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1.  The Sex-Determination Genes fruitless and doublesex Specify a Neural Substrate Required for Courtship Song 
Current Biology  2007;17(17-3):1473-1478.
Summary
Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information [1–6]. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production [7]. Our examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS [8, 9] and are necessary for song production [10–13], we investigated the individual contributions of fru and dsx to the specification of a male CNS and song production. We show a novel requirement for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (FruM), we show a critical requirement for the male isoform of dsx (DsxM), alongside FruM, in the specification of courtship song. Therefore, although FruM expression is sufficient for the performance of many male-specific behaviors [14], we have shown that without DsxM, the determination of a male-specific CNS and thus a full complement of male behaviors are not realized.
doi:10.1016/j.cub.2007.07.047
PMCID: PMC2583281  PMID: 17716899
SYSNEURO
2.  Doublesex establishes sexual dimorphism in the Drosophila central nervous system in an isoform-dependent manner by directing cell number 
Developmental biology  2008;320(2):378-390.
Summary
doublesex (dsx) encodes sex-specific transcription factors (DSXF in females and DSXM in males) that act at the bottom of the Drosophila somatic sex determination hierarchy. dsx, which is conserved among diverse taxa, is responsible for directing all aspects of Drosophila somatic sexual differentiation outside the nervous system. The role of dsx in the nervous system remains minimally understood. Here, the mechanisms by which DSX acts to establish dimorphism in the central nervous system were examined. This study shows that the number of DSX-expressing cells in the central nervous system is sexually dimorphic during both pupal and adult stages. Additionally, the number of DSX-expressing cells depends on both the amount of DSX and the isoform present. One cluster of DSX-expressing neurons in the ventral nerve cord undergoes female-specific cell death that is DSXF-dependent. Another DSX-expressing cluster in the posterior brain undergoes more cell divisions in males than in females. Additionally, early in development, DSXM is present in a portion of the neural circuitry in which the male-specific product of fruitless (fru) is produced, in a region that has been shown to be critical for sex-specific behaviors. This study demonstrates that DSXM and FRUM expression patterns are established independent of each other in the regions of the central nervous system examined. In addition to the known role of dsx in establishing sexual dimorphism outside the central nervous system, the results demonstrate that DSX establishes sex-specific differences in neural circuitry by regulating the number of neurons using distinct mechanisms.
doi:10.1016/j.ydbio.2008.05.543
PMCID: PMC2631280  PMID: 18599032
sexual development; sex hierarchy; nervous system
3.  Genomic and Functional Studies of Drosophila Sex Hierarchy Regulated Gene Expression in Adult Head and Nervous System Tissues 
PLoS Genetics  2007;3(11):e216.
The Drosophila sex determination hierarchy controls all aspects of somatic sexual differentiation, including sex-specific differences in adult morphology and behavior. To gain insight into the molecular-genetic specification of reproductive behaviors and physiology, we identified genes expressed in the adult head and central nervous system that are regulated downstream of sex-specific transcription factors encoded by doublesex (dsx) and fruitless (fru). We used a microarray approach and identified 54 genes regulated downstream of dsx. Furthermore, based on these expression studies we identified new modes of DSX-regulated gene expression. We also identified 90 and 26 genes regulated in the adult head and central nervous system tissues, respectively, downstream of the sex-specific transcription factors encoded by fru. In addition, we present molecular-genetic analyses of two genes identified in our studies, calphotin (cpn) and defective proboscis extension response (dpr), and begin to describe their functional roles in male behaviors. We show that dpr and dpr-expressing cells are required for the proper timing of male courtship behaviors.
Author Summary
The fruit fly Drosophila is an excellent model system to use to understand the molecular-genetic basis of male courtship behavior, as the potential for this behavior is specified by a well-understood genetic regulatory hierarchy, called the sex determination hierarchy. The sex hierarchy consists of a pre-mRNA splicing cascade that culminates in the production of sex-specific transcription factors, encoded by doublesex (dsx) and fruitless (fru). dsx specifies all the anatomical differences between the sexes, and fru is required for all aspects of male courtship behavior. In this study, we measure gene expression differences between males and females, and between sex hierarchy mutants and wild-type animals, to identify genes that underlie the differences between males and females. We have performed these studies on adult head and nervous system tissues, as these tissues are important for establishing the potential for behaviors. We have identified several genes regulated downstream of dsx and fru and more extensively characterized two genes that are more highly expressed in males. One gene regulated downstream of dsx is expressed in the retina and is known to have a function in visual transduction. The other gene, regulated downstream of fru, plays a role in the timing of male courtship behavior.
doi:10.1371/journal.pgen.0030216
PMCID: PMC2082469  PMID: 18039034
4.  Cellular and Behavioral Functions of fruitless Isoforms in Drosophila Courtship 
Current Biology  2014;24(3):242-251.
Summary
Background
Male-specific products of the fruitless (fru) gene control the development and function of neuronal circuits that underlie male-specific behaviors in Drosophila, including courtship. Alternative splicing generates at least three distinct Fru isoforms, each containing a different zinc-finger domain. Here, we examine the expression and function of each of these isoforms.
Results
We show that most fru+ cells express all three isoforms, yet each isoform has a distinct function in the elaboration of sexually dimorphic circuitry and behavior. The strongest impairment in courtship behavior is observed in fruC mutants, which fail to copulate, lack sine song, and do not generate courtship song in the absence of visual stimuli. Cellular dimorphisms in the fru circuit are dependent on FruC rather than other single Fru isoforms. Removal of FruC from the neuronal classes vAB3 or aSP4 leads to cell-autonomous feminization of arborizations and loss of courtship in the dark.
Conclusions
These data map specific aspects of courtship behavior to the level of single fru isoforms and fru+ cell types—an important step toward elucidating the chain of causality from gene to circuit to behavior.
Highlights
•fru A, B, and C isoforms have largely overlapping expression in the male fly CNS•All three fru isoforms contribute to male courtship, with fruC being the most critical•FruC specifies sexual dimorphisms in neuron number and arborizations•FruC is required in defined neuronal classes for male-specific anatomy and behavior
Drosophila express three male-specific isoforms (A, B, and C) of the putative transcription factor fruitless (fru) in an overlapping pattern. von Philipsborn et al. show that isoform-mutant flies exhibit defects in male courtship, copulation success, and song production. FruC specifies sexual dimorphisms in defined neuronal classes potentially crucial for pheromone processing.
doi:10.1016/j.cub.2013.12.015
PMCID: PMC3969150  PMID: 24440391
5.  Turning Males On: Activation of Male Courtship Behavior in Drosophila melanogaster 
PLoS ONE  2011;6(6):e21144.
The innate sexual behaviors of Drosophila melanogaster males are an attractive system for elucidating how complex behavior patterns are generated. The potential for male sexual behavior in D. melanogaster is specified by the fruitless (fru) and doublesex (dsx) sex regulatory genes. We used the temperature-sensitive activator dTRPA1 to probe the roles of fruM- and dsx-expressing neurons in male courtship behaviors. Almost all steps of courtship, from courtship song to ejaculation, can be induced at very high levels through activation of either all fruM or all dsx neurons in solitary males. Detailed characterizations reveal different roles for fruM and dsx in male courtship. Surprisingly, the system for mate discrimination still works well when all dsx neurons are activated, but is impaired when all fruM neurons are activated. Most strikingly, we provide evidence for a fruM-independent courtship pathway that is primarily vision dependent.
doi:10.1371/journal.pone.0021144
PMCID: PMC3120818  PMID: 21731661
6.  Sex and the Single Cell. II. There Is a Time and Place for Sex 
PLoS Biology  2010;8(5):e1000365.
In both male and female Drosophila, only a subset of cells have the potential to sexually differentiate, making both males and females mosaics of sexually differentiated and sexually undifferentiated cells.
The Drosophila melanogaster sex hierarchy controls sexual differentiation of somatic cells via the activities of the terminal genes in the hierarchy, doublesex (dsx) and fruitless (fru). We have targeted an insertion of GAL4 into the dsx gene, allowing us to visualize dsx-expressing cells in both sexes. Developmentally and as adults, we find that both XX and XY individuals are fine mosaics of cells and tissues that express dsx and/or fruitless (fruM), and hence have the potential to sexually differentiate, and those that don't. Evolutionary considerations suggest such a mosaic expression of sexuality is likely to be a property of other animal species having two sexes. These results have also led to a major revision of our view of how sex-specific functions are regulated by the sex hierarchy in flies. Rather than there being a single regulatory event that governs the activities of all downstream sex determination regulatory genes—turning on Sex lethal (Sxl) RNA splicing activity in females while leaving it turned off in males—there are, in addition, elaborate temporal and spatial transcriptional controls on the expression of the terminal regulatory genes, dsx and fru. Thus tissue-specific aspects of sexual development are jointly specified by post-transcriptional control by Sxl and by the transcriptional controls of dsx and fru expression.
Author Summary
Morphologically, fruit flies are either male or female. The specification of sex is a multi-step process that depends on whether the fertilized egg has only one X chromosome (will develop as male) or two X chromosomes (will develop as female). This initial assessment of sex activates a cascade of regulatory genes that ultimately results in expression of either the male or female version of the protein encoded by the doublesex gene (dsx). These sex-specific proteins from the dsx gene direct most aspects of somatic sexual development, including the development of all of the secondary sexual characteristics that visibly distinguish males and females. In flies, as in most animal species, only some tissues are obviously different between the two sexes, so we asked the question of whether all cells in the animal nevertheless know which sex they are. That is, do all cells express dsx? We have developed a genetic tool that lets us visualize the cells in which the dsx is expressed. Strikingly, dsx is only expressed in a subset of tissues. Thus, adult flies of both sexes appear to be mosaics of cells that do know their sex and cells that do not know their sex.
doi:10.1371/journal.pbio.1000365
PMCID: PMC2864297  PMID: 20454565
7.  Neural Circuitry Underlying Drosophila Female Postmating Behavioral Responses 
Current Biology  2012;22(13):1155-1165.
Summary
Background
After mating, Drosophila females undergo a remarkable phenotypic switch resulting in decreased sexual receptivity and increased egg laying. Transfer of male sex peptide (SP) during copulation mediates these postmating responses via sensory neurons that coexpress the sex-determination gene fruitless (fru) and the proprioceptive neuronal marker pickpocket (ppk) in the female reproductive system. Little is known about the neuronal pathways involved in relaying SP-sensory information to central circuits and how these inputs are processed to direct female-specific changes that occur in response to mating.
Results
We demonstrate an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. We uncovered shared circuitry between dsx and a subset of the previously described SP-responsive fru+/ppk+-expressing neurons in the reproductive system. In addition, we identified sexually dimorphic dsx circuitry within the abdominal ganglion (Abg) critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus.
Conclusions
We propose that dsx-specified circuitry is required to induce female postmating behavioral responses, from sensing SP to conveying this signal to higher-order circuits for processing and through to the generation of postmating behavioral and physiological outputs.
Highlights
► dsx circuitry plays a pivotal role in the female postmating switch ► Peripheral dsx neurons detect and respond to sex peptide ► Central dsx neurons convey this signal to higher-order processing and direct postmating responses.
doi:10.1016/j.cub.2012.04.062
PMCID: PMC3396843  PMID: 22658598
8.  The hector G-Protein Coupled Receptor Is Required in a Subset of fruitless Neurons for Male Courtship Behavior 
PLoS ONE  2011;6(11):e28269.
Male courtship behavior in Drosophila melanogaster is controlled by two main regulators, fruitless (fru) and doublesex (dsx). Their sex-specific expression in brain neurons has been characterized in detail, but little is known about the downstream targets of the sex-specific FRU and DSX proteins and how they specify the function of these neurons. While sexual dimorphism in the number and connections of fru and dsx expressing neurons has been observed, a majority of the neurons that express the two regulators are present in both sexes. This poses the question which molecules define the sex-specific function of these neurons. Signaling molecules are likely to play a significant role. We have identified a predicted G-protein coupled receptor (GPCR), CG4395, that is required for male courtship behavior. The courtship defect in the mutants can be rescued by expression of the wildtype protein in fru neurons of adult males. The GPCR is expressed in a subset of fru-positive antennal glomeruli that have previously been shown to be essential for male courtship. Expression of 4395-RNAi in GH146 projection neurons lowers courtship. This suggests that signaling through the CG4395 GPCR in this subset of fru neurons is critical for male courtship behavior.
doi:10.1371/journal.pone.0028269
PMCID: PMC3227663  PMID: 22140564
9.  Enhancer-dependent 5′-Splice Site Control of fruitless Pre-mRNA Splicing* 
The Journal of biological chemistry  2003;278(25):22740-22747.
The Drosophila fruitless (fru) gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5′-splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fru is regulated by TRA and TRA2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13-nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3′-splice site choice in the doublesex (dsx) gene. dsx has provided a useful model system to investigate the mechanisms of enhancer-dependent 3′-splice site choice. However, little is known about enhancer-dependent regulation of alternative 5′-splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant TRA/TRA2 could activate the female-specific 5′-splice site of fru. Mutational analysis demonstrated that one 13-nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for dsx, the fruRE can be replaced by a short element encompassing tandem 13-nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments showed that Ser/Arg-rich proteins facilitate enhancer-dependent 5′-splice site activation. We conclude that splicing enhancers function similarly in activating regulated 5′- and 3′-splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5′- and 3′-splice sites.
doi:10.1074/jbc.M301036200
PMCID: PMC2386364  PMID: 12646561
10.  Effect of the Gene doublesex of Anastrepha on the Somatic Sexual Development of Drosophila 
PLoS ONE  2009;4(4):e5141.
Background
The gene doublesex (dsx) is at the bottom of the sex determination genetic cascade and is transcribed in both sexes, but gives rise to two different proteins, DsxF and DsxM, which impose female and male sexual development respectively via the sex-specific regulation of the so-called sexual cyto-differentiation genes. The present manuscript addressed the question about the functional conservation of the tephritid Anastrepha DsxF and DsxM proteins to direct the sexual development in Drosophila (Drosophilidae).
Methodology
To express these proteins in Drosophila, the GAL4-UAS system was used. The effect of these proteins was monitored in the sexually dimorphic regions of the fly: the foreleg basitarsus, the 5th, 6th and 7th tergites, and the external terminalia. In addition, we analysed the effect of Anastrepha DsxF and DsxM proteins on the regulation of Drosophila yolk protein genes, which are expressed in the fat body of adult females under the control of dsx.
Conclusions
The Anastrepha DsxF and DsxM proteins transformed doublesex intersexual Drosophila flies into females and males respectively, though this transformation was incomplete and the extent of their influence varied in the different sexually dimorphic regions of the adult fly. The Anastrepha DsxF and DsxM proteins also behaved as activators and repressors, respectively, of the Drosophila yolk protein genes, as do the DsxF and DsxM proteins of Drosophila itself. Finally, the Anastrepha DsxF and DsxM proteins were found to counteract the functions of Drosophila DsxM and DsxF respectively, reflecting the normal behaviour of the latter proteins towards one another. Collectively, these results indicate that the Anastrepha DsxF and DsxM proteins show conserved female and male sex-determination function respectively in Drosophila, though it appears that they cannot fully substitute the latter's own Dsx proteins. This incomplete function might be partly due to a reduced capacity of the Anastrepha Dsx proteins to completely control the Drosophila sexual cyto-differentiation genes, a consequence of the accumulation of divergence between these species resulting in the formation of different co-adapted complexes between the Dsx proteins and their target genes.
doi:10.1371/journal.pone.0005141
PMCID: PMC2660442  PMID: 19340310
11.  Genetic feminization of the thoracic nervous system disrupts courtship song in male Drosophila melanogaster 
Journal of neurogenetics  2010;24(4):234-245.
Despite the growing research investigating the sex-specific organization of courtship behavior in Drosophila melanogaster, much remains to be understood about the sex-specific organization of the motor circuit that drives this behavior. To investigate the sex-specification of a tightly patterned component of courtship behavior, courtship song, we used the GAL4/UAS targeted gene expression system to feminize the ventral ganglia in male Drosophila and analyzed the acoustic properties of courtship song. More specifically, we used the thoracic-specifying teashirt promoter (tshGAL4) to express feminizing transgenes specifically in the ventral ganglia. When tshGAL4 drove expression of transformer (tra), males were unable to produce prolonged wing extensions. Transgenic expression of an RNAi construct directed against male-specific fruitless (fruM) transcripts resulted in normal wing extension, but highly defective courtship song, with 58% of males failing to generate detectable courtship song. Of those that did sing, widths of individual pulses were significantly broader than controls, suggesting thoracic fruM function serves to mediate proprioceptive-dependent wing vibration damping during pulse song. However, the most critical signal in the song, the interpulse interval, remained intact. The inability to phenocopy this effect by reducing fruM expression in motor neurons and proprioceptive neurons suggests thoracic interneurons require fruM for proper pulse song execution and patterning of pulse structure, but not for pulse timing. This provides evidence that genes establishing sex-specific activation of complex behaviors may also be used in establishing pattern-generating motor networks underlying these sex-specific behaviors.
doi:10.3109/01677063.2010.519805
PMCID: PMC3056398  PMID: 20919857
Drosophila behavior; Acoustic; fruitless; Sexual dimorphism; Motor control
12.  Male-specific Fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains 
BMC Genomics  2013;14:659.
Background
Drosophila melanogaster adult males perform an elaborate courtship ritual to entice females to mate. fruitless (fru), a gene that is one of the key regulators of male courtship behavior, encodes multiple male-specific isoforms (FruM). These isoforms vary in their carboxy-terminal zinc finger domains, which are predicted to facilitate DNA binding.
Results
By over-expressing individual FruM isoforms in fru-expressing neurons in either males or females and assaying the global transcriptional response by RNA-sequencing, we show that three FruM isoforms have different regulatory activities that depend on the sex of the fly. We identified several sets of genes regulated downstream of FruM isoforms, including many annotated with neuronal functions. By determining the binding sites of individual FruM isoforms using SELEX we demonstrate that the distinct zinc finger domain of each FruM isoforms confers different DNA binding specificities. A genome-wide search for these binding site sequences finds that the gene sets identified as induced by over-expression of FruM isoforms in males are enriched for genes that contain the binding sites. An analysis of the chromosomal distribution of genes downstream of FruM shows that those that are induced and repressed in males are highly enriched and depleted on the X chromosome, respectively.
Conclusions
This study elucidates the different regulatory and DNA binding activities of three FruM isoforms on a genome-wide scale and identifies genes regulated by these isoforms. These results add to our understanding of sex chromosome biology and further support the hypothesis that in some cell-types genes with male-biased expression are enriched on the X chromosome.
doi:10.1186/1471-2164-14-659
PMCID: PMC3852243  PMID: 24074028
Fruitless; Sex hierarchy; Drosophila; Behavior; Genomics; RNA-seq
13.  The Orthologue of the Fruitfly Sex Behaviour Gene Fruitless in the Mosquito Aedes aegypti: Evolution of Genomic Organisation and Alternative Splicing 
PLoS ONE  2013;8(2):e48554.
In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.
doi:10.1371/journal.pone.0048554
PMCID: PMC3572092  PMID: 23418412
14.  Regulation of Sex-Specific Selection of fruitless 5′ Splice Sites by transformer and transformer-2 
Molecular and Cellular Biology  1998;18(1):450-458.
In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specific fru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. This fru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar.
PMCID: PMC121514  PMID: 9418892
15.  Genetic Control of Courtship Behavior in the Housefly: Evidence for a Conserved Bifurcation of the Sex-Determining Pathway 
PLoS ONE  2013;8(4):e62476.
In Drosophila melanogaster, genes of the sex-determination hierarchy orchestrate the development and differentiation of sex-specific tissues, establishing sex-specific physiology and neural circuitry. One of these sex-determination genes, fruitless (fru), plays a key role in the formation of neural circuits underlying Drosophila male courtship behavior. Conservation of fru gene structure and sex-specific expression has been found in several insect orders, though it is still to be determined whether a male courtship role for the gene is employed in these species due to the lack of mutants and homologous experimental evidence. We have isolated the fru ortholog (Md-fru) from the common housefly, Musca domestica, and show the gene’s conserved genomic structure. We demonstrate that male-specific Md-fru transcripts arise by conserved mechanisms of sex-specific splicing. Here we show that Md-fru, is similarly involved in controlling male courtship behavior. A male courtship behavioral function for Md-fru was revealed by the behavioral and neuroanatomical analyses of a hypomorphic allele, Md-traman, which specifically disrupted the expression of Md-fru in males, leading to severely impaired male courtship behavior. In line with a role in nervous system development, we found that expression of Md-fru was confined to neural tissues in the brain, most prominently in optic neuropil and in peripheral sensory organs. We propose that, like in Drosophila, overt sexual differentiation of the housefly depends on a sex-determining pathway that bifurcates downstream of the Md-tra gene to coordinate dimorphic development of non-neuronal tissues mediated by Md-dsx with that of neuronal tissues largely mediated by Md-fru.
doi:10.1371/journal.pone.0062476
PMCID: PMC3632534  PMID: 23630634
16.  Control of Sexual Differentiation and Behavior by the doublesex gene in Drosophila melanogaster 
Nature neuroscience  2010;13(4):458-466.
Doublesex proteins, part of the structurally and functionally conserved Dmrt gene family, play essential roles in sex determination throughout the animal kingdom. We targeted the insertion of GAL4 into the doublesex (dsx) locus of Drosophila melanogaster, allowing visualization and manipulation of dsx cells in various tissues. In the nervous system, significant differences between the sexes were detected in dsx neuronal numbers, axonal projections, and synaptic density. We show that dsx is required for the development of male-specific neurons that co-express fruitless (fru), a key regulator of male sexual behavior. We propose that both dsx and fru act together to form the neuronal framework necessary for male sexual behavior. Significantly, we show that disrupting dsx neuronal function has profound effects on male sexual behavior. Furthermore, we demonstrate a role for dsx neurons in pre- through to post-copulatory female reproductive behaviors.
doi:10.1038/nn.2515
PMCID: PMC3092424  PMID: 20305646
17.  A small cohort of FRUM and Engrailed-expressing neurons mediate successful copulation in Drosophila melanogaster 
BMC Neuroscience  2013;14:57.
Background
In Drosophila, male flies require the expression of the male-specific Fruitless protein (FRUM) within the developing pupal and adult nervous system in order to produce male courtship and copulation behaviors. Recent evidence has shown that specific subsets of FRUM neurons are necessary for particular steps of courtship and copulation. In these neurons, FRUM function has been shown to be important for determining sex-specific neuronal characteristics, such as neurotransmitter profile and morphology.
Results
We identified a small cohort of FRUM interneurons in the brain and ventral nerve cord by their co-expression with the transcription factor Engrailed (En). We used an En-GAL4 driver to express a fruM RNAi construct in order to selectively deplete FRUM in these En/FRUM co-expressing neurons. In courtship and copulation tests, these males performed male courtship at wild-type levels but were frequently sterile. Sterility was a behavioral phenotype as these En-fruMRNAi males were less able to convert a copulation attempt into a stable copulation, or did not maintain copulation for long enough to transfer sperm and/or seminal fluid.
Conclusions
We have identified a population of interneurons necessary for successful copulation in Drosophila. These data confirm a model in which subsets of FRUM neurons participate in independent neuronal circuits necessary for individual steps of male behavior. In addition, we have determined that these neurons in wild-type males have homologues in females and fru mutants, with similar placement, projection patterns, and neurochemical profiles.
doi:10.1186/1471-2202-14-57
PMCID: PMC3664081  PMID: 23688386
Courtship; Copulation; Drosophila; Fruitless; Engrailed; Central nervous system
18.  Male-Specific Fruitless Isoforms Target Neurodevelopmental Genes to Specify a Sexually Dimorphic Nervous System 
Current Biology  2014;24(3):229-241.
Summary
Background
In Drosophila, male courtship behavior is regulated in large part by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation or the role that isoform diversity plays in the formation of a male-specific nervous system.
Results
To characterize the roles of sex-specific Fru isoforms in specifying male behavior, we generated novel isoform-specific mutants and used a genomic approach to identify direct Fru isoform targets during development. We demonstrate that all Fru isoforms directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. We observe that fru behavioral phenotypes are specified by either a single isoform or a combination of isoforms. Finally, we illustrate the utility of these data for the identification of novel sexually dimorphic genomic enhancers and novel downstream regulators of male sexual behavior.
Conclusions
These findings suggest that Fru isoform diversity facilitates both redundancy and specificity in gene expression, and that the regulation of neuronal developmental genes may be the most ancient and conserved role of fru in the specification of a male-specific nervous system.
Highlights
•Isoform-specific fru mutants reveal both functional redundancy and specificity•Fru isoform-specific genomic occupancy is characterized in the Drosophila nervous system•All Fru isoforms directly target neuronal morphogenesis genes•Isoform-specific motifs are associated with specific Fru isoform occupancy
Neville et al. characterize the roles of sex-specific Fruitless isoforms in specifying male behavior in Drosophila by generating novel isoform-specific mutants, along with using a genomic approach to identify direct Fruitless isoform targets during development.
doi:10.1016/j.cub.2013.11.035
PMCID: PMC3969260  PMID: 24440396
19.  Functional Dissection of the Neural Substrates for Sexual Behaviors in Drosophila melanogaster 
Genetics  2011;189(1):195-211.
The male-specific Fruitless proteins (FruM) act to establish the potential for male courtship behavior in Drosophila melanogaster and are expressed in small groups of neurons throughout the nervous system. We screened ∼1000 GAL4 lines, using assays for general courtship, male–male interactions, and male fertility to determine the phenotypes resulting from the GAL4-driven inhibition of FruM expression in subsets of these neurons. A battery of secondary assays showed that the phenotypic classes of GAL4 lines could be divided into subgroups on the basis of additional neurobiological and behavioral criteria. For example, in some lines, restoration of FruM expression in cholinergic neurons restores fertility or reduces male–male courtship. Persistent chains of males courting each other in some lines results from males courting both sexes indiscriminately, whereas in other lines this phenotype results from apparent habituation deficits. Inhibition of ectopic FruM expression in females, in populations of neurons where FruM is necessary for male fertility, can rescue female infertility. To identify the neurons responsible for some of the observed behavioral alterations, we determined the overlap between the identified GAL4 lines and endogenous FruM expression in lines with fertility defects. The GAL4 lines causing fertility defects generally had widespread overlap with FruM expression in many regions of the nervous system, suggesting likely redundant FruM-expressing neuronal pathways capable of conferring male fertility. From associations between the screened behaviors, we propose a functional model for courtship initiation.
doi:10.1534/genetics.111.129940
PMCID: PMC3176112  PMID: 21705753
20.  Environmental Sex Determination in the Branchiopod Crustacean Daphnia magna: Deep Conservation of a Doublesex Gene in the Sex-Determining Pathway 
PLoS Genetics  2011;7(3):e1001345.
Sex-determining mechanisms are diverse among animal lineages and can be broadly divided into two major categories: genetic and environmental. In contrast to genetic sex determination (GSD), little is known about the molecular mechanisms underlying environmental sex determination (ESD). The Doublesex (Dsx) genes play an important role in controlling sexual dimorphism in genetic sex-determining organisms such as nematodes, insects, and vertebrates. Here we report the identification of two Dsx genes from Daphnia magna, a freshwater branchiopod crustacean that parthenogenetically produces males in response to environmental cues. One of these genes, designated DapmaDsx1, is responsible for the male trait development when expressed during environmental sex determination. The domain organization of DapmaDsx1 was similar to that of Dsx from insects, which are thought to be the sister group of branchiopod crustaceans. Intriguingly, the molecular basis for sexually dimorphic expression of DapmaDsx1 is different from that of insects. Rather than being regulated sex-specifically at the level of pre–mRNA splicing in the coding region, DapmaDsx1 exhibits sexually dimorphic differences in the abundance of its transcripts. During embryogenesis, expression of DapmaDsx1 was increased only in males and its transcripts were primarily detected in male-specific structures. Knock-down of DapmaDsx1 in male embryos resulted in the production of female traits including ovarian maturation, whereas ectopic expression of DapmaDsx1 in female embryos resulted in the development of male-like phenotypes. Expression patterns of another D. magna Dsx gene, DapmaDsx2, were similar to those of DapmaDsx1, but silencing and overexpression of this gene did not induce any clear phenotypic changes. These results establish DapmaDsx1 as a key regulator of the male phenotype. Our findings reveal how ESD is implemented by selective expression of a fundamental genetic component that is functionally conserved in animals using GSD. We infer that there is an ancient, previously unidentified link between genetic and environmental sex determination.
Author Summary
Sex determination is a fundamental biological process that can be broadly divided into two major categories. In genetic sex determination (GSD), sex-specific differentiation results from intrinsic genetic differences between males and females, whereas environmental sex determination (ESD) relies on environmental signals to induce male or female sex determination. In contrast to model organisms that utilize GSD system, environmental sex-determining organisms are poor genetic models. Therefore, although candidate genes involved in ESD have been found in vertebrates, their functions have remained largely unknown, impairing our understanding of ESD and making the comparison of sex-determining genes between both systems difficult. Here, we report the identification of a gene responsible for the production of males during environmental sex determination in the crustacean Daphnia. This gene is homologous to the Doublesex gene that is functionally conserved in animals that use GSD. Expression of Doublesex was increased primarily in male-specific structures. Gain- and loss-of-function analyses established that Daphnia Doublesex gene is a major effector that regulates the male phenotype in Daphnia. We infer that there is an ancient, previously unidentified link between genetic and environmental sex determination.
doi:10.1371/journal.pgen.1001345
PMCID: PMC3063754  PMID: 21455482
21.  A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila 
PLoS ONE  2014;9(4):e95472.
We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitlessMale (fruM), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fruM expression or inhibition of synaptic transmission in these fruM(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fruM-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship.
doi:10.1371/journal.pone.0095472
PMCID: PMC3989346  PMID: 24740138
22.  Looking under the lamp post: neither fruitless nor doublesex have evolved to generate divergent male courtship in Drosophila 
Cell reports  2014;8(2):363-370.
SUMMARY
How do evolved genetic changes alter the nervous system to produce different patterns of behavior? We address this question using Drosophila male courtship behavior, which is innate, stereotyped and evolves rapidly between species. D. melanogaster male courtship requires the male-specific isoforms of two transcription factors, fruitless and doublesex. These genes underlie genetic switches between female and male behaviors, making them excellent candidate genes for courtship behavior evolution. We tested their role in courtship evolution by transferring the entire locus for each gene from divergent species to D. melanogaster. We found that, despite differences in Fru+ and Dsx+ cell number in wild type species, cross-species transgenes rescued D. melanogaster courtship behavior, and no species-specific behaviors were conferred. Therefore, fru and dsx are not a significant source of evolutionary variation in courtship behavior.
doi:10.1016/j.celrep.2014.06.023
PMCID: PMC4153947  PMID: 25017068
23.  Evolution of Sex-Specific Traits through Changes in HOX-Dependent doublesex Expression 
PLoS Biology  2011;9(8):e1001131.
Analysis in Drosophila suggests that evolutionary changes in the spatial regulation of the transcription factor doublesex play a key role in the origin, diversification, and loss of sex-specific structures.
Almost every animal lineage is characterized by unique sex-specific traits, implying that such traits are gained and lost frequently in evolution. However, the genetic mechanisms responsible for these changes are not understood. In Drosophila, the activity of the sex determination pathway is restricted to sexually dimorphic tissues, suggesting that spatial regulation of this pathway may contribute to the evolution of sex-specific traits. We examine the regulation and function of doublesex (dsx), the main transcriptional effector of the sex determination pathway, in the development and evolution of Drosophila sex combs. Sex combs are a recent evolutionary innovation and show dramatic diversity in the relatively few Drosophila species that have them. We show that dsx expression in the presumptive sex comb region is activated by the HOX gene Sex combs reduced (Scr), and that the male isoform of dsx up-regulates Scr so that both genes become expressed at high levels in this region in males but not in females. Precise spatial regulation of dsx is essential for defining sex comb position and morphology. Comparative analysis of Scr and dsx expression reveals a tight correlation between sex comb morphology and the expression patterns of both genes. In species that primitively lack sex combs, no dsx expression is observed in the homologous region, suggesting that the origin and diversification of this structure were linked to the gain of a new dsx expression domain. Two other, distantly related fly lineages that independently evolved novel male-specific structures show evolutionary gains of dsx expression in the corresponding tissues, where dsx may also be controlled by Scr. These findings suggest that changes in the spatial regulation of sex-determining genes are a key mechanism that enables the evolution of new sex-specific traits, contributing to some of the most dramatic examples of phenotypic diversification in nature.
Author Summary
Most animals are sexually dimorphic, yet each species has a different set of sex-specific traits. Much of evolutionary biology since Darwin has focused on explaining these differences. In contrast to the well-developed theories of sexual selection (how and why males compete for females) we are still far from understanding the molecular mechanisms underlying the rapid gain and loss of sexually dimorphic phenotypes. In Drosophila melanogaster, the development of most sex-specific traits is controlled by the doublesex transcription factor. One of these traits is the sex comb, a group of modified bristles that develops on the front legs of males, which they use during mating to grasp the female's abdomen and genitalia. Sex combs are a recent innovation that evolved within the genus Drosophila but show dramatic diversity in the relatively few species that have them. In this study, we show that the origin and diversification of sex combs were associated with an evolutionary gain of a new doublesex expression domain and novel regulatory interactions between doublesex and the HOX gene Sex combs reduced, best known for its role in the specification of the labial and first thoracic segments. We find that other sex-specific structures that evolved in separate Drosophila lineages are also linked to new doublesex expression domains, suggesting that changes in the spatial regulation of doublesex may be a general mechanism enabling the evolutionary turnover of sex-specific traits.
doi:10.1371/journal.pbio.1001131
PMCID: PMC3160335  PMID: 21886483
24.  Molecular cloning of doublesex genes of four cladocera (water flea) species 
BMC Genomics  2013;14:239.
Background
The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species.
Results
We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5’ upstream regulatory elements are preserved in D. magna and D. pulex.
Conclusions
The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans.
doi:10.1186/1471-2164-14-239
PMCID: PMC3637828  PMID: 23575357
Cladocera; Doublesex genes; Environmental sex determination; Gene duplication; Annotated regulatory regions
25.  Ecdysone receptor acts in fruitless-expressing neurons to mediate Drosophila courtship behaviors 
Current biology : CB  2009;19(17):1447-1452.
Summary
In Drosophila melanogaster, fruitless (fru) encodes male-specific transcription factors (FRUM; encoded by fru P1) required for courtship behaviors [reviewed in 1]. However, downstream effectors of FRUM throughout development are largely unknown [2-5]. During metamorphosis the nervous system is remodeled for adult function, the timing of which is coordinated by the steroid hormone 20-hydroxy ecdysone (ecdysone) through the ecdysone receptor, a heterodimer of the nuclear receptors EcR (isoforms are EcR-A, EcR-B1, or EcR-B2) and Ultraspiracle (USP) [reviewed in 6]. Here, we show that genes identified as regulated downstream of FRUM during metamorphosis are significantly overrepresented with genes known to be regulated in response to ecdysone or EcR. FRUM and EcR isoforms are co-expressed in neurons in the CNS during metamorphosis in an isoform-specific manner. Reduction of EcR-A levels in fru P1-expressing neurons of males caused a significant increase in male-male courtship activity and significant reduction in size of two antennal lobe glomeruli. Additional genes were identified that are regulated downstream of EcR-A in fru P1-expressing neurons. Thus, EcR-A is required in fru P1-expressing neurons for wild type male courtship behaviors and the establishment of male-specific neuronal architecture.
doi:10.1016/j.cub.2009.06.063
PMCID: PMC2763606  PMID: 19646872
sex hierarchy; fruitless; Ecdysone; courtship; behavior; glomeruli morphology

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