/. Embryo/. exp.Morph. Vol. 61,pp. 303-316, 1981
3Q3
Printed in Great Britain © Company of Biologists Limited 1981
The distribution of regenerative potential
in the wing disc of Drosophila
By JANE KARLSSON 1
From the M.R.C. Laboratory of Molecular Biology,
University Postgraduate Medical School, Cambridge
SUMMARY
The distribution of regulative potential was investigated in the wing disc of Drosophila.
Ten complementary pairs of fragments were tested for their capacity to regenerate or
duplicate. The distribution of positional values resulting from this data was found to be
very unequal; six of the twelve clockface values were tightly clustered round the anteriorposterior compartment boundary. Despite this, complementarity between regeneration and
duplication was generally maintained.
INTRODUCTION
One of the most striking results to come out of Drosophila disc regeneration
work is that of two complementary fragments, one duplicates existing structures
while the other regenerates (for review see Bryant, 1978). Usually the smaller
fragment duplicates, but this is not always the case; for example the upper
medial quarter of the leg disc regenerates while the remaining three-quarters
duplicates (Schubiger, 1971). In the terms of positional information (Wolpert,
1971) and the polar coordinate model (French, Bryant & Bryant, 1976), this
quarter has over half the positional values. This uneven spacing of values
has been found in the haltere disc (van der Meer & Ouweneel, 1974) as well
as the leg disc, and inferred from indirect evidence in axolotl and newt limbs
(French et ai, 1976; Stocum, 1978; Holder, Tank & Bryant, 1980). However
in the wing disc, the evidence has suggested an even spacing (Bryant, 1975).
The present experiments were undertaken to find whether this was in fact
the case.
MATERIALS AND METHODS
Host flies were of ebony11 genotype; donors of ebony11 or occasionally
yellow; multiple wing hairs genotype. For descriptions of these mutants see
Lindsley and Grell (1968). Flies were raised at 25 °C on standard cornmeal/
syrup/agar medium seeded with live yeast. Wing discs were removed from
late-third-instar larvae in insect Ringer. Fragments were cut with tungsten
1
Author's address: Genetics Dept., South Parks Road, Oxford, U.K.
304
J. KARLSSON
PDR1
HP __UP_AS2
Tcgula
AS |
Cord
Dorsal
Fig. 1. Fate map of the wing disc (after Bryant, 1975). Presumptive wing blade
is stippled. Compartment boundaries are shown in broken lines. The position of
the dorsal end of the anterior-posterior boundary was taken from data of GarciaBellido, Ripoll & Morata (1976) and of the ventral end from that of Adler (1978).
Abbreviations:
presumptive anterior,
A, P
posterior
Scutellum
Scu
Humeral Plate
HP
UP
Unnamed Plate
Axillary Sclerites 1-3
AS 1-3
TRow
Triple Row of Chaetes
D Row
Double Row of Chaetes
PRow
PDR
A Lobe
A Cord
YC
PVR
PS
PWP
Posterior Row of Hairs
Proximal Dorsal Radius
Alar Lobe
Axillary Cord
Yellow Club
Proximal Ventral Radius
Pleural Sclerite
Pleural Wing Process
needles and implanted into the body cavities of well-fed 1- to 3-day-old
fertilized adult females where they remained for 5-7 days. They were then
removed and reimplanted into the body cavities of late-third-instar larvae
using the method of Ephrussi and Beadle (Ursprung, 1967). When the hosts
emerged as adults, the metamorphosed implants were removed, mounted in
Hydramount, and scored for the cuticular markers shown in Fig. 1. These
were identified with the aid of the descriptions of Bryant (1975).
RESULTS
In all the experiments reported here, the wing disc was cut into two fragments, both of which were tested for their ability to regenerate. The cuts
used are shown in Figs 2 and 3. Testing of both fragments was done for two
reasons. Firstly, to make sure that regeneration and duplication were indeed
Positional values in the Drosophila wing disc
10
IV
7 -
6 -
Fig. 2. (a) The pairs of fragments used in the first set of experiments. All fragments regenerated at a frequency of 19% or over; lines I-V are therefore lines of
transition between regeneration and duplication (see Results) and must have
6 values on either side, (b) The distribution of values resulting from these data.
Values 2, 10 and 11 were placed equidistant from their neighbours.
IX X
Vlll
a/.
ba/b
Fig. 3. (a) The pairs of fragments used in the second set of experiments, (b) The
final allocation of values.
305
Difference between
regeneration (a) and
duplication (b)
Difference between
regeneration (b) and
duplication (a)
12
L
6
2
if
-v
19
V
62
21
34
'
40
20
25
V
40
37
30
3
1
-v
6
V
18
59
17
8
if
39
29
28
Ilia Illb IVa IV b Va
18
71
_v—>
21
lib
14
3
i
1
__
27
V
53
19
75
Per cent regeneration 78
Per cent duplication 17
Ha
15
Ib
16
...
la
18
Fragment
n. ...
Table 1. Regeneration and duplication frequencies for the fragments shown in Figs. 2 and 3
53
>
21
Vb
19
V
8
83
8
1
T
0
82
-. /
65
5
5
24
0
89
0
54
6
20
60
20
V
8
69
8
9
r
0
60
V
9
55
Via VI b Vila VIIb VIIIaVIIIb IXa IX b X a
11 20
12
19 26 15 13 10 11
Percent duplication refers to the percentage of implants which had at least one structure duplicated and which did
not regenerate according to the criterion outlined in the Results section. The bottom two rows show the difference
between the regeneration frequency of one fragment and the duplication frequency of the other for each pair.
s
-
9
5
V
0
60
Xb
15
o
o
Positional values in the Drosophila wing disc
307
complementary; the frequency of regeneration in one fragment of a pair should
equal the frequency of duplication in the other. This was generally found to be
true (Table 1). Secondly, to check the accuracy of cutting; Bryant (1975) has
shown that duplicating fragments have all the structures, and only those
structures, expected from the fate map. If both fragments of a pair are tested,
and one regenerates while the other duplicates, the position of the cut can be
checked by consideration of the structures present in implants of the duplicating
fragment. If both of a pair regenerate sometimes and duplicate sometimes, the
duplicating implants of both can be used for this purpose. Duplicating implants
were found to have those structures expected from the fate map of Bryant
(1975), showing that the position of the cuts coincided with their intended
location.
Criterion for regeneration
An implant was considered to have regenerated if it contained at least one
structure (other than notum and scutellum, see below) which is not expected
from the fate map and lies at some distance from the cut edge. The structures
used as criteria for regeneration are in bold in Tables 2 and 3.
Complementarity between regeneration and duplication
Tn general it was easy to decide whether a particular implant had regenerated
or duplicated; implants considered to have regenerated by the above criterion
rarely had duplicated structures. If they did, in the majority of implants there
was a single duplicated structure close to the line of cutting, a phenomenon
which can be explained as due to anomalous healing (Reinhardt, Hodgkin &
Bryant, 1977). There were however three cases in which regeneration of one
structure was accompanied by duplication of two or more. The most important
of these was the tendency of presumptive ventral tissue to produce 'adventitious
bristles' (Bryant, 1975) while duplicating; while in most implants these bristles
were few in number (one to ten) and unidentifiable, occasionally there were
many and it was quite clear that they were from the notum. In other words,
duplicating ventral fragments which lack notum have the ability to regenerate
it; this phenomenon is at present under investigation (Karlsson & Smith, in
preparation). Structures other than notum and scutellum appear rarely and
only after much longer culture periods than used in the present experiments.
Notum and scutellum however appeared at high frequency; this meant that
neither of these structures could be used as criteria for regeneration. They
were scored in the following way. If an implant had notum or scutellum in its
fate map, or had regenerated according to other criteria, then these structures
were scored as notum and scutellum. If the implant did not include notum or
scutellum in its fate map and had formed no other structures well outside the
fate map, notum-like bristles were scored as 'adventitious bristles'.
...
Costa
TRow
D Row
PRow
PDR
A Lobe
A Cord
YC/PVR
PS/PWP
WB
Adv
HP
UP
AS1
AS2
AS3
Notum
Scutellum
Tegula
n
Fragment
25
25
75
50
50
25
25
—
75
25
—
—
50
—
—
—
—
100
+
t
jL
\
75
50
—
—
—
—
—
—
—
25
—
—
—
—
—
—
—
—
D
Non-R
4
A
la
86
36
64
36
57
50
50
36
57
93
43
93
79
50
29
71
50
100
R
14
69
77
54
69
46
38
46
31
54
100
46
—
—
—
—
—
—
—
+
i
\
69
—
—
—
8
—
38
—
54
38
—
—
—
—
—
—
—
D
Non-R
13
Ib
100
—
100
100
67
33
33
67
100
67
—
33
67
100
—
67
67
100
R
3
86
29
86
57
43
43
29
—
86
71
71
14
57
—
—
57
14
100
+
i
\
14
14
—
29
14
14
14
—
14
14
—
—
29
—
—
—
—
—
D
Non-R
7
A
Ila
100
38
100
88
88
88
88
88
100
100
63
88
75
63
25
100
88
100
R
8
—r
+
18
—
45
45
91
9
55
36
—
27
100
27
1
55
—
—
—
9
—
36
18
9
45
—
—
—
—
—
—
—
D
67
—
100
67
67
67
67
100
33
67
33
100
—
33
100
67
100
100
56
19
—
—
—
—
78
78
30
11
4
—
—
22
19
100
67
—
+
A
27
^~
t
—
19
22
—
—
—
—
4
4
4
—
—
—
—
56
67
—
D
N
Ilia
Non-R
A
\
^
R
3
^"
Non-R
11
A
lib
100
14
71
43
43
71
71
57
43
86
29
14
57
43
29
71
86
100
—
R
7
Table 2. Structures differentiated by implants of the fragments shown in Fig. 2, cultured in adult females for 5-7 days before
transfer to larval hosts for metamorphosis
R, regenerating implants. Non-R, non-regenerating (mostly duplicating) implants. + , percentage of implants where
the marker (complete or partial) was present singly. D, percentage of implants where the marker (complete or partial)
was present in duplicate (or triplicate or quadruplicate). The structures used as criteria for regeneration are in bold.
WB, wing blade. Adv., adventitious bristles. Other abbreviations as in legend to Fig. 1
O
O
GO
r
>
7*
oo
Positional values in the Drosophila wing disc
t-- o
I r- r- r*-
o t->
OOOOOOOOOOOOOO
oo(Nvo«/)Tfrom^tr-vo«vo>or-
M M i i i ^ i i i n M M
Tf Tt Tf
<s ts ts
8
tn N m I « M
t TtOOVOO
oo r~ vo (N «
oo^-ooooOooov©
O ©O I O
© «n </•> I © © © ©
ill
O
o >n o
°-
o
o
309
WB
Adv
A Lobe
A Cord
YC/PVR
PS/PWP
PDR
Costa
TRow
D Row
P Row
HP
UP
AS1
AS2
AS3
Notum
Scutellum
Tegula
n ...
Fragment
•
50
—
50
50
—
100
50
—
—
—
—
—
—
—
—
—
—
50
—
—
—
—
—
—
—
D
—
100
50
50
50
50
—
50
50
50
100
+
2
Non-R
Via
80
30
70
50
60
50
50
60
70
90
70
70
60
20
40
50
40
100
R
10
27
18
64
55
91
—
64
36
18
45
100
18
—
—
—
—
—
—
+
1]I
—
—
—
—
—
—
64
—
—
—
—
—
9
9
73
36
—
D
Non-R
VI b
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
R
0
86
14
57
57
71
71
71
29
86
71
57
71
71
—
29
29
—
100
+
7
—
—
—
—
—
—
—
—
—
14
—
—
—
—
—
—
—
—
D
Non-R
Vila
92
23
77
69
46
38
38
69
62
46
23
92
62
85
38
77
69
100
R
13
—
—
—
—
—
—
—
—
84
16
89
5
37
42
32
32
100
_A
—
—
—
—
—
—
79
—
—
—
—
—
58
42
26
47
—
D
Non-R
19
.
VII b
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
R
0
96
38
73
27
—
—
—
—
88
4
—
—
—
—
—
23
—
27
T^
26
1
4
—
4
12
—
—
—
—
—
—
—
—
—
—
—
31
—
—
D
Non-R
Villa
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
R
0
Table 3. Structures differentiated by implants of the fragments shown in Fig. 3, cultured in adult females for 5-7 days before
transfer to larval hosts for metamorphosis
R, regenerating implants. Non-R, non-regenerating (mostly duplicating) implants. + , percentage of implants where
the marker (complete or partial; was present singly. D, percentage of implants where the marker (complete or partial)
was present in duplicate (or triplicate or quadruplicate). The structures used as criteria for regeneration are in bold.
Abbreviations as in legends to Fig. 1 and Table 2.
o
C/5
>
—
—
—
—
—
17
17
17
—
—
—
—
17
—
17
—
33
—
33
17
—
—
33
17
17
—
—
83
50
83
33
50
—
17
17
100
78
44
78
78
67
56
78
89
89
11
100
89
100
56
67
67
100
100
75
25
50
50
75
75
75
—
25
75
—
25
75
—
—
25
75
100
25
25
33
56
56
78
67
78
56
56
89
33
89*
89
44
22
56
56
100
100
20
—
—
20
10
10
30
—
—
40
80
50
40
—
—
—
—
100
100
—
—
—
—
—
10
20
40
—
—
—
—
20
40
30
—
—
—
80
on
uu
,
t
—
100
80
20
60
20
—
—
100
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
17
33
33
17
67
67
50
50
50
67
—
67
50
100
83
50
100
27
40
33
60
—
—
27
73
47
40
47
—
—
100
53
47
33
13
20
20
13
—
—
—
7
27
7
—
—
—
27
—
7
—
100
—
—
100
—
—
—
—
—
* Posterior row lies too close to the cut edge in this fragment to be used normally as a criterion for regeneration; however in two implants
which had complete posterior rows it was so used.
WB
Adv
A Lobe
A Cord
YC/PVR
PS/PWP
PDR
Costa
TRow
D Row
PRow
HP
UP
AS1
AS2
AS3
Notum
Scutellum
Tegula
•-i
o
C5
I"
O
on
O
312
J. KARLSSON
The other two cases of simultaneous regeneration and duplication occurred
in fragments la and IVa. 4/18, and 4/30 implants respectively, duplicated
several existing structures and regenerated another; this regenerated structure
was yellow club in fragment la and alar lobe in fragment IVa.
In these three cases, a decision had to be made about whether to consider
the anomalous implants as having regenerated. It was decided not to do so if
the regenerated structure was not always clearly identifiable. Thus duplicating
ventral fragments producing 'adventitious bristles' were considered to have
duplicated, even in those cases in which the bristles were clearly from the
notum. The eight anomalous implants of fragments la and IVa, on the other
hand, were included in the regenerating category.
A second type of deviation from the rule of complementary between regeneration and duplication can be seen in Table 1. Here the regeneration frequency
of one fragment of a pair has been subtracted from the duplication frequency
of the other. Since each fragment can regenerate or duplicate (or both), the
10 pairs produced 20 such calculations. For 15 of these this figure is less than
10%, showing that complementarity is in general very well maintained. Of the
remaining five cases, there are two (pairs II and VII) in which one of the pair
is predominantly posterior and shows a high frequency of duplicated structures.
This high frequency seems to be characteristic of posterior fragments (Karlsson,
unpublished results) and is conceivably due to their having a high concentration
of structures in which duplication is easy to detect.
Two of the remaining cases concern pair III, both fragments of which appear
to have low regeneration frequencies. That this is not an artifact due to poor
growth is shown by the quite high frequency of duplication in both fragments.
Nor is it due to a lack of adequate markers for regeneration as both fragments
have several excellent ones.
Fragment VIII b, the fifth case, has a rather higher level of duplication
than expected from the complete failure of regeneration in its complementary
fragment. That this latter fragment is the only one wholly confined to the
anterior compartment could conceivably have a bearing on this result.
Allocation of positional values
The paradigm of the polar coordinate model (French effl/., 1976) was used,
and the twelve circumferential values were allocated on the following basis:
1. If both fragments of a pair regenerated at a frequency of 20% or over
(19 % in the case of fragment Ib) then the line separating them was considered
to be a line of transition between regeneration and duplication. Both such
fragments were allocated 6 of the 12 values, so that intercalary regeneration by
the 'shortest route' (French et al., 1976) around the disc circumference could
either repeat the existing values (duplicate) or replace missing ones (regenerate).
2. A fragment which never regenerated could not have more than five values.
The method employed for finding the distribution of values was to find
Positional values in the Drosophila wing disc
313
lines of transition between regeneration and duplication at several different
angles. This was done for the following reason: any two lines each having
an equal number of values on either side make four quadrants of which both
opposite pairs must have the same number of values. Thus if several such
lines are found, a distribution of values which obeys this rule for all pairs
must be the only possible one.
Two transition lines were known from the work of Bryant (1975); these
were confirmed (lines IV and V, Fig. 2) and three more were found. Values
were allocated by trial and error until a distribution was found in which all
fragments had six values, and all pairs of opposite quadrants had the same
number of values. Figure 2 shows the cutting lines and the resulting value
distribution. Values 2,10 and 11 were placed equidistant from their neighbours;
they are so closely spaced that more precise localisation would not be possible
within the limits of cutting error. The positions of values 4, 5 and 8 remained
to be determined.
The rest of the experiments were done to localise the remaining values and
confirm the positions of those already found. Fragments Villa, VIb and Xb
were used to allocate values 4, 5 and 8 respectively; none of these three fragments ever regenerated and the three values were therefore placed as shown
in Fig. 3 b.
Fragment IX b was used to confirm the very close spacing of values 9 to 11.
This fragment was cut very close ventrally to fragment Villa; since neither
of these two fragments ever regenerated, at least two values must be crammed
into this very small space.
DISCUSSION
The results show that the positional values in the wing disc are very unevenly
spaced, half of them being tightly clustered round the anterior-posterior
compartment boundary.
The simplest way in which unequal spacing could arise is through differences
in cell size or density, closer spacing corresponding to smaller or denser cells,
and all cells having the same proportion of the values. However imaginal
discs have no such differences which could correspond to differences in value
spacing (Ursprung, 1972). Another way is through unequal growth, which
could cause an initially equal spacing to become unequal during development.
This would provide an explanation for the sparseness of values in most of the
posterior compartment; clonal analysis shows that this compartment grows
faster than the anterior compartment during at least part of development
(Lawrence & Morata, 1976). However if this were the whole story, one would
expect clones lying along the anterior-posterior boundary, where values are
closest, to be smaller than average, and this is not the case (Garcia-Bellido
& Merriam, 1971).
It is possible of course that differences in value spacing have no functional
significance whatever, but the very extreme differences found in the present
314
J, KARLSSON
work seem to require an explanation. The spacing of positional values is
generally considered to represent the slope of a gradient of some cellular
parameter responsible for giving cells information about their position and
hence what to do (Wolpert, 1971). If each structure differentiates at a particular
threshold value of this gradient, close spacing would mean that thresholds
could be closer together; this might be required where particularly complex
structures were to differentiate. Indeed, the most complex structures of the
wing disc, those of the dorsal and ventral hinges, lie along the anteriorposterior compartment boundary where the spacing of values is closest. Again,
though, this cannot be the whole story, as there is no such correlation between
value spacing and structure complexity in the leg disc (Schubiger, 1968).
The close spacing at the anterior-posterior compartment boundary, if it
is not coincidence, could mean that cells at the boundary have some special
property. This could be for example the high point of a gradient; it has been
suggested that this boundary represents the common high point of two identical
gradients of positional information (Crick & Lawrence, 1975). Supposing that
regeneration can only occur down the gradient, fragments lacking boundary
would be unable to regenerate, but could only duplicate, as observed. Such
a theory would have to explain why many fragments having boundary do not
regenerate. The greatest difficulty though would be in explaining why the
anterior-posterior boundary is not required for regeneration in the leg disc
(Schubiger, 1971; Steiner, 1976).
The presence of the anterior-posterior compartment boundary has been
found necessary for distal regeneration to occur in both the wing disc (Wilcox
& Smith, 1980; Karlsson, 1980) and the leg disc (Schubiger & Schubiger, 1978),
and several observations suggest that something special is happening at compartment boundaries in normal development. Simpson (1976) has found that
the phenomenon of cell competition, by which slow-growing cells are eliminated,
operates to a much lesser extent at compartment boundaries than elsewhere,
suggesting that the rules governing growth are different at the boundary and
in the middle. Lawrence & Morata (1976) found that small engrailed clones
could cause a shape distortion of the wing blade if they touched the dorsoventral compartment boundary but not otherwise, and suggested that the
boundary was somehow instrumental in controlling growth. It may also be
significant that all well-documented compartment boundaries, in all appendages, are aligned along the major growth axes. These observations, together
with the finding that the anterior-posterior compartment boundary is important
in regeneration, suggest that the significance of compartments may reside in
their boundaries, and that it is here that the overall shape of appendages is
determined.
It is noteworthy that in spite of wide differences in the spacing of positional
values, the complementarity between regeneration and duplication is in general
very well maintained. It clearly never happens that both fragments of a pair
Positional values in the Drosophila wing disc
315
unequivocally regenerate, or unequivocally duplicate. Whether this applies at
the level of individual discs is not known as this would entail fragments from
single discs being kept separate, an experiment which has not so far been
done.
Complementarity between regeneration and duplication has been elegantly
explained by the polar coordinate model (French et al., 1976) in terms of both
fragments of a pair having the same positional values at their cut edges and
therefore intercalating the same structures. That this is indeed a useful way to
view regeneration is confirmed by the internal consistency of the present
results, without which it would not have been possible to allocate values.
This work was supported by an MRC Studentship and a Beit Memorial Fellowship.
REFERENCES
P. N. (1978). Positional information in imaginal discs transformed by homeotic
mutations. Wilhelm Roux' Arch, devl Biol. 185, 271-292.
BRYANT, P. J. (1975). Pattern formation in the imaginal wing disc of Drosophila melanogaster:
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