/ . Embryol. exp. Morph. Vol. 59,pp. 315-323, 1980
Printed in Great Britain © Company of Biologists Limited 1980
?>\5
Distal regeneration in proximal fragments of the
wing disc of Drosophila
By JANE KARLSSON 1
From the MRC Laboratory of Molecular Biology,
University Postgraduate Medical School, Cambridge
SUMMARY
The rules governing proximo-distal regeneration in the wing disc of Drosophila were
investigated. It was found that proximal fragments confined to either anterior or posterior
compartments could not regenerate distally, although many fragments having tissue from
both compartments could do so even in the absence of circumferential regeneration. Fragments containing the ventral but not the dorsal end of the anterior-posterior border were
able to regenerate distally. The use of a cuticular marker in the posterior compartment very
close to the border permitted precise localization of the tissue required to cause anterio
fragments to regenerate distally; in anterior fragments cut close to the border, there was an
almost perfect correlation between possession of this marker and distal regeneration. It
was found however, that distal regeneration was not an all-or-none phenomenon; its extent
was dependent on the total amount of tissue present from both compartments.
INTRODUCTION
Regeneration in imaginal discs can be conveniently thought of as occurring
in two separable axes, one circumferential and the other radial or proximodistal (French, Bryant & Bryant, 1976). Much of the early work concerned
the circumferential axis (for review see Bryant, 1978), and only recently have
specific questions been asked about the proximo-distal one (Schubiger &
Schubiger, 1978; Haynie & Schubiger, 1979; Strub, 1979; Wilcox & Smith,
1980).
It was originally thought that distal regeneration could not occur without a
complete circumference of proximal tissue (French et al. 1976), but recent
results have shown that some distal regeneration can occur from a partial
circumference (Schubiger & Schubiger, 1978; Wilcox & Smith, 1980). This is in
line with work on amphibia which has shown that distal regeneration can occur
from some surgically constructed double half limbs following amputation
(Slack & Savage, 1978; Holder, Tank & Bryant, 1980). Other double half limbs
fail to regenerate distally, and this has been interpreted to mean that the half
in question had very few circumferential positional values (Stocum, 1978;
Holder et al. 1980). However this interpretation may well be inadequate to
1
Author's address: Department of Zoology, West Mains Road, Edinburgh EH9 3JT, U.K.
316
J. KARLSSON
explain similar failures in Drosophilia imaginal discs. Schubiger & Schubiger
(1978) found in the leg disc that the relationship between the number of circumferential values and the ability to regenerate distally was often the opposite of
what would be expected on the above hypothesis. They suggested instead that
what was required was the presence of dividing cells from both anterior and
posterior compartments (Garcia-Bellido, Ripoll & Morata, 1976).
Wilcox & Smith (1980) have made observations on dissociated wing disc
fragments which largely support this idea. They found that anterior tissue was
unable to regenerate distally although reaggregates containing cells from both
compartments were able to do so. However, they found that posterior tissue
could sometimes regenerate distally, contrary to the suggestion of Schubiger &
Schubiger.
The present experiments were undertaken to find whether intact proximal
wing-disc fragments which did not regenerate circumferentially could regenerate
distally, and if so whether there was a requirement for tissue from specific
parts of the disc which might indicate an involvement of compartments.
Questions of this kind can be asked more precisely in the wing disc than in the
smaller leg disc, and more precisely using intact fragments than dissociations.
Some fragments were chosen because they were capable of circumferential
regeneration, so that conclusions could be drawn about the relationship between
the number of circumferential positional values and the ability to regenerate
distally.
MATERIALS AND METHODS
Host and donor flies were of ebony11 genotype (Lindsley & Grell, 1968) and
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 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 & Beadle (Ursprung,
1967). When these emerged as adults, the metamorphosed implants were
removed and mounted in Hydramount. They were then scored for the cuticular
markers shown in Fig. 1, which were identified with the help of the descriptions
of Bryant (1975). Fate maps were done using the same procedure but missing
out the adult incubation step.
The fragments used in these experiments are depicted in Fig. 1 and were
chosen so as to avoid presumptive wing blade (stippled area) which is therefore
the main marker for distal regeneration. The amount of wing blade present
was estimated by eye on an arbitrary scale of 0-5 where 5 is a completely
regenerated wing blade. That presumptive wing blade actually was avoided in
the anterior and dorsal parts of the disc is shown by its complete absence from
implants of fragments c and/(with the exception of a few complete regenerates,
Distal regeneration in the Drosophila wing disc
317
Fig. 1. Fate map of the wing disc (after Bryant 1975), and the fragments used.
Presumptive ventral tissue is at the top of thefigure,dorsal below. The anterior compartment is to the left of the broken line, posterior to the right. The position of the
ventral end of this border was deduced from the structures present in bithorax and
postbithorax haltere discs (Adler 1978). Presumptive wing blade is stippled. The
pairs a-b, c-d, e-f, and h-i were cut from the same discs. Abbreviations: HP, humeral
plate; PS, pleural sclerite; A Lobe, alar lobe; A Cord, Axillary cord; AS 1-3,
Axillary sclerites 1-3; PDR, Proximal dorsal radius; T Row, triple row of chaetes;
D Row, double row of chaetes; P Row, posterior row of hairs.
see below). Many implants of fragments containing the posterior ventral part
of the disc (b, g and j) had small amounts of wing blade, and a fate map of
fragment b was done to ascertain whether this was regenerated or already present
before culture. This fate map had a similar amount (fate map 1-0 ± 0-2, n = 14;
after culture 0-6 ± 0-1). It was concluded that presumptive wing blade lies closer
to the edge of the disc in the posterior than in the anterior and that this was the
reason for its presence in implants of fragments b, g and/
A few implants of fragments a-i and m regenerated circumferentially, producing a complete disc with no evidence of duplication. Since in these complete
regenerates, distal structures may well have been formed following circumferential regeneration, data from these implants was not included in Tables 1 or
2. Frequencies of the different structures were therefore calculated as a percentage of those implants which did not regenerate circumferentially. The total
number of recovered implants, including these complete regenerates, is shown
in brackets in the top row of Table 1.
Of the remaining implants, none produced proximal structures not expected
21
EMB 59
1 vAlllldl
UULOC1J.
d
e
f
g
hi
37
3
_
4 D
_
_
38 100 _
- 64 14
_ 23 _
_ 72 _
14 27
_
_ _
_
_ _
16
_ _
22
_ _
78
_ _
62
47
_
_
_
_
63
25
3
+ D
_
_
_
_
68
43
36
89
__
75
_
_
_
_
21
57
57
4
+ D
26
A
*T
_
_ _
+ D
4• D
_ _
71 29
13 4 100 _ _
7 52 26
7 35 52
_ _
35 57
__
22 61
__
13 13
14 _ 9
- 7
_
_
4- D
72 24
48 12
_ 8
60 _
_ _
_ _
52 8
48 _
_ 72
_
_
4- D
82 18
73 18
9 36
45 _
9 55
_ _
_ _
_ _
_ _
c
4- D
_ _
37 11
19 22
78 11
30 48
41 41
_ _
_ _
_ _
b
11 (39)
a
27 (30) 32 (33) 22 (25) 28 (29) 23 (24) 14 (15) 25 (29)
i
h2
i
j
k
_
88
44
6
88
50
31
_
_
_
_
6
38
50
6
38
69
_
_
_
4- D
_
94
_
_
_
_
9
47
47
_
4
_
D
3
_
_
_
_
16
38
38
84
8 _
D
_
_
_
_
_
_
25 50
42 42
_ 42
4_
_
_
_
_
_
8 -
4- D
__
_ _
_ _
__
8 8
25 58
__
_ _
_ _
16 (39) 32 (35) 12 (12) 12(12)
32
_
4- D
_ _
_ _
_ _
26 _
16 68
21 63
_ _
_ _
_ _
/
19 (19)
8
23
23
46
54
23
38
38
15 _
23
8
54
54
46
46
8
_
4- D
- -
m
13 (17)
n
50 -
4- D
- 14 - 71 57 21
50 29
- _ __
14 (14)
Proximal structures differentiated by implants of fragments a-n, cultured in adult females for 5-7 days before transfer to larval hosts for
metamorphosis. + , percentage of implants where the marker (complete or partial) was present singly; D, pecrentage of implants where the
marker (complete of partial) was present in duplicate (or triplicate or quadruplicate); hlt those implants of fragment h which did not contain
the pleural sclerite; h2, those implants of fragment h which did contain the pleural sclerite. All gaps mean zero frequency. Numbers in brackets in
the top row denote the total number of recovered implants, including those which regenerated circumferentially (see Materials and Methods).
X
Notum
Tegula
Humeral plate
Costa
Yellow club
Pleural sclerite
Alar lobe
Axillary cord
Axillary sclerite 3
Axillary sclerites
1 and 2
Proximal dnr^ai
radius
Adventitious
bristles
Fragment
n ...
Table 1
t—*
r
o
oo
a
b
+
6
_
-
d
—
100
D
+ D
- 100 - _
_ _
68 29
- 3-8 + 01
c
/
100
—
+ D
+ D
100 - 14 13 52
_ _
30 13
_ _
52 26
- 3-7±0-2 - -
e
12
+ D
56 _ _
_ _
12 0-6±0-2
8
h
i
j
k
/
93
75
17
50
84
+ D
+ D
+ D
D
+ D
+ D
92 - 100 97 - 100 50 11 - - 69 31
- - 11 _ _ 44 - _
9 42 17 _ 50 42 42
78 6
_ 3-6±O-3 2-5±0-2 0-7±0-2 l-5±0-3 21 ±0-2
—
+
9
9
_
_
_
K
100
100 _
54 31
62 23
62 23
3-5 + 0-3
+ D
m
79
vo
I'
on
O
\J
+ D &
86 57 14
7 - S'
14 2-3±0-3 Hi
n
Distal structures differentiated by implants of fragments a-n, cultured in adult females for 5-7 days before transfer to larval hosts for metamorphosis. + , 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). Also shown are average amounts of wing blade on a scale of 0-5,
and the percentage of implants having two or more units on this scale (% distal regeneration). All gaps mean zero.
+ D
+ D
Wing blade 100 - 78 56 33
Triple row
_ _
41 4
Double row
_ _
Posterior row 41 - 59 Amount of 3-2±01 0-6±01
wing blade
% Distal
100
16
regeneration
Fragment
Table 2
*ener
320
J. KARLSSON
from the fate map with the exception of adventitious bristles (Bryant, 1975).
This indicates that the actual cuts coincided with their intended location in the
circumferential axis. Most implants in addition showed evidence of duplication.
Where duplication was not evident, the structures involved were usually those
in which duplication can be difficult to detect, such as costa, tegula and notum.
RESULTS
The results of scoring of the metamorphosed implants are shown in Tables
1 and 2. These data allow the following conclusions to be drawn:
(1) Distal regeneration can occur without circumferential regeneration. Whilst
circumferential regeneration did occur in some fragments, it is clear that the
majority of implants of all fragments except b, c, f g and j regenerated distally
in the absence of circumferential regeneration.
(2) Distal regeneration does not occur in fragments which do not contain the
area in which the ventral end of the the anterior-posterior compartment border
lies. Fragments a, d and e, which all contain this area, always produced two or
more units of wing blade. Fragments b, c,f g andy do not contain this area, and
of these c and / never produced wing blade. Fragments b, g and j often had
small amounts of wing blade, but this is expected from the fate map (see
Materials and Methods).
(3) Anterior fragments cannot regenerate distally without some posterior
ventral tissue. Fragment h was cut as close to the ventral end of the anteriorposterior border as possible, and only produced wing blade if the implant
contained the pleural sclerite, which lies in the posterior compartment close to
the border. In this experiment, all implants showing no evidence of duplication
were discarded as well as those showing clear evidence of circumferential
regeneration. This was done to make quite sure that all circumferentially regenerating implants were eliminated. Of 27 duplicating implants, 11 lacked the
pleural sclerite, and only one of these had any wing blade (1 unit). The remaining
16 had the pleural sclerite and all had wing blade, 15 of them in considerable
amounts (Table 2, fragment h2). Thus a very small amount of posterior tissue
is sufficient to cause extensive distal regeneration in anterior fragments.
(4) Fragments unequivocally confined to the posterior compartment cannot
regenerate distally. Fragment j is confined to the posterior compartment and
produced only those small scraps of wing blade produced by fate maps of this
area. Since Wilcox & Smith (1980) found that this fragment regenerates distally
when dissociated, albeit at low frequency, another experiment was done in
which these fragments were mixed together in pairs (Haynie & Bryant, 1976) on
the assumption that this procedure breaks up the tissue in somewhat the same
way as dissociation does, though to a lesser degree. Again no distal regeneration
was observed (average amount of wing blade 0-7 + 0-2 per fragment, n = 16).
However, fragment /, which according to the fate map shown in Fig. 1 has
Distal regeneration in the Drosophila wing disc
321
no anterior ventral tissue, is able to regenerate distally (Table 2). The exact path
of the compartment border is not known though and it is quite possible that
this fragment does indeed have anterior ventral tissue.
(5) The amount of distal regeneration in fragments containing the ventral end
of the anterior-posterior border is dependent on the amount of proximal tissue
present from both compartments. Fragments k-m show an increase in the
frequency and extent of distal regeneration with increasing amounts of proximal
tissue. The amount of wing blade increases from 1-5 ±0-3 through 2-1 ±0-2 to
3-5 ±0-3 and the percentage of implants having two or more units from 50
through 84 to 100. Comparison of fragments k, a and d shows that both compartments contribute to this effect.
(6) Fragments incapable of circumferential regeneration cannot regenerate all
the way to the distal wing tip. Fragments k, I and n never regenerated circumferentially and very rarely produced double row which lies at the distal wing tip.
In contrast, fragment a, which can regenerate circumferentially, often produced
double row. Fragments a and n are from the same part of the disc and both
regenerate triple row. The numbers of medial triple row bristles were counted
in those implants of these two fragments whose triple row was not duplicated.
The average number of bristles in fragment a was 24 ±4, and in fragment n
14 ± 3. This indicates that distal regeneration in fragment n stops well before it
reaches the distal wing tip.
(7) The character of the distal regenerate is dependent on the provenance of the
proximal tissue. Fragment a, which is mainly from the anterior compartment,
regenerated only anterior wing margin structures (triple row, double row, a few
hairs of posterior row) while fragment d, which is mainly posterior, regenerated
only posterior structures (posterior row). This suggests that these fragments
regenerated half the wing blade each. The[amount of wing blade they produced
was considerably over half (3-2 ±0-1 and 3-8+0-1 respectively on a scale of 0-5),
but this can be explained by complete or partial duplication of the wing blade
in some implants, which is consistent with the duplication of marginal structures
often observed. Fragment e produced a complete wing margin, which suggests it
regenerated a complete wing blade; however since the average amount of wing
blade was no greater than fragment d (3-7 + 0-2), it seems more likely that this
fragment too regenerated about half the wing blade, this time the ventral half.
Each fragment of the pairs a-b, c-d and e-/"regenerated circumferentially at
about the same frequency, indicating that they each have half the circumferential positional values. Thus a fragment with half the values produces
half the wing blade.
DISCUSSION
The results presented here show that intact fragments unequivocally confined
to either anterior or posterior compartments cannot regenerate distally,
although many fragments having tissue from both compartments can do so
322
J. KARLSSON
even in the absence of circumferential regeneration. The excellent correlation
found in fragment h between possession of posterior tissue and distal regeneration indicates that the apparent connection between compartments and distal
regeneration is probably real.
The results differ somewhat from those of Wilcox & Smith (1980) who used
dissociated wing-disc fragments, and found that although anterior tissue could
not regenerate distally, posterior tissue sometimes could. They found in addition, as did Schubiger & Schubiger (1978) on the leg disc, that fragments containing either end of the anterior-posterior border could regenerate distally,
whereas in the present experiments only fragments containing the ventral end
did so. A possible explanation for their finding of occasional distal regeneration
from posterior fragments is that they are able to regenerate anterior tissue at low
frequency. That this might be the case is indicated by the presence of'adventitious
bristles' (Bryant, 1975) in one implant of fragment j in the present experiments.
These bristles are probably from the notum (Karlsson & Smith, in preparation),
in which case such a fragment would contain the dorsal end of the anteriorposterior border, which stimulates distal regeneration in dissociations but not
in intact fragments.
There are two different ways in which distal regeneration might be influenced
by the compartment border; either it is stimulated by an interaction between
anterior and posterior cells, or there is no interaction but border cells have
special properties enabling them to regenerate distally. It has been suggested
(Crick & Lawrence, 1975) that the border represents the high point of two identical gradients of positional information (Wolpert, 1971), and an attractive
extension of this idea would be that the high point is necessary for distal regeneration. This might predict that the entire distal regenerate comes from border
cells. Wilcox & Smith (1980) have shown however that cells some distance from
the border contribute to the distal regenerate, indicating that an interaction
between anterior and posterior is the stimulus for distal regeneration rather
than some special property of border cells. It would be interesting if this interaction reflects something similar in normal development, as compartments are
generally thought of as autonomous units, and no function for them has been
suggested which requires such an interaction.
This interaction seems to have the nature of a trigger, since distal regeneration is not an all-or-none process but dependent in its extent on the total amount
of proximal tissue present from both compartments. Fragments with half the
circumferential positional values (a, b and e) produce the appropriate half of
the wing blade. Fragments with less than half (k, I and n) produce less wing
blade and are unable to regenerate all the way to the distal wing tip. These
results are consistent with proposals put forward by several authors that distal
regeneration is proportional to the number of circumferential positional values
(Stocum, 1978; Bryant & Baca, 1978; Cummings & Prothero, 1978; Bryant,
French & Bryant, in preparation). However none of these authors predicts
Distal regeneration in the Drosophila wing disc
323
any requirement for distal regeneration other than a minimum number of
circumferential positional values. The rinding that compartments are involved
in distal regeneration may mean that these ideas will have to be modified.
This work was supported by an MRC Studentship and done under the supervision of
Dr M. Wilcox for whose help I am very grateful. I thank Tsvi Sachs for help with the manuscript. The paper was written while the author was in receipt of a Beit Memorial Fellowship.
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