It is a general rule that of two complementary Drosophila imaginal disc fragments, one regenerates and the other duplicates. This paper reports an investigation of an exception to this rule. Duplicating fragments from the periphery of the wing disc which lacked presumptive notum were found to regenerate notum structures during and after duplication. The propensity for this was greatest in fragments lying close to the presumptive notum, with the exception of a fragment confined to the posterior compartment, which did not regenerate notum. Structures were added sequentially, and regeneration stopped once most of the notum was present.

These results are not easily explained by the polar coordinate model, which states that regeneration cannot occur from duplicating fragments. Since compartments appear to be involved in this type of regeneration as in others, it is suggested that a new type of model is required, one which permits simultaneous regeneration and duplication, and assigns a major role to compartments.

In general, if the Drosophila imaginal wing disc is cut into two fragments, one duplicates during culture while the other regenerates (Bryant, 1975 ; Karlsson, 1981a). An exception to this rule was found recently; in certain fragments, duplication of structures expected from the fate map was found to be accompanied by regeneration of others (Karlsson, 1981a). The structures regenerated were from the notum, and all fragments which were tested and which lacked presumptive notum were found capable of regenerating notal bristles while duplicating. These are probably the ‘adventitious bristles’ described by Bryant (1975).

A similar phenomenon has been reported by Schubiger (1971) in the leg disc, van der Meer & Ouweneel (1974) in the haltere disc, and Bryant & Hsei (1977) in the genital disc. In all these cases, certain fragments were found to duplicate some structures and regenerate others. Van der Meer & Ouweneel (1974) have suggested for this the term ‘regenerative duplication’.

These observations contravene the first rule of the polar coordinate model (French, Bryant & Bryant, 1976). This rule views the complementarity between regeneration and duplication as the result of the regenerating fragment having more than half, and the duplicating fragment less than half, of the values in a circumferential axis of positional information. Wound healing brings disparate values into apposition, and intercalary regeneration occurs to fill in the missing ones by the shorter of the two possible routes round the circumference. Thus if duplicating fragments regenerate, they must do so by the forbidden longer route.

We have therefore investigated this phenomenon in detail to find whether it had sufficient regularity to warrant description as a new, non-intercalary, type of regeneration.

Host and donor flies were of ebony11 genotype (Lindsley & Grell, 1968) and were raised at 25 °C on standard cornmeal/agar/syrup medium seeded with live yeast. Wing discs were removed from late third instar larvae in insect Ringer’s solution. Fragments were cut with tungsten needles and implanted into the body cavities of well-fed 1 - to 3-day-old fertilized adult females where they remained for varying periods. They were then removed and reimplanted into the body cavities of late third instar larvae using the method of Ephrussi & Beadle (Ursprung, 1967). When the hosts emerged as adults, the metamorphosed implants were removed, mounted in Gurr’s Hydramount, and scored for the structures shown in Fig. 1. Implants cultured for 0 days (Table 1) were implanted directly into mature third instar larvae.

Table 1.

Structures differentiated by implants of the fragments shown in Fig. 1, either implanted directly into larval hosts for metamorphosis (0 days of culture) or cultured in adult females for varying periods before transfer to larval hosts

Structures differentiated by implants of the fragments shown in Fig. 1, either implanted directly into larval hosts for metamorphosis (0 days of culture) or cultured in adult females for varying periods before transfer to larval hosts
Structures differentiated by implants of the fragments shown in Fig. 1, either implanted directly into larval hosts for metamorphosis (0 days of culture) or cultured in adult females for varying periods before transfer to larval hosts
Fig. 1.

Fate map of the wing disc (after Bryant, 1975), and the fragments used. Compartment borders are shown in broken lines. The position of the anteroposterior border was found using clones of a new cell marker (Brower, Lawrence & Wilcox, 1981). Presumptive wing blade is stippled. Large dots represent microchaetes of the notum. A, P, D, V, presumptive anterior, posterior, dorsal, ventral. Abbreviations: APA, anterior post-alar; Jo, joint; Scu, scutellum; PNWP, posterior notal wing process ; AS 1–4, axillary sclerites 1–4 ; A Cord, axillary cord ; A Lobe, alar lobe ; ANWP, anterior notal wing process; HP, humeral plate; YC, yellow club; PVR, proximal ventral radius; PS, pleural sclerite; PWP, pleural wing process; UP, unnamed plate; PDR, proximal dorsal radius.

Fig. 1.

Fate map of the wing disc (after Bryant, 1975), and the fragments used. Compartment borders are shown in broken lines. The position of the anteroposterior border was found using clones of a new cell marker (Brower, Lawrence & Wilcox, 1981). Presumptive wing blade is stippled. Large dots represent microchaetes of the notum. A, P, D, V, presumptive anterior, posterior, dorsal, ventral. Abbreviations: APA, anterior post-alar; Jo, joint; Scu, scutellum; PNWP, posterior notal wing process ; AS 1–4, axillary sclerites 1–4 ; A Cord, axillary cord ; A Lobe, alar lobe ; ANWP, anterior notal wing process; HP, humeral plate; YC, yellow club; PVR, proximal ventral radius; PS, pleural sclerite; PWP, pleural wing process; UP, unnamed plate; PDR, proximal dorsal radius.

Incubation

Short-term cultures were incubated at 25 °C and long-term ones (14 days and over) at 30 °C. It was found that development of Drosophila melanogaster at 30 °C takes 80 % of the time it takes at 25 °C. ‘Days of culture’ in Table 1 refers either to actual days at 25 °C or to the number of days a 30 °C incubation would have taken at 25 °C.

Scoring of notal structures

The different structures were identified with the aid of the descriptions of Bryant (1975). It is possible to identify individual macrochaetes of the notum in implants where much of the notum is present. However, where only a few bristles are present, the only one which can be positively identified is anterior post-alar, which is flanked by two sensilla trichodea. Scutellum is easily recognizable as a vesicle of heavy cuticle with two macrochaetes and no microchaetes. Scutellum makes a joint with posterior notal wing process and axillary sclerite 4; this is also clearly recognizable in implants and is here termed ‘joint’. Prescutum is also usually recognizable as an area of heavy cuticle with about 50 closely spaced microchaetes arranged in rows.

This description agrees with that of Bryant (1975).

Table 1 shows the results of scoring the metamorphosed implants of the fragments depicted in Fig. 1. These results allow the following conclusions to be drawn.

(1) Duplicating fragments lacking presumptive notum have the ability to regenerate it

Most implants of fragments a, c and e showed evidence of duplication (Table 1), and none regenerated a complete disc. Fragments b and d, on the other hand, appeared to belong to the class of fragment which regenerates sometimes and duplicates sometimes (Karlsson, 1981 a); some implants of these fragments regenerated a complete disc with no evidence of duplication. For this reason, only those implants of these two fragments which had at least two duplicated structures were included in Table 1.

No implant had notal bristles after direct implantation into larvae with the exception of a few implants of fragments c and d. 4/14 and 4/13 implants respectively of these two fragments had a few bristles after 0 days of culture; these two fragments were cut very close indeed to presumptive notum.

In those implants which produced bristles after culture, in many cases these bristles could not be positively identified as being derived from the notum. In others, however, they were clearly notal bristles, with a characteristic mixture of micro- and macrochaetes on well-tanned cuticle. It was therefore concluded that all bristles were notal bristles. Fragments b, c and d often produced prescutum, and c and d usually produced scutellum and joint. These latter two structures were often duplicated; notal bristles may have been duplicated as well, but this would be almost impossible to detect.

(2) The time-course of regenerative duplication is longer than that of regeneration

Regeneration of wing disc fragments is complete after 3 or 4 days (Karlsson, 1981b). In fragment a, however, there was a significant (P < 0·05) increase in bristle number between 7 and 14 days of culture. Duplication in this fragment was complete after 4 days; no increase in the number of duplicated structures per implant was observed between 4 and 7 days (1·0 ± 0·2 after 4 days; 1·1 ± 0·2 after 7 days). Fragment c also showed a significant increase in average numbers of bristles during culture periods longer than 7 days.

(3) Regenerative duplication stops once most of the notum is present

Fragment c appeared to undergo more extensive regenerative duplication than the others, and was therefore selected for a 30-day culture to find out whether a whole disc would eventually be produced. These implants were not appreciably different from those cultured for 14 days, except that more notal bristles were present (Table 1). Some structures were lost, as expected in long-term cultures (Bryant, 1978), and so the frequency of duplication decreased. All implants had regenerated a fairly complete notum, but virtually nothing else, indicating that regenerative duplication stops once most of the notum is present.

(4) Regenerative duplication produces first those structures closest to the cut edge, and adds others in sequence; structures located some distance from the periphery of the disc in the fate map are not produced

This is most clearly seen in fragment c. If the above is true, no implant of this fragment should have had prescutum which did not also have scutellum and joint, and none should have had tegula which did not also have prescutum, scutellum and joint (see Fig. 1). Also, anterior post-alar, axillary sclerites 1 and 2, and proximal dorsal radius should not have been present, as these markers do not lie at the periphery of the disc in the fate map (Fig. 1). This was all found to be true. The two most frequently regenerated structures were scutellum and joint, which lie close to the cut edge. These usually appeared together; of 19 implants having one or the other or both, 12 had both. Prescutum was the next most frequent structure, and implants having it almost always (8/9) had scutellum as well. Two implants in the 14-day set had tegula and anterior notal wing process (and one of these had humeral plate and proximal costa too), and both of these had prescutum and either joint or scutellum.

Structures located towards the centre of the disc in the fate map were noticeably lacking. Axillary sclerites 1 and 2, unnamed plate and proximal dorsal radius were never found in any of the experiments, except of course in fragment /, in which these structures are expected from the fate map. The frequency of anterior post-alar did not increase after culture in either fragment c or d, and its absence is even more striking since it lies so close to the cut edge.

It seems likely that regenerative duplication in anterior fragments proceeds sequentially too, in the opposite direction to posterior fragments. Fragment b never produced scutellum, joint or anterior post-alar, although it often had prescutum. Anterior notal wing process, which lies near the cut at a short distance from the edge of the disc in the fate map, was lacking as well, suggesting that regenerative duplication in this fragment too proceeds round the periphery of the disc.

Fragment a seems at first glance not to regenerate sequentially. Notum appears for example before tegula and humeral plate, which lie between this fragment and notum on the anterior edge of the disc. However, there is a structure which lies still closer to the edge of the disc at this location. This is mesopleura, which appears in implants as a semicircular vesicle running from the notum towards the yellow club. It is this vesicle on which notal bristles first appear (Fig. 2a). It is thus possible that mesopleura is regenerated first, followed by notum, in which case regenerative duplication in this fragment too would be sequential.

Fig. 2.

Camera-lucida drawings of implants which have duplicated structures expected from the fate map and regenerated notum. (a) fragment a, cultured for 7 days. Three notal bristles have appeared on the vesicle of mesopleura. (b) fragment d, cultured for 7 days. This implant has regenerated scutellum and joint, and some microchaetes which are probably part of the prescutum. Abbreviations as in legend to Fig. 1.

Fig. 2.

Camera-lucida drawings of implants which have duplicated structures expected from the fate map and regenerated notum. (a) fragment a, cultured for 7 days. Three notal bristles have appeared on the vesicle of mesopleura. (b) fragment d, cultured for 7 days. This implant has regenerated scutellum and joint, and some microchaetes which are probably part of the prescutum. Abbreviations as in legend to Fig. 1.

(5) Regenerative duplication is most frequent and complete in fragments lying very close to presumptive notum in the fate map

Implants of fragments c and d, which were cut very close to presumptive notum, sometimes had a few bristles when implanted directly into larvae. One of these bristles was usually (6/8 cases) recognizable as anterior post-alar. These two fragments shared with fragment b, which was also cut very close to presumptive notum but on the anterior side, the highest bristle frequency and average bristle numbers. The difference in average bristle number between these fragments and fragment a, which lies some distance from presumptive notum in the fate map, is significant (P < 0·01 in both cases).

(6) Tissue confined to the posterior compartment does not undergo regenerative duplication

Fragment e was cut so as to exclude tissue from the anterior comparment (Garcia-Bellido, Ripoil & Morata, 1976), and after 14 days of culture only one implant out of 12 had produced any notal bristles, although all had duplicated. Another experiment was done in which this fragment was cut very slightly smaller so as to be absolutely sure of excluding anterior tissue, and none (0/34) of these implants produced any notal bristles. This second experiment is marked with an asterisk in Table 1.

It is probably not possible to cut a fragment confined to the anterior compartment. If compartment borders are closed lines, as at least early ones must be since clones never cross them, the anteroposterior border probably runs down the anterior edge of the disc. All anterior fragments would therefore contain part of it.

(7) Regenerative duplication is a property of the periphery of the disc; the centre is not involved, nor is the central part of the peripodial membrane

Fragment f is from the centre of the disc and includes presumptive wing blade and dorsal hinge (proximal dorsal radius, axillary sclerites 1 and 2, unnamed plate). Only 5 out of 57 implants of this fragment had notal bristles; none of these had duplicated structures, and all 5 had other regenerated structures as well as notum. These included axillary sclerite 3 (4 cases), axillary cord (3 cases), proximal costa (2 cases), and humeral plate, tegula, yellow club and alar lobe (1 case each). None of these structures was found in implants injected directly into larvae (Table 1). Central fragments do sometimes regenerate (Bryant, 1975), and it was concluded these 5 implants had regenerated normally, and that this type of fragment is not capable of regenerative duplication. However the possibility was not ruled out that the peripodial membrane was involved in regenerative duplication and that it had become detached during culture. Another experiment was therefore done with the same fragment in which tungsten needles were used to fold the peripodial membrane inside the epithelial layer to ensure that they remained together (Haynie & Bryant, 1976). One out of 13 of these implants had notal bristles, and as this implant, like the unmixed ones, had no duplicated structures, and had other peripheral structures as well (axillary sclerite 3, humeral plate, proximal costa and yellow club), it was concluded that this central part is not capable of regenerative duplication.

The results show that many duplicating fragments from the periphery of the wing disc which do not include presumptive notum are able to regenerate it during and after duplication. This process appears to be an orderly and predictable one, structures being produced in sequence from the cut edge as in the more usual type of regeneration (Abbott, Karpen & Schubiger, 1981; Karlsson, 1981b), and appearing in virtually all cases to stop once most of the notum is present. Perhaps the most striking result is that tissue confined to the posterior compartment does not produce notum, although slightly larger fragments which contain a small part of the anteroposterior compartment border do so at high frequency. The posterior fragment which was tested was necessarily rather small, but its size clearly did not prevent growth as the frequency of duplication was very high. This experiment was performed twice, the second time great care being taken to exclude anterior tissue. One implant in the first experiment did produce notum; this was virtually complete, again indicating that small size does not prevent this fragment from regenerating notum. It would be interesting to know whether tissue confined to the anterior compartment can regenerate notum, but it is probably not possible to cut such a fragment (see Results).

Since, then, compartments seem to be involved in this phenomenon, it is perhaps no coincidence that by and large, the structures produced lie along the anteroposterior compartment border (see Fig. 1). This apparent ability of tissue at the anteroposterior border to regenerate proximally is reminiscent of the ability of the other end of this border to regenerate distally. Fragments lacking the ventral end cannot regenerate distally (Karlsson, 1980), just as fragments lacking the dorsal end are apparently unable to regenerate notum. Both phenomena are difficult to explain in terms of the polar coordinate model; according to the revised second rule of this model (Bryant, French & Bryant, 1981), all proximal tissue should be able to regenerate distally, and neither of the two rules makes any provision for regeneration which is non-intercalary and proceeds from distal to proximal. It is quite clear that the regeneration described in this paper is not intercalary. The fragments studied duplicate, and their complementary fragments regenerate (Bryant, 1975; Karlsson, 1981 a). They cannot thus have a sufficient number of values for complete regeneration, and in any case complete regeneration never occurs. If, for example, notum regeneration were initiated by a transdetermination-like event which provided the fragment with over half the values, then intercalary regeneration would be expected to produce a complete disc; also, the newly regenerated structures should not be duplicated, which they often are.

There is at present no model which makes provision for epimorphic regeneration which is not intercalary. The present results therefore suggest that a completely new type of model is required, one in which new positional values can be generated by non-intercalary means, and which assigns a major role to compartment borders.

This work was supported by the MRC and by a Beit Memorial Fellowship to JK.

Abbott
,
L. C.
,
Karpen
,
G. H.
&
Schubiger
,
G.
(
1981
).
Compartmental restrictions and blastema formation during pattern regulation in Drosophila imaginal leg discs
.
Devl Biol. (In press
.)
Bryant
,
P. J.
(
1975
).
Pattern formation in the imaginal wing disc of Drosophila melanogaster’. fate map, regeneration and duplication
.
J. exp. Zool
.
193
,
49
77
.
Bryant
,
P. J.
(
1978
).
Pattern formation in imaginal discs
.
In The Genetics and Biology of Drosophila
(ed.
M.
Ashburner
&
T. R. F.
Wright
), vol.
2c
.
London
:
Academic Press
.
Bryant
,
P. J.
&
Hsei
,
B. W.
(
1977
).
Pattern formation in asymmetrical and symmetrical imaginal discs of Drosophila melanogaster
.
Amer. Zool
.
17
,
595
611
.
Bryant
,
S. V.
,
French
,
V.
&
Bryant
,
P. J.
(
1981
).
Distal regeneration and symmetry in epimorphic fields
.
Science (in press)
.
Brower
,
D. L.
,
Lawrence
,
P. A.
&
Wilcox
,
M.
(
1981
).
Clonal analysis of the undifferentiated wing disc of Drosophila
.
Devl Biol, (in press)
.
French
,
V.
,
Bryant
,
P. J.
&
Bryant
,
S. V.
(
1976
).
Pattern regulation in epimorphic fields
.
Science
193
,
969
981
.
Garci A-Bellido
,
A.
,
Ripoll
,
P.
&
Morata
,
G.
(
1976
).
Developmental compartmentalisation in the dorsal mesothoracic disc of Drosophila
.
Dev! Biol
.
48
,
132
147
.
Haynie
,
J. L.
&
Bryant
,
P. J.
(
1976
).
Intercalary regeneration in the imaginal wing disc of Drosophila melanogaster
.
Nature
259
,
659
662
.
Karlsson
,
J.
(
1980
).
Distal regeneration in proximal fragments of the wing disc of Drosophila
.
J. Embryol. exp. Morph
.
59
,
315
323
.
Karlsson
,
J.
(
1981a
).
The distribution of regenerative potential in the wing disc of Drosophila
.
J. Embryol. exp. Morph
.
61
,
303
316
.
Karlsson
,
J.
(
1981b
).
Sequence of regeneration in the Drosophila wing disc
.
J. Embryol. exp. Morph
.
65
, Supplement
37
-
47
.
Lindsley
,
D. L.
&
Grell
,
E. H.
(
1968
).
Genetic variations of Drosophila melanogaster
.
Carnegie Inst. Washington Publ. No
.
627
.
Schubiger
,
G.
(
1971
).
Regeneration, duplication and transdetermination in fragments of the leg disc of Drosophila melanogaster
.
Devl Biol
.
26
,
277
295
.
Ursprung
,
H.
(
1967
).
In vivo culture of Drosophila imaginal discs
.
In Methods in Developmental Biology
(ed.
F. M.
Wilt
&
N. K.
Wessels
).
New York
:
Crowell
.
van der Meer
,
J. M.
&
Ouweneel
,
W. J.
(
1974
).
Differentiation capacities of the dorsal metathoracic (haltere) disc of Drosophila melanogaster. II. Regeneration and duplication
.
Wilhelm Roux Arch. EntwMech. Org
.
174
,
361
373
.