Cell, Vol. 61, 101-l

11, April 6, 1990, Copyright

0 1990 by Cell Press

Spatial and Temporal Patterns of E74 liranscxiption during DrwophHa Development Carl S. Thummel: Kenneth C. Burtis,? and David S. Hogness Department of Biochemistry Stanford University School of Medicine Stanford, California 94305-5307

Summary The E74 gene occupies one of the early puff loci (74EF) centml to the Ashburner model for the ecdysone-induced puffing pattern in Drosophila. In sup port of this model, we show that the E74A promoter is directly activated by ecdysone and is subsequently mpmaeed by ecdyaone-induced proteins. Further support derives from the correspondence observed between 74EF puff size and the accumulation of nascent transcrtpts on the E74A unit. These tranacrfpts elongate at 1.1 kblmin so that this 60 kb unit acts as a timer, delaying the appearance of its mRNA by 1 hr. E74A tmnscrtption ia induced in a variety of ecdyaone target tissues in late third instar lawas and during each of the ecdysone pulses that mark the six stages of Droaophib development. These msufts support an exteneion of the Ashburner model in which ecdysone pulaea coordinate tiaeue development. The temporal pattern of E74g tmnscription overlaps but is distinct from that of E74A. introduction Drosophila development from fertilized egg to adult fly is divisible into six stages: embryogenesis, three larval instars, and the prepupal and pupal stages. Each of these stages is marked by a pulse of the steroid hormone 20-OH ecdysone, hereafter referred to as ecdysone (reviewed by Richards, 1981a). The developmental effects of the three larval pulses have been studied in some detail (reviewed by Richards, 1961b). During the first two larval instars, ecdysone acts in conjunction with juvenile hormone to induce the cuticle molts that punctuate the ends of these instars. A more profound change in development results from the late third instar pulse of ecdysone, which triggers the larval-to-adult metamorphosis by inducing coordinated changes in the developmental pathways of both larval and imaginal tissues. We previously proposed that these coordinated changes in tissue development are specified by ecdysone-activated genetic regulatory hierarchies (Burtis et al., 1990). These hierarchies are akin to that proposed by Ashburner et al. (1974) to account for the specific pattern of transcription puffs induced by ecdysone in the polytene chromol Present address: Howard Hughes Medical Institute, Department of Human Genetics, University of Utah Medical Center, Salt Lake City, Utah 64132. *Present address: Department of Genetics, University of California, Davis, California 96616.

somes of late third instar salivary glands. In this program, a small set of ~6 “early” puffs appears within minutes of ecdysone exposure and then regresses after a few hours of puff expansion; a large heterogeneous set of -100 “late” puffs begins to appear after the induction of the early puffs. Whereas the induction of the late puffs and regressien of the early puffs are sensitive to inhibition of protein synthesis, the induction of the early puffs is not (Ashburner, 1974). These observations led Ashburner et al. (1974) to propose that transcription of the genes responsible for the early puffs (early genes) is directly induced by ecdysone complexed with its receptor protein, and that regulatory proteins encoded by the early genes repress early gene transcription and induce late gene transcription. Similarly, in our coordination model, the ecdysone-receptor complex directly induces transcription of a small set of early regulatory genes in each target tissue. Tissue-specific, presumably overlapping combinations of the regulatory proteins encoded by these early genes would then control the expression of a much larger set of effector (late) genes, which direct a change in the morphological and functional properties that define the developmental state of the target tissue. Finally, the duration of early gene transcription would be finely tuned by the repression of that transcription by one or more of the early regulatory proteins. We further proposed that this coordination model may apply not only to late third instar larvae but also to other developmental stages characterized by an ecdysone pulse. Three genes that play a pivotal role in this and the antecedent Ashburner model were cloned and structurally defined in this laboratory to provide crucial genetic elements for testing the models. One of these is the EcR gene encoding the Drosophila ecdysone receptor, which was shown to bind to the early puff loci, as predicted by the models (M. Koelle, W. A. Segraves, W. S. Talbot, M. Bender, and D. S. H., unpublished data; M. Koelle, W. A. Segraves, l? Cherbas, and D. S. H., unpubtished data). The other two are ecdysone-inducible genes responsible for two early puffs: the E74 gene, responsible for the 74EF puff (Burtis et al., 1990) and the E75 gene, responsible for the 758 puff (Segraves and Hogness, 1990). The deduced amino acid sequences for the R4 and E75 proteins indicate that they belong, respectively, to the ets protooncogene and steroid receptor superfamilies and suggest that they function as nuclear regulatory proteins, as predicted by the models. In this paper, we further test the models by examining the characteristics of E74 transcription. This gene exhibits a complex structure, summarized in Figure X. E74 contains three overlapping transcription units that derive from unique promoters but share a common polyadenylation site. The E74A unit spans 60 kb of genomic DNA and yields a 6.0 kb mRNA. The two E74B mRNAs, 4.6 and 5.1 kb in length, initiate from within the fifth of the seven E74A introns, 40 kb downstream of the E74A start site. The nested arrangement of the E74 transcription units yields

Cell 102

two related proteins with unique amino-terminal regions joined to a common carboxy-terminal region that includes the sequence homology to proteins encoded by the ets proto-oncogene superfamily (Burtis et al., 1990). We focused, for the most part, on the E74A unit, demonstrating first that the ecdysone-induced appearance of its mRNA results from the activation of the E74A promoter. This is the expected result if E74A is directly induced by an ecdysone-receptor complex, as postulated in the models. Repression of E74A transcription follows its induction in late third instar salivary glands exposed to ecdysone, mimicking the 74EF puff response. Moreover, that repression is dependent upon protein synthesis in a manner indicating that E74A transcription is negatively regulated by one or more proteins encoded by early ecdysoneinduced genes; again, this confirms the models. We also show that the temporal and tissue distribution of E74A transcription is consistent with the coordination model. Thus, each ecdysone pulse during development is acc,ompanied by a burst of E74A transcription, and the late third instar pulse results in E74A transcription in a variety of both larval and imaginal target tissues. Although a detailed investigation of E746 transcription will appear elsewhere, we show here that its temporal distribution is significantly different from that for E74A, indicating that the E74 transcription units are differently regulated. Finally, we show that E74A transcripts elongate at a rate of ~1.1 kblmin and that the 1 hr delay in the appearance of cytoplasmic mRNA following the ecdysone activation of the E74A promoter is mostly due to the time required to transcribe the 60 kb E74A unit. We therefore propose that the lengths of the early ecdysone-inducible genes are significant factors in determining both the timing of the ecdysone response and, because of the repression of these genes by the proteins they encode, the number of mRNAs synthesized during each ecdysone pulse.

Results Transcription of the Long E74A Unit Acts As a 1 Hr Delay Timer Following Ecdysone Activation of Its Promoter We previously showed that the ecdysone-induced appearance of the 6.0 kb E74A mRNA in late third instar salivary glands represents a primary response to this hormone (Burtis et al., 1990; also see Figure 2). Here we show that this induction results from the ecdysone activation of the E74A promoter. In addition, we provide the rationale for the delayed appearance of the E74A mRNA by measuring the rate of E74A transcript elongation and showing that the time required for transcription of the 60 kb E74A unit closely approximates the delay time. Mass-isolated late third instar tissues were exposed to ecdysone for different periods of time, after which nuclear and cytoplasmic RNAs were extracted. Mixed tissues rather than isolated salivary glands were used in these experiments because E74A is transcribed in many third instar tissues, as subsequently shown. The amounts of specific transcripts in the RNA preparations were determined

E74

90 60

45 30 20

15

[-

o

0

rS-

L* Nuclear

Cytoplasmic RNA

RNA

Figure 1. Timing the Synthesis in Response to Ecdysone

of Nuclear

and Cytoplasmic

E74A RNA

Cultures of late third instar larval organs were treated with ecdysone for the period of time shown. RNA was then extracted from nuclear and cyloplasmic fractions prepared from the cultured organs as described in Experimental Procedures. Three amounts of RNA (0.5, 1.0, and 2.0 pg) from each time point were loaded onto a strip of Nytran using a slot blot apparatus, and the strips were probed with different radioactive DNAs. The rp49 DNA probe was derived from a genomic clone (O’Connell and Rosbash, 1984). The E74 DNA probe used to detect cytoplasmic RNA was derived from the 5’-proximal Al exon of E74A (Figure 5C). The DNA probes used to detect nuclear E74A RNA were located in E74A introns either 1, 17, or 30 kb from the start site of transcription.

by quantitative slot blot hybridization with 32P-labeled DNA probes that consisted of sequence8 from different parts of the E74A unit or from the Drosophila ribosomal protein 49 gene (rp49; O’Connell and Rosbash, 1984), which was used as a control to normalize the data for variations in RNA recovery. If ecdysone activates the E74A promoter, probes located at increasing distances from the E74A transcription initiation site should first detect E74A nuclear transcripts at increasing times after exposure of the tissues to ecdysone. Figure 1 shows that this is the case for intronic probes located 1, 17, and 30 kb downstream of the initiation site. No E74A nuclear RNA was detected prior to the addition of ecdysone (0 min). At 15 min, the only probe to detect this RNA was that located 1 kb from the 5’ end; indeed, this probe detected low levels of E74A nuclear RNA as soon as 5 min after ecdysone addition (data not shown). At 30 min, both the 1 kb and 17 kb probes clearly detected nuclear E74A RNA, and by 45 min, all three probes detected this RNA. Probes consisting of intron sequences located more than 45 kb downstream of the E74A initiation site also detected the E74t3 transcripts (Figure 5C). Because one or more of the E74B promoters are activated earlier in third instar larvae (Figure 5B), these probes detected nuclear RNA both before and after ecdysone addition,

@3ophila

E74 Transcriptional

Hours Ecdysone 0.3 2 __---

Figure 2. Time Glands

4

e 8

Course

after

Regulation

addition

early puff locus from which it derives; thus, the ecdysone induction of this puff is followed by its regression in the absence of cycloheximide, whereas in the presence of this inhibitor of protein synthesis, no regression is observed (Ashburner, 1974). We therefore infer that the proteins required for the ecdysone activation of the f74A promoter are present prior to ecdysone addition and that one or more proteins encoded by ecdysone-inducible genes are required to effect the decrease in the E74A mRNA level. We reserve to the Discussion a comparative analysis of E74A transcription and 74EF puffing that leads to the conclusion that this decrease in the E74A mRNA level results from the repression of the E74A promoter by ecdysone-induced proteins in conjunction with a relatively rapid turnover of the E74A mRNA.

Ecdysone + Cycloheximide a3 2 4 0 I ----A

of E74A Transcription

in Cultured

Salivary

Salivary glands isolated from late third instar larvae were maintained in organ culture and treated with (A) ecdysone alone or (8) ecdysone and cycloheximide as described in Experimental Procedures. RNA was extracted at the times shown and analyzed by Northern blot hybridization using a radioactive E74kspecific probe derived from exon A2.

yielding a pattern resembling that for rp49 transcription (data not shown). Densitometric scanning and normalization of the data obtained with the E7tispecific probes yielded an E74A transcription elongation rate of 1.1 kb/min at 25%, which we estimate is accurate to f 30%. This result is consistent with in vivo transcription rates reported by others (Kafatos, 1972; Suzuki, personal communication; Ucker and Yamamoto, 1964). Figure 1 also shows that cytoplasmic E74A mRNA was not detected until 60 min after the addition of ecdysone. This result is in agreement with Northern analyses, where the 6.0 kb E74A mRNA did not appear until 60 min after ecdysone addition to late third instar salivary glands (data not shown; Burtis, 1985). Given that the time required to transcribe the 60 kb E74A unit at the above rate accounts for most, if not all, of the delay in the appearance of the E74A mFtNA, it is clear that the length of ,%A is the primary factor determining the delay time. Proteins Encoded by Ecdysone-Inducible Genes Repress E74A Expmssion Figure 2 shows the kinetics of E74A mRNA accumulation in late third instar salivary glands exposed to ecdysone or to ecdysone and cycloheximide, as determined by Northern blot hybridization. In the absence of cycloheximide, the amount of the 6.0 kb E74A mRNA increases after the expected delay, reaches a peak, and then declines. Additional experiments of this sort demonstrated that this decline, which begins just before 6 hr, continues to the virtual disappearance of the mRNA after 12 hr of ecdysone exposure (data not shown). In contrast, in the presence of cycloheximide, the level of E74A mRNA continually increases. This response of E74A mimics that of the 74EF

The Tissue Distribution of E74A Transcription in Late Third lnstar Larvae According to the coordination model, ecdysone would be expected to induce E74A transcription in other late third instar target tissues as well as in the larval salivary gland. The range of tissues that are targets of this hormone is wide; indeed, almost all late third instar tissues display some response to ecdysone (reviewed by Bodenstein, 1965). Thus, strictly larval tissues, such as the larval gut, muscle, and salivary glands, initiate pathways leading to their own histolysis, while the imaginal discs, brain, abdominal histoblasts, and the imaginal gut and salivary gland cell clusters are induced to begin their complex morphogenetic pathways to the corresponding adult structures. Figure 3 shows that the 6.0 kb E74A mRNA is induced by ecdysone in both classes of tissues, the gut and fat body representing the strictly larval tissues and a mixture of wing and eye-antenna1 disks representing the imaginal tissues. The observation that E74A is transcribed in the fat body further confirms the association of this transcription unit with the 74EF early puff, since Richards (1982) observed puffing at this locus in fat body polytene chromosomes. In addition, we similarly detected the 6.0 kb E74A mRNA in a brain and wing disc dissected from a single late third instar larva after 3 hr of incubation in the presence of ecdysone (data not shown; Burtis, 1985). Because the dissected tissues were at a stage when E74A transcription was not detectable either in mixed tissues (Figure 1) or in whole organisms (Figure 5A), we presume that this E74A mRNA also resulted from ecdysone induction. The tissue distribution of E74A transcription was directly examined in third instar larvae just prior to prepupal formation by in situ hybridization of an f74A-specific probe to tissue sections. The most intense hybridization was observed in the brain and other parts of the larval nervous system, the proventriculus of the larval gut, and the larval salivary glands (Figure 4). The proliferation centers within the larval brain (White and Kankel, 1978) showed more intense labeling than the surrounding nervous tissue. Significant hybridization was also observed in other larval tissues-specifically the lateral muscles, epidermis, gut, and fat body. Strong labeling was also observed in imaginal tissues-specifically the eye-antennal, wing, and leg

Cell

104

Fat Bodies

Gut --------

+

-

Discs +

-

+

Ecdysone

sm

-

E74A

Figure 3. E74A Transcription in Isolated Larval Organs Late third instar larval guts, fat bodies, and imaginal discs were hand dissected and maintained in culture. One set of each tissue type was cultured in the absence of ecdysone, and the other set was treated with ecdysone for 4 hr. Total RNA was then extracted and analyzed by Northern blot hybridization using an E74Aspecific radioactive probe. The 6.0 kb E74A mANA is marked with an arrow. This sample of imaginal disc RNA was somewhat degraded; subsequent samples revealed only the single 6.0 kb E744 mRNA, as expected.

discs. In the larval salivary glands, labeling was located predominantly in the polytene nuclei, as shown in Figure 48. While the reason for this nuclear localization is not understood, it may result from a combination of the large number of nascent transcripts that can be accommodated on the long E74A transcription unit and the rapid turnover of cytoplasmic E74A mRNA. It should be emphasized that a complete study of the tissue distribution of EiX4 expression by in situ hybridization was not attempted, either with respect to the identification and examination of every tissue or with respect to varying the developmental stage of third instar larvae examined. We reserved a more complete determination of the tissue distribution of E74 expression to in situ antibody labeling of the E74 proteins, both because of its greater significance and because the double labeling it affords allows better comparison of the distribution of different proteins encoded by the early genes. This is made obvious by the difficulty in determining the tissue distribution of E74B expression by in situ hybridization, given that the E746 unit is completely overlapped by the E74A unit (Figure 5C). When an attempt was made to determine this distribution using a probe from the exon specific to the E74B unit (Figure 5C), the pattern of hybridization was indistinguishable from that obtained with the f 74A-specific probe, leaving open the question as to whether the distributions are indeed the same or confused by hybridization of the E74B exon probe to E74A intronic RNA sequences (E. CYToole and C. S. T., unpublished data). A Pulse of E74A Transcription Is Associated with Each Ecdysone during Drosophila Development The proposition that the coordination

Pulse model for late third

instar tissue development also applies to other stages of development that are similarly characterized by an ecdysone pulse (see Introduction) prompted a determination of the temporal distribution of E74A transcription. Figure 5A shows the results of such a determination, where the embryonic period was divided into 3 hr intervals and the remainder of development into 12 hr intervals, and the amounts of the 6.0 kb E74A mRNA per 20 pg of total RNA from animals in each of these intervals was determined by Northern blot hybridization. The first ecdysone pulse, which peaks at about 10 hr of embryogenesis (Kraminsky et al., 1980; Gietz and Hodgetts, 1985) is accompanied by a weak burst of E74A transcription that begins in the 9-12 hr interval and extends into the 12-15 hr interval. Similarly, pulses of ecdysone during the first and second larval instars (Ll and L2) are accompanied by E74A transcription bursts during the last half of each instar. The strong ecdysone pulse that peaks at the end of the third larval instar (L3: Richards, 1981a; Handler, 1982) and initiates the puffing pattern that forms the basis of the Ashburner model (Ashburner et al., 1974) is nicely correlated with a strong burst of E74A transcription in whole animals, as expected from the results presented in the preceding sections. While E74A mRNA is clearly present during the prepupal stage (PP), the 12 hr intervals used in Figure 5A are not sufficient to resolve the effects of the prepupal ecdysone pulse that closely follows the L3 pulse (Handler, 1982). Because prepupae can be staged quite precisely, we carefully examined E74A transcription at this time in development;

results

are shown

in Figure

6A. During

the first

6 hr

of prepupal development, the E74A mRNA level is seen to decrease to zero from the peak of the E74A transcription burst induced by the L3 ecdysone pulse. Another E74A transcription burst with a peak at 10 hr is then seen. Both indirect (Richards, 1976a, 197613) and direct (Handler, 1982) analyses of the ecdysone level during this period revealed that it drops initially and then increases by 8 hr, in good agreement with the increasing phase of the E74A transcription pulse. It should also be noted that puffing at the 74EF locus in prepupal salivary glands closely follows the E74A transcription response observed here (Richards, 1976~). Returning to Figure 5A, we see that a high broad peak of E74A transcription occurs during the pupal period between 144 hr and 180 hr after egg laying, which again is correlated with a similar high broad peak of ecdysone. Thus, each of the six ecdysone pulses that occur during D. melanogaster development is associated with a pulse of E74A transcription. There are, however, two periods of strong E74A transcription that do not appear to be associated with ecdysone pulses: the first period is near the end of embryogenesis, and the second is at the end of pupal development, a period extending to the adult stage (192-218 hr). The transcription

of E74A

may

therefore

also

be induced

by

signals other than ecdysone. The late embryonic burst of is shown at high resolution in Figure E74A transcription 68, where the E74A mRNA is seen to rise and fall rapidly over a 6 hr interval with a peak at 18 hr after egg laying. The

kinetics

of this

response

thus

closely

resembles

that

Drosophila 105

Figure

E74 Transcriptional

4. Localization

Regulation

of E74.4 RNA in Histological

Sections

of a Late Third

lnstar

Larva

(A) An 8 em section through the anterior portion of a late third instar larva. In situ hybridization was performed as described (Hafen and Levine, 1986). Both bright- and dark-field illuminations are shown. lmaginal discs (probably leg discs; ID), wing imaginal disc (WD), unstained neuropil (NP) surrounded by the stained larval brain, fat body (F), and a portion of the digestive system, the proventriculus (P), are shown. The arrow points to a proliferation center within the larval brain (White and Kankel. 1978). The intense signal seen above the wing disc is due to nonspecific sticking of radioactive probe to portions of the trachae. The anterior-posterior (A-P) axis is labeled. (6) Nuclear localization of E74A RNA. A higher magnification view showing imaginal discs (ID) and the salivary glands (SG) with their large polytene

observed near the end of the prepupal period (Figure 8A), which we presume is induced by ecdysone. E74B Transcription Exhibits an Overlapping but Different Temporal Distribution from That of E74A The E7419 transcripts initiate from two promoters that are 300 bp apart and located 40 kb downstream of the E74A start site, within the fifth of the seven E74A introns (Figure 5C; Burtis et al., 1990). One might therefore expect that these promoters are differently regulated from the E74A promoter. In an initial examination of this question, we determined the temporal distribution of the E74B mRNAs during development by Northern blot hybridization, using the same RNA samples from staged animals as those used for determining the temporal distribution of the E74A mFiNA (Figure 5). The two E74B mRNAs are 4.8 kb (E7482) and 5.1 kb (E7487) in length and consist of identical sequences, with the exception of the extra 300 nucleotides at the 5’ end of

the E74B7 mRNA, which results from the different location of their promoters. These mRNAs were not resolved by the agarose gel electrophoresis used for the Northern blot hybridizations. We therefore refer to the mRNAs registered by the single band of hybridization seen in Figure 58 as the E74B mRNAs. Initial studies using Sl nuclease protection assays to distinguish the different E74B transcripts revealed that these mRNAs are present in the same relative proportions at different stages of development, with the E7482 mRNA as the more abundant species (F. Karim and C. S. T., unpublished data). Comparison of the E74B mRNA distribution with that of the E74A mRNA reveals overlaps as well as striking differences. Both mRNAs are present in 9-15 hr embryos and in the last half of the Ll and L2 instars, although longer exposures than that shown in Figure 58 are required to detect clearly the E74B mRNA at these stages, each of which is associated with an ecdysone pulse. Both mRNAs also reappear during the 18-21 hr interval of embryogenes/s, although the E74B mRNA disappears by the 21-24 hr

Cdl 106

Figure 5. Profiles of E74A and E746 Transcription during Drosophila Development

Hours Ecdysone

Ii E74A probe

m

E74B probe

0 Al

A2 A3.4.5

t3

67

Hours

after puparium formation

024681012

B

Hours

after

kb

6

interval and the E74A mRNA does not. These observations raise the interesting possibility that the E74A and E748 promoters are regulated by the same signals during these periods of development. They also introduce the question of whether the E74B unit is ecdysone inducible, as is clearly the case for the E74A unit in late third instar larvae. One of the striking differences between the E74A and E74B temporal distributions related to this question is the earlier appearance of the E74B mRNA in late third instar larvae during the 96-108 hr interval, when the ecdysone titer is low and the 74EF and other early puffs have not yet been induced. This observation is consistent with the find-

A

RNA was isolated from staged embryos, first (Ll), second (L2), and third (L3) instar larvae, prepupae (PP), pupae, and adults, as described in Experimental Procedures. The RNA was denatured with formaldehyde, fractionated by agarose gel electrophoresis, transferred to Nytran, and hybridized with radioactive DNA probes specific for either E74A mRNA (A) or the E746 mRNAs (B). The hours and stages of development are shown at the top of the figure, along with stippled boxes marking the times when the ecdysone titer is high (Richards, 1981a). (C) A schematic representation of the E74 gene shows the locations of the labeled EcoRl restriction fragments used to detect the E74 transcripts (Burtis et al., 1990).

egg

ing, noted in the first section of the Results, that one or both of the E74B promoters were already activated in the mixed tissue population used to demonstrate the ecdysone activation of the E74A promoter (Figure 1). These results demonstrate that the transcription of one or both of the E748 units is differently regulated from the E74A unit during L3. While it is possible that the appearance of E74B mRNA during the 98-108 hr interval results from ecdysone induction, if so, that induction must occur at much lower ecdysone titers than are required to induce E74A transcription or the 74EF puff. Finally, we note that the temporal distribution of the E74A and E74B mRNAs exhibits significant differences in

laying

Figure 6. Time Course during Late Embryonic opment

141618202224 --a---

f

E74A

of E744 Transcription and Prepupal Devel-

RNA was extracted from either prepupae at 2 hr intervals following puparium formation (A) or staged embryos (B) as described in Experimental Procedures. The RNA was denatured, fractionated by agarose gel electrophoresis, transferred to either Nytran (A) or ATP paper (8). and hybridized with a single-stranded radioactive f744specific DNA probe.

Drosophila 107

E74 Transcriptional

Regulation

pupae and adults, again indicating that their promoters are differently regulated. Whereas the E74A mRNA levels are high during the pupal ecdysone pulse (144-180 hr), the E74B mRNA levels are relatively low. Furthermore, the E74B mRNA levels peak in late pupae, decreasing at the end of pupal development and disappearing by adulthood, and the E74A mRNA levels increase at the end of pupal development and are high in the adult. Discussion The tissue coordination model for the ecdysone control of Drosophila development and the Ashburner model for polytene chromosome puffing are supported by three conclusions inferred from the characteristics of E74A transcription described here. First, the ecdysone response at the 74EF early puff locus in larval salivary glands results from the ecdysone activation of the E744 promoter and the subsequent repression of E74A transcription by proteins encoded by ecdysone-inducible genes. Second, E74A transcription is similarly induced and repressed in association with pulses of ecdysone at other stages of Drosophila development. Third, E744 transcription is induced in a variety of ecdysone target tissues (both larval and imaginal) in late third instar larvae. Two additional conclusions from this work serve to extend and define these models further: namely, transcription of the long E74A unit acts as a 1 hr delay timer, and signals other than ecdysone activate the f74A promoter at the end of embryonic and pupal development. In the following sections of this Discussion we assess the basis for the above conclusions and their relation to the models, considering first the effects of the sequential induction and repression of E74A transcription. Sequential Induction and Repression of E74A Transcription Generates Bursts of E74A mRNA during Development The Late Third lnstar Burst and 74EF Puffing The initiation of both f74A transcription and 74EF puffing occurs within less than 5 min of the addition of ecdysone to cultured tissues of late third instar larvae (Figure 1; see Figure 14 of Ashburner, 1972). This essentially simultaneous generation of nascent E74A transcripts and detectable 74EF puff expansion provides the initial basis for the proposition made here that the 74EF puff size is a direct indicator of the number of nascent transcripts on the E74A unit. Additional support for this proposition derives from the observation that the expansion of the 74EF puff occurs in two phases. During the first hour after exposure to ecdysone, the puff expands at a maximum constant rate; at the end of this hour, the puff expansion rate decreases abruptly to initiate a second phase of slow expansion of puff size that is terminated by the beginning of puff regression at ~4 hr postexposure to ecdysone (see Figures 4 and 14 of Ashburner, 1972). The first phase corresponds nicely to the first hour of E74A transcription following activation of the promoter by ecdysone. Thus, the rate of increase in the number of nascent transcripts per E74A unit will initially equal the rate at which new transcripts are created at the 5’ end of the unit (the “on-rate”). This maxi-

mum rate will persist until transcripts begin to fall off the 3’end of the 60 kb unit. This will occur after ~1 hr given the ml.1 kb/min elongation rate of nascent transcripts. At this time, the rate of increase in the number of nascent transcripts per unit must drop abruptly, as it will then equal the difference between the on-rate and this “off-rate.” The correspondence between this period when the E74A unit is saturated with nascent transcripts, or slowly approaches saturation, and the second phase of slow puff expansion to a plateau value provides an additional argument that puff size is a good indicator of the number of nascent transcripts per E74.A unit. Inactivation of the E74A promoter by repressors would decrease the on-rate to zero at complete repression, and the number of nascent transcripts per E74A unit would then decrease at a rate equal to the off-rate. The regression of the 74EF puff, which begins at ~4 hr postexposure (Ashburner, 1972), is therefore taken to indicate that the E74A promoter is strongly repressed by this time. The mRNA should continue to be synthesized after the onset of promoter repression for the period required to clear the E74A unit of nascent transcripts. The completion of puff regression by ~6 hr is therefore similarly taken to mark the end of this period and of mRNA synthesis. Because the mRNA level is expected to continue to increase after puff regression has begun, the peak in mRNA level should be delayed in relation to that for puff size, an expectation that is met by the observation that the mRNA level begins to decline just before 6 hr postexposure (Figure 2A). That the mRNA level peaks rather than leveling off at this time results from the relatively short half-life of the EX4 mRNA. We estimated this half-life to be in the order of 1 hr from the rate of decrease in the mRNA level between 6 hr, when mRNA synthesis is presumed to have ceased, and 12 hr postexposure, when the mRNA has virtually disappeared (Results). The effects of cycloheximide on the response of both the E74A mRNA and the 74EF puff are consistent with the above interpretation and indicate that E74A promoter repression results from proteins synthesized in the presence of ecdysone. In the presence of cycloheximide or other inhibitors of protein synthesis, the size of the puff increases normally but levels off rather than decreasing rapidly at 4 hr postexposure to ecdysone (Ashburner, 1974). According to the above analysis, this leveling off could result from either of two causes: failure of E74A promoter repression, in which case the number of nascent transcripts per E74A unit would be maintained at a high steady-state level by equivalent on- and off-rates; or inhibition of the off-rate, in which case the number of transcripts would remain constant regardless of the activity of the promoter, and transcription and mRNA synthesis would cease. The observation that the E74A mRNA level continues to increase (Figure 28) is consistent with the first, but not the second, of these two possibilities and demonstrates that promoter repression requires protein synthesis in the presence of ecdysone. The characteristics of the burst of E74A transcription at the end of the third larval instar therefore demonstrate that this transcription unit satisfies several of the criteria for an

Cell 108

“early” gene in the Ashburner model. First, this burst is initiated by the rapid ecdysone activation of the E74A promoter without need of protein synthesis. This activation is undoubtedly effected by an ecdysone-receptor complex. The ecdysone receptor encoded by the EcR gene is a good candidate for this role as it is present in late third instar salivary glands prior to the ecdysone pulse and binds to the 74EF puff locus (M. Koelle, W. A. Segraves, W. S. Talbot, M. Bender, and D. S. H., unpublished data) and to sites in the first E74A intron (W. S. Talbot and D. S. H., unpublished data). Second, the burst is terminated by repression of the E74A promoter that requires one or more proteins encoded by genes induced, directly or indirectly, by ecdysone. These genes have not yet been identified as early genes, as specified by the model. However, the E75 gene responsible for the 756 early puff is a logical candidate, not only because it is directly induced by ecdysone at this time and encodes nuclear regulatory proteins (Segraves and Hogness, 1990), but also because the protein, encoded by the longer of the two nested transcription units (E75.4) that comprise this gene, has been shown to bind to the 74EF puff locus (FL J. Hill, W. A. Segraves, W. S. Talbot, and D. S. H., unpublished data). Furthermore, this E7a transcription unit is 50 kb in length (Segraves and Hogness, 1990) and the time expected for its transcription at an elongation rate of 1.1 kb/min would contribute significantly to the delay in achieving full repression of the E74A unit. One might suppose that feedback repression by the E74A protein is also a likely possibility, as this protein also exhibits the characteristics of a nuclear regulatory protein (Burtis et al., 1990). We discount this possibility, however, because of the following properties of an E74 mutation (X1001) that results from a simple translocation between the left arms of chromosomes 2 and 3 [T(2;3) 29A-29C; 74E-74Fj, in which the breakpoint in the 74EF locus was mapped within the first intron of the E74A unit (Burtis, 1985). Despite the resulting separation of the E74A promoter from the exons that encode the E74A protein (Figure 5C), the aberrant RNA generated from the E74A promoter in Xl001 homozygotic late third instar tissues exposed to ecdysone exhibits the same kinetics of induction and repression as that shown in Figure 2A for the E74A mRNA in wild-type tissues (Burtis, 1985). Finally, the tight correlation between 74EF puffing and E74A nascent transcript accumulation provides a strong argument that 74EF puffing is a manifestation of E74A transcription, thereby satisfying another criterion for an early gene in the Ashburner model. As noted in the last section of this Discussion, the E74B unit does not satisfy this criterion because its transcription does not appear to be coupled to 74EF puffing. Other Bursts Associated with Ecdysone Pulses Our knowledge of the role played by ecdysone in generating the other bursts of E74A transcription is less complete, because the data is thus far restricted to whole animal analysis of that transcription (Figures 5A and 8). We focus here principally on the prepupal burst shown in Figure 6A, both because it is associated with 74EF puffing activity, which has been examined in considerable detail in iso-

lated salivary glands (Ashburner and Richards, 1978; Richards, 1976b, 1976c), and because it can be compared most accurately to ecdysone titers (Richards, 1976b; Handler, 1982) given that reproducible staging is easily achieved. The high ecdysone titer observed at the beginning of prepupal development represents the peak of the late third instar ecdysone pulse, which begins -6 hr earlier. The titer drops to very low levels during the first ~3 hr of pupal development and stays at this low level until ~8 hr, when it rises at approximately the same time that the prepupal burst of E74A transcription is initiated (Figure 6A; Richards, 1976b; Handler, 1982). Puffing at the 74EF locus is also initiated at this time to yield a puff profile (Ashburner and Richards, 1976; Richards, 1976c) that is approximately coincident with that for the E74A mRNA seen in Figure 6A. Richards’ studies (1976c) on isolated salivary glands show that the prepupal 74EF puff, like the larval puff, is directly induced by ecdysone and that its regression in the continued presence of ecdysone is dependent upon protein synthesis. Given the close correlation between E74A transcription and 74EF puffing described in the preceding section, we therefore presume that the prepupal burst of E74A transcription results from the ecdysone activation of the E74A promoter followed by its repression by proteins encoded by ecdysoneinducible genes. The mRNA profile generated by the prepupal burst of E74A transcription spans only -4 hr of development (Figure 6A), while that generated by the third instar burst spans ~11 hr (Figure 2A and associated text). Our analysis of the third instar burst indicates that the second half of the 11 hr profile (which corresponds to the decreasing levels of E74A mRNA observed in Figure 6A during the first hours of prepupal development) results solely from the turnover of the E74A mRNA. Comparison in Figure 6A of the rate of this decay of E74A mRNA with that generated by the prepupal burst demonstrates that the shorter duration of the prepupal profile results, in large measure, from a faster turnover rate. Because the duration of the prepupal 74EF puff (-4 hr) is also shorter than the third instar puff (~6 hr; Ashburner and Richards, 1976), a part of the difference between the two mRNA profiles may also result from the more rapid repression of the E74A promoter in the prepupal burst, perhaps as a consequence of the carryover of low concentrations of repressor from the preceding ecdysone pulse. In this connection, it should be noted that Richards’ studies (19764 1976~) demonstrate that the ecdysone induction of the prepupal 74EF puff is dependent upon a preceding interval of very low ecdysone concentration and upon protein synthesis during that interval. These results suggest that one purpose of such a dip in the ecdysone concentration may be the inactivation of repressors by proteins encoded by genes that are repressed by ecdysone-a possibility that Richards noted in connection with repressors of 74EF puff activity. The association of ecdysone pulses with the bursts of E74A transcription observed during mid-embryogenesis and the last half of the first and second larval instars, in conjunction with the relatively short duration of these

Drosophila 109

E74 Transcriptional

Regulation

bursts (Figure 8A), suggests that they also result from the ecdysone induction of the E74A promoter and the subsequent repression of that promoter by proteins encoded by ecdysonainducible genes. This, however, does not appear to be the case for the broad peak of E744 mRNA observed during pupal development (144-180 hr, Figure 3A), which is associated with a similarly broad ecdysone peak (Richards, 1981a). While ecdysone may similarly activate the E74A promoter during this period, it apparently does not similarly induce the synthesis of effective repressors of that promoter-providing, of course, that the turnover rate for the E74A mRNA is not extremely low during this period. In any case, this long period of high f74A mRNA levels, in comparison with the preceding short periods, raises interesting questions respecting E744 function that cannot be addressed until the patterns of ,!%A protein expression are determined. It may be, for example, that the translation of the E74A mRNA is regulated during this period-a possibility suggested by its extremely long 5’ leader (1891 nucleotides), which contains 17 short AUGinitiated ORFs (Burtis et al., 1990). Bursts Not Associated wlfh Ecdysone Pulses Careful studies by Hodgetts and his associates (Kraminsky et al., 1980; Gietz and Hodgetts, 1985) of ecdysone titers during embryogenesis have not revealed a significant increase in these titers during or immediately preceding the strong burst in EVA transcription observed during late embryogenesis (18-20 hr; Figure 8B). It appears likely, therefore, that the E74A promoter is activated by elements other than ecdysone at this stage of development. This supposition is consistent with the finding that D. melanogaster strains that contain a hybrid gene consisting of the E74A promoter and flanking DNA fused to a reporter gene exhibit induction of hybrid gene expression during late embryogenesis but not during the ecdysone pulses (C. S. T., unpublished data). The rapid and abrupt termination of this late embryonic burst of E74A transcription, which mimics that for the prepupal burst (Figure 8) suggests the possibility that this termination results from repression of the E74A promoter by the same elements that terminate the ecdysone-induced bursts of E74A transcription. In this respect, we note that the E754 transcription unit of the gene responsible for the 758 early puff (Segraves and Hogness, 1990) is also induced during late embryogenesis (Segraves, 1988). E74A transcription is also induced at the end of pupal development (Figure !5A) in the absence of an increase in the ecdysone titer (Hodgetts et al., 1977; Richards, 1981a). In this case, however, there is no evidence that the transcription is terminated, even within the first 12 hr of adulthood. This observation further suggests that the mechanisms for rapid repression of E74A transcription, employed during each of the first five stages of development, are relaxed during the pupal stage. Tha Wtdespread Distribution of E74A li’anscriptlon in Late Third Instar Larvae Is Consistent with the Tissue Coordination Model As applied to the ecdysone target tissues of the late third larval instar, two fundamental assumptions of the tissue

coordination model are: first, that the ecdysone activation in the larval salivary gland of the genetic regulatory hierarchy depicted by the Ashburner model serves to initiate the next step, or series of steps, in its developmental pathway, and second, that highly related but distinct regulatory hierarchies are similarly activated by ecdysone in each target tissue to initiate the next steps in their developmental pathways. A concept related to these assumptions is the self-arrest of the developmental pathways of the target tissues prior to the ecdysone activation of the next steps. Thus, each tissue would achieve a final larval state from which further development would be blocked in the absence of ecdysone activation. A further assumption of the tissue coordination model, based on genetic regulatory economy, is that the combination of ecdysone-inducible early regulatory genes in each hierarchy derives from a small population of these genes such that overlap is expected among the combinations employed by the different hierarchies. That overlap could be complete or partial, depending upon the level at which tissue-specific hierarchical distinction is introduced. In either case, a given early gene would be expected to be induced in multiple target tissues. That expectation is certainly met by E 74A. The ecdysone induction of its transcription was observed in a variety of isolated target tissues, both imaginal and strictly larval (Figure 3 and associated text), and in situ hybridization of tissue sections at the end of the third larval instar revealed E74A transcription in each of the tissues identified and examined (Figure 4). While not all third instar tissues were examined and no attempt was made to eliminate bias in the selection of those that were, it is clear that E74A transcription is widespread. Considerable variation was, however, observed in the abundance of the E744 transcripts, both among the target tissues and within individual tissues. Clearly, such quantitative differences could result in different regulatory effects. Furthermore, we have yet to determine the distribution of the E74A protein upon which such regulatory effects depend and, as has been noted, the primary structure of the E74A mRNA suggests that it may be subject to specific translational controls. Finally, it should be emphasized that the wide spread distribution of the activity of one member of the early gene population does not imply a similarly widespread distribution of all members of that population. Certain early gene functions may be required in all regulatory hierarchies, whereas others may determine the regulatory specificity of individual hierarchies. Concluding Remarks Our results show that the E74A transcription unit satisfies many of the criteria for an early gene in the Ashburner model. Current research is directed toward testing the remaining criteria. Thus, the role played by the receptor encoded by the EcR gene in the ecdysone activation of the E74A promoter remains to be determined, as does the identification of the proteins that repress that promoter. In addition, we have yet to identify the regulatory targets of the E74A protein. The temporal distribution of E74A transcription during development and the tissue distribution

Cell 110

of that transcription indicate that E74A plays a developmental role beyond the confines of the larval salivary gland and provide a sufficient basis for the further investigation of the tissue coordination model presented here and by Burtis et al. (1990). The discovery that E74A transcription is induced at the ends of embryonic and pupal development not only indicates that the regulation of that transcription is more complex than anticipated by the Ashburner model but also introduces the possibility that genetic regulatory hierarchies of the sort proposed in that model may be triggered by hormonal signals other than ecdysone. Our finding that the 1 hr delay in the appearance of the E74A mRNA is primarily due to the time required to transcribe the 60 kb E74A unit introduces transcription unit length as an important factor in any consideration of the regulatory interactions among the genes of the proposed hierarchies-particularly as these interactions include an induction-repression sequence that determines the burst size of the mRNA produced by the early genes. To appreciate this importance, one has only to imagine the effect on that burst size if a short early gene-say 3 kb in length-encoded the repressor of E74A. Repression of E74A transcription would then be expected to occur within minutes rather than hours after its induction, with an equivalent reduction in the mRNA burst size. It may therefore be no accident that the two other genes responsible for early puffs (E75: Segraves and Hogness, 1990; the gene responsible for the 265 puff: Chao and Guild, 1966, and Guild, personal communication) are also exceptionally long. Finally, we comment on the E746 unit, which is somewhat of an enigma at this initial stage of its characterization. The observation that it is transcribed in third instar larvae several hours before the 74EF puff is induced by the third instar ecdysone pulse (Figure 58) suggests that its transcription is not coupled to 74EF puffing. Furthermore, in isolated third instar tissues at the same stage as those employed in the experiment shown in Figure 1, the E74B mRNA is present before exposure to ecdysone and does not increase after that exposure (C. S. T., unpublished data), indicating that E74B transcription is not induced by ecdysone at this stage of development. In contrast to these results, which dissociate E746 from the early gene class, the overlap between the temporal distributions of E74A and E74B transcription during embryogenesis and the first two larval instars and the overlap in the carboxy-proximal amino acid sequences of the proteins they encode (Burtis et al,, 1990) suggest a close relationship between their functions. The resolution of this enigma should therefore be of considerable interest. Experimental

Procedures

Developmental Staging of Drosophila Large population houses of wild-type Canton S or Oregon R flies (-50,000 flies/house) were used for egg laying. These were maintained in 70%-60% relative humidity at 25OC with a 24 hr light-dark cycle. Plates containing grape agar and a strip of yeast paste were used to collect eggs for the appropriate period of time. Five inch diameter plastic jars containing standard cornmeal agar were used as substrates for growing larvae. Each cage was innoculated with 0.1 g

of dechorionated embryos suspended in 2 ml of 14% sucrose, 0.7% NaCI, 0.05% Triton X-100. Crawling late third instar larvae were removed from the cage walls and used to inoculate fresh containers in order to stage pupal development. For precise staging of prepupal development, white prepupae were individually selected and allowed to age at 25OC for the appropriate period of time. Organ Culture A modification of the procedure of Wolfner and Kemp (1986) was used to extract total larval organs. Late third instar larvae were washed from the cage walls and rinsed several times with water. They were then suspended in Robb’s saline (Robb, 1969) and passed between stainless steel rollers set just close enough to extrude the internal organs. The saline was oxygenated by vigorous agitation in a blender to reduce anoxic shock. Larval cuticles and attached tissues were strained out on a piece of nylon mesh, and the organs were settled through Robtis saline in 30 ml tubes. After allowing the organs to settle for 4 min, the cloudy supernatant was aspirated off and replaced by fresh Robb’s saline. The settling was repeated until the supernatant was clear (3-5 times). The organs were then cultured at 25% in 5 ml of Robb’s saline per 100 mm diameter petri dish. The dishes were prereated with 1% BSA to prevent the tissues from sticking to the plastic. Ecdysone (Sigma) was added to a final concentration of 4 x low6 M. Cycloheximide (Calbiochem), when used, was added to a final concentration of 7 x 10m5 M. Specific organs were individually isolated by sucking them into a glass capillary while observing with a dissecting microscope. lsolatlon of RNA For isolation of small quantities of RNA, tissues were dounced in a 1:l mixture of phenol and extraction buffer (0.3 M NaCI. 0.1 M Tris base, 20 mM EDNA, 1% Sarkosyf). The aqueous and organic phases were separated by centrifugation. and the aqueous phase was reextracted with fresh phenol followed by ether extraction. The RNA was precipitated by the addition of 2 vol of ethanol and incubation for I-60 min at room temperature. The RNA pellet was resuspended in water and stored frozen. Large quantities of RNA free from DNA were prepared by a modification of the method of Chirgwin et al. (1979). Tissues were disrupted in at least a lo-fold excess of 6 M guanidine-HCI (Schwartz-Mann UltraPure), 0.1 M sodium acetate [pH 5.21 using a glass dounce homogenizer with a B pestle. Insoluble material was pelleted by centrifugation, and the supernatant was layered over a cushion of 5 M CsCI, 10 mM EDTA in an ultracentrifuge tube. The RNA was pelleted by centrifugation at 30,600 rpm for 14 hr at 20°C. The supernatant was carefully removed by aspiration, and the clear RNA pellet was resuspended in 0.3 M sodium acetate [pH 5.5) by heating at 7ooC for 5 min. The RNA was then precipitated by the addition of 2 vol of ethanol before being resuspended in water for storage at -6OYZ

Northern Blotting and Hybridization For Figures 2 and SB, RNA was denatured by incubation in 1 M glyoxal, 50% DMSO and fractionated by agarose gel electrophoresis in 10 mM sodium phosphate (pH 6.6; f&Master and Carmichael, 1977). The RNA was then transferred onto ATP paper (Seed, 1962) by blotting. Otherwise, the RNA (up to 20 pg) was dissolved in 4.5 pl of water and mixed with 10 nl of formamide, 3.5 ~1 of formaldehyde, and 2 nl of 10x gel buffer (0.2 M MOPS buffer, 50 mM sodium acetate, 10 mM EDTA [pH 7.01). After heating at 65OC for 5 min, the RNA was fractionated by gel electrophoresis, using an agarose gel prepared with lx gel buffer and 6.6% formaldehyde and a 1x gel buffer (without formaldehyde) as a running buffer. After electrophoresis the gel was rinsed in 10 mM phosphate buffer (pH 66) for 1 hr and then transferred by blotting to Nytran (Schleicher & Schuell) with 20x SSC. The baked filters were prehybridized and hybridized at 42“C in 5g% formamide, 5x SSC, 0.1% SDS, 0.1 mglml sonicated salmon DNA, Ix Denhardt’s solution (Denhardt, 1966) 5%~10% dextran sulfate (Sigma). Radioactive DNA probes were used at a concentration of 1 x 106 cpmlml and were prepared by either nick translation (Rigby et al., 1977) or gel purification of single-stranded DNA synthesized from ml3 inserts (Burtis et al., 1990). The filters were washed in 0.1-0.5~ SSC, 0.1% SDS at 50°C, and the hybridized DNA was visualized by autoradiography.

Drosophila 111

hnscrlptton

E74 Transcriptional

Regulation

Timing

Six 150 mm petri dishes containing mass-isolated larval organs were maintained at 2S’C in Robbs saline as described above. These were incubated for 2 hr to allow recovery from any prior exposure to ecdysane in vivo. Ecdysone wrw then added to each plate at timed intervals. At the end of the time course the organs were chilled and collected by a 30 s centrifugation at 1506 rpm. The organs were disrupted in 5 ml of cofd lysts buffer (10 mM Tris [pH 7.61, 150 mM NaCI, 0.65% NP-40) by 5 strokes with an A pestle in a glass dounce homogenizer. The nuclei were pelleted by centrifugatfon at 7000 rpm for 2 min at 5%. The supernatant cytoplasm was poured into phenol, and the nuclear pellet was resuspended in 6 M guanidine-HCI, 0.1 M sodium acetate (pH 5.2) and treated at described above for RNA extraction. The cytoplasmic RNA was purified by phenol extraction, ether extraction, and ethanol precipitation. These samples were resuspended in 30 mM Tris (pli 7.6) 10 mM MgCls, and 3.5 mg of RNAase-free DNAase (Worthington) was added. After incubation at 37X for 30 min the RNA was purified by phenol extraction, ether extraction, and ethanol precipitation. Nuclear and cytoplasmic RNAs were denatured with formaldehyde as described above and loaded onto Nytran using a plexiglass slot blot apparatus. The Nytran strips were hybridized with single-stranded radioactive DNA probes as described above for Northern blots.

This work was supported by a National Science Foundation grant (to D. S. H.), a National Science Foundation Graduate Fellowship (to K. C. B.), and an American Cancer Society postdoctoral fellowship (to C. S. T). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

January

3, 1990.

ylase expression during Dev. Biol. 107. 142155.

embryogenesis

in Drosophila

melanogaster.

Hafen, E., and Levine, M. (1966). The localization of RNAs in Drusopbi/a tissue sections by in situ hybridization. In Drosophila-A Practical Approach, D. B. Roberts, ed. (Washington, DC.: IRL Press), pp. 139-157. Handler, A. M. (1962). Ecdysteroid titres during pupal and adult development in Drosophila melancgaster. Dev. Biol. 93, 73-82. Hodgetts, R. B., Sage, B., and O’Connor, J. D. (1977). Ecdysone during postemblyonic development of Dmsophila melanogastec Biol. 60, 310-317.

Kafatos, F. C. (197’2). The cocoonase zymogen cells of silk moths: a model of terminal differentiation for specific protein synthesis. Curr. Topics Dev. Biol. 7, 125-191. Kraminsky, G. l?, Clark, W. C., Estelle, M. A., Gietz, R. D., Sage, B. A., O’Connor, J. D., and Hodgetts, R. B. (1960). Induction of translatable mRNA for dopa decarboxylase in Dmsc@i/a: an early response to ecdysterone. Proc. Natl. Acad. Sci. USA 77, 4175-4179. McMaster. G. K., and Carmichael, G. G. (1977). Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. USA 74, 4635-4636. O’Connell, P, and Rosbash, M. (1964). Sequence, codon preference of the Drosophila ribosomal protein Acids Res. 12, 54955513.

structure, 49 gene.

Richards, G. P (1976b). Sequential polytene chromosomes of Drosophila period. Dev. Biol. 54, 256-263.

gene activation melenogsster:

by ecdysone in the mid prepupal

Richards, G. P (19761~). Sequential polytene chromosomes of Drosophile period. Dev. Biol. 54, 264-275.

gene activation melanogaster

by ecdysone in the late prepupal

Richards, G. f? (1961a). The radioimmune assay of ecdysteroid in Drosophila melanogestw Mol. Cell. Endocrinol. 27, 161-197

Ashburner, M. (1972). Patterns of puffing activity in the salivary gland chromosomes of Dmsop/ri/a. VI. Induction by ecdysone in salivary glands of D. melanogaster cultured in vitro. Chromosoma 38,255-281.

Richards, 501X.49.

Ashburner, M., and Richards, G. (1976). The role of ecdysone in the control of gene activity in polytene chromosomes of DmscphA. In Insect Development, P A. Lawrence, ed. (Oxford: Blackwell Scientific Publications), pp. 203-225. Ashburner, M.. Chihara, C., Meltzer. l?. and Richards, G. (1974). Temporal control of puffing activity in polytene chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38, 655-662. Bodenstein, D. (1965). The postembryonic development of Dmsophila. In Biology of Drosophila, M. Demerec, ed. (New York: Hafner Publishing Co.), pp. 275-367. Burtis, K. B. (1965). Isolation and characterization of an ecdysone inducible gene from Drosophila melanogaster. Ph.D. thesis, Stanford University, Stanford, California. Burtis, K. B., Thummel, C. S., Jones, C. W., Karim, F D., and Hogness, D. S. (1990). The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two e&-related proteins. Cell, this issue. Chao, A. T., and Guild, G. M. (1986). Molecular sterone-inducible 285 “early” puff in Drosophila J. 5, 143-150.

analysis of the ecdymelanogastec EMBO

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 78, 5294-5299. Denhardt. D. (1966). A membrane-filter technique for the detection of complementary DNA. B&hem. Biophys. Res. Commun. 23,641-646. Gietz.

R. D.. and Hodgetts,

R. 8. (1965). An analysis

of dopa decarbox-

and Nucl.

Richards, G. P (1976a). The control of prepupal puffing patterns in vitro: implications for prepupal ecdysone titre in Drosophila me/arm gas&r. Dev. Biol. 48. 191-195.

References

Ashburner. M. (1974). Sequential gene activation by ecdysone in polytene chromosomesof Drosophi/ame/anogester. II. The effectsof inhibitors of protein synthesis. Dev. Biol. 39, 141-157.

titres Dev.

G. f? (1961b). Insect hormones

in development.

titres

Biol. Rev. 56,

Richards, G. P (1962). Sequential gene activation by ecdysteroids in polytene chromosomes of Dmsophile melanogaster. VII. Tissue specific puffing. Roux’s Arch. Dev. Biol. 797, 103-111. Rigby, P, Dieckmann, M., Rhodes, C., and Berg, P (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 713, 237-251. Robb, J. A. (1969). Maintenance ogaster in chemically defined

of imaginal discs ot Drosophile melanmedia. J. Cell Biol. 47, 676-685.

Seed, B. (1962). Diazotizable arylamine ling and hybridization of nucleic acids.

cellulose papers for the coupNucl. Acids Res. 70, 1799.

Segraves, W. A. (1966). Molecular and genetic analysis of the E75 ecdysone-responsive gene of Drosophila melanogaste~ Ph.D. thesis, Stanford University, Stanford, California. Segraves, W. A., and Hogness, D. S. (1990). The E75 ecdysoneinducible gene responsible for the 758 early puff in Dmsopfti/a encodes two new members of the steroid receptor superfamily. Genes Dev., in press. Ucker, D. S., and Yamamoto, K. R. (1964). Early events in the stimulation of mammary tumor virus RNA synthesis by glucocorticoids. J. Biol. Chem. 259, 74167420. White, K.. and Kankel, D. R. (1976). Patterns of cell division and cell movement in the formation of the imaginal nervous system in Dmsophi/a melanogaster. Dev. Biol. 65, 296-321. Wolfner. M., and ecdysterone-inducible 104-106.

Kemp, D. J. (1966). A method tissues of D. melancgaster

for mass-isolating Dros. Inf. Serv. 67,