Developmental behaviour of fragments of symmetrical and ...

sentencecopyElectronics - Devices

Oct 13, 2013 (3 years and 8 months ago)

64 views

J. Embryol. exp. Morph., Vol. 16, 2, pp. 355-368, October 1966 355
With 2 plates
Printed in Great Britain
Developmental behaviour of fragments of
symmetrical and asymmetrical imaginal discs of
Drosophila melanogaster (Diptera)
By ROLF NOTHIGER
1
& GEROLD SCHUBIGER
1
From the Zoologisch-vergl. anatomisches Institut der Universitat Zurich
INTRODUCTION
Earlier experiments have demonstrated a very high 'regulative' ability in the
genital disc of Drosophila melanogaster. Medially sectioned discs, even from
mature larvae, are able to form a fully differentiated normal genital apparatus if
the fragments are cultured in vivo in a larval host for a sufficient length of time
before metamorphosis. Even a quarter of a disc has this 'regulative' ability.
However, it should be emphasized that the fragment as a unit is not capable of
'regulation', but only its single anlage elements (Hadorn, Bertani & Gallera,
1949; Hadorn, 1963; Ursprung, 1959). These findings appeared to contradict
the prevailing belief that Drosophila exhibits a mosaic development. This view
was favoured by Geigy (1931), who showed that an anlage pattern of presump-
tive imaginal structures was already present at an early stage of embryogenesis.
Similarly, mosaic development in the leg disc was postulated by Bodenstein
(1941), who found, for example, that when half of a leg disc formed the sex
comb the complementary half did not.
The unpaired genital disc is a bilaterally symmetrical anlage whose left and
right halves contain the same presumptive material (Text-fig, la). In contrast,
in the paired leg discs, the anlage material is distributed asymmetrically (Text-
fig. 2). Thus, medial sectioning of the genital disc results in two equal halves,
while any two halves of the leg disc are unequal with respect to their anlage
material.
These facts have led us to ask the following questions. (1) Is it possible to
induce regulation and/or regeneration in fragments of young leg discs when
transplanted into young larval hosts? (2) What is the behaviour of half of a
genital disc which has been made experimentally asymmetrical with respect to
one of its anlagen when it has sufficient time for 'regulative' growth?
1
Authors' address: Zoologisch-vergl. anatomisches Institut der Universitat Zurich,
Switzerland.
356
R. NOTHIGER & G. SCHUBIGER
MATERIAL AND METHODS
Drosophila stocks
The wild stock 'Sevelen' of Drosophila melanogaster was used in all experi-
ments as larval donor and host. The adult hosts were fertilized females of a
homozygous white stock (w: 1-1-5). The animals were reared on standard food
(maize, sugar, agar, yeast) at 25 °C. The age of donors and hosts in each experi-
mental series is given in hours (h) after egg laying.
Co S
G
A L I
30/i
250-300/;
(a) (b)
Text-fig. 1. (a) Anlage plan of the male genital disc (Ursprung, 1959). G, Hind gut;
A, anal plates; C, claspers; L, lateral plates; PB, peripheral bristles of the genital
arch, (b) Procedure for causing unilateral damage in the genital disc by UV-irradia-
tion. S, Plane of sectioning; Co, non-irradiated control.
Technique
Genital discs and first leg discs of male larvae were dissected in Ringer's
solution and cut with a tungsten needle. The fragments were then transplanted
into larvae or flies (Ephrussi & Beadle, 1936). We tried to obtain the desired
asymmetry of the genital disc by irradiation with a fine UV-beam (Text-fig. 1 b).
A 30x50/*
2
area was irradiated for 15 sec with a 265 m/* wavelength. The
optimal irradiation time must be experimentally determined for each UV-
source. A prolonged local irradiation may result in the death of all cells of the
imaginal disc. The apparatus used for UV-irradiation was described by Ursprung
(1959) who succeeded in obtaining specific defects in the disc. He employed this
method to determine the anlage plan of the genital disc.
,>.„.„, j.
r
, Experimental plan
{a) First leg disc oj male
r
Series A: donors, 96 h (late third-instar larvae); hosts, 96 h. This experiment
was conducted to determine the anlage plan. For this purpose the discs were cut
into several fragments and their developmental capacities were investigated.
Series B: donors, 76-80 h (early third-instar larvae); hosts, 55-58 h (second-
instar larvae). Each half of a disc was implanted into a larval host. The larval
Development ofimaginal discs 357
hosts were reared individually in separate vials. Thus, it was possible to follow
complementary halves of the same disc. This procedure was necessary because
the asymmetrical discs could not be cut in exactly the same plane in each case.
(b) Male genital disc
The genital discs were cut in half and transplanted immediately after irradia-
tion into their respective hosts (Text-fig. 1 b). At first the procedure described for
series B was used to follow complementary halves of the same disc. Later, this
complication was dropped because every median cut resulted in two halves
containing portions of all anlagen (Text-fig. 1). Hence any irradiated half is
complementary to any non-irradiated half.
Series C: donors, 100 h (late third-instar larvae); hosts, 55-58 h.
Series D: donors, 100 h; hosts, 24 h old adult females.
The fragments were allowed to grow in the adult hosts for 3 days. They were
then removed and transplanted into 78 h old larvae where they metamorphosed
simultaneously with their hosts. In spite of the additional work involved in
using an intermediate adult host, this method was employed because the disc
fragments grew sufficiently in the adult milieu so that they could be retrans-
planted into older larval hosts which have a considerably lower death-rate than
young larval hosts. The results of series C and D were the same.
Permanent preparations
We removed the metamorphosed implants from the abdomens of the final
hosts 2-3 days after emergence. Whole mounts in Faure's solution were prepared
according to standard procedures. In the genital apparatus only the presence or
absence of a certain element was recorded and not the number of bristles on the
plates which varied greatly. In some cases the bristle pattern was also altered.
T
,. RESULTS
Leg disc
Series A. In order to determine the anlage plan the discs were cut into different
fragments and transplanted into mature larvae, where they were forced to begin
metamorphosis within a few hours (method described by Hadorn & Buck, 1962).
A simplified anlage plan is shown in Text-fig. 2. It contains only those structures
which we examined in these experiments. A more detailed anlage plan has been
worked out by one of us (G. S.) and will be published elsewhere. Text-fig. 2 also
shows where the discs were cut and demonstrates that the anlagen are unequally
distributed in the two halves. Each of the halves differentiated only those struc-
tures which one would predict from the anlage plan, thus confirming Bodenstein's
results (1941).
Series B. Seventeen complementary halves of single discs were analysed. A
typical pair is shown in Plate 1, fig. A. One can see from this illustration that the
disc fragments made more structures when they had a prolonged period of
358 R. NOTHIGER & G. SCHUBIGER
growth before pupation (some 70 h), than when they metamorphosed immedi-
ately as in series A. But this intensive growth resulted only in a duplication or
enlargement of those anlagen already present in the fragment at the time of
implantation. No regulation or regeneration of the missing anlagen occurred.
Thus, a hah"" disc made either two sex combs or none, depending on whether or
not it contained the corresponding anlage material. This was also true for the
asymmetrically distributed primordia of the proximal leg segments. Four re-
presentative pairs of complementary disc halves are described in Table 1. We
observe that the markers under investigation appear, as a rule, in either the
median (M) or lateral (L) half, but not in both. The structures whose anlage
material is clearly located on the median site (e.g. the transversal rows (TR) of
the tibia and the sex comb (SC)) follow this rule without exception. These
structures are only differentiated by median halves. The differentiated elements
are either double structures or at least contain a greater number of bristles than
normal. In some cases we observed that both the median and lateral halves made
claws (Plate 1, fig. A and Table 1 pairs 8 and 10). Two other examples of this
Table 1. Differentiation of leg disc halves after transplantation into
55-58 h old larval hosts
Tarsal segment
Trochanter
, * , Tibia
No. GSt Stl Sc
+
5 EB Sc'S Sc 3 St 5 Bo-Z TR
8M ++ + + - - + + + + + + + +
T
I
I J
I
9M + ++ + - - + + + + + + + +
L -
10 M + + ^
L -
12 M + + 4
L -
Four complementary pairs are shown: Nos. 8, 9, 10, 12 (series B, page 356). M, Median;
L, Lateral half (Text-fig. 2); +, structure present, -, structure absent, + +, structure doubled
or enlarged; surrounded by lines = structure in both halves. G St, group of sensilla trichodea;
St 1, one single sensillum trichodeum; Sc
+
5, five sensilla campaniformia on hairy cuticle;
EB, edge bristle; Sc~S, eight sensilla campaniformia on hairless cuticle; Sc 3, three sens. camp.;
St 5, row of five sens, trich.; Bo-Z, trochanter bristles other than the edge bristle; TR, bristles
of the transversal
TOWS
of the tibia; SC, sex comb; Cl, claws. The same results were obtained
in the coxa and the femur (not shown).
PLATE 1
Fig. A. Structures differentiated by a half leg disc. Al, Median half; A2, Lateral half. TR,
transversal rows of the tibia; SC, sex comb; Cl, claws; Tib, tibia; TS, tarsal segment.
Fig. B. Symmetrical duplications in the trochanter differentiated by a median half of a leg
disc. EB, Edge bristle; Sc~S, eight sensilla campaniformia.
1.
sc
+
+
5.
Cl
+
+
/. Embryol. exp. Morph., Vol. 16, Part 2 PLATE 1
Tib
-£8
Sc-8
100/i
R. NOTHTGER & G. SCHUBIGER
facing p. 358
J. Embryol. exp. Morph., Vol. 16, Part 2
PLATE 2
PB
R. NOTHIGER & G. SCHUBIGER
facing p. 359
Development of imaginal discs
359
£8
Text-fig. 2. Simplified anlage plan of the male foreleg imaginal disc. Part of the
prothorax and the different leg segments from the coxa to the tarsus are arranged in
'concentric' rings from the outside to the centre. One ring corresponds to one seg-
ment whose structural elements are distributed around the ring. During metamor-
phosis the entire disc is drawn out in a telescopic fashion. M, Median; L, Lateral.
Trochanter: dotted area, bristles; hatched area, hairs; 2G St, two groups of sensilla
trichodea; Sc
+
5, five sensilla campaniformia; St 1, one single sensillum trichodeum;
EB, edge bristle; Sc~$, eight sens, camp.; Sc 3, three sens, camp.; St 5, row of five
sens, trich. Tibia: TR, transversal rows (bristles). First tarsal segment: SC, sex comb.
Fifth tarsal segment: Cl, claws. S, Plane of sectioning.
PLATE 2
Fig. C. Normal and complete male genital apparatus (only chitinous parts), formed by an
implanted untreated whole disc. Pe, Penis apparatus; A, anal plates; C, claspers; L, lateral
plates; PB, peripheral bristles of the genital arch.
Figs. D and E. Symmetrical defects in irradiated halves of genital discs. D, Anal plates missing,
claspers (C) not yet fully separated. The remaining structures are normal. E, lateral plates
missing. The remaining structures are normal, but during handling the topography was
altered. Abbreviations are the same as in Fig. C.
23 JEEM l6
360 R. NOTHIGER & G. SCHUBIGER
regeneration-like phenomenon are shown in Table 1. These are the edge bristles
(EB) and a group of sensilla (So" 8) of pairs 10 and 12. These exceptions occurred
with the following frequencies: claws 6/17, edge bristles 7/17, sensilla 13/17.
These cases will be considered in the discussion.
These results are consistent with the view that the development of leg discs is
mosaic in character. This mosaicism manifests itself even in disc fragments which
are implanted into young larval hosts where they have a long period of growth
before metamorphosis. Under these conditions only a duplication or an en-
largement of the anlagen already present is observed.
Genital disc
(a) Preliminary experiments. Control transplantation experiments confirmed
that non-irradiated whole male genital discs could make a completely normal
sex apparatus (Plate 2, fig. C). In these experiments only the chitinous structures
of the external apparatus were dealt with. The inner soft parts—vasa deferentia,
paragonia, ductus ejaculatorius, sperm pump and hind gut (Ursprung, 1959)—
usually were severely damaged by irradiation and could not be recognized easily.
The method of UV-irradiation employed made it possible to eliminate single
elements more or less selectively. This was a prerequisite for answering our
question as to the behaviour of asymmetrical genital disc halves. We irradiated
30 whole discs and found that none made a normal genital apparatus. In 12
cases all of the structures were present, but either the bristle number was re-
duced or the topography was disturbed. In the remaining 18 cases we succeeded
in completely eliminating one or more elements. The distribution of these
eliminations is summarized in Table 2. For our purposes it was not important
which anlage elements were missing.
Table 2. Element specific damages of unilaterally irradiated whole
genital discs (Text-fig. 1b)
No. of cases with
more than one
No. of cases with one element absent element absent
Pe* A + C+L C+L
Missing on one side 4 0 2 1 1 4
Missing on both sides 6 0 0.0 0
A, Anal plates; C, claspers; L, lateral plates; Pe, penis apparatus (see Plate 2, fig. C).
* Unpaired central element.
We do not know why both anal plates are often lacking. Even in experiments
with non-irradiated genital disc halves the anal plates are the elements most
sensitive to injury (Table
4). Heavier doses of UV caused the irradiated area of the
normally transparent disc to become opaque and to die. We observed under the
microscope that the local irradiation damage spread to an ever increasing area,
Development ofimaginal discs 361
finally causing the death of the entire disc. It may be that even the lower doses
induced a wave of damage which resulted in the elimination of both sensitive
anal plates. To exclude this factor from our experiments we cut the discs im-
mediately after irradiation.
(b) Series C and D. We shall present series C and D together since both gave
Table 3. Differentiation of irradiated and non-irradiated
halves of genital discs
Hosts
Irradiated Non-irradiated
Schematic 55h larvae Adults 55h larvae Adults
Results of differentiation example n = 31 n = 50 n = 56 n = 29
(a) Complete and symmetrical Pe 7 (2) 22(11) 70(39) 62(18)
genital apparatus L L
CC
AA
(b) Symmetrical only for single Pe 13 (4) 4 (2) 20(11) 31 (9)
elements. At least one L L
element of each type C —
present A A
(c) Only one element of every Pe 3 (1) 2 (1) 5 (3) 0
type present L —
C -
A -
(d) Some types of elements com- Pe 22 (7) 12 (6) 0 0
pletely eliminated. Only L —
one element of each of C —
the other types present - -
(e) Some types of elements com- Pe 55(17) 60(30) 5 (3) 7 (2)
pletely eliminated. One or L L
more elements of the other — —
types symmetrically present AA
Total 100(31) 100(50) 100(56) 100(29)
Numerals in bold print are percentages, followed by absolute number of cases in parenthesis.
n designates total number of cases. Pe, Penis apparatus; L, lateral plates; C, claspers; A, anal
plates; -, structure missing. See Text-fig. 3.
the same results. We found that non-irradiated disc halves could differentiate a
complete genital apparatus in most cases (Table 3, row a). Irradiated halves
were seldom able to do this. If we take row a and b together we see that non-
irradiated half-discs could 'regulate' more or less completely in 90-93 % of the
cases, while 'regulation' took place in irradiated halves in only 20-26 % of the
cases. There were instances of special interest in which a symmetrical sex appara-
tus was made which lacked single elements symmetrically (row e). 55-60% of
the irradiated halves belong to this category, in contrast to only 5-7 % for the
362
R. NOTHIGER & G. SCHUB1GER
non-irradiated halves. For our purposes rows a, b, d, and e (Table 3) are the
most important ones, since obvious differences between irradiated and non-
irradiated half-discs are revealed here. Apparently the irradiation had elimi-
nated single anlage districts which then could no longer be replaced even by
intensive growth. This inability resulted in an incomplete genital apparatus
with symmetrical defects as illustrated in Plate 2, figs. D, E.
Table 4. Element specific differentiation of irradiated and non-irradiated
halves of genital discs
No. of elements per implant
1
Anal plates Irradiated
Non-irradiated
Claspers Irradiated
Non-irradiated
Lateral plates Irradiated
Non-irradiated
Penis apparatus:
Complete Irradiated
Non-irradiated
Incomplete Irradiated
Non-irradiated
12
(1)





12
(1)
12
0)


41
(33)
85
(72)
56
(45)
89
(76)
54
(44)
77
(65)
11
(9)
9
(8)
33
(27)
11
(9)
20
(16)
23
(20)
45
(37)
6
(5)
98
(8)


26
(21)


81
85
81
85
81
5
44
(36)
85
(72)
56
(45)
15
(13)
Numerals in bold print are percentages, followed by absolute number of cases in parentheses.
The number of elements in a normal genital apparatus is surrounded by lines, n designates
total number of cases. See also Plate 2, figs. D, E, and Table 3.
Table 4 shows the distribution of the injuries for the single elements of the
genital apparatus. The anal plates are eliminated with the highest frequency and
the claspers with the lowest. This differential sensitivity is consistent with the
findings in our preliminary experiments. The only structures ever eliminated in
non-irradiated discs are the anal plates (Table 3, row e, and Table 4). Only very
rarely does the number of plates increase. The central primordia of the penis
apparatus are only weakly impaired by the lateral irradiation. The observed
defects may also be caused by mechanical injuries induced by cutting the disc
medially.
Development ofimaginal discs 363
DISCUSSION
Interpretation
A medially halved genital disc, implanted into a mature larval host, forms
half of a genital apparatus. Only the central unpaired elements of the penis
apparatus become complete through 'regulative' growth. However, the same
disc fragment differentiates into a whole genital apparatus after transplantation
into a young larval host (55-60 h old). In contrast, half of a leg disc can never
make a complete leg, even when it is allowed to grow for a long period before
metamorphosis (series B). Under such conditions, the disc fragment differentiates
a double structure consisting of two symmetrical half legs containing the same
elements (Plate 1, fig. A). The complementary structures of the other half of the
leg were not made. Hence, when the presumptive anlage material for a
structure such as the sex comb is absent (according to our anlage plan), the
half-disc is unable to make the missing structure even after intensive growth.
This growth results only in a duplication of those structures whose anlagen are
already present in the fragment (Table 1). This fact has also be pointed out by
Gehring (1966) for fragments of the antennal disc. This mechanism of repetition
will simulate production of complementary structures in discs possessing
bilateral symmetry such as the genital disc (Text-fig. 1). Here the repetition
leads to a restoration of totality, while this is impossible for the asymmetrical
leg disc (Text-fig. 2).
The elimination of single elements of the genital disc with UV (Text-fig. 1 b)
has shown that the same mechanism of duplication which occurs in fragmented leg
discs operates here as well. A genital disc which has lost a particular quality
after UV-irradiation cannot restore it. Thus, the genital disc half makes two
half-structures, which appear normally as one symmetrical complete genital
apparatus. This situation is shown schematically in Text-fig. 3. The genital and
leg discs behave in exactly the same way in this respect. However, it should be
noticed that when a disc fragment is cultured for a longer period (months and
years) it may become able by transdetermination to differentiate structures which
were not present before as anlagen in the disc (Hadorn, 1965).
The question now arises as to what extent our findings are general. In fact,
these results were not unexpected. Multiplication of identical structures and
symmetrical duplications have already been demonstrated by Vogt (1946) for
eye-antennal disc fragments, by Hadorn & Buck (1962) for wing disc fragments,
and most recently by Gehring (1966) for antennal disc fragments. One can see
from their results that the fragments never made complementary structures, since
complete antennae or wings were never found. An apparent exception to the
above is shown in Plate 1, fig. A, where both halves of a leg disc made claws. This
could mean that one of the halves regenerated a complementary structure.
However, this phenomenon can easily be explained in another way: the cut was
made through the middle of the claw anlage, which is thus distributed to both
23-2
364
R. NOTHIGER & G. SCHUBIGER
fragments of the disc. Therefore, both halves were able to differentiate claws.
This interpretation also holds true for the other examples of this sort listed in
Table 1 (EB and Sc~8). Gehring (1966) suggested that the observed differentia-
tion of complementary structures in antennal discs which were cultured for
weeks could result from' region specific transdetermination'. This term refers to a
switch in determination from one structure to another whose anlage is normally
present in the complete disc. Under this interpretation, the claws, sensilla and
edge bristles would arise in complementary disc halves as transdeterminations of
cells which were originally determined for other leg structures. However, we
believe that this interpretation does not apply to our results since all of the pri-
mordia which were made by both halves were located in the middle of the disc
where it was cut.
Metamorphosis Metamorphosis
in 55 h old larvae
/
\
AA
Claspers lacking
Text-fig. 3. Schematic illustration of the UV-elimination experiment (see Text-fig. 1;
Plate 2, figs. D, E; Table 3). I. Male genital disc with anlage regions for lateral plates
(L), peripheral bristles (PB), claspers (C), hind gut (G), anal plates (A). S, plane of
sectioning. At the right side: elimination of a clasper anlage by UV-irradiation.
II. Differentiated structures of the non-irradiated (left side) and irradiated (right side)
half-disc. Pe, Penis apparatus. Below this, the result is given in the schematic form
used in Table 3.
Development of imaginal discs 365
, -,
D
.. Regeneration and regulation ?
{a) Regeneration *
6
The term 'regeneration' denotes those morphogenetic processes which result
in a replacement of the missing parts in a structurally and functionally differen-
tiated system (P. Tardent, personal communication). Although imaginal discs
responded to the loss of a part with cell divisions, they did not replace missing
anlagen in these experiments. They could only duplicate those anlagen which
remained. Thus, apart from rare transdetermination (Hadorn, 1965), the divi-
ding cells only repeat their own state of determination. From this we conclude
that the term 'regeneration' should not be applied to the growth of fragments of
imaginal discs, or even to the reconstitution of a single anlage. Although a
division of the claw anlage, for example, results in the formation of two claws by
a proliferation of the anlage material in question (Plate 1, fig. A), this does not
come under the above definition of regeneration, since this growth involved un-
differentiated cells. The production of the new cells is not under obvious quantita-
tive control. If a sufficient amount of time is allowed for growth, more material
will be made than was originally present. The growth extends even to those regions
which are not damaged by the cut. An example of this occurs when a leg disc is cut
in half and the edge bristle and the sensilla located on the trochanter away from the
wound surface double as a result of proliferation and reorganization of their
anlage material (Plate 1, fig. B, EB and Sc~S). The occurrence of duplications of
this sort (Table 1) suggests to us that mitoses take place all over the fragment, and
not only at the wound surface and in the new blastema as was found by Kroeger
(1958) for fragments of wing discs of Ephestia. Whether or not cell migration
plays a role in the process of duplication is not known. Hence, it is possible that
the cell divisions do not produce a typical blastema, which grows out of the
wound, but a general proliferation. These problems are in need of further
investigation.
(b) Regulation
The term 'regulation' denotes those morphogenetic processes which result in
a restitution of wholeness in a structurally and functionally undifferentiated
system. This process occurs without an increased rate of cell division and involves
only a reorganization of the material already present (P. Tardent, personal
communication). At no time do the imaginal discs investigated thus far exhibit
regulation as it is denned above. At the most, the behaviour of a single anlage
(e.g. for claspers, anal plates, sex comb, etc.) may be considered as regulation
(Hadorn et al. 1949; Ursprung, 1959). However, these examples of regulation
involve more than a simple reorganization of the material already present, since
they also require growth. For example, half of a genital disc contains one clasper
anlage; this does not reorganize immediately and form two smaller anlagen, but
first enlarges by cell division, preserving the same state of determination. We
then suppose that a process of organization occurs (homonomous arealization—
23-3
366 R. NOTHIGER & G. SCHUBIGER
Gehring, 1966) which may proceed in the same manner as during normal
development.
Regulation requires that the prospective potency be greater than the prospec-
tive significance (fate). In agreement with earlier workers we have demonstrated
that for all of the structures shown in the anlage plan (Text-figs. 1, 2), the pro-
spective potency is equal to the prospective significance. However, this may not
apply to the individual cells of an anlage, since the specific role that a particular
cell will have in the formation of a clasper, an anal plate, a sex comb, etc., is
probably not determined before metamorphosis. Thus, the prospective potency
of single cells may be greater than their prospective significance, but only within
the confines of a clasper, an anal plate, a sex comb, etc. Under this interpretation
we may speak of this process as regulation. However, since it is preceded by an
increase in cell number, we propose to call this special behaviour 'proliferative
regulation'.
Wildermuth & Hadorn (1965) recently found that an intact labial disc is
capable of forming a complete proboscis, while its prospective significance is
only half a proboscis. Although this appears to be an example of classical regu-
lation, cell divisions are also required here (H. Wildermuth, personal communi-
cation). A further example which could be taken as classical regulation was
described by Hadorn (1953), who found harmoniously reduced anal plates in
fragmented male genital discs of Drosophila seguyi. As Liiond (1961) re-
vealed in a careful study of subsequent stages of this regulation process, a har-
moniously reduced anal plate is formed only when there were more than sixteen
bristles, which is half the normal number. When this number was not reached as
a result of insufficient time for growth before metamorphosis, the bristle pattern
was disproportionate. So here again cell proliferation is necessary for the regu-
lation process. Hence, the term 'proliferative regulation' applies to this case as
well.
As far as we can see now, proliferative regulation comprises the following
processes. (1) A fragmented primordium enlarges by cell multiplication, whereby
the area-specific state of determination is reproduced. (2) When the blastema has
reached a critical mass at the time of metamorphosis, it is arealized (homono-
mous arealization) into anlage districts of like size resulting in two or more
symmetrical and identical structures. Proliferative regulation is followed by
pattern formation in which individual cells are given specific roles (e.g. dif-
ferentiation into a clasper bristle or a clasper epidermal cell).
SUMMARY
1. The developmental behaviour of imaginal discs of Drosophila melanogaster
was investigated. The discs were medially halved and transplanted into 55 h old
larval hosts to allow a sufficient time for cell proliferation.
2. The different anlagen for the male first leg are asymmetrically distributed
Development ofimaginal discs 367
on the leg disc. Its median and lateral halves did not differentiate any structures
of the complementary half even after intensive growth. The proliferation re-
sulted only in an enlargement or duplication of the already present anlagen.
3. In the symmetrical male genital disc certain anlagen could be eliminated by
unilateral local irradiation with UV. This technique allowed the production of
asymmetrical discs. After cutting, the non-irradiated half formed a complete and
symmetrical genital apparatus. The irradiated half differentiated into a sym-
metrical apparatus which lacked, however, single elements on both sides. A
schematic illustration is given.
4. The results are interpreted to mean that the cell divisions initiated by the
fragmentation of the disc always repeat the same state of determination. During
a culture period of 3-4 days before metamorphosis a replacement of missing
anlagen does not occur.
5. The two terms 'regeneration' and 'regulation' are discussed with respect
to imaginal discs. We have proposed the modified term 'proliferative regula-
tion' to describe the special behaviour ofimaginal disc fragments. This pheno-
menon consists of two processes: (1) an intensive cell proliferation whereby the
state of determination is conserved; (2) a division of the overgrown blastema
into areas of a fixed size (homonomous arealization).
The authors gratefully acknowledge the assistance of Dr H. Oberlander in preparing the
English translation of this paper and wish to thank Professor Dr E. Hadorn, Professor Dr
P. Tardent and Dr W. Gehring for their critical reading of the manuscript. The publication
of this paper was supported by the 'Karl Hescheler—Stiftung.'
ZUSAMMENFASSUNG
1. Imaginalscheiben von Drosophila melanogaster wurden halbiert und ihre
Entwicklungsleistungen nach Transplantation in junge 55 h alte Larvalwirte
untersucht. Die Fragmente erhielten so geniigend Zeit zu ausgedehnter Zell-
proliferation.
2. Mediane und laterale Halften mannlicher Vorderbeinscheiben, auf
der die Anlagen fur die verschiedenen imaginalen Bildungen des 1. Beines
asymmetrisch verteilt sind (Text-fig. 2), differenzierten auch nach intensivem
Wachstum keine Strukturen der komplementaren Partnerhalfte. Die starke
Zellproliferation ausserte sich nur in einer Vergrosserung oder Verdoppelung
der vorhandenen Anlagen (Plate 1 und Tab. 1).
3. In der symmetrisch aufgebauten mannlichen Genitalscheibe wurden
bestimmte Anlagen mit UV einseitig ausgeschaltet und die Scheibe dadurch ex-
perimentell asymmetrisch gemacht (Text-fig. 1). Nach der Halbierung bildete die
unbestrahlte Halfte einen vollstandigen und symmetrischen Geschlechtsapparat
(Plate 2). Die bestrahlte Halfte differenzierte ebenfalls einen symmetrischen
Geschlechtsapparat, dem aber einzelne Elemente beiderseitig fehlen (Plate 2).
Die Versuchsanordnung ist schematisch in Text-fig. 3 dargestellt.
368 R. NOTHIGER & G. SCHUBIGER
4. Die Resultate werden dahin interpretiert, dass die Zellteilungen, die im
Anschluss an die Fragmentation einsetzen, in der Regel nur gleiche Determina-
tionszustande repetieren. Etwas Fehlendes wurde unter den gegebenen experi-
mentellen Bedingungen nicht erganzt.
5. Die beiden Begriffe 'Regeneration' und 'Regulation' werden diskutiert
und fiir das spezielle Verhalten der Fragmente von Imaginalscheiben der modi-
fizierte Terminus 'proliferative Regulation' vorgeschlagen. Diese umfasst zwei
Prozesse: 1. Eine intensive Zellvermehrung, wobei der gleiche Determina-
tionszustand beibehalten wird; 2. Eine Gliederung des iibergrossen Blastems in
Areale einer festgesetzten Grosse (homonome Arealisation).
REFERENCES
BODENSTEIN,
D. (1941). Investigation on the problem of metamorphosis. VIII. Studies on leg
determination. /. exp. Zool. 87, 31-53.
EPHRUSSI,
B. &
BEADLE,
G. W. (1936). A technique of transplantation for Drosophila. Am.
Nat. 70, 218-25.
GEHRING,
W. (1966). Obertragung und Aenderung der Determinationsqualitaten in Anten-
nenscheiben-Kulturen von Drosophila melanogaster. J. Embryol. exp. Morph. 15, 77-111.
GEIGY,
R. (1931). Erzeugung rein imaginaler Defekte durch ultraviolette Eibestrahlung bei
Drosophila melanogaster. Wilhelm Roux Arch. EntwMech. Org. 125, 406-47.
HADORN,
E. (1953). Regulation and differentiation within field districts in imaginal discs of
Drosophila. J. Embryol. exp. Morph. 1, 213-16.
HADORN,
E. (1963). Differenzierungsleistungen wiederholt fragmentierter Teitiicke mann-
licher Genitalscheiben von Drosophila melanogaster nach Kultur in vivo. Devi. Biol. 7,
617-29.
HADORN,
E. (1965). Problems of determination and transdetermination. Brookhaven Symp.
Biol. 18, 148-61.
HADORN,
E.,
BERTANI,
G. &
GALLERA,
J. (1949). Regulationsfahigkeit und Feldorganisation
der mannlichen Genital-Imaginalscheiben von Drosophila melanogaster. Wilhelm Roux
Arch. EntwMech. Org. 144, 31-70.
HADORN,
E. &
BUCK,
D. (1962). Ober Entwicklungsleistungen transplantierter Teilstiicke
von Flugel-Imaginalscheiben von Drosophila melanogaster. Rev. suisse Zool. 69, 302-10.
KROEGER,
H. (1958). Ober Doppelbildungen in die Leibeshohle verpflanzter Fliigelimaginal-
scheiben von Ephestia kuhniella. Wilhelm Roux Arch. EntwMech. Org. 150, 401-24.
LUOND,
H. (1961). Untersuchungen zur Mustergliederung in fragmentierten Primordien des
mannlichen Geschlechtsapparates von Drosophila seguyi. Devi Biol. 3, 615-56.
URPSPRUNG,
H. (1959). Fragmentierungs- und Bestrahlungsversuche zur Bestimmung von
Determinationszustand und Anlageplan der Genitalscheiben von Drosophila melanogaster.
Wilhelm Roux Arch. EntwMech. Org. 151, 504-58.
VOGT, M. (1946). Zur labilen Determination der Imaginalscheiben von Drosophila. I.
Verhalten verschiedenaltriger Imaginalanlagen bei operativer Defektsetzung. Biol. Zbl. 65,
223-38.
WILDERMUTH,
H. &
HADORN,
E. (1965).Differenzierungsleistungen der Labialimaginalscheibe
von Drosophila melanogaster. Rev. suisse Zool. 72, 686-94.
(Manuscript received 29 March 1966)