Contrasting modulations of gene expression by a juvenile hormone analog

Insect Bioehem. Vol. 20, No. 2, pp. 105-11 I, 1990 Printed in Great Britain. All rights reserved

0020-1790/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc

CONTRASTING MODULATIONS OF GENE EXPRESSION BY A JUVENILE HORMONE ANALOG JAMESY. BRADFIELD,ROBERTL. BERLINand LARRYL. KEELEY Laboratories for Invertebrate Neuroendocrine Research, Department of Entomology, Texas A&M University, College Station, TX 77843-2475, U.S.A. (Received 2 May 1989; accepted 21 August 1989)

A~tract--Decapitation altered the pattern of in vitro protein synthesis by the fat body of adult female Blaberus discoidalis cockroaches. Several polypeptides showed marked diminution, whereas two polypeptides showed markedly increased synthesis. The polypeptide profile was restored to normal in decapitated cockroaches by treatment with the juvenile hormone analog (JHA) methoprene. Cloned cDNAs that were isolated represented two fat body transcripts: one transcript was strongly stimulated and the other strongly suppressed by JHA. JHA acted on fat bodies in vitro to modulate the levels of the two transcripts. These studies indicate a system where juvenile hormone may provoke vividlycontrasting gene expressions in the same tissue at the same time. Key Word Index: gene expression, molecular cloning, fat body, juvenile hormone, cockroach

INTRODUCTION

MATERIALS AND METHODS

Juvenile hormone (JH) stimulation of protein synthesis in adult insects is widely recognized. This is especially evident in the female fat body of many species, where JH regulates the synthesis of vitellogenins (yolk protein precursors) (Engelmann, 1983). The availability of cloned gene sequences has allowed the locust fat body to be particularly well-studied in terms of JH-stimulated expression of identified genes. In this system, JH induces massive, coordinate accumulation of vitellogenin mRNAs and resulting high rates of vitellogenin synthesis (Wyatt, 1988). Other transcripts and proteins are also enhanced in the locust fat body by JH (Kanost et aL, 1988; Wyatt, 1988). JH stimulates both vitellogenin synthesis and the overall rate of protein synthesis in the fat body of the adult female cockroach, Blaberus discoidalis (Wojchowski and Kunkei, 1987; Keeley et al., 1988). The fat body of this species appears to be another system for explaining accumulation of gene products in response to JH. This paper documents suppression of transcripts and polypeptides by a JH analog (JHA, methoprene) in the fat body of adult female B. discoidalis. The suppression was contemporary with JHA-stimulation of another transcript (likely a vitellogenin message) and other polypeptides. Depending on the hormonal milieu, the suppressed and stimulated transcripts and proteins were either at low levels or among the most abundant gene products of the fat body. JHA acted directly on the fat body to depress the level of one transcript while stimulating another. The data suggest that the adult cockroach fat body is a novel system for examining gene regulation by juvenile hormone. I05

Insects

Virgin female B. discoidalis were maintained as described previously (Keeley et al., 1988). JHA treatment Cockroaches were immobilizedwith CO2 and decapitated on the day of the adult molt (=day 0), and the wound was sealed with a melted, I : 1 beeswax:petrolatum mixture. On days 2, 5, 8 and 11, 10 #g of methoprene (gift from ZoScon Corp.) in 10 #1 acetone were smeared on the dorsal abdominal cuticle. Control decapitated insects received 10/~1 of acetone on the same days. Analysis of protein synthesis in vitro Fat bodies were removed on day 15 (middle of first gonotrophic cycle) and incubated in 2ml insect tissue culture medium (Reddy and Wyatt, 1967) for 9 h as described (Keeley et al., 1988). The medium was prepared without methionine, and at the beginning of incubation I00 #Ci of a 7:2 mixture of [35S]methionine:[35S]cysteine (1100 Ci/mmol, ICN Radiochemicals) was added. Labeled polypeptides secreted into the medium were separated by SDS-PAGE (Laemmli, 1970) and detected with autoradiography. Isolation of cDNAs to differentially regulated transcripts Total fat body RNA was isolated (Chirgwin et al,, 1979) from untreated, day-15 cockroaches, and poly(A)+ RNA was selected by oligodeoxythymidylate--cellulosechromatography (Aviv and Leder, 1972). Doube-strand eDNA (complementary DNA) was prepared according to Gubler and Hoffman (1983), ligated to EcoRI/NotI linker-adaptors (Invitrogen, San Diego, Calif.), and cDNAs i> 0.5 kb (kilobase) were isolated by electrophoresis in agarose and glass powder adsorption (Vogelsteinand Gillespie, 1979). cDNAs were ligated to bacteriophage 2gt 11 DNA (Young and Davis, 1983), packaged with a commercial extract (Stratagene Cloning Systems, La Jolla, Calif.), and propagated on

JAMES Y. BRADFIELDet al.

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Escherichia coli Y1088 lawns. Duplicate plaque lifts were made onto nylon filters (NEN Research Products). To isolate cloned cDNAs representing transcripts differentially expressed in response to JHA, filters were hybridized with 15-day fat body [32p]-cDNA(Schleifand Wensink, 1981) from decapitated JHA-treated cockroaches vs [32p]. cDNA from fat bodies of decapitated controls. Hybridization (106cpm/ml) was in 50% formamide, 4 x SSPE (20 x is 3.6 M NaCI, 0.2 M Na-phosphate buffer pH 7.4, 20 mM EDTA), 2% SDS, 0.5% nonfat dry milk at 42°C overnight, and washes were in 15mM NaC1, 10mM Tris--HC1 (pH 7.4), 1% SDS, 1 mM EDTA at 60°C. Plaques hybridizing to one cDNA preparation, but not to the other, were detected with autoradiography. One cloned cDNA from each of the two classes was used as a hybridization probe in experiments below. Northern blot analysis Total fat body RNA was isolated on day 15, denatured with MeHgOH (Bailey and Davidson, 1976), and separated by electrophoresisin 1.2% agarose. RNA was transferred to nylon filters and hybridized with nick-translated (Rigby et al., 1977) cloned cDNAs under conditions described above. Relative levels of JHA-stimulated and suppressed transcripts Steady-state levels of JHA-stimulated and suppressed fat body transcripts were monitored from days 0 to 15 by dot-blot hybridization. Total fat body RNA was quantified with an orcinol reaction (Dawson et aL, 1986) because A260 values were unreliable according to visualization of stained gels. One/~g samples were formaldehyde-denatured (White and Bancroft, 1982), applied to nylon filters using a hybridization manifold (Bethesda Research Laboratories) and hybridized with cloned cDNAs. Extent of hybridization was determined by liquid scintillation counting. Transcript responses in vitro Cockroaches were decapitated on day 0. On day 10, insects were immersed in 1% HgC12 and squeezed firmly several times to pump the HgC12 into the abdominal tracheae. Insects were then squeezed several times under 70% ethanol. Dissections were with sterile instruments in a laminar flow hood. Gut rupture was avoided. Fat bodies were rinsed several times with sterile saline and divided along the dorso-ventral midline: one-half was placed in culture medium alone, the other in medium adjusted to contain 100vg/ml methoprene suspended by sonication. The medium contained gentamicin (50 #g/ml) and pimaricin (25/~g/ml) to retard bacterial and fungal growth. Culture vessels had been siliconized (Schleif and Wensink, 1981) to minimize JHA adsorption to glass. Incubation was for 72 h as described (Keeley et al., 1988), with medium change at 6, 24 and 48 h. Samples with cloudy medium or noticeable tissue breakdown at the end of incubation were discarded. Extent of hybridization of cloned cDNAs to dot-blots (1 v g each) of total RNA was determined by liquid scintillation counting. RESULTS J H A -regulated f a t body protein synthesis The profile of polypeptide synthesis and secretion was determined at 15 days for in vitro fat bodies of untreated and 0-day decapitated females [Fig. I(A) and (B)]. Decapitation suppressed the synthesis of several polypeptides (90->200kDa), whereas two polypeptides (70-80 kDa) were markedly stimulated. Repeated topical application of JHA to decapitated insects restored the normal 15-day polypeptide profile [Fig. I(C)] and showed that JHA replaced head

factors that control protein synthesis/secretion by the B. discoidalis fat body. These results suggest that JH stimulates the syntheses of some fat body proteins while suppressing others. Isolation o f cDNAs representing differentially expressed transcripts To isolate cDNAs representing JHA-regulated fat body transcripts, we constructed a 2gt 1i library from fat body poly(A) + RNA of 15-day normal females. The library was screened with fat body [32p]-cDNA from 15-day, decapitated, JHA-treated cockroaches vs cDNA from decapitated controls. Of 4000 recombinant plaques screened with the two probes, 300 hybridized only with cDNA from JHA-treated decapitated insects, and 5 hybridized only with cDNA from decapitated controls. One cloned cDNA from each class was selected for further study. The two cDNAs were ~ 1 kb in length. Northern analysis Total fat body RNA was isolated at 15 days, separated by electrophoresis and transferred to nylon. Figure 2(A) shows hybridization with the cloned cDNA that represented a JHA-dependent transcript. The cloned probe hybridized to a 6.5 kb transcript from normal female body (lane 1) that was not detected in RNA from decapitated animals (lane 2) but was restored in decapitated insects by JHA application (lane 3). Hybridization of the three fat body RNA samples with the second cloned cDNA followed a different pattern. A 2.4 kb transcript was much more abundant in fat body from decapitated control cockroaches than in normal or decapitated, JHA-treated insects [Fig. 2(B)]. These experiments showed that JHA both enhanced and suppressed accumulation of different transcripts in adult B. discoidalis fat body. JHA-enhanced and suppressed transcripts were visualized by ethidium bromide staining of fat body poly(A) ÷ RNA separated in agarose [Fig. 2(C)]. RNA from decapitated, JHA-treated cockroaches contained a prominent 6.5 kb transcript (lane 1) not seen in the sample from decapitated controls (lane 2). By contrast, there were two clearly visible RNAs around 2.4 kb in decapitated insects that disappeared after JHA treatment. This visualization of the three transcripts shows that, in each case, they were remarkably abundant fat body RNAs with contrasting responses to JHA. The 6.5 kb JHA-stimulated transcript and one of the two suppressed 2.4kb transcripts seen in stained gel are likely represented by the two cloned cDNAs. Hybridization of the cloned cDNA to only one of the two suppressed transcripts suggests that the suppressed RNAs are not closely related to sequence. Transcript level time course Dot-blot hybridization monitored levels of the two fat body transcripts during the first half of the reproductive cycle. In fat body from females starved from day 0 to eliminate effects of food and water intake, the 6.5 kb transcript was not detected on day 0, appeared by day 3 and then accumulated steadily to a high level by day 15 [Fig. 3(A)]. The 2.4 kb RNA

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Fig. 1. In vitro synthesis and secretion of polypeptides by adult female B. discoidalis fat body. Day-15 fat bodies were incubated in culture medium containing [35S]methionine: [35S]cysteine for 9 h at 27°C. Labeled polypeptides in medium were separated by SDS-PAGE (7.5% gel) and detected with autoradiography, (A) Two normal females. (B) Females that had been decapitated on day 0. (C) Day 0-decapitated females treated topically with 10 pg methoprene on days 2, 5, 8 and 11.

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Fig. 2. (A) and (B) Northern hybridization of total fat body RNA (5 #g/lane) from 15-day adult female cockroaches. (A) Hybridization with cloned cDNA selected to represent a JH-stimulated transcript. Lane 1, normal females; 2, day 0-decapitated females; 3, day 0-deciptated females treated repeatedly with JHA. (B) Same samples hybridized with the cDNA selected to represent a JH-suppressed transcript. (C) Ethidium bromide-stained agarose gel of 15 day fat body poly(A) + RNA (5 pg/lane). Lane 1, females decapitated on day 0 and treated with JHA; 2, decapitated controls. The arrows indicate a 6.5 kb JHA-stimulated transcript and two ~ 2.4 kb JHA-suppressed transcripts, r indicates ribosomal RNA. The higher molecular weight material in panel (C), lane 2 is contaminating DNA.

108

JH-regulated gene expression

Table I, In vitro responsesof 6.5-2.4kb fat bodytranscripts to JHA. B. discoidalis femalesweredecapitatedon day 0. On day 10, one-halffat body was placed in culture medium adjusted to contain 100/~g]mlmethoprene (+JHA), the other half in medium alone (-JHA). Incubationwas for 72h at 27°C. Dot-blots of total RNA (l#g) were hybridized with cloned eDNAs representingthe 6.5 and 2.4 kb transcripts. Hybridizationwas measured by liquid scintillation 6.5 kb RNA 2.4 kb RNA (cpm) (cpm) Fat body -JHA + J H A -JHA +JHA 1 7 378 571 33 2 9 336 834 41 3 7 393 1152 21 4 7 267 1142 12 5 5 255 1386 7 6 20 695 1228 93 7 2 477 904 38

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DAYS AFTER EMERGENCE

Fig. 3. Day 0-15 levels of 6.5 kb JHA-enhanced and 2.4 kb JHA-suppressed fat body transcripts. For each point, 1#g total RNA from five pooled fat bodies was dot-blotted and hybridized with a cloned eDNA representing one of the two transcripts. Hybridization was measured by liquid scintillation. O, JHA-stimulated transcript; O, JHA-suppressed transcript. (A) Starved females. (B) Females decapitated on day 0. (C) Decapitated females treated repeatedly with JHA.

level was highest on day 0, declined strongly from days 0 to 3, and remained low but detectable thereafter. The 6.5 kb RNA was always rare or absent after decapitation [Fig. 3(B)]. In contrast, after an initial decline and lag, the 2.4 kb transcript rose dramatically between days 6-12 in decapitated females and declined after day 12. Repeated JHA treatment of decapitated cockroaches resulted in transcript patterns virtually identical to those in starved intact insects [Fig. 3(C)]. The responses of the two transcripts to JHA were pronounced. The 6.5 kb transcript, essentially absent in decapitated insects, became dominant in response to JHA treatment [see Fig. 2(C)]. The contrasting response of the 2.4 kb RNA to JHA was also striking. Based on titration (not shown), the day 12 level in decapitated insects was at least 100-fold higher than in starved or decapitated, JHA-treated animals.

Transcript responses in vitro

We tested direct action of JHA on the fat body to stimulate and suppress transcripts. Females were decapitated on day 0, and on day 10 [during presumed rapid accumulation of the 2.4 kb RNA; Fig. 3(B)] fat bodies were divided and incubated in absence and presence of JHA. After incubation, 6.5 and 2.4 kb transcript levels in total RNA were measured by dot-blot hybridization. We detected no in vitro response to JHA by either transcript within 24 h. By 48 h, both transcripts had responded in five of six fat bodies, but with varying magnitude (not shown). We observed strong, consistent responses at 72 h (Table 1). These responses agreed with the in vivo data and showed direct action of JHA on the fat body to regulate the two transcripts. We note that JHA not only prevented accumulation of the 2.4 kb RNA [see Fig. 3(B) and (C)], but also appeared to cause a drastic decline in steady-state levels.

DISCUSSION The studies described here show down-regulation of fat body transcripts and proteins by JH activity in an adult insect. They show direct action of JHA on the fat body to regulate specific transcripts. More importantly, they suggest dramatically contrasting modulations of gene expression by JH in the same tissue at the same time. The high-magnitude, contrasting responses of gene products of the B. discoidalis fat body will facilitate further molecular analysis of JH action. Among several systems being exploited for explaining JH action at the molecular level (Pau et al., 1986; Riddiford, 1986; Jones et aL, 1987, 1988; Wyatt, 1988), the cockroach fat body may offer an exceptional opportunity to study differential regulation of gene expression by JH. JH action on biosynthesis in the adult fat body is generally considered to be stimulatory. This is the case for B. discoidalis, where JH stimulates viteilogenesis and general protein synthesis (Wojchowski and Kunkel, 1987; Keeley et aL, 1988). The JHdependent accumulation of a fat body transcript and stimulation of polypeptide synthesis shown here is therefore not surprising. However, in contrast to the

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JAMESY. BRADFIELDet al.

stimulated products were two RNAs and polypeptides that were drastically suppressed by JH activity. Whether selective, marked JH suppression of gene expression in the adult insect fat body is widespread or restricted to only a few species remains to be determined. We do not know functions of the JH-stimulated and suppressed gene products. We suspect that the stimulated 6.5 kb RNA is a vitellogenin message because it is conspicuously large (see Gemmill et al., 1986; Locke et al., 1987); JH-dependent; first seen just before the onset of JH-dependent ovarian maturation (see Fig. 3 and Keeley et al., 1988); among the dominant transcripts in the mid-cycle fat body; apparently sex-limited (although inducible to a low level in males by JHA; not shown). Role(s) for the two JH-suppressed 2.4 kb transcripts are obscure. The 2.4 kb RNAs became very abundant in response to decapitation and were the right size to code the two abundantly-synthesized, 70-80 kDa polypeptides secreted by fat bodies of decapitated females. The 2.4 kb fat body messages and 70-80 kDa hemolymph polypeptides are also abundant in normal adult males (not shown). The 6.5 kb fat body message was always essentially absent in females deprived of JH. It was detected at a low level by 24 h after the first JHA application and accumulated in response to repeated JHA treatment with a pattern consistent with intact females. The pattern of the suppressed 2.4 kb transcript was also similar among JHA-treated decapitated insects and intact females. However, the 2.4 kb message did not respond to decapitation alone for several days. We do not know reasons for the long interval before accumulation of the 2.4 kb transcript in decapitated cockroaches. Presence of a small amount of JH in the hemolymph at the time of decapitation is likely, since corpora aUata of newly-emerged B. discoidalis synthesize JH at a low rate (G. Bhaskaran, personal communication). In vitro studies demonstrated that JHA acted on fat bodies to modulate the 6.5 and 2.4 kb RNAs. The transcript responses agreed with in vivo results and indicate that JH alone acts directly on the fat body to regulate gene expression at the pretranslational level. The data are consistent with direct action of JH on locust fat body to regulate protein synthesis (Wyatt et al., 1987). Since we monitored transcript responses by assessing steady-state levels only, we do not know whether JH regulates the 6.5-2.4 kb transcripts at the level of transcription or RNA stability. Another factor that must be addressed is the lag of a day or more between JHA administration and responses of the stimulated and suppressed RNAs. A comparably long lag is observed in the locust fat body (Dhadialla et al., 1987), where JH-regulated expression of heterologous genes is required for stimulation of vitellogenin mRNA (G. R, Wyatt, personal communication). Regulatory sequences linked to the genes represented in this paper may respond as a consequence of JH-chromatin interaction(s) elsewhere in the nucleus. Acknowledgements--We thank Drs E. P. Marks and H. Oberlander for advice on establishing long-term fat body

cultures, and Drs G. Bhaskaran and S. M. Rankin for manuscript review. This work was supported by the Texas Advanced Technology Research Program, the Robert J. Kleberg Jr and Helen C. Kleberg Foundation and National Science Foundation grant DCB 8511058 REFERENCES

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