BACKGROUND
many hormones and neurotransmitters act by binding to g-protein-coupled receptors  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  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  are a family of gtpase activating proteins  that trigger the intrinsic gtpase activity of the gα subunits  <cit> .
although a great deal is known about the regulation of g-protein-coupled receptor signalling in a variety of organisms  <cit>  less is known in drosophila and more importantly the involvement of g-protein-coupled receptor signalling in developmental decisions.
in drosophila nine genes encoding for rgs proteins have been identified  <cit> , however protein function has only been studied in three of them, axin, gprk <dig> and loco.
daxin, the drosophila orthologue of axin  <cit> , is a scaffold protein, that in the absence of wnt signaling, negatively regulates cytosolic armadillo by aiding its proteosome-dependent degradation  <cit> .
the negative regulation of armidillo by axin is inhibited by the interaction of axin with the gαs subunit of prostaglandin e2-stimulated in colon cancer cells  <cit> .
gprk <dig>  g-protein-coupled receptor kinase  <dig>  maintains camp levels in the ovary and is required for embryonic anterior patterning  <cit> .
the drosophila loco gene encodes a number of loco protein isoforms, all of which contain the rgs domain and also a goloco motif that acts as a guanine nucleotide dissociation inhibitor , .
previous studies suggest that loco might play an important role during early drosophila development.
for example during asymmetric cell division of the drosophila neuroblast, loco may act as a gtpase activating protein of the gαi protein via its rgs domain as well as a  through its goloco motif  <cit> .
in the drosophila embryo loco is essential for the formation, extension, and migration of glial cells, and plays a role in asymmetric cell division of drosophila neuroblasts  <cit> .
rare adult flies lacking the loco c <dig> and loco c <dig> transcripts have locomotive defects  <cit> .
previously, we showed that loco is expressed in the nurse cells and in specific subsets of the follicle cells of the drosophila egg chamber, and that it is required for cytoplasmic dumping during oogenesis and for dorsal-ventral axis formation of the egg chamber and embryo  <cit> .
the drosophila loco gene encodes a number of isoforms of an rgs protein .
loco c <dig> and loco c <dig> were the first transcripts to be identified and were found to be differentially expressed during embryogenesis  <cit> , subsequently we identified a third transcript, loco c <dig>  and showed that it was required for egg and embryo development  <cit> .
the sequence data on loco c <dig> has been extended to show that more sequence, including a start site, was upstream of the original start site identified for loco c <dig>  the two start sites are in frame, but neither has been shown to be functionally active  <cit> .
to remain in line with the published nomenclature, we will call this extended transcript loco c <dig>
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  <cit>  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  <cit> .
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  <cit> .
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.
RESULTS
screens for male sterility
previously we carried out a p element mediated mutagenesis screen using a p element located between exons ii- <dig> and i- <dig> of the loco gene   <cit> .
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  <dig> 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  <dig> 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.
in this study we wanted to determine if any of the semi-lethal lines which generated some adult progeny were male sterile.
we found that  <dig> of the loco mutant lines generated a few adult males.
the fertility of the rare adult males was investigated by crossing the homozygous loco mutant males to orr virgin-females.
five lines produced none or very rare progeny; loco <dig>  loco <dig>  loco <dig> , loco <dig>  and loco <dig>  the first three of these are red-eyed lines, which have either resulted from a partial excision or from a mobilised p element.
the latter two lines are white eyed and therefore lack at least part of the original p insertion and possibly some flanking genomic dna.
one of these lines  subsequently ceased producing any adult males and was not further investigated.
complementation analysis showed that the mutations of the semi-sterile males fell into the same complementation group .
the rare heteroallelic males produced in these experiments generated very few progeny when crossed to orr females .
flies heterozygous for each of the male semi-sterile lines and one of the df lines that affects loco  were crossed and rare heteroallelic mutant males were collected.
groups of 2– <dig> males crossed with 3– <dig> orr females would normally generate several hundred progeny if the males are also orr.
each experiment was repeated twice.
no adult males were obtained for loco <dig>
none of the loco mutants heterozygous with orr showed significantly reduced male fertility, indicating that the semi-sterile loco mutant males were fully recessive .
to further characterise the loco mutants we analysed male fertility in two other strains lacking loco.
the two deficiency strains used were df15ce <dig> , which lacks the cytogenetic region 93f-;94c-94d, and df17d <dig> , which lacks 93e-94c, both lack the loco gene which is at position 94b6-94b <dig>  the deficiency lines were crossed to orr and the resulting heterozygous males were crossed to orr to assess the effect of the mutation on fertility .
only df15/+ showed a significant reduction in fertility when compared to orr, this is likely to be due to the loss of genes present in the region 94c-d as df17/+, when crossed to orr, did not show any significant reduction in male fertility .
furthermore locoΔ <dig>  a mutant strain, which was previously reported to lack part of the loco gene and  <dig> kb of downstream sequence  <cit>  also showed no significant reduction in fertility when crossed to orr .
this suggests that heterozygous loss of loco does not affect male fertility.
heterozygous flies, containing the loco mutation and the deficiency chromosome 15ce <dig>  df17d <dig>  locoΔ13) or the orr, chromosome, were crossed with orr.
the ratio of eggs laid and eggs hatched is represented as a percentage.
significance was calculated by calculating chi <dig> values between heterozygous mutant males and orr, and the hemizygous mutants to the heterozygous deficiency lines.
chi <dig> >  <dig>  suggests the two lines are closely linked.
s: significant, ns: not significant.
in order to assess whether our loco mutants were in the same or different complementation groups to the deficiency lines  <cit> , 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 .
despite all of the heteroallelic loco mutants showing reduced fertility not all were a statistically significant reduction .
however all of the mutant loco alleles  hemizygous with the locoΔ <dig> allele, showed a statistically significant reduction in male fertility .
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 loco in the testis
the semi-sterile males obtained from the loco mutants isolated in this screen suggested that loco may have a role within the testes.
to investigate whether loco was expressed in the male gonads a uas-lacz reporter strain was crossed to the original p insertion strain , which contained a gal <dig> insertion in the loco gene  <cit> .
the resulting male adult gonads were stained for β-galactosidase activity.
the testis and seminal vesicles showed very strong lacz expression .
the loco gene is alternatively spliced to give rise to several isoforms loco c <dig>  loco c <dig>  loco c <dig>  and loco c <dig>  <cit> .
upon database searching we revealed that there was a fifth transcript, isolated from an adult est testis library .
the sequence had no conserved domains relating to loco and mapped to the genomic region located in the intron between exon ii- <dig> and exon i- <dig> .
this suggested that there was an additional transcript, not previously described, but expressed in the testis.
this new exon has been labelled exon iv- <dig> and the transcript produced is loco c <dig> .
in order to analyse the expression of the various loco splice variants in the drosophila testis primers were designed to specifically amplify loco c <dig>  loco c <dig>  loco c <dig> and loco c <dig>  it should be noted that the primers used to amplify loco c <dig> could not discriminate between loco c <dig> and loco c <dig>  furthermore it is possible that these two transcripts form the same transcriptional unit.
rt-pcr revealed that loco c <dig>  loco c <dig>  loco c <dig>  and loco c <dig> were expressed in the orr male testis .
detection of loco c <dig> using the primer pair fp <dig> and rp <dig>  produced a specific product at approximately  <dig> kb and two non-specific bands at lower molecular weights .
cloning and sequencing revealed that the  <dig> kb band was specific to loco c <dig>  the  <dig>  kb band aligned to cabp <dig>  and the  <dig>  kb band aligned to myosin binding subunit .
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  <cit> .
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  <cit>  and has more recently been shown to contribute to asymmetric cell division  <cit> .
furthermore it is expressed in the nurse cells and oocyte and is present in various adult nerve cells  <cit> .
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  <cit> .
gα49b, a gq subunit involved in phospholipase c activation, is involved in the drosophila visual system  <cit> .
gα49b is known to be expressed in the adult testis  <cit> , and thus acted as a positive control.
pcr showed that g-oα47a, gα73b and gα49b were expressed in the testis .
this raises the possibility that in the adult testis there are additional gα subunits that may interact with loco.
analysis of mutant phenotypes
we have shown that loco and various gα subunits are expressed in the testis and have identified, genetically, that mutation in loco can lead to significantly reduced fertility in males.
we therefore went on and analysed adult gonad morphology in the loco mutants.
all of the loco mutants had abnormal reproductive systems and showed a variety of defects .
the phenotype was almost 100% penetrant, there was a range in severity within the same line, and only the very occasional male reproductive system was normal.
often the accessory glands were abnormal and the ejaculatory ducts appeared swollen.
neither testes from virgin wild-type males or aged wild-type males show this peculiar phenotype, suggesting that this phenotype was not attributable to the mating status .
there were no obvious differences between the mutant lines, which may be attributable to all mutants being in the same complementation group.
the testes, even those very abnormal in shape and size, contained mobile sperm .
this suggested that the differentiation of the sperm does not require the loco isoform affected in this group of mutants, but possibly differentiation and morphogenesis of the derivatives of the genital discs and gonad are altered and often fails in these mutants.
identification of motile sperm in the male mutants may explain why some offspring were produced; it seems likely that the abnormal morphology of the gonads is the causative effect of reduced fertility.
expression of loco during male development
the phenotypes of the mutant testes and reproductive system suggested that organogenesis and not necessarily spermatogenesis was abnormal.
since differentiation of the testis and reproductive system occurs during metamorphosis we investigated the expression of loco in early and late pupae .
we divided the pupae into early pupae and late pupae and performed rt-pcr on the various loco transcripts .
the loco c <dig>  loco c <dig>  and loco c <dig> transcripts were expressed during the early stages and late stages of pupal development.
the newly identified transcript, loco c <dig>  was found to be differentially expressed and seemed to be detectable only in early staged pupae .
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 gal <dig> in the loco gene  <cit> , was crossed to a uas-lacz reporter strain.
β-galactosidase activity was detected in the larval male gonads  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 loco in 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 c <dig>  loco c <dig>  loco c <dig> and loco c <dig> in the mutant pupae of three of the male semi-sterile lines, loco <dig>  loco <dig>  and loco <dig> .
we detected loco c <dig>  the 5'end of loco c <dig> and the loco c <dig> transcripts in the loco <dig> mutant.
however loco <dig> completely lacked expression of the loco c <dig> transcript .
furthermore loco <dig> only expressed the 5'end of loco c <dig>  the 3'end of loco c <dig> was not detected  as only the  <dig>  kb non-specific band was detected.
pupal expression from the loco gene in the loco <dig> mutant produced loco c <dig>  loco c <dig> and loco c <dig>  however like loco <dig> the loco <dig> mutant lacked loco c <dig> .
pupal expression of loco c <dig>  loco c <dig>  loco c <dig> and loco c <dig> was detected in the loco <dig> mutant .
the testis expression of loco <dig> was also found to lack any detectable loco c <dig> 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 .
furthermore detection of loco c <dig>  loco c <dig> and loco c <dig> from the same cdna samples which lacked loco c <dig> suggests that the rna transcribed from the loco gene was intact.
taken together this data suggests that there is a requirement for loco c <dig> 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 c <dig> strain  <cit> .
pcr utilising a p element primer directed to the 5' end of the p element  in combination with a gene specific primer revealed that the p element was in reverse orientation to the loco gene .
sequencing of the pcr product revealed that the p element had inserted  <dig> bp upstream of exon i- <dig> .
pcr analysis of the loco <dig> mutant revealed that the p element primer directed to the 5' end of the p element  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 <dig> 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  and the second p element was present at the same position but in the opposite direction .
the sequencing of the pcr products from loco <dig> also revealed a  <dig> bp duplication of genomic dna on either side of the p elements .
it is possible that two p elements  <dig> bp upstream of exon  <dig> alters pre-mrna length such that there is premature dissociation of rna polymerase 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 <dig> failed to produce pcr products in the mutant line loco <dig>  pcr across the original insertion site revealed that the p element had excised and had not deleted any genomic sequence .
the mutant line loco <dig> expresses white, it lacks expression of loco c <dig>  and the mutation genetically maps to the loco gene .
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 fp <dig> produced a fragment of  <dig> kb, suggesting that the p element was  <dig> kb upstream of exon  <dig>  genomic pcr was performed across all introns in the loco gene.
pcr failed only between exon i- <dig> and exon  <dig>  whereas a pcr product across this region in wild-type genomic dna was detected .
this further suggested that the partial p element in the mutant line loco <dig> was located between exon i- <dig> and exon  <dig> and thus produced a pcr product too long to be detected by the pcr programme.
direct sequencing of the insertion site in loco <dig> revealed that no sequence had been deleted .
the loco <dig> line was found to express loco c <dig>  loco c <dig>  loco c <dig>  and loco c <dig> .
the reduction in male sterility when loco <dig> is hemizygous with df <dig>  df <dig> or locoΔ <dig> was statistically significant  and the adult reproductive tissue of loco <dig> is morphologically abnormal .
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  <dig> bp, therefore if a large inversion or rearrangement had occurred in loco <dig> 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.
discussion
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.,   <cit>  showed that loco mutants died as embryos showing abnormalities in the contacts between glial cells  <cit> .
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  <cit> .
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  <cit> .
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 c <dig>  loco c <dig>  loco c <dig>  and loco c <dig>  <cit> .
here we describe the expression of a fifth transcript, loco c <dig>  we have analysed the expression of loco c <dig>  loco c <dig>  loco c <dig>  and loco c <dig> in the wild-type testis and developing pupae and show that there is developmental regulation of loco c <dig> 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  <cit> .
the g protein gαi  binds to loco c <dig>  <cit>  and it is also co-expressed with loco in a variety of cell types  <cit> .
we have shown by pcr that other gα subunits are expressed  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 c <dig> 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 c <dig> 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.
CONCLUSIONS
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 c <dig>  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 c <dig>  we propose that loco c <dig> 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  <cit> .
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.

 many hormones and neurotransmitters act by binding to g-protein-coupled receptors 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 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 are a family of gtpase activating proteins that trigger the intrinsic gtpase activity of the gα subunits. although a great deal is known about the regulation of g-protein-coupled receptor signalling in a variety of organisms less is known in drosophila and more importantly the involvement of g-protein-coupled receptor signalling in developmental decisions. in drosophila nine genes encoding for rgs proteins have been identified, however protein function has only been studied in three of them, axin, gprk and loco. daxin, the drosophila orthologue of axin, is a scaffold protein, that in the absence of wnt signaling, negatively regulates cytosolic armadillo by aiding its proteosome-dependent degradation. the negative regulation of armidillo by axin is inhibited by the interaction of axin with the gαs subunit of prostaglandin e2-stimulated in colon cancer cells. gprk g-protein-coupled receptor kinase maintains camp levels in the ovary and is required for embryonic anterior patterning. the drosophila loco gene encodes a number of loco protein isoforms, all of which contain the rgs domain and also a goloco motif that acts as a guanine nucleotide dissociation inhibitor,. previous studies suggest that loco might play an important role during early drosophila development. for example during asymmetric cell division of the drosophila neuroblast, loco may act as a gtpase activating protein of the gαi protein via its rgs domain as well as a through its goloco motif. in the drosophila embryo loco is essential for the formation, extension, and migration of glial cells, and plays a role in asymmetric cell division of drosophila neuroblasts. rare adult flies lacking the loco c and loco c transcripts have locomotive defects. previously, we showed that loco is expressed in the nurse cells and in specific subsets of the follicle cells of the drosophila egg chamber, and that it is required for cytoplasmic dumping during oogenesis and for dorsal-ventral axis formation of the egg chamber and embryo. the drosophila loco gene encodes a number of isoforms of an rgs protein. loco c and loco c were the first transcripts to be identified and were found to be differentially expressed during embryogenesis, subsequently we identified a third transcript, loco c and showed that it was required for egg and embryo development. the sequence data on loco c has been extended to show that more sequence, including a start site, was upstream of the original start site identified for loco c 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 c 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. 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- and i- of the loco gene. 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 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 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. in this study we wanted to determine if any of the semi-lethal lines which generated some adult progeny were male sterile. we found that of the loco mutant lines generated a few adult males. the fertility of the rare adult males was investigated by crossing the homozygous loco mutant males to orr virgin-females. five lines produced none or very rare progeny; loco loco loco, loco and loco the first three of these are red-eyed lines, which have either resulted from a partial excision or from a mobilised p element. the latter two lines are white eyed and therefore lack at least part of the original p insertion and possibly some flanking genomic dna. one of these lines subsequently ceased producing any adult males and was not further investigated. complementation analysis showed that the mutations of the semi-sterile males fell into the same complementation group. the rare heteroallelic males produced in these experiments generated very few progeny when crossed to orr females. flies heterozygous for each of the male semi-sterile lines and one of the df lines that affects loco were crossed and rare heteroallelic mutant males were collected. groups of 2– males crossed with 3– orr females would normally generate several hundred progeny if the males are also orr. each experiment was repeated twice. no adult males were obtained for loco none of the loco mutants heterozygous with orr showed significantly reduced male fertility, indicating that the semi-sterile loco mutant males were fully recessive. to further characterise the loco mutants we analysed male fertility in two other strains lacking loco. the two deficiency strains used were df15ce, which lacks the cytogenetic region 93f-;94c-94d, and df17d, which lacks 93e-94c, both lack the loco gene which is at position 94b6-94b the deficiency lines were crossed to orr and the resulting heterozygous males were crossed to orr to assess the effect of the mutation on fertility. only df15/+ showed a significant reduction in fertility when compared to orr, this is likely to be due to the loss of genes present in the region 94c-d as df17/+, when crossed to orr, did not show any significant reduction in male fertility. furthermore locoΔ a mutant strain, which was previously reported to lack part of the loco gene and kb of downstream sequence also showed no significant reduction in fertility when crossed to orr. this suggests that heterozygous loss of loco does not affect male fertility. heterozygous flies, containing the loco mutation and the deficiency chromosome 15ce df17d locoΔ13) or the orr, chromosome, were crossed with orr. the ratio of eggs laid and eggs hatched is represented as a percentage. significance was calculated by calculating chi values between heterozygous mutant males and orr, and the hemizygous mutants to the heterozygous deficiency lines. chi > suggests the two lines are closely linked. s: significant, ns: not significant. 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. despite all of the heteroallelic loco mutants showing reduced fertility not all were a statistically significant reduction. however all of the mutant loco alleles hemizygous with the locoΔ allele, showed a statistically significant reduction in male fertility. 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 loco in the testis the semi-sterile males obtained from the loco mutants isolated in this screen suggested that loco may have a role within the testes. to investigate whether loco was expressed in the male gonads a uas-lacz reporter strain was crossed to the original p insertion strain, which contained a gal insertion in the loco gene. the resulting male adult gonads were stained for β-galactosidase activity. the testis and seminal vesicles showed very strong lacz expression. the loco gene is alternatively spliced to give rise to several isoforms loco c loco c loco c and loco c. upon database searching we revealed that there was a fifth transcript, isolated from an adult est testis library. the sequence had no conserved domains relating to loco and mapped to the genomic region located in the intron between exon ii- and exon i-. this suggested that there was an additional transcript, not previously described, but expressed in the testis. this new exon has been labelled exon iv- and the transcript produced is loco c. in order to analyse the expression of the various loco splice variants in the drosophila testis primers were designed to specifically amplify loco c loco c loco c and loco c it should be noted that the primers used to amplify loco c could not discriminate between loco c and loco c furthermore it is possible that these two transcripts form the same transcriptional unit. rt-pcr revealed that loco c loco c loco c and loco c were expressed in the orr male testis. detection of loco c using the primer pair fp and rp produced a specific product at approximately kb and two non-specific bands at lower molecular weights. cloning and sequencing revealed that the kb band was specific to loco c the kb band aligned to cabp and the kb band aligned to myosin binding subunit. 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. 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 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. 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. this raises the possibility that in the adult testis there are additional gα subunits that may interact with loco. analysis of mutant phenotypes we have shown that loco and various gα subunits are expressed in the testis and have identified, genetically, that mutation in loco can lead to significantly reduced fertility in males. we therefore went on and analysed adult gonad morphology in the loco mutants. all of the loco mutants had abnormal reproductive systems and showed a variety of defects. the phenotype was almost 100% penetrant, there was a range in severity within the same line, and only the very occasional male reproductive system was normal. often the accessory glands were abnormal and the ejaculatory ducts appeared swollen. neither testes from virgin wild-type males or aged wild-type males show this peculiar phenotype, suggesting that this phenotype was not attributable to the mating status. there were no obvious differences between the mutant lines, which may be attributable to all mutants being in the same complementation group. the testes, even those very abnormal in shape and size, contained mobile sperm. this suggested that the differentiation of the sperm does not require the loco isoform affected in this group of mutants, but possibly differentiation and morphogenesis of the derivatives of the genital discs and gonad are altered and often fails in these mutants. identification of motile sperm in the male mutants may explain why some offspring were produced; it seems likely that the abnormal morphology of the gonads is the causative effect of reduced fertility. expression of loco during male development the phenotypes of the mutant testes and reproductive system suggested that organogenesis and not necessarily spermatogenesis was abnormal. since differentiation of the testis and reproductive system occurs during metamorphosis we investigated the expression of loco in early and late pupae. we divided the pupae into early pupae and late pupae and performed rt-pcr on the various loco transcripts. the loco c loco c and loco c transcripts were expressed during the early stages and late stages of pupal development. the newly identified transcript, loco c was found to be differentially expressed and seemed to be detectable only in early staged pupae. 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 gal in the loco gene, was crossed to a uas-lacz reporter strain. β-galactosidase activity was detected in the larval male gonads 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 loco in 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 c loco c loco c and loco c in the mutant pupae of three of the male semi-sterile lines, loco loco and loco. we detected loco c the 5'end of loco c and the loco c transcripts in the loco mutant. however loco completely lacked expression of the loco c transcript. furthermore loco only expressed the 5'end of loco c the 3'end of loco c was not detected as only the kb non-specific band was detected. pupal expression from the loco gene in the loco mutant produced loco c loco c and loco c however like loco the loco mutant lacked loco c. pupal expression of loco c loco c loco c and loco c was detected in the loco mutant. the testis expression of loco was also found to lack any detectable loco c 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. furthermore detection of loco c loco c and loco c from the same cdna samples which lacked loco c suggests that the rna transcribed from the loco gene was intact. taken together this data suggests that there is a requirement for loco c 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 c strain. pcr utilising a p element primer directed to the 5' end of the p element in combination with a gene specific primer revealed that the p element was in reverse orientation to the loco gene. sequencing of the pcr product revealed that the p element had inserted bp upstream of exon i-. pcr analysis of the loco mutant revealed that the p element primer directed to the 5' end of the p element 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 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 and the second p element was present at the same position but in the opposite direction. the sequencing of the pcr products from loco also revealed a bp duplication of genomic dna on either side of the p elements. it is possible that two p elements bp upstream of exon alters pre-mrna length such that there is premature dissociation of rna polymerase 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 failed to produce pcr products in the mutant line loco pcr across the original insertion site revealed that the p element had excised and had not deleted any genomic sequence. the mutant line loco expresses white, it lacks expression of loco c and the mutation genetically maps to the loco gene. 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 fp produced a fragment of kb, suggesting that the p element was kb upstream of exon genomic pcr was performed across all introns in the loco gene. pcr failed only between exon i- and exon whereas a pcr product across this region in wild-type genomic dna was detected. this further suggested that the partial p element in the mutant line loco was located between exon i- and exon and thus produced a pcr product too long to be detected by the pcr programme. direct sequencing of the insertion site in loco revealed that no sequence had been deleted. the loco line was found to express loco c loco c loco c and loco c. the reduction in male sterility when loco is hemizygous with df df or locoΔ was statistically significant and the adult reproductive tissue of loco is morphologically abnormal. 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 bp, therefore if a large inversion or rearrangement had occurred in loco 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. discussion 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., 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 c loco c loco c and loco c. here we describe the expression of a fifth transcript, loco c we have analysed the expression of loco c loco c loco c and loco c in the wild-type testis and developing pupae and show that there is developmental regulation of loco c 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. the g protein gαi binds to loco c and it is also co-expressed with loco in a variety of cell types. we have shown by pcr that other gα subunits are expressed 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 c 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 c 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 c 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 c we propose that loco c 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. 
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 c thus, we conclude that specific isoforms of loco are required for the differentiation of the male gonad and genital disc. we identified a number of lethal loco mutants and four semi-lethal lines, which generate males with reduced fertility. 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 have characterised the expression pattern of all loco transcripts during pupal development in the adult testes, both in wild type and loco mutant strains. we have identified a fifth loco transcript and show that it is differentially expressed in developing pupae.
 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 c thus, we conclude that specific isoforms of loco are required for the differentiation of the male gonad and genital disc. 