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Molecular and Cellular Biology, December 2003, p. 8495-8504, Vol. 23, No. 23
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.23.8495-8504.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Drosophila Selenoprotein BthD Is Required for Survival and Has a Role in Salivary Gland Development

So Yeon Kwon,^1,2 Paul Badenhorst,³ F. Javier Martin-Romero,^1, Bradley A. Carlson,¹ Bruce M. Paterson,⁴ Vadim N. Gladyshev,⁵ Byeong Jae Lee,^2* and Dolph L. Hatfield^1*

Basic Research Laboratory,¹ Laboratory of Molecular Cell Biology,³ Laboratory of Biochemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892,⁴ Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588,⁵ Laboratory of Molecular Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea²

Received 31 March 2003/ Returned for modification 9 May 2003/ Accepted 21 August 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Selenium is implicated in many diseases, including cancer, but its function at the molecular level is poorly understood. BthD is one of three selenoproteins recently identified in Drosophila. To elucidate the function of BthD and the role of selenoproteins in cellular metabolism and health, we analyzed the developmental expression profile of this protein and used inducible RNA interference (RNAi) to ablate function. We find that BthD is dynamically expressed during Drosophila development. bthD mRNA and protein are abundant in the ovaries of female flies and are deposited into the developing oocyte. Maternally contributed protein and RNA persist during early embryonic development but decay by the onset of gastrulation. At later stages of embryogenesis, BthD is expressed highly in the developing salivary gland. We generated transgenic fly lines carrying an inducible gene-silencing construct, in which an inverted bthD genomic-cDNA hybrid is under the control of the Drosophila Gal4 upstream activation sequence system. Duplex RNAi induced from this construct targeted BthD mRNA for destruction and reduced BthD protein levels. We found that loss of BthD compromised salivary gland morphogenesis and reduced animal viability.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Selenium is an important micronutrient in the diet of many life forms, including vertebrates (reviewed in reference 16), Drosophila (28), and the lower plant Chlamydomonas reinhardtii (33). Selenium has been said to be a cancer chemopreventive agent, to prevent heart disease and other cardiovascular and muscle disorders, to inhibit viral expression, to delay the progression of AIDS in human immunodeficiency virus-positive patients, to slow the aging process, and to have roles in immune function, mammalian development, and male reproduction (see reviews in reference 15).

During protein synthesis, selenium is incorporated into nascent selenopeptides as the amino acid selenocysteine (Sec) (16). Sec is specified by the codon UGA that normally terminates protein synthesis. However, in selenium-containing proteins, a specialized stem-loop structure located in the mRNA (designated the Sec insertion sequence [SECIS] element) (27) stipulates UGA in the open reading frame to incorporate Sec.

The identification and characterization of selenoproteins are essential to understanding the role of this class of proteins in development and in cellular metabolism. Computer programs designed to find SECIS elements in expressed sequence tags and genomic DNA have been developed as a means of identifying selenoprotein genes (22, 23). Such in silico searches for SECIS elements have identified three selenoprotein genes in the Drosophila genome (8, 28). One of the selenoproteins is selenophosphate synthetase 2 (SPS2), a 43-kDa protein that is involved in the biosynthesis of Sec through the generation of selenophosphate, the selenium donor compound. SPS2 in Drosophila has previously been described (17) and is the homolog of a protein previously found in mammals (12, 21, 26). In addition to SPS2, two novel proteins were found (8, 28): a 28-kDa protein designated BthD and a 12-kDa, glycine-rich protein designated G-rich (28). Since SPS2 is involved in Sec biosynthesis, BthD and G-rich should be responsible for the biological effects of selenium in fruit flies. However, the specific functions of these proteins are not known.

RNA interference (RNAi), which is also called posttranscriptional gene silencing, has proven to be a powerful tool for disrupting gene expression. This technique was initially developed in plants (3, 10) and nematodes (9) and more recently in Drosophila (19, 20, 32), zebra fish (24), and mice (6, 13, 14). RNAi can be used to interfere with gene expression both transiently (by introducing double-stranded RNA [dsRNA] directly into cells) and stably (by inserting an appropriate construct that produces the targeting dsRNA into the genome). The advantage of using inheritable or inducible RNAi is that the phenomenon becomes a permanent feature of an organism, and the effect can often be triggered under a variety of different conditions and/or at different times of development.

We have characterized the expression profile of BthD and find that it is expressed dynamically during Drosophila development. BthD is abundant in the developing ovary of female flies, and large amounts of RNA and protein are deposited into the early embryo. At the later stages of development, BthD expression reinitiates in the developing salivary gland. The subcellular distribution of BthD at these stages suggests that BthD might have roles in protein secretion or processing. To address this, we have used inducible RNAi to ablate BthD expression. Transgenic lines that express dsRNA as an extended hairpin loop (19) were generated and used to interfere specifically with BthD expression. We find that BthD is required for viability and normal salivary gland morphogenesis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Northern analysis. Total RNA was isolated from staged embryos, larvae, and adult flies according to the protocol of Montell et al. (29). A total of 30 µg of total RNA from each stage was fractionated on a 2.2 M formaldehyde-1.2% agarose gel and transferred to a nylon membrane. Probes for hybridization were labeled using Ready-To-Go DNA labeling beads (Amersham), and hybridization was performed at 68°C in Quickhyb hybridization solution (Stratagene). To allow reprobing of the membrane, bound probe was removed by washing the blot with boiling 0.1% sodium dodecyl sulfate. Removal of the probe was confirmed before the next hybridization. A PCR-amplified fragment of full-length bhtD cDNA was used as a probe, and rp49 was used as a loading control.

Antibody production. To allow expression of BthD in Escherichia coli, full-length bthD cDNA was mutated to convert the Sec-encoding TGA codon to TGC (Cys codon). Mutagenesis was performed using a PCR-directed mutagenesis strategy (18) and the following primer sets: 5'-GGAATTCCATATGCCACCAAAACGGAACA-3', 5'-ACGACGAAAGACTCGGCAGGATCGGCAGTGCTC-3', 5'-GAGCACTGCCGATCCTGCCGAGTCTTTCGTCGT-3', and 5'-CCGCTCGAGTTAGGCATGATGATGATGATGATGCCTCCTCCGCTTGGCACCG-3' (the underlined characters designate the mutation sites). The altered cDNA was cloned into pET-15b (Novagen) to generate a six-histidine-tagged fusion derivative of BthD. The His-tagged BthD fusion protein was overexpressed in E. coli strain BL21(DE3)/pLysS. The fusion protein was purified employing Qiagen Ni-nitrilotriacetic acid (NTA) beads and used to immunize rabbits (Spring Valley, Woodbine, Md.).

Denaturing and nondenaturing gel electrophoresis. Drosophila SL2 cells were harvested and resuspended in phosphate-buffered saline (PBS) containing protease inhibitor cocktail (Sigma). Cells were lysed by sonication on ice, and 10 µg of total protein was electrophoresed on Novex Tris-glycine-10% polyacrylamide gels (Invitrogen) under either denaturing or nondenaturing conditions (as described in the manufacturer's instructions).

Cell culture and transfection. Drosophila SL2 cells (Invitrogen) were maintained in HyQ CCM-3 medium (HyClone) containing 50 U of penicillin and streptomycin (Gibco/BRL). SL2 cells were plated in six-well plates and transfected using Lipofectin (Invitrogen). In each transfection, 10 µg of bthD dsRNA was used. At 72 h after the transfection, cells were harvested and used for Western blotting.

Genetics and Drosophila strains. Flies were raised at 25°C. daGal4 (39) or fkhGal4 (31) was used to drive ubiquitous or salivary gland-specific expression, respectively. The enhancer trap line NT33 (35) was used as a marker for the embryonic salivary gland.

Inducible RNAi construct. For the bthD RNAi construct, genomic and cDNA fragments were amplified using PCR with primers containing unique restriction sites. The genomic fragment was isolated by PCR as an EcoRI-to-BglII fragment and cloned into pUAST (5). The inverted cDNA sequence digested with KpnI and XbaI was ligated sequentially to the genomic fragment-containing vector. Ligated RNAi constructs were transformed into DH5 cells (Invitrogen). Primer sequences for RNAi constructs were as follows: bthD genomic forward, 5'-CCGGAATTCATGCCACCAAAACGGAACAA-3'; bthD genomic reverse, 5'-GGAAGATCTGAAGTTGGTGCCTTCGTTTTCCTGTGCTTCC-3'; bthD cDNA forward, 5'-CGGGGTACCCCTCGTTTTCCTGTGCTTCC-3'; and bthD cDNA reverse, 5'-CTAGTCTAGAATGCCACCAAAAACGGAACAA-3'. The construct was transformed into w¹¹¹⁸ flies by P-element-mediated transformation (36). Adult G₀ transformants were identified by outcrossing to w¹¹¹⁸ and balanced over SM5 or TM3 balancer chromosomes. Four different homozygous, viable UAS-BthDRNAi lines were used in all experiments. No reduction in viability or morphological abnormalities was observed in any of the upstream activation sequence (UAS) lines in the absence of a GAL4 driver.

Western blot analysis. Cells and embryos were lysed in PBS buffer containing a complete protease mix (Roche). Anti-BthD antisera were used at a 1:2,000 dilution for Western analysis. Antitubulin antibody (Developmental Studies Hybridoma Bank) was used as a control at a 1:1,000 dilution. Each antigen was detected using an enhanced chemiluminescence Western blotting detection kit (Amersham Bioscience).

Immunohistochemistry and immunofluorescence. Embryo fixation and immunohistochemistry were performed as described previously (2). Immunoassaying of ovaries was performed as described previously (25). For immunofluorescence, fluorescein isothiocyanate- and Cy3-conjugated anti-immunoglobulin G secondary antibodies (Jackson ImmunoResearch) were used. Samples were mounted in Vectashield (Vector Laboratories) and viewed using a confocal microscope (Bio-Rad model MRC 1024). For immunohistochemistry, biotinylated anti-immunoglobulin G secondary antibodies and a Vectastain ABC kit (Vector Laboratories) were used. Antibodies were used at the following dilutions: for mouse anti-ß-galactosidase (anti-ß-Gal) (Promega), 1:10,000, and for rabbit anti-BthD, 1:200 (immunofluorescence) or 1:1,000 (immunocytochemistry). DNA was stained using SYTOX Green (Molecular Probes). Embryos were staged as given previously (7).

Salivary glands, imaginal disks, and the gut and trachea were dissected from third-instar larvae and incubated in fixative (PBS, 3.7% formaldehyde, 0.1% Triton X-100) for 20 min on ice. After extensive washes in PBS, samples were mounted in Vectashield containing DAPI (4',6'diamidino-2-phenylindole) (Vector Laboratories). To visualize the actin cytoskeleton, dissected third-instar salivary glands were incubated in 37% formaldehyde for 4 min, washed extensively with PBT (PBS, 0.1% Triton X-100), and then incubated overnight in PBS containing 0.1% Triton X-100, 10% fetal calf serum, and 0.8 U of rhodamine-phalloidin (Molecular Probes)/ml. After four washes in PBT, samples were mounted in Vectashield.

Top Abstract Introduction Materials and Methods Results Discussion References

 
BthD RNA and protein distribution. The expression of BthD during development and in adult flies was first examined by Northern blotting. Total RNA was isolated from adult male and female flies and from staged collections of embryos and larvae that spanned development. As shown in Fig. 1A, bthD mRNA appeared to be expressed at high levels during early embryogenesis (0-5 hours lane) and in adult females (Female adult lane) and at moderate levels in mid- and late-phase embryogenesis (5-10 hours lane and 10-15 hours lane, respectively). bthD mRNA was poorly expressed in the larval stages (L1 larvae, L2 larvae, and L3 larvae lanes) and in adult males (Male adult lane). Quantitation of bthD mRNA levels relative to those of rp49 (used as a loading control) confirmed these observations, as shown in Fig. 1B.

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FIG. 1. Expression of bthD mRNA. (A) Northern blot analysis. Total RNA was extracted from adults and staged embryo and larval collections and was hybridized with probes specific to bthD mRNA or rp49 as shown. (B) mRNA quantization. bthD mRNA levels were normalized (using a PhosphorImager) to rp49 as a control and plotted as shown.

We also observed the expression of BthD in adult female, but not male, flies by ⁷⁵Se labeling (28). To visualize selenoproteins, adult flies were maintained overnight on ⁷⁵Se-containing medium (28) to allow incorporation into nascent polypeptides. Three selenoproteins, BthD, SPS2, and G-rich, which had previously been observed by using ⁷⁵Se labeling in Drosophila (28), were observed in adult females, but only BthD was not detected in males (data not shown).

As a tool to characterize the expression of BthD in more detail, we generated specific antibodies against the recombinant protein. The resultant antisera specifically detected one band in extracts from SL2 cells (Fig. 2A). This band had the expected molecular weight of BthD (see denaturing gel in Fig. 2A) and comigrated with the protein labeled by ⁷⁵Se (data not shown). To further confirm the specificity of anti-BthD antibody, we treated SL2 cells with dsRNA directed against BthD (Fig. 2B). The abundance of BthD was markedly decreased relative to that of untreated, control cells. Tubulin was used as a gel loading control. Interestingly, when BthD was run on a nondenaturing gel it migrated as a larger protein (Fig. 2C), suggesting that it might occur as a dimer.

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FIG. 2. Antibody detection of BthD in SL2 cells. (A) Western blot analysis in extracts. (B) Western blot analysis in RNAi-treated cells. SL2 cells were treated with bthD dsRNA, and an extract was prepared and compared (using gel electrophoresis) to an extract from untreated cells as shown. (C) Western blot analysis of denaturing and nondenaturing gels. SL2 cell extracts were electrophoresed on polyacrylamide gels as shown.

Anti-BthD antibodies were used to examine protein expression in ovaries of adult female flies. Ovaries were dissected from adult flies, and BthD was visualized by confocal microscopy (Fig. 3). BthD, which is shown in red in Fig. 3A, is abundantly expressed in ovarioles from the earliest stages (see upper-left portion of the panel) until the completion of oocyte development (see right-hand portion of the panel). The results of higher magnification of stage 8 of the developing oocyte suggest that BthD occurs in the cytoplasm (Fig. 3D). To visualize nuclei, DNA was labeled with Sytox Green dye, as shown by the green staining in Fig. 3B and E. Simultaneous visualization of BthD and DNA revealed that the protein is found exclusively in the cytoplasm of the developing oocyte and not in nurse cell nuclei or in the nuclei of the surrounding follicle cells, as shown in Fig. 3C and F.

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FIG. 3. Localization of BthD in developing egg chambers. BthD antibodies (-BthD) and DNA dye were used (as described in Materials and Methods) to visualize BthD expression (shown in red) (A and D) and DNA (shown in green) (B and E). (C and F) Merged images. (D to F) Higher-magnification images of the stage 8 egg chamber show that BthD is expressed throughout the cytoplasm of the follicle (fc) and nurse cells (nc). Magnification for panels A to C is x100 and for panels D to F is x200.

Embryonic expression profile of BthD. We examined BthD protein distribution in embryos. Consistent with the high levels of expression of BthD protein and RNA in the ovary of adult female flies, antibody staining using anti-BthD antibodies revealed that BthD is expressed ubiquitously, and at high levels, in the early embryo (Fig. 4A; a syncytial blastoderm stage 2 embryo is shown). The abundance of maternally contributed BthD declined after cellularization of the blastoderm (stage 4) and was much reduced by the onset of germband retraction (stage 10). At later stages of development, BthD expression appeared to initiate in the salivary gland. BthD was first detected at stage 14, and this accumulation was fully apparent by stages 15 and 16.

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FIG. 4. BthD embryonic expression profile. (A) Expression during embryogenesis. Embryos were stained with anti-BthD antibodies, and BthD expression was revealed using horseradish peroxidase-coupled secondary antibodies. Magnification is x100. (B) Salivary gland-specific expression. Embryos containing the pasilla-enhancer trap (NT33), which expresses ß-Gal under the control of the pasilla promoter, were stained with antibodies against ß-Gal (-ßgal) (red) and BthD (-BthD) (green). Magnification of embryos is x100. Higher-magnification (x630) images of the salivary glands from stage (St) 14 and stage 15 embryos are also shown alongside the merged embryo images. In all panels, embryos are oriented with the anterior side to the left and the dorsal side up.

Salivary gland expression of BthD was confirmed by comparing BthD distribution to that of Pasilla, a known salivary gland marker (35). As shown in Fig. 4B, double-label immunofluorescence (using antibodies against ß-Gal [shown in red] and BthD [shown in green]) of pasilla-enhancer trap line N33 embryos confirmed that BthD is expressed in the salivary gland. BthD expression, however, initiated relatively late in salivary gland development. Although robust Pasilla expression was observed in stage 12 embryos, BthD was absent. BthD expression was apparent at stage 14. Higher-magnification images of these embryos (see panels labeled "Salivary gland" in Fig. 4B) reveal that BthD expression was primarily cytoplasmic. At stage 14, significant staining was noted in the lumen of the salivary gland. By stage 15, lumenal staining was decreased, although BthD staining was still observable in the cytoplasm of the cells that flanked the lumen.

Subcellular distribution of BthD. We analyzed in greater detail the subcellular distribution of BthD in the early embryo and the salivary gland. Interestingly, subcellular distribution of BthD was quite dynamic. At the earliest stages, the majority of BthD was distributed in large sphere-like structures (Fig. 5A). These were located in the cortical cytoplasm, just beneath the cell membrane, at the extreme periphery of the embryo (see inset in Fig. 5A). As embryonic development proceeded and the cortical cytoplasm expanded, the major accumulation of BthD tracked the cortical cytoplasm into the interior of the embryo. At this stage, large accumulations of BthD appeared to break down and were absorbed into punctate granules that encircled regions once occupied by spheres.

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FIG. 5. BthD subcellular distribution. (A) High-magnification (x1,260) confocal images of BthD protein distribution in syncytial embryos (stages [St] 2 and 3). Insets are confocal sections revealing the cortical cytoplasm (immediately below the membrane). (B) BthD distribution in the salivary gland. BthD is localized to granules that encircle the nuclei of the salivary gland cells (asterisks signify nuclei that were stained with DAPI [data not shown]). Punctate BthD staining can be seen in confocal sections that traverse the lumen of the salivary gland or traverse the exterior of the gland. The lumen of the salivary gland is indicated by arrows. (C) Distribution in SL2 cells. SL2 cells were stained with anti-BthD antibodies (red) and NBD-C6-ceramide (green), which reveals the Golgi apparatus. Insets show a top view of stained cells, and arrows indicate colocalization of BthD and the Golgi marker.

This particulate distribution was also seen in salivary glands (Fig. 5B). The majority of BthD was located in punctate granules that surrounded the nuclei in salivary gland cells. This was seen in both stage 14 and stage 16 embryos. In stage 14 embryos, high BthD staining was also detected in the lumen of the salivary gland. By stage 16, lumenal staining was much reduced. We attempted to use known markers for subcellular compartments for identification of these granules. Staining of SL2 cells with anti-BthD antibodies recapitulated the punctate staining profile. Costaining with NBD-C6-ceramide, which marks the Golgi apparatus, indicated that BthD staining overlaps the Golgi (Fig. 5C).

Inducible RNAi targets BthD. We used an inducible RNAi strategy to ablate BthD function. Our approach is summarized in Fig. 6A. A construct was synthesized in which an inverted bthD genomic-cDNA hybrid was placed under the control of the GAL4-UAS. Expression from this construct led to the synthesis of a hybrid RNA which, after splicing of the two introns from the genomic sequence, snapped back to form a dsRNA. This construct was transformed into flies, and four independent transgenic lines were recovered. All lines showed equivalent results.

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FIG. 6. BthD ablation using inducible RNAi. (A) Strategy for inducible dsRNA synthesis. (B) Induction of dsRNA using the salivary gland-specific fkhGAL4 driver. Extracts from stage 16 fkhGAL4/+ or fkhGAL4/UAS-BthDRNAi embryos were analyzed by Western blotting using anti-BthD antibodies and antitubulin antibodies as a loading control.

Induction of the bthD dsRNA synthesis was able to reduce BthD protein levels. As shown in Fig. 6B, overexpression (using the salivary gland-specific fkhGAL4 driver) of the UAS-RNAi construct decreased BthD protein levels in late-stage-16 embryos. The daGAL4 driver was also used to direct ubiquitous expression of the UAS-RNAi construct. daGAL4-mediated expression also reduced bthD mRNA and protein levels (data not shown).

BthD is required for survival and specifically for salivary gland morphogenesis. We next examined how ablation of BthD affects survival and viability. Induction using either daGAL4 or fkhGAL4 did not significantly affect embryo survival. However, the number of adult flies that were recovered was significantly reduced. We collected defined numbers of first-instar larvae and assayed survival by counting the number of adult flies to eclose. As shown in Fig. 7A, both daGAL4- and fkhGAL4-mediated dsRNA induction reduced the number of adult flies that were recovered from approximately 100 to 21.5 and 28, respectively. Larval survival was not significantly affected, but most animals died shortly after pupariation (Fig. 7B).

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FIG. 7. Viability and pupae phenotypes in BthD-deficient organisms. (A) Viability after induction of bthD dsRNA. Homozygous daGAL4 or fkhGAL4 virgin female flies were crossed to either w1118 or UAS-BthDRNAi males. Progeny first-instar larvae were collected and placed in vials (150 each). The number of adult flies of each genotype that eclosed was recorded. Data are the means and standard deviations of five independent determinations. (B) daGAL4/+ and daGAL4/UAS-BthDRNAi pupae.

As we had shown that BthD is expressed in the embryonic salivary gland, we examined whether RNAi induction affected salivary gland development. The salivary gland is an interesting model organ system, as salivary gland cells are set aside during embryonic stages and the final cell number for each gland is established by the completion of embryogenesis. During larval life, the cell number does not increase and growth is mediated only by an increase in cell size.

Induction of bthD RNAi either (using fkhGAL4) specifically in the salivary gland or (using daGAL4) throughout the entire embryo had profound effects on larval salivary gland morphogenesis. Total cell number was not decreased, as the same number of nuclei was still detected by DAPI staining of third-instar larval salivary glands (Fig. 8). However, cell size was dramatically reduced. Glands were significantly smaller, and nuclei were more closely packed. This is clearly seen in the insets to Fig. 8B and C. The effects of bthD RNAi appear to be specific to the salivary gland. Although the daGAL4 driver directed expression throughout the embryo from early stages of development, no significant effects on embryo development, or on the development of other larval tissues, were noted. For example, no differences in imaginal disk morphology were seen between daGal4/+ and daGal4/UAS-BthdRNAi larvae (Fig. 8D and E). In addition, no differences were seen in the size or morphology of the third-instar larval gut between daGal4/+ and daGal4/UAS-BthdRNAi larvae (Fig. 8F and G) or in the developing trachea of these fly lines (data not shown).

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FIG. 8. Salivary gland morphogenesis. Salivary glands were dissected from third-instar daGAL4/+ (A), daGAL4/UAS-BthDRNAi (B), and fkhGAL4/UAS-BthDRNAi (C) larvae, fixed, and stained with DAPI to visualize nuclei (x50 magnification). The insets in panels B and C show images of daGAL4/UAS-BthDRNAi and fkhGAL4/UAS-BthDRNAi salivary glands, respectively, at higher magnification (x100). As controls, leg imaginal disks from third-instar daGAL4/+ (D) and daGAL4/UAS-BthDRNAi (E) larvae were dissected, fixed, and stained with DAPI (x100 magnification) and the gut from third-instar daGAL4/+ (F) and daGAL4/UAS-BthDRNAi (G) larvae was fixed and stained with DAPI (x50 magnification).

The reduction in salivary gland size was confirmed by staining with rhodamine-phalloidin to visualize cortical actin. The size of individual cells was dramatically reduced in daGal4/UAS-BthdRNAi salivary glands (data not shown). Although in vivo generation of bthD dsRNA had a profound effect on larval salivary gland morphology, staining with antibodies against the embryonic salivary gland marker Pasilla (35) showed little or no effect on embryonic salivary gland development (data not shown). This observation is consistent with the expression of BthD late in embryonic development and suggests that BthD does not have a function in the early embryonic phases of salivary gland development.

Top Abstract Introduction Materials and Methods Results Discussion References

 
BthD is one of three selenoproteins identified in Drosophila (8, 28). As part of a program to understand the role of selenoproteins in health and development, we examined the biological function of BthD. Homologs of this protein are present in a variety of organisms, including humans, but none of the members of this family have been previously characterized. Our analysis shows that BthD is expressed dynamically during Drosophila development. High levels are detected in the adult ovary, and a large maternal dowry of BthD protein and RNA is contributed to early embryos. At late stages of embryogenesis, BthD accumulates in the developing salivary gland. Using an inducible RNAi system, we ablated BthD expression in the salivary gland and found that BthD is required for proper salivary gland morphogenesis. Loss of BthD reduces animal viability.

We had anticipated that the BthD expression profile would provide important clues to function. To a certain extent this expectation was realized. The most noticeable feature of BthD expression is its relatively dynamic nature. Both RNA and protein abundance and distribution fluctuate considerably. Such an expression profile argues against BthD having a general function in cellular metabolism. This is in marked contrast to the other two Drosophila selenoproteins, SPS2 and G-rich. Analyses using antibodies against either SPS2 or G-rich indicate that these proteins are expressed ubiquitously and constantly throughout development (unpublished data). While the actions of G-rich are unclear, the functions of SPS2 are well established. SPS2 underpins Sec biosynthesis, as it catalyzes the synthesis of selenophosphate for its donation to the acceptor intermediate in Sec biosynthesis (12, 21, 26), and it also indirectly serves in Drosophila as an antioxidant (30).

In tissues where it is expressed, BthD is localized in the cytoplasm and is not detected in the nucleus. BthD staining in SL2 cells colocalizes with that of a known Golgi marker. It is tempting to speculate, therefore, that BthD protein traffics through the Golgi to another compartment. The gross expression profile of BthD suggests that BthD has some function in protein secretion or processing. Both regions where BthD is most highly expressed correspond to high biosynthetic activity. In the developing egg chambers, nurse cells abutting the developing oocyte synthesize large quantities of protein that drive the early postfertilization development of the Drosophila embryo. The salivary glands synthesize digestive enzymes and pupal glue proteins (4).

In early embryos, the cytoplasmic distribution of BthD is dynamic. At the earliest stages that we examined, the majority of BthD was found in large spherical structures. These are located immediately below the plasma membrane in the cortical cytoplasm and do not correspond to the known distributions of yolk particles or lipid vesicles (38). As development proceeds these structures appear to break down or be absorbed into small vesicles or particles. In the salivary gland, this particulate staining is recapitulated. It is also interesting that when BthD can first be detected in the salivary gland, high levels are seen in the lumen. At later stages, lumenal staining is decreased. It is possible that decreased lumenal staining corresponds with gland activity. Salivary glands are believed to be functional and to actively secrete by larval hatching (1).

We used an inducible RNAi strategy to selectively remove BthD expression and elucidate its function in salivary glands. Inducible RNAi has been successfully employed to target gene function in Drosophila (11, 19, 32, 37). Analysis using two-color fluorescent-protein imaging indicates that the effects of RNAi are cell autonomous.

We used two GAL4 drivers to induce RNAi expression: daGAL4, which drives ubiquitous expression from early embryonic stages (39), and fkhGAL4, which drives salivary gland-specific expression (31). We were able to reduce BthD levels in late-stage embryos and observed similar phenotypes with both drivers. Quantitative reverse transcription-PCR showed that bthD mRNA levels were also reduced (data not shown). Ubiquitous expression of bthD dsRNA did not have general effects on cell viability and morphology. The major effect we observed was on salivary gland morphogenesis. Removal of BthD caused a dramatic reduction in the size of the third-instar larval salivary gland. Cell number was not decreased. However, cell size was greatly affected.

We were unable to address the functions of BthD in ovary or early embryo development, as our constructs were based on pUAST, which does not express well in the female germ line (34). Moreover, the daGAL4 driver initiates expression after early embryonic BthD levels have started to decline. The elucidation of the expression of BthD and its effect on salivary gland morphogenesis in Drosophila, however, appears to suggest a novel role for selenoproteins in development. It will be of considerable interest to elucidate the specific role of BthD action in salivary gland morphogenesis and survival.

 
We express our sincere appreciation to D. Andrew, JHMI, for the ET ps(N33) Drosophila line and the Pasilla antibody and to Susan H. Garfield, CCR Confocal Microscopy Core, LEC, NCI, NIH, for her expert assistance with confocal microscopy.

This work was supported in part by NIH GM061603 (V.N.G.), 21C Frontier Functional Human Genome Project from the Ministry of Science and Technology of Korea (B.J.L.), and the Wellcome Trust (P.B.).

 
* Corresponding author. Mailing address for B. J. Lee: Laboratory of Molecular Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea. Phone: 82-2-880-6775. Fax: 82-2-872-9019. E-mail: imbglmg{at}plaza.snu.ac.kr.. Mailing address for D. L. Hatfield: National Cancer Institute, National Institutes of Health, Building 37, Room 2D09, Bethesda, MD 20892. Phone: (301) 496-2797. Fax: (301) 435-4957. E-mail: hatfield{at}dc37a.nci.nih.gov.

Present address: Departmento de Bioquimica y Biologia Molecular, Facultat de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, December 2003, p. 8495-8504, Vol. 23, No. 23
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.23.8495-8504.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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