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Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster,
DEVELOPMENTAL
BIOLOGY
Sequential
tid,
241-255 (1976)
Gene Activation by Ecdysone in Polytene Chromosomes Drosophila melanogaster
of
III. Consequences of Ecdysone Withdrawal MICHAEL Department
of Genetics,
ASHBURNER University
of
AND GEOFFREY
Cambridge,
Accepted
July
Downing
RICHARDS Street,
Cambridge,
England
16,1976
Ecdysone induces a sequence of changes in puffing activity of the polytene salivary gland chromosomes of Drosophila melnnogaster. In the continuous presence of ecdysone this sequence takes about 12 hr to complete. Salivary glands were incubated in the presence of ecdysone for various times and then transferred to hormone-free medium to study the effects of the hormone’s removal on the sequence of puffing activities. The early ecdysone-induced puffs immediately regress on removal of hormone. They can be fully reinduced if exposed to hormone again, but only if the initial period of exposure to the hormone was 2 hr or less; with longer periods of initial exposure the early puffs become progressively refractory to reinduction. This process is inhibited by cycloheximide. A group of the late puffs may be induced prematurely by removal of the hormone -the extent of their premature induction is correlated with the size of the early puffs during the period of ecdysone exposure. Ecdysone inhibits the premature induction of these late puffs. INTRODUCTION
There occurs at the end of larval development of Drosophila melanogaster, and many other Diptera, a sequence of puffing activity in the polytene chromosomes of larval tissues. In D. melanogaster, as in Chironomus tentans, this sequence commences with the induction of a small number of early puffs (Clever, 1964; Ashburner, 1972) and with the regression of some puffs previously active. As the puff sequence progresses, the early puffs regress and many late puffs become active (Clever, 1964; Ashburner, 1972). This sequence of puffing activity results from an action of the hormone ecdysone. The late larval-early ‘prepupal’ puffing sequence of the salivary gland chromosomes of D. melanogaster can be seen when glands from third instar larvae are cultured in a fully defined medium in the presence of hormone (Ashburner, 1972). Previous experiments led to the conclusion that ecdysone acts, in some way, as a trigger for this puffing sequence (Ashburner,
1973). Data to be presented in this paper support this conclusion by showing that the sequence becomes independent of exogenous hormone. It does so, however, in a rather unexpected, and fascinating, way. MATERIALS
(a) Third instar larvae of the Canton-S ‘wild-type’ stock of D. melanogaster were used in all experiments. Their salivary glands were cultured by the technique previously described (Ashburner, 1972). All glands were at Puff Stage 1 (PSl) at the initiation of their culture. This was determined by scoring the put% of one, of the paired salivary gland lobes which was fxed immediately on dissection: its contralateral lobe was cultured. Unless stated otherwise, all gland culture experiments were done at room temperature (20-22”(J). The technique used to score the puffs is described by Ashburner (1972). The measured puffs, with their typical sizes when uninduced and fully induced, are listed in Table 1 of Ashburner, 1974. 24Ll
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
VOLUME 54, 1976
DEVELOPMENTAL BIOLOGY
242
(b) The only technique novel to the experiments presently reported is that used to wash cultured glands. To do so, a gland was removed from the culture medium by picking it up by its duct with watchmaker’s forceps. It was then transferred to 100 ~1 of the appropriate wash medium (e.g., minus-ecdysone medium) and allowed to remain there for about 15-30 sec. It was then picked up again and transferred to 50 or 100 ~1 of the new incubation medium. Although no direct experiments (e.g., with labelled ecdysones) have been done to quantify the release of ecdysone by glands washed in this way, it is clear from the results that it is both rapid and effectively complete. (c) /3-Ecdysone (referred to as ‘ecdysone’ in the text) (Rohto Pharmaceutical Co., Osaka, Japan, lots 016 and 018) was used in all experiments. Unless stated otherwise, it was used at final concentrations in the medium of between 6.10+ and 6.10e6 M, sufficient for optimal puff induction. Cycloheximide was purchased (as actidione) from Koch-Light Ltd., and was used at a concentration of 7.10d5 M. Data showing the inhibition of salivary gland protein synthesis caused by cycloheximide and the normal induction of puffs after prolonged cycloheximide inhibition have been published before (Ashburner, 1974; also, Richards, 1976). Grace’s insect tissue culture medium was purchased commercially from Grand Island Biological Supply Co., from Flow Laboratories, or prepared in the laboratory. It was modified as described in earlier papers (Ashburner, 1972, 1973).
25AC and 68C, clear cut: removal of ecdysone never results in their reinduction. Having regressed in culture in the presence of hormone they remain inactive. The behaviour of 3C is different. This is a puff active in young larvae during the intermoult PSl. The rate of its regression in cultured glands could not be correlated with the ecdysone concentration (Ashburner, 1973). During the first hour or so of culture, 3C regresses in the presence or absence of hormone. However, in the continued presence of hormone it remains regressed; in the complete absence of hormone a puff at 3C reappears after 5-6 hr of culture (Fig. 1). Similarly, if ecdysone is withdrawn from cultured glands, the puff at 3C is reactivated. This can be seen from the experiment shown in Fig. 1. This observation confirms, in effect, the results of Becker (1959); see Discussion. 2. The Early Puffs: 23E, 74EF, and 75B
(i) Removal of hormone from salivary glands at any time during the first 4 hr of their exposure to it results in the rapid regression of the three early puffs 23E, 74EF, and 75B. Data, for 23E and 74EF, in a typical series of experiments, are shown in Fig. 2. (ii) The normal regression of 74EF and 75B, that is to say their regression after 4
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RESULTS
1. The Intermoult 6X
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----p-J
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+
Puffs: 3C, 25AC, and
We have studied the regression in culture of three puffs active in PSl animals. The consequences of ecdysone withdrawal after a period of culture of glands in the presence of ecdysone are, for two of them,
4
1.q 0
,
,
,
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FIG. 1. Puffing activity at the intermoult puff locus 3C in (iI the continuous presence of ecdysone (O--3), (ii) the continuous absence of ecdysone (O----O), and (iii) 2 hr with ecdysone, then no ecdysone (D-0). Ordinate, p&size; abscissa, hours.
ASHLIURNER
AND RICHARDS
Sequential
Gene
Activation
243
ZZZ
2.62.42.*to1.e Ia,At*-
FIG. 2A. The effect of removal of ecdysone on puffing activity at 23E. The solid circles are the sizes of the puffafier 1,2,3,4, and 5 hr of continuous ecdysone (abscissa) and the open circles are the sizes after removal of the hormone at these times. Ordinate, puff size. FIG. 2B. The effect of removal of ecdysone on puffing activity at 74EF. The solid squares are the sizes of the puff after 1, 2, 3, 4, and 5 h of continuous ecdysone (abscissa) and the open squares are the sizes aRer removal of the hormone at these times. Ordinate, puff size.
REGRESSION
TABLE 1 OF THE EARLY PTJFIW 74EF AND 75B IN GLANDS TRANSFERRED
TO HORMONE-FREE
MEDIUM
E;p;zreyt
INDUCED BY ECDYKINE FOR A FURTHER 2, 3, OR
Size of puff Ecdysone C?
1. 74EF 75B 2. 74EF
75B
FOR 3 HR AND THEN 4 HR”
No ecdysone (wash)
3 hr
C?
5 hr
6 hr
No
2.357 + 0.093
No Yes
1.147 2 0.037 1.212 + 0.044
1.164 k 0.046 1.287 f 0.029
7hr 1.105 5 0.026 1.318 2 0.044
No
2.612 2 0.10
No Yes
1.166 2 0.042 1.130 k 0.037
1.124 k 0.027 1.088 2 0.087
1.135 k 0.026 1.033 Yk0.019
No
2.396 + 0.055
No Yes
-
1.132 k 0.023 1.171 f 0.031
-
Yes
2.214 + 0.127
No Yes
-
1.143 f 0.041 1.218 f 0.036
-
No
2.678 f 0.145
No Yes
-
1.110 k 0.052 1.182 + 0.036
Yes
2.598 f 0.152
No Yes
1.192 2 0.053 1.220 f 0.041
-
a In experiment 1, cycloheximide was present, if at all, only during the second (wash) period; in experiment 2, cycloheximide was present (i) during induction only, (ii) during wash only, (iii) during neither period, or (iv) during both periods. C? = cycloheximide present?
hr in the continuous presence of ecdysone, is inhibited by cycloheximide or other inhibitors of protein synthesis (Ashburner, 1974). However, the regression of these puffs, resulting from hormone withdrawal,
is not inhibited by cycloheximide (Table 1). Indeed, puffs 74EF and 75B may be induced by ecdysone in the presence of cycloheximide and will then regress on transfer of the glands to medium contain-
244
DEVELOPMENTAL
BIOLOGY
ing the antibiotic alone (Tables 1 and 3 and Fig. 5). (iii) Having regressed as a consequence of ecdysone washout, the early puffs 23E, 74EF, and 75B may be reinduced if the glands are reexposed to ecdysone. The size to which these three puffs may be reinduced depends upon the time the glands were first exposed to hormone. For example, in glands cultured with hormone for 0.5 hr, cultured in hormone-free medium for 2 hr, and then reexposed to hormone for a further 2 hr, 74EF reached a size of approximately 2.24. When, however, the period of first exposure to ecdysone was 5 hr, again followed by a 2-hr wash and a 2-hr reinduction, 74EF only reached a size of approximately 1.43 during the second induction period. In both experiments, parallel control glands were cultured without hormone for a period equivalent to the first induction and wash periods and were then induced. For the two experiments quoted above, the control 74EF sizes on induction at 0.5 + 2 hr and 5 + 2 hr, respectively, were 2.36 and 2.38. Similar results were found for 23E and 75B. Thus, these early puffs become progressively less sensitive to ecdysone as the time of their exposure to hormone increases. This is especially so after 2 hr of exposure to ecdysone. The control experiments show this not to be an artifact of the culture conditions. Figure 3 illustrates the reinduction of 23E, 74EF, and 75B after 1 or 5 hr of initial induction with ecdysone and the control inductions for glands incubated without hormone for 1 + 2 or 5 + 2 hr, respectively, before incubation with hormone for the first time. Results for 23E and 75B from experiments over a range from 0 to 7 hr of initial induction are summarised in Figs. 6a and 6b. It is clear that after 3 hr of exposure to hormone, these puffs rapidly become refractory to reinduction after 2 hr of hormone-free culture. Increasing the period of ecdysone-free
54, 1976
VOLUME 1.*-
A
t\\ \ \ \ \
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\ \ p.
\ \
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+
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+-I
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‘.O___,--,-FIG. 3. The reinduction of the early puffs 23E, 74EF, and 75B after their regression resulting from ecdysone’s withdrawal at either 1 or 5 hr. Glands were cultured for either 1 or 5 hr with hormone (Un ) and then transferred to hormone-free medium for a further 2 hr (W----W) before culture for a further 2 hr with hormone (W-B); control glands were cultured for either 3 or 7 hr in the absence of hormone (O----O) before being induced for the first time (OCl). (A) 23E; (B) 74EF, (C) 75B. Ordinate, puff&e; abscissa, hours.
ASHBURNER
Sequential
AND RICHARDS
TABLE OF EARLY PUFFS AVER 2 HR OF INITIAL 2-HR OR A 4-HR WASH PERIOD
Period of wash (hr)
245
III
2 EXP~EURE TO ECDYSONE FOLLOWED AND A 2-HR REINDUCTION
BY EITHER
A
Size of puff after reinduction 23E
2 4
1.834 1.645
2 f.
74EF 2.331 2 0.056 2.240 + 0.048
0.050 0.051 TABLE
REINDUCTION
Activation
(u) Although the early puffs become increasingly insensitive to reinduction following a wash, with increasing time of initial exposure to ecdysone, this is not so for initial induction periods of 2 hr or less (2.iii, above, and Fig. 6). This enabled the demonstration of the fact that puff size on reinduction is not necessarily a function of the size to which the puffs were first induced. This was shown by inducing 74EF
culture from 2 to 4 hr does not alter the sizes of the early puffs on reinduction by ecdysone (Table 2). (iv) The reinduction, after an ecdysone washout caused regression, of puffs 74EF and 75B is not sensitive to cycloheximide (Table 3 and Fig. 5). The puff at 23E is, just as it is sensitive to inhibitors of protein synthesis for its initial induction (Table 3b).
REINDUCTION
Gene
75B 2.271 k 0.073 2.148 + 0.059
3a
OF EARLY PUFFS 74EF AND 75B FOLLOWING AN INITIAL INDUCTION FOR 3 HR, CULTURE ABSENCE OF ECDY~~NE FOR 2 HR, AND THEN REEXPOSURE TO ECDYSONE FOR 2 HR~
IN THE
Size of puff Ecdysone 74EF
75B
C? No
No ecdysone
3hr 2.329 f 0.083
No
2.664 2 0.132
a C? = cycloheximide
C? No
5 hr 1.278 k 0.047
Yes
1.211
No
1.215 + 0.041
Yes
1.211 ?z 0.036
EXPERIMENT
Cycloheximide Initial ind$owntf ecd’ysone No
TO THAT
ILLUSTRATED
+ 0.041
7 hr 2.151 2.181 2.178 2.070
2 k + 2
0.062 0.097 0.068 0.051
No Yes No Yes
1.921 2.181 2.456 2.438
2 0.091 k 0.097 rc_0.105 f 0.065
3b
IN TABLE 3a BUT WITH FOR 1 HR ONLY
AN INITIAL
EX~~~IJRE
TO ECDYSONE
Size of puff at 5 hr
present?
Wash (2 Reinduchr), no ec- tiqn (2 hr), with ecdydysone sone No No Yes Yes
C? No Yes No Yes
present? TABLE
A SIMILAR
Ecdysone
No Yes
23E
74EF
75B
1.684 “_ 0.070 1.267 k 0.070
2.424 k 0.087 2.108 -t 0.086
2.522 k 0.070 2.423 f 0.100
1.080
1.971 f 0.048 2.106 k 0.075
2.356 f 0.054 2.458 -c 0.053
2 0.017
1.133 ‘- 0.030
a Data for three early puffs, 23E, 74EF, and 75B, are given.
246
BIOLOGY
DEVELOPMENTAL
and 75B to various sizes by using suboptima1 ecdysone concentrations for 2 hr. All series of glands were, after a standard 2 hr in hormone-free medium, reinduced but now with a single, high hormone concentration. The result of this experiment, Fig. 4, shows that the size on reinduction was the same for all glands, regardless of the fact that puff size on initial induction varied over a considerable range. (vi) In section 2.iii above, experiments were described which point to the conclusion that the early puffs 74EF and 75B become progressively refractory to ecdysone. These data are quite consistent with those that show these puffs to regress after 4 hr under conditions where the ecdysone concentration remains high (Ashburner, 1973). The following experiment indicates that the development of insensitivity to ecdysone which accompanies the activity of 74EF and 75B requires protein synthesis. The experiment reported in Section 2.iii (above) was repeated with the change that now cycloheximide was present in all culture media; that is, in the medium used for initial induction, that used to wash the glands free of hormone and to culture
2.* 2.1 1 1
I
I.0 0
1
2
3
1
s
6
I
I
FIG. 4. Induction of 74EF by 8 x lo-” M ecdysone (A-A), 6 x lo-‘M ecdysone (M-W), or 2.4 X 10-O M ecdysone (O-O) for 2 hr followed by 2 hr of hormone-free culture (--1 and then a second induction with 2.4 x 10m6 M ecdysone. Ordinate, puff size; abscissa, hours.
VOLUME
54, 1976
FIG. 5. The reinduction of the early puffs 74EF and 75B in the presence of cycloheximide. The experimental protocol was identical to that illustrated in Fig. 3 with the difference that cycloheximide was present in all culture media. (A) 74EF; (B) 75B. Ordinate, puff size; abscissa, hours.
them in hormone-free medium for 2 hr thereafter, and in the ecdysone reinduction medium. The results of the l- and 5-hr experiments are shown in detail in Fig. 5, and these and other experiments are summarised in Fig. 6. Now we see that there is no difference between the experimental, i.e., preinduced, series of glands and the control uninduced series. This is true even after 5 hr of initial induction. The absolute puff sizes are smaller in these experiments than in the equivalent experiments described in section 2.iii. This may reflect a general decline in the gland’s health after prolonged exposure to cycloheximide. 23E could not, of course, be followed in these experiments since its induction is cycloheximide sensitive.
ASHBURNER
AND RICHARDS
Sequential
Gene Activation
III
247
dium these puffs are reinduced on reexposure to hormone and reach their normal sizes. Data for 78D are shown in Fig. 7a. 4. The Late Puffs 22C, 63E, and 82F
FIG. 6A and 6B. A summary of the reinduction data for (A) 23E and (B) 75B. In the lower part of the figures the size (as a percentage of the corresponding control puff size) of the puffs on reinduction after an initial exposure to hormone for the times indicated (followed by a 2-hr hormone-free culture) is shown. The upper part of the figure gives the actual sizes of the puffs in the control experiments i.e., in glands cultured without ecdysone fort + 2 hr before a 2-hr ecdysone induction. In Fig. 6B the solid symbols are the experiments under normal culture conditions and the open symbols are from experiments in which protein synthesis was inhibited, by cycloheximide, throughout culture (see text). Left ordinate, percentage control activity; right ordinate, puff size; abscissa, hours.
3. The Late Puffs 62E and 780 These two late puffs are, in the continuous presence of ecdysone, maximal at 5 hr (62E) and at 6 hr (78D). If puffing has commenced at these sites by the time ecdvsone is withdrawn from the glands (e.g., at 4 hr), then they regress. After culture of the glands for 2 hr in hormone-free me-
During a continuous ecdysone experiment, with an optimal hormone concentration, these put& reach their maximal sizes as follows: 22C at 8-10 hr, 63E at 8-10 hr, and 82F at 10 hr (very rarely at 8 hr). They then regress. Their behaviour in glands from which ecdysone has been withdrawn is rather surprising. (9 All three puffs immediately appear in glands exposed to ecdysone for 4 hr and then washed free of hormone. That is to say that these late puffs are prematurely induced by ecdysone’s withdrawal (Fig. 7). (ii) The size reached by the prematurely induced puffs is a function of the extent of puffing, during the initial exposure to ecdysone, of the early puff sites. This can be shown by two experiments: (a) Glands were exposed to ecdysone for varying periods of time (from 0.5 to 4 hr) before being transferred to hormonefree medium. The extent of puffing, during the period of culture in hormone-free medium, of these three late puffs is a function of the time of ecdysone exposure. Even after 1 hr of ecdysone they appear, albeit rather small and after a lag of some hours. After longer periods in ecdysone (e.g., 4 hr) the prematurely induced puffs approach their normal maximum sizes and with no lag (Fig. 7). (b) Glands were exposed to different ecdysone concentrations for a standard period of time (2 hr) before being transferred to hormone-free medium. The extent of premature induction of 22C, 63E, and 82F was a function of the ecdysone concentration, and hence size of the early puffs, during the period of exposure to hormone. Data from both of these, and other, experiments are plotted for the three late puffs 22C, 63E, and 82F in Fig. 8. The correlation between the size of 75B (or with 74EF, not shown) during the induc-
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FIG. 7A-C. The behaviour of 78D, cultured in hormone for 1 (upper panel), of hormone for up to 6 hr (O----O). To culture (H-W). The lower panel also of ecdysone. (A) 78D; (B) 63E, and (C)
\ \ I -__-
-;
63E, and 82F on ecdysone withdrawal and re-addition. Glands were 2 (middle panel), or 4 hr (lower panel) (O-O) and then washed free half of the glands, hormone was re-added after 2 hr of hormone-free shows (0-O) the activities of these puffs in the continuous presence 82F. Ordinate, puff size; abscissa, hours. 248
ASHBURNER
Sequential
AND RICHARDS
++
1.5- * tom-
:
+
t +++
+++
9 22c h=
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++ +$+ t
-. 10-
*
+
++
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03E
4: +&'
,I$
++++
+
FIG. BA-C. The size of prematurely induced late puffs (ordinate) as a function of the size of an early puff (75B) (abscissa) at the time of ecdysone’s removal in washout experiments. The arrows indicate the sizes of these puff sites when inactive. (A) 82F; (B) 22C, and (C) 63E.
TABLE SIZE OF PREMATURELY HR PERIOD FOLLOWING
Ecdysone concentration in second period (Ml 7.5 7.5 1.8 3.8 7.5
0 x 1.0-g x 1.0-a x I-O-’ x l.O-’ x 10-1
4
INDUCED LATE PUFFS AS A FUNCTION OF THE CONCENTRATION AN INITIAL EXPOSURE FOR 3 HR OF GLANDS TO A HIGH (6 x OF ECDYSONE
74EF 1.163 1.065 1.181 1.202 1.591 1.871
2 0.053 k 0.034 k 0.059 k 0.055 rf: 0.086 k 0.086
249
III
finally regress. These points are illustrated by the data of Fig. 7. The inhibition, by ecdysone, of premature induction of the late puffs is also seen in the data given in Table 4. For this experiment., glands were cultured (for 3 hr) in a high concentration of ecdysone before being transferred (for 2.5 hr) to media containing ecdysone over a wide range of concentrations. Full premature induction only occurs in the complete absence of ecdysone. Even as little as 7.5 x 10eg M ecdysone in the second period causes some inhibition. (iv) In the continuous presence of ecdysone, cycloheximide inhibits late puff induction (Ashburner, 1974). This is a specific inhibition, since premature induction of the late puffs still occurs, albeit to a reduced extent, if glands are transferred from ecdysone medium to ecdysone-free cycloheximide medium. No premature induction occurs if cycloheximide is present only during the ecdysone treatment period or throughout the experiment. Data for three independent. experiments are shown in Table 5. The behaviour of the puffs 22C and 82F in these experiments will be discussed first, that of 63E is rather a special case. In all three experiments the absence of cycloheximide in both the ecdysone and wash periods results in premature induction of 22C and 82F. Addition of cycloheximide to the wash alone does not inhibit this premature induction, although the sizes of the prematurely induced puf’fs are smaller than in the controls lacking inhibitor throughout the experiments. Ad-
tion phase and the size attained by the prematurely induced late puffs on ecdysone washout is clear. (iii) Ecdysone inhibits premature induction. This is shown by the fact that if glands are reexposed to ecdysone, after the period of hormone-free culture, then the prematurely induced late puffs regress (if active) or are inhibited from appearing (if the ecdysone was re-added before the end of their lag period). As the second exposure to ecdysone continues, the late puffs are induced -for the second time-and then
20-
Gene Activation
75B 1.108 1.063 1.127 1.178 1.552 1.988
” k k 2 ” k
63E 0.044 0.041 0.074 0.056 0.063 0.079
2.199 1.710 1.520 1.498 1.429 1.422
k 0.059 2 0.064 -r- 0.077 k 0.057 k 0.052 k 0.089
OF ECDYSONE
IN A 2.5
lo-’ M) CONCENTRATION 82F 2.734 2.177 2.053 1.633 1.534 1.414
-t k k k k 2
0.083 0.081 0.106 0.106 0.139 0.096
250
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE THE EFFECTS
5
OF CYCLOHEXIMIDE ON THE PREMATURE INDUCTION OF THE LATE PUFFS 22C, DURING A WASH PERIOD FOLLOWING Exrosuas TO ECDYSONE~
Ecdysone incubation CC?)
Wash
No
No
Expt
Yes
No
63E, AND 82F
Size of puff 63E
2.103k 0.079 1.8362 0.043 1.628k 0.035
22c 1.702A 0.083 1.5052 0.078 1.352f 0.036
82F 2.442f. 0.112
2 3
2.083f 0.052 1.954 2 0.085 1.621e 0.043
1.478? 0.051 1.475+- 0.049 1.2472 0.038
1.639 f 0.051 1.439 k 0.077 1.536f 0.051
1
1.620k 0.098
2 3
1.291 f 0.065 1.548+ 0.052
1.162f 0.047 1.128A 0.034 1.061k 0.022
1.269 ? 0.046 1.210k 0.052 1.213” 0.021
CC?) 1
2 3
Yes
54, 1976
1
1.798 + 0.112 1.767zt 0.039
k 0.058 1.159 f 0.028 1.281 + 0.038 1.219 f 0.084 1.1132 0.047 1.206k 0.102 1.560f 0.034 1.086k 0.021 1.254zt 0.026 a Three experiments are shown, the period of ecdysone induction being 3 hr in the first and 3.5 hr in the second and third; the period of wash was 3 hr in experiments 1 and 2 and 4 hr in experiment 3. C? = cycloheximide present?
Yes
1
1.552
2 3
dition of inhibitor during the ecdysone period, whether or not it is present during the wash period, completely inhibits the appearance of prematurely induced puffs at either 22C or 82F. The behaviour of puff 63E is anomolous and not entirely consistent between these experiments. Interpretation of the data for this puff must take note of the fact (Ashburner, 1974) that cycloheximide alone (i.e., in the absence of ecdysone) results in the appearance of a pufY at this site. The puff induced by this drug (or other inhibitors of protein synthesis) is always smaller than that induced by ecdysone (see Table 5 of Ashburner, 1974). In the current experiments the induction of 63E is seen whether or not cycloheximide was present during the ecdysone induction period (though less markedly in experiment 2), but was much larger when cycloheximide was absent during this ecdysone period (whether or not present during the wash) in two of the three experiments (i.e., 1 and 2 ofTable 5). We noted before (Ashburner, 1974) that the induction of 63E by cycloheximide alone was in contrast to the fact that cycloheximide inhibited the induction of this
puff in the continuous presence of ecdysone. This experimental result may be reworded in the form ‘ecdysone inhibits the induction of 63E by cycloheximide.’ Seen in this way, it is then obvious that we should ask whether or not this inhibition by ecdysone is physiological-as assayed, for example, by the shape of the doseresponse curve for inhibition. To study this, salivary glands were incubated in media containing cycloheximide and various concentrations of ecdysone for 3 hr at 25°C. The sizes of three early puffs (23E, 74EF, and 75B) and of 63E were then measured. Confirming earlier results, cycloheximide completely inhibits the induction of 23E. Both 74EF and 75B respond according to the ecdysone concentration, the dose response curve for them being almost identical to that previously published (Ashburner, 1973) despite the presence of cycloheximide in the current experiments and the difference in culture temperature. In the absence of ecdysone, a large puff at 63E was induced by cycloheximide. As the ecdysone concentration increased, the size of this puff gradually decreased so that by 7.5 x lo-’ M ecdysone
ASHBURNER
AND RICHARDS
Sequential
the puff was almost completely inhibited (Fig. 9a). When the percentage of maximum induction of 74EF (or 75B) is plotted together with the percentage of inhibition of 63E (Fig. 9b) it can be seen that the ecdysone dose-response curve for the inhibition of the cycloheximide induction of 63E is identical to those for the ecdysone induction of 74EF and 75B. DISCUSSION
The results presented in this paper, and in three previous papers (Ashburner, 1972, 1973, and 1974), demonstrate the apparent complexity of the control of the late larval puffing cycle in D. melanogaster. As a step toward a synthesis, the present data will be discussed together with those previously published. The reader is referred to the earlier papers and to the Results section of this paper for the full details of the experiments.
-‘6
’
’ “““’
-6
’
“““”
Gene
Activation
(a) The Inter-mod
ZZZ
251
Puffs
Three intermoult puffs have been considered in detail: 3C, 25AC, and 68C. All are active during the long PSl and regress as the ecdysone-induced cycle gets under way. The detailed behaviour, and hence presumably the control, of each puff differs. 3C. Unlike 25AC and 68C, this puff is active at another stage of development in addition to PSl. It is active from PS15 to PS20 in ‘prepupae.’ Indeed, in 8-hr (PS18) prepupae it is larger than at any time during the analysable period of larval development (Becker, 1959; Ashburner, 1969). In culture, this puff regresses regardless of the ecdysone status of the medium. However, when ecdysone is absent it reappears at 5-6 hr. It also reappears on ecdysone washout. In fact, this was observed
-1
’
’ “““’
-6
’
“““”
-5
FIG. 9A. The sizes of puffs (ordinate) at 23E (O----O), 63E (O----O), 74EF (O--O), and 75B (W--a) as E function of the log,, ecdysone concentration (abscissa) in the continuous presence of cycloheximide. FIG. 9B. Normalized dose-response curves for the induction of 74EF (0) and 75B (m) by ecdysone in the presence of cycloheximide and for the inhibition, by ecdysone, of the induction of 63E by cycloheximide (01. Ordinate, size of puffs as a percentage of the corresponding control puff size; abscissa, log,, ecdysone concentration.
252
DEVELOPMENTAL
BIOLOGY
by Becker (1959): when glands of up to PS5/6 (equivalent to 5 hr in culture under the conditions of our experiments) were explanted from larvae into a saline solution for an hour or more, a puff at 3C appeared. Becker interpreted this observation to mean that up to a critical point in the puffing cycle (i.e., PS5-6) the puffing pattern actually reversed on removal of the glands from the animal and their culture in saline. Recently, Korge (1975) has presented evidence which strongly implicates the 3C puff in coding for a polypeptide component of the salivary gland’s glue secretion. This secretion begins to accumulate as electrondense granules in the cytoplasm a few hours after the moult from the second instar, and glue synthesis probably ceases on ecdysone release late in the third instar. The behaviour, both in uiuo and in vitro, of 3C suggests that it may be activated, in either larvae or prepupae (Richards, 1975), by a fall in ecdysone concentration. 25AC. This intermoult puff regresses in culture and does so faster at high ecdysone concentrations than at low, or zero, concentrations. This regression is unaffected by inhibiting protein synthesis. No reinduction occurs on ecdysone washout. 68C. 68C also regresses either in the presence or absence of ecdysone but does so much faster at high ecdysone concentrations. The ecdysone-facilitated regression of 68C is slowed by cycloheximide but its ecdysone independent regression is not. Like 25AC, 68C is not reinduced on hormone withdrawal. Each intermoult puff is unique in its response to the experimenter’s conditions. For 25AC and 68C, at least, the absence of ecdysone would appear to be a necessary, but not sufficient, condition for their activity during PSl. In D. virilis, puff 55E behaves in a similar manner, during development, as the intermoult puffs 25AC and 68C of D. melanoguster. Kress (1973) has reported that 55E can be caused to regress
VOLUME
54,
1976
prematurely, in uiuo, by injecting animals with ecdysone. Interestingly, accelerated regression of 55E also results from the injection of glucosamine into larvae. Attempts to speed up the slow regression of 25AC and 68C, seen when PSl glands ofD. melanogaster are cultured without ecdysone, by adding aminosugars to the medium have so far been unsuccessful. However, the regression of 55E induced by injecting glucosamine into larvae takes a much longer time than does the regression of either 25AC or 68C in glands cultured in the absence of ecdysone. While confirming Becker’s observation on the behaviour of 3C on ecdysone withdrawal, we question the reality of what Becker calls ‘Ruckwartslaufen’ of the puffing pattern. We have shown that 3C behaves ‘oddly’ even when PSl glands are cultured without hormone; furthermore, other puffs quite characteristic of PSl (e.g., 25AC and 68C) are never reinduced. The pattern of puffing in washed glands, either from cultured material or in material taken directly from larvae (i.e., a repeat of Becker’s original experiment; M. Ashburner, unpublished), only superflcially resembles a PSl pattern. The importance of this observation is that in Chironomus species, Kroeger (1964) described a particular type of puffing response ‘rejuvenation’ which, he proposes, is analogous to the phenomenon discussed above in Drosophila. Rejuvenation of the puffing pattern in Chironomus is thought to be a response of the chromosomes to various factors, all of which lead to an influx of sodium ions into the cells of the salivary gland. As such, the phenomenon forms one of the pieces of evidence for the ‘ion’ model of control of gene activity (e.g., Kroeger, 1968). Our point is that there is no evidence for any similar phenomenon in D. melanogaster. (b) The Early
Puffs
The three early puffs studied in detail (23E, 74EF, and 75B) respond within min-
ASIXEWRNER
AND RICHARDS
utes to ecdysone (Ashburner, 1972). Their dose-response curves are broad: a 600-fold range of ecdysone concentration spans their threshold to maximal response. 23E. This is the only early puff known to be sensitive to inhibitors of protein synthesis. It is different from other early puffs in three other respects: (i) It is often induced, to a rather small size, when PSl glands are cultured without ecdysone, (ii) it shows three peaks of activity in the continuous presence of an optimal ecdysone concentration, and (iii) it has a higher sensitivity to ecdysone. Once induced, withdrawal of ecdysone results in its regression. It may then be induced a second time on readdition of hormone to the culture medium. Reinduction, but not its regression on ecdysone withdrawal, is, like its initial induction, inhibited by cycloheximide. 74EF and 75B. These two puffs may be considered together, as their behaviour is nearly always the same. Their induction by ecdysone is very rapid and, at optimal hormone concentrations, they regress, having attained their maximum sizes at 4 hr. Regression, in the presence of ecdysone, is cycloheximide inhibited. Their activity depends upon the presence of exogenous hormone. Removal of hormone causes them to regress immediately, they can be reinduced if the glands are reexposed to hormone. Neither their regression on ecdysone washout nor their reinduction is cycloheximide sensitive. Evidence has been obtained that the normal regression of these puffs, that is, their regression after 4 hr in the presence of hormone, is itself a response to ecdysone. Having so regressed, they are refractory to ecdysone. This insensitivity will not develop if protein synthesis is inhibited during the inducing phase. The insensitivity to ecdysone after ecdysone exposure is not a ‘ticketing’ phenomenon. By this we mean that a class of model to explain the behaviour of these puff sites is that each puff site possesses a finite
Sequential
Gene
Activation
III
253
number of ‘tickets’ allowing transcription. Once these have been exhausted, the puff must regress and become refractory. This appears to be ruled out by the fact that the puff size on second induction is independent of puff size during a 2-hr first induction period. Instead, the most economic model to explain the behaviour of these puffs is to suppose that they are in fact turned off by their own products or those of another early puff such as 23E (Cherbas and Kafatos, 1976). In the detailed model published (Ashburner et al., 1974), we consider that these early puffs respond initially to the concentration of hormone complexed to its receptor protein(s) (ER). Removal of the ecdysone dissociates the ER complex and the puffs then regress. When active, however, the puffs synthesize a class of products (P) which are either proteins or depend upon protein synthesis for their activity. These products compete, at the puff site, with the bound ER complex. With time, in the presence of ecdysone, the concentration of P rises to an extent capable of displacing all of the ER complex from the puff site and the puff, therefore, regresses. Thus, we can interpret the curve showing the development of insensitivity of these puffs to ecdysone (Fig. 6) as a function of the rise in P concentration. (c) The Late Puffs All five late puffs studied (62E, 78D, 22C, 63E, and 82F) appear after a lag of several hours in the presence of hormone. All have very steep, almost all-or-none, dose-response curves and all are inhibited by cycloheximide. Yet, they fall into two groups. 62E and 780. These puffs behave in ecdysone withdrawal experiments rather like 74EF and 75B. If active on ecdysone withdrawal, they regress; they are then reinducible. That is to say, they are late puffs for which the contemporary presence of ecdysone is necessary for activity. We may call them the ‘early lutes.’
254
DEVELOPMENTAL
BIO~CY
22C, 63E, and 82F. The three ‘late lates’ may be considered together, although the anomalous behaviour of 63E in response to inhibitors of protein synthesis in the absence of ecdysone (Schoon and Rensing, 1973; Ashburner, 1974 and this paper) should be recalled. All three show the phenomenon of premature induction. Their premature induction is ecdysone inhibited. Yet, ecdysone is not inhibitory to their activity per se. This is shown by their normal activity in a continuous ecdysone experiment and their second phase of activity on reinduction by ecdysone. The extent of their premature induction is correlated with the extent of induction of the early puffs 74EF and 75B during the initial ecdysone treatment. We stress that this is but a correlation of the observed phenomena. Since we are concerned with finding the simplest model that will account for the experimental data and serve as a useful basis for further experiments we have considered the possibility that these late puffs are, in fact, responding to a product of the early puffs (Ashburner et al., 1974). The lag in their normal appearance is accounted for by proposing that the ER complex, in addition to inducing the early puffs, acts negatively on the late latepuffs. With the activity of the early puffs, the concentration of their products, P rises in the cell and competes for the ER complex at the late late-puff sites. Eventually, it rises sufficiently to displace the ER complex from these sites and to turn on the puffs. Cycloheximide does not allow P to be made. Therefore, the late puffs are not induced [the anomalous behaviour of 63E in the absence of ecdysone and presence of cycloheximide cannot be adequately accounted for as yet, although the fact that ecdysone inhibits this induction with dose-response characteristics identical to those of early puff induction (results) is clearly quite consistent with our model]. However, if the ecdysone is washed out
VOLUME
54,
1976
after, say, 2 hr, a certain amount of P has already been made as a consequence of early puff activity. The ecdysone concentration having fallen, ER dissociates and the available P can immediately, and prematurely, induce the late late-puffs. If ecdysone is re-added, the ER now displaces this P from the late late-sites and they regress. But with the renewed activity of the early puffs, the P concentration begins to rise further, until it is suffkient to displace the ER at the late late-puff sites and activate them once again. As expected on this model, premature induction of the late late-puffs is not inhibited (at least not completely) by transferring glands from ecdysone into cycloheximide medium. The necessary P was made during the ecdysone period of culture. Whatever the precise details of this interpretation, it is very clear that the temporal sequence of late late-puffs is independent of ecdysone’s presence and that, therefore, the total sequence does become independent of hormone once initiated. The fuller implications of this model have been discussed in the paper of Ashburner et al. (1974), to which the reader is referred. The experiments described in the present paper, and in those previously published, are not to be used to ‘support’ this particular model. The model is only of prospective, not retrospective, value. Many details of the experimental results, for example, the intermoult puffs and the behaviour of the early puff 23E and of the two early late-puffs, are not considered in the model at all. In a formal way, the model, or simple variants upon it (and there are many), can account for the sequential cycle of gene activation and regression seen in polytene chromosomes of D. melanogaster at the end of larval life. How close an approximation the model is to the real situation can only be judged by a further analysis of its elements, by genetic and biochemical techniques. This analysis is in progress.
A~HBURNER
AND RICHARDS
This work has been supported by grants from the Science Research Council (B/SR/9750 and B/RG/ 7429/7) to M.A. We thank Peter Cherbas for many stimulating discussions and Trevor Littlewood for looking after our flies. REFERENCES ASHBURNER, M. (1969). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. II. The X-chromosome puffing patterns of D. melanogaster and D. simulans. Chromosoma 27, 4763. ASHBUFINER, M. (1972). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. VI. Induction by ecdysone in salivary glands of D. melanogaster cultured in vitro. Chromosoma 38, 255-281. ASHBURNER, M. (1973). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration. Develop. Biol. 35, 47-61. AIJHBURNER, M. (1974). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibition of protein synthesis. Develop. Biol. 39, 141-157. ASHBURNER, M., CHIHARA, C., MELTZER, P., and RICHAIU, G. (1974). On the temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38,655-662. BECKER, H. J. (1959). Die Puffs der Speicheldriisenchromosomen von Drosophila melanogaster. I. Beobachtungen zum Verhalten des Puffmusters im Normalstamm und bei zwei Mutanten, giant und lethal-giant-larvae. Chromosoma 10,654-678. CHERBAS, P. T., and KAFATOS, F. C. (1976). Temporal programs of gene expression during cell dif-
Sequential
Gene
Activation
ZZZ
255
ferentiation. Dahlem Workshop on “Molecular Basis of Circadian Rhythms.” Berlin, West Germany. CLEVER, U. (1964). Actinomycin and puromycin: Effects on sequential gene activation by ecdysone. Science 146, 794-795. KORGE, G. (1975). Chromosome puff activity and protein synthesis in the larval salivary glands of Drosophila melanogaster. PFOC. Nat. Acad. Sci. USA 72, 4550-4554. KRESS, H. (1973). Specific repression of a puff in the salivary gland chromosomes of Drosophila virilis after injection of glucosamine. Chromosoma 40, 379-386. KROEGER, H. (1964). Zellphysiologische Mechanismen bei der Regulation von Genaktivittiten in den Riesenchromosomen von Chironomus thummi. Chromosoma 15, 36-70. KROEGER, H. (1968). Gene activities during metamorphosis and their control by hormones. In “Metamorphosis: A Problem for Developmental Biology” (W. Etkin and L. I. Gilbert, eds.). Appleton-century-crofts, New York. RICHARDS, G. (1975). The hormonal control of chromosomal puffing in salivary glands of Drosophila melanogaster. Ph.D. thesis, University of Cambridge. RICHARDS, G. (1976). Sequential gene activation by ecdysone in polytene chromosomes of Drosophiiu melanogaster. V. The late prepupal puffs. Develop. Biol. 54, 000-000. SCHOON, H., and RENSING, L. (1973). The effect of protein synthesis-inhibiting antibiotics on the puffing pattern of Drosophila salivary glands in vitro. Cell Diff. 2, 97-106.
BIOLOGY
Sequential
tid,
241-255 (1976)
Gene Activation by Ecdysone in Polytene Chromosomes Drosophila melanogaster
of
III. Consequences of Ecdysone Withdrawal MICHAEL Department
of Genetics,
ASHBURNER University
of
AND GEOFFREY
Cambridge,
Accepted
July
Downing
RICHARDS Street,
Cambridge,
England
16,1976
Ecdysone induces a sequence of changes in puffing activity of the polytene salivary gland chromosomes of Drosophila melnnogaster. In the continuous presence of ecdysone this sequence takes about 12 hr to complete. Salivary glands were incubated in the presence of ecdysone for various times and then transferred to hormone-free medium to study the effects of the hormone’s removal on the sequence of puffing activities. The early ecdysone-induced puffs immediately regress on removal of hormone. They can be fully reinduced if exposed to hormone again, but only if the initial period of exposure to the hormone was 2 hr or less; with longer periods of initial exposure the early puffs become progressively refractory to reinduction. This process is inhibited by cycloheximide. A group of the late puffs may be induced prematurely by removal of the hormone -the extent of their premature induction is correlated with the size of the early puffs during the period of ecdysone exposure. Ecdysone inhibits the premature induction of these late puffs. INTRODUCTION
There occurs at the end of larval development of Drosophila melanogaster, and many other Diptera, a sequence of puffing activity in the polytene chromosomes of larval tissues. In D. melanogaster, as in Chironomus tentans, this sequence commences with the induction of a small number of early puffs (Clever, 1964; Ashburner, 1972) and with the regression of some puffs previously active. As the puff sequence progresses, the early puffs regress and many late puffs become active (Clever, 1964; Ashburner, 1972). This sequence of puffing activity results from an action of the hormone ecdysone. The late larval-early ‘prepupal’ puffing sequence of the salivary gland chromosomes of D. melanogaster can be seen when glands from third instar larvae are cultured in a fully defined medium in the presence of hormone (Ashburner, 1972). Previous experiments led to the conclusion that ecdysone acts, in some way, as a trigger for this puffing sequence (Ashburner,
1973). Data to be presented in this paper support this conclusion by showing that the sequence becomes independent of exogenous hormone. It does so, however, in a rather unexpected, and fascinating, way. MATERIALS
(a) Third instar larvae of the Canton-S ‘wild-type’ stock of D. melanogaster were used in all experiments. Their salivary glands were cultured by the technique previously described (Ashburner, 1972). All glands were at Puff Stage 1 (PSl) at the initiation of their culture. This was determined by scoring the put% of one, of the paired salivary gland lobes which was fxed immediately on dissection: its contralateral lobe was cultured. Unless stated otherwise, all gland culture experiments were done at room temperature (20-22”(J). The technique used to score the puffs is described by Ashburner (1972). The measured puffs, with their typical sizes when uninduced and fully induced, are listed in Table 1 of Ashburner, 1974. 24Ll
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND METHODS
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DEVELOPMENTAL BIOLOGY
242
(b) The only technique novel to the experiments presently reported is that used to wash cultured glands. To do so, a gland was removed from the culture medium by picking it up by its duct with watchmaker’s forceps. It was then transferred to 100 ~1 of the appropriate wash medium (e.g., minus-ecdysone medium) and allowed to remain there for about 15-30 sec. It was then picked up again and transferred to 50 or 100 ~1 of the new incubation medium. Although no direct experiments (e.g., with labelled ecdysones) have been done to quantify the release of ecdysone by glands washed in this way, it is clear from the results that it is both rapid and effectively complete. (c) /3-Ecdysone (referred to as ‘ecdysone’ in the text) (Rohto Pharmaceutical Co., Osaka, Japan, lots 016 and 018) was used in all experiments. Unless stated otherwise, it was used at final concentrations in the medium of between 6.10+ and 6.10e6 M, sufficient for optimal puff induction. Cycloheximide was purchased (as actidione) from Koch-Light Ltd., and was used at a concentration of 7.10d5 M. Data showing the inhibition of salivary gland protein synthesis caused by cycloheximide and the normal induction of puffs after prolonged cycloheximide inhibition have been published before (Ashburner, 1974; also, Richards, 1976). Grace’s insect tissue culture medium was purchased commercially from Grand Island Biological Supply Co., from Flow Laboratories, or prepared in the laboratory. It was modified as described in earlier papers (Ashburner, 1972, 1973).
25AC and 68C, clear cut: removal of ecdysone never results in their reinduction. Having regressed in culture in the presence of hormone they remain inactive. The behaviour of 3C is different. This is a puff active in young larvae during the intermoult PSl. The rate of its regression in cultured glands could not be correlated with the ecdysone concentration (Ashburner, 1973). During the first hour or so of culture, 3C regresses in the presence or absence of hormone. However, in the continued presence of hormone it remains regressed; in the complete absence of hormone a puff at 3C reappears after 5-6 hr of culture (Fig. 1). Similarly, if ecdysone is withdrawn from cultured glands, the puff at 3C is reactivated. This can be seen from the experiment shown in Fig. 1. This observation confirms, in effect, the results of Becker (1959); see Discussion. 2. The Early Puffs: 23E, 74EF, and 75B
(i) Removal of hormone from salivary glands at any time during the first 4 hr of their exposure to it results in the rapid regression of the three early puffs 23E, 74EF, and 75B. Data, for 23E and 74EF, in a typical series of experiments, are shown in Fig. 2. (ii) The normal regression of 74EF and 75B, that is to say their regression after 4
2.0,’ +‘.
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IJ-
1
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'.
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-
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RESULTS
1. The Intermoult 6X
'\
----p-J
/'
+
Puffs: 3C, 25AC, and
We have studied the regression in culture of three puffs active in PSl animals. The consequences of ecdysone withdrawal after a period of culture of glands in the presence of ecdysone are, for two of them,
4
1.q 0
,
,
,
,
,
,
,
, 1
I
2
3
4
5
6
7
I
FIG. 1. Puffing activity at the intermoult puff locus 3C in (iI the continuous presence of ecdysone (O--3), (ii) the continuous absence of ecdysone (O----O), and (iii) 2 hr with ecdysone, then no ecdysone (D-0). Ordinate, p&size; abscissa, hours.
ASHLIURNER
AND RICHARDS
Sequential
Gene
Activation
243
ZZZ
2.62.42.*to1.e Ia,At*-
FIG. 2A. The effect of removal of ecdysone on puffing activity at 23E. The solid circles are the sizes of the puffafier 1,2,3,4, and 5 hr of continuous ecdysone (abscissa) and the open circles are the sizes after removal of the hormone at these times. Ordinate, puff size. FIG. 2B. The effect of removal of ecdysone on puffing activity at 74EF. The solid squares are the sizes of the puff after 1, 2, 3, 4, and 5 h of continuous ecdysone (abscissa) and the open squares are the sizes aRer removal of the hormone at these times. Ordinate, puff size.
REGRESSION
TABLE 1 OF THE EARLY PTJFIW 74EF AND 75B IN GLANDS TRANSFERRED
TO HORMONE-FREE
MEDIUM
E;p;zreyt
INDUCED BY ECDYKINE FOR A FURTHER 2, 3, OR
Size of puff Ecdysone C?
1. 74EF 75B 2. 74EF
75B
FOR 3 HR AND THEN 4 HR”
No ecdysone (wash)
3 hr
C?
5 hr
6 hr
No
2.357 + 0.093
No Yes
1.147 2 0.037 1.212 + 0.044
1.164 k 0.046 1.287 f 0.029
7hr 1.105 5 0.026 1.318 2 0.044
No
2.612 2 0.10
No Yes
1.166 2 0.042 1.130 k 0.037
1.124 k 0.027 1.088 2 0.087
1.135 k 0.026 1.033 Yk0.019
No
2.396 + 0.055
No Yes
-
1.132 k 0.023 1.171 f 0.031
-
Yes
2.214 + 0.127
No Yes
-
1.143 f 0.041 1.218 f 0.036
-
No
2.678 f 0.145
No Yes
-
1.110 k 0.052 1.182 + 0.036
Yes
2.598 f 0.152
No Yes
1.192 2 0.053 1.220 f 0.041
-
a In experiment 1, cycloheximide was present, if at all, only during the second (wash) period; in experiment 2, cycloheximide was present (i) during induction only, (ii) during wash only, (iii) during neither period, or (iv) during both periods. C? = cycloheximide present?
hr in the continuous presence of ecdysone, is inhibited by cycloheximide or other inhibitors of protein synthesis (Ashburner, 1974). However, the regression of these puffs, resulting from hormone withdrawal,
is not inhibited by cycloheximide (Table 1). Indeed, puffs 74EF and 75B may be induced by ecdysone in the presence of cycloheximide and will then regress on transfer of the glands to medium contain-
244
DEVELOPMENTAL
BIOLOGY
ing the antibiotic alone (Tables 1 and 3 and Fig. 5). (iii) Having regressed as a consequence of ecdysone washout, the early puffs 23E, 74EF, and 75B may be reinduced if the glands are reexposed to ecdysone. The size to which these three puffs may be reinduced depends upon the time the glands were first exposed to hormone. For example, in glands cultured with hormone for 0.5 hr, cultured in hormone-free medium for 2 hr, and then reexposed to hormone for a further 2 hr, 74EF reached a size of approximately 2.24. When, however, the period of first exposure to ecdysone was 5 hr, again followed by a 2-hr wash and a 2-hr reinduction, 74EF only reached a size of approximately 1.43 during the second induction period. In both experiments, parallel control glands were cultured without hormone for a period equivalent to the first induction and wash periods and were then induced. For the two experiments quoted above, the control 74EF sizes on induction at 0.5 + 2 hr and 5 + 2 hr, respectively, were 2.36 and 2.38. Similar results were found for 23E and 75B. Thus, these early puffs become progressively less sensitive to ecdysone as the time of their exposure to hormone increases. This is especially so after 2 hr of exposure to ecdysone. The control experiments show this not to be an artifact of the culture conditions. Figure 3 illustrates the reinduction of 23E, 74EF, and 75B after 1 or 5 hr of initial induction with ecdysone and the control inductions for glands incubated without hormone for 1 + 2 or 5 + 2 hr, respectively, before incubation with hormone for the first time. Results for 23E and 75B from experiments over a range from 0 to 7 hr of initial induction are summarised in Figs. 6a and 6b. It is clear that after 3 hr of exposure to hormone, these puffs rapidly become refractory to reinduction after 2 hr of hormone-free culture. Increasing the period of ecdysone-free
54, 1976
VOLUME 1.*-
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.
1
‘.O___,--,-FIG. 3. The reinduction of the early puffs 23E, 74EF, and 75B after their regression resulting from ecdysone’s withdrawal at either 1 or 5 hr. Glands were cultured for either 1 or 5 hr with hormone (Un ) and then transferred to hormone-free medium for a further 2 hr (W----W) before culture for a further 2 hr with hormone (W-B); control glands were cultured for either 3 or 7 hr in the absence of hormone (O----O) before being induced for the first time (OCl). (A) 23E; (B) 74EF, (C) 75B. Ordinate, puff&e; abscissa, hours.
ASHBURNER
Sequential
AND RICHARDS
TABLE OF EARLY PUFFS AVER 2 HR OF INITIAL 2-HR OR A 4-HR WASH PERIOD
Period of wash (hr)
245
III
2 EXP~EURE TO ECDYSONE FOLLOWED AND A 2-HR REINDUCTION
BY EITHER
A
Size of puff after reinduction 23E
2 4
1.834 1.645
2 f.
74EF 2.331 2 0.056 2.240 + 0.048
0.050 0.051 TABLE
REINDUCTION
Activation
(u) Although the early puffs become increasingly insensitive to reinduction following a wash, with increasing time of initial exposure to ecdysone, this is not so for initial induction periods of 2 hr or less (2.iii, above, and Fig. 6). This enabled the demonstration of the fact that puff size on reinduction is not necessarily a function of the size to which the puffs were first induced. This was shown by inducing 74EF
culture from 2 to 4 hr does not alter the sizes of the early puffs on reinduction by ecdysone (Table 2). (iv) The reinduction, after an ecdysone washout caused regression, of puffs 74EF and 75B is not sensitive to cycloheximide (Table 3 and Fig. 5). The puff at 23E is, just as it is sensitive to inhibitors of protein synthesis for its initial induction (Table 3b).
REINDUCTION
Gene
75B 2.271 k 0.073 2.148 + 0.059
3a
OF EARLY PUFFS 74EF AND 75B FOLLOWING AN INITIAL INDUCTION FOR 3 HR, CULTURE ABSENCE OF ECDY~~NE FOR 2 HR, AND THEN REEXPOSURE TO ECDYSONE FOR 2 HR~
IN THE
Size of puff Ecdysone 74EF
75B
C? No
No ecdysone
3hr 2.329 f 0.083
No
2.664 2 0.132
a C? = cycloheximide
C? No
5 hr 1.278 k 0.047
Yes
1.211
No
1.215 + 0.041
Yes
1.211 ?z 0.036
EXPERIMENT
Cycloheximide Initial ind$owntf ecd’ysone No
TO THAT
ILLUSTRATED
+ 0.041
7 hr 2.151 2.181 2.178 2.070
2 k + 2
0.062 0.097 0.068 0.051
No Yes No Yes
1.921 2.181 2.456 2.438
2 0.091 k 0.097 rc_0.105 f 0.065
3b
IN TABLE 3a BUT WITH FOR 1 HR ONLY
AN INITIAL
EX~~~IJRE
TO ECDYSONE
Size of puff at 5 hr
present?
Wash (2 Reinduchr), no ec- tiqn (2 hr), with ecdydysone sone No No Yes Yes
C? No Yes No Yes
present? TABLE
A SIMILAR
Ecdysone
No Yes
23E
74EF
75B
1.684 “_ 0.070 1.267 k 0.070
2.424 k 0.087 2.108 -t 0.086
2.522 k 0.070 2.423 f 0.100
1.080
1.971 f 0.048 2.106 k 0.075
2.356 f 0.054 2.458 -c 0.053
2 0.017
1.133 ‘- 0.030
a Data for three early puffs, 23E, 74EF, and 75B, are given.
246
BIOLOGY
DEVELOPMENTAL
and 75B to various sizes by using suboptima1 ecdysone concentrations for 2 hr. All series of glands were, after a standard 2 hr in hormone-free medium, reinduced but now with a single, high hormone concentration. The result of this experiment, Fig. 4, shows that the size on reinduction was the same for all glands, regardless of the fact that puff size on initial induction varied over a considerable range. (vi) In section 2.iii above, experiments were described which point to the conclusion that the early puffs 74EF and 75B become progressively refractory to ecdysone. These data are quite consistent with those that show these puffs to regress after 4 hr under conditions where the ecdysone concentration remains high (Ashburner, 1973). The following experiment indicates that the development of insensitivity to ecdysone which accompanies the activity of 74EF and 75B requires protein synthesis. The experiment reported in Section 2.iii (above) was repeated with the change that now cycloheximide was present in all culture media; that is, in the medium used for initial induction, that used to wash the glands free of hormone and to culture
2.* 2.1 1 1
I
I.0 0
1
2
3
1
s
6
I
I
FIG. 4. Induction of 74EF by 8 x lo-” M ecdysone (A-A), 6 x lo-‘M ecdysone (M-W), or 2.4 X 10-O M ecdysone (O-O) for 2 hr followed by 2 hr of hormone-free culture (--1 and then a second induction with 2.4 x 10m6 M ecdysone. Ordinate, puff size; abscissa, hours.
VOLUME
54, 1976
FIG. 5. The reinduction of the early puffs 74EF and 75B in the presence of cycloheximide. The experimental protocol was identical to that illustrated in Fig. 3 with the difference that cycloheximide was present in all culture media. (A) 74EF; (B) 75B. Ordinate, puff size; abscissa, hours.
them in hormone-free medium for 2 hr thereafter, and in the ecdysone reinduction medium. The results of the l- and 5-hr experiments are shown in detail in Fig. 5, and these and other experiments are summarised in Fig. 6. Now we see that there is no difference between the experimental, i.e., preinduced, series of glands and the control uninduced series. This is true even after 5 hr of initial induction. The absolute puff sizes are smaller in these experiments than in the equivalent experiments described in section 2.iii. This may reflect a general decline in the gland’s health after prolonged exposure to cycloheximide. 23E could not, of course, be followed in these experiments since its induction is cycloheximide sensitive.
ASHBURNER
AND RICHARDS
Sequential
Gene Activation
III
247
dium these puffs are reinduced on reexposure to hormone and reach their normal sizes. Data for 78D are shown in Fig. 7a. 4. The Late Puffs 22C, 63E, and 82F
FIG. 6A and 6B. A summary of the reinduction data for (A) 23E and (B) 75B. In the lower part of the figures the size (as a percentage of the corresponding control puff size) of the puffs on reinduction after an initial exposure to hormone for the times indicated (followed by a 2-hr hormone-free culture) is shown. The upper part of the figure gives the actual sizes of the puffs in the control experiments i.e., in glands cultured without ecdysone fort + 2 hr before a 2-hr ecdysone induction. In Fig. 6B the solid symbols are the experiments under normal culture conditions and the open symbols are from experiments in which protein synthesis was inhibited, by cycloheximide, throughout culture (see text). Left ordinate, percentage control activity; right ordinate, puff size; abscissa, hours.
3. The Late Puffs 62E and 780 These two late puffs are, in the continuous presence of ecdysone, maximal at 5 hr (62E) and at 6 hr (78D). If puffing has commenced at these sites by the time ecdvsone is withdrawn from the glands (e.g., at 4 hr), then they regress. After culture of the glands for 2 hr in hormone-free me-
During a continuous ecdysone experiment, with an optimal hormone concentration, these put& reach their maximal sizes as follows: 22C at 8-10 hr, 63E at 8-10 hr, and 82F at 10 hr (very rarely at 8 hr). They then regress. Their behaviour in glands from which ecdysone has been withdrawn is rather surprising. (9 All three puffs immediately appear in glands exposed to ecdysone for 4 hr and then washed free of hormone. That is to say that these late puffs are prematurely induced by ecdysone’s withdrawal (Fig. 7). (ii) The size reached by the prematurely induced puffs is a function of the extent of puffing, during the initial exposure to ecdysone, of the early puff sites. This can be shown by two experiments: (a) Glands were exposed to ecdysone for varying periods of time (from 0.5 to 4 hr) before being transferred to hormonefree medium. The extent of puffing, during the period of culture in hormone-free medium, of these three late puffs is a function of the time of ecdysone exposure. Even after 1 hr of ecdysone they appear, albeit rather small and after a lag of some hours. After longer periods in ecdysone (e.g., 4 hr) the prematurely induced puffs approach their normal maximum sizes and with no lag (Fig. 7). (b) Glands were exposed to different ecdysone concentrations for a standard period of time (2 hr) before being transferred to hormone-free medium. The extent of premature induction of 22C, 63E, and 82F was a function of the ecdysone concentration, and hence size of the early puffs, during the period of exposure to hormone. Data from both of these, and other, experiments are plotted for the three late puffs 22C, 63E, and 82F in Fig. 8. The correlation between the size of 75B (or with 74EF, not shown) during the induc-
1.e ,.I/
Ii?-
I.6 B ,”A.‘. ‘. / I.4 -4 // 1.2 &./ / 1.- t__---
/&--a -
(,. .4-----i
T ,.6/
I.,,.*.-(
_----
+-/ ____
4
4-H--
w2% t 2.h /
,a-
t \
3.P 1.b
\
,[
1.2co-
+-+--t
‘3-
/
--__
FIG. 7A-C. The behaviour of 78D, cultured in hormone for 1 (upper panel), of hormone for up to 6 hr (O----O). To culture (H-W). The lower panel also of ecdysone. (A) 78D; (B) 63E, and (C)
\ \ I -__-
-;
63E, and 82F on ecdysone withdrawal and re-addition. Glands were 2 (middle panel), or 4 hr (lower panel) (O-O) and then washed free half of the glands, hormone was re-added after 2 hr of hormone-free shows (0-O) the activities of these puffs in the continuous presence 82F. Ordinate, puff size; abscissa, hours. 248
ASHBURNER
Sequential
AND RICHARDS
++
1.5- * tom-
:
+
t +++
+++
9 22c h=
M-
++ +$+ t
-. 10-
*
+
++
C 20-
t tt
03E
4: +&'
,I$
++++
+
FIG. BA-C. The size of prematurely induced late puffs (ordinate) as a function of the size of an early puff (75B) (abscissa) at the time of ecdysone’s removal in washout experiments. The arrows indicate the sizes of these puff sites when inactive. (A) 82F; (B) 22C, and (C) 63E.
TABLE SIZE OF PREMATURELY HR PERIOD FOLLOWING
Ecdysone concentration in second period (Ml 7.5 7.5 1.8 3.8 7.5
0 x 1.0-g x 1.0-a x I-O-’ x l.O-’ x 10-1
4
INDUCED LATE PUFFS AS A FUNCTION OF THE CONCENTRATION AN INITIAL EXPOSURE FOR 3 HR OF GLANDS TO A HIGH (6 x OF ECDYSONE
74EF 1.163 1.065 1.181 1.202 1.591 1.871
2 0.053 k 0.034 k 0.059 k 0.055 rf: 0.086 k 0.086
249
III
finally regress. These points are illustrated by the data of Fig. 7. The inhibition, by ecdysone, of premature induction of the late puffs is also seen in the data given in Table 4. For this experiment., glands were cultured (for 3 hr) in a high concentration of ecdysone before being transferred (for 2.5 hr) to media containing ecdysone over a wide range of concentrations. Full premature induction only occurs in the complete absence of ecdysone. Even as little as 7.5 x 10eg M ecdysone in the second period causes some inhibition. (iv) In the continuous presence of ecdysone, cycloheximide inhibits late puff induction (Ashburner, 1974). This is a specific inhibition, since premature induction of the late puffs still occurs, albeit to a reduced extent, if glands are transferred from ecdysone medium to ecdysone-free cycloheximide medium. No premature induction occurs if cycloheximide is present only during the ecdysone treatment period or throughout the experiment. Data for three independent. experiments are shown in Table 5. The behaviour of the puffs 22C and 82F in these experiments will be discussed first, that of 63E is rather a special case. In all three experiments the absence of cycloheximide in both the ecdysone and wash periods results in premature induction of 22C and 82F. Addition of cycloheximide to the wash alone does not inhibit this premature induction, although the sizes of the prematurely induced puf’fs are smaller than in the controls lacking inhibitor throughout the experiments. Ad-
tion phase and the size attained by the prematurely induced late puffs on ecdysone washout is clear. (iii) Ecdysone inhibits premature induction. This is shown by the fact that if glands are reexposed to ecdysone, after the period of hormone-free culture, then the prematurely induced late puffs regress (if active) or are inhibited from appearing (if the ecdysone was re-added before the end of their lag period). As the second exposure to ecdysone continues, the late puffs are induced -for the second time-and then
20-
Gene Activation
75B 1.108 1.063 1.127 1.178 1.552 1.988
” k k 2 ” k
63E 0.044 0.041 0.074 0.056 0.063 0.079
2.199 1.710 1.520 1.498 1.429 1.422
k 0.059 2 0.064 -r- 0.077 k 0.057 k 0.052 k 0.089
OF ECDYSONE
IN A 2.5
lo-’ M) CONCENTRATION 82F 2.734 2.177 2.053 1.633 1.534 1.414
-t k k k k 2
0.083 0.081 0.106 0.106 0.139 0.096
250
DEVELOPMENTAL
BIOLOGY
VOLUME
TABLE THE EFFECTS
5
OF CYCLOHEXIMIDE ON THE PREMATURE INDUCTION OF THE LATE PUFFS 22C, DURING A WASH PERIOD FOLLOWING Exrosuas TO ECDYSONE~
Ecdysone incubation CC?)
Wash
No
No
Expt
Yes
No
63E, AND 82F
Size of puff 63E
2.103k 0.079 1.8362 0.043 1.628k 0.035
22c 1.702A 0.083 1.5052 0.078 1.352f 0.036
82F 2.442f. 0.112
2 3
2.083f 0.052 1.954 2 0.085 1.621e 0.043
1.478? 0.051 1.475+- 0.049 1.2472 0.038
1.639 f 0.051 1.439 k 0.077 1.536f 0.051
1
1.620k 0.098
2 3
1.291 f 0.065 1.548+ 0.052
1.162f 0.047 1.128A 0.034 1.061k 0.022
1.269 ? 0.046 1.210k 0.052 1.213” 0.021
CC?) 1
2 3
Yes
54, 1976
1
1.798 + 0.112 1.767zt 0.039
k 0.058 1.159 f 0.028 1.281 + 0.038 1.219 f 0.084 1.1132 0.047 1.206k 0.102 1.560f 0.034 1.086k 0.021 1.254zt 0.026 a Three experiments are shown, the period of ecdysone induction being 3 hr in the first and 3.5 hr in the second and third; the period of wash was 3 hr in experiments 1 and 2 and 4 hr in experiment 3. C? = cycloheximide present?
Yes
1
1.552
2 3
dition of inhibitor during the ecdysone period, whether or not it is present during the wash period, completely inhibits the appearance of prematurely induced puffs at either 22C or 82F. The behaviour of puff 63E is anomolous and not entirely consistent between these experiments. Interpretation of the data for this puff must take note of the fact (Ashburner, 1974) that cycloheximide alone (i.e., in the absence of ecdysone) results in the appearance of a pufY at this site. The puff induced by this drug (or other inhibitors of protein synthesis) is always smaller than that induced by ecdysone (see Table 5 of Ashburner, 1974). In the current experiments the induction of 63E is seen whether or not cycloheximide was present during the ecdysone induction period (though less markedly in experiment 2), but was much larger when cycloheximide was absent during this ecdysone period (whether or not present during the wash) in two of the three experiments (i.e., 1 and 2 ofTable 5). We noted before (Ashburner, 1974) that the induction of 63E by cycloheximide alone was in contrast to the fact that cycloheximide inhibited the induction of this
puff in the continuous presence of ecdysone. This experimental result may be reworded in the form ‘ecdysone inhibits the induction of 63E by cycloheximide.’ Seen in this way, it is then obvious that we should ask whether or not this inhibition by ecdysone is physiological-as assayed, for example, by the shape of the doseresponse curve for inhibition. To study this, salivary glands were incubated in media containing cycloheximide and various concentrations of ecdysone for 3 hr at 25°C. The sizes of three early puffs (23E, 74EF, and 75B) and of 63E were then measured. Confirming earlier results, cycloheximide completely inhibits the induction of 23E. Both 74EF and 75B respond according to the ecdysone concentration, the dose response curve for them being almost identical to that previously published (Ashburner, 1973) despite the presence of cycloheximide in the current experiments and the difference in culture temperature. In the absence of ecdysone, a large puff at 63E was induced by cycloheximide. As the ecdysone concentration increased, the size of this puff gradually decreased so that by 7.5 x lo-’ M ecdysone
ASHBURNER
AND RICHARDS
Sequential
the puff was almost completely inhibited (Fig. 9a). When the percentage of maximum induction of 74EF (or 75B) is plotted together with the percentage of inhibition of 63E (Fig. 9b) it can be seen that the ecdysone dose-response curve for the inhibition of the cycloheximide induction of 63E is identical to those for the ecdysone induction of 74EF and 75B. DISCUSSION
The results presented in this paper, and in three previous papers (Ashburner, 1972, 1973, and 1974), demonstrate the apparent complexity of the control of the late larval puffing cycle in D. melanogaster. As a step toward a synthesis, the present data will be discussed together with those previously published. The reader is referred to the earlier papers and to the Results section of this paper for the full details of the experiments.
-‘6
’
’ “““’
-6
’
“““”
Gene
Activation
(a) The Inter-mod
ZZZ
251
Puffs
Three intermoult puffs have been considered in detail: 3C, 25AC, and 68C. All are active during the long PSl and regress as the ecdysone-induced cycle gets under way. The detailed behaviour, and hence presumably the control, of each puff differs. 3C. Unlike 25AC and 68C, this puff is active at another stage of development in addition to PSl. It is active from PS15 to PS20 in ‘prepupae.’ Indeed, in 8-hr (PS18) prepupae it is larger than at any time during the analysable period of larval development (Becker, 1959; Ashburner, 1969). In culture, this puff regresses regardless of the ecdysone status of the medium. However, when ecdysone is absent it reappears at 5-6 hr. It also reappears on ecdysone washout. In fact, this was observed
-1
’
’ “““’
-6
’
“““”
-5
FIG. 9A. The sizes of puffs (ordinate) at 23E (O----O), 63E (O----O), 74EF (O--O), and 75B (W--a) as E function of the log,, ecdysone concentration (abscissa) in the continuous presence of cycloheximide. FIG. 9B. Normalized dose-response curves for the induction of 74EF (0) and 75B (m) by ecdysone in the presence of cycloheximide and for the inhibition, by ecdysone, of the induction of 63E by cycloheximide (01. Ordinate, size of puffs as a percentage of the corresponding control puff size; abscissa, log,, ecdysone concentration.
252
DEVELOPMENTAL
BIOLOGY
by Becker (1959): when glands of up to PS5/6 (equivalent to 5 hr in culture under the conditions of our experiments) were explanted from larvae into a saline solution for an hour or more, a puff at 3C appeared. Becker interpreted this observation to mean that up to a critical point in the puffing cycle (i.e., PS5-6) the puffing pattern actually reversed on removal of the glands from the animal and their culture in saline. Recently, Korge (1975) has presented evidence which strongly implicates the 3C puff in coding for a polypeptide component of the salivary gland’s glue secretion. This secretion begins to accumulate as electrondense granules in the cytoplasm a few hours after the moult from the second instar, and glue synthesis probably ceases on ecdysone release late in the third instar. The behaviour, both in uiuo and in vitro, of 3C suggests that it may be activated, in either larvae or prepupae (Richards, 1975), by a fall in ecdysone concentration. 25AC. This intermoult puff regresses in culture and does so faster at high ecdysone concentrations than at low, or zero, concentrations. This regression is unaffected by inhibiting protein synthesis. No reinduction occurs on ecdysone washout. 68C. 68C also regresses either in the presence or absence of ecdysone but does so much faster at high ecdysone concentrations. The ecdysone-facilitated regression of 68C is slowed by cycloheximide but its ecdysone independent regression is not. Like 25AC, 68C is not reinduced on hormone withdrawal. Each intermoult puff is unique in its response to the experimenter’s conditions. For 25AC and 68C, at least, the absence of ecdysone would appear to be a necessary, but not sufficient, condition for their activity during PSl. In D. virilis, puff 55E behaves in a similar manner, during development, as the intermoult puffs 25AC and 68C of D. melanoguster. Kress (1973) has reported that 55E can be caused to regress
VOLUME
54,
1976
prematurely, in uiuo, by injecting animals with ecdysone. Interestingly, accelerated regression of 55E also results from the injection of glucosamine into larvae. Attempts to speed up the slow regression of 25AC and 68C, seen when PSl glands ofD. melanogaster are cultured without ecdysone, by adding aminosugars to the medium have so far been unsuccessful. However, the regression of 55E induced by injecting glucosamine into larvae takes a much longer time than does the regression of either 25AC or 68C in glands cultured in the absence of ecdysone. While confirming Becker’s observation on the behaviour of 3C on ecdysone withdrawal, we question the reality of what Becker calls ‘Ruckwartslaufen’ of the puffing pattern. We have shown that 3C behaves ‘oddly’ even when PSl glands are cultured without hormone; furthermore, other puffs quite characteristic of PSl (e.g., 25AC and 68C) are never reinduced. The pattern of puffing in washed glands, either from cultured material or in material taken directly from larvae (i.e., a repeat of Becker’s original experiment; M. Ashburner, unpublished), only superflcially resembles a PSl pattern. The importance of this observation is that in Chironomus species, Kroeger (1964) described a particular type of puffing response ‘rejuvenation’ which, he proposes, is analogous to the phenomenon discussed above in Drosophila. Rejuvenation of the puffing pattern in Chironomus is thought to be a response of the chromosomes to various factors, all of which lead to an influx of sodium ions into the cells of the salivary gland. As such, the phenomenon forms one of the pieces of evidence for the ‘ion’ model of control of gene activity (e.g., Kroeger, 1968). Our point is that there is no evidence for any similar phenomenon in D. melanogaster. (b) The Early
Puffs
The three early puffs studied in detail (23E, 74EF, and 75B) respond within min-
ASIXEWRNER
AND RICHARDS
utes to ecdysone (Ashburner, 1972). Their dose-response curves are broad: a 600-fold range of ecdysone concentration spans their threshold to maximal response. 23E. This is the only early puff known to be sensitive to inhibitors of protein synthesis. It is different from other early puffs in three other respects: (i) It is often induced, to a rather small size, when PSl glands are cultured without ecdysone, (ii) it shows three peaks of activity in the continuous presence of an optimal ecdysone concentration, and (iii) it has a higher sensitivity to ecdysone. Once induced, withdrawal of ecdysone results in its regression. It may then be induced a second time on readdition of hormone to the culture medium. Reinduction, but not its regression on ecdysone withdrawal, is, like its initial induction, inhibited by cycloheximide. 74EF and 75B. These two puffs may be considered together, as their behaviour is nearly always the same. Their induction by ecdysone is very rapid and, at optimal hormone concentrations, they regress, having attained their maximum sizes at 4 hr. Regression, in the presence of ecdysone, is cycloheximide inhibited. Their activity depends upon the presence of exogenous hormone. Removal of hormone causes them to regress immediately, they can be reinduced if the glands are reexposed to hormone. Neither their regression on ecdysone washout nor their reinduction is cycloheximide sensitive. Evidence has been obtained that the normal regression of these puffs, that is, their regression after 4 hr in the presence of hormone, is itself a response to ecdysone. Having so regressed, they are refractory to ecdysone. This insensitivity will not develop if protein synthesis is inhibited during the inducing phase. The insensitivity to ecdysone after ecdysone exposure is not a ‘ticketing’ phenomenon. By this we mean that a class of model to explain the behaviour of these puff sites is that each puff site possesses a finite
Sequential
Gene
Activation
III
253
number of ‘tickets’ allowing transcription. Once these have been exhausted, the puff must regress and become refractory. This appears to be ruled out by the fact that the puff size on second induction is independent of puff size during a 2-hr first induction period. Instead, the most economic model to explain the behaviour of these puffs is to suppose that they are in fact turned off by their own products or those of another early puff such as 23E (Cherbas and Kafatos, 1976). In the detailed model published (Ashburner et al., 1974), we consider that these early puffs respond initially to the concentration of hormone complexed to its receptor protein(s) (ER). Removal of the ecdysone dissociates the ER complex and the puffs then regress. When active, however, the puffs synthesize a class of products (P) which are either proteins or depend upon protein synthesis for their activity. These products compete, at the puff site, with the bound ER complex. With time, in the presence of ecdysone, the concentration of P rises to an extent capable of displacing all of the ER complex from the puff site and the puff, therefore, regresses. Thus, we can interpret the curve showing the development of insensitivity of these puffs to ecdysone (Fig. 6) as a function of the rise in P concentration. (c) The Late Puffs All five late puffs studied (62E, 78D, 22C, 63E, and 82F) appear after a lag of several hours in the presence of hormone. All have very steep, almost all-or-none, dose-response curves and all are inhibited by cycloheximide. Yet, they fall into two groups. 62E and 780. These puffs behave in ecdysone withdrawal experiments rather like 74EF and 75B. If active on ecdysone withdrawal, they regress; they are then reinducible. That is to say, they are late puffs for which the contemporary presence of ecdysone is necessary for activity. We may call them the ‘early lutes.’
254
DEVELOPMENTAL
BIO~CY
22C, 63E, and 82F. The three ‘late lates’ may be considered together, although the anomalous behaviour of 63E in response to inhibitors of protein synthesis in the absence of ecdysone (Schoon and Rensing, 1973; Ashburner, 1974 and this paper) should be recalled. All three show the phenomenon of premature induction. Their premature induction is ecdysone inhibited. Yet, ecdysone is not inhibitory to their activity per se. This is shown by their normal activity in a continuous ecdysone experiment and their second phase of activity on reinduction by ecdysone. The extent of their premature induction is correlated with the extent of induction of the early puffs 74EF and 75B during the initial ecdysone treatment. We stress that this is but a correlation of the observed phenomena. Since we are concerned with finding the simplest model that will account for the experimental data and serve as a useful basis for further experiments we have considered the possibility that these late puffs are, in fact, responding to a product of the early puffs (Ashburner et al., 1974). The lag in their normal appearance is accounted for by proposing that the ER complex, in addition to inducing the early puffs, acts negatively on the late latepuffs. With the activity of the early puffs, the concentration of their products, P rises in the cell and competes for the ER complex at the late late-puff sites. Eventually, it rises sufficiently to displace the ER complex from these sites and to turn on the puffs. Cycloheximide does not allow P to be made. Therefore, the late puffs are not induced [the anomalous behaviour of 63E in the absence of ecdysone and presence of cycloheximide cannot be adequately accounted for as yet, although the fact that ecdysone inhibits this induction with dose-response characteristics identical to those of early puff induction (results) is clearly quite consistent with our model]. However, if the ecdysone is washed out
VOLUME
54,
1976
after, say, 2 hr, a certain amount of P has already been made as a consequence of early puff activity. The ecdysone concentration having fallen, ER dissociates and the available P can immediately, and prematurely, induce the late late-puffs. If ecdysone is re-added, the ER now displaces this P from the late late-sites and they regress. But with the renewed activity of the early puffs, the P concentration begins to rise further, until it is suffkient to displace the ER at the late late-puff sites and activate them once again. As expected on this model, premature induction of the late late-puffs is not inhibited (at least not completely) by transferring glands from ecdysone into cycloheximide medium. The necessary P was made during the ecdysone period of culture. Whatever the precise details of this interpretation, it is very clear that the temporal sequence of late late-puffs is independent of ecdysone’s presence and that, therefore, the total sequence does become independent of hormone once initiated. The fuller implications of this model have been discussed in the paper of Ashburner et al. (1974), to which the reader is referred. The experiments described in the present paper, and in those previously published, are not to be used to ‘support’ this particular model. The model is only of prospective, not retrospective, value. Many details of the experimental results, for example, the intermoult puffs and the behaviour of the early puff 23E and of the two early late-puffs, are not considered in the model at all. In a formal way, the model, or simple variants upon it (and there are many), can account for the sequential cycle of gene activation and regression seen in polytene chromosomes of D. melanogaster at the end of larval life. How close an approximation the model is to the real situation can only be judged by a further analysis of its elements, by genetic and biochemical techniques. This analysis is in progress.
A~HBURNER
AND RICHARDS
This work has been supported by grants from the Science Research Council (B/SR/9750 and B/RG/ 7429/7) to M.A. We thank Peter Cherbas for many stimulating discussions and Trevor Littlewood for looking after our flies. REFERENCES ASHBURNER, M. (1969). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. II. The X-chromosome puffing patterns of D. melanogaster and D. simulans. Chromosoma 27, 4763. ASHBUFINER, M. (1972). Patterns of puffing activity in the salivary gland chromosomes of Drosophila. VI. Induction by ecdysone in salivary glands of D. melanogaster cultured in vitro. Chromosoma 38, 255-281. ASHBURNER, M. (1973). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration. Develop. Biol. 35, 47-61. AIJHBURNER, M. (1974). Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibition of protein synthesis. Develop. Biol. 39, 141-157. ASHBURNER, M., CHIHARA, C., MELTZER, P., and RICHAIU, G. (1974). On the temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38,655-662. BECKER, H. J. (1959). Die Puffs der Speicheldriisenchromosomen von Drosophila melanogaster. I. Beobachtungen zum Verhalten des Puffmusters im Normalstamm und bei zwei Mutanten, giant und lethal-giant-larvae. Chromosoma 10,654-678. CHERBAS, P. T., and KAFATOS, F. C. (1976). Temporal programs of gene expression during cell dif-
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