Genome-wide survey of V-ATPase genes in Drosophila reveals a conserved renal phenotype for lethal alleles

Adrian K. Allan, Juan Du, Shireen A. Davies and Julian A. T. Dow

Institute of Biomedical and Life Sciences Division of Molecular Genetics, University of Glasgow, Glasgow, United Kingdom


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
V-ATPases are ubiquitous, vital proton pumps that play a multiplicity of roles in higher organisms. In many epithelia, they are the major energizer of cotransport processes and have been implicated in functions as diverse as fluid secretion and longevity. The first animal knockout of a V-ATPase was identified in Drosophila, and its recessive lethality demonstrated the essential nature of V-ATPases. This article surveys the entire V-ATPase gene family in Drosophila, both experimentally and in silico. Adult expression patterns of most of the genes are shown experimentally for the first time, using in situ hybridization or reporter gene expression, and these results are reconciled with published expression and microarray data. For each subunit, the single gene identified previously by microarray, as upregulated and abundant in tubules, is shown to be similarly abundant in other epithelia in which V-ATPases are known to be important; there thus appears to be a single dominant "plasma membrane" V-ATPase holoenzyme in Drosophila. This provides the most comprehensive view of V-ATPase expression yet in a multicellular organism. The transparent Malpighian tubule phenotype first identified in lethal alleles of vha55, the gene encoding the B-subunit, is shown to be general to those plasma membrane V-ATPase subunits for which lethal alleles are available, and to be caused by failure to accumulate uric acid crystals. These results coincide with the expression view of the gene family, in which 13 of the genes are specialized for epithelial roles, whereas others have spatially or temporally restricted patterns of expression.

reverse genetics; Malpighian tubule; functional genomics


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
V-ATPASES ARE LARGE, multisubunit pumps that transport hydrogen ions in exchange for energy, in the form of ATP. They are present in the endomembranes of all cells and in the plasma membranes of many specialized eukaryotic cells (28). The V-ATPases of animals, plants, and fungi are structurally very similar and are composed of two functional domains, V1 and Vo. The V1 domain, located on the cytoplasmic side of the membrane, is composed of eight different subunits (A–H) and is responsible for ATP hydrolysis. The Vo domain is a membrane-bound, proton-conducting complex composed of at least four, possibly five, subunits (a–e). In insects, V-ATPases are localized in the apical membranes of nearly all epithelial tissues (such as salivary glands, midgut, and Malpighian tubules), where they energize secondary active transport processes across the epithelium (54). Tissue specificity has also been demonstrated for V-ATPase isoforms for the a- and c-subunits in Caenorhabditis elegans (32–34) as well as the B-, C-, d-, E-, and G-subunits in mammals (26, 29, 35, 46, 47). This may reflect distinct targeting of isoforms in various tissues; indeed, differential localization of isoforms has been observed for the a-subunit in mice, where isoform-a1 resides in synaptic vesicles (24) and -a3 in multinucleate osteoclasts (23).

Although much of the functional characterization of V-ATPases has been performed in yeast, with its powerful genetic advantages, a genetically tractable animal model is essential for study of plasma membrane V-ATPases. The first animal knockout of a V-ATPase subunit (vha55, encoding the B-subunit) was identified in Drosophila, where it conferred a larval lethal phenotype (7). Drosophila, with rich genetic resources and a completed genome project, is extremely suited to dissection of genes involved in plasma membrane functions of the V-ATPase on an organismal scale. To study the V-ATPase multigene family in Drosophila, as well as its localization and function, we used a combination of bioinformatics, reverse genetics, and expression analysis to characterize the genes required for plasma membrane V-ATPase function and the consequences of their disruption. This is the most detailed mutational characterization of the V-ATPase gene family in an animal to date.


    METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drosophila.
Drosophila melanogaster strains were reared on standard cornmeal-yeast-agar medium and maintained on a 12:12-h light-dark cycle at 25°C. l(2)1339 (vha44sh), l(2)1248 (vha14sh), and l(2)1634 (vhaAC45sh) flies were supplied by Steven Hou (31). All other flies were obtained from stock centers at Szeged and Bloomington. For lethal-phase analysis, mutant fly lines were crossed to GFP balancer lines [w*; In(2LR)noc4LScorv9R, b1/CyO, P{w+mC = ActGFP}JMR1 for chromosome II, and w*;; Sb1/TM3, P{w+mC = ActGFP}JMR2, Ser1 for chromosome III; Bloomington stock nos. 4533 and 4544]. These balancers have characteristic patterns of green fluorescence in the embryonic midgut and so allow (nonfluorescent) embryos or larvae, homozygous for a V-ATPase mutation, to be distinguished from (fluorescent) siblings carrying one or two copies of the balancer.

In situ hybridization.
The in situ procedure is derived from those described by Ref. 40 and the Berkeley Drosophila Genome Project (BDGP) 96-well in situ protocol (http://www.fruitfly.org/about/methods/RNAinsitu.html). We designed in situ probes directed toward the 3'-UTR of each V-ATPase gene to minimize cross-hybridization between related V-ATPase genes. PCR products encoding the V-ATPase genes were cloned into pBluescript or PCRII vectors, and DIG-labeled RNA probes were generated by in vitro transcription. Adult tissues composed of gut, testes, ovaries, and Malpighian tubules were dissected in Schneider’s medium (Invitrogen) and placed into wells of a Millipore 96-well plate (MAGVN22 or MAGVS22) with 100 µl of Schneider’s medium. Schneider’s medium was removed by use of a vacuum pump, and postfix solution [10 mM potassium phosphate buffer (pH 7.0) containing 140 mM NaCl, 0.1% (vol/vol) Tween-20, and 5% (vol/vol) formaldehyde] was added for 20 min, followed by three washes with PBT [10 mM potassium phosphate buffer (pH 7.0) containing 140 mM NaCl and 0.1% (vol/vol) Tween-20]. The tissues were incubated with proteinase K in PBT (4 µg/ml) for 3 min at room temperature; the reaction was stopped with two washes of PBT containing 2 mg/ml glycine. The samples were washed twice with PBT before incubation with postfix for a further 20 min at room temperature. The tissues were washed with five changes of PBT, followed by one wash with 50% hybridization buffer [5x SSC containing 50% (vol/vol) formamide, 10 mM KPO4, 140 mM NaCl, 1 mg/ml glycogen, 0.2 mg/ml sheared salmon sperm DNA, and 0.1% Tween-20 (pH 7.0)] plus 50% (vol/vol) PBT. The samples were washed once with hybridization buffer before a 1-h preincubation with hybridization buffer at 55°C and subsequently incubated for 43 h at 55°C with 100 µl of hybridization buffer containing 10–500 ng of either the sense or antisense riboprobe, taking care to seal the wells with parafilm to prevent evaporation. After hybridization, the samples were washed four times with hybridization buffer at 55°C, followed by a final wash overnight with hybridization buffer at 55°C. Samples were washed once with 50% (vol/vol) hybridization buffer and 50% (vol/vol) PBT, followed by four washes with PBT, and then incubated overnight at room temperature with 100 µl of preabsorbed alkaline phosphatase-conjugated anti-digoxigenin Fab fragment (Roche Molecular Biochemicals) diluted 1:2,000 with PBT. The unbound antibody was removed with extensive washing in PBT (at least 10 times for ~5–10 min). We incubated the samples with DIG detection buffer (100 mM Tris·HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 5 min and then repeated again. The color reaction was initiated by the addition of DIG detection buffer + 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium and left from 10 min to 2 h at room temperature. Development was stopped with extensive washing with PBT containing 50 mM EDTA, and the tissues were removed from the wells and mounted on slides with 70% glycerol and viewed with the Axiocam imaging system (Zeiss, Welwyn Garden City, UK).

PCR.
Total RNA or genomic DNA was prepared from freshly dissected tubules with the use of TRIzol reagent (Invitrogen), digested with DNase1 and reverse transcribed with Superscript II RNase reverse transcriptase (Invitrogen) to produce cDNA. Primer sequences for V-ATPase gene amplification are shown in Supplementary Table S1 (available at the Physiological Genomics web site).1 Sequences were amplified using a high-fidelity Taq polymerase from cDNA and were cloned into plasmid vectors pBluescript or pCRII. The clones were sequenced to verify their identity, using sequencing primers specific to the cloning vector, before use to transcribe probes for in situ hybridization.

Cyberscreening and in silico analysis.
Cyberscreening was performed using National Center for Biotechnology Information (NCBI) and BDGP databases with basic local alignment search tool (BLAST)N, BLASTP, BLASTX or TBLASTN searches as appropriate. For each subunit, the prototypic human or yeast peptide was downloaded (as listed in Table 1) and used for a BLASTP search against the deduced Drosophila proteome (http://flybase.net/). Low-complexity filtering was turned off, and other parameters were defaults. Because these searches presume that the relevant Drosophila genes are both identified and correctly translated, the search was repeated as a TBLASTN search against the entire Drosophila genome (again, with filtering turned off, but with other parameters set to default values). However, no further genes were identified in this way, suggesting that gene calling by the genome project is now of a high standard. Genes were selected from the BLASTP outputs on the basis of stringent e-values (<10–10), and their deduced peptides were then re-BLASTed against the full NCBI protein database to ensure that their closest matches were indeed to the V-ATPase subunit in question. This step was necessary because of the close evolutionary similarities between V- and F-ATPases, and between some V-ATPase subunits. For example, BLASTP searches with the human A-subunit also turn up very close (e <10–20) matches to bellwether, an F-ATPase subunit, and vha55, the V-ATPase B-subunit. For comparative analysis with other species, the Saccharomyces genome database (http://www.yeastgenome.org/) and WormBase (http://www.wormbase.org/) were searched using the BLASTP strategy documented above. These results were also compared with published tabulations of known V-ATPase families for human, mouse, and Arabidopsis [Smith et al. (42) no. 119, Sze et al. (48) no. 112].


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Table 1. Curation of the V-ATPase gene family in Drosophila

 
Once Drosophila genes had been identified, matching expressed sequence tag (EST) clones and transcripts were identified from their FlyBase genome annotations (http://flybase.net). This procedure also allowed for the identification of putative alleles of these genes; all such stocks in public databases were ordered as they became available.

Detailed EST data are available (http://www.fruitfly.org/EST/EST.shtml) for more than 240 000 clones. Even though these ESTs are a mixture of 5'- and complete sequences, it is possible to identify in silico the putative transcripts with some authority for those genes with multiple EST hits.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The V-ATPase multigene family is encoded by 33 genes.
It previously was shown that at least 24 genes encode the 14 subunits of the V-ATPase in Drosophila (9), and we have recently extended this to 31 genes (52). Here, we now describe 33 genes that encode the presently known V-ATPase subunits or accessory proteins (Table 1). Apart from five subunits in the V1 sector that are encoded by single genes (B, C, E, G, and H) and the accessory subunits vhaAC45 and vhaM8.9, each V-ATPase subunit is encoded by more than two genes, and as many as five genes encode the Vo subunits-a and -c. To add further complexity, seven subunits have been annotated by the genome project as encoding alternatively spliced genes, although splice variants have only been shown in the 5'-untranslated regions of the ESTs for four subunits. Therefore, splice variants that affect protein sequence may only occur for subunits-C, -H, and -a. Pseudogenes are thought to be rare in Drosophila, with perhaps only 100 in total (17), so it is likely that most of these genes will be authentic. EST corroboration of these transcripts is discussed below.

Genomic organization of the V-ATPase gene family.
In some simple organisms, V-ATPase genes are found as an operon, allowing simple coordination of their expression (8, 44). In Drosophila, however, the genes for most subunits are spread at apparently random chromosomal locations throughout the genome (Table 1), although some subunits that are encoded by multiple genes show evidence of local gene duplication. For example, the A-subunit has a cluster of three genes at 34A, vha100-2 and vha100-4 are at 91A, vha16-2 and vha16-3 are at 68C, vhaPPA-1 and vhaPPA-2 are at 88D, and vhaM9.7-2 and vhaM9.7-3 are at 64B. Interestingly vha36-1 (CG8186) on the second chromosome has been described as a young retrotransposed gene derived from its parental gene vha36-3 (CG8310) on the X chromosome (5). The parental gene vha36-3 has three exons whereas the retrotransposed vha36-1 gene has one exon, presumably due to integration of reverse-transcribed mRNA into a new genomic position. Other V-ATPase subunits encoded by multiple genes have similar characteristics; the F-, c-, and e-subunits are encoded by multiple genes, of which one has multiple exons and the rest have one.

ESTs as evidence of bona fide expression.
In parallel with the Drosophila genome project, a relatively comprehensive program of cDNA sequencing to produce ESTs allows the authenticity and approximate abundance of genes to be documented. Analysis of the V-ATPase ESTs shows that all of the genes except eight have been detected as ESTs to date. The single gene subunits all have high numbers of ESTs (>48). As observed previously (12), where several genes encode a single subunit, there is a highly expressed ubiquitous gene with a comparatively higher number of ESTs. In the case of multigene subunits we know to be present in epithelial tissues, such as vha68-2 and vha16-1 (Ref. 12 and unpublished observations), these have a high number of ESTs compared with other genes coding the same subunit.

Recently, a comprehensive Affymetrix comparison of gene expression between Malpighian tubule and whole fly has been undertaken (52). The Malpighian tubule is a classic insect transporting epithelium in which the plasma membrane role of V-ATPases in principal cells is well documented (4, 7, 9, 11, 30, 45). It thus provides an ideal tissue in which to seek an expression "signature" for those V-ATPase genes that contribute to the epithelial plasma membrane holoenzyme. Such a signature is readily apparent (Table 1); it was observed that for each subunit, exactly one gene was both highly abundant and strongly upregulated in tubules: it was thus predicted that these genes encoded the subunits of the plasma membrane V-ATPase in tubule (52). However, to validate these plausible predictions, we adopted here an independent method for assessing expression levels in multiple tissues.

A common in situ expression signature for plasma membrane V-ATPase subunit genes.
To map the expression of V-ATPase subunits, a 96-well in situ hybridization method was employed, using RNA probes directed against the 3'-UTR of each gene to minimize cross-hybridization. The results are informative at several levels. First, because the holoenzyme must be assembled with at least one gene product encoding each subunit, all genes that uniquely encode a subunit (e.g., B) are very widely expressed, as one would expect. Second, for each subunit, at least one gene showed strong hybridization signals in all major epithelial tissues (Malpighian tubules, midgut, hindgut, and rectum) (Table 2 and Figs. 1 and 2). In every case, these were the genes that had been implicated in Malpighian tubule function by virtue of tubule enrichment in our earlier microarray analysis (52). (The only exception is the accessory subunit vhaM8.9, thought to participate in vesicle trafficking; despite repeated efforts we were unable to obtain a convincing in situ result for this gene.) Taken together, the microarray and in situ data thus allow us to assert that the plasma membrane V-ATPase holoenzyme, not just in Malpighian tubule but in the major transporting epithelia, is a single isozyme composed of polypeptides from vha68-2, vha55, vhaSFD, vha44, vha36-1, vha26, vha14-1, vha13, vha100-2, vha16-1, vhaPPA1-1, vhaM9.7-2, vhaAC39-1, and vhaAC45. To our knowledge, this is the first time that a plasma membrane V-ATPase has been genetically characterized at this level.


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Table 2. Expression profiles of the V-ATPase gene family in Drosophila

 


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Fig. 1. Expression of cytoplasmic V1 and accessory V-ATPase isoforms in Drosophila tissues. In situ hybridization was performed on whole mount adult Drosophila tissues. Expression of V-ATPase subunits encoding components of the cytoplasmic V1 sector are shown in Malpighian tubules (mt), midgut (mg), hindgut (hg), rectum (re), testes (te), ovarioles (ov), ejaculatory bulb (eb), and duct (ed). Leucokinin receptor (LKR) was used a positive control: it marks stellate cells of the Malpighian tubule (37).

 


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Fig. 2. Expression of Vo V-ATPase isoforms in Drosophila tissues. In situ hybridization for V-ATPase subunits in Malpighian tubules (mt), ovarioles (ov), larval brain (lb), testes (te), hindgut (hg), rectum (re), copper cells of the midgut (Cu), and midgut (mg). Sense negative control probe signal is demonstrated for vha100-2.

 
Genes with spatially restricted expression patterns.
Where, if anywhere, are the other genes expressed? Although our adult in situ screen may not pick up genes with extremely restricted expression patterns, it was possible to identify sites for most genes. In particular, specialized transcripts were found in brain, testes, or ovaries (Table 2). These results are consistent with a previous EST and microarray analysis of testes, which identified a relatively large percentage of genes that were either entirely novel or which did not appear to be abundantly expressed elsewhere (1).

Although we did not do in situ hybridization analysis in embryos, there is an online resource (50) that currently displays embryonic expression patterns for hundreds of Drosophila genes, including 12 of the V-ATPase genes (http://www.fruitfly.org/cgi-bin/ex/insitu.pl). Interestingly, of the available genes, those considered to be plasma membrane V-ATPase subunits (vha68-2, vha55, vhaSFD, vha44, vha36-1, vha26, vhaM9.7-2, vhaAC39-1, and vhaAC45) generally show ubiquitous embryonic expression, with strongest staining in the gut and tubules. The other three genes (vha68-1, vha100-1, and vha100-4) show distinctly different patterns. In contrast to vha68-2, which shows expression in gut, tubules, caeca, and muscle, vha68-1 is restricted to hindgut, ventral nerve cord, and central nervous system (Fig. 3). The fact that the majority of ESTs for vha68-1 are from head libraries (30/42), while head ESTs make up only one-third of vha68-2 ESTs (30/84) supports the assignment of vha68-1 as a head isoform. The gene vha68-3 has 56 ESTs that are exclusively derived from testes, and we observe strong expression for this isoform in the testicular ducts, accessory gland, ejaculatory duct, and bulb. The vha68 genes encoding the A-subunit thus show very clearly distinct roles for each gene.



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Fig. 3. V-ATPase isoform expression shows temporal and spatial specificity in embryos. V-ATPase in situ hybridization images were kindly reproduced with permission from the Berkeley Drosophila Genome Project in situ website (http://www.fruitfly.org/cgi-bin/ex/insitu.pl). Top: vha68-1 shows expression in central nervous system, nerve, and midgut, whereas vha68-2 shows expression in Malpighian tubules, hindgut, gastric caeca, and midgut. vha100-4 displays expression in copper cells of the midgut. Middle: vha55 shows maternal mRNA expression at stages 1–3 (left), no signal at stages 7–8 (middle), and gut and tubule staining at stages 13–16 (right). Bottom: microarray-based expression profile of vha55 during embryogenesis is consistent with the embryonic in situs.

 
In common with humans (Table 2), the Drosophila a-subunit is encoded by multiple vha100 genes, and again there is some evidence for differential expression. The ubiquitous/epithelial isoform is encoded by vha100-2 (Tables 1 and 3), while vha100-1 appears to be enriched in head, testes, and ovaries (Table 2). Consistent with this, vha100-1 is most closely similar to the mammalian a1-isoform: in mouse, the a1-1-transcript is brain specific (27). All six vha100-3 ESTs obtained are from testes, suggesting tissue specificity for this isoform. The genes vha100-4 and vha100-5 are likely to have restricted embryonic expression: 12 of the 13 ESTs for vha100-5 are of embryonic origin, as well as the single EST for vha100-4. The expression pattern of vha100-4 observed from the online embryo expression database displays restricted expression in what appears to be copper cells of the gut. Thus the vha100 genes appear to be similar to worms, mice, Arabidopsis, and humans (Table 3) in that they have at least four isoforms with varying tissue expression. Because the a-subunit contributes to the hemichannels that couple rotation to proton transport by the holoenzyme (13), it is possible that selection of different a-subunits may provide different kinetic, or "gearing," properties appropriate to different tissues (25, 36).


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Table 3. Comparative analysis of V-ATPase gene families

 
Our in situ results, taken together with community in situ, lacZ, and EST data, show that the large majority of V-ATPase genes in Drosophila are genuinely expressed as mRNAs. For each subunit, there is a single gene that is widely expressed and that is also highly expressed in epithelia. Other genes appear to have developmentally or spatially much more restricted expression patterns, typically embryonic only, or specific for testes or head.

Lethal phase of vha55 mutations.
It has been previously shown that a known and well-characterized existing genetic locus (SzA) (14) corresponds to the gene (vha55) encoding the B-subunit, thus making vha55 the first animal "knockout" for a V-ATPase subunit (7). For this prototypic knockout, an allelic series was characterized (14). True nulls (deleted for the entire locus) survived to hatching but failed to thrive, whereas point mutant homozygotes could die midway through embryonic development (14). We have suggested that these results are consistent with a large maternal investment of V-ATPase mRNA in the embryo, sufficient to permit hatching without further zygotic mRNA. By contrast, a wave of zygotic expression in midembryogenesis would be antimorphic if the mRNA encoded a defective protein (9, 10). There is now gene expression evidence to support this dual-peak mRNA model. Using the BDGP gene expression website (http://www.fruitfly.org/cgi-bin/ex/insitu.pl), we observed that the plasma membrane V-ATPases have a large (necessarily maternal) mRNA expression from stages 1–5, followed by a sharp decrease in mRNA (Fig. 3). Expression of zygotic V-ATPase mRNAs starts to rise again at stage 9. These results are exactly consistent with our earlier predictions (9, 10).

A visible tubule phenotype is shared by all plasma membrane V-ATPase gene disruptants.
A characteristic of lethal alleles of vha55 is a transparent Malpighian tubule phenotype that is autonomous when mutant tubules are transplanted into abdomens of healthy flies (14). We have suggested that this phenotype is due to a defect in urinary acidification (9, 11). In insects, purine metabolites are excreted as uric acid, to conserve water. Urate ions are transported in soluble form into the tubule lumen, where they are acidified to precipitate uric acid. In normal embryos, birefringent uric acid crystals first become visible just before hatching (41).

If this explanation is correct, then the lethal clear tubule phenotype should be general to null alleles of all the plasma membrane V-ATPase subunits. Indeed, this phenotype has already been shown by null alleles of vha68-2 (12). The genetic resources available for Drosophila allow the generality of our hypothesis to be tested more comprehensively than has previously been possible for such a large gene family. We obtained P-element insertions for all the plasma membrane V-ATPase subunits except one (vhaAC39-1), as well as further EMS mutants for vha55, and established both the lethality of the insertions and the lethal phases where appropriate, and scored for a transparent tubule phenotype. All the P-element insertions or EMS point mutations in V-ATPase genes except one (vha100-2) resulted in a lethal phenotype, mostly at the embryonic or early larval stage, and generally displayed clear tubule phenotypes (Table 4 and Fig. 4). The only exceptions were P-element insertions in vha44, vhaPPA-1, and vhaAC45, which had much later lethal phases, suggesting that they were hypomorphs that expressed sufficient zygotic mRNA to survive hatching. This work thus shows that the transparent tubule phenotype is associated with disruption of any gene encoding a subunit of the tubule plasma membrane V-ATPase.


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Table 4. Clear tubule lethal phenotype is conserved among V-ATPase mutant alleles

 


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Fig. 4. Conservation of a clear tubule phenotype in V-ATPase mutants. Flies with P-element insertions in vhaSFD and vha14 are homozygous lethal at the late embryo/late larval stage and display a transparent tubule phenotype compared with heterozygous siblings. Top: transmitted light shows opaque tubule in heterozygotes (above) compared with vhaSFD and vha14 homozygotes (below). Middle: polarized light microscopy shows increased uric acid crystal deposits in heterozygotes compared with homozygotes. Bottom: polarized light and GFP fluorescence image. Embryos and larvae were genotyped using GFP balancer chromosomes: heterozygotes show widespread green fluorescence in the gut, characteristic of the balancer chromosome, whereas homozygotes show no fluorescence due to absence of the GFP balancer chromosome. By inspection, birefringent uric acid crystals are seen to be far more abundant in the heterozygote tubules.

 
Although the alleles we obtained are annotated (by FlyBase) as mutants of V-ATPase subunits by virtue of their genomic insertion sites, it should be stressed that this falls short of rigorous genetic proof. Specifically, to confirm that the phenotype observed is due to disruption of a particular gene, it is normally necessary to demonstrate rescue by transgenesis with the wild-type cDNA. For so large a collection of mutants, this workload would prove prohibitive; nonetheless, we are confident that these alleles are authentic V-ATPase mutants. This is because we have previously demonstrated reversion of lethality by precise excision of the P-elements in alleles of both the B- and A-subunits: vha55j2e9 (7) and vha68-2k02508 (15, 16), confirming that lethality was due to the insertions. Additionally, we have rescue of lethality and of the transparent tubule phenotype for vha55 alleles by overexpression of a vha55::GFP fusion protein (A. K. Allan, unpublished observations). Furthermore, the transparent tubule phenotype is rare; a search for "transparent Malpighian" across 93,777 alleles documented in FlyBase (http://flybase.bio.indiana.edu/) only matches five alleles, all of vha55. Such close association of transparent Malpighian tubules with putative V-ATPase disruptants is thus extremely persuasive and in turn provides experimental support for the automated assignment of these insertions as V-ATPase mutants.

It is possible also to confirm our previous model for the tubule phenotype. The tubule contains two kinds of apical concretion, spherites of calcium phosphate (53) and crystals of uric acid (41); only the latter of these is birefringent under polarized light. The transparent tubule phenotype we describe is invariably associated with loss of birefringence in the tubule lumen (Fig. 4); therefore, it results from failure to excrete uric acid.

Are all V-ATPase genes essential?
The distribution of available lethal alleles among the V-ATPase genes is itself worthy of comment. Together with the stocks available to us, other workers have shown that P-element insertions in vha44 (18) and vha100-2 (6) are lethal. There are thus lethal alleles for all the subunits we have defined as plasma membrane, except vhaAC39-1, and there are no lethal alleles for any of the other genes. There are thus no mutants currently available to use as a negative control; so, while we have shown that lethal alleles of plasma membrane subunits do display the transparent tubule phenotype, we cannot assert the contrary. Many tens of thousands of P-element insertion lines have now been generated across the world, and 40% of genes now have associated insertions (3). It would thus be tempting to suppose that the nonplasma membrane subunits do not have associated lethal alleles because their function is not essential. However, P-elements have notoriously nonuniform insertion patterns within the genome (3) and prefer to insert in the first introns of abundantly expressed genes. Most of the nonplasma membrane subunits have only one exon and are thus difficult to disrupt by P-element insertions. It will be of interest to follow the progress of Drosophila gene disruption projects that utilize other classes of transposon, such as piggyBac (49).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The completion of the Drosophila genome project and the uniquely powerful associated informatic and mutant resources associated with it have allowed us to curate the V-ATPase gene family in unprecedented detail for a multicellular organism. Combined with the EST data available, exon predictions can be confirmed, splice variants annotated, and the relative abundance of each gene estimated in various stages in tissues using EST number. Out of the 14 subunits identified, 8 are encoded by multiple genes. These subunits display tissue specificity, with each subunit having a gene enriched in epithelial cells usually accompanied by other isoforms, the expression of which is restricted to reproductive tissues or the nervous system. Consistent with this, microarray analysis from other groups has demonstrated that there is a tightly regulated gene battery for the genes coding for the plasma membrane subunits during development (2) and in epithelial tissue (22), which is typical for a group of genes that code for proteins incorporated into the same complex. Finally, when these genes are disrupted, they display a clear tubule phenotype that is conserved across mutant alleles for almost all the V-ATPase subunits. This renal phenotype is particularly interesting, as defects in renal acidification are also observed in human patients with mutations in the genes coding for subunit-B (21) and subunit-a (43).

Although we have now shown a really tight correlation between V-ATPase mutation and transparent tubules, it does not follow that these are the only mutants that may prove to display such a phenotype. For example, defects in a gene encoding the cognate urate transporter should also prevent the accumulation of apical uric acid crystals. Perhaps more interestingly, we have predicted that any accessory protein that participates in the assembly, apical targeting, or anchoring of the plasma membrane holoenzyme should show such a defect (11). This could be important, because it would provide the first screen for such proteins in an animal.

The role of the V-ATPase subunits whose expression is not enriched in epithelial tissues is unclear. Some have ESTs from testes, head, and embryo; others have no ESTs at all. Some show in situ hybridization signals in ovaries, testes, and larval brain. Comparisons with V-ATPase isoforms from other animals may give some clues. In mice there is a testis-specific isoform for subunit-E (46) that is localized to acrosomes in sperm and has been proposed to be required for acrosome acidification, which is necessary for processing protease zymogen essential for fertilization. Neuron-specific isoforms of subunits-a1 in C. elegans and Torpedo marmorata (24, 33), -e in rodents (51), and -G in mice (26) have been reported. Within neurons, V-ATPase isoforms can be localized to synaptic vesicles and the presynaptic membrane (24, 26). Thus it is entirely feasible the nonepithelial V-ATPases may be performing similar tissue-specific roles.

The transcriptional regulation of V-ATPase gene battery has been shown to be tightly regulated during development, with typically a large maternal expression at stages 1–3 followed by a sharp decrease. At stage 9 is a rise in expression, peaking at larval stage, followed by a decrease at pupation. Expression returns as flies emerge from the pupae. The decrease in expression is attributed to the effect of the molting hormone ecdysone, acting on ecdysone-responsive elements in V-ATPase promoters, and it has been shown in Manduca sexta that ecdysone can transcriptionally downregulate V-ATPase transcription when injected as well as when assayed with a reporter system to analyze promoters (38). We also identified putative ecdysone-responsive elements 1–2 kb upstream of all the plasma membrane promoters in Drosophila (not shown). Another interesting observation was the absence of TATA boxes or downstream promoter elements in all the V-ATPase genes except vha16-1, which is typically a characteristic of housekeeping genes. These characteristics are also shared by the promoter for pyruvate kinase, which is also ecdysone responsive and has no TATA box or downstream promoter element (20).

The completion of the Drosophila genome project, the availability of online databases with microarray and gene expression data, plus the rich abundance of existing mutant stocks and genetic tools, unique to this species, have enabled a detailed characterization of the V-ATPase gene family in a model organism. Further challenges lie ahead in understanding the transcriptional regulation, assembly, targeting, and activity of plasma and endomembrane V-ATPases in multicellular eukaryotes.


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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by a Genomics in Animal Function Grant from the Biotechnology and Biological Sciences Research Council of the United Kingdom.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: J. A. T. Dow, IBLS Division of Molecular Genetics, Univ. of Glasgow, Glasgow G11 6NU, UK (e-mail: j.a.t.dow{at}bio.gla.ac.uk).

10.1152/physiolgenomics.00233.2004

1 The Supplemental Material for this article (Supplemental Table S1) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00233.2004/DC1. Back


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