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Genetics, Vol. 172, 253-264, January 2006, Copyright © 2006
doi:10.1534/genetics.105.046631
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,2,
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* Department of Entomology, Ohio State University, Columbus, Ohio 43210, Department of Neurology, University of Arizona, Tucson, Arizona 85721-0077, ARL Division of Neurobiology, University of Arizona, Tucson, Arizona 85721-0077, and Interdisciplinary Program in Insect Science, University of Arizona, Tucson, Arizona 85721-0077
1 Corresponding author: Department of Entomology, 318 W. 12th Ave., Ohio State University, 400 Aronoff Laboratory, Columbus, OH 43210.
E-mail: wilson.1457{at}osu.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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In Drosophila melanogaster, metamorphosis is controlled by several waves of 20E secretion: the first at the end of the third larval instar driving pupararium formation, the second 10–12 hr after pupararium formation (APF) triggering pupation, and finally a large wave beginning 25–30 hr APF (HANDLER 1982; RIDDIFORD 1993). JH has been detected in Drosophila larvae but not pupae (BOWNES and REMBOLD 1987; SLITER et al. 1987) when its absence is thought to permit 20E control of metamorphosis. Application of exogenous JH or JH analog insecticides, such as methoprene and pyriproxyfen which act as JHA (STAAL 1975; WILSON 2004), does not block pupararium formation or pupation. Rather, it results in disruptions of the adult abdominal epidermis, male genitalia orientation, and many internal tissues, resulting in death during the late pupal (pharate adult) stage (ASHBURNER 1970; MADHAVAN 1973; POSTLETHWAIT 1974; WILSON and FABIAN 1986; RIDDIFORD and ASHBURNER 1991; RESTIFO and WILSON 1998).
The primary response gene BR-C is composed of three lethal complementation groups: broad (br), reduced bristles on the palpus (rbp), and 2Bc, mutations of which cause death during prepupal or pupal stages (BELYAEVA et al. 1980; KISS et al. 1988). Null alleles of the entire gene, called nonpupariating1 (npr1), cause death in late third-instar larvae (KISS et al. 1988; GONZY et al. 2002), demonstrating that BR-C function is essential for metamorphic onset. BR-C uses alternative splicing to encode a small family of transcription factors with amino-terminal BTB–POZ domains linked to one of four possible C2H2 zinc-finger domains: BRC–Z1, BRC–Z2, BRC–Z3, and BRC–Z4 (DIBELLO et al. 1991; BAYER et al. 1996). During metamorphosis BRC isoforms regulate numerous downstream effector genes (GUAY and GUILD 1991; KARIM et al. 1993; VON KALM et al. 1994; HODGETTS et al. 1995; BAYER et al. 1996, 1997; CROSSGROVE et al. 1996; LIU and RESTIFO 1998; MUGAT et al. 2000; DUBROVSKY et al. 2001; DUNNE et al. 2002; SEMPERE et al. 2003). Although there is some functional redundancy among the isoforms, in general, BRC–Z1 provides the function for rbp, BRC–Z2 for br, and BRC–Z3 for 2Bc (CROSSGROVE et al. 1996; BAYER et al. 1997; SANDSTROM et al. 1997; LIU and RESTIFO 1998; CONSOULAS et al. 2005).
BR-C metamorphosis phenotypes include failures of larval tissue histolysis (LEE and BAEHRECKE 2001; KUCHAROVA-MAHMOOD et al. 2002), epidermal morphogenesis (KISS et al. 1988), and internal tissue remodeling (RESTIFO and WHITE 1991, 1992; SANDSTROM et al. 1997; CONSOULAS et al. 2005). Several phenotypes, such as a split-brain abnormality (RESTIFO and WHITE 1991) were common to all BR-C mutants, while others, such as failure of thoracic muscle attachment or persistence of larval salivary glands (RESTIFO and WHITE 1992) were restricted to one or two complementation groups. This suggested that BRC proteins operate in two pathways, "common," requiring all three functions and "restricted," requiring a subset of them (RESTIFO and WHITE 1991, 1992; RESTIFO and WILSON 1998). Following metamorphosis BR-C is expressed in egg chamber follicle cells to function during oogenesis (HUANG and ORR 1992; DENG and BOWNES 1997; TZOLOVSKY et al. 1999).
Our previous work showed that lethal pharate adults developing from wild-type methoprene-treated larvae showed a selective BR-C phenocopy, including disruptions of the central nervous system and salivary glands (RESTIFO and WILSON 1998). The methoprene syndrome was striking in that it included none of the restricted-pathway defects, which is inconsistent with methoprene simply blocking the 20E induction of BR-C expression during the larval-to-pupal transition. We interpreted the pathological effects of methoprene to reflect dysfunction of BR-C and probably additional primary response genes during metamorphosis, resulting in aberrant expression of secondary response genes. In contrast, other investigators found that the JHA pyriproxyfen caused abnormal pupal cuticle gene expression, apparently due to abnormal BR-C expression (ZHOU and RIDDIFORD 2002).
Methoprene-tolerant (Met) is essential for the manifestation of the toxic and morphogenetic effects of JH/JHA in D. melanogaster (WILSON and FABIAN 1986; RIDDIFORD and ASHBURNER 1991; WILSON 1996; RESTIFO and WILSON 1998). Met mutants are resistant to these effects of methoprene (WILSON and FABIAN 1986). MET can bind JH III with specificity and nanomolar affinity (SHEMSHEDINI and WILSON 1990; MIURA et al. 2005), suggesting that it is a component of a JH receptor. Met encodes a bHLH–PAS transcriptional regulator family member (ASHOK et al. 1998) and MET can activate a reporter gene in transfected Drosophila S-2 cells (MIURA et al. 2005).
We hypothesize that BR-C and Met function together in one or more aspects of development. If methoprene disrupts 20E-mediated metamorphic development or oogenesis by acting on BR-C or its downstream genes, one would expect double mutants to show synergistic genetic interaction, such as synthetic lethality of viable alleles, shifts in lethal phase, or enhanced oogenesis defects. We found that animals carrying both Met and BR-C mutations showed just such synergistic interactions. However, we did not detect any major disruption in BRC protein accumulation following methoprene treatment, suggesting that the MET and BRC interact to regulate expression of downstream effector gene(s).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Double mutants were constructed by genetic recombination, and each was maintained heterozygous with an FM7 balancer chromosome. Each double-mutant chromosome was tested to verify the presence of each mutation and the absence of inadvertently introduced lethal mutations.
D. melanogaster culture and methoprene treatment:
Stocks and crosses were cultured on one of three standard media with mold inhibitors, at 25° with a 12:12 L:D photoperiod unless otherwise specified. For experiments comparing phenotypes, the same culture medium and other conditions were used for all genotypes. For progeny phenotype analysis, cultures were performed in glass vials (Capital Vial) without larval crowding, and all progeny were censused and examined, either as adults or as uneclosed pupae.
Cultures were tested for methoprene resistance on diagnostic doses given in Table 5 of ZR-2008, the biologically active isomer of methoprene [isopropyl-(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienonate], as previously described (WILSON 1996; WILSON and ASHOK 1998). At lethal concentrations, mortality occurs in Met+ typically during the pharate adult stage. Eclosing survivors were examined for methoprene-induced morphogenetic defects of malrotated male genitalia and defective sternal bristles, particularly on the posterior sternites. For analysis of BRC proteins, 100–120 OreRC or v Met27 eggs were transferred to glass bottles containing Drosophila Instant Food (Carolina Biologicals) with either a high-lethal dose of methoprene or vehicle (acetone) alone, as described in RESTIFO and WILSON (1998). Control and methoprene-treated cultures were reared in parallel.
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Ovipositional rate and ovary examination:
Females isolated within 4–6 hr after eclosion were provided with wild-type (OreRC) males in food vials sprinkled with baker's yeast. Egg counts were made at 2-day intervals when the medium was changed, and fertility of the eggs was noted. Oogenesis was assessed by dissecting ovaries from females at several times after eclosion and examining for the presence of stages 8–14 vitellogenic oocytes as previously described (WILSON and ASHOK 1998).
Analysis of BRC protein accumulation by immunoblotting:
White prepupae (WPP) were collected and either homogenized immediately or placed in humid chambers for further development. In experiments with late-pupal stages, animals were resynchronized at head eversion. Protein extraction was based on the method of EMERY et al. (1994). For each time point, 5–10 animals were homogenized in 50–100 µl sample buffer with a Teflon pestle in a microcentrifuge tube. Sample buffer consisted of 75 mM Tris-HCl, pH 6.8, 6% SDS, 15% glycerol, 10% ß-mercaptoethanol and protease inhibitors (0.1 µg/µl pepstatin A, 0.5 µg/µl leupeptin, and 10 mM PMSF, Sigma). Following centrifugation for 10 min at 14,000 rpm (Eppendorf 5415C), the supernatant was used immediately for electrophoresis or stored at –80° for up to 3 weeks, which did not compromise BRC protein stability (data not shown).
Extracts representing 0.25- or 0.5-animal equivalents were heated for 5 min at 90°, quick chilled on ice for 10 min, and separated by SDS–PAGE (TOWBIN et al. 1979). For optimal band separation and size assessment, we used large (16 x 18 cm) 10% acrylamide gels on a Hoefer SE 600 Ruby electrophoresis apparatus at constant current (30 mA) for 5 hr. Otherwise, 12% acrylamide gels (7 x 8 cm) were run on a Bio-Rad mini-PROTEAN II apparatus at constant voltage (195 V) for 50 min. Proteins were transferred to nitrocellulose membranes (Protran, 0.45 µm, Schleicher & Schuell) by electroblotting at 4° overnight. Overall protein pattern was detected by staining the membrane with 0.5% Ponceau-S (Sigma, St. Louis).
Nonspecific binding sites were blocked with phosphate-buffered saline (PBS) plus 0.1% Tween 20 (TPBS) and 5% w/v nonfat dry milk powder (Bio-Rad) for 90 min at room temperature. Blots were probed with either anti-BRcore (mAb25E9) or anti-Z1 (mAb3C11) mouse monoclonal antibodies (EMERY et al. 1994), diluted 1:2,500 or 1:100, respectively, in PBS + 5% w/v milk powder, for 2 hr at room temperature or overnight at 4°. After three 10-min washes in TPBS, the blots were incubated with HRP-conjugated goat anti-mouse IgG (Cappel, ICN) at 1:10,000 (with anti-BRcore) or 1:2,500 (with anti-Z1) for 1 hr at room temperature. After another three 10-min washes, the signal was revealed by chemilumiscent detection of HRP (ECL detection kit; Amersham-Pharmacia) and exposure to X-ray film (Kodak X-OMAT AR). To evaluate lane loading, the blot was stripped (by serial 10-min washes in water, 0.2N NaOH, water, and TPBS), reblocked, and reprobed with anti-actin (mAb1501; Chemicon) at 1:20,000 (MACKLER and REIST 2001).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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60% eclosion (Figure 1). Careful examination of rbp2/npr13 mutants, starting at puparium formation and continuing throughout metamorphosis, showed that 100% of them were able to pupate, after which 15% died as early pupae (Figure 1). Eighty percent of the original cohort survived to late pharate adult stage, with 20% failing to eclose. In contrast, when the hypomorphic-viable allele Met3 was crossed onto each of the BR-C mutant chromosomes, the survival curve of the resulting heteroallelic double mutants was shifted (Figure 1), showing a dramatic reduction in viability.
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15% mortality during pupal development (Figure 1). Flies homozygous for rbp2 and carrying one copy of Met27 showed good survival to adults (Table 1). In contrast, survival of rbp2 Met27 homozygotes to the adult stage was poor, dying typically during the pharate adult stage (Table 1).
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Lethality in rbp2 Met27 homozygotes or rbp2 Met27/rbp1 Met27 heteroallelic pupae could be rescued by one copy of Met+ as the p[EN71] transgene (Table 1).
Interaction between Met and br alleles:
We next tested for interaction between Met and br. A viable br allele, br1, exists (MORGAN et al. 1925; KISS et al. 1988). Individuals homozygous for br1 Met27 were usually lethal, but escaper adults eclosed in small numbers (Table 1), and hemizygotes showed slightly higher viability (Table 3) and were fertile. Examination of br1 Met27 pupae showed lethality in both pupal and especially pharate adult stages. Transgenic br1 Met27; p[EN71]/+ flies readily survived (Table 1). Individuals homozygous or hemizygous for the severe br5 allele were lethal in early pupal development (KISS et al. 1988) and double mutants of Met27 with br5 were likewise lethal in prepupal/early pupal development. Heteroallelic br5 Met27/br1 pupae survived well, but br5 Met27/br1 Met27 heteroallelic individuals were lethal in prepupal/early pupal development (Table 1), an effect of Met27 similar to that seen in rbp2 Met27/rbp1 Met27.
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Altered BR-C complementation patterns in the absence of MET:
BR-C complementation group mutants complement one another to varying extents, with rbp partially complementing br, and 2BC fully complementing both rbp and br (BELYAEVA et al. 1980; KISS et al. 1988). To determine if Met27 influences complementation for viability, double mutants for both the weak and severe alleles of br and rbp and for 2Bc were crossed and survival of the F1 examined. The absence of Met+ resulted in failure of br5 and rbp1 to complement one another as well as reduced complementation between br5 and rbp2 (Table 1). Complementation was reduced between 2Bc1 and either br5 or rbp1. However, complementation in the Met27 double mutants continued to be robust between either of the weak alleles br1 or rbp2 with 2Bc1 (Table 1). Therefore, an absence of MET reduced complementation to varying degrees, depending on the alleles examined, being less evident with the weak alleles and with the 2Bc1 mutation.
Oogenesis in BR-C Met double mutants:
Both of these genes are involved in oogenesis in Drosophila (HUANG and ORR 1992; WILSON and ASHOK 1998; TZOLOVSKY et al. 1999). Normally, vitellogenic oocyte development begins within 12 hr following eclosion, and mature eggs begin to be oviposited within 48 hr (KING 1970; WILSON and ASHOK 1998). BR-C homozygotes for the weak alleles br1 and rbp2 showed good oviposition, although below that of v/v females (Table 3). Met27 females show oogenesis reduced to 20% of wild type (WILSON and ASHOK 1998). To determine if interaction between BR-C and Met is apparent during this process in adults, oviposition was examined in BR-C Met homozygotes at 2-day intervals following eclosion. Homozygous br1 Met27 and rbp2 Met27 females occasionally survived to adulthood, and survivors showed strong reductions in oviposition: both br1 Met27 and rbp2 Met27 homozygotes laid only a few eggs during an 8-day examination period, and casual examination of these females for another week showed no change in the ovipositional pattern.
To determine if the ovipositional failure was due to a defect in oogenesis or in oviposition, both br1 Met27 and rbp2 Met27 females were dissected at 5–7 days following eclosion and their vitellogenic oocytes staged and censused. Despite having access to ample yeast and courting wild-type males, there were few (usually 0–3) vitellogenic oocytes in the ovaries of these females, showing that the defect resides in oogenesis, not oviposition. However, no degenerating oocytes were present, a condition suggestive of hormonal disruption (WILSON 1982; SOLLER et al. 1999).
Oviposition was also measured in the br1 Met double-mutant combinations of the various Met alleles. Some of the allele combinations resulted in severe disruption of oviposition (Table 2), presumably due to defects in oogenesis, as seen for Met27. Other combinations resulted in ovipositional rates higher than those of br1 Met27. In summary, the severe depression in oviposition seen for some Met allele combinations represents a strong allele interaction with br1 and is not specific for the Met27 allele.
The link between BRC and methoprene:
We addressed the enigmatic relationship between BR-C and methoprene in three contexts: (i) the overlap between methoprene-induced defects and BR-C common phenotypes, (ii) the influence of BR-C function on sensitivity to methoprene, and (iii) the effect of methoprene on BR-C expression.
The BR-C common phenotypes include malrotation of male genitalia:
In our previous study, we showed that methoprene treatment of wild type causes a specific partial phenocopy of BRC-associated internal defects (RESTIFO and WILSON 1998). Here, we tested whether BR-C mutants of each complementation group show the well-known methoprene-induced malrotation of the male genitalia (POSTLETHWAIT 1974; WILSON and FABIAN 1986). In some cases, this required combining strong mutations with moderate wild-type transgene activity, an established method for revealing late developmental functions (HALL and THUMMEL 1998). The malrotation phenotype is of particular interest because a genetic interaction between Met and spin, a Fas2 mutation with a malrotation phenotype, has recently been described (ADAM et al. 2003).
For rbp, we found that 100% of rbp1/Y hemizygotes have malrotated genitalia (Table 4). This fully penetrant phenotype was rescued by a BR-C+ Y-borne duplication y2Y67g and uncovered by the Y-borne duplication with an interstitial deletion y2YSz280 that lacks all BR-C sequences, confirming that it maps to the BR-C region. To generate br mutant males that die as pharate adults (when the genitalia are pigmented), we partially rescued br-null mutants br5/Y using two transgenic copies of heat-shock-inducible BRC–Z2. Optimal heat-shock protocols rescue lethality (BAYER et al. 1997), gene expression (LIU and RESTIFO 1998), and CNS morphogenesis (R. F. SPOKONY and L. L. RESTIFO, unpublished data). To obtain partial rescue, we heat-shocked unsynchronized third-instar larvae once (37°, 1 hr), and then twice more 18 and 23 hr later. This resulted in very small numbers of pharate adults, 93% of which (14/15) had malrotated genitalia (Table 4). For 2Bc, we first observed malrotation in two very rare, late-dying 2Bc2/Y mutants (RESTIFO and WHITE 1991, 1992; CONSOULAS et al. 2005). To examine larger numbers, we used a "leaky" BRC–Z3 transgene whose expression is dependent on temperature (see MATERIALS AND METHODS). We found malrotated genitalia, inversely related to rearing temperature: 14% at 25° and 40% at 22–23° (Table 4).
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Sensitivity of BR-C mutants to methoprene:
Met mutations confer semidominant resistance to both the toxic and morphogenetic effects of methoprene (WILSON and FABIAN 1986; RESTIFO and WILSON 1998). To determine if BR-C mutations, either singly or in double-mutant combination with Met, affect the response to methoprene treatment, larvae were raised in the presence of one of four diagnostic concentrations of methoprene and evaluated for survival and the external morphology of surviving adults. Viable BR-C mutations do not change the susceptibility to methoprene from that of wild type (Table 5). Similarly, reducing the dose of 2Bc+ or BR-C+ by 50% (2Bc1/FM7 or npr13/FM7, respectively) did not shift the sensitivity to methoprene. Moreover, in the double mutant, br1 did not affect the resistance conferred by Met3. Hence, BR-C+ function does not appear to impact the MET-dependent methoprene-sensitivity mechanism.
Effect of Met and methoprene on BRC protein accumulation:
We investigated BRC protein expression for two reasons. First, a plausible mechanistic explanation for the genetic interaction between Met and BR-C would be that Met+ upregulates BR-C expression, and that reduced BRC levels in Met mutants would enhance the lethality of partial-loss-of-function BR-C genotypes. Second, reported effects on BR-C transcript levels caused by JH/JHA treatment (ZHOU et al. 1998; ZHOU and RIDDIFORD 2002) have failed to provide an explanation for the methoprene phenocopy of BR-C common defects, especially those involving internal structures of the head and thorax (RESTIFO and WILSON 1998).
The BRC family of proteins, which migrate as three size groups (EMERY et al.,1994; MUGAT et al. 2000), is readily detected by immunoblotting of proteins extracted from whole white prepupae (Figure 2A). The largest group, Emery's p118, is thought to represent BRC–Z4; the middle group, Emery's p91 and p81 contains BRC–Z1 and –Z3; the smallest group, Emery's p64 and p57 contains BRC–Z2. Over the subsequent 24 hr, especially after head eversion (12 hr APF), BRC protein levels declined (Figure 2B). In Met27 mutants, the pattern and relative abundance of BRC isoforms detected over this first day of metamorphosis was indistinguishable from those of wild type (Figure 2B). Likewise, methoprene treatment of wild-type animals did not change the overall quantities and isoform patterns of BRC proteins (Figure 2C).
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75% of metamorphosis, during which animals were resynchronized at head eversion. At 24 hr APF (12 hr after head evesion), BRC proteins were detectable in the methoprene-treated animals, but not in the controls (Figure 3A). Similarly, methoprene-treated animals showed mild persistence of BRC–Z1 during midpupal stages, following a peak in accumulation at the normal time, 8 hr APF. There was no reappearance of BRC–Z1 or any other BRC isoforms during mid-to-late pupal stages (Figure 3A), even on very long exposures of the immunoblots (data not shown). In summary, chronic larval treatment with lethal doses of the JH agonist and mimic methoprene does not block BRC protein accumulation during the first day of metamorphosis, a developmental window in which BR-C function is essential.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Met interacted with both the weak-viable alleles br1 and rbp2 and with the severe alleles br5 and rbp1 (Table 1) during pupal development. Each of the weak alleles possesses sufficiently functional gene product to permit completion of pupal development; but this amount is insufficient when MET is absent or defective. The more severe rbp1 homogygotes are pupal lethal, but only at late metamorphosis, in the pharate adult stage (BELYAEVA et al. 1980). Lethality was shifted in rbp1 Met27 pupae to prepupal/early pupal development, suggesting that MET absence causes the residual rbp1 function to be insufficient during these earlier stages in pupal development. Homozygotes of br5 and 2Bc die in the early and late prepupal stage, respectively (KISS et al. 1988), and the double mutants with Met27 showed a similar phenotype, demonstrating that the interaction cannot shift lethality to an earlier stage, late third-instar larvae. Our observations are consistent with the interaction between BR-C and Met beginning in prepupal or early pupal development. While we interpret the Met–BR-C interaction as enhancing the lethality of br and rbp mutations, it is also possible that Met becomes an essential gene when BR-C function is reduced, or that the interaction is mutual, such that both mutations become more severe in phenotype when they are present together.
Genetic interaction became strikingly evident when complementation failures between mutant alleles from different BR-C complementation groups occurred in the presence of Met27 (Table 1). Without MET, developing animals may be less able to make use of the partial functional redundancy among BRC isoforms that has been documented previously (BAYER et al. 1997).
The interaction between mutant alleles of BR-C and Met was also evident in the adult stage when oogenesis was examined. Both the rate of oviposition and the paucity of vitellogenic oocytes in ovaries of br1 Met27 and rbp2 Met27 females reflects almost complete failure of oogenesis, with only a few eggs oviposited during the lifetime of the female.
Previous studies have also detected BR-C interaction with other genes. Double mutants of BR-C with another primary response gene E74 show interaction for some but not all of the phenotypic characters (FLETCHER and THUMMEL 1995). In addition to interactions among transcription regulators of the ecdysone cascade, br alleles interact with genes involved in imaginal disc morphogenesis, including those encoding an atypical serine protease, Stubble-stubboid (BEATON et al. 1988; APPEL et al. 1993), nonmuscle myosin II heavy chain (zipper), the Drosophila serum response factor transcription factor [blistered: (GOTWALS and FRISTROM 1991)], the small GTPase Rho1, cytoplasmic tropomyosin, and 22 others (WARD et al. 2003).
Although BR-C expression and function overlap the JH/JHA-sensitive period, data presented above demonstrate that methoprene treatment does not block BRC expression in either wild-type or Met-null mutants (Figures 2 and 3). Furthermore, the methoprene phenocopy, which excludes complementation group-specific defects (e.g., larval salivary gland persistence, which is rbp-restricted), is not consistent with methoprene simply reducing BRC expression (RESTIFO and WILSON 1998). We propose that JH application results in abnormal function of BRC proteins, thus phenocopying certain characteristics common to all BR-C mutants. Therefore, the link between BR-C mutant phenotypes and JH-induced defects could be abnormal regulation of target genes, resulting in the phenotypic characteristics observed (Figure 4). Previously, we suggested several possibilities to explain methoprene pathology and BR-C phenocopy, including BRC interaction with an unidentified partner, perhaps MET (RESTIFO and WILSON 1998). We believe that the Met–BR-C genetic interaction reported in this work can be explained best by this hypothesized protein–protein interaction between MET and BRC to regulate one or more target genes. Supporting our hypothesis are the following findings: first, both proteins are located in the nucleus (EMERY et al. 1994; RESTIFO and HAUGLUM 1998; PURSLEY et al. 2000; RENAULT et al. 2001), so there is no compartmental barrier to interaction. Second, both proteins appear to be transcription factors: BRC isoforms bind specific DNA sequences (VON KALM et al. 1994; DUBROVSKY et al. 2001) and regulate transcription (DUBROVSKY et al. 1994; HODGETTS et al. 1995; CROSSGROVE et al. 1996; MUGAT et al. 2000). BR-C mutants have misexpressed secondary-response and other target genes (GUAY and GUILD 1991; KARIM et al. 1993; MUGAT et al. 2000; RENAULT et al. 2001; DUNNE et al. 2002; SEMPERE et al. 2003). MET is a member of the bHLH–PAS family of transcription factors (ASHOK et al. 1998) and was recently shown to act as one (MIURA et al. 2005). Third, both are found at common times during development, such as prepupae (KARIM et al. 1993; ASHOK et al. 1998) and during vitellogenic oocyte development (TZOLOVSKY et al. 1999; PURSLEY et al. 2000). Finally, PAS domains in bHLH–PAS proteins are thought to promote protein–protein interaction (HEERY et al. 1997), either with other PAS proteins or as coactivators with nuclear receptor proteins (XU et al. 1999), and the BTB/POZ domain of BRC has been implicated in protein–protein interaction (ZOLLMAN et al. 1994; MELNICK et al. 2002).
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70% amino acid identity) homology to Met (MOORE et al. 2000) that could substitute for MET to rescue larval and/or pupal development. However, this substitute does not appear to be satisfactory if BR-C is mutant. When a gce mutant becomes available, its phenotype could help evaluate this hypothesis. How does the application of exogenous JH act to phenocopy BR-C? It is clear that the action of these compounds occurs through MET, probably acting as a JH receptor component (WILSON and FABIAN 1986; SHEMSHEDINI et al. 1990; SHEMSHEDINI and WILSON 1990; ASHOK et al. 1998; MIURA et al. 2005). JH is present during larval development when it presumably acts to prevent premature metamorphosis resulting from each wave of 20E secretion that triggers a molt. This failsafe mechanism may occur by JH binding by and conformational change of MET, resulting in regulation of genes necessary for molting or perhaps simply blocking expression of metamorphic genes. Studies with Drosophila S-2 cells have implicated the transcription factor E75A in promoting JH regulation of larval development (DUBROVSKY et al. 2004). At metamorphosis, when little or no JH is present (BOWNES and REMBOLD 1987; SLITER et al. 1987), BR-C is expressed, and we propose that BRC dimerizes with the nonliganded MET protein to regulate a different set of target genes, promoting the initiation of metamorphosis. If exogenous JH is present during this time, it binds to MET and results in a more larval conformation, resulting in inappropriate binding to BRC and leading to a change in target-gene expression patterns consequently seen as defects characteristic of BR-C mutants.
Other work has implicated BR-C in the action of the JH agonist pyriproxyfen during metamorphic disruption. ZHOU and RIDDIFORD (2002) showed that application of this compound to white prepupae resulted in re-expression of BRC–Z1 in the abdomen during late pupal development, which in turn caused abnormal development of abdominal epidermis, including bristle disturbances. Those findings differ from ours with methoprene in two significant ways. First, a lethal dose of methoprene caused a mild enhancement and prolongation of BRC protein accumulation in young pupae, but no re-expression at later times (Figure 3A). Second, the modest effect of methoprene on BRC protein profiles cannot mediate the developmental effects of this JHA because the same mild persistence of BRC was seen in Met27 mutants (Figure 3B), which are protected against methoprene-induced defects. It is not clear what underlies the difference in response of BR-C to methoprene and pyriproxyfen. We note that pyriproxyfen is a more powerful JH agonist than methoprene (RIDDIFORD and ASHBURNER 1991), but qualitative differences in the actions of the two compounds may exist as well.
In summary, our results provide genetic evidence that supports other studies implicating BR-C as a focal point for interaction of JH and 20E signaling pathways, and they suggest that BRC and MET interact to regulate expression of one or more effector genes involved in metamorphic development.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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3 Present address: Department of Entomology, University of Georgia, Athens, GA 30602-2603.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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