/ . Embryo!. exp. Morph. Vol. 57, pp. 155-165,1980
Printed in Great Britain © Company of Biologists Limited 1980
155
The initiation of pupariation in Drosophila:
dependence on growth of the imaginal discs
By P. SIMPSON, 1 P. BERREUR 2 AND
J. BERREUR-BONNENFANT 2
From the Centre de Genetique Moleculaire, Gif-sur-Yvette, France
SUMMARY
Regeneration was induced in the imaginal discs in situ following lesions caused by heatsensitive cell-lethal mutations. A clonal analysis of this event demonstrated that the subsequent delay in pupariation was correlated with the amount of extra growth that occurred
during the regeneration. Pupariation of heat-treated gynandromorphs bearing the mutations
was also retarded, and the duration of larval development increased with greater amounts
of mutant tissue, it was therefore correlated with the extent of the lesions in the imaginal
discs. Elimination of entire imaginal discs, or the presence of very small amounts of lethal
tissue, did not result in prolonged larval life.
INTRODUCTION
It is well known that pupariation in Diptera is brought about by the moulting
hormone, 20-OH-ecdysone. Ecdysone is secreted by the ring gland under the
influence of the neurosecretory cells of the brain. It is later converted by
peripheral tissues (for review, see Doane, 1973). Very little is known, however,
about how the timing of pupariation is controlled. We are currently investigating
the relationship between growth of the imaginal discs and the onset of metamorphosis. Regeneration interferes with the latter. A delay in pupariation has
been reported for temperature-sensitive cell-lethal mutations in Drosophila after
heat pulses (Russell, 1974; Simpson & Schneiderman, 1975), and a similar
delay in larval moulting or metamorphosis has been observed in other insects
undergoing regeneration of differentiated appendages or imaginal discs
(O'Farrell & Stock, 1954; Pohley, 1967; Rahn, 1972; Dewes, 1975; Kunkel,
1977).
The delayed pupariation of regenerating animals implies some sort of
communication between the imaginal discs and the hormonal centres that
bring about metamorphosis. It seems likely that imaginal discs that have not
completed their growth are somehow able to inhibit pupariation. We have
1
Author's address: Centre de Genetique Moleculaire, C.N.R.S., 91190 Gif-sur-Yvette,
France.
2
Authors' address: Equipe de Recherche 229, C.N.R.S., 91190 Gif-sur-Yvette, France.
156 P. SIMPSON, P. BERREUR AND J. BERREUR-BONNENFANT
designed experiments to answer the following questions: First, is the observed
delay in pupariation correlated with the amount of extra growth required to
regenerate the missing parts? Second, what is the minimal amount of damaged
imaginal disc tissue that is capable of retarding the onset of metamorphosis?
MATERIAL AND METHODS
The following mutations and stocks were used: the sex-linked recessive
lethals l(l)ts 504 (Simpson & Schneiderman, 1975) and I(l)ts 5697 (Arking,
1975), hereafter abbreviated to 504 and 5697. These mutations have temperaturesensitive periods extending throughout the entire larval and early pupal stages.
They both cause extensive cell death in the imaginal discs at 30 °C, the restrictive
temperature. The chromosome 504 sn3 5697 was balanced with M-5 (see
Lindsley & Grell, 1968, for a description of mutations). We used the mutations
singed, (sn3) and multiple wing hairs, (mwh) as cell markers; they affect bristles
or trichomes, respectively. sn3 is on the X chromosome and served to reveal
the male patches on the gynandromorphs. mwh is on the third chromosome
and was used to mark clones induced by mitotic recombination in both male
and female tissue.
Gynandromorphs were produced by means of the unstable ring-A" chromosome, R(l)2wvC (Hinton, 1955). This chromosome is frequently lost during the
early divisions in the egg, but remains stable throughout larval development.
Ring-X bearing males were mated to 504 sn3 5697; mwh females. Loss of the
ring-X chromosome in ring X - r o d X female zygotes forms XO male tissue,
which in this case expresses the recessive mutant phenotypes of 504, 5697 and
sn3.
Flies were grown on a corn meal-yeast-agar medium, and were allowed to
lay eggs in split bottles at 23 °C for 2 h periods. Care was taken to avoid overcrowding of the culture bottles. A heat pulse at 30 °C was applied between
48 and 96 h after egg laying. Larvae were irradiated (dose 1000 R, Theta X-ray
machine, machlett tube, 1 mm Al filter, 40 kVP, 20 mA) at 120 h in order to
induce mitotic recombination (Becker, 1957) and the formation of mwh clones.
On the wing blade each cell secretes one trichome (Dobzhansky, 1929), and
clone sizes are therefore given as number of cells per clone.
For a chronology of events at 23 °C, see Fig. 1.
Pupae were collected from the sides of the culture bottles twice a day. In one
experiment, the time of eclosion of the flies was recorded, once each day. No
attempt was made to dissect uneclosed flies from their pupal cases. Gynandromorphs were selected under the dissecting microscope. Animals were cooked
in 10% KOH and mounted in Euparal for examination under the compound
microscope.
Gynandromorphs were examined for the distribution of male XO 504 sn3
.5697 mutant tissue. The derivatives of 15 imaginal discs were scored: 2 eye-
157
Time of pupariation and growth of imaginal discs
30°C
E
LI
L2
r
-rays
Stage of development
at23°C
rt^T.
A
L3
11
13
15
Days
Fig. 1. Stages of development at 23 °C, the permissive temperature for 504 sns 5697
animals. The times of the heat pulse given at 30 °C (48-96 h after oviposition) and of
X-irradiation (120 h) are indicated.
Table 1. Number and mean size of clones on wing blade, and time of eclosion of
male flies from the cross M-5//(l)ts 504 sn3 /(l)ts 5697 xmwh, grown at 23 °C,
heat-pulsed at 30 °C, and irradiated at 120 h
Clones
Genotype
3
Temperature
pulse (h
after
oviposition)
504 sn 5697; mwh/mwh+ (Control)
48—96
M-5: mwh/mwh+
(Control)
48—96
Mean day
eclosion
±S.E.
13 6 ± 0 1
14-4 ± 0 1
13-5 + 0-2
13-4±0-2*
FreAverage
quency size in cell
per wing numbers
disc
±S.E.
Number
89
35
103
129
1-2
0-4
37±5
272 ±35
1-5
1-3
28±3
3O±3
* One might expect the heat pulse to advance pupariation but it did not, probably because
M-5 flies are at a slight disadvantage at higher temperatures: at 29 °C M-5/M-5 females
hatch 0-6 days later than M-5/504 sn3 5697 females.
antennal, 2 proboscal, 2 dorsal prothoracic, 2 wing-thoracic, 6 leg and 1 genital
discs. The tergites 1-6 were also scored for left and right sides of the body.
Mosaic discs were counted as one half.
RESULTS
Heat treatment causes the death of a variable number of cells of the mutant
imaginal discs, which are presumably regenerated by the remaining cells after
the return to permissive temperature (Simpson & Schneiderman, 1975). Clone
sizes allow a rough estimation of the amount of cell death. Irradiation of heatpulsed male larvae resulting from the cross M-5/504 sn3 5697 x mwh <$ causes
the appearance of mwh clones (Table 1). Clones on the wings of control M-5
flies averaged 30 cells. If we assume the final wing contains approximately
II
BIB
57
158 P. SIMPSON, P. BERREUR AND J. BERREUR-BONNENFANT
30000 cells (Garcia-Bellido & Merriam, 1971a) then it follows that the wing
region of the discs contained approximately 1000 cells at the time of irradiation.
By the same reasoning the mutant discs contained about 110 cells at this time.
Therefore over 80% of the cells were killed yet regulation still occurred and
the majority of the discs regenerated and metamorphosed into normal wings.
Haynie & Bryant (1977) and Clark & Russell (1977) also reported complete
regulation following cell death in imaginal discs. The loss of cells is also
reflected in the lower frequency of clones per mutant wing. It will be noted that
this frequency is nevertheless higher than would be expected from our estimation
of the number of remaining cells. We have no explanation for this discrepancy
that has been repeatedly observed. Perhaps following heat shock, the mutant
cells are weaker and more susceptible to X-irradiation or perhaps a greater
number of them are dividing.
Regeneration and delayed eclosion. In order to determine whether the delay
of metamorphosis is proportional to the amount of regeneration, the duration
•of development and the extent of growth were measured after heat-pulseinduced lesions. Male flies hatching from the cross M-5/504 snz 5697 xmwh <$
were counted each day. It can be seen from Table 1 that eclosion of the ts
mutant male flies occurred 1 day later than that of the M-5 male controls.
The clone size directly indicates the amount of growth that occurred between
initiation of the clone and cytodifferentiation (Bryant & Schneiderman, 1969).
The mean clone size observed after heat treatment was 272 cells in the wings
of ts mutant flies and 30 cells in the wings of the M-5 controls, thus confirming
the supposition that cell death is followed by regeneration in many discs.
During this regeneration the remaining cells of the mutant wing disc must have
undergone on average three more cell divisions than the cells of the undamaged
control discs (272/30 = 211; n = 3-18). Doubling time has been estimated at
approximately 8-5 h in the developing wing disc at 25 °C (Bryant, 1970; GarciaBellido & Merriam, 1971a). Thus a 24 h delay in development of the ts mutant
flies provides times for approximately three additional cell divisions and suggests
that pupariation was retarded by the amount of time necessary to repair the
lesions. This would only be the case, of course, if cell division remained constant
and was not accelerated in the regenerating discs.
Frequency o/XO mutant tissue on gynandromorphs and delay of pupariation.
Gynandromorphs produced by loss of the R(l)2wrC chromosome present
variable frequencies and distributions of XO tissue. Heat treatment (48-96 h
after oviposition) of these larvae caused cell lesions only in the XO mutant
imaginal disc territories, the XX female territories being unaffected. After the
return to permissive temperature, regeneration can occur by division of either
the remaining XO cells or the XX cells in the case of mosaic discs. Growth was
monitored by comparing the sizes of mwh clones induced by X-irradiation at
120 h. In a first experiment (in which the time of pupariation was not recorded)
gynandromorphs with bilaterally mosaic thoraces were selected. An average
Time of pupariation
and growth of imaginai
discs
120 - 1 4 4 h ( « = 47)
1-5%
A
144-168 h (n = 38)
15-1%
0
1
2
3
4
5
6
7
8
10 11 12 13 14 15
9
3
N u m b e r of à sn discs
120-144 h (n = 50)
B
0
1
2
3
4
5
6
7
8
3
N u m b e r of d sn
9
10 11 12 13 14 15
discs
3
Fig. 2. T h e extent of male 504 sn 5697 cuticle present in the imaginal disc derivatives
of 504 sn 56971R{l)2w^
mwh/+
g y n a n d r o m o r p h s that p u p a r i a t e d o n the 6th,
7th, 8th day or later, after oviposition. (A) animals were grown at 23 °C, subjected to
a heat pulse at 30 °C from 48 to 96 h a n d X-irradiated at 1 2 0 h . I n the control
series, t o o few g y n a n d r o m o r p h s p u p a r i a t e d after 168 h to provide an accurate study.
T h e n u m b e r of g y n a n d r o m o r p h s scored for each series is given in parentheses. T h e
percentage represents the average a m o u n t of male sn cuticle. It c a n be seen in (A)
t h a t the majority of g y n a n d r o m o r p h s that p u p a r i a t e d early h a d very few or n o male
sn disc derivatives, i.e. only the a b d o m e n s bore male sn cuticle.
3
3
3
3
160
P. S I M P S O N , P. B E R R E U R
A N D J. B E R R E U R - B O N N E N F A N T
clone size of 53-7 ±9-0 was found on the entirely male wings (70 clones) and
10-3 ±1-9 on entirely female wings (106 clones). This result is similar to that
just described for entirely mutant flies compared with M-5 controls. The
mutant discs underwent nearly three divisions more than the heterozygous
female ones in the same animals (54/10 = 2 ; n = 2-5).
It has previously been shown that the heat-pulse-induced delay in eclosión
of 504 flies is the direct result of a comparable delay in pupariation (Simpson
& Schneiderman, 1975). For greater precision we have measured the time of
pupariation in 504 sn 5697 gynandromorphs. Those subjected to a heat pulse
from 48 to 96 h pupariated at 7-5 ±1-1 (S.D.) days (n = 157), compared to
6-4 ±0-4 (S.D.) days (n = 112) for control non heat-treated gynandromorphs. It
can be seen that the onset of pupariation of the experimental gynandromorphs
varied considerably.
The percentages of male sn tissue found on gynandromorphs pupariating at
different times are presented in Figs. 2 and 3. Although there is considerable
individual variation, earlier authors have recorded that on an average approxi­
mately 50 % of the adult cuticle is male due to early loss of the ring chromosome
(Hotta & Benzer, 1972; Ripoll, 1972). While this is true of the control gynandro­
morphs it will be noticed that addition of all the experimental series does not
reveal an average of 5 0 % male tissue in the imaginal discs (34%). Presumably
one category of the gynandromorphs did not survive. They may have failed to
hatch because of pattern abnormalities in the head and thorax (see Russell,
1974; Arking, 1975; Simpson & Schneiderman, 1975).
In the experimental series, animals that pupariated on the 6th and 7th day
bore no or very small areas of lethal tissue in the imaginal disc derivatives. We
therefore conclude that a minimal amount of regenerating tissue (on average
two or more discs), is necessary to retard pupariation (see Fig. 2). Those
animals that pupariated later resembled the controls and presented nearly
50 % male sn mutant cuticle.
A high frequency of experimental gynandromorphs were observed with very
small amounts of male disc tissue, 1 5 % of the experimental series had male
tissue only on the abdomen and genitalia (Class ' O'), compared to 7 % in the
controls. This is undoubtedly an overestimation of the actual number of such
animals originally present in the population. Frequencies in the experimental
series are not directly comparable with those of the control series, since the
entire population of gynandromorphs (n) was not recovered. Since the missing
flies most likely bore male discs, if they had been included, this would have
increased the number («), and therefore lowered the frequency of class ' O '
animals. Furthermore a number of these gynandromorphs were found to have
one or more missing imaginal disc derivatives (altogether 2-8 % of the discs
were absent). Presumably in these cases the entire disc was composed of XO
mutant tissue that died. Dissection of such animals did not reveal the presence
of unevaginated cuticle. Missing territories were not included in our study, but
n
3
3
3
Time of pupariation
and growth of imaginai discs
1 2 0 - 1 4 4 h {n = 4 4 )
C
42 A
A
1 4 4 - 1 6 8 h (n = 39)
45-5%
0
1
2
3
4
5
6
7
3
N u m b e r of 6 sn
8
9
10
11
12
tergites
120
144 h (n = 50)
B
g
1 4 4 - 1 6 8 h ( « = 50)
I
45-8%
0 1
2
3
4
5
6
N u m b e r of 6 sn
3
7 8 9
3
10
11
12
tergites
Fig. 3. T h e extent of m a l e 504 sn 5697 cuticle present o n the dorsal a b d o m e n of
504 sn 5697/R(l)2w ;
mwh/+
g y n a n d r o m o r p h s t h a t p u p a r i a t e d on the 6th, 7th,
8th day o r later after oviposition. (A) animals were g r o w n at 23 °C, subjected t o a
heat pulse from 48 to 96 h a n d X-irradiated at 120 h . (B) C o n t r o l series, animals
grown at 23 °C a n d X-irradiated at 120 h. T h e n u m b e r of g y n a n d r o m o r p h s scored
for each series is given in parentheses. T h e percentage represents the average a m o u n t
of male sn cuticle.
3
vC
3
161
162 P. SIMPSON, P. BERREUR AND J. BERREUR-BONNENFANT
if they had been scored as male, they too would have contributed to a decrease
in the frequency of class ' O ' flies.
It is worth noting that those gynandromorphs bearing male tissue only on
the abdomen, but missing some imaginal discs, were only observed amongst
flies that pupariated on days 6 or 7.
In flies in which pupariation was retarded because of lesions in only some of
the imaginal discs, the other intact discs had an extended period of time for
growth before the onset of metamorphosis. They did not however differentiate
organs or appendages that were any larger than the usual size. One may suppose
that either growth is completed at a slower rate in such discs (or at a normal
rate, accompanied by cell death), or that growth stops and the discs 'wait*
until the other damaged discs have completed their growth, and metamorphosis
takes place. A comparison of mwh clone sizes between male and female wings
of gynandromorphs with bilaterally mosaic thoraces that pupariate at different
times, should enable a distinction to be made between these possibilities.
Insufficient clones were obtained in the present experiment for us to draw a
clear conclusion.
The abdomen was relatively unaffected by the heat pulse (Fig. 2), 55 % of
the gynandromorphs hatching on the 6th day displayed male J/I3 cuticle solely
on the abdomen. Previous studies have revealed that cell death occurs in the
histoblasts when the animals are exposed to the restrictive temperature during
the time of pupariation (Simpson & Schneiderman, 1975), i.e. at the stage at
which they undergo cell division (Garcia-Bellido & Merriam, 19716; Guerra,
Postlethwait & Schneiderman, 1973). Heat treatment during the larval period
either does not affect the non-dividing histoblast cells, or else the lesions are
sufficiently repaired before pupariation.
DISCUSSION
Previous reports on other holometabolous insects have shown that implantation of imaginal disc fragments can retard pupariation of larval hosts which
contain their own full complement of discs (Pohley, 1967; Rahn, 1972; Dewes,
1975). Furthermore a correlation was observed between the duration of larval
development and the stage of completion of the implanted regenerate (Rahn,
1972; Dewes, 1979). The results of the clonal analysis presented here, are
consistent with this result. In mutant animals undergoing regeneration, pupariation was found to be delayed by more or less the amount of time needed for
completion of the extra growth in the imaginal discs.
Three of our observations of gynandromorphs are pertinent to the question
of whether imaginal disc growth is linked to the timing of pupariation:
(1) First, a number of gynandromorphs bearing male tissue only on the
tergites, but deficient for one or more imaginal discs was observed. As such
flies pupariated at the normal time, absence of imaginal discs is clearly without
Time of pupariation and growth of imaginal discs
163
effect. This observation supports a theory in which growing or regenerating
discs inhibit pupariation.
(2) Second, it appears that a certain minimal amount of tissue must be
regenerating in order to effectively delay metamorphosis. Gynandromorphs
with only mosaic discs or very small quantities of lethal tissue, pupariated at
the normal time.
(3) Third, the presence of XO mutant tissue on the abdominal tergites was
not correlated with delayed pupariation. Heat treatment during the larval stages
either does not affect the histoblasts, or the lesions were repaired prior to
pupariation and were without effect. It seems unlikely in any case that the
abdominal histoblasts play a role in the timing of metamorphosis since these
cells multiply only at pupariation (Garcia-Bellido & Merriam, 1971 b; Guerra
et a/. 1973; Wieschaus & Gehring, 1976; Madhavan & Schneiderman, 1977;
Lawrence, Green & Johnston, 1978). For our purposes they provide a convenient internal control.
Although we cannot entirely exclude the possibility that the cell lethal
mutations used in this study may also affect cells of the endocrine organs,
several observations indicate their malfunctioning is probably not the cause of
the delay in pupariation. (1) Previous results have shown that heat treatment
applied continuously after the beginning of the third larval instar is without
effect on pupariation and therefore does not prevent normal functioning of the
endocrine glands (Simpson & Schneiderman, 1975; Arking, 1975). (2) In the
Diptera, the endocrine organs, like the nervous system, are condensed in the
anterior region of the animal (at the level of the ring gland). If it is the genotype
of the endocrine organs that determines the time of pupariation, then pupariation would be delayed when the anterior part of the fly, including the anterior
discs, is male. The genotype of the genital disc, however, would be independent
of the genotype of the endocrine glands as the two organs are widely separated.
One would therefore expect the genital discs to be frequently male in early
pupariating gynandromorphs, and furthermore morphologically abnormal or
deficient since they were not provided with sufficient time to regenerate. (Earlier
experiments have shown that late heat-treated mutant animals, that do not
experience a delayed pupariation, have deficient imaginal discs, Simpson &
Schneiderman, 1975). This was not the case. Only 8% of gynandromorphs
pupariating between 120 and 144 h, and 18% of those pupariating between
144 and 168 h, had male genitalia. About half of these male genitalia were
incomplete although many were associated with female genital elements. (3) A
number of late pupariating gynandromorphs was observed, in which the head
and thorax, and presumably also the endocrine organs, were female, but the
genitalia were male. We can only assume that pupariation of these animals was
retarded as a result of regeneration of the male genital disc.
Earlier experiments have shown that experimentally produced lesions or
reduced growth in Drosophila imaginal discs, only lead to a retarded pupariation
164 P.SIMPSON, P. BERREUR AND J. BERREUR-BONNENFANT
when induced at or before a certain critical stage in development, around the
beginning of the third larval instar (Simpson & Schneiderman, 1975, 1976). A
similar critical period has been described in the last larval instar in. Ephestia
(Pohley, 1967; Muth, 1961), and also in the larval instars of cockroaches
(O'Farrell & Stock, 1954; Kunkel, 1977). In cockroaches this critical period
coincides with an increase in ecdysone titre. It has recently been shown that the
critical period in Drosophila coincides with the release of a small peak of
ecdysteroid, and that experimentally delayed pupariation is accompanied by a
similarly retarded secretion of this ecdysteroid peak (Berreur, Porcheron,
Berreur-Bonnenfant & Simpson, 1979). We would like to propose that there is
a direct relationship between growth of the imaginal discs and the timing of
pupariation, and that discs that have not completed a certain amount of growth
are able to inhibit pupariation, possibly by, in some way, preventing the release
of a small peak of ecdysone at the beginning of the third larval instar. The
nature of this feedback in the Diptera is unknown. Kunkel (1977) has produced
evidence that in Blattela orientalis, leg autotomy may result in a nervous
stimulation of the brain affecting the subsequent release of brain hormone.
We would like to thank Mmes Dupre, Duchene et Fried for their technical help and
Dr R. Arking for the mutation l(l)ts 5697. Comments on the manuscript from Drs M. Gans,
P. A. Lawrence and N. Prud'homme are gratefully acknowledged. This work was supported
by the Centre National de la Recherche Scientifique, the Fondation Medicale and the
Universite Pierre et Marie Curie.
REFERENCES
R. (1975). Temperature sensitive cell lethal mutants of Drosophila: Isolation and
characterization. Genetics 80, 519-537.
BECKER, H. J. (1957). Uber Rontgenmosaikfiecken und Defektmutationen am Auge von
Drosophila und die Entwicklungsphysiologie des Auges. Z. indukt: Abstamm.- u. Vererbhehre. 88, 333-373.
BERREUR, P., PORCHERON, P., BERREUR-BONNENFANT, J. & SIMPSON, P. (1979). Ecdysone
levels and pupariation in a temperature-sensitive mutation of Drosophila melanogaster.
J. exp. Zool. 210, 347-352.
BRYANT, P. J. (1970). Cell lineage relationships in the imaginal wing disc of Drosophila
melanogaster. Devi Biol. 22, 389-411.
BRYANT, P. J. & SCHNEIDERMAN, H. A. (1969). Cell lineage, growth and determination in
the imaginal leg discs of Drosophila melanogaster. Devi Biol. 20, 263-290.
CLARK, W., RUSSELL, M. A. (1977). The correlation of lysosomal activity and adult phenotype in a cell lethal mutant of Drosophila. Devi Biol. 57, 160-173.
DEWES, E. (1975). Entwicklungsleistungen implantierterganzer und halbierter mannlicher
Genitalimaginalscheiben von Ephestia kuhniella. Z. und Entwicklungsdauer der Wirtstiere.
Wilhelm Roux's Arch, devl Biol. 178, 167-183.
DEWES, E. (1979). LTber den Einfluss der kulturdauer im larvalen Wirt auf die Entwicklungsleistungen implantierter mannlicher Genitalimaginalscheiben von Ephestia kuhniella Z.
Wilhelm Roux's Arch, devl Biol. 186, 309-331.
DOANE, W. W. (1973). Role of hormones in insect development. In Developmental Systems:
Insects, vol. 2 (ed. S. J. Counce & C. H. Waddington), pp. 291-466.
DOBZHANSKY, Th. (1929). The influence of the quantity and quality of chromosomal material
on the size of the cells in Drosophila melanogaster. Wilhelm Roux Arch. EntwickMech.
Org. 115, 363-379.
ARKING,
Time of pup aviation and growth of imaginal discs
165
GARCIA-BELLIDO, A. & MERRIAM, J. R. (1971 a). Parametersof thewingimaginaldiscdevelop-
ment of Drosophila melanogaster. Devl Biol. 24, 61-87.
GARCIA-BELLIDO, A. & MERRIAM, J. R. (1971 b). Clonal parameters of tergite development
in Drosophila. Devi Biol. 26, 264-276.
GUERRA, M., POSTLETHWAIT, J. H. & SCHNEIDERMAN, H. A. (1973). The development of the
imaginal abdomen of Drosophila melanogaster. Devi Biol. 32, 361-372.
HAYNIE, j . L. & BRYANT, P. J. (1977). The effects of X-rays on the proliferation dynamics of
cells in the imaginal wing disc of Drosophila melanogaster. Wilhelm Roux's Arch, devl
Biol. 183, 85-100.
HINTON, C. W. (1955). The behaviour of an unstable ring chromosome of Drosophila
melanogaster. Genetics 40, 951-961.
HOTTA, Y. & BENZER, S. (1972). Mapping of behaviour in Drosophila mosaics. Nature,
Lond. 240, 527-535.
KUNKEL, J. G. (1977). Cockroach molting. IL The nature of regeneration-induced delay of
molting hormone secretion. Biol. Bull. mar. biol. Lab., Woods Hole 153, 145-162.
LAWRENCE, P. A., GREEN, S. & JOHNSTON, P. (1978). Compartmentalisation and growth of
the Drosophila abdomen. / . Embryol. exp. Morph. 43, 233-245.
LINDSLEY, D. L. & GRELL, E. H. (1968). Genetic variations of Drosophila melanogaster.
Publs Carnegie Jnstn no. 627.
MADHAVAN, M. & SCHNEIDERMAN, H. A. (1977). Histological analysis of the dynamics of
growth of imaginal discs and histoblast nests during the larval development of Drosophila
melanogaster. Wilhelm Roux's Arch. devl. Biol. 183, 269-305.
MUTH, F. W. (1961). Untersuchungen zur Wirkungsweise der Mutante 'kfl' beider Mehlffotte
Ephestia kuhniella Z. Wilhelm Roux Arch. EntwickMech. Org. 153, 370-418.
O'FARRELL & STOCK, A. (1954). Regeneration and the moulting cycle in Blatella germanica
L. III. Successive regeneration of both metathoracic legs. Aust. J. biol. Sci. 7, 525-536.
POHLEY, H. J. (1967). Regenerationsexperimente und kritische Perioden. Wilhelm Roux
Arch. EntwickMech. Org. 158, 341-357.
RAHN, P. (1972). Untersuchungen zur Entwicklung von Ganz und Teilimplantaten der
Flugelimaginalscheibe von Ephestia kuhniella Z. Wilhelm Roux Arch. EntwickMech.
Org. 170, 48-82.
RIPOLL, P. (1972). The embryonic organisation of imaginal wing disc of Drosophila melanogaster. Wilhelm Roux Arch. EntwickMech. Org. 169, 200-21.
RUSSELL, M. (1974). Pattern formation in imaginal discs of a temperature-sensitive cell
lethal mutant of Drosophila melanogaster. Devl Biol. 40, 24-39.
SIMPSON, P. & SCHNEIDERMAN, H. A. (1975). Isolation of temperature sensitive mutations
blocking clone development in Drosophila melanogaster, and the effects of a temperature
sensitive cell lethal mutation on pattern formation in imaginal discs. Wilhelm Roux's
Arch, devl Biol. 178, 247-275.
SIMPSON, P. & SCHNEIDERMAN, H. A. (1976). A temperature sensitive mutation that reduces
mitotic rate in Drosophila melanogaster. Wilhelm Roux's Arch, devl Biol. 179, 215-236.
WIESCHAUS, E. & GEHRING, W. (1976). Clonal analysis of primordial disc cells in the early
embryo of Drosophila melanogaster. Devl Biol. 50, 249-263.
(Received 1 November 1979, revised 24 January 1980)