The RGS gene loco is essential for male reproductive system differentiation in Drosophila melanogaster
© McGurk et al; licensee BioMed Central Ltd. 2008
Received: 25 June 2007
Accepted: 03 April 2008
Published: 03 April 2008
The loco gene encodes several different isoforms of a regulator of G-protein signalling. These different isoforms of LOCO are part of a pathway enabling cells to respond to external signals. LOCO is known to be required at various developmental stages including neuroblast division, glial cell formation and oogenesis. Less is known about LOCO and its involvement in male development therefore to gain further insight into the role of LOCO in development we carried out a genetic screen and analysed males with reduced fertility.
We identified a number of lethal loco mutants and four semi-lethal lines, which generate males with reduced fertility. We have identified a fifth loco transcript and show that it is differentially expressed in developing pupae. We have characterised the expression pattern of all loco transcripts during pupal development in the adult testes, both in wild type and loco mutant strains. In addition we also show that there are various G-protein α subunits expressed in the testis all of which may be potential binding partners of LOCO.
We propose that the male sterility in the new loco mutants result from a failure of accurate morphogenesis of the adult reproductive system during metamorphosis, we propose that this is due to a loss of expression of loco c3. Thus, we conclude that specific isoforms of loco are required for the differentiation of the male gonad and genital disc.
Many hormones and neurotransmitters act by binding to G-protein-coupled receptors (GPCRs) which transduce the signal via second messengers such as cAMP. The heterotrimeric G proteins comprise of one member from each of the Gα, Gβ and Gγ families. In the absence of an external signal the GPCRs are associated with an inactive heterotrimer complex, Gα-GDP/Gβ/Gγ. When a specific ligand binds a GPCR, the intrinsic nucleotide exchange factor (GEF) activity is activated; the resultant Gα-GTP subunit dissociates from Gβ/Gγ, leaving the Gβ/Gγ heterodimer and the Gα-GTP to spread the signal to downstream target molecules. The GTP is slowly hydrolysed by Gα, and the Gα-GDP then returns and binds to the Gβ/Gγ complex rendering the receptor inactive. RGS proteins (Regulator of G-protein signalling) are a family of GTPase activating proteins (GAP) that trigger the intrinsic GTPase activity of the Gα subunits [1, 2]. Although a great deal is known about the regulation of G-protein-coupled receptor signalling in a variety of organisms [3, 4] less is known in Drosophila and more importantly the involvement of G-protein-coupled receptor signalling in developmental decisions.
The Drosophila loco gene encodes a number of isoforms of an RGS protein (Figure 1A). loco c1 and loco c2 were the first transcripts to be identified and were found to be differentially expressed during embryogenesis , subsequently we identified a third transcript, loco c3, and showed that it was required for egg and embryo development . The sequence data on loco c3 has been extended to show that more sequence, including a start site, was upstream of the original start site identified for loco c3. The two start sites are in frame, but neither has been shown to be functionally active . To remain in line with the published nomenclature, we will call this extended transcript loco c4.
Until now nothing was known about the involvement of RGS in G protein signalling in the male reproductive system, we therefore set out to determine if LOCO was also essential for male development. We isolated male fertility mutants from a P element mobilisation screen  and have shown that the male sterility is due to mutations mapping in the loco gene. Gene expression analysis has not only identified disrupted gene expression in the loco mutant lines but it has also revealed a fourth loco transcript required for correct male development. This alongside the phenotypic analysis of the semi-sterile males suggests a role for loco in the differentiation of the testis from the male gonads and genital discs. Furthermore we analyse loco expression in male gonads and the male adult reproductive tissue in both wild-type and loco mutant lines.
Finally, RGS proteins, such as LOCO, negatively regulate signalling mediated by G-protein coupled receptors, by reducing the time that the Gβ/Gγ subunit is available to signal. However with an additional GoLoco motif, LOCO can also increase the initiation rate of G protein signalling . LOCO may well regulate this signalling pathway in the follicle cells of the Drosophila egg and glial cells of the embryo by binding to the Drosophila Gαi subunit [14, 16, 17]. In order to analyse the presence of Gα-proteins, which could potentially interact with LOCO in the Drosophila testis, we undertook a candidate PCR approach and identified a further two Gα subunits expressed in the testis.
Screens for male sterility
Previously we carried out a P element mediated mutagenesis screen using a P element located between exons II-1 and I-1 of the loco gene (Figure 1A) . We established that perfect excision of this element led to fully viable fertile lines, indicating that there were no other mutations in the stock. Most of the 399 lines that we generated were homozygous lethal and many of the viable lines produced very few homozygous adults, indicating a requirement for loco during development. Many of the lines, which generated some adults, also showed reduced fertility in females. Complementation analysis of the 399 lines showed that the mutants fell into two different complementation groups, however, two mutant lines fell into both complementation groups. The complex splicing of loco transcripts makes it likely that both of the complementation groups affect different essential transcripts of the loco gene.
Complementation tests for male fertility
Average number of progeny produced per cross
loco 358 /loco 318
loco 387 /loco 318
loco 387 /loco 358
loco 370 /loco 358
loco 370 /loco 318
loco T1 /loco 318
loco Δ113 /loco 387
loco Δ113 /loco 358
loco Δ113 /loco 318
Complementation of male semi-sterile lines with deficiency lines
loco 318 /OrR
loco 358 /OrR
loco 370 /OrR
loco 387 /OrR
loco 318 /Df15
loco 318 /Df17
loco 318 /loco Δ13
loco 358 /Df15
loco 358 /Df17
loco 358 /loco Δ13
loco 370 /Df15
loco 370 /Df17
loco 370 /loco Δ13
loco 387 /Df15
loco 387 /Df17
loco 387 /loco Δ13
In order to assess whether our loco mutants were in the same or different complementation groups to the deficiency lines , the four loco mutant lines were crossed to each of the above mentioned deficiency lines. All heteroallelic mutants had reduced fertility when compared to the heterozygous mutants (Figure 1C). Despite all of the heteroallelic loco mutants showing reduced fertility not all were a statistically significant reduction (Table 2). However all of the mutant loco alleles (loco 318 , loco 358 , loco 370 , and loco 387 ) hemizygous with the loco Δ113 allele, showed a statistically significant reduction in male fertility (Table 2 and Figure 1C). This not only suggests that the mutations isolated here fall into the same complementation group but, that the mutations we have isolated reside within the loco gene.
Expression of locoin the testis
In order to analyse the expression of the various loco splice variants in the Drosophila testis primers were designed to specifically amplify loco c1, loco c2, loco c3 and loco c5. It should be noted that the primers used to amplify loco c3 could not discriminate between loco c3 and loco c4, furthermore it is possible that these two transcripts form the same transcriptional unit. RT-PCR revealed that loco c1, loco c2, loco c3, and loco c5 were expressed in the OrR male testis (Figure 2C). Detection of loco c2 using the primer pair FP1 and RP8 (Figure 1A) produced a specific product at approximately 1 kb and two non-specific bands at lower molecular weights (Figure 2C, 3'c2). Cloning and sequencing revealed that the 1 kb band was specific to loco c2, the 0.9 kb band aligned to CaBP1 (CG5809) and the 0.4 kb band aligned to myosin binding subunit (CG32156). We also show later in the paper that the novel transcript identified from the testis EST library is not unique to the testis, as it is also expressed in pupae. Thus what we have identified and described is a further novel loco transcript which is expressed during development.
G-protein α subunits are also expressed in the adult reproductive system
LOCO is a regulator of G-protein signalling and has been shown to interact with various Gα subunits [14, 17]. Searching the Drosophila genome and various EST databases we found several Gα transcripts and proteins. We wanted to investigate if different Gα subunits were expressed in the testis providing potential binding partners for the isoforms of LOCO that are generated. The three Gα genes we choose to analyse were G-oα47A, Gα73B, and Gα49B.
G-oα47A the Drosophila homologue of the mammalian Goα, is needed for embryonic development [18–20] and has more recently been shown to contribute to asymmetric cell division . Furthermore it is expressed in the nurse cells and oocyte and is present in various adult nerve cells . Gα73B encodes a further Gα subunit called Gfα, it is expressed in the embryonic midgut and in the nurse cells after which it is transported to the oocyte . Gα49B, a Gq subunit involved in phospholipase C activation, is involved in the Drosophila visual system [24, 25]. Gα49B is known to be expressed in the adult testis , and thus acted as a positive control. PCR showed that G-oα47A, Gα73B and Gα49B were expressed in the testis (Figure 2D). This raises the possibility that in the adult testis there are additional Gα subunits that may interact with LOCO.
Analysis of mutant phenotypes
Expression of locoduring male development
We have now shown that loco is expressed during metamorphosis. In order to ascertain whether loco is expressed during organogenesis of the male gonad, the original P element insertion line, which contains GAL4 in the loco gene , was crossed to a UAS-lacZ reporter strain. β-galactosidase activity was detected in the larval male gonads (Figure 2B, arrow) as well as in the fat body tissue surrounding the gonad. The observed β-galactosidase activity in the gonad confirms that expression of the loco gene takes place in the male gonad during development.
Expression of locoin mutant pupae and testes
We have demonstrated that loco is expressed in the developing male and adult gonads, furthermore we have shown that loss of loco expression resulted in reduced fertility and testis with abnormal morphology. In order to ascertain which transcript was affected by the mutation we performed RT-PCR for loco c1, loco c2, loco c3 and loco c5 in the mutant pupae of three of the male semi-sterile lines, loco 318 , loco 358 , and loco 387 (Figure 4C–E). We detected loco c1, the 5'end of loco c2 and the loco c5 transcripts in the loco 318 mutant. However loco 318 completely lacked expression of the loco c3 transcript (Figure 4C). Furthermore loco 318 only expressed the 5'end of loco c2. The 3'end of loco c2 was not detected (Figure 4C, 3'c2) as only the 0.9 kb non-specific band was detected. Pupal expression from the loco gene in the loco 358 mutant produced loco c1, loco c2 and loco c5, however like loco 318 the loco 358 mutant lacked loco c3 (Figure 4D). Pupal expression of loco c1, loco c2, loco c3 and loco c5 was detected in the loco 387 mutant (Figure 4E). The testis expression of loco 358 was also found to lack any detectable loco c3 transcript.
The RT-PCR analysis was carried out on multiple different RNA samples and the same result was consistently achieved. Whenever the quality of the RNA was checked on a formaldehyde agarose gel it was found to be intact (data not shown). Furthermore detection of loco c1, loco c2 and loco c5 from the same cDNA samples which lacked loco c3 suggests that the RNA transcribed from the loco gene was intact. Taken together this data suggests that there is a requirement for loco c3 in adult reproductive tissue.
Analysis of the genomic sequence in the mutant lines
To gain further insight into molecular nature of the loco mutations, PCR was performed on genomic DNA isolated from the original c139 strain . PCR utilising a P element primer directed to the 5' end of the P element (pgawb5a inv) in combination with a gene specific primer revealed that the P element was in reverse orientation to the loco gene (the 5' end of the P element was orientated toward the 3'end of the loco gene). Sequencing of the PCR product revealed that the P element had inserted 322 bp upstream of exon I-1 (Figure 1A).
PCR analysis of the loco 318 mutant revealed that the P element primer directed to the 5' end of the P element (pgawb5a inv) worked in combination with gene specific primers in the forward and reverse orientation. This suggested that there was more than one P element present in loco 318 and that they were in the opposite orientation to one another. Direct sequencing of these products revealed that one P element was present at the original position (322 bp upstream of exon I-1) and the second P element was present at the same position but in the opposite direction (Figure 1A). The sequencing of the PCR products from loco 318 also revealed a 9 bp duplication of genomic DNA on either side of the P elements (Figure 1A). It is possible that two P elements 322 bp upstream of exon 2 alters pre-mRNA length such that there is premature dissociation of RNA polymerase II (RNAPol II), hindrance of the folding of the pre-mRNA molecule which prevents the joining of the splice sites, or it may disrupt important splice factor binding sites.
The P element primer used to reveal the position of the P element in the mutant line loco 318 failed to produce PCR products in the mutant line loco 358 . PCR across the original insertion site revealed that the P element had excised and had not deleted any genomic sequence (Figure 1B). The mutant line loco 358 expresses white, it lacks expression of loco c3 (Figure 4D) and the mutation genetically maps to the loco gene (Table 2). This suggested that a partial P element, which lacks the P element primer site, was present in the loco gene. PCR with the P element primer and FP8 produced a fragment of 2 kb, suggesting that the P element was 2 kb upstream of exon 2. Genomic PCR was performed across all introns in the loco gene. PCR failed only between exon I-1 and exon 2, whereas a PCR product across this region in wild-type genomic DNA was detected (data not shown). This further suggested that the partial P element in the mutant line loco 358 was located between exon I-1 and exon 2 and thus produced a PCR product too long to be detected by the PCR programme.
Direct sequencing of the insertion site in loco 387 revealed that no sequence had been deleted (Figure 1B). The loco 387 line was found to express loco c1, loco c2, loco c3, and loco c5 (Figure 4E). The reduction in male sterility when loco 387 is hemizygous with Df15, Df17 or loco Δ13 was statistically significant (Table 2) and the adult reproductive tissue of loco 387 is morphologically abnormal (Figure 3E). This suggests that a mutation resides within the loco gene. Large rearrangements can occur during P element mobilisation. The PCR product across the loco insertion site was approximately 500 bp, therefore if a large inversion or rearrangement had occurred in loco 387 it would not be detected by this simple PCR. These data alongside the genetic data strongly suggests that the mutations lie within the loco gene.
We isolated a number of homozygous lethal mutant lines of loco. These lines die at a variety of developmental stages, however, among them four lines were able to generate some homozygous adult males, which were semi-sterile. We suggest that this is most likely to be due to a failure of the correct morphogenesis of the testis and reproductive organ derivatives of the larval gonad. This adds another role to the wide range of developmental decisions that are known to be dependent upon loco. Granderath et al., (1999)  showed that loco mutants died as embryos showing abnormalities in the contacts between glial cells . Our previous studies illustrated that there is a requirement for loco in cytoplasmic dumping from the nurse cells to the oocyte and that loco is required for correct patterning of the eggshell and embryo . There is also a large maternal supply of loco in the embryo probably explaining why the embryos die so late in embryogenesis. Finally, it was shown more recently that loco contributes to asymmetric cell division of neuroblasts . These findings suggest that G-protein signalling may be important at wide variety of developmental stages in Drosophila.
The loco gene expresses several splice variants loco c1, loco c2, loco c3, and loco c4 [13–15]. Here we describe the expression of a fifth transcript, loco c5. We have analysed the expression of loco c1, loco c2, loco c3, and loco c5 in the wild-type testis and developing pupae and show that there is developmental regulation of loco c5 expression during morphogenesis. In addition we show that several G proteins are expressed in the male gonads and are therefore potential binding partners for the various LOCO isoforms. It is possible that the protein isoforms, expressing different conserved domains, will have different binding specificities and preferences for different G-proteins [27–31]. The G protein Gαi (G-oα65A) binds to loco c2  and it is also co-expressed with loco in a variety of cell types [16, 32]. We have shown by PCR that other Gα subunits are expressed (Goα47A, Gα49B, and Gα73B) in the testis and thus there is potential for LOCO to interact with other Gα subunits.
The analysis of the final morphology of the adult reproductive system in all of the flies analysed, strongly suggests that there is a failure in male gonad and genital morphogenesis It is possible that loco c3 expression could be the underlying reason for this phenotype. However the variability in testes morphology between flies may hint that there is some level of redundancy between the loco transcripts. Thus, whilst loco is clearly essential, a lack of or reduction of loco c3 expression does not cause a complete failure of gonad and genital differentiation. The loco mutants we isolated still express several loco transcripts, so further mutants will be needed which disrupt different transcripts or sets of transcripts to discover the role of loco and G-protein signalling in spermatogenesis and to further investigate it in imaginal discs and in the somatic cells of the gonad.
We show that all of the known loco spliceforms are expressed in the pupae and testis. In addition to this we have identified a fifth loco transcript, loco c5. We also show that there are a variety of Gα proteins expressed in the testis that may interact with the various LOCO isoforms. We have generated a set of new alleles of loco that affect the expression of specific loco transcripts. These deletions seem to be highly deleterious to Drosophila, as only a few adults hatch and the majority die as larvae. Mutant pupae and adult gonads of the few males that hatch show a loss of loco c3. We propose that loco c3 is needed for correct morphogenesis of the male gonad and the reproductive system derived from the male genital disc during metamorphosis. The role we have observed for loco in morphogenesis is in some ways similar to its role in glial cells where it has been proposed that G-protein signalling is important for shape changes . Although the reproductive system is derived from the genital disc and the testis from the gonad, both tissues are affected. It, therefore, seems likely that loco is involved in cell-cell interactions during evagination and morphogenesis. During these processes cell and tissue shape changes are crucial.
These results support the well-documented findings that G-protein signalling is crucial throughout development. An extensive investigation is needed to identify the binding specificities of different loco isoforms, the temporal and spatial distribution of different loco transcripts and which Gα subunits co-localise with loco in the gonad and genital discs and in the adult male testis. With this information it will be possible to design genetic and molecular experiments to investigate the developmental mechanisms in which loco participates.
Wild-type flies were OrR. Df(3R)17D1 Df(3R)15CE1, loco Δ13 and loco T1 were obtained from Christian Klambt. The original P insertion line was c139; it has an insertion of GAL4 in the loco gene . The mutant lines were generated and described in Pathirana et al 2001 . All Drosophila strains were raised on standard cornmeal-yeast-agar medium at 25°C.
β-galactosidase staining of testes and gonads
Testes and male gonads were dissected from the progeny from c139 crossed to the UAS-lacZ reporter line. Staining was carried out as described by Deng et al 1999 .
The dideoxy chain determination method was used initially in the form of a Sequenase 3.1 kit (US Biochemicals), followed by automated sequencing on Perkin Elmer ABI 373A and 377A machines using dye labeled primers, then dye labeled terminator reactions. Sequenced fragments were assembled using GCG and GENE-JOCKEY software. Sequence analysis was done with GCG GAP, MAP, FASTA, TFASTA and PILEUP software. Conserved domains were predicted using SMART  and the NCBI conserved domain database .
Reverse transcription (RT)-PCR
Reverse transcription-PCR was carried out as described in Deng et al 1999 . The primers used to identify transcripts were:
The primer pairs used to identify Gα subunits were:
Gα 73B (G-αF), accession number: CG12232-RA
Gα 49B (Gqα-3) Accession number: U31092
Goα 47A (G-αO) accession number: M30152
Preparation of and analysis of genomic DNA
Homozygous mutants were homogenized in 100 mM Tris pH 8.5, 80 mM NaCl, 50 mM EDTA pH 8, 0.5% (w/v) sucrose, 0.5% (w/v) SDS. The samples were incubated at -70°C for 30 minutes and then 65°C for 30 minutes. Potassium acetate was added to a final concentration of 1 M, the cell debris was discarded and the supernatant precipitated with isopropanol.
Primers designed to the intronic sequence were:
The P element primer used was designed to the inverted repeat:
pgawb5a inv CCACCTTATGTTATTTCATCATG
Leeanne McGurk was supported by the MRC. Stephen Pathirana was supported by a BBSRC studentship. Thorsten Trimbuch was a visiting Biotechnology student from the University of Applied Sciences Berlin and Paulo Columbini was a visiting student from the University of Modena and Reggio Emilia, Italy on a Biological Sciences course. Kathleen Rothwell was supported by a BBSRC grant to Mary Bownes and by the University of Edinburgh. We are grateful to George Tzolovsky and Fabio Acquaviva for comments on the manuscript and to Hilary Anderson for help with the manuscript preparation.
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