Apr 25, 1983 - roxy-ecdysone and were analyzed (A) at 0 h (PSI unincubated control),. (B) after I h (PS2), (C) after 2 h (PS4), (D) after 4 h (PS7), (E) after 6 h.
The EMBO Joumal Vol.2 No.8 pp. 1323-1330, 1983
Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster B. Dworniczak1, R. Seidel and 0. Pongs* Ruhr-Universitat Bochum, Lehrstuhl fur Biochemie, 4630 Bochum, FRG Communicated by M. Ashburner Received on 25 April 1983
Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10-6 M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/ prepupal puffing cycle characterized by puff stages 1- 10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxyecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae. Key words: ecdysone/puffs/chromosomes/immunofluorescence Introduction As a consequence of an increase in the concentration of the moulting hormone ecdysone in the haemolymph, third instar Drosophila larvae initiate pupariation (Becker, 1969). Shortly before and during pupariation, the pattern of chromosome puffing changes from that characteristic of the intermolt to that characteristic of the metamorphosing insect. The salivary glands of D. melanogaster third instar larvae have polytenized interphase chromosomes, and are an excellent experimental system in which to study puffing patterns and, hence, gene activities. Changes in puffing activity of polytene chromosomes are sequential, transient and, in many instances, are controlled by ecdysteroids (Ashburner, 1972a). This allows analysis of the control of a complex set of genes whose activities change as development proceeds. It is possible in vitro to reproduce the hormonal effects on the puffing pattern of larval salivary gland chromosomes by culturing isolated salivary glands in defined media in the presence of 20-hydroxy-ecdysone (Ashburner, 1972b). The changes in puffing activity that occur in vitro parallel the changes that 'Present address: Universite de Geneve, Department de Biologie mol&ulaire, Sciences 11, 30, quai Ernest-Ansermet, CH-1211 Geneve 4, Switzerland. *To whom reprint requests should be sent.
(C IRL Press Limited, Oxford, England.
would have occurred in the intact animal in the 10 h period preceding puparium formation. The temporal and sequential changes in puffing activity have been described as a sequence of puff stages (PS), each defined by a unique set of puffs (Ashburner, 1972a; Ashburner and Berendes, 1978); PS1 to PSII cover the development of third instar larvae from the characteristic of intermolt to that of prepupae. There is a causal relationship between the ecdysteroid concentration in the haemolymph, or in the incubation media, and puffing activity. Recently, we showed that this puffing activity is directly related to the binding of ecdysteroid to respective loci of polytene chromosomes (Gronemeyer and Pongs, 1980; Gronemeyer et al., 1981). Ecdysteroid was photocross-linked in situ to polytene chromosomes of salivary glands of D. melanogaster. Covalently bound hormone was then localized by an indirect immunofluorescence microscopy assay. Based on the relative timing and nature of the response of the puffs to 20-hydroxy-ecdysone, they can be grouped into three classes: (i) intermolt puffs active before hormone action; some of these regress under the influence of 20-hydroxyecdysone, (ii) early puffs, induced within minutes by 20-hydroxy-ecdysone, and (iii) late puffs, induced only after a lag of 3 h or longer (Ashburner, 1972b, 1973). We have shown that ecdysteroid present in vivo can be covalently linked to early as well as to late puffs by photoactivation (Gronemeyer and Pongs, 1980). Now, we have cultured salivary glands in vitro and correlated the sequential puffing activity between PSI and PS 1I with the sequential binding of ecdysteroid to respective loci. The results show that there is a good correlation between ecdysteroid binding to chromosomal loci and their temporal puffing activity. Results Intermolt glands have a puffing pattern described as PSI (Ashburner, 1972a, 1972b). PSI glands were cultured in vitro in the presence of 5 x 10-6 M ecdysterone. In accordance with previous studies, this in vitro culture causes some of the puffs active at PSI to regress. Early puffs, on the other hand, are induced rapidly and reach their maximum size between 1 and 4 h before regressing. Late puffs do not appear before 3 h of incubation (Ashburner, 1972b). Ecdysteroid can only be detected on polytene chromosomes after covalently binding the hormone to chromosomal sites. This was accomplished by irradiating salivary glands of D. melanogaster third instar larvae with light of >320 nm wavelength in a suitable apparatus for 5 min. A systematic correlation between irradiation time and fluorescence intensities showed that 5 min irradiation was optimal. Irradiating glands for 5 min did not visibly damage them neither did it impede the development of the normal larval/prepupal puffing cycle (Dworniczak, 1982). Photocross-linked 20-hydroxyecdysone was localized by indirect immunoflourescence microscopy of squashed salivary glands. Figures 1-5 show correlations of fluorescence images with phase contrast pictures of chromosomes of salivary glands which were irradiated and squashed at various puff stages. Since at least 10 1323
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Fig. 1. Photocross-linked 20-hydroxy-ecdysone in the distal region of chromosome X at various puff stages by indirect immunofluorescence microscopy. Salivary glands of D. melanogaster third instar larvae were explanted at puff stage (PSI) into Grace's insect tissue culture medium containing 5 x 10-6 M 20-hydroxy-ecdysone and incubated in depression slides at 22°C. (A) At PSI (unincubated 0 h control), (B) after I h incubation (PS2), (C) after 2 h (PS4), (D) after 4 h (PS7), (E) after 6 h (PS9- 10), glands were irradiated for 5 min, fixed, squashed, incubated with rabbit antisera directed against 20-hydroxy-ecdysone and with fluorescent goat antibodies directed against rabbit lgG. Glands of PS3 were treated as above. Squashes, however, were incubated with a monoclonal anti-ecdysteroid mouse immunoglobulin and with fluorescent goat anti-mouse IgGs. Chromosome squashes were photographed in the fluorescence microscope (right panels), and finally were stained for phase-contrast microscopy (left
panels,
x
980).
independent incubations were carried out for each puff stage, Figures 1-5 are examples of a total of -1000 pairs of phase contrast/fluorescence pictures. The intermolt puffs The intermolt puffing pattern (PS1) is characterized by a few large puffs visible at the following sites: 3C, 25AC, 42A, 43E, 58DE, 68C, 85F and 90B (Ashburner, 1972a, 1972b, 1974). Upon incubation of salivary glands in vitro in the presence of hormone, puffs at 3C, 25AC and 68C regress rapidly, within the first hour. The other puffs regress at later puff stages (PS7 PS9) except those at 43E and especially at 85F, which are active during the whole larval:prepupal puffing cycle. The regression of puffs is accelerated 2-fold by 20-hydroxy-ecdysone at locus 25AC and 4-fold at locus 68C. The photoaffinity labeling procedure localized ecdysteroid hormone on most of the intermolt puffs, which regress in response to hormone. The exceptions are the puffs 1324 -
-
Fig. 2. Photocross-linked 20-hydroxy-ecdysone in the distal region of chromosome 2L at various puff stages by indirect immunofluorescence microscopy (see Figure 1). PSI salivary glands of D. melanogaster third instar larvae were cultured in vitro in the presence of 5 x 10-6 M 20-hydroxy-ecdysone and were analyzed (A) at 0 h (PSI unincubated control), (B) after I h (PS2), (C) after 2 h (PS4), (D) after 4 h (PS7), (E) after 6 h (PS9- 10). Squashes of glands of PS5 were incubated with a monoclonal anti-ecdysteroid mouse immunogiobulin as described for Figure IF. Phasecontrast patterns (x 980) are on the left and corresponding fluorescence images on the right.
Table 1. Binding of ecdysteroid hormone to intermolt puffs of polytene chromosomes of third instar larvae of D. melanogaster Locus
Puff stagea PSI PS2
3C
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aPuff stages and puffing activity of intermolt puffs (+) are listed according to Ashburner (1972a). Circles indicate at which puff site and puff stage the ecdysteroid hormone was localized by indirect immunofluorescence microscopy as described in Materials and methods. Data are shown for the puffs at 3C in Figure 1, at 25AC in Figure 2, at 58DE and at 85F in Figure 5. The other data were obtained by Dworniczak (1982). at 25AC (Figure 2) and at 43E (data not shown). Apparently, 20-hydroxy-ecdysone indirectly brings about the regression of puffs at these two sites. Table I summarizes the results, which
Ecdysteroid binding and polytene chromosome puffing
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Fig. 3. Photocross-linked 20-hydroxy-ecdysone in the proximal region of chromosome 3L at various puff stages by indirect immunofluorescence microscopy (see Figure 1). PSI salivary glands of D. melanogaster third instar larvae were cultured in vitro in the presence of 5 x 10-6 M 20-hydroxy-ecdysone and were analyzed (A) at 0 h (PSI) unincubated control, (B) after 1 h (PS2), (C) after 2 h (PS4), (D) after 3 h (PS5), (D) after 4 h (PS6) (E) after 5 h (PS7), (F) after 6 h (PS9- 10). Phase-contrast patterns (x 1460) are on the left and corresponding fluorescence images on the right.
were obtained for the other intermolt puffs between PSI and PS10. There is a good correlation between puffing activity and the presence of ecdysteroid at the respective puffing sites. Concomitant with the regression of these intermolt puffs, ecdysteroid hormone was no longer detected at these chromosomal loci after irradiation.
Early puffs Puffs are induced within 15 min in response to 20-hydroxy-ecdysone at 23E, 74EF and 75B and also rapidly at
chromosomal sites: 2B5.6, 47A, 63F, 74C, 83E, 88D, 88EF, 89B, 93D. Puffs at 23E, 74EF and 75B, which have been studied in considerable detail by Ashburner (1972a, 1972b, 1973, 1974), are described here as representatives of this group. Whereas the hormonal induction of early puffs is generally not blocked by cycloheximide, the induction of the puff at 23E is. Moreover, this puff does not exhibit the simple induction/regression behaviour of the other early puffs; instead it displays two to three peaks of activity during the larval/pre1325
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Fig. 4. Photocross-linked 20-hydroxy-ecdysone in the distal region of chromosome 3L at various puff stages by indirect immunofluorescence microscopy (see Figure 1). PSI salivary glands of D. melanogaster third instar larvae were cultured in vitro in the presence of 5 x 10-6 M 20-hydroxy-ecdysone and were analyzed (A) at 0 h (PSI unincubated control), (B) after 1 h (PS2), (C) after 2 h (PS4), (D) after 4 h (PS6), (E) after 6 h (PS9- 10). Phase-contrast patterns (x 980) are on the left and corresponding fluorescence images on the right.
pupal puffing cycle. Figure 2 shows the tip of chromosome 2L, where 23E is located, from glands at stages PSI to PS10. In agreement with our in vivo experiments (Gronemeyer and Pongs, 1980) 23E did not show a significant fluorescence despite its puffed status. Other early puffs exhibited a hormone binding pattern, which was correlated with their puffing activity, for example the puffs at 74EF and 75B (Figure 3). Ecdysteroid hormone was not detectable at sites 74EF and 75B at PSI. After incubating PSI glands for 1 h in the presence of hormone, large puffs were induced at these sites, accompanied by a strong fluorescence indicative of ecdysteroid binding. The puffs are active through PS7 and then regress so that ecdysteroid binding was not detected at these sites at PS9/10, following regression. It is interesting to note that the fluorescence pattern clearly shows an ecdysterone inducible site at 74C, which had been tentatively proposed previously (Ashburner, 1972a). The fluorescence intensity at 74EF is very bright and densely spread throughout the whole puff. On the other hand, the fluorescence intensity at 75B is diffuse and rather dim. This suggests that the photoreaction between ecdysteroid and chromatin constituents and/or the binding of hormone to the puff at 74EF is different from that to the puff at 75B. Late puffs The activity of five puffs of this group has been studied previously (Ashburner, 1972b; Ashburner et al., 1974). They are induced by 20-hydroxy-ecdysone at 22C, 62E, 63E, 78D, and at 82F. The puffs at 62E (maximum activity at PS6 -8) at 63E (maximum activity at PS1O), at 78D (maximum activity at PS7- 10), and at 22C (maximum activity at PS9- 11)
1326
are shown in Figures 2- 4). Fluorescence was not detected at these loci at PSI as is also the case for early puff sites. The later the puffs become active, the later ecdysteroid-dependent fluorescence was detected. However, fluorescence was detectable at these sites before they had reached their maximum puffing activity. The puff at 62E is the first late puff to reach maximum activity and the puff at 22C is the latest of these four to become active. Fluorescence was first observed at 62E at stage PS2, at 63E at PS6, at 78D at stage PS5 and at 22C at stage PS6/7. This
shows that 20-hydroxy-ecdysone bound sequentially to late puff sites and hormone was detected at these chromosomal loci in register with puff induction. Figures 2-4 also show that hormone binding was detected at a late puff site well before maximum puffing activity. This observation also holds for all the other late puffs which we have investigated. Apparently, maximum puffing activity is not a prerequisite for binding hormone, nor is there an obvious correlation between puff size and the quantity of fluorescence intensity at a puffed site. Incubation puffs So far, chromosomal loci have been considered, at which puffs regress or are induced in response to 20-hydroxy-ecdysone. The culture of PSI salivary glands also affects the activity of a number of puffs independently of the presence of 20-hydroxy-ecdysone in the medium (Ashburner, 1972b). The appearance of these 'incubation puffs' is characteristic for each puff stage. Figure 5 shows phase contrast pictures of several incubation puffs (26A, 29CD, 47AD, 50CD, 57D, 62C, 84E, 94C, 95AB, 99B) at various puff stages. The corresponding immunofluorescence images show that, except for the two complex puffs at 50CF and 57CD, none exhibited ecdysteroid-dependent fluorescence. Two other incubation puffs, 5B and 73B, are indicated in Figure 1 and Figure 3, respectively. The puff at 57CD reaches maximum size in culture at PS2/3, regardless of whether or not 20-hydroxy-ecdysone is present. However, in the presence of 20-hydroxy-ecdysone, the puff regresses at the later puff stages, whereas in the absence of hormone it remains active (Ashburner, 1972b). Accordingly, fluorescence at 57CD was detected at PS2 (Figure 5) and at PS4 (data not shown), but not at earlier or later stages. Heat-shock puffs Transfer of salivary glands from 25°C to 37°C for 30 min results in the rapid induction of a few specific heat-shock puffs located at 33B, 63BC, 64F, 67B, 70A, 87A, 87B, 93D and 95D (Ashburner and Bonner, 1979). Figure 6 shows that neither the heat-shock puffs nor any other chromosomal loci contained detectable amounts of ecdysteroid-dependent fluorescence after heat-shock. This is in accordance with our previous findings (Gronenmeyer and Pongs, 1980; Schaltmann and Pongs, 1982), involving heat-shock of the intact animal and assays for cross-linked steroid hormone. Discussion Our method of mapping in vitro ecdysteroid binding sites on polytene chromosomes involves the incubation of glands in the presence of 5 x 10-6 M 20-hydroxy-ecdysone. Squash preparations are then irradiated and incubated first with rabbit anti-ecdysterone antibodies and then with fluoresceinlabeled goat anti-rabbit IgG (Gronemeyer and Pongs, 1980).
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Fig. 5. Correlation of fluorescence images (lower) with phase-contrast patterns (upper) of chromosomal regions where puffs appear independently of 20-hydroxy-ecdysone after incubation of PSI salivary glands in vitro for (A) 2 h (PS4, proximal part of chromosome 2L), (B) I h (PS2, distal part of chromosome 3R), (c) I h (PS2, middle part of chromosome 2R), (D) I h (PS2, distal part of chromosome 2R), (E) 6 h (PS9- 10), proximal part of chromosome 3R).
This indirect approach has several pitfalls. It is known that steroids like 20-hydroxy-ecdysone undergo rearrangements upon irradiation (Bellus et al., 1969). Under our conditions, however, 20-hydroxy-ecdysone only undergoes substantial rearrangements or changes in structure, as judged by t.l.c. (data not shown), after irradiation times of >20 min. This change in structure is concomitant with the decrease of fluorescence intensity, which was observed after irradiating glands for >20 min. After photoactivation, 20-hydroxyecdysone can undergo a dark reaction (Reum et al., 1982) which is very slow, unspecific and requires high concentrations of protein targets. When salivary glands were heatshocked, neither ecdysteroid regulated puffs nor heat-shock puffs (Figure 6) reacted with ecdysteroid upon photoactivation. This shows that an unspecific reaction between chromosomal components and irradiated ecdysteroid hormone does not take place under our experimental conditions (in this respect, see also the incubation puffs of Figure 5). The specificity of the rabbit anti-ecdysterone antibodies, which were used for immunofluorescence microscopy, was measured by determining binding constants from Scatchard plots (Scatchard, 1949). As Table II shows, the binding con-
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Fig. 6. Indirect immunofluorescence microscopy of chromosome 3 at puff stage 2. PS2 salivary glands of D. melanogaster third instar larvae were cultured in vitro in the presence of 5 x 10-6 M 20-hydroxyecdysone for 30 min at 37°C before irradiation and analysis for photocross-linked hormone (see Figure 1) (upper). Phase-contrast pattern of chromosome 3 (x 600) and (lower) fluorescence image of the same chromosome. Heat-shock puffs are indicated by arrows.
1327
B. Dwomiczak, R. Seidel and 0. Pongs
Table II. Binding constants of steroid hormones to rabbit anti-20-hydroxyecdysone antibodies Steroid hormone
K (1010 M-1)
Ecdysone 20-Hydroxy-ecdysone 20-Hydroxy-ecdysoneb Ponasterone A Testosterone Dexamethasone
1.0 3.1 i 1.0 1.5 + 1.0 1.0 3.9 n.m.a n.m.a 2.1
Binding of steroid hormone to rabbit anti-20-hydroxy-ecdysone antibodies was measured after charcoal treatment according to Korenmann (1968). Binding constants were calculated from Scatchard plots (Scatchard, 1949). The standard deviation from the mean ecdysteroid binding constant was calculated from seven independent Scatchard plot data. aBinding of testosterone and dexamethasone was not measurable after charcoal treatment, i.e., binding constants were < 10-5 M. bA 5 x 10-6 M 20-hydroxy-ecdysone solution was irradiated for 5 min in phosphate buffered saline before the antibody binding experiments.
stants for ecdysteroids were all of the order of 10-10 M. Small differences between the binding constants are probably not significant, as they are within experimental error. The antibodies did not bind unrelated steroid hormones such as dexamethasone or testosterone to an extent which could be measured in the charcoal assay system. Previously, we had shown that irradiation was necessary to obtain fluorescent chromosomal sites (Gronemeyer and Pongs, 1980; Gronemeyer et al., 1981) and that protein extracts of salivary glands did not react with our rabbit antibody preparations (Schaltmann and Pongs, 1980). Moreover, squash preparations of polytene chromosomes of ecdl third instar larvae, which contain only low levels of 20-hydroxy-ecdysone (Lepesant et al., 1978), did not reveal significant immunofluorescence upon irradiation (Dworniczak et al., 1982). Together, these data show that the rabbit antibodies specifically recognized irradiated ecdysteroid hormone on the squash preparations of polytene chromosomes and that unspecific binding was
negligible. In addition, we have used monoclonal antibodies, which were raised against haemocyanin-20-hydroxy-ecdysone conjugates (Seidel, 1982). Figures IF and 2F show that very similar immunofluorescence images were obtained with the monoclonal mouse anti-ecdysterone antibodies and the rabbit anti-ecdysterone antiserum. We have also used an antiecdysterone antiserum, raised by Reum et al. (1982), which has a 10-fold higher affinity for 20-hydroxy-ecdysone than for ecdysone. Again, the immunofluorescence images were very similar to the ones shown in Figures 1-5. However, the fluorescence intensities with these antibodies were generally weaker and they were not used for the detailed analysis of the hormone-regulated larval/prepupal puffing cycle. We have shown that irradiation of salivary glands results in the efficient covalent attachment of [3H]20-hydroxy-ecdysone to one protein with the characteristics of a hormone receptor (Schaltmann and Pongs, 1982). Since our antisera, however, did not distinguish between different ecdysteroids, we cannot say with certainty whether 20-hydroxy-ecdysone or a metabolite of it is the immunoreactive antigen responsible for the immunofluorescence images of the polytene chromosome squashes. Although all the available data implies that 20-hydroxy-ecdysone is indeed the ecdysteroid hormone which we 1328
have localized at chromosomal sites, this conclusion is not unequivocal. Therefore, while our data show that ecdysteroid hormone binding and puffing activity are correlated, they do not show whether the ecdysteroid hormone at all the puff sites is of identical structure. Immunofluorescence at a particular chromosomal locus is generally indicative of an ecdysteroid hormone cross-linked to a component of this locus, but does not prove that it is 20-hydroxy-ecdysone. Intermolt puffs are active before the rise in hormone titre in the hemolymph of third instar larvae (Ashburner and Berendes, 1978). Since we detected hormone in intermolt puffs, these represent high affinity ecdysteroid binding sites. Yet, we cannot be absolutely sure that ecdysteroid hormone regulates the puffing activity of intermolt puffs, rather than only their regression. We cannot exclude with certainty the possibility that the PSI glands dissected from the larvae had already been exposed, albeit for a very short time, to a high concentration of hormone. This, however, does not invalidate the observation that ecdysteroid hormone is present at intermolt puffs before the onset of the larval/prepupal puffing cycle. The results of immunofluorescence experiments depend on the accessibility of antigen to antibody and the reactivity of the hormone at its binding site. Thus, failure to detect ecdysteroid at a chromosomal site does not necessarily indicate that ecdysteroid was absent. It is also not possible to use fluorescence intensity as a direct measure of the amount of hormone at a particular site. However, it is important to note that the degree of chromosome decondensation is not correlated to the degree of fluorescence intensity. Compare, for instance, the fluorescence intensities of Figure 3D or those of Figure 4D. Figures 1 -4 show that ecdysteroid binding to puffs is sequential as well as temporal. The affinity of ecdysteroid hormone for intermolt, early or late puffs is apparently not regulated by the 20-hydroxy-ecdysone concentration, but by the time of incubation, i.e., the puff stage. At present, we do not know whether the binding of hormone to a particular chromosomal locus is a prerequisite for puff induction. Since sequential puffing and hormone binding to puffs are so analogous, ecdysteroid-hormone does not seem to have solely a trigger function for the development of a temporal puffing pattern as previously discussed (Ashburner, 1973), but rather the presence of ecdysteroid in the nucleus appears to be necessary for gene activity throughout the whole larval/prepupal puffing cycle. When salivary glands are transferred to an appropriate puff stage (e.g., PS4) to a hormone-free culture medium, late puffs at 22C, 63E, and 82F are prematurely induced (Ashburner et al., 1974; Ashburner and Richards, 1976). These 'washout' experiments implied that during early puff stages the presence of 20-hydroxy-ecdysone in the medium inhibits induction of late puffs. Our immunofluorescence experiments suggest that this inhibitory action of 20-hydroxy-ecdysone at early puff stages is not mediated, at least visibly, by ecdysteroid binding to late puff sites. Similarly, the influence of 20-hydroxy-ecdysone on the puffing activities at 23E and at 25AC could not be correlated with ecdysteroid binding at these sites. A rise and fall in 20-hydroxyecdysone concentration in the incubation medium might create other signals important for puff induction and for regulation of gene activity. The cycloheximide sensitivity of the puff at 23E (Ashburner, 1974) could be indicative of such a situation. Moreover, the early puffs at 74EF/75B are
Ecdysteroid binding and polytene chromosome puffing
responsible for factors which might regulate the timing of early puff regression as well as late puff induction (Walker and Ashburner, 1981). Similar factors might also be involved in the activity and the timing of intermolt puffs (Belyaeva et al. 1981). The observed accumulation of ecdysteroid at late puff sites after induction does not necessarily contradict the results of the 'washout' experiments (Ashburner et al., 1974; Ashburner and Richards, 1976). As pointed out, it is not clear whether we have detected, at late puff sites, 20-hydroxy-ecdysone or another ecdysteroid, which cannot be easily washed out, and the lifetimes of nuclear ecdysteroid-receptor complexes are not yet known. The experiments of Ashburner and co-workers and our immunofluorescence experiments might, in fact, have yielded results on two quite different aspects of ecdysteroid action in salivary glands of Drosophila larvae. Generally, the terms puffing activity and gene activity are used synonymously. Puffs may represent a genetic unit, but there is ample evidence that they contain more than one gene and that genes in a particular puff are not necessarily coordinately transcribed. Puff mutants (e.g., 3°C and 64°C) which show synapsis-dependent non-autonomy have been described (Korge, 1975; Ashburner, 1977). They are probably mutants at the level of the unfolding of the condensed chromatin to an extended configuration on which transcription can occur. Therefore, it is necessary to distinguish puff induction and gene transcription. Thus, we can envisage the situation in which the steroid hormone regulates puff induction, but not the transcription of the genes therein (intermolt puffs?), or that it regulates puffing activity and gene activity (early puffs), or, alternatively, that puffs are induced by factors other than the hormone, yet the transcription of genes is regulated by the hormone (late puffs?). It is clearly necessary to attempt to separate puff induction and hormone binding in future experiments. Materials and methods Antibodies Anti-ecdysteroid antiserum with half-maximal antibody binding capacity of -0.05 was used in the immunofluorescence experiments (Gronemeyer and Pongs, 1980). Binding of steroid hormones to antibody preparations was measured by incubating 250 A1 of 1:50 diluted antibody solution at 4°C overnight with various amounts of tritiated hormone (10-9 -107 M). Dextrancoated charcoal was added to a final concentration of 2.507o (Korenmann, 1968). Incubations were continued for 5 min on ice. Charcoal was removed by centrifugation (10 000 g for 5 min at 4°C). Radioactivity in the supernatant was determined in a liquid scintillation counter (Beckmann LS 7000). In parallel with each incubation, tritiated hormone was added in the presence of 10 -5M unlabelled hormone as competitor for determining unspecific binding. [3H]Ecdysone binding (40 Ci/mmol) (New England Nuclear, Dreiech) was competed with ecdysone (Fluka AG, Neu-Ulm); [3H]20-hydroxy-ecdysone (2.2 Ci/mmol) and [3H]ponasterone A (120 Ci/mmol) binding with 20-hydroxy-ecdysone (Simes, Milano, Italy). [3H]20-hydroxy-ecdysone and [3Hlponasterone A were a generous gift of K.-D. Spindler, Dusseldorf. All hormones were purified by h.p.l.c. prior to use. The B/F ratios (bound versus unbound hormone) varied for ecdysone and 20-hydroxy-ecdysone between 0.5 and 4.0 and for ponasterone A between 0.05 and 0.5. Binding constants were calculated from Scatchard plots (Scatchard, 1949). Each plot was constructed from six measurements. Monoclonal antibodies were prepared by R. Seidel (1982). Briefly, BALB/c mice were immunized with 20-hydroxyecdysone-haemocyanin conjugates. Subsequently, spleen cells were fused with P3x63Ag8.653 myeloma cells (Galfre and Milstein, 1981; Kearney et al., 1979). Hybridoma clones, producing ecdysteroid antibodies, were selected and screened by solid-phase radioimmunoassays (Reth et al., 1978) with succinylated thyroglobulin versus ecdysteroid-thyroglobulin conjugates as antigens. Irradiation of salivary glands Salivary glands of third instar larvae of D. melanogaster Oregon R wild-
type strain from 4- to 5-day-old cultures were dissected in 1/5 diluted Grace's medium. After removing as much fat body as possible without damaging the glands, one lobe of the paired organ was fixed immediately (see below) while the contralateral lobe was cultured at 22°C in 200 Al of 5 x 10-6 M 20-hydroxy-ecdysone containing medium on a depressed slide (Ashburner, 1972b). After incubating for suitable times, the gland was transferred into a small glass capillary containing 75 Il culture medium. The capillary was mounted in a 3 ml cuvette for cooling during the 5 min irradiation. The irradiation apparatus was as described (Gronemeyer and Pongs, 1980). The longpass filter (WG320, Schott, Mainz) had 8007o transmission at 320 nm. Phase contrast and immunofluorescence microscopy Glands were incubated after irradiation on a siliconized coverslip for 1 min in phosphate buffered saline containing 107o Triton X-100. They were fixed and squashed in a 45070 acetic acid solution containing 3.607o formaldehyde. Post-fixation was done in 9507o ethanol. Squash preparations were either directly processed by immediate staining with lacto-acetic acid/orcein for phase contrast microscopy and puff stage analysis (Ashburner, 1967), or, for immunofluorescence microscopy, were incubated at first with 1:20 diluted anti-ecdysterone antiserum and then with fluorescein isothiocyanate-coupled goat anti-rabbit immunoglobulin (Miles-Yeda, Frankfurt, FRG) as described. Specimens were photographed by incident u.v. illumination in a Zeiss standard microscope with an incident u.v. attachment and fluorescence optics (25/0.8 Neofluar, Zeiss). The camera factor was 0.5. Exposure times were 30 s. Phase contrast photographs were taken after the preparations were stained with dye. Heat-shock of salivary glands Salivary glands were cultured as above, but were incubated for 30 min at 37°C. Afterwards, glands were irradiated, squashed and incubated with antibodies as described above.
Acknowledgements The expert help of K. Grabert in preparing the photographs is gratefully acknowledged. This study was supported by grants from the Deutsche Forschungsgemeinschaft and Land Nordrhein-Westfalen to 0. Pongs.
References Ashburner,M. (1967) Chromosoma, 21, 398-428. Ashburner,M. (1972a) in Beerman,W. (ed.), Results and Problems in Cell Differentiation, Vol. 4, Springer, Berlin, pp. 102-151. Ashburner,M. (1972b) Chromosoma, 38, 255-281. Ashburner,M. (1973) Dev. Biol., 35, 47-61. Ashburner,M. (1974) Dev. Biol., 39, 141-157. Ashburner,M. (1977) in DeLaChapelle,A. and Sorsa,M. (eds.), Chromosomes Today, Vol. 6, Elsevier, Amsterdam, pp. 213-222. Ashburner,M., Chihara,C., Meltzer,P. and Richards,G. (1974) Cold Spring Harbor Symp. Quant. Biol., 38, 655-662. Ashburner,M. and Richards,G. (1976) Dev. Biol., 54, 241-255. Ashburner,M. and Berendes,H.D. (1978) in Ashburner,M. and Wright, T.R.F. (eds.), The Genetics and Biology of Drosophila, Vol. 2, Academic Press, London, pp. 315-395. Ashburner,M. and Bonner,J. (1979) Cell, 17, 241-254. Becker,H.J. (1959) Chromosoma, 10, 654-678. Bellus,D., Kearns,D.R. and Schaffner,K. (1969) Helv. Chim. Acta, 52, 971-985. Belyaeva,E.S., Vlassova,I.E., Biyasheva,Z.M., Kakpakov,V.T., Richards,G. and Zhimulev,I.F. (1981) Chromosoma, 84, 207-219. Dworniczak,B. (1982) Ph.D. Thesis, Ruhr-Universitat Bochum. Dworniczak,B., Kobus,S., Eiteljorge,K. and Pongs,O. (1982) in Jaenicke,L. (ed.), Biochemistry of Differentiation and Morphogenesis, Springer, Berlin, pp. 163-176. Galfre,G. and Milstein,C. (1981) Methods Enzymol., 73, 3Gronemeyer,H. and Pongs,O. (1980) Proc. Natl. Acad. Sci. USA, 74, 20792083. Gronemeyer,H., Hameister,H. and Pongs,O. (1981) Chromosoma, 82, 543559. Korenmann,S.G. (1968) J. Clin. Endocrinol. Metab., 28, 127-130. Kearney,J.F., Radbruch,A., Liesegang,B. and Rajewski,K. (1979) J. Immunol., 123, 1548-1550. Korge,G. (1975) Proc. NatI. Acad. Sci. USA, 72, 45504554. Lepesant,J.A., Lepesant,J.R. and Garen,A. (1978) Proc. Natl. Acad. Sci. USA, 75, 5570-5574. Reth,M., Hammerling,G. and Rajewski,K. (1978) Eur. J. Immunol., 8, 393400. Reum,L., Kauser,G., Enderle,U. and Koolman,J. (1982) Z. Naturforsch., 37c, 967-976.
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Scatchard,G. (1949) Ann. N. Y. Acad. Sci., 51, 660-672. Schaltmann,K. and Pongs,O. (1980) Hoppe-Seyler's Z. Physiol. Chem., 361, 207-210. Schaltmann,K. and Pongs,O. (1982) Proc. Natl. Acad. Sci. USA, 79, 6-10. Seidel,R. (1982) Diplomarbeit, Ruhr-Universitat Bochum. Walker,V.K. and Ashburner,M. (1981) Cell, 26, 269-277.
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