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Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster,
54, 256-263
(1976)
DEVELOPMENTAL
BIOLOGY
Sequential
Gene Activation by Ecdysone in Polytene Drosophila melanogaster IV. The Mid Prepupal GEOFFREY
Department
of Genetics,
University
July
256 Q 1976 by Academic Press, Inc. of reproduction in any form reserved.
Street,
Cambridge,
England
16,1976 melanogaster two major changes in prepupal puffs (e.g., 63E, 75CD) are 75B, and 93F) acquire the competence vitro. Both require that the ecdysone upon protein synthesis.
prepupal
INTRODUCTION
Copyright All rights
Period
Downing
In the mid prepupal period of development of Drosophila gene activity occur in the salivary glands: (a) The mid induced and (b) the late prepupal puffs (e.g., 62E, 74EF, to respond to ecdysone. These events can be studied in titre be very low (~5 x 10m9M) and both events depend
In the previous papers of this series (Ashburner 1973, 1974; Ashburner and Richards, 1976) we have described the control by ecdysone of the complex changes in puffing activity seen in the polytene chromosomes of the salivary glands of the late third instar larvae of Drosophila melanogaster. The puffing sequence induced by continuous culture of larval glands with ecdysone does not progress beyond that stage characteristic of the 3-4-hr prepupa. During normal development, changes in puffing activity continue for a further 9-11 hr until the histolysis of the larval salivary gland in 12-14-hr prepupae. Some of the puffs active in prepupae have previously been active in larvae, while others are active only in prepupae (Becker, 1959; Ashburner, 1967). I have reported (Richards, 1976a) preliminary experiments with prepupal glands which suggested that these further changes were also controlled by ecdysone. This paper will describe a study of the events that occur in viva between 3 and 8 hr after pupariation (the mid prepupal period). This period is important for two processes: (i) the activity of the mid prepupal puffs (e.g., 63E and 75CD) and (ii) the acquisition of competence to respond to ecdysone by the late
of
RICHARDS
of Cambridge, Accepted
Chromosomes
puffs (e.g., 74EF, 75B, and 93F). MATERIALS
AND METHODS
Animals. Animals from the Canton-S wild-type stock, grown at 25 f 1°C on yeast glucose medium, were used in all experiments. Prepupae were staged at pupariation. This can be determined within 15 min. Zero-hour prepupae are soft white immobile animals with everted anterior spiracles which show no cuticle tanning and will not resume wandering when provoked. Chemicals. P-ecdysone (Rohto) was used at a concentration of 2.4 x lo+ M unless otherwise stated. Grace’s tissue culture medium was made up in the laboratory. Culture and cytological methods. These have been described previously (Ashburner, 1972a; Ashburner and Richards, 1976; Richards, 1976a). All times given are at 25°C and all the experiments of this paper were performed at 25 & 1°C (unlike those described in earlier papers of this series, which were done at 20-22°C). Puffs in this study were (with reference band): 52A (52B2.3), 52C (52B2.3), 6331.3 (63A1.2), 69A (68E1.2), 74EF (74A1.2), 75B (74A1.2), and 93F (93F1.2). The method for evaluating puff size was described by Ashburner (1972).
GEOFFREY
Sequential
RICHARDS
Gene
Activation
IV
257
RESULTS
A. The Mid Prepupal
Puffs
(i) Normal development. In normal development, these puffs increase their activity from 4-5 hr after pupariation. They reach their maximum sizes at 8 hr, whereafter they regress (Fig. 1). I have studied the activity of 63E and 75CD in detail; 63E has two periods of activity, the first during the larval ecdysone sequence and the second in the mid prepupal period, whilst 75CD is active only in prepupae. (ii) The activity of the mid prepupal puffs in vitro. I have shown (Richards, 1976a) that both 63E and 75CD are repressed by ecdysone throughout the prepupal period. In the present experiments, Ohour prepupal glands were cultured for up to 6 hr either with or without ecdysone (Fig. 2). In the presence of ecdysone, 63E regresses at the same rate as it would have done in vivo and remains inactive. In the absence of ecdysone, the behaviour of 63E is more complex. In the first hour of culture the activity of 63E decreases (but never to the extent seen in vivo) and the puff remains small until 5 hr. It then in-
-I
15w
I
FIG. 1. The activity of 63E and 75CD in normal development in late larval and prepupal glands. The timing of larval glands as hours before puparium formation is based upon observations both in uiuo and in larval glands cultured with ecdysone (Ashburner and Richards, personal observations; see also Richards, 1976b). Prepupal glands were staged at puparium formation. The abscissa is time, in hours at 25”C, before and after puparium formation. The ordinate is puff size (i.e., the ratio between the diameter of the puff and an inactive reference band).
FIG. 2. The activity of 63E and 75CD in glands from 0-hr prepupae cultured with (closed squares) or without (open squares) ecdysone (2.4 x 10-O M) for O-6 hr. The activity of these puffs in uiuo is also shown (open circles). Ordinate, puff size; abscissa, hours at 25°C.
creases in size. This increase in activity at 6 hr is similar to that observed in vivo. The behaviour of 75CD is similar with respect to ecdysone; in the presence of ecdysone it is never active. In the absence of ecdysone it becomes active after 3 hr of culture; this activation occurs 2 hr earlier than that observed in vivo. Culture of larval glands in the absence of ecdysone does not lead to puffing at either 63E or 75CD (Table 1). (iii) Sensitivity of mid prepupal puffs to ecdysone. In 6-hr prepupae the mid prepupal puffs (e.g., 63E and 75CD) are active, though are not yet at the peak of their activity. If 6-hr prepupal glands are cultured in the presence of ecdysone (2.4 x lop6 Ml, these puffs regress; when cultured in ecdysone-free medium they increase in size and remain active longer than in vivo (Fig. 3). In order to determine the sensitivity of these puffs to ecdysone, glands from 6-hr prepupae were cultured in the presence of various concentrations of ecdysone. The sizes of 63E and 75CD, after 4 hr of culture,
258
DEVELOPMENTAL TABLE
BIOLOGY
VOLUME
54, 1976
1
THE ACTIVITY OF 63E AND 75CD IN LARVAL AND PREPUPAL GLANDS CULTURED 4 HR WITH OR WITHOUT ECDYSONE Age of glands Larval
(PSl)
Prepupal hr)
(0
Ecdysone
63E
75CD
+ -
1.16 2 0.05 1.02 r 0.01
1.00 2 0.00 1.00 T 0.00
+ -
1.11 ‘- 0.03 1.34 2 0.04
1.00 2 0.00 1.24 f 0.03
14L, ..
,,,,,,,
-9
‘b ,_..,, -? .-.-.-...-.~.-.-C.-. .,,,,,,, ,,,,,,, -, -7 -6
..J
-5
FIG. 4. The size of 63E and 75CD in 6-hr prepupal glands cultured for 4 hr in various concentrations of ecdysone. Ordinate, puff size; abscissa, log ecdysone concentration.
M 0
1.eb----, 1
4
B
FIG. 3. The activity of 63E and 75CD in glands from 6-hr prepupae cultured with (closed squares) or without (open squares) ecdysone (2.4 x 1O-6 M) for O-6 hr. The activity of these puffs in uiuo from 6-12 hr after puparium formation is also shown (open circles). Ordinate, puff size; abscissa, hours at 25°C.
are shown as a function of the ecdysone concentration in Fig. 4. Fifty percent inhibition of puffing at these loci is seen at the surprisingly low concentration of 1 x lo-8 M, while ecdysone concentrations as low as 5 x 1O-8 M cause the complete regression of these puffs. (iv) Requirements for protein synthesis. Zero-hour prepupal glands were cultured for up to 6 hr in the presence of an inhibitor of protein synthesis, cycloheximide (7.5 x 10e5 M). The activity of 63E and the induction of 75CD, previously observed when 0-hr prepupal glands are cultured in the absence of ecdysone, are inhibited (Fig. 5). It thus appears that protein synthesis is essential for the activity of the mid prepupal puffs. (v) Other mid prepupal puffs. Several other puffs are, like 63E and 75CD, at their maximum activity in 8-hr prepupae,
J Ml FIG. 5. The inhibition of puffing activity at 63E and 75CD by cycloheximide. Zero-hour prepupal glands were cultured for O-6 hr with (closed squares) or without (open squares) cycloheximide (7.5 x 10m5 M). Ordinate, puff size; abscissa, hours at 25°C.
e.g., 52A, 52C, and 69A. The responses to ecdysone of these puffs are very similar to those described for 75CD. That is, they are repressed in the presence of the hormone and induced in its absence. The activities of these puffs in 6-hr prepupal glands cultured for 2 hr with or without ecdysone are shown in Table 2. B. The Acquisition of Competence (to Respond to Ecdysone) by Late Prepupal Puffs During normal development, several puffs become active in the lo-hr prepupa. I have shown (Richards, 1976a) that they do so as a response to ecdysone. The charac-
GEOFFREY
RICHARDS
Sequential
Gene
TABLE THE SIZES OF MID Puff
PREPUPAL
PUFFS
Size in 6-hr
prepupae
Activation
2
IN 6-HR PREPUPAL GLANDS CULTURED ECDYSONE (2.4 x 10m6 M) Size in 6-hr
prepupal glands hr -tecdysone
+ 52A 52c 63E 69A 75CD
1.24 1.30 1.27 1.01 1.17
+ 2 r t k
0.05 0.04 0.03 0.00 0.02
1.01 1.00 1.05 1.00 1.00
teristics of this response are described in the following paper (Richards, 1976b). Glands taken from 0-hr prepupae are not competent to respond to ecdysone by puffing at these sites. Glands taken from 6-hr prepupae are competent and, when cultured with ecdysone, these puffs (74EF, 75B, and 93F) show a maximal response within 2 hr of culture. I will now describe the experiments which define the conditions necessary for glands to become competent to respond to ecdysone. The standard assay for competence used was the activity of 74EF, 75B, and 93F after culture of glands in ecdysone for 2 hr. (i) In vitro acquisition. Salivary glands dissected from prepupae aged 0,2, and 4 hr will not respond, by puffing at 74EF, 75B, and 93F, to ecdysone -even after culture for as long as 10 hr. However, if these glands are first incubated in medium lacking hormone and then transferred to hormone containing medium, these puffs are induced. Hence, competence to respond to ecdysone can be acquired in vitro. The length of time salivary glands require to be cultured without ecdysone before they become fully competent varies with their age (Fig. 6 and Table 3). For 0-hr glands it is 3 hr, for 2-hr glands it is 3 hr, and for 4hr glands it is 2 hr. This suggests that whatever is happening in vitro, in the absence of ecdysone, resulting in competence being acquired, is similar to a process occurring in uiuo during the mid prepupal period; indeed, once started in vivo this process can be completed in vitro. The time course of the ecdysone induc-
k k k + 5
259
IV
FOR 2 HR WITH OR WITHOUT
cultured
2
Size in 8-hr prepupae
0.01 0.00 0.01 0.00 0.00
1.37 1.42 1.59 1.24 1.51
k 5 -c + rt
0.03 0.03 0.05 0.04 0.04
1.58 1.52 1.75 1.46 1.68
+ + f c +
0.04 0.04 0.05 0.03 0.05
FIG. 6. The in vitro acquisition of competence to respond to ecdysone at 74EF and 93F in 0-, 2-, and 4hr prepupal glands. Zero- (closed circles), 2- (open circles), and 4- (closed squares) hr prepupal glands were cultured in the absence of ecdysone for n hours before transfer to ecdysone (2.4 x lo-@ M) for 2 hr. The response during the ecdysone culture is shown against the period of ecdysone-free culture (n, n-2, and n-4 hours, respectively, for 0-, 2-, and 4-hr prepupal glands). Ordinate is puff size, abscissa is hours of ecdysone-free culture at 25°C (see above).
tion of 74EF, 75B, and 93F in salivary glands from 0-hr prepupae, which had previously acquired competence by culture for 3 hr without ecdysone, is very similar to that in competent 6-hr prepupal glands and to that seen from 8-12 hr of normal development (Fig. 7). (ii) The sensitivity of the acquisition of competence to ecdysone inhibition. It has
been shown that competence is acquired in
260
DEVELOPMENTAL
the absence of ecdysone, but not in the presence of a high concentration of ecdysone (2.4 x lo+ M). To study the effect of ecdysone further, 0-hr prepupal glands TABLE THE ACQUISITION ECDY~ONE Puff
3
OF COMPETENCE IN 4-Ha PREPUPAL 2 hr +/dysone
TO RESPOND GLANDS” ec-
TO
Size of puff in 4-hr prepupal glands
After
74EF
1.01 + 0.01
(+) (-)
1.03 + 0.02 1.00 + 0.00
1.01 2 0.01 2.11 2 0.05
93F
1.01 f 0.01
(+) (6)
1.09 ” 0.02 1.05 2 0.02
1.03 2 0.01 1.53 + 0.04
After a further 2 hr in ecdysone
93F
were cultured for 3 hr in various concentrations of ecdysone before transfer to a high ecdysone concentration (2.4 x lop6 M) for 2 hr to assay for the induction of 74EF, 75B, and 93F. The data (Fig. 8) show the acquisition of competence to be very sensitive to ecdysone. Fifty percent inhibition of acquisition of competence occurs at 1 x 1O-8 M p-ecdysone, and the process is effected quantitatively over a very wide concentration range.
required for glands from in the presor cyclohexiand transfer
WEF
FIG. 8. The inhibition of acquisition of competence by ecdysone. Zero-hour prepupal glands were preincubated for 3 hr in various ecdysone concentrations (abscissa) before being challenged with ecdysone (2.4 x 10-O M) for 2 hr. The ordinate is the percentage of inhibition of the induction of 74EF (closed circles), 75B (open circles), and 93F (closed squares).
TABLE
74EF
1976
macromolecular synthesis is the acquisition of competence, 0-hr prepupae were cultured ence of either actinomycin D mide for 3 hr, before washing
FIG. 7. The activity of 74EF and 93F (a) in vivo (solid circles) from 8-12 hr after puparium formation, (b) in vitro (open circles) in 6-hr prepupal glands cultured with ecdysone (2.4 x 10eBM) and (c) in vitro (closed squares) in 0-hr prepupal glands cultured with ecdysone (2.4 x 1O-6 kf) after an initial culture of 3 hr in the absence of ecdysone. Ordinate is puff size, abscissa is hours at 25°C.
Puff
54,
(iii) Requirements for macromolecular synthesis. In order to study whether or not
n Glands were cultured for 2 hr with, or without, ecdysone and then for a further 2 hr with ecdysone.
b.
VOLUME
BIOLOGY
INHIBITION 0-hr
OF ACQUISITION prepupal glands
1.03 r 0.01
1.06 -c 0.02
a Zero-hour prepupal 2 hr with ecdysone with
glands were no inhibitor.
OF COMPETENCE
4 BY ACTINOMYCIN
3 hr zero Control + actinomycin + cycloheximide Control + actinomycin + cycloheximide cultured
D (8.9 x lo-’ M) (7.5 x W5 M)
D
for 3 hr without
D AND CYCL~HEXIMIDE’
ecdysone
ecdysone
2 hr ecdysone 1.00 f 0.00 1.00 -t 0.00 1.00 + 0.00
1.96 2 0.04 1.00 2 0.00 1.01 2 0.01
1.01 f 0.01 1.01 r+ 0.00 1.02 -t 0.01
1.61 k 0.06 1.03 f 0.01 1.03 % 0.01
and kinhibitor
and then cultured
for
GEOFFREY RICHARDS
Sequential
to ecdysone medium for 2 hr. The results of these experiments are shown in Table 4; both drugs inhibit the subsequent induction of the puffs 74EF, 75B, and 93F. To test whether or not these drugs inhibit the induction of these puffs in competent glands, the following experiments were done. Glands from 0-hr prepupae were cultured for 3 hr without ecdysone and then transferred to ecdysone medium containing one of the inhibitors. For actinomycin D, the result (Table 5) is both clear and expected since actinomycin D inhibition of RNA synthesis inhibits puff induction (Clever and Romball, 1966; Berendes, 1968; Ashburner, 197213). The effects of cycloheximide in this experiment were less clear. The drug appeared to inhibit 74EF, 75B, and 93F induction, but by less than 50% (Table 6). However, 0-hr glands cultured for 4 hr in the absence of ecdysone and then transferred to ecdysone and cycloheximide showed no inhibition of 74EF, 75B, and 93F induction (Fig. 9). It thus appears that a consequence of inducing these puffs in the presence of cycloheximide is to extend TABLE
5
ACTINOMYCIN D INHIBITION OF INDUCTION AND 93F IN COMPETENT GLANDS Puff
74EF
93F
OF 74EF
0-hr prepupal glands cultured for 3 hr in zero ecdysone
Actinomytin D (8.9 x lo-‘M)
2 hr ecdysone
1.00 :t 0.00
+
1.98 f 0.06 1.02 + 0.01
+
1.51 2 0.05 1.08 + 0.02
1.01
THE INHIBITION 0-hr prepupal glands cultured in the absence of ecdysone (hr)
+ 0.00
ECDYSONE INDUCTION GLANDS FIRST CULTURED Ecdysone culture 2 hr + cycloheximide OF
Gene
Activation
261
IV
t FIG. 9. The response of 74EF, 75B, and 93F in Ohr prepupal glands cultured for n hours in ecdysonefree, cycloheximide-free medium before culture with ecdysone (open squares) or ecdysone plus cycloheximide (closed squares) for 2 hr. Ordinate is puff size, abscissa is hours at 25°C of initial culture.
by about 1 hr the time required for full competence to be acquired. This suggests that in previous experiments, that is, ecdysone inductions in the absence of cycloheximide, competence is still being acquired at the time of the initial induction by ecdysone. DISCUSSION
These studies have revealed an unsuspected phase in the prepupal period of D.
TABLE 6 OF 74EF, 75B, AND 93F, BY CYCLOHEXIMIDE IN 0-HR FOR 3 OR 4 HR IN THE ABSENCE OF ECDYSONE 74EF
75B
93F
PREPUPAL
3
+ -
1.31 r 0.04 1.98 r 0.06
1.61 + 0.05 2.00 k 0.05
1.31 k 0.04 1.51 f 0.05
4
+ -
1.96 2 0.06 2.06 + 0.04
2.13 ? 0.05 2.10 r 0.04
1.56 k 0.04 1.55 k 0.04
262
DEVELOPMENTAL
BIOLOGY
melanogaster development, a phase when the moulting hormone ecdysone is not only unneccessary for, but in fact inhibits, the processes of normal development. This conclusion is based upon two series of observations: (i) that the puffs characteristic of the 8-hr prepupa (e.g., 52A, 52C, 63E, 69A, and 75CD) will only be active in vitro if ecdysone is absent; their activity is 50% inhibited by 1 x 10e8 it4 P-ecdysone, and (ii) that the puffs of the lo-hr prepupa (e.g., 74EF, 758 and 93F) are induced by ecdysone (Richards, 1976a), but only if the glands have previously been at least 3 hr in the absence of ecdysone. In some way, this 3-hr period is essential for these puffs to become competent to respond to ecdysone. We presume that, in ho, the ecdy sone-free periods necessary for 63E and 75CD activity and for the acquisition of competence of 74EF, 75B, and 93F are one and the same. The induction of mid prepupal puffs following ecdysone withdrawal in 0-hr prepupal glands occurs after a delay of 2-3 hr and requires protein synthesis. If, however, these puffs are active in glands transferred to ecdysone medium, they regress immediately and this process does not require protein synthesis. This suggests that there are two different control functions of ecdysone in regulating the activity of the mid prepupal puffs. While both act to prevent puffing activity, the former requires protein synthesis while the latter does not. The effect of ecdysone on the acquisition of competence for the late prepupal puffs appears similar to its effect on the induction of the mid prepupal puffs, in that there is a delay following ecdysone withdrawal, and protein synthesis is also necessary. It must be noted that the acquisition of competence occurs at the same time as the induction of the mid prepupal puffs. It is therefore unlikely that the activity of the mid prepupal puffs is necessary for the activity of the late prepupal puffs. This is supported by the experiments
VOLUME
54, 1976
with 0-hr prepupal glands. These glands are fully competent after 3 hr of culture in the absence of ecdysone (Fig. 61, but show little puking activity at either 63E or 75CD (Fig. 2). The dose-response experiments (both upon the inhibition of 63E and.75CD activity and the inhibition of the acquisition of competence at 74EF, 75B, and 93F) suggest that the maximum effective ecdysone concentration during this mid prepupal period is ca. 5 x lo+ M. Such a low concentration has not been found by radioimmune assay (Borst et al., 1974; De Reggi et al., 1975), but this may be due to the fact that the period of low titre is short (see below) relative to the variance in age within samples assayed by these workers. The precise period during which the ecdysone titre is low can be estimated from the present experiments. It must end between 8 and 9 hr after pupariation, for at that time, 74EF, 75B, and 93F are induced and 63E and 75CD regress, both processes presumably a consequence of an increase in ecdysone titre. The fact that 2-hr prepupal glands require 3 hr of ecdysone-free culture to become competent, whilst 4-hr prepupal glands require 2 hr, suggests that by 4 hr this process has already been going on for 1 hr in uiuo. This suggests that the period of low ecdysone titre begins at about 3 hr after pupariation. This is supported by the observation that in 0-hr prepupal glands cultured without ecdysone, 75CD requires 5 hr to reach its maximum size. In uiuo it reaches this size 8 hr after pupariation or 5 hr after the time (3 hr) we propose the ecdysone titre falls. It is apparent that if such rapid changes in hormone titre occur in normal development then the metabolism of ecdysone must play an important regulatory role. While the metabolism of exogenous hormone has been studied in various insects, the ways in which ecdysone titres are regulated in uiuo are not yet understood. Injected ecdysone has a short half-life (values between ~20 min and 3 hr have been
GEOFFREY
RICHARDS
Sequential
reported for third instar larvae of CaZZiphoru and Sarcophuga (Karlson and Bode, 1969; Shaaya, 1969; Heinrich and Hoffmeister, 1970; Ohtaki et al., 1970)): This is evidence of a potent inactivating system, which is presumably located in the insect fat body (King, 1972). If the peak ecdysone titre is 5 x 10-T M (Ashburner, 1974; Ashburner and Richards, 1976; Borst et al., 1974; De Reggi et al., 1975) and is reached 2 hr before pupariation, then even a conservative estimate of ecdysone’s half-life of 1 hr brings the ecdysone titre to 8 x lo+ M by 4 hr following pupariation (assuming no synthesis). I am grateful to Dr. M. Ashbumer for advice and encouragement and to Professor J. M. Thoday and Dr. P. Cherbas for their interest in this study, which was supported by a Medical Research Council Scholarship. REFERENCES ASHBURNER, M. (1967). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. I. Autosomal puffing patterns in a laboratory stock of Drosophila melanogaster. Chromosoma 21, 398-428. ASHBURNER, M. (1972a). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. VI. Induction by ecdysone in salivary glands of D. melanagaster cultured in vitro. Chromosoma 38, 255-281. ASHBURNER, M. (1972b). Ecdysone induction of puffing in polytene chromosomes of Drosophila melanogaster. Effects of inhibitors of RNA synthesis. Exp. Cell Res. 71, 433-440. ASHBURNER, M. (1973). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon Ecdysone Concentration. Develop. Biol. 35, 47-61. ASHBURNER, M. (1974). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibitors of protein synthesis. Develop. Biol. 39, 141-157. ASHBURNER, M., and RICHARDS, G. (1976). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. III. Conse-
Gene
Activation
IV
263
quences of ecdysone withdrawal. Develop. Biol. 54, 241-255. BECKER, H-J. (1962). Die Puffs der SpeicheldriisenSpeicheldrusenchromosomen n van von Drosophila melanogaster. I. Beobachtungen zumm Verhalten des Puffmusters im Normalstamm und bei zwei Mutanten, giant und lethal-giant-larvae. Chromosoma 10, 654-678. BERENDES, H. D. (1968). Factors involved in the expression of gene activity in polytene chromosomes. Chromosoma 24, 418-437. BORST, D. W., BOLLENBACHER, W. E., O’CONNOR, J. D., KING, D. S., and FRISTROM, J. W. (1974). Ecdysane levels during metamorphosis of Drosophila melanogaster. Develop. BioZ. 39, 308-316. CLEVER, U., and ROMBALL, C. G. (1966). RNA and protein synthesis in the cellular response to a hormone, ecdysone. Proc Nat. Acad. Sci . USA 56, 1470-1476. DE REGGI, M. L., HIRN, M. H., and DELAUQE, M. A. (1975). Radioimmunoassay of ecdysone on application to Drosophila larvae and pupae. Biochim. Biophys. Res. Comm. 66, 1307-1315. HEINRICH, G., and HOFFMEISTER, H. (1970). Insektenhaiitungshormone und ihre Wirkungweise: Bildung von Hormonglykosiden als inaktivierungsmechanismus bei Calliphora erythrocephala. 2. Naturforsch 25b, 358-361. KARLSON, P., and BODE, C. (1969).
Die Inaktivierung des Ecdysons bei der Schmeissfliege CaZZiphora erythrocephala Meigen. J. Insect Physiol. 15, 111-118. KING, D. S. (1972). Metabolism of a-ecdysone and possible precursors by insects in uivo and in vitro. Gen. Camp. Endocrinol. (suppl.) 3, 221-227. OHTAKI, T., and WILLIAMS, C. M. (1970). Inactivation of a-ecdysone and cyasterone by larvae of the fleshfly, Sarcophaga peregrina, and pupae of the silkworm, Samia Cynthia. BioZ. Bull. 138,326-333. RICHARDS, G. (1976a). Control of prepupal puffing patterns in uitro, implications for prepupal ecdysone titres in Drosophila melanogaster. Develop. BioZ. 48, 191-195. RICHARDS, G. (1976b). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. V. The late prepupal puffs. Develop. BioZ. SHAAYA, E.
54, 264-275.
(1969). Untersuchungen iiber die Verteilung des Ecdysons in verschiedenen Geweben von Calliphora erythrocephala und iiber seine biologische Halbwertszeit. 2. Naturforsh. 24b, 718-721.
(1976)
DEVELOPMENTAL
BIOLOGY
Sequential
Gene Activation by Ecdysone in Polytene Drosophila melanogaster IV. The Mid Prepupal GEOFFREY
Department
of Genetics,
University
July
256 Q 1976 by Academic Press, Inc. of reproduction in any form reserved.
Street,
Cambridge,
England
16,1976 melanogaster two major changes in prepupal puffs (e.g., 63E, 75CD) are 75B, and 93F) acquire the competence vitro. Both require that the ecdysone upon protein synthesis.
prepupal
INTRODUCTION
Copyright All rights
Period
Downing
In the mid prepupal period of development of Drosophila gene activity occur in the salivary glands: (a) The mid induced and (b) the late prepupal puffs (e.g., 62E, 74EF, to respond to ecdysone. These events can be studied in titre be very low (~5 x 10m9M) and both events depend
In the previous papers of this series (Ashburner 1973, 1974; Ashburner and Richards, 1976) we have described the control by ecdysone of the complex changes in puffing activity seen in the polytene chromosomes of the salivary glands of the late third instar larvae of Drosophila melanogaster. The puffing sequence induced by continuous culture of larval glands with ecdysone does not progress beyond that stage characteristic of the 3-4-hr prepupa. During normal development, changes in puffing activity continue for a further 9-11 hr until the histolysis of the larval salivary gland in 12-14-hr prepupae. Some of the puffs active in prepupae have previously been active in larvae, while others are active only in prepupae (Becker, 1959; Ashburner, 1967). I have reported (Richards, 1976a) preliminary experiments with prepupal glands which suggested that these further changes were also controlled by ecdysone. This paper will describe a study of the events that occur in viva between 3 and 8 hr after pupariation (the mid prepupal period). This period is important for two processes: (i) the activity of the mid prepupal puffs (e.g., 63E and 75CD) and (ii) the acquisition of competence to respond to ecdysone by the late
of
RICHARDS
of Cambridge, Accepted
Chromosomes
puffs (e.g., 74EF, 75B, and 93F). MATERIALS
AND METHODS
Animals. Animals from the Canton-S wild-type stock, grown at 25 f 1°C on yeast glucose medium, were used in all experiments. Prepupae were staged at pupariation. This can be determined within 15 min. Zero-hour prepupae are soft white immobile animals with everted anterior spiracles which show no cuticle tanning and will not resume wandering when provoked. Chemicals. P-ecdysone (Rohto) was used at a concentration of 2.4 x lo+ M unless otherwise stated. Grace’s tissue culture medium was made up in the laboratory. Culture and cytological methods. These have been described previously (Ashburner, 1972a; Ashburner and Richards, 1976; Richards, 1976a). All times given are at 25°C and all the experiments of this paper were performed at 25 & 1°C (unlike those described in earlier papers of this series, which were done at 20-22°C). Puffs in this study were (with reference band): 52A (52B2.3), 52C (52B2.3), 6331.3 (63A1.2), 69A (68E1.2), 74EF (74A1.2), 75B (74A1.2), and 93F (93F1.2). The method for evaluating puff size was described by Ashburner (1972).
GEOFFREY
Sequential
RICHARDS
Gene
Activation
IV
257
RESULTS
A. The Mid Prepupal
Puffs
(i) Normal development. In normal development, these puffs increase their activity from 4-5 hr after pupariation. They reach their maximum sizes at 8 hr, whereafter they regress (Fig. 1). I have studied the activity of 63E and 75CD in detail; 63E has two periods of activity, the first during the larval ecdysone sequence and the second in the mid prepupal period, whilst 75CD is active only in prepupae. (ii) The activity of the mid prepupal puffs in vitro. I have shown (Richards, 1976a) that both 63E and 75CD are repressed by ecdysone throughout the prepupal period. In the present experiments, Ohour prepupal glands were cultured for up to 6 hr either with or without ecdysone (Fig. 2). In the presence of ecdysone, 63E regresses at the same rate as it would have done in vivo and remains inactive. In the absence of ecdysone, the behaviour of 63E is more complex. In the first hour of culture the activity of 63E decreases (but never to the extent seen in vivo) and the puff remains small until 5 hr. It then in-
-I
15w
I
FIG. 1. The activity of 63E and 75CD in normal development in late larval and prepupal glands. The timing of larval glands as hours before puparium formation is based upon observations both in uiuo and in larval glands cultured with ecdysone (Ashburner and Richards, personal observations; see also Richards, 1976b). Prepupal glands were staged at puparium formation. The abscissa is time, in hours at 25”C, before and after puparium formation. The ordinate is puff size (i.e., the ratio between the diameter of the puff and an inactive reference band).
FIG. 2. The activity of 63E and 75CD in glands from 0-hr prepupae cultured with (closed squares) or without (open squares) ecdysone (2.4 x 10-O M) for O-6 hr. The activity of these puffs in uiuo is also shown (open circles). Ordinate, puff size; abscissa, hours at 25°C.
creases in size. This increase in activity at 6 hr is similar to that observed in vivo. The behaviour of 75CD is similar with respect to ecdysone; in the presence of ecdysone it is never active. In the absence of ecdysone it becomes active after 3 hr of culture; this activation occurs 2 hr earlier than that observed in vivo. Culture of larval glands in the absence of ecdysone does not lead to puffing at either 63E or 75CD (Table 1). (iii) Sensitivity of mid prepupal puffs to ecdysone. In 6-hr prepupae the mid prepupal puffs (e.g., 63E and 75CD) are active, though are not yet at the peak of their activity. If 6-hr prepupal glands are cultured in the presence of ecdysone (2.4 x lop6 Ml, these puffs regress; when cultured in ecdysone-free medium they increase in size and remain active longer than in vivo (Fig. 3). In order to determine the sensitivity of these puffs to ecdysone, glands from 6-hr prepupae were cultured in the presence of various concentrations of ecdysone. The sizes of 63E and 75CD, after 4 hr of culture,
258
DEVELOPMENTAL TABLE
BIOLOGY
VOLUME
54, 1976
1
THE ACTIVITY OF 63E AND 75CD IN LARVAL AND PREPUPAL GLANDS CULTURED 4 HR WITH OR WITHOUT ECDYSONE Age of glands Larval
(PSl)
Prepupal hr)
(0
Ecdysone
63E
75CD
+ -
1.16 2 0.05 1.02 r 0.01
1.00 2 0.00 1.00 T 0.00
+ -
1.11 ‘- 0.03 1.34 2 0.04
1.00 2 0.00 1.24 f 0.03
14L, ..
,,,,,,,
-9
‘b ,_..,, -? .-.-.-...-.~.-.-C.-. .,,,,,,, ,,,,,,, -, -7 -6
..J
-5
FIG. 4. The size of 63E and 75CD in 6-hr prepupal glands cultured for 4 hr in various concentrations of ecdysone. Ordinate, puff size; abscissa, log ecdysone concentration.
M 0
1.eb----, 1
4
B
FIG. 3. The activity of 63E and 75CD in glands from 6-hr prepupae cultured with (closed squares) or without (open squares) ecdysone (2.4 x 1O-6 M) for O-6 hr. The activity of these puffs in uiuo from 6-12 hr after puparium formation is also shown (open circles). Ordinate, puff size; abscissa, hours at 25°C.
are shown as a function of the ecdysone concentration in Fig. 4. Fifty percent inhibition of puffing at these loci is seen at the surprisingly low concentration of 1 x lo-8 M, while ecdysone concentrations as low as 5 x 1O-8 M cause the complete regression of these puffs. (iv) Requirements for protein synthesis. Zero-hour prepupal glands were cultured for up to 6 hr in the presence of an inhibitor of protein synthesis, cycloheximide (7.5 x 10e5 M). The activity of 63E and the induction of 75CD, previously observed when 0-hr prepupal glands are cultured in the absence of ecdysone, are inhibited (Fig. 5). It thus appears that protein synthesis is essential for the activity of the mid prepupal puffs. (v) Other mid prepupal puffs. Several other puffs are, like 63E and 75CD, at their maximum activity in 8-hr prepupae,
J Ml FIG. 5. The inhibition of puffing activity at 63E and 75CD by cycloheximide. Zero-hour prepupal glands were cultured for O-6 hr with (closed squares) or without (open squares) cycloheximide (7.5 x 10m5 M). Ordinate, puff size; abscissa, hours at 25°C.
e.g., 52A, 52C, and 69A. The responses to ecdysone of these puffs are very similar to those described for 75CD. That is, they are repressed in the presence of the hormone and induced in its absence. The activities of these puffs in 6-hr prepupal glands cultured for 2 hr with or without ecdysone are shown in Table 2. B. The Acquisition of Competence (to Respond to Ecdysone) by Late Prepupal Puffs During normal development, several puffs become active in the lo-hr prepupa. I have shown (Richards, 1976a) that they do so as a response to ecdysone. The charac-
GEOFFREY
RICHARDS
Sequential
Gene
TABLE THE SIZES OF MID Puff
PREPUPAL
PUFFS
Size in 6-hr
prepupae
Activation
2
IN 6-HR PREPUPAL GLANDS CULTURED ECDYSONE (2.4 x 10m6 M) Size in 6-hr
prepupal glands hr -tecdysone
+ 52A 52c 63E 69A 75CD
1.24 1.30 1.27 1.01 1.17
+ 2 r t k
0.05 0.04 0.03 0.00 0.02
1.01 1.00 1.05 1.00 1.00
teristics of this response are described in the following paper (Richards, 1976b). Glands taken from 0-hr prepupae are not competent to respond to ecdysone by puffing at these sites. Glands taken from 6-hr prepupae are competent and, when cultured with ecdysone, these puffs (74EF, 75B, and 93F) show a maximal response within 2 hr of culture. I will now describe the experiments which define the conditions necessary for glands to become competent to respond to ecdysone. The standard assay for competence used was the activity of 74EF, 75B, and 93F after culture of glands in ecdysone for 2 hr. (i) In vitro acquisition. Salivary glands dissected from prepupae aged 0,2, and 4 hr will not respond, by puffing at 74EF, 75B, and 93F, to ecdysone -even after culture for as long as 10 hr. However, if these glands are first incubated in medium lacking hormone and then transferred to hormone containing medium, these puffs are induced. Hence, competence to respond to ecdysone can be acquired in vitro. The length of time salivary glands require to be cultured without ecdysone before they become fully competent varies with their age (Fig. 6 and Table 3). For 0-hr glands it is 3 hr, for 2-hr glands it is 3 hr, and for 4hr glands it is 2 hr. This suggests that whatever is happening in vitro, in the absence of ecdysone, resulting in competence being acquired, is similar to a process occurring in uiuo during the mid prepupal period; indeed, once started in vivo this process can be completed in vitro. The time course of the ecdysone induc-
k k k + 5
259
IV
FOR 2 HR WITH OR WITHOUT
cultured
2
Size in 8-hr prepupae
0.01 0.00 0.01 0.00 0.00
1.37 1.42 1.59 1.24 1.51
k 5 -c + rt
0.03 0.03 0.05 0.04 0.04
1.58 1.52 1.75 1.46 1.68
+ + f c +
0.04 0.04 0.05 0.03 0.05
FIG. 6. The in vitro acquisition of competence to respond to ecdysone at 74EF and 93F in 0-, 2-, and 4hr prepupal glands. Zero- (closed circles), 2- (open circles), and 4- (closed squares) hr prepupal glands were cultured in the absence of ecdysone for n hours before transfer to ecdysone (2.4 x lo-@ M) for 2 hr. The response during the ecdysone culture is shown against the period of ecdysone-free culture (n, n-2, and n-4 hours, respectively, for 0-, 2-, and 4-hr prepupal glands). Ordinate is puff size, abscissa is hours of ecdysone-free culture at 25°C (see above).
tion of 74EF, 75B, and 93F in salivary glands from 0-hr prepupae, which had previously acquired competence by culture for 3 hr without ecdysone, is very similar to that in competent 6-hr prepupal glands and to that seen from 8-12 hr of normal development (Fig. 7). (ii) The sensitivity of the acquisition of competence to ecdysone inhibition. It has
been shown that competence is acquired in
260
DEVELOPMENTAL
the absence of ecdysone, but not in the presence of a high concentration of ecdysone (2.4 x lo+ M). To study the effect of ecdysone further, 0-hr prepupal glands TABLE THE ACQUISITION ECDY~ONE Puff
3
OF COMPETENCE IN 4-Ha PREPUPAL 2 hr +/dysone
TO RESPOND GLANDS” ec-
TO
Size of puff in 4-hr prepupal glands
After
74EF
1.01 + 0.01
(+) (-)
1.03 + 0.02 1.00 + 0.00
1.01 2 0.01 2.11 2 0.05
93F
1.01 f 0.01
(+) (6)
1.09 ” 0.02 1.05 2 0.02
1.03 2 0.01 1.53 + 0.04
After a further 2 hr in ecdysone
93F
were cultured for 3 hr in various concentrations of ecdysone before transfer to a high ecdysone concentration (2.4 x lop6 M) for 2 hr to assay for the induction of 74EF, 75B, and 93F. The data (Fig. 8) show the acquisition of competence to be very sensitive to ecdysone. Fifty percent inhibition of acquisition of competence occurs at 1 x 1O-8 M p-ecdysone, and the process is effected quantitatively over a very wide concentration range.
required for glands from in the presor cyclohexiand transfer
WEF
FIG. 8. The inhibition of acquisition of competence by ecdysone. Zero-hour prepupal glands were preincubated for 3 hr in various ecdysone concentrations (abscissa) before being challenged with ecdysone (2.4 x 10-O M) for 2 hr. The ordinate is the percentage of inhibition of the induction of 74EF (closed circles), 75B (open circles), and 93F (closed squares).
TABLE
74EF
1976
macromolecular synthesis is the acquisition of competence, 0-hr prepupae were cultured ence of either actinomycin D mide for 3 hr, before washing
FIG. 7. The activity of 74EF and 93F (a) in vivo (solid circles) from 8-12 hr after puparium formation, (b) in vitro (open circles) in 6-hr prepupal glands cultured with ecdysone (2.4 x 10eBM) and (c) in vitro (closed squares) in 0-hr prepupal glands cultured with ecdysone (2.4 x 1O-6 kf) after an initial culture of 3 hr in the absence of ecdysone. Ordinate is puff size, abscissa is hours at 25°C.
Puff
54,
(iii) Requirements for macromolecular synthesis. In order to study whether or not
n Glands were cultured for 2 hr with, or without, ecdysone and then for a further 2 hr with ecdysone.
b.
VOLUME
BIOLOGY
INHIBITION 0-hr
OF ACQUISITION prepupal glands
1.03 r 0.01
1.06 -c 0.02
a Zero-hour prepupal 2 hr with ecdysone with
glands were no inhibitor.
OF COMPETENCE
4 BY ACTINOMYCIN
3 hr zero Control + actinomycin + cycloheximide Control + actinomycin + cycloheximide cultured
D (8.9 x lo-’ M) (7.5 x W5 M)
D
for 3 hr without
D AND CYCL~HEXIMIDE’
ecdysone
ecdysone
2 hr ecdysone 1.00 f 0.00 1.00 -t 0.00 1.00 + 0.00
1.96 2 0.04 1.00 2 0.00 1.01 2 0.01
1.01 f 0.01 1.01 r+ 0.00 1.02 -t 0.01
1.61 k 0.06 1.03 f 0.01 1.03 % 0.01
and kinhibitor
and then cultured
for
GEOFFREY RICHARDS
Sequential
to ecdysone medium for 2 hr. The results of these experiments are shown in Table 4; both drugs inhibit the subsequent induction of the puffs 74EF, 75B, and 93F. To test whether or not these drugs inhibit the induction of these puffs in competent glands, the following experiments were done. Glands from 0-hr prepupae were cultured for 3 hr without ecdysone and then transferred to ecdysone medium containing one of the inhibitors. For actinomycin D, the result (Table 5) is both clear and expected since actinomycin D inhibition of RNA synthesis inhibits puff induction (Clever and Romball, 1966; Berendes, 1968; Ashburner, 197213). The effects of cycloheximide in this experiment were less clear. The drug appeared to inhibit 74EF, 75B, and 93F induction, but by less than 50% (Table 6). However, 0-hr glands cultured for 4 hr in the absence of ecdysone and then transferred to ecdysone and cycloheximide showed no inhibition of 74EF, 75B, and 93F induction (Fig. 9). It thus appears that a consequence of inducing these puffs in the presence of cycloheximide is to extend TABLE
5
ACTINOMYCIN D INHIBITION OF INDUCTION AND 93F IN COMPETENT GLANDS Puff
74EF
93F
OF 74EF
0-hr prepupal glands cultured for 3 hr in zero ecdysone
Actinomytin D (8.9 x lo-‘M)
2 hr ecdysone
1.00 :t 0.00
+
1.98 f 0.06 1.02 + 0.01
+
1.51 2 0.05 1.08 + 0.02
1.01
THE INHIBITION 0-hr prepupal glands cultured in the absence of ecdysone (hr)
+ 0.00
ECDYSONE INDUCTION GLANDS FIRST CULTURED Ecdysone culture 2 hr + cycloheximide OF
Gene
Activation
261
IV
t FIG. 9. The response of 74EF, 75B, and 93F in Ohr prepupal glands cultured for n hours in ecdysonefree, cycloheximide-free medium before culture with ecdysone (open squares) or ecdysone plus cycloheximide (closed squares) for 2 hr. Ordinate is puff size, abscissa is hours at 25°C of initial culture.
by about 1 hr the time required for full competence to be acquired. This suggests that in previous experiments, that is, ecdysone inductions in the absence of cycloheximide, competence is still being acquired at the time of the initial induction by ecdysone. DISCUSSION
These studies have revealed an unsuspected phase in the prepupal period of D.
TABLE 6 OF 74EF, 75B, AND 93F, BY CYCLOHEXIMIDE IN 0-HR FOR 3 OR 4 HR IN THE ABSENCE OF ECDYSONE 74EF
75B
93F
PREPUPAL
3
+ -
1.31 r 0.04 1.98 r 0.06
1.61 + 0.05 2.00 k 0.05
1.31 k 0.04 1.51 f 0.05
4
+ -
1.96 2 0.06 2.06 + 0.04
2.13 ? 0.05 2.10 r 0.04
1.56 k 0.04 1.55 k 0.04
262
DEVELOPMENTAL
BIOLOGY
melanogaster development, a phase when the moulting hormone ecdysone is not only unneccessary for, but in fact inhibits, the processes of normal development. This conclusion is based upon two series of observations: (i) that the puffs characteristic of the 8-hr prepupa (e.g., 52A, 52C, 63E, 69A, and 75CD) will only be active in vitro if ecdysone is absent; their activity is 50% inhibited by 1 x 10e8 it4 P-ecdysone, and (ii) that the puffs of the lo-hr prepupa (e.g., 74EF, 758 and 93F) are induced by ecdysone (Richards, 1976a), but only if the glands have previously been at least 3 hr in the absence of ecdysone. In some way, this 3-hr period is essential for these puffs to become competent to respond to ecdysone. We presume that, in ho, the ecdy sone-free periods necessary for 63E and 75CD activity and for the acquisition of competence of 74EF, 75B, and 93F are one and the same. The induction of mid prepupal puffs following ecdysone withdrawal in 0-hr prepupal glands occurs after a delay of 2-3 hr and requires protein synthesis. If, however, these puffs are active in glands transferred to ecdysone medium, they regress immediately and this process does not require protein synthesis. This suggests that there are two different control functions of ecdysone in regulating the activity of the mid prepupal puffs. While both act to prevent puffing activity, the former requires protein synthesis while the latter does not. The effect of ecdysone on the acquisition of competence for the late prepupal puffs appears similar to its effect on the induction of the mid prepupal puffs, in that there is a delay following ecdysone withdrawal, and protein synthesis is also necessary. It must be noted that the acquisition of competence occurs at the same time as the induction of the mid prepupal puffs. It is therefore unlikely that the activity of the mid prepupal puffs is necessary for the activity of the late prepupal puffs. This is supported by the experiments
VOLUME
54, 1976
with 0-hr prepupal glands. These glands are fully competent after 3 hr of culture in the absence of ecdysone (Fig. 61, but show little puking activity at either 63E or 75CD (Fig. 2). The dose-response experiments (both upon the inhibition of 63E and.75CD activity and the inhibition of the acquisition of competence at 74EF, 75B, and 93F) suggest that the maximum effective ecdysone concentration during this mid prepupal period is ca. 5 x lo+ M. Such a low concentration has not been found by radioimmune assay (Borst et al., 1974; De Reggi et al., 1975), but this may be due to the fact that the period of low titre is short (see below) relative to the variance in age within samples assayed by these workers. The precise period during which the ecdysone titre is low can be estimated from the present experiments. It must end between 8 and 9 hr after pupariation, for at that time, 74EF, 75B, and 93F are induced and 63E and 75CD regress, both processes presumably a consequence of an increase in ecdysone titre. The fact that 2-hr prepupal glands require 3 hr of ecdysone-free culture to become competent, whilst 4-hr prepupal glands require 2 hr, suggests that by 4 hr this process has already been going on for 1 hr in uiuo. This suggests that the period of low ecdysone titre begins at about 3 hr after pupariation. This is supported by the observation that in 0-hr prepupal glands cultured without ecdysone, 75CD requires 5 hr to reach its maximum size. In uiuo it reaches this size 8 hr after pupariation or 5 hr after the time (3 hr) we propose the ecdysone titre falls. It is apparent that if such rapid changes in hormone titre occur in normal development then the metabolism of ecdysone must play an important regulatory role. While the metabolism of exogenous hormone has been studied in various insects, the ways in which ecdysone titres are regulated in uiuo are not yet understood. Injected ecdysone has a short half-life (values between ~20 min and 3 hr have been
GEOFFREY
RICHARDS
Sequential
reported for third instar larvae of CaZZiphoru and Sarcophuga (Karlson and Bode, 1969; Shaaya, 1969; Heinrich and Hoffmeister, 1970; Ohtaki et al., 1970)): This is evidence of a potent inactivating system, which is presumably located in the insect fat body (King, 1972). If the peak ecdysone titre is 5 x 10-T M (Ashburner, 1974; Ashburner and Richards, 1976; Borst et al., 1974; De Reggi et al., 1975) and is reached 2 hr before pupariation, then even a conservative estimate of ecdysone’s half-life of 1 hr brings the ecdysone titre to 8 x lo+ M by 4 hr following pupariation (assuming no synthesis). I am grateful to Dr. M. Ashbumer for advice and encouragement and to Professor J. M. Thoday and Dr. P. Cherbas for their interest in this study, which was supported by a Medical Research Council Scholarship. REFERENCES ASHBURNER, M. (1967). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. I. Autosomal puffing patterns in a laboratory stock of Drosophila melanogaster. Chromosoma 21, 398-428. ASHBURNER, M. (1972a). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. VI. Induction by ecdysone in salivary glands of D. melanagaster cultured in vitro. Chromosoma 38, 255-281. ASHBURNER, M. (1972b). Ecdysone induction of puffing in polytene chromosomes of Drosophila melanogaster. Effects of inhibitors of RNA synthesis. Exp. Cell Res. 71, 433-440. ASHBURNER, M. (1973). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon Ecdysone Concentration. Develop. Biol. 35, 47-61. ASHBURNER, M. (1974). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibitors of protein synthesis. Develop. Biol. 39, 141-157. ASHBURNER, M., and RICHARDS, G. (1976). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. III. Conse-
Gene
Activation
IV
263
quences of ecdysone withdrawal. Develop. Biol. 54, 241-255. BECKER, H-J. (1962). Die Puffs der SpeicheldriisenSpeicheldrusenchromosomen n van von Drosophila melanogaster. I. Beobachtungen zumm Verhalten des Puffmusters im Normalstamm und bei zwei Mutanten, giant und lethal-giant-larvae. Chromosoma 10, 654-678. BERENDES, H. D. (1968). Factors involved in the expression of gene activity in polytene chromosomes. Chromosoma 24, 418-437. BORST, D. W., BOLLENBACHER, W. E., O’CONNOR, J. D., KING, D. S., and FRISTROM, J. W. (1974). Ecdysane levels during metamorphosis of Drosophila melanogaster. Develop. BioZ. 39, 308-316. CLEVER, U., and ROMBALL, C. G. (1966). RNA and protein synthesis in the cellular response to a hormone, ecdysone. Proc Nat. Acad. Sci . USA 56, 1470-1476. DE REGGI, M. L., HIRN, M. H., and DELAUQE, M. A. (1975). Radioimmunoassay of ecdysone on application to Drosophila larvae and pupae. Biochim. Biophys. Res. Comm. 66, 1307-1315. HEINRICH, G., and HOFFMEISTER, H. (1970). Insektenhaiitungshormone und ihre Wirkungweise: Bildung von Hormonglykosiden als inaktivierungsmechanismus bei Calliphora erythrocephala. 2. Naturforsch 25b, 358-361. KARLSON, P., and BODE, C. (1969).
Die Inaktivierung des Ecdysons bei der Schmeissfliege CaZZiphora erythrocephala Meigen. J. Insect Physiol. 15, 111-118. KING, D. S. (1972). Metabolism of a-ecdysone and possible precursors by insects in uivo and in vitro. Gen. Camp. Endocrinol. (suppl.) 3, 221-227. OHTAKI, T., and WILLIAMS, C. M. (1970). Inactivation of a-ecdysone and cyasterone by larvae of the fleshfly, Sarcophaga peregrina, and pupae of the silkworm, Samia Cynthia. BioZ. Bull. 138,326-333. RICHARDS, G. (1976a). Control of prepupal puffing patterns in uitro, implications for prepupal ecdysone titres in Drosophila melanogaster. Develop. BioZ. 48, 191-195. RICHARDS, G. (1976b). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. V. The late prepupal puffs. Develop. BioZ. SHAAYA, E.
54, 264-275.
(1969). Untersuchungen iiber die Verteilung des Ecdysons in verschiedenen Geweben von Calliphora erythrocephala und iiber seine biologische Halbwertszeit. 2. Naturforsh. 24b, 718-721.