J. Embryo/, exp. Morph. Vol. 49, pp. 103-113, 1979 ]Q3Printed in Great Britain © Company of Biologists Limited 1979

Acquisition of differentiative capacity in imaginalwing discs of Drosophila melanogaster

By MARY BOWNES1 AND SARAH ROBERTS1

From the Department of Biology. University of Essex

SUMMARYThe wing discs from larvae undergoing the moult fiom 1st to 2nd instar are able to dif-

ferentiate some parts of the adult wing when forced to undergo a premature metamorphosis.The first structures which differentiate are parts of the wing hinge and the wing blade. Asdevelopment proceeds and older discs are forced through metamorphosis, the capacity todifferentiate moves out both proximally and distally until gradually all of the derivatives of amature wing disc are formed. Individual structures often differentiate from young discs in anincomplete form and pattern elements, such as bristles or sensilla, are added as older discsare tested.

INTRODUCTION

The imaginal disc of a late 3rd instar larva, when implanted into anotherlarva of the same age will metamorphose along with its host, in response to thehormonal environment, and produce all of the adult structures it would normallyproduce in situ. Eye-antennal and leg imaginal discs of younger larvae havefewer cells and when they are forced to metamorphose prematurely by trans-plantation into an older host only some of the normal derivatives of these discsdifferentiate (Mindek, 1972; Mindek & Nothiger, 1973; Schubiger, 1974;Gateff & Schneiderman, 1975). The time at which cells are first able to differ-entiate during larval growth, and the sequence which is followed within the discof cells acquiring the ability to differentiate particular structures is different ineach of the previously studied discs. Adult structures first differentiate from60 to 66 h old eye-antennal discs (Mindek, 1972; Gateff & Schneiderman, 1975)and from 74 to 80 h old leg discs (Schubiger, 1974). Within the eye disc thevarious structures are sequentially differentiated following a proximo-distalsequence within the eye-antennal disc (when the base of the optic stalk is con-sidered proximal). In the antenna a proximo-distal sequence is also followed withfirst the 3rd antenna! segment differentiating and finally the arista forming(Gateff & Schneiderman, 1975). In the leg, however, Schubiger (1974) foundthat both proximal and distal, i.e. coxa and tarsus, cells differentiate first andthen the intermediate regions form.

Authors' address: Department of Biology, University of Essex, Wivenhoe Park, Colchester,CO4 3SQ, Essex, U.K.

104 M. BOWNES AND S. ROBERTS

In this paper the acquisition of differentative capacity in the wing disc hasbeen followed. Differentiation was first observed in discs from 48 to 54 h oldlarvae and generally the appearance of structures followed a pattern movingproximally and distally from the wing hinge.

MATERIALS AND METHODS

Oregon-R larvae were used as both hosts and donors and were reared at25 °C on a cornmeal-yeast-sugar medium. Larvae were reared from eggscollected over a 1 h period. The age of the larvae is referred to throughout thispaper in hours after oviposition. In our experiments embryogenesis lasts 24 h.The moult from 1st to 2nd instar occurs at 48 h and from 2nd to 3rd instar at72 h. Pupariation begins 120 h after oviposition. Discs were dissected fromlarvae at 24, 36, 48, 60, 72, 96 and 115 h after oviposition for implantation. Asa further check on larval age we picked out larvae in the process of hatching ormoulting for the 24, 48 and 72 h discs and took some of these larvae and keptthem for a further 12 or 24 h for the 36, 60 and 96 h discs.

Transplan tat ions

The anterior third of the larva was transplanted for the 24 and 36 h donorlarvae. At 48 h the region of tracheae with the wing disc attached was injected,but no attempt was made to dissect it free or remove the leg and haltere discfrom this complex. For all older stages the wing disc was dissected out intoinsect Ringer (Chan & Gehring, 1971). The appropriate larval tissue wasinjected into late 3rd instar larval hosts using the transplantation, technique ofEphrussi & Beadle (1936). All hosts pupated within 4-5 h, thus allowing theminimum possible further growth of the disc within the larva.

The implants were dissected from the hosts when they emerged as adults andmounted between two coverslips in Gurrs water mounting medium.

RESULTS

Wing discs isolated from various ages of larvae, from the time when thelarvae hatch from the egg until just prior to pupariation, were forced throughmetamorphosis by transplantation into a host about to pupate (Fig. 1). Thirtystructures were then scored in the resulting implants (Table 1 and Fig. 2). Thestructures chosen were those which could consistently be accurately scored,especially in implants with few structures. It is impossible to identify withcertainty the macrochaetes (Fig. 3) in the thoraces differentiated from youngerdiscs, we therefore counted the number of macrochaetes and microchaetespresent in each implant.

Differentiated structures were first obtained from wing discs of 48 h oldlarvae, when the moult from 1st to 2nd instar occurs. From this time onwards

Imaginal wing discs of Drosophila 105

Abbreviations used in all figures are listed in the legend to Table 1.Fig. 1. Wing discs at (a) 65 h, (b) 72 h (c) 96 h, and (d) 120 h. The insets show

them at the same magnification.

106 M. BOWNES AND S. ROBERTS

Table 1.

Macro, Macrochaetes; micro, microchaetes; Teg, tegula; HP. humeral plate;UP, unnamed plate; AS1, first axillary sclerite; AS2, second axillary sclerite;AS3, third axillary aclerite; AS4, fourth axillary sclerite; PCo, proximal costa;MCo, Medial costa; DCo, distal costa; TR, triple bristle row; DR, double bristlerow; PR, posterior row of hairs; Sc4d, group of 4 sensilla campaniformia onproximal dorsal radius; Sc25, group of 25 sensilla campaniformia on proximaldorsal radius; Sepl, first septum on proximal dorsal radius; Scl2, group of 12sensilla campaniformia on proximal dorsal radius; AL, alar lobe; AC, axillary cord;YC, yellow club; PVR, proximal ventral radius; PWP, pleural wing piocess; PS,pleural sclerite; AP, axillary pouch; Sc4v, group of 4 sensilla campaniformia onproximal ventral radius; Sc3, group of 3 sensilla campaniformia on proximalventral radius; Sc5, group of 5 sensilla campaniformia on proximal ventral radius;Wing, wing blade hairs.

Age of larvae in hours after oviposition

Total number implants scoiedAverage number structures perimplant

Average number macro per implantAverage number micro per implantAverage number bristles on PCoAverage number sensillae on Sc25Average size YC (x 01 mm)

Structures

Untanned cuticleThoracic tissueWing tissue

TegHPUPAS1AS2AS3AS4PCoMCoDCoTRDRPRSc4dSc25SeplScl2ALACYCPVRPWPPSAPSc4vSc3Sc5

48

327-9

0-880309-40-48

65

24130

2-913-94-38-40-48

72

4221-5

4-42804-911-30-56

Percentage of implants with

8444473734283737226192525919316473134634141444137663

4692100634238586342833382113254217171631344279715854404

A

0100100868169887979408171795262297490767943219395957990171212

96

2522-6

5-843-26-416-20-50

115

2627-7

6-852-27-3190-60

each structure

0100100100100928810088648084845656438084928854288888929288494840

010010092928810010010088929292929285921001001009285100100969696696269

Imaginal wing discs o/Drosophila 107

Table 2. Classification of implants by number of structures

(Abbreviations as in Table 1)

Number of structures

1-5

500-4—3—

6-10

50-45-2-6-0-

6055

Percentage1004080——————————————2020——————————

80401006020204020——202020————40404020—2040806020——20

11-15

131-391106-90-48

of implants2385100773823544623—152323—15—8545446——9262856977——

16-20

29312013-81120-33

21-25

434-28'3'90

666757

26-30

366-8

49-66-716-50-57

with each structure693100797659909079315555413559215590908610386798679663—

59310091937795100844488869156633784981009335289893989591282121

310010010097941001001009497100100979483979497979775100100979797696772

Total number in classAverage number macro per implantAverage number micro per implantAverage number bristles on PCoAverage number sensillae on Sc25Average size YC (x 0-1 mm)

StructuresUntanned cuticleThoracic tissueWing tissue

TegHPUPAS1AS2AS3AS4PCoMCoDCoTRDRPRSc4dSc25SeplScl2ALACYCPVRPWPPSAPSc4vSc3Sc5

the average number of scored structures per implant and the number of thoracicmacro and microchaetes present increase. Only 50 % of 48 h discs produceddifferentiated structures, while from 65 h onwards all of the implants containeddifferentiated structures. Untanned cuticle was present in 84 % of the implantsobtained from 48 h discs and 46 % from 65 h discs, but none was found inimplants from older discs. As can be seen from Table 1 there is a gradual

108 M. BOWNES AND S. ROBERTS

Ventral

Fig. 2. Fate map of a wing disc to show the location of the structures scored in theimplants (from Bryant, 1971).

increase in the percentage occurrence of each structure as the age of the donordisc increases. Generally the dorsal and ventral hinge appear most frequentlyalong with the wing blade and costa. The later appearing structures are thegroups of campani forme sensilla on the proximal ventral radius and the wingmargin.

It is evident that although the discs were of similar age the number of struc-tures they could differentiate was very variable. Some of these differences mayhave been due to minor differences in the age of larvae but it is possible that theactual number of cells in a disc varies from larva to larva. Table 2 classifies thedata according to the number of structures found in each implant. The patternthen becomes clearer. The number of implants containing untanned cuticledecreases as more structures differentiate. Implants containing 1-5 structuresmake only wing tissue, some thoracic tissue, and part of the proximal dorsalradius. It should be noted that all of the implants in this class also had somehinge tissue present but the individual structures could not be identified. In thenext two classes spanning 6-15 structures most of the other wing structures arepresent, those appearing occasionally in the smaller implants are present at a

Fig. 3. The thoracic tissue from an implant derived from a 48 h disc to show that itis not possible to always identify with certainty which macrochaetes are present.Fig. 5. The proximal dorsal radius differentiated from a 65 h disc to show thatthere are only a few sensilla campaniformia.Fig. 6. The proximal dorsal radius differentiated from 96 h disc lo show that thereare many sensilla campaniformia.Fig. 7. A yellow club differentiated from a 72 h disc.

Imaginal wing discs o/Drosophila 109

Thorax

Macro

Sc25(22 sensillac)

Sc25

(5sensillae)

YC

E M B 49

110 M. BOWNES AND S. ROBERTS

(a) (1-5 structures) (/>) (6 -10 structures)

(c) (11-15 structures) (</) (16-20 structures)

Fig. 4. The sequence of appearance of structures in implants containing increasingnumbers of structures (a-d). To simplify the data a structure is only added if itappeared in all the subsequent sizes of implants. The numerals on the thoraxrepresent the number of macrocheates and microcheates.

greater frequency in the larger implants. Much of the dorsal and ventral hinge iscomplete in these implants and the costa begins to appear. The last structuresto differentiate in implants are the wing margins, the fourth axillary sclerite, thealar lobe, the axillary cord and the groups of sensillae campani forme on theproximal ventral radius. (The pattern of appearance of structures is shown in

Imaginal wing discs o/Drosophila 111Fig. 4.) The macrochaetes and microchaetes gradually increase in number as theage of the donor larva and as the number of structures per implant increase(Tables 1 and 2), but because of the difficulty of identification in young implantsthe numbers only are represented in Fig. 2 and we cannot follow exactly howthis pattern is filled in.

We counted the number of bracted bristles on the proximal costa and foundthat they gradually increased in relation to both the age of the donor larva andthe number of structures differentiated per implant. For example, there wereonly 3-8 bracted bristles per proximal costa in implants with 16-20 structuresas compared to 6-7 bristles in implants with 26-30 structures. The number ofsensillae in the group of 25 on the proximal dorsal radius was also counted andgradually increased during development (Tables 1 and 2, Figs. 5, 6). Thus itappears that not only are new structures added with time, but individualstructures which are only partially differentiated from younger discs aregradually completed with time. The yellow club (Fig. 6), a small structure whichwas well formed in implants of all sizes was measured and found to increaseslightly in its size from 0-048 to 0-060 mm from 48 h discs to 115 h discs.

Suggesting that small structures may increase in size during disc growth,although their pattern is complete in all the ages of discs tested.

DISCUSSION

The results presented here agree well with the findings of other authors thatthe imaginat discs of Drosophila are first able to differentiate some structuresunder the correct hormonal conditions at around the beginning of the 2ndlarval instar (Mindek, 1972; Mindek & Nothiger, 1973; Schubiger, 1974;Gateff & Schneiderman, 1975).

As in other discs there is a specific order in which the structures from wingdiscs of increasing age differentiate. The pattern followed, however, seems to beunique for each disc. Gateff & Schneiderman (1975) found that the eye andantenna gradually acquired competence in a proximo-distal sequence, whereasSchubiger (1974) found that in leg discs both proximal and distal structuresoccurred first and then the middle parts were filled in. If one considers the wingblade margin to be distal and the notum proximal, then neither of thesesequences is found in the wing disc, but the capacity to differentiate wouldgenerally be acquired first near the middle and move out both proximally intothe notum and distally into the wing.

When part of a wing disc undergoes regeneration during a period of growthin the female abdomen cells must again be formed which have the correctpositional information and are competent to produce the structures which hadpreviously been removed. Under these circumstances the first pattern elementsto be differentiated were those just proximal in the disc to the original cuttingline, then those most distal to it and finally structures between gradually filled

112 M. BOWNES AND S. ROBERTS

in (James, Bownes & Glenn, 1979). This pattern was not followed during thedevelopment of the whole wing disc and may indicate that there are differentmechanisms for setting up the original pattern elements in the wing and re-generating them during growth after damage to the disc. It is interesting that agroup of cells from a young disc is able to differentiate a structure which isrecognizable, though incomplete. There are possibly two processes occurringduring disc development: (1) the setting up of positional information and thecompetence to respond to hormones to produce a given structure, (2) somestructures are initially incomplete and as older discs are used the pattern ele-ments of that structure are increased. For example, the number of sensillae inthe Sc25 group increases as discs are taken from older larvae. The incompletestructure formed may result either from the pattern being only partially specifiedor there may be insufficient cells to make a complete structure and the cellsmake a partial pattern rather than a miniature but complete structure.

Clonal analysis (Garcia-Bellido & Merriam, 1971) and direct observation ofDNA replication in discs (Bownes, unpublished) indicate that growth and celldivisions continue after pupariation. Thus late 3rd instar discs do not neces-sarily represent the final pattern of the disc, although all structures are oftenpresent. There appears to be no further addition of pattern elements to struc-tures with extra developmental time. There was an average of 19 sensillae in theSc25 group when discs from 115 h larvae were metamorphosed and afterculturing fragments of imaginal discs for 5 days in an adult before metamor-phosis, there was an average of 19-7 sensillae in this group (Bownes and James,unpublished).

It is important to discuss the two things that we may be observing as the wingdiscs acquire the ability to differentiate more structures as they increase in age.It is possible that what we are measuring is the gradual establishment ofpositional information in the cells of the disc, or the cells could already have thepositional information but would gradually acquire the competence to respondto the hormonal signals to differentiate. It is most likely that what is beingmeasured is the acquisition of competence to respond to the hormones, as discsare able to duplicate and regenerate in response to damage very early in develop-ment (Bryant, 1971, Postlethwait & Schneiderman, 1973; Bownes, 1975) sug-gesting that they have at least some positional information. However, it isdifficult to be sure that the discs in these defect experiments had the whole rangeof positional values; they could have had the general duplication or regenerationproperties and only have completed the details of all the positional valuesduring the subsequent development. At present, then, it is not possible to assessthe relative contributions of these two processes to the sequence of appearanceof structures observed.

If young discs are cultured in larvae, adults or pupae and allowed to divideand grow, then the process of completing the pattern in the disc is continued(Ginter & Kuzin, 1970; Mindek & Nothiger, 1973). This suggests that the

Imaginal wing discs 0/Drosophila 113

acquisition of differentiative capacity observed is autonomous within the discand not the result of a specific hormonal stimulus occurring at a precisedevelopmental time.

We would like to thank Susan Proctor for her excellent technical assistance with thephotography. This research was supported by the Science Research Council.

REFERENCESBOWNES, M. (1975). Adult deficiencies and duplications of head and thoracic structures

resulting from microcautery of blastoderm stage Drosophila embryos. / . Embryol. exp.Morph. 34, 33-54.

BRYANT, P. J. (1971). Regeneration and duplication following operations in situ in theimaginal discs of Drosophila melanogaster. Devi Biol. 26, 637-65].

BRYANT, P. J. (1975). Pattern formation in the imaginal wing disc of Drosophila melanogaster:fate map, regeneration and duplication. / . exp. Zool. 193, 49-78.

CHAN, L. N. & GEHRING, W. (1971). Determination of blastoderm cells in Drosophilamelanogaster. Proc. natn. Acad. Sci., U.S.A. 68, 2217-2221.

EPHRUSSI, B. & BEADLE, G. W. (1936). A technique for transplantation for Drosophila.Amer. Nat. 70, 218-225.

GARCIA-BELLIDO, A. & MERRIAM, J. R. (1971). Parameters of wing imaginal disc developmentof Drosophila melanogaster. Devi Biol. 24, 61-87.

GATEFF, E. A. & SCHNEIDERMAN, H. A. (1975). Developmental capacities of immature eye-antennal imaginal discs of Drosophila melanogaster. Wilhelm Roux Arch. EntwMech. Org.176,171-189.

GINTER, E. K. & KUZIN, B. A. (1970). Readiness of eye and antennal imaginal discs inDrosophila melanogaster of different instars for differentiation. Ontogenese 1, 492-500.

JAMES, A., BOWNES, M. & GLENN, S. (1978). The re-establishment of pattern elements inregenerating imaginal discs of Drosophila melanogaster (submitted to Devi Biol.).

MINDEK, G. (1972). Metamorphosis of imaginal discs of Drosophila melanogaster. WilhelmRoux Arch. EntwMech. Org. 169, 353-356.

MINDEK, G. & NOTHIGER, R. (1973). Parameters influencing the acquisition of competencefor metamorphosis in imaginal discs of Drosophila. J. Insect Physiol. 19, 1711-1720.

POSTLETHWAIT, J. H. & SCHNEIDERMAN, H. A. (1973). Pattern formation in imaginal discsof Drosophila melanogaster after irradiation of embryos and young larvae. Devi Biol. 32,345-360.

SCHUBIGER, G. (1974). Acquisition of differentiative competence in the imaginal leg disc ofDrosophila. Wilhelm Roux Arch. EntwMech. Org. 174, 303-311.

{Received 30 June 1978, revised 11 August 1978)