J. Embryo/, exp. Morph. Vol. 49, pp. 103-113, 1979
Printed in Great Britain © Company of Biologists Limited 1979
]Q3
Acquisition of differentiative capacity in imaginal
wing discs of Drosophila melanogaster
By MARY BOWNES1 AND SARAH ROBERTS 1
From the Department of Biology. University of Essex
SUMMARY
The wing discs from larvae undergoing the moult fiom 1st to 2nd instar are able to differentiate 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. As
development proceeds and older discs are forced through metamorphosis, the capacity to
differentiate moves out both proximally and distally until gradually all of the derivatives of a
mature wing disc are formed. Individual structures often differentiate from young discs in an
incomplete form and pattern elements, such as bristles or sensilla, are added as older discs
are tested.
INTRODUCTION
The imaginal disc of a late 3rd instar larva, when implanted into another
larva of the same age will metamorphose along with its host, in response to the
hormonal environment, and produce all of the adult structures it would normally
produce in situ. Eye-antennal and leg imaginal discs of younger larvae have
fewer cells and when they are forced to metamorphose prematurely by transplantation into an older host only some of the normal derivatives of these discs
differentiate (Mindek, 1972; Mindek & Nothiger, 1973; Schubiger, 1974;
Gateff & Schneiderman, 1975). The time at which cells are first able to differentiate during larval growth, and the sequence which is followed within the disc
of cells acquiring the ability to differentiate particular structures is different in
each of the previously studied discs. Adult structures first differentiate from
60 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 the
various structures are sequentially differentiated following a proximo-distal
sequence within the eye-antennal disc (when the base of the optic stalk is considered proximal). In the antenna a proximo-distal sequence is also followed with
first the 3rd antenna! segment differentiating and finally the arista forming
(Gateff & Schneiderman, 1975). In the leg, however, Schubiger (1974) found
that both proximal and distal, i.e. coxa and tarsus, cells differentiate first and
then 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 has
been followed. Differentiation was first observed in discs from 48 to 54 h old
larvae and generally the appearance of structures followed a pattern moving
proximally and distally from the wing hinge.
MATERIALS AND METHODS
Oregon-R larvae were used as both hosts and donors and were reared at
25 °C on a cornmeal-yeast-sugar medium. Larvae were reared from eggs
collected over a 1 h period. The age of the larvae is referred to throughout this
paper 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 at
72 h. Pupariation begins 120 h after oviposition. Discs were dissected from
larvae at 24, 36, 48, 60, 72, 96 and 115 h after oviposition for implantation. As
a further check on larval age we picked out larvae in the process of hatching or
moulting for the 24, 48 and 72 h discs and took some of these larvae and kept
them 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 donor
larvae. 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 disc
from this complex. For all older stages the wing disc was dissected out into
insect Ringer (Chan & Gehring, 1971). The appropriate larval tissue was
injected into late 3rd instar larval hosts using the transplantation, technique of
Ephrussi & Beadle (1936). All hosts pupated within 4-5 h, thus allowing the
minimum possible further growth of the disc within the larva.
The implants were dissected from the hosts when they emerged as adults and
mounted between two coverslips in Gurrs water mounting medium.
RESULTS
Wing discs isolated from various ages of larvae, from the time when the
larvae hatch from the egg until just prior to pupariation, were forced through
metamorphosis by transplantation into a host about to pupate (Fig. 1). Thirty
structures were then scored in the resulting implants (Table 1 and Fig. 2). The
structures chosen were those which could consistently be accurately scored,
especially in implants with few structures. It is impossible to identify with
certainty the macrochaetes (Fig. 3) in the thoraces differentiated from younger
discs, we therefore counted the number of macrochaetes and microchaetes
present in each implant.
Differentiated structures were first obtained from wing discs of 48 h old
larvae, when the moult from 1st to 2nd instar occurs. From this time onwards
Imaginal wing discs of Drosophila
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.
105
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 bristle
row; PR, posterior row of hairs; Sc4d, group of 4 sensilla campaniformia on
proximal dorsal radius; Sc25, group of 25 sensilla campaniformia on proximal
dorsal radius; Sepl, first septum on proximal dorsal radius; Scl2, group of 12
sensilla 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 on
proximal ventral radius; Sc3, group of 3 sensilla campaniformia on proximal
ventral radius; Sc5, group of 5 sensilla campaniformia on proximal ventral radius;
Wing, wing blade hairs.
Age of larvae in hours after oviposition
65
72
96
115
7-9
24
130
42
21-5
25
22-6
26
27-7
0-8
80
30
9-4
0-48
2-9
13-9
4-3
8-4
0-48
4-4
280
4-9
11-3
0-56
5-8
43-2
6-4
16-2
0-50
6-8
52-2
7-3
19
0-60
48
Total number implants scoied
Average number structures per
implant
Average number macro per implant
Average number micro per implant
Average number bristles on PCo
Average number sensillae on Sc25
Average size YC (x 01 mm)
32
Percentage of implants with each structure
Structures
A
Untanned cuticle
Thoracic tissue
Wing tissue
Teg
HP
UP
AS1
AS2
AS3
AS4
PCo
MCo
DCo
TR
DR
PR
Sc4d
Sc25
Sepl
Scl2
AL
AC
YC
PVR
PWP
PS
AP
Sc4v
Sc3
Sc5
84
44
47
37
34
28
37
37
22
6
19
25
25
9
19
3
16
47
31
34
6
3
41
41
44
41
37
6
46
92
100
63
42
38
58
63
42
8
33
38
21
13
25
4
21
71
71
63
13
4
42
79
71
58
54
4
6
0
3
4
0
100
100
86
81
69
88
79
79
40
81
71
79
52
62
29
74
90
76
79
43
21
93
95
95
79
90
17
12
12
0
100
100
100
100
92
88
100
88
64
80
84
84
56
56
43
80
84
92
88
54
28
88
88
92
92
88
49
48
40
0
100
100
92
92
88
100
100
100
88
92
92
92
92
92
85
92
100
100
100
92
85
100
100
96
96
96
69
62
69
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
Total number in class
Average number macro per implant
Average number micro per implant
Average number bristles on PCo
Average number sensillae on Sc25
Average size YC (x 0-1 mm)
Structures
Untanned cuticle
Thoracic tissue
Wing tissue
Teg
HP
UP
AS1
AS2
AS3
AS4
PCo
MCo
DCo
TR
DR
PR
Sc4d
Sc25
Sepl
Scl2
AL
AC
YC
PVR
PWP
PS
AP
Sc4v
Sc3
Sc5
5
0
0-4
—
3
—
6-10
5
0-4
5-6
2-0
6-5
0-5
11-15
16-20
21-25
13
1-3
91
10
6-9
0-48
29
31
201
3-8
112
0-33
43
4-6
28' 6
3'6
97
0 57
26-30
36
6-8
49-6
6-7
16-5
0-57
Percentage of implants with each structure
100
80
3
5
23
6
100
93
40
85
93
40
100
100
100
80
100
100
100
—
77
60
79
91
97
—
76
20
93
38
94
77
—
20
23
59
100
95
—
54
90
40
—
100
100
90
20
46
—
—
100
84
23
79
—
—
—
94
44
31
—
97
20
88
15
55
—
100
86
55
20
23
—
100
20
23
41
91
97
—
—
—
56
35
—
—
94
63
15
59
—
83
—
37
—
21
—
—
97
84
8
55
94
98
54
90
40
20
97
100
54
90
40
20
40
97
—
93
46
86
20
97
—
35
—
10
—
75
—
—
28
3
100
—
20
98
92
86
—
100
93
62
40
79
—
97
98
85
80
86
—
97
95
60
79
69
97
—
77
91
66
20
—
—
—
28
69
3
—
—
—
—
67
21
72
21
20
the average number of scored structures per implant and the number of thoracic
macro and microchaetes present increase. Only 50 % of 48 h discs produced
differentiated structures, while from 65 h onwards all of the implants contained
differentiated structures. Untanned cuticle was present in 84 % of the implants
obtained from 48 h discs and 46 % from 65 h discs, but none was found in
implants 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 the
implants (from Bryant, 1971).
increase in the percentage occurrence of each structure as the age of the donor
disc increases. Generally the dorsal and ventral hinge appear most frequently
along with the wing blade and costa. The later appearing structures are the
groups of campani forme sensilla on the proximal ventral radius and the wing
margin.
It is evident that although the discs were of similar age the number of structures they could differentiate was very variable. Some of these differences may
have been due to minor differences in the age of larvae but it is possible that the
actual number of cells in a disc varies from larva to larva. Table 2 classifies the
data according to the number of structures found in each implant. The pattern
then becomes clearer. The number of implants containing untanned cuticle
decreases as more structures differentiate. Implants containing 1-5 structures
make only wing tissue, some thoracic tissue, and part of the proximal dorsal
radius. It should be noted that all of the implants in this class also had some
hinge tissue present but the individual structures could not be identified. In the
next two classes spanning 6-15 structures most of the other wing structures are
present, 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 it
is 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 that
there are only a few sensilla campaniformia.
Fig. 6. The proximal dorsal radius differentiated from 96 h disc lo show that there
are many sensilla campaniformia.
Fig. 7. A yellow club differentiated from a 72 h disc.
Imaginal wing discs o/Drosophila
109
Sc25
(5sensillae)
Thorax
Macro
YC
Sc25
(22 sensillac)
EMB
49
110
M. BOWNES AND S. ROBERTS
(a) (1-5 structures)
(c) (11-15 structures)
(/>) (6 -10 structures)
(</) (16-20 structures)
Fig. 4. The sequence of appearance of structures in implants containing increasing
numbers of structures (a-d). To simplify the data a structure is only added if it
appeared in all the subsequent sizes of implants. The numerals on the thorax
represent the number of macrocheates and microcheates.
greater frequency in the larger implants. Much of the dorsal and ventral hinge is
complete in these implants and the costa begins to appear. The last structures
to differentiate in implants are the wing margins, the fourth axillary sclerite, the
alar lobe, the axillary cord and the groups of sensillae campani forme on the
proximal ventral radius. (The pattern of appearance of structures is shown in
Imaginal wing discs o/Drosophila
111
Fig. 4.) The macrochaetes and microchaetes gradually increase in number as the
age 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 implants
the numbers only are represented in Fig. 2 and we cannot follow exactly how
this pattern is filled in.
We counted the number of bracted bristles on the proximal costa and found
that they gradually increased in relation to both the age of the donor larva and
the number of structures differentiated per implant. For example, there were
only 3-8 bracted bristles per proximal costa in implants with 16-20 structures
as compared to 6-7 bristles in implants with 26-30 structures. The number of
sensillae in the group of 25 on the proximal dorsal radius was also counted and
gradually increased during development (Tables 1 and 2, Figs. 5, 6). Thus it
appears that not only are new structures added with time, but individual
structures which are only partially differentiated from younger discs are
gradually completed with time. The yellow club (Fig. 6), a small structure which
was well formed in implants of all sizes was measured and found to increase
slightly 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 that
the imaginat discs of Drosophila are first able to differentiate some structures
under the correct hormonal conditions at around the beginning of the 2nd
larval 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 wing
discs of increasing age differentiate. The pattern followed, however, seems to be
unique for each disc. Gateff & Schneiderman (1975) found that the eye and
antenna gradually acquired competence in a proximo-distal sequence, whereas
Schubiger (1974) found that in leg discs both proximal and distal structures
occurred first and then the middle parts were filled in. If one considers the wing
blade margin to be distal and the notum proximal, then neither of these
sequences is found in the wing disc, but the capacity to differentiate would
generally be acquired first near the middle and move out both proximally into
the notum and distally into the wing.
When part of a wing disc undergoes regeneration during a period of growth
in the female abdomen cells must again be formed which have the correct
positional information and are competent to produce the structures which had
previously been removed. Under these circumstances the first pattern elements
to be differentiated were those just proximal in the disc to the original cutting
line, 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 the
development of the whole wing disc and may indicate that there are different
mechanisms for setting up the original pattern elements in the wing and regenerating them during growth after damage to the disc. It is interesting that a
group of cells from a young disc is able to differentiate a structure which is
recognizable, though incomplete. There are possibly two processes occurring
during disc development: (1) the setting up of positional information and the
competence to respond to hormones to produce a given structure, (2) some
structures are initially incomplete and as older discs are used the pattern elements of that structure are increased. For example, the number of sensillae in
the Sc25 group increases as discs are taken from older larvae. The incomplete
structure formed may result either from the pattern being only partially specified
or there may be insufficient cells to make a complete structure and the cells
make a partial pattern rather than a miniature but complete structure.
Clonal analysis (Garcia-Bellido & Merriam, 1971) and direct observation of
DNA replication in discs (Bownes, unpublished) indicate that growth and cell
divisions continue after pupariation. Thus late 3rd instar discs do not necessarily represent the final pattern of the disc, although all structures are often
present. There appears to be no further addition of pattern elements to structures with extra developmental time. There was an average of 19 sensillae in the
Sc25 group when discs from 115 h larvae were metamorphosed and after
culturing fragments of imaginal discs for 5 days in an adult before metamorphosis, 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 wing
discs acquire the ability to differentiate more structures as they increase in age.
It is possible that what we are measuring is the gradual establishment of
positional information in the cells of the disc, or the cells could already have the
positional information but would gradually acquire the competence to respond
to the hormonal signals to differentiate. It is most likely that what is being
measured is the acquisition of competence to respond to the hormones, as discs
are able to duplicate and regenerate in response to damage very early in development (Bryant, 1971, Postlethwait & Schneiderman, 1973; Bownes, 1975) suggesting that they have at least some positional information. However, it is
difficult to be sure that the discs in these defect experiments had the whole range
of positional values; they could have had the general duplication or regeneration
properties and only have completed the details of all the positional values
during the subsequent development. At present, then, it is not possible to assess
the relative contributions of these two processes to the sequence of appearance
of structures observed.
If young discs are cultured in larvae, adults or pupae and allowed to divide
and 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 disc
and not the result of a specific hormonal stimulus occurring at a precise
developmental time.
We would like to thank Susan Proctor for her excellent technical assistance with the
photography. This research was supported by the Science Research Council.
REFERENCES
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 the
imaginal 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 Drosophila
melanogaster. 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 development
of Drosophila melanogaster. Devi Biol. 24, 61-87.
GATEFF, E. A. & SCHNEIDERMAN, H. A. (1975). Developmental capacities of immature eyeantennal 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 in
Drosophila melanogaster of different instars for differentiation. Ontogenese 1, 492-500.
JAMES, A., BOWNES, M. & GLENN, S. (1978). The re-establishment of pattern elements in
regenerating imaginal discs of Drosophila melanogaster (submitted to Devi Biol.).
MINDEK, G. (1972). Metamorphosis of imaginal discs of Drosophila melanogaster. Wilhelm
Roux Arch. EntwMech. Org. 169, 353-356.
MINDEK, G. & NOTHIGER, R. (1973). Parameters influencing the acquisition of competence
for metamorphosis in imaginal discs of Drosophila. J. Insect Physiol. 19, 1711-1720.
POSTLETHWAIT, J. H. & SCHNEIDERMAN, H. A. (1973). Pattern formation in imaginal discs
of 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 of
Drosophila. Wilhelm Roux Arch. EntwMech. Org. 174, 303-311.
BOWNES,
{Received 30 June 1978, revised 11 August 1978)