Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 2008 Jan;178(1):273–281. doi: 10.1534/genetics.107.080754

rosy Function Is Required for Juvenile Hormone Effects in Drosophila melanogaster

Xiaofeng Zhou 1,1, Lynn M Riddiford 1,2
PMCID: PMC2206077  PMID: 18202373

Abstract

Application of a high dose of juvenile hormone (JH) III or its mimics (JHM) to Drosophila at the white puparium stage causes the formation of a pupal-like abdomen with few or no short bristles. We report here that the rosy (ry) gene encoding the enzyme xanthine dehydrogenase (XDH), which catalyzes the final two-step oxidation in purine catabolism, is required for this effect of JH on the epidermis. In ry506 (null allele) homozygotes or hemizygotes, JH III or pyriproxifen (a JHM) had little effect on abdominal bristle or cuticle formation, but disrupted the development of the central nervous system as in wild-type flies. Wild-type ry rescued the JH sensitivity of the abdominal epidermis in ry506 mutants. Inhibition of XDH activity phenocopied the ry null mutant's insensitivity to JH. Larvae fed on hypoxanthine or xanthine showed a decreased JH sensitivity. ry506 clones were sensitive to JH, indicating that ry is required non-cell autonomously for the JH effects. Normally JH applied at pupariation causes the aberrant reexpression of the transcription factor broad in the abdominal epidermis during adult development, but in the ry506 mutant most of the cells in the dorsal tergite showed no broad reexpression, indicating that ry is upstream of broad in the JH signaling pathway.

JUVENILE hormone (JH), an insect sesquiterpenoid related to retinoids, has a wide range of functions in regulating development and physiological processes such as metamorphosis, caste determination, ovarian maturation, diapause, and migration in insects (Riddiford 1994, 1996; Wyatt and Davey 1996; Goodman and Granger 2005). A holometabolous insect (complete metamorphosis) molts several times during the larval stages and then undergoes metamorphosis, first into a pupa and then into an adult. These processes are largely regulated by JH and the steroid hormone ecdysone (Riddiford et al. 2001). In the presence of JH, ecdysone causes a molt to a similar stage (thus, a larva molts to another larval stage, and experimentally a pupa given JH molts to a second pupa); but in the absence of JH, ecdysone causes a metamorphic molt (Riddiford 1994). This effect of JH is defined as its “status quo” action (Williams 1952). Typically, the JH titer in a final instar larva drops to an undetectable level, which allows ecdysone to initiate metamorphosis. No JH is produced in the early pupa, and consequently ecdysone causes an adult molt.

Unlike Lepidoptera and most other holometabolous insects, higher Diptera such as Drosophila melanogaster have lost most of their sensitivity to JH. Topical JH application cannot prevent the transition from larva to pupa in D. melanogaster (Ashburner 1970; Postlethwait 1974). Nevertheless, application of high amounts of JH or a JH mimic (JHM) before or at the time of pupariation causes the formation of a pupal–adult mosaic: a transparent abdomen covered with pupal cuticle with no or a few short bristles and a relatively normal appearance of adult head and thorax (Figure 1, B and C) (Ashburner 1970; Postlethwait 1974; Riddiford and Ashburner 1991; Zhou and Riddiford 2002).

Figure 1.—

Figure 1.—

Open in a new tab

JHM given at pupariation does not prevent outgrowth of the abdominal bristles in rosy (ry)-deficient mutants as it does in wild-type flies. Pyriproxifen (a JHM) and acetone (control) treatment were as described in materials and methods. (A) A control wild-type Canton-S (CS) pharate adult was removed from its puparium just before eclosion. (B–K) JHM-treated pharate adults of wild-type, P-element insertion lines (KG00431 and KG06479) and various ry alleles failed to eclose; each was removed from its puparium on day 5 after pupariation for scoring of abdominal bristles. The pictures are typical of a total of treated puparia for each line: (A) 15, (B) 18, (C) 42, (D) 43, (E) 45, (F) 27, (G) 47, (H) 15, (I) 14, (J) 14, and (K) 27. KG00431 and KG06479, P-element insertion lines in a ry 506 background; ry 506, a null mutant of rosy; and ry1, ry2, ry8, and ry26 are all hypomorphic alleles of rosy.

We previously showed that the molecular basis of this status quo action of JH on the Drosophila abdomen was due to the reexpression of the broad (br) gene in the epidermis during adult development (Zhou and Riddiford 2002). Those studies showed that Broad (BR) specified pupal development and inhibited both larval and adult development. BR is a member of the broad complex Tramtrack bric-á-brac/pox virus and zinc finger (BTB/POZ) domain-containing transcription factor family (Dibello et al. 1991; Bardwell and Treisman 1994; Zollman et al. 1994). These factors are known to interact with chromatin regulatory proteins via the BTB/POZ domain and stabilize a particular configuration (Melnick et al. 2000). One member of this family, promyelocytic leukemia zinc finger (PLZF), is a transcriptional repressor that interacts with the histone deacetylase corepression complex (Lin et al. 1998). Its fusion with the retinoic acid receptor α (RARα) protein due to a chromosomal translocation leads to acute promyelocytic leukemia since the fusion protein is unable to bind to the RAR response element (Lin et al. 2001).

Here we show that the rosy (ry) gene is required for the juvenilizing effects of JH in the abdominal epidermis of D. melanogaster. The ry gene encodes the enzyme xanthine dehydrogenase (XDH), which is necessary for the formation of uric acid (Chovnick et al. 1990). XDH plays an important role in nucleic acid degradation in all organisms; i.e., it catalyzes the oxidation of hypoxanthine to xanthine and then to uric acid. In both primates and insects, uric acid is the final product of purine degradation, but in most other mammals, uric acid is further oxidized to the more soluble allantoin as the end product. Uric acid is a powerful antioxidant that directly scavenges reactive oxygen species and chelates iron in humans and Drosophila (Ames et al. 1981; Davies et al. 1986; Hilliker et al. 1992; Vorbach et al. 2003; Glantzounis et al. 2005). In xanthinuria type I patients, mutations of the human XDH gene cause loss of the enzyme function, resulting in high plasma levels of hypoxanthine and xanthine, which may lead to urinary tract calculi, acute renal failure, or myositis (Simmonds et al. 1995). Our finding of the requirement of ry for the effects of high JH on the developing abdominal epidermis links the purine degradation pathway to a growth regulatory hormonal signal.

MATERIALS AND METHODS

D. melanogaster strains:

All flies were raised at 25° on standard cornmeal–molasses-based medium (Sullivan et al. 2000). Canton-S was used as wild type. JH-insensitive P-element insertion fly lines were KG00431(FBst0013662) and KG06479 (FBst0014492) (Bellen et al. 2004). The deficiency chromosomes used were Df(3R)Exel8157 (deleted region 87D8; 87D10), Df(3R)ry615 (deleted region 87B11–13; 87E8–11), and Df(3R)ry506-85C (deleted region 87D1–2; 88E5–6). The ry alleles used in the present study include ry1, ry2, ry8, ry26, and ry506. Fly strains carrying wild-type ry+t7.2 in a ry null background were P{ry11}fs(2)41, P{ry11}fs(2)111, and P{ry11}fs(2)121 (Berg and Spradling 1991). The following strains were used as a starter set to generate ry506 clones: UAS-FLP yw, arm-GAL4, FRT 75A, yw; +; P{wHy}DG25107, and ry506. All the fly strains were obtained from the Bloomington Stock Center (http://flystocks.bio.indiana.edu/).

JH application:

One hundred nanograms or otherwise designated amounts of the JHM pyriproxifen (Sumitomo, Osaka, Japan) or 500 ng JH III (Sci Tech, Praha, Czech Republic) in 0.2 μl acetone (J. T. Baker) were topically applied to the dorsum of a white puparium (within 30 min of pupariation). Control puparia each received 0.2 μl acetone. The JH III- or JHM-treated animals were kept at 25° in a humidified chamber for 5 days, and then their abdomens were scored as follows: long bristles (normal, Figure 1A), short bristles (Figure 1B), and bristleless (Figure 1C).

RNA extraction and Northern analysis:

Total RNA was extracted from the whole abdomen of D. melanogaster at timed stages by the proteinase K/phenol method (Andres et al. 1993). Ten micrograms of denatured RNA were separated on a 1% agarose gel containing 2.2 m formaldehyde and transferred to a Duralon UV membrane (Stratagene, La Jolla, CA), which was then cross-linked in an UV Stratalinker (Stratagene). Specific cDNA probes labeled with [α-32P]dATP (Amersham, Arlington Heights, IL) were used to detect the transcripts of the br core region, Edg78E and Acp65A as described previously (Zhou and Riddiford 2002).

Feeding experiments:

Thirty milliliters standard cornmeal–molasses-based medium were melted in a microwave oven and a designated amount of compound was mixed into the medium at 60°, to make a medium containing 250 ng/ml allopurinol [4-hydroxypyrazolo (3,4-d) pyrimidine; Sigma, St. Louis], 100 mg/ml hypoxanthine (Sigma), or 100 mg/ml xanthine (Sigma). Approximately 6-ml portions were dispensed into 25 × 95-mm vials. About 15 pairs of wild-type adult flies were placed in each vial and allowed to lay eggs for 2–3 days.

ry− clonal analysis:

We used the FLP/FRT system to produce ry− clones in the abdominal epidermis (Xu and Rubin 1993). To generate flies capable of producing clones, we crossed ∼30 males of yw/Y; +; FRT75A, ry506 to ∼30 females of yw, UAS-FLP; arm-GAL4; FRT75A, P{wHy}DG25107. In their progeny, the ubiquitous expression of arm-GAL4 drove FLPase to generate ry506 clones in various tissues including the abdominal epidermis. The ry506 clones were marked with yellow−, so that they could be easily identified.

Immunocytochemistry:

Abdominal integument and central nervous systems (CNS) were dissected in phosphate-buffered saline (PBS) (9.7 mm phosphate, 154 mm NaCl, pH 7.2) and fixed in 3.7% formaldehyde and processed as described previously (Zhou and Riddiford 2002). Abdominal integuments were stained with an antibody against the BR core region (1:250) (Emery et al. 1994) followed by 1:500 FITC-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA). The brains were stained with Fasciclin II (1:100) followed by FITC-conjugated donkey anti-mouse secondary antibody and Texas red phalloidin (1:100). Images were obtained using a Bio-Rad (Hercules, CA) MRC-600 confocal laser scanning microscope and processed using NIH Image, Photoshop, and Canvas software.

RESULTS

The effects of JH on mutants with loss of rosy (ry) function:

Topical application of a high dose of JH III or JHM to D. melanogaster at pupariation caused the formation of a pupal–adult mosaic fly that failed to eclose, with a normal adult head and thorax and a pupal-like abdomen distinguished by a new layer of pupal cuticle with no or a few short bristles (Figure 1, B and C) (Postlethwait 1974; Riddiford and Ashburner 1991; Zhou and Riddiford 2002). The short bristles usually appeared laterally on the dorsal tergite; no bristles were found in the dorsal midline (Figure 1B). Lower doses of JH produced flies with progressively more normal bristles, but the flies failed to eclose. The lowest effective dose of JH had no adverse effect on eclosion but the males had malrotated genitalia (Postlethwait 1974; Riddiford and Ashburner 1991).

We found that two fly lines with P-element insertions on the ry506 (a ry null allele) chromosome formed relatively normal abdominal bristles after exposure to a high dose of JH III or pyriproxifen (a JHM). Normally, topical application of 1 ng pyriproxifen on wild-type Canton-S (CS) was sufficient to cause the formation of a mostly bald abdomen (data not shown), but application of 100-fold more pyriproxifen on either KG00431 (N = 43) or KG06479 (N = 45) had little effect on abdominal bristle development (Figure 1, D and E). Even 80 μg pyriproxifen were unable to prevent the abdominal bristle formation in the two lines (N = 6 each). Interestingly, females of both KG lines formed somewhat shortened bristles, indicating that they were more sensitive to JH (data not shown) than the males. Both KG lines were also insensitive to 500 ng of a natural hormone JH III (N = 21 and 5, respectively), which is ∼12 times the ED50 for JH III in wild-type flies (Wozniak et al. 2004). Nevertheless, these JH-treated pharate adults failed to eclose and all the males had malrotated genitalia (N = 26). When white puparia of either KG line were treated with a low dose of the JHM pyriproxifen, both eclosion and rotation of male genitalia were affected. At a dose of 0.025 ng pyriproxifen, ∼30% of wild-type CS (N = 35) or KG00431 (N = 43) eclosed. Thus, in these KG flies the abdominal epidermal cells appeared to be insensitive to JH, while their internal tissues, such as the nervous system that is necessary for eclosion, were apparently affected by JH.

We therefore further examined the effect of JH on the CNS of a KG line. Either a low (0.025 ng) or a high (100 ng) dose of pyriproxifen was applied to both KG00431 and CS white puparia and the CNSs of the pharate adults were examined. In the JH-treated CS puparia, the subesophageal ganglion failed to fuse completely with the central brain and it remained large rather than condensing into the adult size (Figure 2, A–C). In addition, we found substantial degeneration within the optic lobe, especially in the part of medulla close to the first optic chiasm and in the second optic chiasm (Figure 2, G–I). This phenotype has previously been reported for methoprene (a JHM)-treated Oregon RC puparia (Restifo and Wilson 1998). In KG00431 flies, both the abnormal subesophageal ganglion and the degeneration of optic lobe were also seen although the abdomen appeared normal externally (Figure 2, D–F and J–L). These data show that the JHM had penetrated the puparium and affected the development of the CNS similarly in CS and KG00431 flies. Yet the abdominal epidermal cells of the KG00431 flies apparently are resistant to the applied hormone.

Figure 2.—

Figure 2.—

Open in a new tab

The rosy mutant nervous system is sensitive to JHM treatment at pupariation. Frontal confocal optical sections through pharate adult central brains of wild-type Canton-S (A–C) or the KG00431 line (D–F) that received no (control), 0.025, or 100 ng pyriproxifen (a JHM) at pupariation are shown. The brains were stained with Texas red-conjugated phalloidin. (A) CS control (no JHM treatment). The neuropil of the subesophageal ganglion (seg) is tightly packed beneath the esophagus (arrowhead). eb, ellipsoid body; mb, mushroom body. (B) CS, after a low dose (0.025 ng) of JHM. Note the abnormal shape and depth of the subesophageal ganglion (seg). (C) CS, after a high dose of the JHM (100 ng). (D–F) JHM has similar effects on the KG00431 subesophageal ganglion (seg): (D) KG00431, no JHM treatment; (E) KG00431, treated with a low dose (0.025 ng) of the JHM; (F) KG00431, treated with a high dose (100 ng) of the JHM. (G–L) Frontal sections through the optic lobe of control or JHM-treated CS or KG00431 pharate adults. The tissues were double labeled with Fasciclin II (green) and Texas red-conjugated phalloidin (red). In both CS and KG00431 flies, JHM treatments caused selective degeneration (labeled with asterisks) within the optic lobe. (G–I) CS; (J–L) KG00431. me, medulla; och1, the first optic chiasma; och2, the second optic chiasma; lo, lobula.

Our chromosomal deficiency and gene expression analyses showed that the P-element insertions in the KG flies were not responsible for the epidermal insensitivity to JH (see supplemental Figure 1 at http://www.genetics.org/supplemental/). Instead, it was the “genetic background” ry506 that caused the abnormal response of the epidermis to JH. The JHM pyriproxifen had little effect on abdominal bristle formation in ry506 males (Figure 1F), similar to the phenotype seen in KG00431 (Figure 1D). But as in wild type and in the KG00431 insertion line, the development of the rosy506 CNS was disrupted by JHM application (both low and high doses as above) at the time of pupariation (data not shown). Furthermore, the abdominal epidermis of KG00431/ry506 and KG06479/ry506 animals was also insensitive to JHM (data not shown). When deficiency chromosomes of Df(3R)Exel8157 (deleted region 87D8; 87D10), Df(3R)ry615 (deleted region 87B11–13; 87E8–11), or Df(3R)ry506-85C (deleted region 87D1–2; 88E5–6) were paired with the ry506 chromosome, the abdominal epidermis of hemizygous flies was also not sensitive to pyriproxifen [see Figure 1G for ry506/Df(3R)Exel8157]. Several ry alleles were tested for their sensitivity to the JHM. Figure 1, H–K, shows the varying degrees of resistance to pyriproxifen of four other alleles, which may be related to the levels of residual gene function. These results strongly suggest that ry function is necessary for the effects of JH on the abdominal histoblasts when it is given at pupariation.

The wild-type ry gene restores the JH sensitivity of ry null mutants:

If the loss of the sensitivity of abdominal epidermis to JH is due to the lack of ry function, then introduction of the wild-type ry gene into a ry null mutant should restore the animal's sensitivity to JH. Fortunately, due to the use of the ry gene as a scored marker to facilitate gene transformation (Rubin and Spradling 1983), many chromosomes carrying a genomic DNA fragment of the wild-type ry gene (ry+t7.2) are available in the public fly stock centers. ry506 flies cannot synthesize the red drosopterin eye pigments, and consequently they have brownish eye color (Chovnick et al. 1990). We used three fly lines carrying ry+t7.2 on the second chromosome and ry506 on the third chromosome (see materials and methods) to cross with ry506 flies, respectively (Figure 3A). The progeny with the genotype of P{ry+t7.2}/+; ry506 had normal red eye color (Figure 3B) and showed sensitivity to JHM as only a few short abdominal bristles were formed after pyriproxifen treatment (Figure 3D). By contrast, the progeny of +/Cyo; ry506 retained a brownish ry− eye color (Figure 3C) and formed primarily normal bristles after the JHM treatment (Figure 3E). This result together with the null mutant phenotype clearly shows that ry function is required for the effect of JH on the abdominal epidermis.

Figure 3.—

Figure 3.—

Open in a new tab

The wild-type ry gene rescues both JH sensitivity and eye color. The flies were treated with 100 ng pyriproxifen (a JHM) as described in materials and methods. (A) A cross scheme to produce both ry506 homozygotes and ry506 mutants carrying ry+t7.2. (B and C) Eye colors of (B) ry+t7.2/+; ry506 (N = 19) and (C) +/Cyo; ry506 (N = 45). (D and E) Abdominal bristles of (D) ry+t7.2/+; ry506 (N = 19) and (E) +/Cyo; ry506 (N = 45) after JHM treatment.

XDH is important for JH sensitivity of D. melanogaster abdominal epidermis:

The gene ry encodes XDH (EC 1.17.1.4), which converts hypoxanthine to xanthine and then to uric acid in the purine degradation pathway (Chovnick et al. 1990). Allopurinol is an isomer of hypoxanthine, and it can be converted to alloxanthine by XDH (Keller and Glassman 1965; Rundles 1966). Both allopurinol and alloxanthine inhibit XDH enzyme activity. To test if XDH activity is important for JH signaling, we fed wild-type Canton-S larvae on a diet containing 250 ng/ml allopurinol and then applied 100 ng of pyriproxifen to the white puparia. Fifty-two percent of the JHM-treated, allopurinol-fed larvae (N = 90) metamorphosed to pharate adults with normal abdominal bristles (Figure 4, A and B). The pharate adults with normal bristles also had a brownish eye color, a typical ry mutant eye phenotype (Figure 4C). None of the JHM-treated Canton-S larvae that had fed on normal medium had normal bristles on the abdomen (Figure 4B, N = 60); 70% formed bare pupal cuticle and the remainder formed some short bristles (Figure 4A). Therefore, the lack of functional XDH leads to a phenocopy of the ry mutant's epidermal insensitivity to JH, indicating that XDH function is required for JH sensitivity.

Figure 4.—

Figure 4.—

Open in a new tab

XDH activity is necessary for JH action on the abdominal epidermis during pupal and adult development. (A) Larvae were fed on the designated diet, then 100 ng pyriproxifen was applied to the white puparia, and the adults or pharate adults were scored as to abdominal bristle length 5 days later as described in materials and methods. The bars represent the percentage having each score [mean ± standard deviation (SD)]. An asterisk (*) indicates that a bar is significantly different from its counterpart of normal diet-fed animals (P ≤ 0.02). The numbers following each type of diet on the x-axis indicate the numbers of white puparia treated with 100 ng pyriproxifen. (B) A dorsolateral view of allopurinol-fed and normal diet-fed wild-type pharate adults after JHM treatment. (C) Close-up of the eye colors of the animals shown in B.

The loss of XDH function in the ry null mutant causes the accumulation of both hypoxanthine and xanthine (Hilliker et al. 1992). To test if the accumulation of these XDH substrates in ry mutants is responsible for the JH insensitivity, we raised wild-type larvae on medium containing a high concentration of either hypoxanthine or xanthine (100 mg/ml) and then assayed the sensitivity of the puparia to a high dose of the pyriproxifen. Although none of the XDH substrate-fed puparia formed normal-length adult abdominal bristles after the JHM treatment, the percentage forming short bristles was significantly increased (Figure 4A, Student's t-test: P = 0.015 or P = 0.002, respectively). Thus, excess dietary hypoxanthine or xanthine causes a decrease in the sensitivity of wild-type abdominal epidermis to JHM.

ry is required non-cell autonomously for JH effects:

ry is necessary for normal pigment formation in the adult eyes, but this requirement is not cell autonomous (Reaume et al. 1989). To test if ry is also required for JH effects in the epidermal cells in a non-cell-autonomous manner, we generated ry− clones on the abdominal epidermis using the FRT/FLP system (Xu and Rubin 1993). Males of yw/Y; +; FRT75A ry506 were crossed to females of yw UAS-FLP; arm-GAL4; FRT75A P{wHy}DG25107. In the progeny, ry506 clones were marked with yellow− (y−), which could be identified by the yellow bristles (Figure 5A). If ry is required cell autonomously for JH effects, one would expect to see clusters of long yellow bristles from y− ry− clones surrounded by short black bristles or bare pupal cuticle. Instead we found that the yellow bristles were as short as the surrounding black wild-type bristles (Figure 5B, 25/25 clones), indicating that the requirement of ry for JH effects is not cell autonomous. Note that our experimental system was not able to identify those y− ry− bristles that were completely blocked by JH. This non-cell-autonomous function of ry together with the reduction of JH sensitivity by excessive dietary hypoxanthine/xanthine leads us to propose that the accumulation of the XDH substrates, but not the XDH enzyme itself, is the basis of insensitivity to JH.

Figure 5.—

Figure 5.—

Open in a new tab

ry is required non-cell autonomously for JH action on the abdominal epidermis. (A) A ry506 clone marked with yellow bristles (arrows) in a control adult abdomen. (B) A typical ry506 clone generated short yellow bristles (arrows) similar to the nearby short wild-type black bristles after JHM treatment (25 clones were examined). An arrowhead points to a thoracic macrochaete that is extending posteriorly in this preparation.

The effect of JH on br and cuticle gene expression in ry null mutants:

We previously found that JH treatment at pupariation causes continuous expression of br in the abdominal epidermal cells, and this presence of BR protein after a surge of ecdysone causes the deposition of a second pupal cuticle (Zhou and Riddiford 2002). Figure 6A shows that considerably lower levels of the br transcripts, especially the Z1 isoform [the 4.4-kb band (Bayer et al. 1996)], were found in the abdomen of the JHM-treated ry506 mutant at 40 hr after pupariation (AP) as compared to the JHM-treated wild-type CS developing adults. No br mRNA was detected in untreated wild-type or mutant abdomens at that time (Figure 6A). In JHM-treated CS developing adults at 42 hr after puparium formation, BR protein was seen in most of the imaginal cells of the anterior compartment of the abdominal tergite and in the posteriormost cells of the posterior compartment (Figure 6B, left) (see Minakuchi et al. 2007 for details). In contrast, only the posteriormost cells in the anterior compartment of the abdominal segment expressed br in JHM-treated ry506 mutants. Furthermore, BR was not present in the trichogen nuclei of the tergal macrochaetes in the JHM-treated ry506 mutants, in contrast to its presence in these cells in treated wild-type animals (Figure 6B, insets).

Figure 6.—

Figure 6.—

Open in a new tab

ry is upstream of br and cuticle genes in the JH signaling pathway. All the flies were treated with 100 ng pyriproxifen (JHM) at pupariation except the two control lanes in A. (A) Total abdominal RNA was isolated at 40 hr AP, and a cDNA br core region probe was used to detect br transcripts on Northern blots. Methylene blue staining of rRNA on the Northern blots indicates similar loading of total RNA in each lane. Note the high level of the 4.4-kb band that represents the transcripts of the Z1 isoform; the top 7.0-kb band is likely a mixture of other isoforms (Bayer et al. 1996). (B) BR core immunostaining in abdominal dorsal epidermis of JHM-treated wild-type CS and ry506 at 42 hr AP. The arrowhead denotes a nucleus of a trichogen cell forming a macrochaete in the row at the posterior of the anterior compartment. Bar, 50 μm. (C) Total abdominal RNA was isolated at 48 hr AP (for detecting Edg78E transcripts) or 72 hr AP (for detecting Acp65A transcripts) for Northern analysis. CS, Canton-S wild-type strain; Edg78E, a pupal-specific cuticle gene; Acp65A, an adult-specific cuticle gene.

In JHM-treated CS flies, the pupal cuticle gene Edg78E was reexpressed at 48 hr AP, but in the ry506 and the KG00431 insertion lines, no Edg78E mRNA was present (Figure 6C). Also, in wild type, the adult cuticle Acp65A gene is normally expressed at 72 hr AP (Figure 6C). This expression was nearly completely suppressed after JHM treatment. By contrast in ry506 and the KG00431 mutants, the expression of Acp65A was only slightly suppressed by JHM (Figure 6C). These results further confirmed that normal adult epidermal development in the ry506 mutants is little affected by JHM treatment. Apparently, the role of ry in the epidermal response to JH lies upstream of br and the cuticle genes.

DISCUSSION

This study has shown a novel relationship between JH and purine metabolism controlled by the ry gene that encodes XDH. The Drosophila XDH function is required in a non-cell-autonomous manner for JH to disrupt the development of the abdominal epidermis during adult development. Our evidence is threefold: (1) loss of ry function causes abdominal epidermal insensitivity to JH treatment in terms of bristle formation and cuticle production, but does not prevent the effect of JH on eclosion, the rotation of the male genitalia, or development of the adult brain; (2) introduction of the wild-type ry gene into a ry null mutant restores its epidermal sensitivity to JH; and (3) pharmacological inhibition of XDH phenocopies the ry− mutant's epidermal insensitivity to JH. Since ry is a widely used genetic marker, one of the practical important implications of this study is to urge caution in interpretation of data from hormonal manipulation of lines carrying a ry mutation.

JH when given during larval development or to the white puparium of D. melanogaster prevents rotation of the male genitalia at the lowest dose, adult eclosion at a slightly higher dose, and adult differentiation of the abdominal epidermis at much higher doses (e.g., for pyriproxifen, ≥0.01, 0.03, and ∼1–100 ng, respectively, on the wild-type CS line), but has little effect on cuticular development of the adult head and thorax (Ashburner 1970; Postlethwait 1974; Riddiford and Ashburner 1991). The high dose of JH apparently does not interfere with proliferation followed by spreading of the imaginal cells derived from the abdominal histoblasts after pupariation but prevents bristle outgrowth and causes the deposition of pupal rather than adult cuticle in the abdomen (Zhou and Riddiford 2002). This effect of JH is mediated by the prolongation of the expression of the transcription factor BR in the epidermis during most of adult development. We show here that this effect of JH on the development of the adult abdominal epidermis requires the presence of a functional ry gene.

Interestingly, the requirement of ry function for JH effects appears to be restricted to the abdominal integument that includes the epidermis, the intersegmental muscles, and the sensory nerves that spread over the epidermis. In JH- or JHM-treated ry506 mutants, the intersegmental muscles and the sensory nerve net (Williams and Truman 2005) were found to be in their normal adult configuration (data not shown). Adult development of both these muscles (D. Curry and L. M. Riddiford, unpublished results) and the ingoing epidermal sensory nerves (D. W. Williams and L. M. Riddiford, unpublished results) is usually disrupted when JH is applied to the white puparium. In contrast, the effects of JH on the CNS apparently do not require rosy since developing adult brains from wild-type, rosy506, and KG00431 flies that had a ry506 mutant background were equally affected by the JH, irrespective of whether it was the high or the low dose of JH. This observation is consistent with the observation that after application of low doses of JHM, ry mutants failed to eclose and to rotate the male genitalia similarly to wild-type flies given JHM. Thus, the role of ry in JH action appears to be tissue specific rather than systemic. Therefore, it is unlikely that JH requires modification by xanthine dehydrogenase and/or its products before exerting its status quo action on the abdominal histoblasts and their derivative imaginal epidermal cells.

ry action in purine degradation and modification of JH effects:

The role of ry in mediating the sensitivity of the abdominal epidermis to JH is not cell autonomous. Thus, ry is behaving just as it does in eye pigmentation (Reaume et al. 1989). Normally, ry is mainly expressed in the fat body and Malpighian tubules where XDH converts xanthine and hypoxanthine, products of purine metabolism, into uric acid for excretion. In ry null mutants, both hypoxanthine and xanthine accumulate to high levels and uric acid is absent (Hilliker et al. 1992). These ry mutants are much more sensitive to stress, including chemical (Glassman 1965), radiation (Hilliker et al. 1992), hyperoxygen (Hilliker et al. 1992), and starvation (C. Mirth and L. M. Riddiford, unpublished results). Hilliker et al. (1992) suggested that urate may be important to prevent mass tissue oxidations during metamorphosis. Since then uric acid has been found to be an important antioxidant in humans (Glantzounis et al. 2005) and in innate immunity throughout the animal kingdom (Vorbach et al. 2003). The enzyme XDH itself can be converted post-translationally to xanthine oxidase (XO) that generates reactive oxygen and reactive nitrogen species (Hille and Nishino 1995). Therefore, XDH is an important regulator of the cellular redox potential and also involved in detoxification in addition to its role in purine catabolism.

One possible reason for the lack of the effectiveness of the applied JH on the ry null mutant abdominal epidermis was its inactivation by tissue oxidation during the prepupal period in the absence of the normal high levels of uric acid. Yet the finding that it had its normal antimetamorphic action in the nervous system even at low doses indicated that the applied JH was still active within the animal. Furthermore, thin-layer chromatographic analysis of the metabolism of topically applied [3H]JH III 1 hr after application showed no significant difference in the amount of intact [3H]JH III remaining in wild-type CS and in ry506 puparia (data not shown). Finally, feeding ry506 larvae high doses of uric acid had no effect on their sensitivity to applied JH (data not shown), although how much of this dietary uric acid was taken up by the gut into the hemolymph is not known. Therefore, it is unlikely that the applied JH was inactivated prematurely in the ry506 mutants.

Alternatively, the accumulation of hypoxanthine and xanthine may be responsible for the insensitivity of the abdominal epidermis to JH. Our finding that excessive dietary ingestion of hypoxanthine or xanthine decreased wild-type sensitivity to the JHM supports this hypothesis. We have not determined how much of the dietary hypoxanthine or xanthine is absorbed by the gut and enters into the epidermis or whether this exogenous substrate is rapidly metabolized by XDH in wild-type animals. Therefore, further biochemical studies are necessary to test this hypothesis.

ry and JH effects:

Our study has revealed the existence of both ry-dependent and ry-independent JH actions in different tissues. In the ry-dependent JH signaling pathway in the epidermal cells, ry appears to act upstream of br. JH treatment of wild-type D. melanogaster at pupariation causes the reexpression of br in abdominal epidermis (Zhou and Riddiford 2002). This reexpression occurred in most of the epidermis throughout the segment including the trichogen nuclei; only a very few specific anterior and posterior cells lacked it (see Minakuchi et al. 2007 for details). In JH-treated ry506 flies, the number of BR-positive cells was dramatically reduced, and notably all the trichogen cells forming the macrochaetes lacked BR even though the surrounding cells at the posterior of the anterior compartment showed br reexpression. The basis for this difference in patterning of the JH-induced reexpression in the XDH-deficient flies is unknown.

The mechanism whereby ry influences the JH regulation of br expression is still a mystery. The JH receptor is still unidentified although two putative JH receptors have been proposed: ultraspiracle (USP) and methoprene tolerant (MET). USP is the insect ortholog of the vertebrate retinoid X receptor (RXR) and is the heterodimeric partner of the ecdysone receptor (EcR) (Yao et al. 1993). JH III binds to USP with low affinity in vitro and induces transcription of a reporter gene in cell culture (Jones and Sharp 1997; Xu et al. 2002). Methyl farnesoate (the immediate precursor of JH III) found in high levels in larval hemolymph binds to USP with higher, nanomolar affinity (Jones et al. 2006; Jones and Jones 2007). An in vivo demonstration that USP is required for JH action is, however, lacking.

Met encodes a putative transcription factor that has a bHLH–PAS domain having ∼40% identity to the aryl hydrocarbon nuclear translocator (Arnt) that combines with the dioxin receptor (AhR) to transport it to the nucleus (Ashok et al. 1998). JH III binds to in vitro transcribed and translated MET with high affinity and activates a reporter gene in vitro in a JH-dependent manner (Miura et al. 2005). Met null mutant flies are ∼10 times more resistant than wild-type flies to the morphogenetic and lethal effects of both the natural JH III and the JH mimics methoprene and pyriproxifen (Wilson and Ashok 1998). Larval life and metamorphosis occur normally but fecundity is reduced and egg maturation is delayed. Since JH is necessary for oogenesis in flies (Jowett and Postlethwait 1980; Soller et al. 1999), the delayed maturation and loss of fecundity have been attributed to the decreased JH sensitivity of these mutants. In addition, a second Pas domain gene with 70–86% identity in conserved regions, germ cell-expressed (gce), is present in the D. melanogaster genome and could account for the incomplete loss of JH sensitivity in Met null mutants (Godlewski et al. 2006). Importantly, MET and GCE form heterodimers except in the presence of JH, leading Godlewski et al. (2006) to speculate that MET may act differently during larval life than at metamorphosis where it interacts with BR, primarily the Z1 and Z2 isoforms (Wilson et al. 2006). Silencing the orthologous gene TcMet in the beetle Tribolium castaneum (in which there is only one gene) during early larval stages causes the formation of precocious pupae even when given JH III or a JHM, indicating that these MET-depleted beetles cannot respond to JH (Konopova and Jindra 2007). Thus, the met/gce gene products are critical for JH action in prevention of metamorphosis.

Interestingly, dioxin induces XDH activity about threefold through the AhR/Arnt complex in mice (Sugihara et al. 2001). Furthermore, hypoxic conditions increase XDH transcripts twofold and hyperoxia decreases XDH mRNA about threefold (Lanzillo et al. 1996). We found that a high dose of JH applied at pupariation caused a twofold increase in ry mRNA accumulation in the integument 4 hr later (normally rosy expression is very low after pupariation; data not shown), presumably leading to an increase in XDH that somehow is necessary for JH to have its effect on the histoblasts and the imaginal epidermis that they generate. Both MET (Pursley et al. 2000) and BR (Zhou and Riddiford 2002) are normally present in prepupal and pupal abdominal histoblasts; whether GCE is present has not been studied. If GCE is present and its heterodimerization with MET is necessary for the interaction of MET and BR (particularly BR–Z1) to regulate the normal pupal development of the imaginal abdominal epidermis, we speculate that the disruption of the MET–GCE heterodimer by the applied JH leads to the upregulation of XDH by MET and later to the observed prolongation of broad expression in the abdominal epidermis during adult development (Zhou and Riddiford 2002). Further studies of the genetic interactions among met/gce, broad, and rosy and the effects of JH on these interactions are clearly warranted.

In summary, these studies have discovered an important novel effect of the ry gene; i.e., it is required in a non-cell-autonomous manner for JH to disrupt normal adult development of the abdominal epidermis of D. melanogaster, but has no role in the effects of JH on the metamorphosing CNS. In ry-dependent JH signaling, ry appears to be acting upstream of the br gene. Our data suggest that the accumulation of XDH substrates in ry mutants may be the cause of JH insensitivity of the epidermis.

Acknowledgments

We thank James W. Truman for his help in analyzing JHM effects on the nervous system and Michael Ashburner and two anonymous reviewers for comments on the manuscript. We also thank the Bloomington Stock Center for fly stocks for this study and Daniel Shaw for maintaining the fly stock and setting up crosses. This study was supported by National Institutes of Health grant R01 GM060122 to L.M.R.

References

  1. Ames, B. N., R. Cathcart, E. Schwiers and P. Hochstein, 1981. Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc. Natl. Acad. Sci. USA 78: 6858–6862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andres, A. J., J. C. Fletcher, F. D. Karim and C. S. Thummel, 1993. Molecular analysis of the initiation of insect metamorphosis: a cooperative study of Drosophila ecdysone-regulated transcription. Dev. Biol. 160: 388–404. [DOI] [PubMed] [Google Scholar]
  3. Ashburner, M., 1970. Effects of juvenile hormone on adult differentiation of Drosophila melanogaster. Nature 227: 187–189. [DOI] [PubMed] [Google Scholar]
  4. Ashok, M., C. Turner and T. G. Wilson, 1998. Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators. Proc. Natl. Acad. Sci. USA 95: 2761–2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bardwell, V. J., and R. Treisman, 1994. The POZ domain: a conserved protein-protein interaction motif. Genes Dev. 8: 1664–1677. [DOI] [PubMed] [Google Scholar]
  6. Bayer, C. A., B. Holley and J. W. Fristrom, 1996. A switch in Broad-Complex zinc-finger isoform expression is regulated posttranscriptionally during the metamorphosis of Drosophila imaginal discs. Dev. Biol. 177: 1–14. [DOI] [PubMed] [Google Scholar]
  7. Bellen, H. J., R. W. Levis, G. Liao, Y. He, J. W. Carlson et al., 2004. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics 167: 761–781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berg, C. A., and A. C. Spradling, 1991. Studies on the rate and site-specificity of P element transposition. Genetics 127: 515–524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chovnick, A., S. H. Clark, F. L. Dutton, Jr. and A. G. Reaume, 1990. The rosy locus and xanthine dehydrogenase in Drosophila melanogaster. Prog. Clin. Biol. Res. 344: 1–18. [PubMed] [Google Scholar]
  10. Davies, K. J., A. Sevanian, S. F. Muakkassah-Kelly and P. Hochstein, 1986. Uric acid-iron ion complexes. A new aspect of the antioxidant functions of uric acid. Biochem. J. 235: 747–754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DiBello, P. R., D. A. Withers, C. A. Bayer, J. W. Fristrom and G. M. Guild, 1991. The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics 129: 385–397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Emery, I. F., V. Bedian and G. M. Guild, 1994. Differential expression of Broad-Complex transcription factors may forecast tissue-specific developmental fates during Drosophila metamorphosis. Development 120: 3275–3287. [DOI] [PubMed] [Google Scholar]
  13. Glantzounis, G. K., E. C. Tsimoyiannis, A. M. Kappas and D. A. Galaris, 2005. Uric acid and oxidative stress. Curr. Pharm. Des. 11: 4145–4151. [DOI] [PubMed] [Google Scholar]
  14. Glassman, E., 1965. Genetic regulation of xanthine dehydrogenase in Drosophila melanogaster. Fed. Proc. 24: 1243–1251. [PubMed] [Google Scholar]
  15. Godlewski, J., S. Wang and T. G. Wilson, 2006. Interaction of bHLH-PAS proteins involved in juvenile hormone reception in Drosophila. Biochem. Biophys. Res. Commun. 342: 1305–1311. [DOI] [PubMed] [Google Scholar]
  16. Goodman, W. G., and N. A. Granger, 2005. The juvenile hormones, pp. 319–408 in Comprehensive Molecular Insect Science, Vol. 3, edited by L. I. Gilbert, K. Iatrou and S. S. Gill. Elsevier, Oxford.
  17. Hille, R., and T. Nishino, 1995. Flavoprotein structure and mechanism. 4. Xanthine oxidase and xanthine dehydrogenase. FASEB J. 9: 995–1003. [PubMed] [Google Scholar]
  18. Hilliker, A. J., B. Duyf, D. Evans and J. P. Phillips, 1992. Urate-null rosy mutants of Drosophila melanogaster are hypersensitive to oxygen stress. Proc. Natl. Acad. Sci. USA 89: 4343–4347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jones, D., and G. Jones, 2007. Farnesoid secretions of dipteran ring glands: what we do know and what we can know. Insect Biochem. Mol. Biol. 37: 771–798. [DOI] [PubMed] [Google Scholar]
  20. Jones, G., and P. A. Sharp, 1997. Ultraspiracle: an invertebrate nuclear receptor for juvenile hormones. Proc. Natl. Acad. Sci. USA 94: 13499–13503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones, G., D. Jones, P. Teal, A. Sapa and M. Wozniak, 2006. The RXR ortholog, ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand. FEBS J. 273: 4983–4996. [DOI] [PubMed] [Google Scholar]
  22. Jowett, T., and J. H. Postlethwait, 1980. The regulation of yolk polypeptide synthesis in Drosophila ovaries and fat body by 20-hydroxyecdysone and a juvenile hormone analog. Dev. Biol. 80: 225–234. [DOI] [PubMed] [Google Scholar]
  23. Keller, Jr., E. C., and E. Glassman, 1965. Phenocopies of the ma-1 and ry mutants of Drosophila melanogaster: inhibition in vivo of xanthine dehydrogenase by 4-hydroxypyrazolo(3,4-d)pyrimidine. Nature 208: 202–203. [DOI] [PubMed] [Google Scholar]
  24. Konopova, B., and M. Jindra, 2007. Juvenile hormone resistance gene Methoprene-tolerant controls entry into metamorphosis in the beetle Tribolium castaneum. Proc. Natl. Acad. Sci. USA 104: 10488–10493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lanzillo, J. J., F. S. Yu, J. Stevens and P. M. Hassoun, 1996. Determination of xanthine dehydrogenase mRNA by a reverse transcription-coupled competitive quantitative polymerase chain reaction assay: regulation in rat endothelial cells by hypoxia and hyperoxia. Arch. Biochem. Biophys. 335: 377–380. [DOI] [PubMed] [Google Scholar]
  26. Lin, R. J., L. Nagy, S. Inoue, W. Shao, W. H. Miller, Jr. et al., 1998. Role of the histone deacetylase complex in acute promyelocytic leukaemia. Nature 391: 811–814. [DOI] [PubMed] [Google Scholar]
  27. Lin, R. J., T. Sternsdorf, M. Tini and R. M. Evans, 2001. Transcriptional regulation in acute promyelocytic leukemia. Oncogene 20: 7204–7215. [DOI] [PubMed] [Google Scholar]
  28. Melnick, A., K. F. Ahmad, S. Arai, A. Polinger, H. Ball et al., 2000. In-depth mutational analysis of the promyelocytic leukemia zinc finger BTB/POZ domain reveals motifs and residues required for biological and transcriptional functions. Mol. Cell Biol. 20: 6550–6567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Minakuchi, C., X. Zhou and L. M. Riddiford, 2007. Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster. Mech. Dev. (in press). [DOI] [PMC free article] [PubMed]
  30. Miura, K., M. Oda, S. Makita and Y. Chinzei, 2005. Characterization of the Drosophila Methoprene-tolerant gene product. Juvenile hormone binding and ligand-dependent gene regulation. FEBS J. 272: 1169–1178. [DOI] [PubMed] [Google Scholar]
  31. Postlethwait, J. H., 1974. Juvenile hormone and the adult development of Drosophila. Biol. Bull. 147: 119–135. [DOI] [PubMed] [Google Scholar]
  32. Pursley, S., M. Ashok and T. G. Wilson, 2000. Intracellular localization and tissue specificity of the Methoprene-tolerant (Met) gene product in Drosophila melanogaster. Insect Biochem. Mol. Biol. 30: 839–845. [DOI] [PubMed] [Google Scholar]
  33. Reaume, A. G., S. H. Clark and A. Chovnick, 1989. Xanthine dehydrogenase is transported to the Drosophila eye. Genetics 123: 503–509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Restifo, L. L., and T. G. Wilson, 1998. A juvenile hormone agonist reveals distinct developmental pathways mediated by ecdysone-inducible Broad Complex transcription factors. Dev. Genet. 22: 141–159. [DOI] [PubMed] [Google Scholar]
  35. Riddiford, L. M., 1994. Cellular and molecular actions of juvenile hormone. I. General considerations and premetamorphic actions. Adv. Insect Physiol. 24: 213–274. [Google Scholar]
  36. Riddiford, L. M., 1996. Juvenile hormone: the status of its “status quo” action. Arch. Insect Biochem. Physiol. 32: 271–286. [DOI] [PubMed] [Google Scholar]
  37. Riddiford, L. M., and M. Ashburner, 1991. Effects of juvenile hormone mimics on larval development and metamorphosis of Drosophila melanogaster. Gen. Comp. Endocrinol. 82: 172–183. [DOI] [PubMed] [Google Scholar]
  38. Riddiford, L. M., P. Cherbas and J. W. Truman, 2001. Ecdysone receptors and their biological actions. Vitam. Horm. 60: 1–73. [DOI] [PubMed] [Google Scholar]
  39. Rubin, G. M., and A. C. Spradling, 1983. Vectors for P element-mediated gene transfer in Drosophila. Nucleic Acids Res. 11: 6341–6351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rundles, R. W., 1966. Metabolic effects of allopurinol and allo-xanthine. Ann. Rheum. Dis. 25: 615–620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Simmonds, H. A., S. Reiter and T. Nishino, 1995. Hereditary xanthinuria, pp. 1781–1797 in The Metabolic and Molecular Basis of Inherited Disease, edited by C. R. Scriver, A. L. Beaudet, W. S. Sly and D. Vallee. McGraw-Hill, New York.
  42. Soller, M., M. Bownes and E. Kubli, 1999. Control of oocyte maturation in sexually mature Drosophila females. Dev. Biol. 208: 337–351. [DOI] [PubMed] [Google Scholar]
  43. Sugihara, K., S. Kitamura, T. Yamada, S. Ohta, K. Yamashita et al., 2001. Aryl hydrocarbon receptor (AhR)-mediated induction of xanthine oxidase/xanthine dehydrogenase activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. Biophys. Res. Commun. 281: 1093–1099. [DOI] [PubMed] [Google Scholar]
  44. Sullivan, W., M. Ashburner and R. S. Hawley, 2000. Drosophila Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  45. Vorbach, C., R. Harrison and M. R. Capecchi, 2003. Xanthine oxidoreductase is central to the evolution and function of the innate immune system. Trends Immunol. 24: 512–517. [DOI] [PubMed] [Google Scholar]
  46. Williams, C. M., 1952. Morphogenesis and the metamorphosis of insects. Harvey Lect. Ser. 47: 126–155. [PubMed] [Google Scholar]
  47. Williams, D. W., and J. W. Truman, 2005. Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons. Development 132: 3631–3642. [DOI] [PubMed] [Google Scholar]
  48. Wilson, T. G., and M. Ashok, 1998. Insecticide resistance resulting from an absence of target-site gene product. Proc. Natl. Acad. Sci. USA 95: 14040–14044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wilson, T. G., Y. Yerushalmi, D. M. Donnell and L. L. Restifo, 2006. Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between Methoprene-tolerant and Broad-Complex. Genetics 172: 253–264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wozniak, M., Y. Chu, F. Fang, Y. Xu, L. Riddiford et al., 2004. Alternative farnesoid structures induce different conformational outcomes upon the Drosophila ortholog of the retinoid X receptor, ultraspiracle. Insect Biochem. Mol. Biol. 34: 1147–1162. [DOI] [PubMed] [Google Scholar]
  51. Wyatt, G. R., and K. G. Davey, 1996. Cellular and molecular actions of juvenile hormone. 2. Roles of juvenile hormone in adult insects. Adv. Insect Physiol. 26: 1–155. [Google Scholar]
  52. Xu, T., and G. M. Rubin, 1993. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117: 1223–1237. [DOI] [PubMed] [Google Scholar]
  53. Xu, Y., F. Fang, Y. Chu, D. Jones and G. Jones, 2002. Activation of transcription through the ligand-binding pocket of the orphan nuclear receptor ultraspiracle. Eur. J. Biochem. 269: 6026–6036. [DOI] [PubMed] [Google Scholar]
  54. Yao, T. S., B. M. Forman, Z. Jiang, L. Cherbas, J. D. Chen et al., 1993. Functional ecdysone receptor is the product of EcR and Ultraspiracle genes. Nature 366: 476–479. [DOI] [PubMed] [Google Scholar]
  55. Zhou, X., and L. M. Riddiford, 2002. Broad specifies pupal development and mediates the ‘status quo’ action of juvenile hormone on the pupal-adult transformation in Drosophila and Manduca. Development 129: 2259–2269. [DOI] [PubMed] [Google Scholar]
  56. Zollman, S., D. Godt, G. G. Prive, J. L. Couderc and F. A. Laski, 1994. The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc. Natl. Acad. Sci. USA 91: 10717–10721. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES