HADORN, E., BERTANI, G., and GALLERA, J. (1949). Regulationsfahigkeit und Feldorganisation der mtinlichen Genital-Imaginalscheibe von Drosophila melanogaster. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 144, 31-70. HADORN, E., HAURI, H., MUELLER,P., and STEINER, E. (1969). Transplantationsexperimente mit Imaginalanlagen von Tenebrio molitor L. Rev. Suisse 2001. 76, 642-649. KROEGER, H. (1958). l?ber Doppelbildungen in die Liebeshiihle verpflanzter Fliigelimaginalschieben von Ephestia ktihnielia 2. Wilhelm Roux’ Arch. Entwicklnngsmech. Organismen 150, 401-424. LAWRENCE, P. A. (1970). Polarity and patterns in the postembryonic development of insects. Aduan. Insect Physiol. 7, 197-226. LEES, A. D. (1941). Operations on the pupal wing of Drosophila melanogaster. J. Genet. 42, 115-142. MIND, H. (1961). Untersuchungen zur Mustergliederung in fragmentierten Primordien des m&mlichen Geschlectsapparates von Drosophila seguyi Deuelop. Biol. 3, 615-656. MURPHY, C. (1967). Determination of the dorsal mesothoracic disc in Drosophila. Deuelop. Biol. 15, 368-394. NGTHIGER, R., and SCHUBIGER, G. (1966). Developmental behaviour of fragments of symmetrical and asymmetrical imaginal discs of Drosophila melanogaster (Diptera). J. Embryol. Exp. Morphol. 16, 355-368. PANTELOURIS, E. M., and WADDINGTON, C. H. (1955). Regulation capacities of the wing and haltere discs of wild type and bithorax Drosophila. Wilhelm Roux’ Arch. Entwicklungsmech. Organismen 147, 539-546. PENZLIN, H. (1965). Experimentelle Erzeugung von Dreifachbildungen bei einem heterometabolen Insekt. Naturwissenschaften 52, 217. POODRY, C. A., and SCHNEIDERMAN, H. A. (1970). The ultrastructure of the developing leg of Drosophila melanogaster. Wilhelm Roux’ Arch. Entwicklangsmech. Organismen 166, l-44. POSTLETHWAIT, J., POODRY, C. A., and SCHNEIDERMAN, H. A. (1971). Cellular dynamics of pattern duplication in imaginal discs of Drosophila melanogaster. Deuelop. Biol. in press. Laws in Animal PRZIBRAM, H. (1931). “Connecting Morphology.” Univ. of London Press, London. BOSE, S. M. (1962). Tissue-arc control of regeneration in the amphibian limb. Symp. Sot. Study Deuelop. Growth 20. SCHUBIGER, G. (1968). Anlageplan, Determinationsz&and und Transdeterminationsleistungen der mannlichen Vorderbeinscheibe von Drosophila mekmogaster. Wilhelm Roax’ Arch. Entwicklungsmech. Organismen 160, 9-40.
BRYANT SCHUBIGER, G. (1971). Regeneration,
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