InteractiveFly: GeneBrief

fruitless : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - fruitless

Synonyms -

Cytological map position - 91B2--91B3

Function - transcription factor

Keyword(s) - Sex determination hierarchy, brain, CNS

Symbol - fru

FlyBase ID:FBgn0004652

Genetic map position - 3-[64]

Classification - C2H2 zinc finger - BTB domain

Cellular location - nuclear

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Dweck, H.K., Ebrahim, S.A., Thoma, M., Mohamed, A.A., Keesey, I.W., Trona, F., Lavista-Llanos, S., Svatos, A., Sachse, S., Knaden, M. and Hansson, B.S. (2015). Pheromones mediating copulation and attraction in Drosophila. Proc Natl Acad Sci U S A 112(21): E2829-35. PubMed ID: 25964351
Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. This study shows that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fruM)-positive Or47b-expressing OSNs detected ML exclusively, and Or47b- and Or47b-expressing OSNs were required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male was sufficient to provide a competitive mating advantage. Further, the vigorous male courtship displayed toward oenocyte-less flies was attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, Or88a-expressing OSNs responded to all three compounds, and that these neurons were necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants was transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, both Or47b- and Or88a-based systems and their ligands were remarkably conserved over a number of drosophilid species. Taken together, these results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved.

Sellami, A. and Veenstra, J. A. (2015). SIFamide acts on Fruitless neurons to modulate sexual behavior in Drosophila melanogaster. Peptides [Epub ahead of print]. PubMed ID: 26469541
The Drosophila gene fruitless expresses male and female specific transcription factors which are responsible for the generation of male specific neuronal circuitry for courtship behavior. Mutations in this gene may lead to bisexual behavior in males. Bisexual behavior in males also occurs in the absence of the neuropeptide SIFamide. SIFamide neurons do not express fruitless. However, when fruitless neurons are made to express RNAi specific for the SIFamide receptor, male flies engage in bisexual behavior, showing that SIFamide acts on fruitless neurons. If neurons expressing a SIFaR-gal4 transgene are killed by the apoptotic protein Reaper or when these neurons express SIFamide receptor RNAi, males also show male-male courtship behavior. This transgene was used to localize neurons that express the SIFamide receptor. Such neurons are ubiquitously present in the central nervous, and two neurons were also found in the uterus that project into the central nervous system.

Wang, Q., Taliaferro, J.M., Klibaite, U., Hilgers, V., Shaevitz, J.W. and Rio, D.C. (2016). The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila. Proc Natl Acad Sci U S A [Epub ahead of print]. PubMed ID: 27114556
Fruitless alternative pre-mRNA splicing (AS) isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, this study shows that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ∼1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

Newell, N.R., New, F.N., Dalton, J.E., McIntyre, L.M. and Arbeitman, M.N. (2016). Neurons that underlie Drosophila melanogaster reproductive behaviors: detection of a large male-bias in gene expression in fruitless-expressing neurons. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27247289
Male and female reproductive behaviors in Drosophila melanogaster are vastly different, but neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How this set of neurons can generate such different behaviors between the two sexes is an unresolved question. A particular challenge is that fru P1-expressing neurons comprise only 2-5% of the adult nervous system, and so studies of adult head tissue or whole brain may not reveal crucial differences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons allowing a sensitive, cell-type-specific assay. It was also found that TRAP mRNAs from fru P1-expressing neurons exhibit four times more male-biased than female-biased genes. This suggests a potential mechanism to generate dimorphism in behavior. The male-biased genes may direct male behaviors by establishing cell fate in a similar context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise from a shared set of neurons.

Rezaval, C., Pattnaik, S., Pavlou, H. J., Nojima, T., Bruggemeier, B., D'Souza, L. A., Dweck, H. K. and Goodwin, S. F. (2016). Activation of latent courtship circuitry in the brain of Drosophila females induces male-like behaviors. Curr Biol 26: 2508-2515. PubMed ID: 27568592
Courtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors. Fruit fly males exhibit an elaborate courtship display toward a potential mate. Females never actively court males, but their response to the male's display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru). Activation of male-specific dsx/fru+ P1 neurons in the brain initiates the male's courtship display, suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx+ neurons play a pivotal role in sexual receptivity and post-mating behaviors. This study manipulated the function of dsx+ neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, it was found that activation of female dsx+ neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. Specific dsx+ neurons critical for driving male courtship were uncovered and pheromones were identified that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, this study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.
Chen, D., Sitaraman, D., Chen, N., Jin, X., Han, C., Chen, J., Sun, M., Baker, B. S., Nitabach, M. N. and Pan, Y. (2017). Genetic and neuronal mechanisms governing the sex-specific interaction between sleep and sexual behaviors in Drosophila. Nat Commun 8(1): 154. PubMed ID: 28754889
Animals execute one particular behavior among many others in a context-dependent manner, yet the mechanisms underlying such behavioral choice remain poorly understood. This research studied how two fundamental behaviors, sex and sleep, interact at genetic and neuronal levels in Drosophila. An increased need for sleep was shown to inhibits male sexual behavior by decreasing the activity of the male-specific P1 neurons that coexpress the sex determination genes fruM and dsx, but does not affect female sexual behavior. Further, a sex-specific neuronal circuit was delineated wherein the P1 neurons encoding increased courtship drive suppressed male sleep by forming mutually excitatory connections with the fruM -positive sleep-controlling DN1 neurons. In addition, FRUM was found to regulates male courtship and sleep through distinct neural substrates. These studies reveal the genetic and neuronal basis underlying the sex-specific interaction between sleep and sexual behaviors in Drosophila, and provide insights into how competing behaviors are co-regulated. Genes and circuits involved in sleep and sexual arousal have been extensively studied in Drosophila. This study has identified the sex determination genes fruitless and doublesex, and a sex-specific P1-DN1 neuronal feedback that governs the interaction between these competing behaviors.
Tanaka, R., Higuchi, T., Kohatsu, S., Sato, K. and Yamamoto, D. (2017). Optogenetic activation of the fruitless-labeled circuitry in Drosophila subobscura males induces mating motor acts. J Neurosci 37(48): 11662-11674. PubMed ID: 29109241
It remains an enigma how the nervous system of different animal species produces different behaviors. The neural circuitry for mating behavior was studied in Drosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic model D. melanogaster, in which the core courtship circuitry has been identified. The D. subobscura fruitless (fru) gene, a master regulator for the courtship circuitry formation in D. melanogaster, was disrupted resulting in complete loss of mating behavior. frusoChrimV was also developed that expresses the optogenetic activator Chrimson fused with a fluorescent marker under the native fru promoter. The fru-labeled circuitry in D. subobscura visualized by frusoChrimV revealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification.
Chowdhury, Z. S., Sato, K. and Yamamoto, D. (2017). The core-promoter factor TRF2 mediates a Fruitless action to masculinize neurobehavioral traits in Drosophila. Nat Commun 8(1): 1480. PubMed ID: 29133872
In fruit flies, the male-specific fruitless (fru) gene product FruBM plays a central role in establishing the neural circuitry for male courtship behavior by orchestrating the transcription of genes required for the male-type specification of individual neurons. This study identified the core promoter recognition factor gene Trf2 as a dominant modifier of fru actions. Trf2 knockdown in the sexually dimorphic mAL neurons leads to the loss of a male-specific neurite and a reduction in male courtship vigor. TRF2 forms a repressor complex with FruBM, strongly enhancing the repressor activity of FruBM at the promoter region of the robo1 gene, whose function is required for inhibiting the male-specific neurite formation. In females that lack FruBM, TRF2 stimulates robo1 transcription. These results suggest that TRF2 switches its own role from an activator to a repressor of transcription upon binding to FruBM, thereby enabling the ipsilateral neurite formation only in males.

Male courtship behavior is regulated by the fruitless gene. Drosophila courtship is an interaction wherein males hound females until copulation takes place. Each partner contributes to the enticement: the male engages in a series of actions that include orienting toward and following the female, tapping her with his forelegs, singing a species-specific courtship song by extending and vibrating one of his wings, licking the genitalia of the female, and curling his abdomen to attempt copulation. The behavior of the female, although it largely consists of running away, is not altogether discouraging: females produce a chemical aphrodesiac compound, and if she has not recently mated, and is sufficiently stimulated by his courtship, she will slow down, open her vaginal plates, and allow copulation. (reviewed by Hall, 1994).

fruitless is found near the bottom of the sex determination hierarchy (see Schematic of the sex determination hierarchy in Control of male sexual behavior in Drosophila by the sex determination pathway, Billeter, 2006). At the top is Sex lethal. The Sex lethal gene is functional only in the female, acting to splice the mRNAs from two different transformer genes, transformer and tra-2. Since Sex lethal is not transcribed in males, its action on the transformers is restricted to females. Transformer is also a splice factor, acting in turn on downstream RNAs that require sex-specific splicing (Sosnowski, 1994). Transformer proteins, determines female developmental fate, and in their absence, a male fate ensues. Below Transformer, the sex determination hierarchy bifurcates, with different roles played by doublesex and fruitless. In Fruitless mRNA splicing, the absence of Transformer proteins results in a male fate. In males, splicing is carried out by splicing machinery that is not sex specific.

Before discussing fruitless mutant phenotypes, we will digress for a minute to provide a little information about doublesex. External sexual morphologies for both sexes and aspects of their internal biochemisty are controlled by the doublesex gene. The products of the transformer and tra-2 genes regulate the splicing of the pre-mRNA of Doublesex, leading to the production of a female-specific DSX protein. In males, where Sxl and Tra do not make functional products, DSX pre-mRNA is spliced in its default pattern resulting in a male-specific DSX protein that differs from the female form at its carboxy-terminus. DSX protein transforms females into morphologically wild-type males, but they do not court (Burtis, 1989 and 1993 and Taylor, 1994).

Sexual orientation, as well as courtship behavior in Drosophila, is regulated by fruitless, the first gene in the sex-determination hierarchy functioning specifically in the central nervous system (CNS). The phenotypes of new fru mutants encompass nearly all aspects of male sexual behavior. Alternative splicing of FRU transcripts produces sex-specific proteins belonging to the BTB-ZF family of transcriptional regulators.

It is thought that Fruitless acts through modification of nervous system function. Fruitless regulates the development of a large, paired, male-specific muscle, the "muscle of Lawrence" (MOL), spanning the fifth abdominal segment in adult males and named after Peter Lawrence, who first described it. There are other sexual dimorphisms in the external abdomen, including pigmentation, and dimorphism in intergite musculature associated with male versus female reproductive organs, but only the dimorphism of the MOL is regulated by Fruitless. Fruitless also regulates many aspects of male courtship.

Fruitless modifies the MOL by controlling the presence of additional muscle nuclei, as compared with neighboring muscles of MOL-homologs in females. Since the number and distribution of muscle precursors is the same in both fruitless mutants and wild-type animals, it is thought that one fruitless function is to direct the male-specific recruitment of myoblasts into the MOL-myotubes (Taylor, 1995). How does Fruitless function to recruit myoblasts? Mosaic analysis reveals that MOL development is dependent on its motoneuronal innervation: MOL development proceeds independently of any signal from the myogenic tissue or from the ectodermal tissue at the cuticle where the MOL inserts. In a mosaic in which these tissues are genotypically female and the innervating motoneuron is genotypically male, a MOL develops (Lawrence 1986).

Not all Fruitless effects locate to the fly's abdomen. fruitless mutants show aberrant mating behavior. Many mutant alleles result in male flies that court indiscriminately, fail to copulate and have MOL defects. Some mutant males show very little wing extension and during wing displays generate no song pulse signals. This defect is specific to courtship, as these mutants are normal for flight and are able to flick their wings when rejecting advances made by another male. When fru mutant males are grouped together, they form male-male courtship chains in which each male is simultaneously both courting and being courted. All mutant combinations show some male-male chaining (Ryner, 1996 and Ito, 1996).

Fruitless is expressed in as many as 500 neurons of the brain. One such neuronal cluster is a prominently labeled group of cells in the dorsal-posterior protocerebral region; using gynandromorphs, early steps in courtship (following and wing extension) have been found to map to this region. Sex specific transcripts of fruitless are also found in groups of primary sensory interneurons in the antennal lobe involved in the processing of chemosensory information. These appear to be relay interneurons, known to project to higher brain centers such as the calyx of the mushroom body (Ryner, 1996).

It is concluded therefore that fruitless regulates neural aspects of sexual dimorphism while doublesex regulates morphological aspects. One interesting aspect of this research are the ramifications concerning the regulation of sexual orientation. Whereas wild-type males normally recognize only females as appropriate courtship objects, fruitless mutants court males and females indiscriminately. There is a variety of evidence in vertebrates, including humans, suggesting that male sexual behavior, including sexual orientation, also has a genetic component (Hu, 1995). It is clear that sex determination and sexual behavior is a complex phenomenon regulated by many genes (easily more than 10 in Drosophila). The illusion of the dual nature of sexual characters is belied by the complexity of their regulation.

Functional analysis of fruitless gene expression by transgenic manipulations of Drosophila courtship: fruitless controls singing behavior in identified neurons

A gal4-containing enhancer-trap called C309, which is expressed broadly in central-brain and VNC regions, has been shown to cause subnormal courtship of Drosophila males toward females and courtship among males when driving a conditional disrupter of synaptic transmission (shiTS). These manipulations have been extended to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shiTS at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, C309/traF combination was also found to induce high levels of courtship between pairs of males and 'chaining' behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRUM protein is regulated by Tra, it was hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fruMIR) would mimic the effects of traF; but C309/fruMIR males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRUM in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of traF or fruMIR effects on courtship song, because C309 was found to be coexpressed with FRUM within the same 10 neuronal clusters in pupae. Thus, it is hypothesized that elimination of singing behavior by the C309/shiTS combination involves neurons acting downstream of FRUM cells (Villella, 2005).

Various portions of the CNS in Drosophila are inferred to control separate elements of normal male courtship, in part by analysis of abnormal behavior. Some such studies have involved brain-behavioral analyses of the fruitless (fru) gene and its mutants. Different fru mutants exhibit courtship subnormalities to varying degrees and at separate stages of the courtship sequence, depending on the mutant allele. Most fru mutants court other males substantially above levels normally exhibited by pairs or groups of wild-type males. The original fruitless mutation leads to spatially nonrandom decreases of fru-product presence within particular subsets of the normal CNS expression pattern, which may be causally connected with the breakdown of recognition that is a salient effect of fru1 on male behavior. fru-like courtship can be induced by the effects of a transgene that encodes GAL4 (a transcription factor derived from yeast). When this broadly expressed C309 enhancer trap was combined with a GAL4-drivable factor containing a dominant-negative, conditionally expressed variant of the shibire gene (shiTS), heat treatment of doubly transgenic males caused them to court females subnormally and to court other males vigorously. Although this strain had been termed a mushroom body enhancer trap in terms of the gal4 sequence it contains, being expressed 'predominantly' within that dorsal-brain structure, C309 drives marker expression in a widespread manner. Therefore, attempts were made to correlate various CNS regions in which this transgene is expressed with its effects on male behavior, emphasizing a search for 'C309 neurons' that might overlap with elements of the FRUM pattern (Villella, 2005).

The possibility was also entertained that the C309/shiTS combination causes a mere caricature of fruitless-like behavior. Therefore, what would be the courtship effects of C309 driving a transgene that produces the female form of the transformer gene product? This Tra protein participates in posttranscriptional control of fru's primary 'sex transcript,' so that FRUM protein is not produced in females. If C309 and traF are naturally coexpressed in a subset of the to-be-analyzed neurons, feminization of the overlapping cells should eliminate this protein. These transgenic experiments were extended to target fruitless expression specifically by gal4 driving of an inhibitory RNA (IR) construct, which was generated with fru DNA. Their experiments furnish one object lesson as to how 'enhancer–trap mosaics' can delve into the neural substrates of a complex behavioral process, an approach commonly taken to manipulate brain structures and functions in courtship experiments. Because few genetic loci putatively identified by such transposons have been specified, the tactics applied are in the context of CNS regions in which expression of a 'real gene' is hypothesized to underlie well defined behaviors (Villella, 2005).

Mutations at the fruitless locus and the C309/UAS-shiTS transgene combination each cause similar courtship subnormalities and anomalies. In this context, the C309 enhancer trap is expressed in many CNS neurons that contain male-specific FRUM protein. One element of the courtship effects of C309/UAS-shiTS involves subnormal interactions between males and females. From correlating C309/FRUM coexpression with the fact that fruitless mutations lead to lower-than-normal male–female courtship, it is speculated that FRUM brain regions 2, 8, 13, and 14 are connected with the deleterious effects of shiTS. As to why two additional neuronal groups that coexpress FRUM and C309 are not noted here (groups 5 and 7), see below (Villella, 2005).

With respect to males courting females, special attention might be paid to clusters 13 and 14. These posteriorly located groups of FRUM neurons are likely to include brain regions within which genetic maleness is required if sex mosaics are to exhibit orientation toward and following of females. A problem with this interpretation is that feminizing substantial proportions of the C309/FRUM coexpressing cells led to no decrements in male-female interactions, despite the 7- to 10-fold coexpression reductions caused by XY/C309/UAS-traF within clusters 13 and 14. Perhaps the relevant 'overlap percentages' would have had to drop from 47 and 31 to 0 for both of these clusters if a traF-affected courtship decrement were to be realized. An alternative to viewing this matter in the context of C309/FRUM coexpression is that certain neurons in which this gal4 driver is active could be anatomically downstream of the fru-expressing brain cells that influence a male's ability to initiate and sustain courtship of a female (Villella, 2005).

This conception is relevant to the striking elimination of courtship song in recordings of C309/UAS-shiTS males. Once again, UAS-traF has no such effect. Neurogenetic findings pertinent to this matter are that C309 is expressed in imaginal thoracic ganglia; fru is 'song-involved'; this gene makes its products within several regions of the ventral nerve cord, coexpressing C309 within most of them; and genetic maleness within Drosophila's VNC has been implicated in song control. Thus, turning off synaptic transmission emanating from one or more subsets of the C309/UAS-shiTS neurons in the thoracic ganglia could be the etiology of heat-induced songlessness exhibited by these doubly transgenic males. Regarding the absence of a traF effect, what if C309 was not expressed in any FRUM-containing song-relevant neurons during metamorphosis? In other words, C309 expression in VNC neurons underlying song control could be activated late in the life cycle, allowing for the shiTS effect to take hold after adult males are heated; however, the progenitors of such cells might not express C309 during an earlier 'feminization-relevant' stage, so that post-metamorphic activation of traF would occur too late to affect singing ability. However, substantial coexpression of FRUM and C309 was found within the pupal VNC. In this respect, it is submitted that assessing the C309's expression pattern throughout the life cycle is a valuable object lesson as to what must be done properly to interpret the biological effects of a given enhancer trap (Villella, 2005).

As to the divergent effects C309-driven shiTS vs. traF, recall that the former factor seems broadly to impinge on VNC functioning, in that the fly's general ability to vibrate its wings is shut down by the synaptic disruptor; in contrast, songless fruitless mutants fly normally. Thus, consider a scenario in which C309 neurons would include those that mediate wing vibrations during flight, and that this transgene is expressed in separate VNC cells hypothetically dedicated to such vibrations during courtship. Therefore, it is speculated that the expression domain of C309 includes inter- or motor-neurons functioning within and downstream of a 'command center' for flight as well as neurons located in relatively distal regions of a separate anatomical pathway. The latter would originate where fru-expressing cells exert the gene's crucial regulation of courtship song (Villella, 2005).

Turning to anomalous courtship interactions among males, focus shifts back to the brain: FRUM clusters 5 and 7, where fru1 causes an apparent absence of this protein. This mutation minimally affects the gene's expression in other brain regions. It is notable that the C309/UAS-traF combination knocked down driver/FRUM coexpression to ~10% of normal in cluster 5. Cluster 7 was similarly affected, but special attention should be paid to cluster 5. One reason that this group was thought to be the etiology of frantic courtship among fru1 males is that cluster 5 is located near the antennal lobes; and transgenically mediated feminization of a brain region near these structures induces intermale courtships, although none of the gal4 drivers in that study included C309. Therefore, if proper male-specific structure or function of cluster 5 is involved in normal sex recognition, the mutation's demasculinizing effect on this brain region, or transgene-effected feminization of it, could cause this aspect of courtship to break down (Villella, 2005).

Elements of the current findings suggest that abnormal formation of the brain region in question is not necessary for it to mediate anomalous interactions between males; this is because deactivating synaptic transmission in cluster 5, after CNS development has been completed in a male manner, is sufficient to induce intermale courtship. Perhaps this behavioral effect of driving UAS-shiTS involves removal of inhibitory neurotransmission relevant to the functioning of this brain region, which in normal males would block their wherewithal to sustain courtship between males. Therefore, the fru1 effect on cluster 5 and that of driving Tra production in this region might not involve the formation of a sex recognition center (such that a hypothetical circuit involved in inhibiting intermale courtship is not present or miswired), but instead the intracellular quality and function of neurons in the mature brain (Villella, 2005).

Considering further that certain cluster 5 neurons comprise the subset of FRUM's spatial domain for shiTS- or traF-induced intermale courtship, the relevant cells would be those in which both fruitless and C309 generate their gene products (20% of the 35 neurons within this group). One problem with this supposition is that C309/UAS-traF flies elicit fairly high levels of courtship. Thus, groups of C309/UAS-traF males may form chains for reasons extending beyond a given fly's inappropriate 'motivation' to court another male: The extent to which a C309/UAS-traF fly is feminized could include self-stimulation that might contribute to intermale courting. However, recall the case of C309/UAS-traF/Cha-gal80 males, a transgenic type that is similarly feminine externally and elicits courtship. The diminished extent to which C309's gal4 is effective when combined with Cha-gal80 led to weakened homosexual courtship in single-pair tests and dramatically reduced chaining behavior, although there was essentially no effect of Cha-gal80 on the basic courtship ability of these triply transgenic males. Thus, the effects of this 'neurons-only' manipulation suggest that hypothetical self-stimulation, which did not cause C309/UAS-traF/Cha-gal80 males vigorously to court other ones, is minimally operating to induce the homosexual courtships performed by XY/C309/UAS-traF flies. Males carrying C309 and UAS-fruMIR are also not feminized externally; however, they courted other males robustly in single-pair tests, an effect that was diminished by adding Cha-gal80. Therefore, it is surmised that flies carrying a given fruitless-affecting transgene exhibit intermale courtship because the relevant CNS neurons are demasculinized (Villella, 2005).

However, what about neural structures not analyzed in the current study that could be involved in the behavioral effects of C309 driving either traFor fruMIR? Thus, consider that Tra affects the primary transcript emanating from the doublesex (dsx) gene and that dsx null mutations cause XY flies to exhibit modest levels of intermale courtships. C309 driving of traF could lead to the female (F) form of DSX (thus, no DSXM, as in dsx) within brain cells connected to sex recognition other than those analyzed. Indeed, dsx+ is expressed in the brain; however, the functional significance of these cells is unknown, let alone whether any of them also express fru+. In this regard, it was important to home in on disruption of fruitless's CNS expression alone by combining C309 with the UAS-fruMIR transgenes; this was sufficient to induce courtship between a given pair of doubly transgenic males but led to no chaining. Thus, anomalously high levels of courtship between two males has been disconnected from courtship chaining. [The same disconnect between these different kinds of intermale courtship occurred when Cha-gal80 was added to the C309/UAS-traF combination. It is as if the broad neural effects of a genetic abnormality such as a fruitless mutation, or combining C309 with UAS-traF, is necessary to cause sustained courtship among several variant males; however, if the impingement on fru+ expression is more limited, only courtship between a pair of males can occur (Villella, 2005).

In this regard, the C309/UAS-fruMIR flies were substantially less affected in terms of numbers of brain neurons within which FRUM became undetectable, compared with the effect of the same driver combined with UAS-traF. This brings us to the matter of additional neurons that are potentially relevant to courtship and should be analyzed in context of the C309 effects. Here, the many PNS cells recently discovered to express fruitless in external sensory structures are referred to. It is unknown whether any of these neurons coexpress C309, such that sensory inputs relevant to courtship may have been impinged upon by combining that transgene with UAS-shiTS or with the sex-affecting transgenes. However, fru+ expression in external appendages is not required for a fly to recognize, follow, and perform wing extension at a female: when these structures are genetically female in certain gynandromorphs, maleness within the brain is sufficient to trigger mosaic-with-female courtship (Villella, 2005).

The current study aimed to delve into various regions of the male CNS in which the fruitless gene is expressed: Do certain subsets of the spatial pattern govern a male's ability to perform a discrete feature of the reproductive sequence? Using the gal4-containing C309 enhancer trap was valuable, because it leads to impersonations of certain fru-mutant behaviors when this driver is combined with a shiTS-containing factor that broadly disrupts neural functioning. By limiting C309's efficacy to disrupt by causing it to drive sex-related transgenes succeeded in provisionally partitioning fru-related 'sex recognition' neurons to a subset of the normal brain pattern. By subtraction, the partitioning was further delimited by knocking out the driver's efficacy in a subset C309's spatial domain: adding a neurally driven gal80 transgene substantially attenuated anomalous intermale courtships. A pleasant surprise occurred when the C309/UAS-fruMIR combination was found not to mimic the effects on courtship among males of combining the driver with UAS-traF. Thus, the broader pattern of FRUM expression, unaffected by the IR compared with the substantial decrement caused by traF, takes the analysis a further step. For example, the manner by which fru mutations and related factors influence courtship between two males, as opposed to the much more complicated behavioral dynamics that can occur in a group of such Drosophila, are now being teased out (Villella, 2005).

However, inferences about the potentially relevant subsets of a given brain cluster do not approach specifically identifiable neurons. For this, it will be necessary to do more than quantify the cells in which a transgene driver and fruitless are coexpressed. Further brain-behavioral dissections will require assessing the differential connectivity patterns defining a given class of FRUM neurons, along with variations of cellular content that are likely to discriminate one category of such neurons from another. The relevant object lessons stem from analyses of, so far, only the posterior-most component of fruitless's expression domain in the male CNS: partitioning certain abdominal-ganglion neurons that differentially connect with either a male-specific muscle or with internal reproductive organs, and discovering that the latter type of FRUM cells uniquely contain serotonin. Neurons containing another neurotransmitter, acetylcholine, are on point; but not all of the C309 effects can be ascribed to neurons affected by Cha-gal80, because certain courtship defects were found to remain when analyzing males that carried this transgene along with C309 and UAS-shiTS. This finding reinforces that notion that additional neuronal qualities must be uncovered with regard to cells expressing this enhancer-trap, the fruitless gene, or both (Villella, 2005).

The sex-determination genes fruitless and doublesex specify a neural substrate required for courtship song

Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production. 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 and are necessary for song production, the individual contributions of fru and dsx to the specification of a male CNS and song production was investigated. A novel requirement is shown 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), a critical requirement is shown 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, this study has shown that without DsxM, the determination of a male-specific CNS and thus a full complement of male behaviors are not realized (Rideout, 2007).

Courtship behavior in Drosophila consists of a sequence of behaviors performed by males to interest females in copulation. The male orients to the female, follows her, taps her abdomen with his foreleg, sings a species-specific courtship song, licks her genitals, attempts copulation, and finally copulates. Sex mosaic studies have shown that the sex of the central nervous system (CNS) is critical to the performance of these behaviors, suggesting that sex determination in the CNS is required for male sexual behavior in flies. In particular, one sex-determination gene, fruitless (fru), is a key regulator of many steps in the courtship ritual (Rideout, 2007 and references therein).

Transcripts derived from the fru P1 promoter are spliced in females by the sex-specific splice factor Transformer (Tra) in conjunction with the non-sex-specific Transformer-2 (Tra-2), introducing a premature stop codon into female P1 transcripts. In males, a default splice occurs, giving rise to a class of male-specific fru isoforms (FruM proteins) that are expressed in the CNS and peripheral nervous system (PNS) in regions associated with male-specific behaviors (Rideout, 2007).

The constitutive expression of FruM isoforms in females triggers many male-specific courtship behaviors. However, these females perform subnormal amounts of courtship and do not attempt copulation, suggesting that fru alone cannot specify all male courtship behaviors (Rideout, 2007).

The role of doublesex (dsx), another sex-determination gene, was examined in the specification of male sexual behavior. dsx transcripts also undergo sex-specific splicing by Tra, producing male- and female-specific isoforms: DsxM and DsxF, respectively. dsx is responsible for somatic sexual differentiation and aspects of sex-specific development in the CNS. dsx is also expressed in the CNS and is necessary for wild-type courtship song in males. dsx has been shown to act alongside fru in the differentiation of male-specific neurons in the abdominal ganglion; however, few other studies have examined the relative contributions of both fru and dsx in specifying a male-specific CNS and regulating male sexual behavior. Therefore, this study examined the individual contributions of both genes in specifying courtship song (Rideout, 2007).

Courtship song in Drosophila is male-specific and is critical to stimulating the female. It consists of a humming sound called sine song, and a rhythmically patterned pulse song, which together stimulate the female to mate, reducing the time to copulation. Pulse song also communicates species-specific information, allowing females to recognize conspecific males (Rideout, 2007).

FruM mutant males lack pulse song, and constitutive FruM expression in the CNS of fruM and fruΔtra females induces the performance of many steps of the male courtship ritual, suggesting an important role for FruM in specifying courtship song. To determine the contribution of FruM in the specification of courtship song, song production was analyzed in females of genotype fruM and fruΔtra (Rideout, 2007).

Song analysis was based on 29 fruM and fruΔtra females because most fruM and fruΔtra females did not perform sufficient courtship behavior or song for analysis. The wing-extension indices (WEIs) of these 29 fruM and fruΔtra females were not significantly different from wild-type and control fruM and fruΔtra males; however, a significant decrease was foudn in the song index (SI) (the percentage of time spent singing during wing extension). Also, the fruM and fruΔtra females' pulse song was highly aberrant. The number of pulse trains per minute (PTPM), mean pulses per train (MPPT) and interpulse interval (IPI) were all significantly lower compared to wild-type and control males. Most striking, however, was the complete absence of sine song in these females. Although the fruM and fruΔtra females were capable of wild-type wing extension, they spent significantly less time singing during courtship and produced song of poor quality. Thus, FruM expression alone cannot specify wild-type song production (Rideout, 2007).

To dissect the individual contributions of both FruM and Dsx to the specification of courtship song, males were analyzed lacking FruM and Dsx [genotype fru3,In(3R)dsx23/fru3,Df(3R)dsx15]. These double mutants had a courtship index (CI) of 0 toward females and no song. FruM expression in females is not sufficient for courtship song. Likewise, the expression of DsxM in females is also not sufficient for song. Thus neither fru nor dsx alone can specify courtship song. In fact, only the presence of both FruM and DsxM, as in transformer (tra) mutant females, renders females capable of wild-type courtship song. Together, these results demonstrate a previously unrecognized requirement for DsxM, in conjunction with FruM, in specifying courtship song (Rideout, 2007).

Studies with male-female mosaics have shown that in gynandromorphs with a male head, the ventral thoracic ganglia of the adult CNS (including the mesothoracic ganglion [Msg]) must also be male for courtship song. This suggests that the neural foci of courtship song are located in the ventral thoracic ganglia and that the sex of this region is critical to song. fru and dsx are both expressed in neurons located in this region, and mutations in both genes cause song defects. In the abdominal ganglion (Abg) of the CNS, FruM and Dsx were shown to colocalize in a proportion of neurons and play critical roles in the development of male-specific clusters of serotonergic neurons. Therefore, it was asked whether FruM and Dsx were also coexpressed in the thoracic ganglia, and whether they act in parallel (if expressed in different neurons) or in concert (if expressed in the same neurons) to determine the neuronal substrate for courtship song in the CNS (Rideout, 2007).

It was determined that Dsx and FruM colocalize in the Msg of the CNS. Colocalization occurred in a subset of Dsx-expressing neurons (TN1 cluster. The number of neurons coexpressing Dsx and FruM in 2-day-old male pupae was 17.4 ± 1.7 per hemisegment. Colocalization occurred in a further two subsets of Dsx-expressing neurons in the posterior brain, pC1 and pC2, in addition to previously reported colocalization in the Abg. Given the critical importance of the sex of the ventral ganglia (including the Msg) to song production, the colocalization of FruM and Dsx in this region suggests that sexually dimorphic developmental mechanisms might be operating in the Msg, contributing to the sex-specific nature of courtship song production (Rideout, 2007).

Electrophysiological studies show that the activity of seven of the direct flight muscles (DFMs) is directly related to the beating of the wing during song. These seven DFMs are the basalar muscles B1-B4, the anterior muscles of the first and third axillaries AX1a and AX3a, and the sternobasalar muscle SB. The axonal morphology and cell-body location of the motor neurons innervating six of these DFMs (mnDFMs) has also been reported. The cell bodies of these six mnDFMs lie in the ventral thoracic ganglia, five having cell bodies in the Msg. Therefore whether male-specific song production could be attributed to fru- and/or dsx-regulated sexually dimorphic characteristics of these motor neurons was investigated (Rideout, 2007).

First, it was asked whether any of the mnDFMs were fru or dsx expressing. By using fruGAL4, a GAL4 driver expressing in all fru neurons, it was determined that only mnB3/B4 (a single motor neuron innervating both B3 and B4 was fruGAL4 positive, and thus is a fru neuron. This neuron was fruGAL4 positive in both males and females, and the innervation was not obviously sexually dimorphic. However, because some dsx-expressing neurons in the Msg are not fru expressing, the axonal morphology of all mnDFMs was examined to eliminate the possibility of sex-specific DFM innervation (Rideout, 2007).

The axonal morphology and expression of common neurotransmitters at the neuromuscular junction (NMJ) of all seven DFMs were examined, and no obvious differences between the sexes were found. Type I and type II synaptic terminals were present on all mnDFMs, where type I terminals expressed glutamate and type II terminals expressed octopamine (see Tyramine β hydroxylase), in accordance with previous reports of neurotransmitter expression at the adult NMJ. Moreover, no obvious differences were found in either axonal morphology or common neurotransmitters were observed in either fru or dsx mutant males. Therefore, the sexually dimorphic production of song is not likely to be a result of an obvious dimorphism in the neuronal morphology of the mnDFMs or in the neurotransmitter expression at the NMJs. Where might the critical difference(s) then lie (Rideout, 2007)?

It has been showm that FruM expression prevented reaper-mediated programmed cell death in a cluster of cells, resulting in more neurons in this cluster in males. DsxM, in contrast, prolongs neuroblast divisions in the Abg of males, again resulting in more neurons in males. Thus, sexual dimorphisms might be present in regions in which Dsx and FruM colocalize, as suggested by the ability of FruM and Dsx to generate sexually dimorphic neuronal populations. Given that this investigation found no obvious sex-specific dimorphisms in the mnDFMs, the dimorphism might lie in a population of interneurons. Therefore, the Msg was examined so that it could be determined whether a sexually dimorphic population of neurons was present (Rideout, 2007).

By using fruGAL4, which expresses in both males and females, to drive a GAL4-responsive UAS-LacZ.NZ reporter, the number of β-Gal-positive neurons was quantified in males and females. The number of β-Gal-positive neurons was significantly higher in males, with 136.4 ± 3.3 cells per hemisegment (n = 10) versus 111.6 ± 3.1 cells per hemisegment in females. A sexual dimorphism has been reported in the number of neurons expressing fru P1 transcripts in the Msg. Together, these results suggest that a sexually dimorphic population of neurons is present in the Msg; therefore, the individual contributions of FruM and Dsx in the creation of this difference was examined in fruGAL4-positive neuron number in the Msg (Rideout, 2007).

A sexually dimorphic number of fruGAL4-expressing neurons was found in the Msg, a region of the CNS central to song production and in which FruM and Dsx colocalize. To determine the individual contributions of dsx and fru in the creation of this sexually dimorphic number of neurons, fruM and fruΔtra females were examined to see if FruM expression alone abolishes the observed difference in neuronal number in the Msg between the sexes. It was found that the number of FruM-expressing neurons in the Msg of these females was significantly reduced in comparison to wild-type and control males. Furthermore, this decrease in FruM-expressing neurons was comparable to the difference in neuron number observed in the Msg of fruGAL4 males and females driving the UAS-LacZ.NZ reporter (Rideout, 2007).

These results demonstrate that the difference in neuronal populations of males and females in the Msg lies in a subpopulation of FruM-expressing neurons, and that FruM expression alone cannot eliminate this difference. Thus FruM expression cannot, by itself, dictate the creation of the sexually dimorphic population of neurons in the Msg. It was therefore asked whether Dsx, which colocalizes with FruM in the Msg, plays a role in the specification of this sexually dimorphic population of neurons, helping to determine the full complement of FruM neurons (Rideout, 2007).

dsx affects the sex-specific development of other regions of the CNS. To determine whether dsx contributes to creating the sex-specific population of neurons in the Msg, the number of FruM-expressing neurons was tabulated in the Msg of dsx null and dsx heterozygote control males. It was found that dsx mutant males had significantly fewer FruM-expressing neurons in the Msg than did wild-type and control males, demonstrating that Dsx is indeed required to obtain a full complement of FruM-expressing neurons. Because fruM and fruΔtra females (who express the female-specific isoform of dsx, DsxF) do not have a full complement of FruM-expressing neurons in the Msg, this study has demonstrated a critical role for DsxM in the creation of a sexually dimorphic Msg. In fact, only when both FruM and DsxM are present, as in tra mutant females, can a full complement of FruM-expressing neurons in the Msg be obtained. Thus, this study has demonstrated a previously unrecognized requirement for DsxM in the specification of a population of FruM-expressing neurons in the Msg (Rideout, 2007).

DsxM prolongs the division of neuroblasts in the Abg of males, resulting in more neurons in the male Abg. Also in the Abg, DsxM plays a critical role alongside FruM in the differentiation of a male-specific serotonergic population of neurons. The current findings suggest that DsxM operates in a similar manner in the Msg and the posterior brain to create sexually dimorphic neuronal numbers. These differences in neuronal populations suggest a common developmental theme in colocalization regions, where DsxM generates a sexually dimorphic population of neurons, which is exploited by FruM to fashion a male-specific behavioral neural network (Rideout, 2007).

It is not clear why the absence of a sexually dimorphic population of FruM-expressing neurons in the Msg is associated with striking defects in courtship song because the results suggest that this population of FruM-expressing neurons does not directly innervate the DFMs. It is proposed that the FruM-expressing neurons form at least part of a male-specific neural network responsible for controlling the production of courtship song (Rideout, 2007).

Thus, although FruM expression can specify many male-specific behaviors, this study showd that without DsxM, the determination of a complete male-specific CNS, and therefore a full complement of male behaviors, is not realized. This additional gene function is critical to understanding complex sex-specific phenotypes compared to previous interpretations of function, where fru has been described as the only gene needed for a 'genetic switch' to male sexual behavior in Drosophila. Significantly, it adds to the growing evidence that fru and dsx are both necessary for a complete male courtship repertoire, in both neural and nonneural tissues (Rideout, 2007).

A bidirectional circuit switch reroutes pheromone signals in male and female brains

The Drosophila sex pheromone cVA elicits different behaviors in males and females. First- and second-order olfactory neurons show identical pheromone responses, suggesting that sex genes differentially wire circuits deeper in the brain. Using in vivo whole-cell electrophysiology, this study has shown that two clusters of third-order olfactory neurons have dimorphic pheromone responses. One cluster responds in females; the other responds in males. These clusters are present in both sexes and share a common input pathway, but sex-specific wiring reroutes pheromone information. Regulating dendritic position, the Fruitless transcription factor both connects the male-responsive cluster and disconnects the female-responsive cluster from pheromone input. Selective masculinization of third-order neurons transforms their morphology and pheromone responses, demonstrating that circuits can be functionally rewired by the cell-autonomous action of a switch gene. This bidirectional switch, analogous to an electrical changeover switch, provides a simple circuit logic to activate different behaviors in males and females (Kohl, 2013).

This study reveals principles of neural circuit organization and development that are of general significance. First, it was shown that two populations of neurons, present in both sexes, show reciprocal, sex-specific responses to the same stimulus. Second, it was demonstrated that these responses result from differential wiring of a common input to different outputs. Together, these results define an elegant principle of neural circuit organization: a developmental circuit switch directly analogous to an electrical changeover (or single pole, double throw, SPDT) switch that efficiently reroutes a common input signal to one of two possible outputs. This model appears directly applicable to sex-specific processing of mouse pheromones, including ESP1 and Darcin (Haga, 2010; Stowers, 2010), but not to Caenorhabditis elegans ascarosides, where recent data suggest wiring differences may not be required. The electrical changeover switch is the prototype for a wide-range of electrical switches in which concerted changes involving three or more contacts reroute signals; it is very likely that neural circuits, including those involved in pheromone processing, contain more complex switches or assemblies of multiple switches that elaborate on the basic mechanism that are described in this study. Indeed, over 700 sites of dimorphic neuronal overlap have been identified that may form such switches in other sensory pathways, multimodal interneurons, or motor circuits across the fly brain (Cachero, 2010). Third, sex-specific placement of target neuron dendrites were identified as the primary cellular basis of the switch that is described in this study. This contrasts with earlier studies of this circuit that proposed that axonal dimorphism or neurons present only in one sex were the key dimorphic element. Regarding axonal dimorphism, Datta (2008) hypothesized that a male-specific extension of DA1 PN axon terminals is the basis of differential wiring in this system, and Ruta (2010) subsequently proposed that this extension synapses with the dendrites of aSP-f LHNs in males. The large shifts in dendritic position that were observed in aSP-f and aSP-g neurons mean the male-specific extension of DA1 PNs cannot be sufficient for rewiring. Is it necessary? In mosaic masculinization experiments, aSP-f and aSP-h neurons adopt male morphology and pheromone responses in a brain in which other neurons (including DA1 PNs) are female. Therefore, the male-specific ventral extension is either not necessary for differential wiring or is a secondary consequence of changes in the dendrites of post-synaptic LHNs. Of course, this extension may increase contact between DA1 PNs and aSP-f and aSP-h LHNs, strengthening responses of those LHNs in males. All three mechanisms (dendritic and axonal dimorphisms, dimorphic cell numbers) are likely relevant to different degrees in different circuits (Kohl, 2013).

Fourth, having defined this bi-directional switch, it was demonstrated that its male form is specified by the fruitless gene. This transcription factor has a dual function, coordinating the disconnection of one group of target neurons and the connection of the other. Fifth, it was shown that masculinization of third-order neurons alone is sufficient for functional rewiring. Although previous studies have demonstrated a cell-autonomous effect of fruitless on neuronal morphology, this study now demonstrates a difference in functional connectivity. This is surprising because many would predict that connectivity changes would depend on coordinate regulation of genes in synaptic partner neurons. Such simplicity has evolutionary implications: it may allow variation in circuit structure and ultimately in behavior, through evolution of cis-regulatory elements, as previously shown for somatic characters, such as wing spots (Kohl, 2013).

Sixth, studies of pheromone processing in general and cVA processing in particular have emphasized a labeled line processing model. However, the current data indicate that both narrowly (aSP-f) and broadly tuned (aSP-h) cVA-responsive neurons coexist in males. Likewise in females, aSP-g neurons respond to cVA and general odors, such as vinegar, but only cVA responses depend on the Or67d receptor. It will be very interesting to determine the circuit origin and behavioral significance of this integration of odor channels. For example, it seems reasonable to speculate that coincidence of cVA and food odors could interact in a supralinear way to promote female courtship or egg laying. This parallels the convergence in the lateral horn of a labeled line responsive to non-cVA fly odors (Or47b/VA1lm neurons) and one responsive to a specific food odorant, phenylacetic acid, that acts as a male aphrodisiac (Kohl, 2013).

This study naturally raises additional questions. The action of fruitless within fewer than 5% of the neurons in the fly brain can specify behavior, and this study now shows that it can reroute pheromone signals within those neurons. But what is the behavioral relevance of this particular bidirectional switch? Testing this will require the development of sensitive behavioral assays of cVA processing and a reliable genetic approach to control this switch without affecting the many other dimorphic elements in sensory and motor circuits. Indeed, it remains to be seen whether flipping a single switch in sensory processing is sufficient to engage motor behavior typical of the opposite sex without masculinizing downstream circuitry. It is noted that it is possible to force the production of courtship song by activating fruitless-positive neurons in headless females, but this was almost never successful in intact females (Kohl, 2013).

Another open question concerns the functional significance of female aSP-f and male aSP-g neurons, which do not respond to cVA or other tested odors. Do they receive input at all? One possibility, based on in silico analysis of the brain-wide 3D maps is that they receive gustatory input, perhaps from contact pheromones, although further work is necessary to test this hypothesis. Finally, which genes does fruitless regulate in order to differentially wire the switch? Clonal transformation experiments strongly support the earlier proposal that male and female aSP-f/g/h clusters are generated by neuroblasts common to both sexes but that those neurons develop in a sex-specific manner. Therefore, cell-surface molecules required for dendritic guidance are plausible targets. It will be intriguing to see if the same fru-dependent factor(s) direct(s) male aSP-f and female aSP-g dendrites to the ventral lateral horn and, more generally, whether fruitless acts on conserved downstream targets across all the dimorphic neurons in the fly brain (Kohl, 2013).


cis-Regulatory Sequences and Functions

A fruitless upstream region that defines the species specificity in the male-specific muscle patterning in Drosophila

The muscle of Lawrence (MOL) is a male-specific muscle present in the abdomen of some adult Drosophila species. Formation of the MOL depends on innervation by motoneurons that express fruitless, a neural male determinant. Drosophila melanogaster males carry a pair of MOLs in the 5th abdominal segment, whereas D. subobscura males carry a pair in both the 5th and 4th segments. It was hypothesized that the fru gene of D. subobscura but not that of D. melanogaster contains a cis element that directs the formation of the additional pair of MOLs. Successively extended 5' DNA fragments to the P1 promoter of D. subobscura or the corresponding fragments that are chimeric (i.e., containing both melanogaster and subobscura elements) were introduced into D. melanogaster and tested for their ability to induce the MOL to locate the hypothetical cis element. A 1.5-2-kb genomic fragment located 4-6-kb upstream of the P1 promoter in D. subobscura but not that of D. melanogaster was found to permit MOL formation in females, provided this fragment is grafted to the distal approximately 4-kb segment from D. melanogaster, demonstrating that this genomic fragment of D. subobscura contains a cis element for the MOL induction (Takayanagi, 2014).

Transcriptional Regulation

Mutation in the Drosophila retained/dead ringer (retn) gene leads to female behavioral defects and alters a limited set of neurons in the CNS. retn is implicated as a major repressor of male courtship behavior in the absence of the fruitless (fru) male protein. retn females show fru-independent male-like courtship of males and females, and are highly resistant to courtship by males. Males mutant for retn court with normal parameters, although feminization of retn cells in males induces bisexuality. Alternatively spliced RNAs appear in the larval and pupal CNS, but none shows sex specificity. Post-embryonically, retn RNAs are expressed in a limited set of neurons in the CNS and eyes. Neural defects of retn mutant cells include mushroom body ß-lobe fusion and pathfinding errors by photoreceptor and subesophageal neurons. It is posited that some of these retn-expressing cells function in females to repress a male behavioral pathway activated in males by fruM (Ditch, 2005). retn females show one behavior not shown by dsf, dsx or fru females: male-like courtship of females and males, especially as they age. retn females follow, tap and appear to sing. Although not as robust as male courtship (following is not as sustained, full wing extension and vibration are not seen, and copulatory bending is weak or absent), these behaviors highly resemble courtship. These behaviors vary between and within allelic combinations, but when the behaviors are seen they are striking and continue for hours. retnz2-428/retndri8 females, which show the most consistent behaviors, with maximum penetrance at 3-4 weeks post-eclosion, averaged 42 courtship events per 5-minute observation period, while control females display fewer than three courtship-like events in the same period. Although male behaviors are evident, the fruM-dependent Muscles of Lawrence are not seen in retn females (Ditch, 2005).

Aspects of the retn female behaviors are similar to wild-type female defenses of food and egg-laying resources. One study on Drosophila aggressive behaviors indicated that aggression in wild-type females increases if females are raised individually before pairing for observation. No increase was found in male-like behaviors in females kept separately from eclosion until testing. This suggests that these behaviors are not an exaggerated defense response. Other indications that these behaviors are not based on access to food come from observations of wild-type females starved overnight on moistened filter paper and transferred back onto food. These females showed short head-to-head and head-to-side interactions, but did not show behavior resembling male courtship. Courting retn females, by contrast, primarily show posterior orientation, and will follow other females on and off a food source for minutes at a time (Ditch, 2005).

retn is expressed in the CNS during pupal stages when sexual behavior is hardwired. To map retn expression in the CNS, retn-driven GFP expression was mapped using retn-Gal4 insertions that rescue retn phenotypes with the retn cDNA. These Gal4 enhancer traps, in addition to rescuing retn viability and behaviors, exactly reproduce Retn antibody patterns in embryos and larval eye tissue; therefore, they should represent the later CNS expression to a high degree of accuracy. Expression and projections were monitored using membrane-associated UASCD8::GFP (UAS-mGFP). retn expression in the CNS begins in the embryo, and continues through adulthood, in specific subsets of neurons. Focus was placed on expression of retn in the periods before and during metamorphosis, when adult neurons are born and larval neurons are remodeled into adult-specific forms. Notably, expression is seen in the mushroom bodies, subesophageal ganglion, ventral ganglion and developing photoreceptors. These patterns are essentially the same in both sexes (Ditch, 2005).

In the third instar, MB expression is seen in the Kenyon cell (KC) bodies lying in the dorsoposterior of the central brain, with staining in the calyx, containing KC dendrites, and the pedunculus and lobes, containing KC axons. Between 12 and 18 hours after puparium formation (APF), the calyx retracts, the alpha and ß lobes narrow and what appears to be axonal debris can be seen at the lobe tips. At this stage there are slightly more retn cells in females than in males, perhaps reflecting the greater axon number in female MBs. By 36 hours APF, the adult alpha, alpha', ß, ß', and gamma lobe projections are visible, although retn expression is stronger in alpha/ß projections. Between 24 and 48 hours APF, expression in all lobes except alpha/ß gradually fades, and by 48 hours only the alpha/ß lobes can be seen. This pattern remains through the rest of metamorphosis (Ditch, 2005).

In the larval Subesophageal ganglion (SOG), two central groups of six or seven neurons and two anterior groups of five neurons send projections towards the protocerebrum and ventral nerve cord. Laterally to these neurons are four additional neurons per side. The projections of these neurons form a dense pattern, and individual projections cannot be discerned. Retraction of larval-specific processes can be seen beginning six hours APF; by 36 hours APF, new processes are evident. The number of SOG neurons expressing retn remains constant, but projections become increasingly dense through the pupal period (Ditch, 2005).

retn-Gal489 is expressed posterior to the morphogenetic furrow, in photoreceptor cells R1-R6, which project to the lamina and R8, which projects to the medulla, as is also seen with Retn antibody staining. Beyond 48 hours APF, R8 expression and projections fade, although lamina projections remain. Expression in the eye, MB, SOG and ventral nerve cord is still visible post-eclosion (Ditch, 2005).

MB-specific abnormalities are seen in three different retn mutant genotypes: retn-Gal489/retnZ2-428 larvae and pupae; retndri8/retnZ2-428, and retnRo44/retnRO44 adults. MB neurons diverge within the nerve tracks and ß-lobe neurons cross the midline and join with the opposite ß-lobe neurons, causing ß-lobe fusion, compared with retn-Gal489/+. This is more common in females than males, but phenotypes of retn; fru males indicate that retn functions in male neurons. Using antibodies to Fas2, which is expressed in MB axons projecting to the alpha- and ß-lobes in retndri8/retnZ2-428 and retnR044/retnR044 adults, it was found that in a subset of mutant females, axons in the posterior part of the ß-lobe crossed the midline, resulting in ß-lobe fusion. In addition, in those animals with ß-lobe fusion, there were fewer Fas2-positive axons in the alpha-lobe. These MB fusion phenotypes are similar to the ß-lobe fusion phenotypes reported in other mutants, such as linotte/derailed, Drosophila fragile X mental retardation 1, fused lobes, ciboulot and alpha-lobe absent. Resistance is shown by the vast majority of females of these genotypes, thus MB fusion is unlikely to be causal for resistance (Ditch, 2005).

To determine retn neuronal birth dates and the neural phenotypes of dri-class alleles, the MARCM system, which can simultaneously create homozygous mutant cells and allow them to express Gal4-regulated marker genes, was used. retn-expressing MB neurons are born throughout the larval and pupal stages and eye clones appear at all embryonic and larval stages. The VNC neurons are born only within 48 hours of egg laying, and SOG retn neurons are born in 8-hour-old or younger embryos (Ditch, 2005).

Homozygous retn-Gal489 clones show striking mis-projection phenotypes in SOG neurons. The normal elaboration and symmetry of arbors in mid-pupae is diminished; ventral dendritic branches do not show normal density, and anterior projections wander and fail to extend. Neurons also fail to fasciculate normally. A central SOG midline-crossing tract, visible throughout metamorphosis, contains tightly bundled projections. In mutant clones, projections stray from this tract, apparently losing some adherent ability. Photoreceptor neurons also mis-project. In retndri clones, induced in the embryo, R1-R6 cells overshoot the lamina, and a number now target the medulla. Although retn mutations alter neuronal projection patterns, and projection differences are consistent with changes in behavior, retn behavioral functions have not yet been mapped to a particular set of neurons, nor has it been demonstrated that the projection differences, as opposed, for example, to retn-induced reductions in neural activity, are responsible for behavioral changes (Ditch, 2005).

It has been concluded that retn functions in multiple, separable processes during development. It acts in differentiation and control of gene expression along the anterior posterior and dorsal ventral axes in embryos. It also acts in the production of various tube structures such as salivary ducts and gut. Failures in these or other embryonic processes with dri-class (null or near null) alleles lead to embryonic death. retn-class (hypomorphic missense) alleles can perform the embryonic functions but show defects in neural development and projections. Correlating with this are changes in female behavior, including resistance to male courtship and, strikingly, generation of male-like courtship behaviors. Additional functions in development of internal genital ducts and fertility (Ditch, 2005).

retn neural and behavioral phenotypes are substantially different from those of dsf or fru. dsf females, like retn-females, are sterile and resist male courtship. For dsf, sterility results from loss of motor synapses on the circular muscles of the uterus. By contrast, these synapses are intact in retn females. dsf females show no male behaviors, while retn females do. dsf males are bisexual and slow to copulate, owing to inefficient abdominal bending, correlated with abnormal synapses on the muscles of ventral abdominal segment 5. retn males court and mate with normal kinetics and have normal A5 synapses. This suggests that retn and dsf have largely separate functions (Ditch, 2005).

retn and fru also have different phenotypes. In a wild-type background retn behavioral phenotypes are restricted to females. fru behavioral phenotypes are restricted to males and include failure to attempt copulation, bisexual and homosexual courtship, and, in the strongest allelic combinations, complete lack of male courtship. In addition, fru males lack the male-specific muscles of Lawrence in dorsal abdominal segment 5. retn males have normal muscles of Lawrence, and retn females do not have muscles of Lawrence. In addition, the larval and pupal expression patterns of retn and the sex-specific products of the fru P1 promoter, notably the active male-specific fru proteins, show little or no overlap. This all suggests that fru and retn are unlikely to interact intracellularly and would be expected to be involved in different aspects of behavioral control (Ditch, 2005).

The latter conclusion seems to be contradicted by the male-like courtship generated by retn females, since previous work demonstrates that otherwise wild-type males require Fru-M to generate male behavior. It has been operationally and molecularly shown that the male behavior generated by retn females occurs even in the absence of fru P1 transcripts (Ditch, 2005).

A plausible working model has been developed that reconciles the data on the necessity of fruM in males and male-like courtship by retn females. The largely non-overlapping expression patterns of fru and retn suggests that the formal interactions of this model will result from interactions between networks of fru- and retn-influenced neurons rather than by intracellular regulatory interactions involving Fru-M and Retn, although the model can accommodate either situation (Ditch, 2005).

The model posits that in the absence of fruM and retn the nervous system has an inherent tendency to set down some rudiments of neural pathways for male courtship behavior. When retn is wild type and fruM is not expressed, as in wild-type females, retn, or cells expressing retn [perhaps in conjunction or parallel with other factors such as dsxF], act to suppress the basal male courtship pathway. This blocks male courtship behaviors. This is the case in wild-type females (Ditch, 2005).

Finally, in wild-type males, fruM or cells expressing fruM, perhaps along with other factors such as dsxM, act to strengthen the male courtship pathway such that the repressive action of retn-expressing cells is overpowered. This makes fru the switch that results in male behavior and captures both the requirement for fru+ in males, and the male-like courtship by retn females (Ditch, 2005).

This model does not rule out involvement of other components. For example, it has been suggested that dsxF can suppress male behaviors in a retn+ background. This can be fitted into the model as an additional female-specific block to male behavior. A simple prediction of such a role for dsx is that reduction of dsx expression in a retn mutant background will enhance the retn phenotype. Recent work involving expression of fru RNAi in a subset of fru neurons suggests a role for temporally repression in the sequencing of male behaviors in courtship (Ditch, 2005).

An extensive series of experiments is in progress to test predictions of this model. Experiments are also in progress to determine if dsx participation fits within the context of the model, and to identify the molecules and mechanisms downstream of retn in the control of behavior (Ditch, 2005).

Targets of Activity

The Drosophila somatic sex-determination regulatory pathway has been well studied, but little is known about the target genes that it ultimately controls. In a differential screen for sex-specific transcripts expressed in fly heads, a highly male-enriched transcript was identified encoding Takeout, a protein related to a superfamily of factors that bind small lipophilic molecules. Sex-specific takeout transcripts derive from fat body tissue closely associated with the adult brain and are dependent on the sex determination genes doublesex (dsx) and fruitless (fru). The male-specific Doublesex and Fruitless proteins together activate Takeout expression, whereas the female-specific Doublesex protein represses takeout independently of Fru. When cells that normally express takeout are feminized by expression of the Transformer-F protein, male courtship behavior is dramatically reduced, suggesting that male identity in these cells is necessary for behavior. A loss-of-function mutation in the takeout gene reduces male courtship and synergizes with fruitless mutations, suggesting that takeout plays a redundant role with other fru-dependent factors involved in male mating behavior. Comparison of Takeout sequences to the Drosophila genome reveals a family of 20 related secreted factors. Expression analysis of a subset of these genes suggests that the takeout gene family encodes multiple factors with sex-specific functions (Dauwalder, 2002).

To identify genes under the control of the sex-determination regulatory pathway, a PCR-based subtractive hybridization screen was carried out for sex-specific RNAs expressed in adult fly heads. Head RNA of tra-2/tra-2+ phenotypically wild-type XX adult females was subtracted against the head RNA of sibling XX tra-2/tra-2 mutants, and vice versa. The latter flies are transformed into males both somatically and behaviorally. One cDNA clone that hybridized preferentially with sequences from phenotypic males was isolated and studied in more detail. Northern blot hybridizations confirmed that this sequence represents a highly male-specific 1.1-kb mRNA that was expressed primarily in adult heads. Expression of this mRNA was repressed by Tra-2 in females, since XX tra-2 mutants expressed levels similar to wild-type males. The sequence of the clone was later found to be identical to that of takeout, an independently identified gene responsive to circadian rhythms and starvation. The takeout gene encodes a secreted protein related to circulating carrier proteins of lipophilic factors, such as the juvenile hormone-binding proteins of other insects. Analysis of RNA prepared at different times during the day failed to reveal any significant variation in takeout levels (Dauwalder, 2002).

Although courtship of the above mutant flies is quantitatively reduced, it is not absent. The mutant males are capable of all steps of courtship, but perform them less frequently, and seem to lack motivation to court. Unlike homozygous fru mutants, takeout mutants displayed no male-chaining behavior, suggesting that these males are capable of distinguishing between males and females as potential mates. A simultaneous reduction of fruitless and takeout function interferes with male courtship. The original fru3 and fru4 mutant alleles were generated in a ry506 genetic background. Prior to the above studies, it was noticed that the third chromosomes in the fru3 and fru4 strains carry not only these fru and ry mutations, but also the to1 mutant allele. This led to a test of whether previously observed courtship phenotypes associated with these fru alleles might have been enhanced by the presence of the takeout mutation. The courtship of the double mutant flies (to1, fru4) was compared with a recombinant fru4 line (to+, fru4) carrying the allele of takeout from the wild-type strain Canton-S. The presence of the takeout mutation causes a statistically significant reduction in courtship index. However, this effect is small in relation to that of the fru4 mutation alone (Dauwalder, 2002).

Analysis of fru mutants demonstrated that it affects takeout expression only in males. Repeated Northern analysis of RNA from XY fru adults showed that the levels of takeout RNA present in these individuals was consistently reduced by about 32% relative to fru/+ males. Expression of takeout was not increased by loss of fru function in XX females. This is consistent with the recent finding that functional sex-specific Fru protein is not present in females. Taken together, the above results indicate that both Fru and Dsx function to specify male-specific expression of takeout RNA (Dauwalder, 2002).

Functional analysis of Dsx and Fru has led to the suggestion that they have distinct and complementary roles, with Fru specifying sexual identity of tissues in the CNS that are responsible for courtship behavior, and Dsx specifying sex in other somatic tissues. However, given the observation that dsx mutants also have minor effects on courtship behavior, it is believed that a clear delineation of the roles played by Dsx and Fru will require more information about the specific genes and cell types whose sexual identity these factors specify (Dauwalder, 2002).

This study has provided evidence that the takeout gene is a target of regulation by the somatic sex-determination pathway. Although takeout expression in some tissues is nonsex-specific, the vast majority of takeout RNA derives from fat body within the adult head and is specific to males. Surprisingly, analysis of RNA from mutant flies indicates that sex-specific takeout expression depends on the function of both Dsx and Fru. Although this would seem to contradict the expected restriction of Fru function to the CNS, it is worth noting that the effect of Fru on takeout expression could be mediated indirectly by diffusible factors. In situ hybridization studies localized fru RNA to a variety of specific neurons, but not the fat body cells in which male-specific takeout RNA is most prominently expressed. Interestingly, the only other instance of sexual differentiation outside of the CNS, where sex-specific Fru function is known to be required, is in the formation of the Muscle-of-Lawrence, a male-specific abdominal muscle in which sexual fate is determined through inductive signals that originate from the innervating motor neuron (Dauwalder, 2002).

Both the dsx and fru genes encode alternatively spliced transcripts that encode distinct forms of the Dsx and Fru proteins in males and females. Thus, both genes could potentially play a role in either activating takeout in males or repressing it in females. Full activation of takeout is not achieved in either dsx null or fru hypomorphic mutant XY individuals and, instead, takeout RNA is present at levels intermediate between those found in males and females. In chromosomal females, only Dsx is required for repression of takeout. The fact that fru mutants do not affect takeout expression is consistent with experiments suggesting that the female-specific form of fru mRNA is not translated into a functional protein. Moreover, all sex-specific Fru functions so far identified have been found in males. Therefore, although a sex-specific Fru mRNA is produced in females that potentially encodes a protein, there is currently no evidence that it functions to regulate sexual differentiation (Dauwalder, 2002).

The yellow (y) gene is shown to be genetically downstream of fru in the third-instar larval brain. Yellow protein is present at elevated levels in neuroblasts, which also show expression of male-specific Fre proteins, compared to control neuroblasts without Fre. A location for y downstream of fru in a genetic pathway was experimentally demonstrated by analysis of fru mutants lacking transcription of zinc-finger DNA binding domains, and of animals with temporal, spatial, or sexual mis-expression of male-specific Fru. A subset of fru and y mutants is known to reduce levels of a specific behavioral component of the male courtship ritual, (wing extension), and Fre and Yellow are detected in the general region of the brain where maleness is necessary for development of that behavior. It is therefore hypothesized that ectopic expression of Yellow in the third-instar brain, in a y null background, would rescue low levels of wing extension and male competitive mating success, and this was found to be the case. Overall, these data suggest that y is a downstream member of the fru branch of the D. melanogaster sex determination hierarchy, where it plays a currently unknown role in the development of adult male wing extension during courtship (Drapeau, 2003).

Sex- and clock-controlled expression of the neuropeptide F gene in Drosophila

Drosophila neuropeptide F (NPF), a homolog of vertebrate neuropeptide Y, functions in feeding and coordination of behavioral changes in larvae and in modulation of alcohol sensitivity in adults, suggesting diverse roles for this peptide. To gain more insight into adult-specific NPF neuronal functions, how npf expression is regulated in the adult brain was studied. npf expression is regulated in both sex-nonspecific and male-specific manners. The data show that male-specific npf (ms-npf) expression is controlled by the transformer (tra)-dependent sex-determination pathway. Furthermore, fruitless, one of the major genes functioning downstream of tra, is apparently an upstream regulator of ms-npf transcription. Males lacking ms-npf expression (through traF-mediated feminization) or npf-ablated male flies display significantly reduced male courtship activity, suggesting that one function of ms-npf neurons is to modulate fruitless-regulated sexual behavior. Interestingly, one of the ms-npf neuronal groups belongs to the previously defined clock-controlling dorsolateral neurons. Such ms-npf expression in the dorsolateral neurons is absent in arrhythmic ClockJrk and cycle02 mutants, suggesting that npf is under dual regulation by circadian and sex-determining factors. Based on these data, it is proposed that NPF also plays a role in clock-controlled sexual dimorphism in adult Drosophila (Lee, 2006).

Sexual dimorphism of brain structure and function generates differential neural circuitries, ultimately leading to the production of gender-specific behaviors. In Drosophila, fru is an essential neural sex determinant responsible for male courtship behavior. Because fru-encoded FRUM protein is a BTB-Zn-finger transcription factor, FRUM likely regulates expression of an array of genes to establish neural substrates controlling male behavior. However, such downstream targets of the FRUM are poorly known, hampering understanding of the molecular mechanisms underlying fru-controlled establishment of male-specific neural circuitry. Intriguingly, these studies identified npf as an at least indirect target of the FRUM, suggesting that NPF is a neurochemical factor mediating FRUM functions (Lee, 2006).

Recent studies on the expression of sex-specific fru transcripts and of reporter expression driven by fru-gal4 suggest that fru acts in establishing sexually dimorphic anatomical differences and in rendering male-specific functions to neurons that are commonly present in both. Thus, one important question is whether ms-npf is due to the lack of corresponding neurons in the female brains or to cell-specific transcriptional activation of npf by FRUM in males. The data support the argument that the latter is the case, at least for L1-s neurons, because a comparable number of such neurons independently marked by anti-TIM was observed in both males and females, and because FRUM is persistently present in well differentiated ms-npf neurons. Therefore, it is suggested that one way of masculizing neurons directed by fru is to establish sex-specific production of neurosignaling molecules, which are likely to deliver male-specific neuronal functions. In line with this suggestion, it is notable that male-specific serotonin production in a group of eight neurons in the abdominal ganglion is also controlled by fru, and such serotonergic neurons innervate male reproductive organs to control appropriate male mating activities. These data indicate that ms-NPF is another neurosignaling molecule for fru-controlled male courtship. Although the neuronal targets of ms-npf are unknown, prolonged courtship-initiation latencies and general attenuation of courtship activities caused by the absence of ms-npf suggest that ms-NPF is involved in the central processing of courtship-activating stimuli or in an 'output pathway' that mediates courtship actions (Lee, 2006).

Sexually dimorphic NPY expression has been described in the rat hypothalamus. Large populations of hypothalamic NPY mRNA-producing cells are localized within the arcuate nucleus. Interestingly, the caudal region of the arcuate nucleus contains significantly more NPY cells in males than in females. Further studies suggested that the male gonadal hormone testosterone is a positive regulator of male-biased NPY expression. Similar sexually dimorphic NPY/NPF expression in the brains of distantly related species suggests that these neuropeptides play conserved roles associated with male-specific CNS functions that underlie sexual behavior (Lee, 2006).

Dual regulation of npf by sex and clock factors within a subset of male LNd neurons suggests that NPF is associated with clock-controlled sexually dimorphic behavioral performance. In light/dark cycles, the circadian timing system directs bimodal daily peaks of locomotion, occurring at lights-on (morning) and at lights-off (evening) transitions in WT flies. Interestingly, a distinct sexual difference was observed in the peak of the morning locomotion, which occurs ~1 h earlier in males than in females. However, this male-specific phase of the morning activity was unaffected by npf-ablation, suggesting that npf is not associated with this aspect of sexual dimorphism (Lee, 2006).

The brain-behavioral system is also capable of anticipating photic transitions, as demonstrated by the gradual increase in activity levels before lights-on or lights-off. Among six groups of clock neurons defined in the Drosophila adult brain, clock-relevant functions are relatively well studied for the s-LNvs and the LNds. In the former cell type, clock-controlled pigment-dispersing factor production is essential for circadian locomotor activity rhythms as well as lights-on anticipation, whereas the LNds are required for anticipation of lights-off. The data implicate NPF as a neuromodulatory substance within a subset of the LNds, involved in this 'late-day' component of the locomotor cycle in males (Lee, 2006).

Although the biological meaning of LNd-regulated lights-off anticipation remains unknown, it is notable that the flies’ increasing locomotion at dusk is temporally coincident with especially vigorous mating activities of fruit flies. This finding, then, raises the possibility that anticipatory activity at dusk is causally connected with maximum mating propensity. In this respect, that there are sex- and clock-controlled npf expressions in the LNds provides the first glimpse of this neuropeptide as a putative output factor, which would participate in certain aspects of the clock-controlled reproductive behavior. These actions are intimately connected to evolutionary fitness, which may be one reason for the circadian system of Drosophila to have evolved and been refined (Lee, 2006).

Fruitless mRNA splicing

The alternatively spliced transcripts from the distal promoter are generated by use of 5' splice sites 1590 nucleotides apart. Splice site selection is sex-specific. A downstream splice site for the transcript of the distal promoter is present in FRU mRNA in female, but not male heads, indicating that this specific splice site is used only in females. Analysis of the upstream 5' splice site reveals that the transcript class missing the repeats region is only detected in males. Thus, the upstream 5' splice site is used only in males and the downstream 5' splice site is used only in females. In chromosomally XX females mutant for tra or tra-2, the male form of FRU transcripts is produced, while the FRU splice pattern is unaffected in XY flies mutant for either tra or tra-2. These results demonstrate that sex-specific splicing of FRU transcripts is indeed controlled by tra and tra-2. A genetic test also reveals that FRU mRNA splicing is controlled by tra and tra-2. If FRU splicing is controlled by tra and tra-2, then XX flies double mutant for fru and tra (or tra-2) should exhibit the mutant fruitless Muscle of Lawrence and behavioral phenotypes. Double mutants have an abnormal MOL muscle like that of fruitless mutant males, and demonstrate abnormal behavior as well (Ryner, 1996).

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. 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 (SS). 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' SS 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 (Heinrichs, 1998).

To determine whether the tra/tra-2 repeat elements are essential for regulation of FRU splicing by Tra and Tra-2, a construct, fruM+FREmut, was tested in which the sequence of the conserved part of the tra/tra-2 repeat elements was changed from TCAATCAACA to GGCAGCTTAC. In construct fruM+FREmut, switching to female FRU splicing by Tra and Tra-2 is almost completely blocked, as indicated by the presence of significant amounts of male splicing product M in the presence of cotransfected tra and tra-2. In contrast, deletion of a 1-kb fragment between the repeat elements and the male-specific 5' SS, as in construct fruM+FB-M, does not affect regulation of FRU splicing by Tra and Tra-2. These findings show that the tra/tra-2 repeat elements are required for regulation of FRU splicing by Tra and Tra-2, suggesting that Tra and Tra-2 promote female-specific FRU splicing by acting through these elements (Heinrichs, 1998).

Is the tra/tra-2 repeat region in FRU sufficient to promote the activation of a 5' SS by tra/tra-2? Since the male-specific FRU 5'SS is normally unaffected by tra/tra-2, a 300-bp fragment of fru containing the tra/tra-2 repeats was inserted 4 nt upstream of the male-specific FRU 5' SS (construct fruM+REwt). Interestingly, spliced male product is detected upon cotransfection with tra/tra-2, suggesting activation of the male-specific Fru 5' SS by tra/tra-2 in this hybrid construct. Thus, the tra/tra-2 repeat elements are essential to promote the activation of a heterologous 5' SS by tra/tra-2. Lack of usage of the male-specific 5' SS in the constructs fruM+REwt and fruM+REmut in the absence of cotransfected tra/tra-2 was found to be due to the deletion of a stretch of sequence upstream of the male-specific 5' SS in these constructs. The insertion of the FRU repeat region in either orientation does not affect the usage of the male-specific FRU 5' SS in the absence of cotransfected tra/tra-2 (Heinrichs, 1998).

The Drosophila fruitless 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 fruitless 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 gene. Doublesex 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 fruitless. Mutational analysis has demonstrated that at least one 13 nucleotide repeat element within the fruRE is required and sufficient to activate the regulated female-specific splice site. As was established for doublesex, 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 show that SR proteins facilitate enhancer-dependent 5' splice site activation. It is concluded 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 (Lam, 2003).

fruitless splicing specifies male courtship behavior in Drosophila

All animals exhibit innate behaviors that are specified during their development. Drosophila melanogaster males (but not females) perform an elaborate and innate courtship ritual directed toward females (but not males). Male courtship requires products of the fruitless (fru) gene, which is spliced differently in males and females. Alleles of fru have been generated that are constitutively spliced in either the male or the female mode. Male splicing is essential for male courtship behavior and sexual orientation. More importantly, male splicing is also sufficient to generate male behavior in otherwise normal females. These females direct their courtship toward other females (or males engineered to produce female pheromones). The splicing of a single neuronal gene thus specifies essentially all aspects of a complex innate behavior (Demir, 2005).

Development endows an animal with the morphology and instinctive behaviors characteristic for its species, preparing it for survival and reproduction in the environment into which it is likely to be born. An animal's instinctive behaviors are just as stereotyped and just as characteristic for its species as its morphology, and so one might expect to find a similar logic underlying the genetic programs that specify morphology and behavior. Yet, whereas morphological development has now largely succumbed to the attack of classical forward genetics in a few model organisms, the same approach has made only modest inroads into the developmental origins of complex innate behaviors. Does this reflect a fundamental difference in the ways behavior and morphology are specified during development or just a lack of attention to the problem of behavioral development (Demir, 2005)?

One of the lessons from the genetic analysis of morphological development is that anatomical features are often specified by switch genes, the actions of which are both necessary and sufficient to direct the formation of a particular feature. A striking example of such a morphological switch gene is the eyeless gene of Drosophila, that is both necessary and sufficient for eye development. If analogous genetic principles guide the emergence of both morphology and behavior, then it should also be expected that at least some innate behaviors are specified by switch genes. The action of such a behavioral switch gene would be both necessary and sufficient to hardwire the potential for the behavior into the nervous system. Until now, such behavioral switch genes have been elusive. This study demonstrates that the fruitless gene of Drosophila is a switch gene for a complex innate behavior: the elaborate ritual of male courtship (Demir, 2005).

fru has long been known to be required for male courtship behavior. In this regard, however, fru is not particularly unusual. Many other genes have also been implicated in male courtship behavior, and in one way or another, a substantial fraction of the genome is likely to be required for a male to be capable of and inclined to court a female. fru only assumed its more prominent position when it was molecularly characterized, revealing that some of its transcripts are spliced differently in males and females. This led to the hypothesis that splicing of fru specifies male courtship behavior. Although widely discussed, this hypothesis has remained untested for almost a decade. This study now confirms the key predictions of this hypothesis by showing that male splicing is indeed necessary for male courtship behavior and is also sufficient to generate male behavior by an otherwise normal female (Demir, 2005).

Male courtship behavior performed by fruM and fruΔtra females is a remarkable mimic of courtship by wild-type or control fruC males. Some courtship steps, such as initiation, orientation, following, and wing extension, are indistinguishable in fruM (and fruΔtra) females and fruC males. Other steps are clearly abnormal. fruM females do not, for obvious reasons, copulate. But licking, which should be anatomically possible, is also significantly reduced. Qualitatively, this pattern of courtship resembles that of dsx males. This is perhaps not surprising, since fruM females resemble dsx males in that they lack male-specific Dsx isoforms (DsxM) and hence are anatomically female, yet they express the male-specific Fru isoforms (FruM) (Demir, 2005).

The distinct roles of fru and dsx in sexual development are clearly illustrated by the differences between animals that produce either only FruM or only DsxM. Animals that express DsxM but not FruM (either fruF males or dsxM females) resemble normal males but do not court. Conversely, animals that express FruM but not DsxM (either fruM females or dsx males) do court, even though they resemble normal females. Thus, FruM is both necessary and sufficient for male courtship, whereas DsxM is neither necessary nor sufficient. The role of DsxM in courtship may simply be to provide the gross male anatomy needed for its optimal execution. This anatomical contribution of DsxM includes the formation of male reproductive organs and external genitalia, the generation of the neurons that innervate these organs, and the formation of male-specific taste sensilla on the forelegs that may house pheromone-detecting neurons (Demir, 2005).

An open question is whether fru specifies male-like behavioral patterns more generally or is exclusively involved in male courtship behavior. This study focused on courtship behavior because this is the most dramatic, most robust, and best understood of the sexually dimorphic behaviors in Drosophila. But other behavioral patterns, such as aggression, are also sexually dimorphic, and it will be interesting to determine to what extent these behaviors depend on fru (Demir, 2005).

Evidence has been presented (Stockinger, 2005) that Fru positive neurons form a neural circuit that is largely dedicated to male courtship behavior. As the same circuit seems to be present in the female, it was reasoned that FruM most likely exerts its effect by modulating the function rather than the assembly of this circuit. Nevertheless, the critical period for FruM to do so is evidently during development, since adult males begin courting soon after eclosure, without any prior exposure to another fly. Moreover, experiments involving conditional expression of tra have suggested that male behavior is irreversibly programmed during the early- to midpupal-stages, coincident with the onset of FruM expression in increasing numbers of neurons in the male nervous system (Demir, 2005 and references therein).

By analogy to other members of the BTB-zinc finger family, FruM proteins are thought to be transcription factors and as such would specify sexual behavior by regulating the expression of one or more target genes. In the simplest scenario, fru may regulate one and the same target gene in all of the neurons in which it is expressed, in effect acting merely as a switch that sets another switch. Alternatively, fru might directly regulate a large number of target genes, with different targets in different neurons. Several observations favor this latter scenario. The set of FruM proteins includes isoforms with at least four different DNA binding domains, which are likely to homo- and hetero-dimerize through their common BTB domain. FruM may also interact with other BTB-domain-containing zinc finger transcription factors such as Lola, which itself has at least 20 different DNA binding domains. Thus, FruM has the potential to form a large set of distinct regulatory complexes, as might be expected if it is to regulate different genes in different neurons. That at least some of this potential is utilized is suggested by the fact that a fru mutant could not be rescued with cDNAs encoding just a single isoform (even when using fruGAL4 to drive expression in the correct neurons, and that mutations have already been isolated in two different DNA binding domains in an ongoing screen for revertants of the gain-of-function fruΔtra phenotype (Demir, 2005).

The fru target genes themselves are unknown, as are, for the most part, their effects. The few cellular functions so far ascribed to fru are the regulation of the number or size of synaptic terminals in specific glomeruli of the antennal lobe and at the MoL, as well as the production of serotonin in certain male-specific neurons of the abdominal ganglion. A fascinating question for the future is whether profound differences in sexual behavior arise as the sum of many subtle differences such as these, or are instead primarily due to a still unknown action of FruM in a few key 'decision' neurons (Demir, 2005).

Genome wide identification of Fruitless targets suggests a role in upregulating genes important for neural circuit formation

The fruitless gene (fru) encodes a set of transcription factors (Fru) that display sexually dimorphic gene expression in the brain of Drosophila. Behavioural studies have demonstrated that fru is essential for courtship behaviour in the male fly and is thought to act by directing the development of sex-specific neural circuitry that encodes this innate behavioural response. This study reports the identification of direct regulatory targets of the sexually dimorphic isoforms of the Fru protein using an in vitro model system. Genome wide binding sites were identified for each of the isoforms using Chromatin Immunoprecipitation coupled to deep sequencing (ChIP-Seq). Putative target genes were found to be involved in processes such as neurotransmission, ion-channel signalling and neuron development. All isoforms showed a significant bias towards genes located on the X-chromosome, which may reflect a specific role for Fru in regulating x-linked genes. Taken together with expression analysis carried out in Fru positive neurons specifically isolated from the male fly brain, it appears that the Fru protein acts as a transcriptional activator. Understanding the regulatory cascades induced by Fru will help to shed light on the molecular mechanisms that are important for specification of neural circuitry underlying complex behaviour (Vernes, 2014).

In order to identify the direct regulatory targets of Fru, each of the sexually dimorphic Fru protein encoding isoforms (FruA, FruB and FruC) were cloned to carry a tag that would allow their specific isolation during biochemical studies. This tag contains a biotin-ligase recognition peptide (BLRP) that undergoes biotinylation when co-expressed with the bacterial biotin ligase protein. Once biotinylated, these tagged proteins (and associated complexes) can be efficiently isolated using streptavidin coupled beads, due to the extremely high affinity streptavidin has for biotin. Chromatin immunoprecipitation of these tagged fru protein-DNA complexes coupled to high throughput sequencing allowed the identification of fruitless binding sites throughout the fly genome (Vernes, 2014).

Drosophila S2 cells were co-transfected with BirA and one of the BLRP tagged versions of Fru (FruA, FruB or FruC). Streptavidin coupled to magnetic beads was used to immunoprecipitate the tagged, biotinylated Fru protein isoforms. To confirm both the expression, the tagging of the protein and to validate the pull down technique, the immunoprecipitated samples were detected via western blotting using an antibody specific for the male specific epitope (FruM). Chromatin Immunoprecipitation coupled to high throughput sequencing (ChIP-Seq) was performed, in order to identify the direct regulatory targets of each of the Fru isoforms in this model system. Peaks of enrichment, indicative of Fru binding, were identified via the Model-based Analysis of ChIP-Seq (MACS) program. Using a p-value cutoff of p < 10−10 there were 791, 449 and 662 peak regions identified that were enriched over input DNA for FruA, FruB and FruC, respectively (Vernes, 2014).

Peaks identified via ChIP-Seq were screened to discover proximal target genes, defined as having transcriptional start sites that lay within 2 kb of the peak region. This generated putative target gene lists for the isoforms of 263, 217 and 291 genes, respectively. These lists were first assessed for the presence of any genes that were already thought to be regulated, directly or indirectly by Fru. Previously, dpr and a number of its family members had been shown to reduce their expression when fru is mutated, suggesting that these genes are normally upregulated by the Fru protein. Futhermore dpr mutations have been shown to have a phenotypic effect on wing extension initiation, an early aspect of courtship behaviour. The ChIP-Seq datasets contained 8 of the dpr family genes, many of which were represented in more than one isoform gene list - including dpr itself, suggesting that Fru directly regulates the transcription of dpr and some dpr family members (Vernes, 2014).

To understand the molecular functions of the genes regulated by fruitless, gene ontology analysis was carried out on each individual target gene list. Significant over representation of a number of related gene categories was observed. All three isoforms demonstrated enrichment for genes involved in 'ion gated channel activity' and many related ontology categories were enriched in one or more of the isoform gene lists, including 'voltage-gated cation channel activity' and 'extracellular-glutamate-gated ion channel activity'. These categories included putative Fru target genes such as an NMDA receptor (Nmdar2), multiple nicotinic Acetylcholine Receptor subunits (nAcRalpha-96Aa & nAcRalpha-7E) and an ionotropic glutamate receptor (GluRIB). FruA and FruC lists were enriched for genes reported to have 'receptor activity' and the FruA list was further enriched for 'transmembrane signalling receptor activity' including genes such as sevenless (sev) and white (w). The FruC gene list was also enriched for 'neurotransmitter receptor activity' due to the inclusion of genes such as Dopamine 2-like receptor (Dop2R) and sex peptide receptor (SPR). All three gene lists also showed a highly statistically significant enrichment for genes that carried an Immunoglobulin-like domain and Immunoglobulin-like fold, consistent with findings from previously published data that recently reported Fru dependent gene expression changes in the fly brain and saw similar protein domain enrichment (Vernes, 2014).

The highly significant ontology categories that were identified across these datasets suggests that Fru mediated transcriptional regulation is important for cellular communication mediated by ion channels. The appropriate expression of combinations of ion channels in neuronal subtypes is essential for the correct formation of and signalling through neuronal circuits. For example, NMDA receptors have been shown to be important for synapse refinement, an essential process required during circuit development to produce the appropriate connectivity. In total, four acetylcholine receptor subunits were identified across the datasets (nAcRalpha-7E, nAcRalpha-30D, nAcRalpha-80B & nAcRalpha-96Aa). Signalling mediated via acetylcholine is central to insect nervous system function and nicotinic acetylcholine receptors have been implicated in instinctive behaviours such as the escape reflex in Drosophila as well as the integration of information in the visual system. Thus the control of expression of specific ion channels such as Nmdar2, GluRIB and nAc receptor subunits by fruitless may represent an important mechanism by which sex-specific circuitry develops downstream of Fru (Vernes, 2014).

Despite the differences in the DNA binding domains of the three isoforms, a high degree of overlap was observed between the three gene lists. Many genes were identified as putative targets for more than one isoform and 60 genes were shared across all three datasets, which equates to more than 20% of each individual gene list being common to all isoforms tested. This common list contained a number of interesting genes including genes implicated in courtship behaviour such as the Sex peptide receptor (SPR) and Shaker (Sh), as well as genes implicated in synaptic transmission (Snap-25), and axon outgrowth (Dscam3). The common gene list showed significant enrichment of a number of the same gene ontology categories as the individual isoforms including 'ion gated channel activity' but also 'passive transmembrane transporter activity'. The target genes in the common list were also significantly enriched for protein domains including 'immunoglobulin-like domain' and 'p53/RUNT-type transcription factor, DNA-binding domain'. Thus, despite very different DNA binding domains, a core set of genes involved in common pathways seem to be targeted by all three Fru isoforms (Vernes, 2014).

During review of this manuscript, an in vivo study identifying targets of FruM isoforms (FruA, FruB, FruC) at different timepoints (larvae, pupae and adult) in neurons was published by Neville (2014). To estimate the biological relevance of the dataset described in this study, the S2 Fru-ChIP targets were compared with the neuronal Fru targets (Neville, 2014). A high degree of overlap was observed, despite the differences in model systems used. Since S2 cells do not represent any particular developmental timepoint, the complete list of targets for each isoform (larvae, pupae and adult) identified by Neville (2014). were overlapped with the S2 Fru-ChIP targets for the corresponding isoform. 29% percent of the S2 FruMA-ChIP targets were represented in the in vivo dataset, while 45% and 49% of the S2 ChIP targets overlapped for FruMB and FruMC, respectively. Interestingly the overlapping genes included Dpr family members (dpr and dpr6, 8, 10, 11, 13 and 16), nicotinic Acetylcholine Receptor subunits (nAcRalpha-7E, -30D, 80B and -96Aa), sevenless (sev), white (w), sex peptide receptor (SPR), shaker (sh) and Dscam3. This high degree of overlap in targets independently identified using an in vivo system, supports the biological relevance of the genes identified in this study (Vernes, 2014).

Of particular interest, when the Fru binding sites were assessed for genomic distribution, a highly significant enrichment of binding on the X chromosome was observed. ChIP-Seq is an unbiased screen for transcription factor binding, and although accessibility of the epitope/tag or local chromatin structure may affect the ability to pull down protein-DNA complexes, it is not expected that this would result in a chromosome specific bias. Indeed ChIP-Seq studies using other transcription factors have not observed this sort of X-chromosome specific bias. Rather, this over-representation of target sites may represent a specific role for X-linked genes in fruitless directed gene networks. Indeed this finding is consistent with results from another paper that was published while this manuscript was in preparation (Dalton, 2013). Dalton showed that fru overexpression resulted in changes in expression for hundreds of genes in neurons of the male fly. A significant over-representation of genes encoded on the X chromosome was observed for those genes that increased due to Fru overexpression, but not for down-regulated genes. Male-specific fruitless isoforms have different regulatory roles conferred by distinct zinc finger DNA binding domains (Vernes, 2014).

To demonstrate that the binding sites identified in S2 cells could translate to real gene expression changes in the fly brain, a method was employed to specifically isolate RNA from Fru positive neurons. This system utilised the GAL4/UAS system to drive expression of a membrane bound GFP signal (CD8-GFP) in subsets of neurons. Here, the GFP signal was expressed in all Fru positive neurons by coupling the Fru-GAL4 driver line with the UAS-CD8-GFP line, however it would be possible to drive expression in subsets of Fru positive neurons by using combinations of driver lines in an intersectional approach. The expression of a cell surface CD8-GFP protein, allowed dissociated neurons to be isolated via antibody coupled magnetic cell sorting. Techniques such as FACS (Fluorescence activated cell sorting) have also been used to isolate tagged populations of cells for analysis, however FACS is a more harsh technique that can cause stress and/or damage to the cells which could affect the results of molecular assays such as transcript analysis. Following magnetic cell sorting, RNA was extracted from the two populations of neurons harvested from the fly; Fru positive neurons (that express CD8-GFP) and all other neurons in the brain. If Fru acts to upregulate a target gene, it would be expected to have enriched levels of transcript in Fru positive neuron sample compared to the baseline (the sample containing all other neurons in the brain). First, the enrichment of fru and GFP transcripts in the cell sorted samples compared to baseline was confirmed via qPCR. Next a small number of target genes were chosen for validation. Two genes Dop2R (Dopamine 2 receptor) and Dscam3 (Down syndrome cell adhesion molecule 3) showed significant enrichment in the Fru positive neurons compared to baseline, suggesting that Fru binding results in their upregulation. Two further genes were also tested (Shaker and Nmdar2) however the transcripts levels were too low to be reliably detected and were thus excluded. In addition to validating targets identified in the S2 Fru-ChIP experiments, these results demonstrate the utility of this method, particularly when coupled to an intersectional genetic approach, to specifically isolate intact populations of neurons for biochemical study, eg., RNA-Seq or ChIP-Seq. In this way, the regulatory cascades that are necessary to specify different aspects of the sexually dimorphic circuitry underlying courtship behaviour could be defined (Vernes, 2014).

A recent study by Dalton (2013) interrogated mRNA changes in the fly brain resulting from Fru isoform overexpression via RNA-Seq. The gene lists identified therein are expected to include genes that are downstream of Fru, but that may represent either direct transcriptional targets or indirectly regulated genes. By contrast, the work detailed int this study reports exclusively those genes putatively targeted by Fru via direct interaction with DNA. By comparing these two datasets, it can be determined which of the RNA-Seq genes are directly regulated by Fru isoforms, and in turn, further validate the ChIP-Seq targets in an in vivo system. A very high degree of overlap was observed between these independent datasets, much more than would be expected based on chance alone. Between ~23%-27% of the direct ChIP-Seq targets were shown to change their expression in Fru P1-expressing neurons of the male fly brain in response to Fru isoform overexpression. Of particular note, the vast majority of S2 Fru-ChIP targets that are also represented in the RNA-Seq data (more than 90%) were upregulated in the fly brain. Only a handful of direct targets in each list were downregulated. This suggests that when Fru binds to a gene promoter it acts as a transcriptional activator, inducing expression of the target gene unlike some other BTB-ZnF transcription factors such as ttk that mediate gene repression. This is further supported by finding that in the in vivo RNA-Seq experiments both Fru binding motif enrichment and X-chromosome enrichment were only observed for those genes that were upregulated in the fly brain (Dalton, 2013). The finding that putative Fru target genes identified via ChIP-Seq were upregulated in the fly brain both in this study as well as in independent studies of gene expression together with the enrichment of Fru binding motifs in upregulated target genes supports the hypothesis that direct Fru binding induces the expression of target genes in the fly brain (Vernes, 2014).

The ChIP target genes that overlap with the Dalton (2013) RNA-Seq experiments represent a subset of high confidence target genes, in that these genes have a ChIP-Seq signal indicative of Fru protein binding and also change their expression in neurons in response to the presence of one or more of the Fru isoforms. Indeed the two target genes (Dscam3 and Dop2R) that showed enrichment in Fru positive neurons, also showed upregulation by all three isoforms tested in the Dalton et al study. Thus, to better understand the pathways regulated by Fru, the genes that overlapped between the ChIP-Seq and RNA-Seq experiments were explored via gene ontology and protein domain enrichment analaysis. The FruA overlap list was significantly enriched for categories relating to neuron development [GO:0048666] and differentiation [GO:0030182], and more specifically axonogenesis [GO:0007409] and axon guidance [GO:0007411]. Significant over-representation was also observed for genes involved in cell projection organisation [GO:0030030] (FruC overlap), as well as cell communication [GO:0007154] (FruC overlap), synapse assembly [GO:0007416] (FruB overlap) and synaptic target attraction [GO:0016200] (FruB and FruC overlap) (Vernes, 2014).

The FruB overlap list was significantly enriched for genes involved in neuromuscular junction development [GO:0007528]. This enrichment was observed for both the FruB overlap list, as well as the FruB ChIP-Seq list, but not for other isoform lists. Genes shared between the two datasets include Nlg1 (Neuroligin 1), futch and cac (cacophony), which have previously been implicated in synapse development at neuromuscular junctions and were also identified as Fru targets in vivo. In addition to courtship behaviour, the male specific functions of fru include directing the formation of the Muscle of Lawrence. The MOL is a large abdominal muscle found exclusively in male flies and its development is dependent upon direct innervation by masculinised, fru positive, glutamatergic motor neurons. Thus, the putative target genes identified in this study that are involved in neuromuscular junction development, such as Nlg1, futch and cac may contribute to the molecular mechanism by which fru is able to affect MOL innervation (Vernes, 2014).

The overlap between the in vitro lists identified in this study and the recent in vivo studies is particularly striking when considering the vastly different model systems used. In this study an in vitro model (S2 cells) was used to investigate the binding of the Fru protein throughout the genome. By contrast the Fru-neuron target identification was performed in CNS tissue and Fru RNA-Seq transcript analysis was prepared from fly heads and thus both reflect the changes occurring in Fru positive neurons in vivo. S2 cells are derived from late stage D.melanogaster embryos and grow as a monolayer of cells with epithelial-like morphology and as such do not reflect a neuronal identity. An advantage of using a cell line such as this is that protein constructs can be tagged and overexpressed to allow high occupancy rates throughout the genome (even at low affinity sites) and efficient isolation of protein-DNA complexes, which reduces background. Hence despite these cells not being neuronal in origin, it might be possible to identify some biologically relevant target sites using this methodology. For each isoform, between 30–50% of the targets identified in S2 cells were identified as Fru targets in the CNS and around one quarter showed FruM dependent neuronal gene expression changes suggesting that a subset of the targets identified in this study might be important for the sexual dimorphism induced in the developing fly nervous system and thus warrant further, in vivo investigation. Some of the genes identified in this study that did not show overlap with the in vivo ChIP or expression data may represent non-neuronal or developmental, non-sexually dimorphic targets, or reflect the technical limitations of the model system. Although some may be true neuronal targets of Fru that could not be detected by the in vivo assays. Given that Fru is expressed in a range of different neurons, such as mAL, median bundle and descending neurons it is likely that the genes regulated by Fru in these neuronal subsets will differ. Thus, targets that are regulated only in small subsets of Fru positive neurons are unlikely to show expression changes dramatic enough to be detected as significant when considering whole head RNA samples, as was done by Dalton (2013). In order to discover these changes, it may be necessary to directly assay the transcripts from only these specific subsets of neurons, using a technique such as the cell sorting method described herein (Vernes, 2014).

Dalton (2013) also assayed gene expression changes induced when FruM isoforms were introduced into the female fly. In contrast to the male fly, here they observed more genes being repressed, rather than upregulated, and only a small proportion of the genes that were regulated in the male were replicated in the female brain (14% overlap between male/female), suggesting that the regulatory activity of FruM is in part determined by its environment. To determine if the S2 Fru-ChIP targets identified in the current study reflected the genes that were regulated in the male, female or overlapping male/female dataset, the respective datasets were compared. In stark contrast to the high degree of overlap observed with the male RNA-Seq gene lists, very little overlap was observed with the female derived RNA-Seq data. Only 1.5%, 3.2% and 3.8% of genes were shared for the FruA, FruB and FruC datasets, respectively. This was unexpected given that the ChIP-Seq data was generated in a cell model system rather than a sexually dimorphic brain. Chromosome analysis of S2 cells have shown that they are male in origin and have an X/A ration of 0.5, as is found in male Drosophila, demonstrating similar dosage compensation as is seen for X-linked genes in the male fly This might, in part, explain the bias towards target genes that are specifically regulated in the male brain. In any case, this study has demonstrated that the in vitro S2 model system represents a powerful starting point providing ease of manipulation for investigating the activity of transcription factors such as Fru, that are involved in complex behavioural programs (Vernes, 2014).

Taken together, the in vitro and in vivo studies suggest that fruitless is able to initiate a cascade of expression changes of genes spread throughout the genome. As shown by Dalton (2003), the majority of these changes result in increased gene expression, however a small subset of genes are down-regulated. More than 90% of the genes that were shown to be both direct targets in this study and downstream of Fru in Dalton, were upregulated, suggesting that the Fruitless protein normally binds to promoter regions in order to upregulate target genes. This is further supported by the finding that there was a significant enrichment of genes encoded on the X-chromosome for all isoforms tested and the corresponding X-chromosome enrichment observed for upregulated genes in the RNA-Seq study. Thus it can be hypothesised that Fru may play a specific role in directly upregulating genes located on the X-chromosome. More work is needed to determine in which subsets of neurons and for which particular functions of Fru these targets play a role. By combining transcript profiling for specific neuronal subpopulations with neural circuit tracing and behavioural studies, true insight can be gained into the molecular mechanisms underlying Fru directed courtship behaviour (Vernes, 2014).

This study has identified a set of direct regulatory targets for each of the sexually dimorphic isoforms of the fruitless gene (FruA, FruB & FruC). It has been hypothesised that Fru was able to directly control gene expression by binding to target DNA and the work in this study directly demonstrates this capacity, suggesting that Fru upregulates a range of genes implicated in neural circuit formation. Understanding the regulatory cascades induced by Fru will help to shed light on the molecular mechanisms that are important for specification of neural circuitry underlying complex behaviours (Vernes, 2014).


Fruitless specifies sexually dimorphic neural circuitry in the Drosophila brain

Fruitless has been postulated to be a neural sex determination factor that directs development of the central nervous system (CNS), thereby producing male-typical courtship behaviour and inducing male-specific muscle. Male-specific Fru protein is expressed in small groups of neurons scattered throughout the CNS of male, but not female. Collectively, these observations suggest that Fru 'masculinizes' certain neurons, thereby establishing neural substrates for male-typical behaviour. However, specific differences between neurons resulting from the presence or absence of Fru are unknown. Previous studies have suggested that Fru might result in sexual differences in the CNS at the functional level, because no overt sexual dimorphism in CNS structure was discernible. This study identifies a subset of fru-expressing interneurons in the brain that show marked sexual dimorphism in their number and projection pattern. This study also demonstrates that Fru supports the development of neurons with male-specific dendritic fields, that are programmed to die during female development as a result of the absence of Fru. Thus, Fru expression can produce a male-specific neural circuit, probably used during heterosexual courtship, by preventing cell death in identifiable neurons (Kimura, 2005).

Assuming that Fru is involved in development of neural circuits crucial for the generation of male-typical behaviour, it is plausible that Fru-expressing neurons show some degree of sexual dimorphism structurally and/or functionally. Unambiguous identification of Fru-expressing neurons is necessary for evaluating this possibility. In a search for enhancer trap lines that express Gal4 in Fru-expressing cells, a conspicuous sex difference was found in the localization of the Gal4 reporter mouse (m)CD8-GFP (green fluorescent protein) in the NP21 strain. The NP21 strain has a P-element insertion in the second intron of the fru gene (Kimura, 2005).

The following histological experiments were carried out primarily with flies heterozygous for the described P-element insertion. Sexually dimorphic reporter expression in NP21 flies was found in two discrete locations: the optic lobe and the region dorsal to the antennal lobe. In the optic lobe, a subset of neurons in the distal medulla expressed mCD8-GFP only in males. In the region dorsal to the antennal lobe, approximately 30 neurons expressed mCD8-GFP in males, compared with approximately 5 neurons in females. The somata of these neurons form a cluster called 'neurons medially located, just above antennal lobe', or mAL (Kimura, 2005).

To identify projection patterns of mAL neurons without interference from surrounding neurons, the MARCM method, which allows mCD8-GFP to be expressed in a single precursor cell and/or its progeny, was used. Somatic chromosomal recombination is induced during development by the stochastic action of a heat-shock-inducible flippase. By changing the timing of the heat-shock, chromosomal recombination can be induced at different stages of development, resulting in a different number of mCD8-GFP-expressing cells (Kimura, 2005).

Early heat-shock resulted in mCD8-GFP expression in about 30 cells in male flies and 5 cells in female flies, indicating the clonal nature of the mAL cluster. In males, the mAL neurons extended neurite projections bilaterally. In females, the neurons had only contralateral projections. In both sexes, the contralateral neurite bifurcated after crossing the midline, with one neurite extending dorsally and terminating in the superior lateral protocerebrum, and the other neurite extending ventrally, terminating in the subesophageal ganglion. The male-specific ipsilateral neurite also terminated in the s ganglion. When single-cell labelling was carried out, the 5 cells in females were devoid of this ipsilateral projection. A further difference between the sexes was observed in the branching pattern of these neurons in the s ganglion: the mAL neurons had forked arborization only in females. The forked arborization typical of female mAL neurons was never observed in males. However, the male mAL had two classes of neurons: one with bilateral projections and the other with only contralateral projections. It was possible to differentiate 8 types among male neurons on the basis of their shape (Kimura, 2005).

The terminal structures in the superior lateral protocerebrum were decorated with many varicosities that were nearly absent from the terminal structures of branches in the s ganglion. This arrangement suggests that the superior lateral protocerebrum is the output site of these interneurons, and the s ganglion is the input site. This hypothesis is supported by the observation that a presynaptic marker (synaptotagmin-HA) was transported to the terminals in the superior lateral protocerebrum but not to those in the s ganglion (Kimura, 2005).

How the timing of somatic recombination by heat-shock application affected the number of labelled neurons was investigated. When a heat-shock was applied to the embryo, the maximal number of mAL neurons was labelled (that is, about 30 in males and 5 in females, indicating that the neuroblast that produced the sexually dimorphic neurons was proliferating in the embryonic stage in both sexes. In contrast, heat-shock activation at the late third instar larval to early pupal stage (that is, 4-5 days after egg collection) failed to label any mAL cells in females, but did result in labelling in males. The number of labelled cells in these cases was very low (typically one cell), but indicates that the neuroblast continued to proliferate in males at this stage. These observations are compatible with any of three alternative explanations for the production of fewer neurons in females compared with males: (1) the female neuroblast stops dividing and becomes quiescent during the late third instar larval to early pupal stage; (2) the female neuroblast dies earlier than the male neuroblast; (3) the female neuroblast continues to proliferate as the male counterpart does, but the daughter cells are eliminated by programmed cell death only in females. To differentiate the second and third possibilities from the first, the effect of mutations that block cell death was examined (Kimura, 2005).

head involution defective (hid, also known as Wrinkled or W), grim and reaper (rpr) are the predominant cell-death genes in Drosophila. They are aligned in tandem in the genome, and therefore can be removed by a single deficiency, Df(3L)H99. In viable rpr mutants of Df(3L)H99/Df(3L)XR38, the mAL cluster had, on average, 10 mAL neurons in females and 30 in males. This result is consistent with the idea that, in females, rpr-mediated cell death eliminates some cells that otherwise contribute to the mAL cluster. In contrast, viable hidA206/Df(3L)H99 mutants had approximately 5 mAL neurons in females and 30 in males. To observe the effect of removing hid, grim and rpr simultaneously, cells homozygous for Df(3L)H99 were generated in a heterozygous background by the MARCM method [a homozygous Df(3L)H99 deficiency causes lethality]. Now the mutant females had as many as 29 mAL neurons. Again, the number of mCD8-GFP-positive cells in mutant males was about 30. These results show that programmed cell death occurs specifically in female flies, reducing the number of mAL neurons (Kimura, 2005).

It was predicted that the cell death programme was probably operating in the daughter cells rather than in the neuroblast itself. This is because Df(3L)H99 homozygous neurons could be produced and labelled by heat-shock-induced activation of recombination at the late larval to early pupal stage even in females, which normally reveal no sign of labelling at this stage. Thus, in wild-type females, the cell death mechanism eliminates late-born neural progeny, whereas in neural progeny homozygous for Df(3L)H99, these cells survive and differentiate into neurons (Kimura, 2005).

When cell death was inhibited at the embryonic stage by heat-shock-induced recombination of the Df(3L)H99-harbouring chromosome, the number of mAL neurons was increased in adult females. Such neurons had the contralateral forked arborization typical of females. Notably, the ipsilateral projections never seen in normal females were formed in those female mAL neurons protected by the Df(3L)H99 deletion. A single neuron with bilateral projections typical of males was stained in a female mAL cluster homozygous for Df(3L)H99. The contralateral projection of the neuron showed neither the typical female nor the typical male arborization pattern. This indicates that sex-specific cell death is not the sole mechanism for establishing sexually dimorphic neuronal projections, since the pattern of H99-protected neurons in the s ganglion was not necessarily male-like (Kimura, 2005).

The NP21 strain has a P-element insertion in the second intron of the fru gene. Across the entire male brain, >80% of Gal4-expressing cells expressed Fru. All of the mAL neurons expressing mCD8-GFP in males were Fru-positive. NP21 homozygous males and NP21/frusat transheterozygous males showed similar abnormalities: loss of the male-specific muscle of Lawrence, no expression of Fru and no detectable expression of intact male-specific fru transcripts by polymerase chain reaction with reverse transcription (RT-PCR) analysis. Markedly reduced courtship behaviour towards males as well as females is also observed. These results show that NP21 is a loss-of-function allele of fru (Kimura, 2005).

Whether mutations in fru could affect the sexually dimorphic development of the mAL cluster was examined. NP21/frusat males had only about 5 mAL neurons, a number comparable with that in females. The males of two other fru mutants also showed a significant reduction in the number of mAL neurons. The similar reduction in the number of mAL cells in fru mutant males was confirmed by cell labelling using the MARCM method. Single-progeny labelling was performed in NP21/NP21 flies, and about 5 mAL neurons were counted in male flies (Kimura, 2005).

In NP21 homozygous males, no neurons with ipsilateral projection were observed. Instead, all mAL neurons in NP21 homozygous males had contralateral, bifurcating projections reminiscent of their female counterparts. For males of other heteroallelic fru mutants, most mAL neurons had 'female-like' unilateral projections, but there were also neurons that had the extended ipsilateral projection typical of males. It should be emphasized that the forked projections typical of females were formed in male fru mutants. To determine whether the Fru protein is required cell-autonomously in mAL neurons for male-typical development, NP21/NP21 homozygous cells were generated in male flies heterozygous for NP21/ + . mAL neurons with the NP21/NP21 genotype had forked projections typical of females, indicating that cell-autonomous fru+ function is necessary for male-type development of mAL neurons (Kimura, 2005).

According to the hypothesis that Fru suppresses cell death specifically in males, Fru expression in female mAL neurons should inhibit the death of mAL neurons in females. The experiments show that this is indeed the case: tra1 homozygous females express Fru in mAL neurons with male-like bilateral projections, which also have male-like arborizations in the s ganglion. Thus, the tra1 mutation not only blocks cell death but also masculinizes the neuron branching pattern, presumably by inducing Fru expression in female mAL neurons. Such tra1 mutant females vigorously courted partner females (Kimura, 2005).

This study has demonstrated that Fru expression is essential for the establishment of a 'male-typical' neural network. The observations support the model that those neurons programmed to extend male-typical projections are eliminated in females by cell death. Fru inhibits cell death in male neurons at the pupal stage, allowing neurons to develop male-typical projections. A similar mechanism would operate in the formation of sexually dimorphic neurons in the optic lobe. Fru could thus be considered to function as a male-female switch in the CNS neural circuit (Kimura, 2005).

The male-typical projections of mAL neurons in the s ganglion have terminals that probably function as input sites. The s ganglion is known to include the association centre for gustation, and the central projections of both tarsal and labellar gustatory neurons terminate in the s ganglion. Contact (gustatory) pheromones are also known to be involved in partner choice in Drosophila mating. Thus, an intriguing possibility is that the male-typical projections of mAL neurons in males act as the input sites of pheromonal information conveyed by gustatory neurons. The enhanced homosexual preference observed in fru mutant males might be due to a failure in integrating the pheromonal information crucial for partner recognition in mating behaviour, as a consequence of the loss of ipsilateral projections from the mAL neurons. The study of Fru-expressing neurons and their circuits may therefore help to identify fundamental principles about the construction of sex-specific structures and functions in the CNS (Kimura, 2005).

Drosophila switch gene Sex-lethal can bypass its switch-gene target transformer to regulate aspects of female behavior

The switch gene Sex-lethal (Sxl) was thought to elicit all aspects of Drosophila female somatic differentiation other than size dimorphism by controlling only the switch gene transformer (tra). This study shows instead that Sxl controls an aspect of female sexual behavior by acting on a target other than or in addition to tra. The existence of this unknown Sxl target was inferred from the observation that a constitutively feminizing tra transgene that restores fertility to tra- females failed to restore fertility to Sxl-mutant females that were adult viable but functionally tra-. The sterility of these mutant females was caused by an ovulation failure. Because tra expression is not sufficient to render these Sxl-mutant females fertile, this pathway is referred to as the tra-insufficient feminization (TIF) branch of the sex-determination regulatory pathway. Using a transgene that conditionally expresses two Sxl feminizing isoforms, it was found that the TIF branch is required developmentally for neurons that also sex-specifically express fruitless, a tra gene target controlling sexual behavior. Thus, in a subset of fruitless neurons, targets of the TIF and tra pathways appear to collaborate to control ovulation. In most insects, Sxl has no sex-specific functions, and tra, rather than Sxl, is both the target of the primary sex signal and the gene that maintains the female developmental commitment via positive autoregulation. The TIF pathway may represent an ancestral female-specific function acquired by Sxl in an early evolutionary step toward its becoming the regulator of tra in Drosophila (Evans, 2013).

Developmental regulatory pathways are rarely as simple as they first appear, but as the twist to the Drosophila sex-determination pathway this study reports here suggests, complications can provide clues to evolution. It was shown that Sxl, the rapidly evolved target of the Drosophila primary sex-determination signal, no longer can be regarded as transmitting all its feminizing orders other than size dimorphism to the soma exclusively through its well-known switch-gene target tra. Instead, one must distinguish between a major pathway branch, TSF, in which tra is sufficient to dictate feminization, and a minor branch, TIF, in which it is not (Evans, 2013).

Evidence for the TIF branch derives from female-viable but masculinizing combinations of partial-loss-of-function Sxl alleles that fail to induce either TSF or TIF in diplo-X individuals, so that when TSF-branch activity is restored by constitutively feminizing transgene U2af-traF or, even more definitively, by a constitutively feminizing mutant endogenous tra allele, mutant females remain TIF defective and hence sterile. Although TIF-mutant sterility superficially resembles sterility in TSF-mutant transgenics, in that both phenotypes include a failure to lay eggs, the TIF-mutant block to egg laying occurs at ovulation, whereas that in TSF-defective transgenics occurs later at oviposition. The possibility that the kind of branch in the TIF pathway that is reported in this study might exist was suggested first in a previous paper reporting the behavior of some U2af-traF-feminized gynandromorphs (coarse-grained XX//XO mosaics) in which the failure of Sxl to activate what is now known to be the TIF pathway was a consequence of the absence of a female primary sex-determination signal in TRA-F-feminized Sxl+ XO cells (Evans, 2007) rather than a consequence of Sxl mutations in TRA-F–feminized XX cells. Because 38% of the feminized egg-producing gynandromorphs failed to lay their eggs, it is concluded that there must be some functionally Sxl- XO somatic cells that cannot substitute for the XX somatic cells required for egg laying, even when feminized by TRA-F. Although gynandromorphs are not nearly as convenient as Sxl-mutant females for studying TIF, they do strengthen the argument that TIF-defective sterility is not caused either by a upset in dosage compensation or by some idiosyncrasy of U2af-traF in Sxl-mutant females (Evans, 2013).

Strong evidence is necessary to legitimize the TIF claim because of the surprising finding that SXL-F functioning in the TIF pathway takes place in a subset of neurons that sex-specifically express fru mRNAs. Because fru sex-specific splicing is controlled entirely by TRA-F, the simplest model would suggest that any deficiency in the sex-specific functioning of these neurons reflects a TSF defect. Of course, just because fru is sex-specifically regulated in these neurons does not require that fru be solely or even partially responsible for their feminization in every case (Evans, 2013).

At this point the 'I' in TIF necessarily stands for 'insufficient' rather than 'independent.' Because conditions under which the TIF phenotype was studied were all ones in which TRA-F activity for the TSF pathway was provided at a level sufficient to rescue the sterility of tra− females, no evidence for or against independence could be generated. If, as the fru neuron results might suggest, tra works with one or more unknown Sxl targets to achieve full feminization in some neurons, the name ultimately might have to be changed to something like 'tra-partnered feminization.' Discovering the identity of the Sxl TIF-gene targets and the specific neurons in which they are required would provide the tools necessary to resolve this question about the relationship between TSF and TIF. The recent availability of an enormous panel of well-characterized neuronal GAL4 drivers should be a great help in this connection, particularly in view of the finding that GAL4-driven SXL-F expression can rescue the TIF-mutant phenotype in females while causing little damage to males. The gene female-specific-independent-of-transformer seemed to be a promising candidate TIF-pathway target until it was shown that, contrary to a previous study, it is firmly in the TSF pathway (and hence is in need of renaming) (Evans, 2013).

The ovulation block should be particularly amenable to future genetic and developmental analyses designed to identify targets of the TIF because it is particularly suited to positive genetic selection in a suppression screen. Arguing for the potential of such a suppression screen is the fact mentioned above that fertility could be restored to TIF-defective females by a GAL4 driver/ SXL-F target combination that had relatively little adverse effect on male viability or fertility. Such sex specificity suggests that the set of neurons responsible for the TIF ovulation defect may not be very large and that disruption of their normal controls is unlikely to disrupt non–sex-specific aspects of development (Evans, 2013).

This report introduces several genetic tools, among which the GAL4 target UAS-Sxlalt5-C8 is perhaps the most broadly useful. That this transgene, which conditionally generates both exon-5 alternative SXL-F isoforms, provides relatively strong Sxl+ function while having no adverse effect on females indicates that the adverse effect on females caused by the Sxl GAL4 target previously reported, a transgene that encodes only a single exon-5 isoform, may not reflect a normal activity of SXL-F protein. Another useful tool is Dp(1;1)SxlΔPm, which can expand the utility of various partial-loss-of-function Sxl alleles. This tool is a chromosomal duplication of Sxl truncated at its 5′ end so that it lacks the gene's maintenance promoter but retains an intact establishment promoter and all the activities that transiently active promoter elicits. The response of this truncated Sxl allele to the female X-chromosome dose signal, a response that ends during the early blastoderm stage, can facilitate engagement of the Sxl positive-feedback loop for various Sxl-mutant alleles without otherwise influencing their Sxl-mutant phenotype. For example, Dp(1;1)SxlΔPm is particularly useful in combination with the intriguing double mutant Sxlf18,f32 because together they can generate thoroughly masculinized Sxl-mutant adult females (pseudomales) with far higher viability and longevity than any previously described masculinizing Sxl genotype. Last, two dominant temperature-sensitive lethal balancers that were introduced in this study should be generally useful, because they allow crosses to be designed so that daughters with one combination of a maternal and paternal X chromosome of choice are the only progeny to survive (Evans, 2013).

Sex determination for flies in the family Drosophilidae is unlike that for most other higher insects in many fundamental respects, including having Sxl rather than tra as the target of their primary sex-determination signal and having Sxl rather than tra as the gene whose positive-feedback loop on its own pre-mRNA splicing maintains the female developmental pathway commitment. Although the TIF branch could be a recent addition to the Drosophila sex-determination pathway made well after Sxl had taken over tra's role as the master feminizing gene, a more intriguing possibility is that TIF instead may reflect an ancestral function that Sxl acquired in the earliest step on its evolutionary path toward usurping tra's role as master sex switch. Because both TIF and TSF function in neurons that sex-specifically express fru, perhaps the first female- specific function that Sxl acquired was to modify the developmental functioning of fru in some neurons. Initially this function may have been achieved without the need for a sex-specific Sxl product, with sex-specific products coming only later as fine-tuning of that particular function under the control of tra. The switch from tra as a regulator of Sxl to Sxl as a regulator of tra (a switch that could have been facilitated by the development of redundancy in the positive-feedback circuits for the two genes) would make any female-specific gene target of Sxl that existed before the switch be independent of tra regulation today if its control by Sxl persisted. Of course there are many important questions about the remarkable path taken by Sxl functional evolution and the forces that drove those changes for which an understanding of the TIF pathway might not be relevant. How did Sxl come to respond to an X-chromosome dose signal? How did it come to control X-chromosome dosage compensation? Why is Sxl's control of germ-line sex determination so different from its control of sex determination in the soma? On the other hand, because next to nothing is known about any of these questions, it is hard to predict where clues might lead regarding an early female-specific Sxl function that the TIF pathway might help reveal. Regardless of whether the TIF pathway is ancestral or recent, further analysis leading to the discovery of the SXL-F targets in this regulatory branch undoubtedly will advance understanding how genes control behavior and how SXL-F proteins control RNA functioning (Evans, 2013).

The Drosophila pheromone cVA activates a sexually dimorphic neural circuit

Courtship is an innate sexually dimorphic behaviour that can be observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate this behaviour are developmentally programmed. In Drosophila, courtship involves a complex yet stereotyped array of dimorphic behaviours that are regulated by FruM, a male-specific isoform of the fruitless gene. FruM is expressed in about 2,000 neurons in the fly brain, including three subpopulations of olfactory sensory neurons and projection neurons (PNs). One set of Fru+ olfactory neurons expresses the odorant receptor Or67d and responds to the male-specific pheromone cis-vaccenyl acetate (cVA). These neurons converge on the DA1 glomerulus in the antennal lobe. In males, activation of Or67d+ neurons by cVA inhibits courtship of other males, whereas in females their activation promotes receptivity to other males. These observations pose the question of how a single pheromone acting through the same set of sensory neurons can elicit different behaviours in male and female flies. Anatomical or functional dimorphisms in this neural circuit might be responsible for the dimorphic behaviour. This study reports a neural tracing procedure that employs two-photon laser scanning microscopy to activate the photoactivatable green fluorescent protein. Using this technique it was found that the projections from the DA1 glomerulus to the protocerebrum are sexually dimorphic. A male-specific axonal arbor was observed in the lateral horn whose elaboration requires the expression of the transcription factor FruM in DA1 projection neurons and other Fru+ cells. The observation that cVA activates a sexually dimorphic circuit in the protocerebrum suggests a mechanism by which a single pheromone can elicit different behaviours in males and in females (Datta, 2008).

In initial experiments, photoactivatable green fluorescent protein (PA-GFP) was expressed in flies in which the GAL4 enhancer-trap GH146 drives the expression of UAS-PA-GFP in 60% of the PNs that innervate most glomeruli in the antennal lobe. PA-GFP exhibits low-level fluorescence, sufficient to identify individual glomeruli, that is enhanced 100-fold after photoconversion with high-energy light. The PA-GFP was photoactivated with a two-photon laser scanning microscope to localize 710-nm light with submicrometre three-dimensional precision. Photoactivation of the antennal lobe neuropil, encompassing all glomeruli, results in intense labelling of the dendritic arbors of GH146 PNs. Diffusion of PA-GFP from the illuminated dendritic arbors allowed revealation of the cell bodies and axonal projections of the multiple GH146 PNs. Photoactivation of individual glomeruli (VM3 and DA1) reveals the dendritic arbors, cell bodies and projections of the subpopulation of GH146 PNs that innervate a single glomerulus (Datta, 2008).

An approach was devised to allow the tracing of individual PNs that innervate identified glomeruli. The DA1 glomerulus was exposed to low levels of photoconverting light and then the antennal lobe was rapidly imaged to identify the PN cell bodies that show modest increases in fluorescence intensity. Under these limiting conditions of photoactivation, diffusion of PA-GFP into axonal projections was not observed. Next a single weakly labeled PN cell body was strongly photoactivated at higher light intensity to reveal the axonal projections of an individual PN that innervates the DA1 glomerulus. Thus, two-photon laser scanning microscope-mediated activation of PA-GFP provides sufficient spatial resolution and photoconversion energy to reveal the neuronal processes of defined neuronal populations as well as individual neurons in the fly brain (Datta, 2008).

The development of a combined genetic and optical neural tracing method permits comparison of the topography of projections from Fru+ PNs that innervate the cVA-responsive DA1 glomerulus in male and female flies. Flies in which GAL4 is expressed under the control of the P1 fruitless promoter responsible for generating FruM (fruGAL4) were crossed with flies harbouring the UAS-PA-GFP transgene. P1 transcripts from the modified fruGAL4 allele do not undergo the sexually dimorphic splicing observed for the wild-type fru allele, and they therefore allow marking of Fru+ cells in both sexes. Unilateral photoactivation of the fly brain reveals many Fru+ cells, including neurons in the antennal lobe. Specific photoactivation of the DA1 glomerulus reveals six Fru+ PNs in both male and female flies that innervate this glomerulus. The cell bodies of these neurons reside in the lateral PN cluster, not the dorsal cluster as previously suggested (Datta, 2008).

It is possible that the sex-specific behavioural responses to cVA result from different functional responses of the DA1 glomerulus in the two sexes despite there being no apparent difference in the number or location of Fru+ DA1 PNs. Therefore the Ca2+-sensitive fluorescent protein GCaMP was expressed in Fru+ neurons, and two-photon imaging was used to examine increases in Ca2+ in the DA1 glomerulus in response to cVA. Large increases in Ca2+ within the DA1 glomerulus were detected by two-photon imaging after exposure of an intact, behaving fly to cVA. However, no differences were observed between male and female responses over a broad range of cVA concentrations (Datta, 2008).

These imaging experiments report local changes in the concentration of Ca2+ in both the presynaptic and postsynaptic compartments, because both Or67d-expressing neurons and DA1 PNs are Fru+. Therefore whether the electrophysiological properties of Fru+ DA1 PNs were sexually dimorphic was examined. The DA1 glomerulus was photoactivated to identify Fru+ DA1 PNs and the enhanced fluorescence was used to guide a patch electrode to the cell bodies. Recordings were made from Fru+ DA1 PNs in the loose patch configuration in an intact fly preparation and no significant difference was noted in the spike frequency or response kinetics between males and females when tested at several concentrations of cVA. These responses are comparable to those previously observed in whole-cell recordings of female DA1 PNs. This result demonstrates that male and female DA1 PNs show similar electrophysiological responses to cVA despite the previously noted dimorphism in the size of the DA1 glomerulus (Datta, 2008).

Next the projection patterns of Fru+ DA1 PNs were examined in the two sexes. Photoconversion of the DA1 glomerulus allowed the projection patterns of the population of DA1 PNs to be revealed in the lateral horn in living brains. Despite significant similarity in the axonal arbors of DA1 PNs in males and females, an increase was observed in the density of ventral axonal branches in the male. Quantification of differences in branch patterns in multiple individual male and female flies was hampered by variations in the orientation of the live brain during microscopy. Therefore the approach was altered to employ fixed brains stained with the antibody nc82 to label the synaptic neuropil of the lateral horn. An image registration algorithm was used to first 'warp' the nc82 channel of individual brains onto a reference brain and then map the PA-GFP fluorescence onto this reference brain. The registration error averaged less than 2μm in any dimension when measured at the neuropil edge. It was observed that the projections from the DA1 glomerulus target the anterior ventromedial region of the LH. The projection pattern is triskelion-shaped, with ventral, lateral and dorsal branches. Fru+ DA1 projections from males have additional axonal branches that extend ventromedially. Superposition of the DA1 projections taken from ten male and ten female flies confirms this observation, indicating that information carried by Fru+ DA1 PNs is differentially segregated in the lateral horn of the two sexes. As a control a similar analysis of the PN projections from the Fru- glomerulus VM3, which responds to alcohols and acetates, was performed. Superposition of the projections from VM3 reveals no consistent differences in the pattern of axonal projections in the lateral horn between the two sexes. These observations show that the image alignment procedure does not introduce sex-specific biases in projection patterns and that the dimorphic projection patterns that were observe for the Fru+ glomerulus DA1 are not a general feature of projections from all glomeruli (Datta, 2008).

The anatomical dimorphism observed at the level of the population of axons is also shown by the axons of single identified neurons. Tracing individual Fru+ DA1 neurons after warping revealed that the ventral axonal branches of male PNs define a male-specific region of protocerebral space (about 600 μm3). Each individual male in the data set sends at least one axon branch into this area. This area seems to partly overlap a region of neuropil in the lateral horn that was recently shown to be larger in male flies than in female flies. In addition, the total density of ventrally oriented axonal branches is significantly greater in males than in females. In contrast, the total innervation of the dorsal axonal arbor showed no statistically significant differences between sexes. No similar female-specific area was identified, although there are several smaller areas (particularly laterally) that appear to have an increased density of female axons. The data from single-axon tracing, along with observations from populations of DA1 neurons, indicate that DA1 PN projections are sexually dimorphic (Datta, 2008).

Fru mutant males court other males with high frequency. If the male-specific arbor contributes to the dimorphic behavioural response, it is expected that the DA1 PN projection patterns will be regulated by the fruitless gene. Therefore the axonal projections of single DA1 PNs were made visible in fru mutant males, and it was observed that DA1 PNs lack the characteristic male-specific axonal branches and exhibit a branching pattern more characteristic of wild-type females. However, the feminization is not complete in that the male-specific ventral axonal branches are significantly reduced but not completely eliminated in fru mutant males. Thus, the male pattern of projections of Fru+ DA1 PNs requires the male-specific isoform of fru, FruM (Datta, 2008).

It was also shown that the ectopic expression of FruM in females masculinizes the axonal arbor of their DA1 PNs. Projections of single Fru+ DA1 PNs in female flies that express FruM (fruGAL4/fruUAS-FruM) exhibit a striking increase in axonal projections to the ventral male-enhanced area. Quantitative analysis of these branches reveals that expression of FruM in females renders their ventral axon branch pattern statistically indistinguishable from that of males. The innervation patterns of individual neurons are sufficient for a computational discrimination algorithm to effectively distinguish individual females from FruM-expressing females with 100% accuracy, and individual males from fru mutant males with more than 91% accuracy. Thus, analysis of the PN projections of both single defined neurons and populations of neurons reveal that Fru+ DA1 PNs project to different regions of the protocerebrum in male and female flies. Moreover, this anatomical dimorphism in the neural circuit is controlled by the dimorphic transcription factor, FruM (Datta, 2008).

Next, whether the formation of the male-specific arbor requires the action of FruM in DA1 projection neurons was examined. The enhancer-trap MZ19 drives the expression of GAL4 in six DA1 PNs, about ten additional PNs that innervate two Fru- glomeruli, and 25 extrinsic neurons of the mushroom body. Flies harbouring fruGAL4, MZ19 or MZ19;fruGAL4 all reveal expression of PA-GFP in six DA1 PNs. This suggests that the six lateral DA1 neurons labelled by the MZ19 and fruGAL4 lines are identical. In accord with this observation, male and female DA1 neurons in MZ19 flies have a sexually dimorphic pattern of projections that closely resembles the dimorphic branching observed for Fru+ DA1 PNs. Therefore FruM expression was eliminated in male MZ19 neurons by expression of Tra, which directs the female-specific splicing of fruitless transcripts. Genetic feminization of male DA1 PNs in MZ19/UAS-tra flies results in two anatomical classes of DA1 projection neurons. Half of the genetically feminized DA1 PNs show a reduction in the male-specific arbor and closely resemble male DA1 projection neurons defective for FruM. The remaining genetically feminized neurons exhibit the wild-type male-specific branching patterns. Within a single male MZ19/UAS-tra fly, neurons of both anatomical classes were observed. These data suggest that FruM is required in DA1 PNs to generate a male-specific projection pattern, but its action in this genetic context is partly penetrant (Datta, 2008).

Also, whether the expression of FruM in female DA1 PNs masculinizes the DA1 axon arbor was examined. DA1 PNs in female MZ19; fruUAS-FruM flies do not significantly innervate the male-specific area, although most send minor branches into the ventral region of the lateral horn. This is in contrast with observations with fruGAL4/fruUAS-FruM strains that exhibit a transformation of the female DA1 PN branching pattern into a complete male-specific arbor. Taken together, these results suggest that FruM is required in both DA1 PNs and in other Fru+ neurons to generate the male-specific pattern of ventral axon arborization in the lateral horn (Datta, 2008).

In Drosophila, courtship behaviour is governed by pheromonal excitation of peripheral olfactory pathways that ultimately activate behavioural circuits in higher brain centres. One pheromone elaborated by the male, cVA, suppresses male-male courtship but in females enhances receptivity to courting males. cVA activates the DA1 glomerulus, which is innervated by PNs that have sexually dimorphic projections in the lateral horn. This dimorphic circuit is under control of the transcription factor FruM, a male-specific isoform of fruitless. Moreover, the dimorphism in this circuit correlates with behaviour. In males mutant for FruM, cVA no longer suppresses male-male courtship and males exhibit a feminized pattern of DA1 projections. In females that express FruM, DA1 PNs exhibit a male pattern of axonal arbors in the lateral horn, and these females show reduced sexual receptivity. These observations are in accord with a mechanism in which the anatomical differences observed in Fru+ DA1 projection neurons contribute to the distinct behaviours elicited by cVA in the two sexes. In Drosophila, dimorphism in the Fru+ SP2 and mAL neurons has been observed, but the behavioural function of these circuits is unknown (Datta, 2008).

The anatomical dimorphism observed may be translated into a behavioural dimorphism if the connections between DA1 PNs and third-order neurons differ between the sexes. Third-order neurons whose dendrites innervate the ventral lateral horn may either receive greater input from male PNs or may restrict their synapses to the male-specific region of the DA1 axon arbor. The relatively small size of the male-specific arbor, about the volume of a glomerulus, implies a precision of connectivity in higher processing centres in the fly brain. The stereotyped and local precision of synaptic connections is an organizing principle in the antennal lobe and may be a common feature of invertebrate nervous systems (Datta, 2008).

Characterization of specific neural circuits that may mediate behaviour, as described in this study for the pheromone-responsive DA1 pathway, requires the development of tracing approaches that label defined populations of neurons. The distinction between genetic approaches -- including MARCM, Flp-Out and PA-GFP-based tracing -- and the histological approaches of Golgi and Cajal 100 years ago is the ability to use genetic markers to identify partners in the neural circuit more precisely. The targeted illumination of PA-GFP permits non-random, optically guided labelling of individual neurons from either anatomically or genetically defined subsets of neurons. Moreover, PA-GFP can be photoactivated in neurons in the living brain and allows electrophysiological recordings of labelled cells. This approach to neural tracing and recording in a defined circuit can be readily adapted to other brain regions in both the fly and mouse (Datta, 2008).

A dimorphic pheromone circuit in Drosophila from sensory input to descending output

Drosophila show innate olfactory-driven behaviours that are observed in naive animals without previous learning or experience, suggesting that the neural circuits that mediate these behaviours are genetically programmed. Despite the numerical simplicity of the fly nervous system, features of the anatomical organization of the fly brain often confound the delineation of these circuits. This study identified a neural circuit responsive to cVA, a pheromone that elicits sexually dimorphic behaviours. Neural tracing using an improved photoactivatable green fluorescent protein (PA-GFP) was combined with electrophysiology, optical imaging and laser-mediated microlesioning to map this circuit from the activation of sensory neurons in the antennae to the excitation of descending neurons in the ventral nerve cord. This circuit is concise and minimally comprises four neurons, connected by three synapses. Three of these neurons are overtly dimorphic and identify a male-specific neuropil that integrates inputs from multiple sensory systems and sends outputs to the ventral nerve cord. This neural pathway suggests a means by which a single pheromone can elicit different behaviours in the two sexes (Ruta, 2010).

The male pheromone 11-cis-vaccenyl acetate (cVA) elicits male-male aggression and suppresses male courtship towards females as well as males. A single class of olfactory neurons mediates behavioural responses to a Drosophila sex pheromone. In females, cVA activates the same sensory neurons to promote receptivity to males. cVA-induced aggregation behaviour is shown by both sexes. What neural circuits permit a single pheromone acting through the same set of sensory neurons to elicit several distinct and sexually dimorphic behavioural responses? (Ruta, 2010).

The sensory neurons that express the odorant receptor Or67d respond to cVA, and these neurons converge on the DA1 glomerulus in the antennal lobe. Projection neurons (PNs) that innervate the DA1 glomerulus terminate in the lateral horn of the protocerebrum. Comprehensive maps of Drosophila higher olfactory centers: spatially segregated fruit and pheromone representation. Previous experiments showed that the DA1 axons are sexually dimorphic and reveal a male-specific ventral axonal arborization in the lateral horn (Datta, 2008). This dimorphism by itself might explain the sexually dimorphic behaviours or, alternatively, it might presage iterative anatomical dimorphisms at each stage in the circuit to descending output. Therefore, a neural circuit was characterized that transmits information from the DA1 PNs to the ventral nerve cord (see Photoactivation identifies dimorphic lateral horn neurons). The analysis was restricted to neurons that express the sexually dimorphic transcription factor fruitless (FruM). FruM is expressed in both Or67d-expressing sensory neurons and DA1 PNs and governs the development of dimorphic neural circuitry including the male-specific axonal arborization of DA1 PNs. In addition FruM specifies many male-specific behaviours, including those that are mediated by cVA (Ruta, 2010).

In initial experiments PA-GFP, a photoactivatable GFP, was used to identify Fru+ third-order neurons whose dendritic processes are closely apposed to DA1 axon termini. A strategy was developed in which two-photon photoactivation is restricted to a small, circumscribed region of a neuron's axonal arborization with the expectation that this would label the postsynaptic cells by photoconversion of PA-GFP in their dendrites. To ensure that this limited activation could produce sufficient signal from the photoconverted fluorophore to illuminate third-order neurons and their most distal processes, two new enhanced PA-GFPs were generated, namely C3PA-GFP and SPA-GFP (Ruta, 2010).

Photoconversion of the DA1 glomerulus in flies expressing C3PA-GFP or SPA-GFP under the control of fruGAL4 readily identified the axonal arborizations of the DA1 PNs. Then the volume of neuropil circumscribing the DA1 axon termini was photoactivated and four clusters of presumptive third-order neurons were reproducibly labelled in the lateral horn of male flies. Labelling of the two dorsal clusters, DC1 and DC2, was observed only in males; the clusters were either absent in the female or lacked projections into the ventral lateral horn. The lateral cluster LC1 was present in the two sexes but was dimorphic in both number and projection pattern. LC2 did not show an apparent numeric or anatomical dimorphism. Photoactivation of DA1 axon terminals in male flies that express C3PA-GFP pan-neuronally labelled few additional neurons and suggests that these four Fru+ clusters constitute the major potential recipients of DA1 input (Ruta, 2010).

These photoactivation experiments identify clusters of third-order neurons in the lateral horn that are anatomically poised to propagate dimorphic responses to cVA. However, anatomical proximity does not ensure functional connectivity. Therefore a method was developed to specifically activate individual glomeruli and simultaneously record from presumptive downstream neurons to determine whether the lateral horn clusters that were identified receive excitatory input from DA1 PNs. DA1 PNs were selectively stimulated by positioning a fine glass electrode in the centre of the DA1 glomerulus and iontophoresing acetylcholine, the neurotransmitter that excites PNs, into the glomerular neuropil. Varying the iontophoretic voltage allowed variation of the frequency of elicited action potentials systematically in DA1 PNs up to 250, a value close to the upper limit of cVA-elicited responses measured in these PNs (Datta, 2008; Schlief, 2007). Activation of the DA1 glomerulus over this voltage range excited DA1 PNs specifically and elicited no response in PNs innervating other glomeruli in the antennal lobe. Stimulation of the neighbouring glomeruli, VA1d and VA1lm, similarly elicited the specific excitation of their cognate PNs but did not activate DA1 PNs (Ruta, 2010).

Next, whether stimulation of the DA1 glomerulus would result in the excitation of neurons within the four clusters in the lateral horn that were identified, a result indicative of functional synaptic connections with DA1 PNs, was examined. The genetically encoded calcium indicator GCaMP3 was examressed in Fru+ neurons in male flies and two-photon imaging was used to monitor increases in Ca2+ concentration in the lateral horn clusters in response to DA1 excitation. Stimulation of the DA1 glomerulus elicited large increases in Ca2+ in neurons within the DC1 and LC1 clusters, with a far weaker response being observed in LC2. The small DC2 cluster is difficult to identify reliably because of the low basal fluorescence of GCaMP3; it was therefore not examined by optical imaging. The Ca2+ response in DC1 was specific for DA1 activation and was not observed when the stimulating electrode was repositioned in two neighbouring glomeruli, VA1d and VA1lm. These optical imaging experiments demonstrate that neurons within the DC1 and LC1 clusters extend processes in anatomical proximity to the DA1 axons and receive excitatory input from DA1 PNs. Immunostaining indicated that neurons within the LC1 cluster produce the inhibitory neurotransmitter GABA (γ-aminobutyric acid). Electrophysiological experiments suggested that DC1 neurons are excitatory but the neurotransmitter remains unknown (Ruta, 2010).

Focused was placed on the male-specific DC1 neurons to define a cVA-responsive circuit. The DC1 cluster consists of ~19.7 cell bodies (n = 10) in a spatially stereotyped location in the dorsal aspect of the anterior protocerebrum. Double labelling experiments revealed that the DC1 processes interdigitate richly with DA1 axons in the lateral horn. Photoactivation of single DC1 cell bodies indicated that the cluster is composed of several anatomical classes of neurons characterized by distinct branch patterns within the protocerebrum that are likely to receive and integrate inputs from both olfactory and non-olfactory brain centres (Ruta, 2010).

Electrophysiological recordings were performed to examine the response of DC1 neurons to both DA1 stimulation and cVA exposure. Selective stimulation of the DA1 glomerulus evoked action potentials in 66% of male-specific DC1 neurons recorded in the loose patch configuration. Among responsive DC1 neurons, it was observed that the sensitivity to DA1 stimulation differed. This functional heterogeneity within the DC1 cluster observed by both electrical and optical recording was consistent with the anatomical heterogeneity of dendritic fields in the lateral horn observed for single DC1 neurons (Ruta, 2010).

In accord with the imaging experiments, the electrophysiological response of DC1 neurons is selectively tuned to DA1 input. After recording the response of a DC1 neuron to DA1 stimulation, the stimulating electrode was repositioned into 6-11 other superficial glomeruli located throughout the antennal lobe. DC1 neurons activated by minimal DA1 stimulation were either weakly excited or unresponsive to strong stimulation of other glomeruli. Stimulation of the Fru+ VA1lm glomerulus failed to excite DC1 neurons despite the close proximity of DA1 and VA1lm axons (Jefferis, 2007). These observations demonstrate the specificity of glomerular excitation and reveal that olfactory input to DC1 is mediated largely by the DA1 glomerulus and not by the activation of at least 11 other glomeruli, suggesting that DC1 neurons receive olfactory stimulation only from cVA. Next cVA-evoked responses from DC1 neurons were recorded in an intact fly preparation. It was observed that 62% of DC1 neurons were responsive to cVA over a range of concentrations. The input-output relationship of DC1 neurons was similar whether action potentials were evoked in DA1 PNs through direct glomerular stimulation or by pheromonal excitation of the antenna, suggesting that DC1 neurons are excited primarily by means of DA1 input. Both Or67d-expressing sensory neurons and DA1 PNs have been shown to be selectively tuned to cVA. DC1 neurons showed similar odorant selectivity and fired only weakly in response to stimulation of the antenna with a cocktail of ten fruit-derived odorants that excite a majority of glomeruli. Thus, DC1 neurons are likely to receive direct excitatory feedforward input from DA1 PNs and respond selectively to cVA (Ruta, 2010).

Photoactivation of PA-GFP in presynaptic DA1 axonal arborizations, in concert with electrophysiology, has identified postsynaptic third-order neurons in the lateral horn that are responsive to cVA. The iterative use of this strategy could allow definition of the complete cVA circuit from sensory input to descending output. Tracing of photoactivated DC1 axons revealed that they terminate proximally within a triangular neuropil in the lateral protocerebrum (the lateral triangle) and extend distal processes to a previously uncharacterized tract within the superior medial protocerebrum (the SMP tract). The lateral triangle and SMP tract are sexually dimorphic neuropils that are absent in females (Ruta, 2010).

Photoactivation of the terminal arborizations of DC1 axons was performed to identify neurons innervating the lateral triangle and SMP tract. Dense labelling was observed in these structures arising from the rich male-specific projections of multiple classes of Fru+ neurons. Dimorphic LC1 neurons that receive direct innervation from DA1 PNs send inhibitory projections to the lateral triangle and SMP tract. Dimorphic mAL neurons were also observed extending from the s ganglion (SOG) and terminating within these neuropils. In addition, these neuropils are innervated by male-specific P1 interneurons implicated in the initiation of male courtship behaviour. Thus, the lateral triangle and SMP tract receive dimorphic projections from several brain regions including other sensory processing areas, suggesting that these neuropils may integrate sex-specific information from multiple sensory systems (Ruta, 2010).

Several neurons that innervate the lateral triangle and SMP tract also extend processes that descend into the ventral nerve cord, suggesting that these potential fourth-order descending neurons may transmit information from cVA-responsive sensory neurons to the ganglia of the ventral nerve cord. Descending neurons that innervate the lateral triangle and SMP tract were characterized by photoactivation of the cervical connectives conveying neural signals from the brain to the ventral nerve cord. In the brain, the processes of these descending neurons showed a marked dimorphism that was apparent in their extensive innervation of the male-specific SMP tract and lateral triangle. A descending neuron, DN1, absent in females, was observed in the ventral posterior aspect of the male brain, at the midline. Labelling of this male-specific cell body revealed short processes terminating within the lateral triangle and SMP tract, and a long descending process entering the ventral nerve cord and terminating within the thoracic and abdominal ganglia. Electroporation of DN1 with Texas Red dextran, followed by photoactivation of the DC1 cluster, revealed extensive intermingling of the green DC1 axons with the red dendrites of the descending neuron. This suggests that this descending neuron is anatomically poised to make direct synaptic contacts with third-order, cVA-responsive DC1 neurons (Ruta, 2010).

Whole-cell patch clamp recordings were performed on DN1 to discern whether it transmits pheromonal information to the ventral nerve cord. In response to either exposure of the antenna to cVA or direct stimulation of the DA1 glomerulus, DN1 received a barrage of excitatory postsynaptic potentials (EPSPs) bringing its membrane potential close to or past threshold. To determine whether this response was mediated by DC1 neurons, a microlesion technique was devised exploiting the spatial precision of a two-photon laser to effectively sever DC1 inputs into the lateral triangle and SMP tract. Optical recordings revealed that microlesioning of DC1 dendrites resulted in the immediate and selective loss of DC1 responses to DA1 stimulation without affecting the excitation of neighbouring LC1 dendrites and cell bodies. Severing the connections between DA1 and DC1 resulted in an almost complete loss of the response of DN1 to stimulation of DA1. The response of this descending neuron was far weaker than the response of early neural participants in this circuit. However, the observation that two-photon-mediated microlesions in DC1 resulted in a decrease of more than 70% in the DN1 response to stimulation of DA1 suggests that, despite its weak excitation, DN1 is a component of this circuit. A more potent response may require a more natural setting that integrates pheromonal input with other sensory signals. Taken together, these experiments suggest that male-specific DC1 neurons excite the male-specific DN1 through synaptic connections within the dimorphic lateral triangle and SMP tract. Thus, olfactory information may be processed by as few as three synapses within the brain before descending to initiate motor programs within the ganglia of the ventral nerve cord. Although a behaviour elicited by this circuit cannot yet be defined, it is presumed that it mediates a component of the innate behavioural repertoire initiated by cVA (Ruta, 2010).

This cVA-responsive circuit provides insights into the mechanism by which sensory information received by the antenna may be translated into motor output. First, the circuit is concise: as few as four neuronal clusters and three synapses bring pheromonal signals from the periphery to the ganglia of the nerve cord. This minimal circuit assumes monosynaptic connections between the neurons that were identified. This circuit is shallow but seems to include adequate synaptic connections to permit the integration of olfactory and non-olfactory information. Third-order lateral horn neurons reveal a capacity for multisensory integration with inputs to the DC1 cluster from the SOG and from the optic lobe. The lateral triangle and SMP tract also integrate sensory inputs from DC1 and LC1 as well as inhibitory projections from the SOG. This integration provides the opportunity for other sensory signals emanating from a cVA-scented fly to modulate the response to the pheromone (Ruta, 2010).

Second, multiple neural components within the circuit are anatomically dimorphic, and this could explain the different behaviours elicited by cVA in males and females. The initial neural components of the circuit, Or67d-expressing sensory neurons and DA1 PNs, are dedicated to the receipt of a singular olfactory stimulus, cVA, and are equally responsive to the pheromone in the two sexes. However, dimorphisms are observed in the synaptic connections between the PNs and the third-order lateral horn neurons and define a node from which sex-specific neural pathways emanate. The DA1 PNs reveal dimorphic axon arborizations, but this dimorphism is only one component of a highly dimorphic circuit. These dimorphic arborizations synapse with male-specific DC1 neurons that send axons to a male-specific neuropil (the lateral triangle and SMP tract). One output of this neuropil is a male-specific descending neuron, DN1. This circuit is likely to participate in the generation of cVA-elicited behaviours observed only in males. The identification of a sex-specific circuit including extensive neuropils present only in males suggests pathways for dimorphic behaviours that differ from earlier proposals that invoke the differential activation of circuits that are common to the two sexes. DA1 PNs also synapse onto the cluster of LC1 neurons that are present in both sexes but are numerically and anatomically dimorphic. The multiple dimorphic targets of a singular olfactory input could explain how a pheromone acting through the same sensory inputs may elicit different behaviours in the two sexes (Ruta, 2010).

Control of sexual differentiation and behavior by the doublesex gene in Drosophila melanogaster

Doublesex proteins, which are part of the structurally and functionally conserved Dmrt gene family, are important for sex determination throughout the animal kingdom. Gal4 was inserted into the doublesex locus of Drosophila, allowing visualization and manipulation of cells expressing dsx in various tissues. In the nervous system, differences between the sexes were detected in dsx-positive neuronal numbers, axonal projections and synaptic density. dsx was found to be required for the development of male-specific neurons that coexpressed fruitless (fru), a regulator of male sexual behavior. It is proposed that dsx and fru act together to form the neuronal framework necessary for male sexual behavior. Disrupting dsx neuronal function had profound effects on male sexual behavior. Furthermore, these results suggest that dsx-positive neurons are involved in pre- to post-copulatory female reproductive behaviors (Rideout, 2010).

Sexual dimorphism in the fly brain

Sex-specific behavior may originate from differences in brain structure or function. In Drosophila, the action of the male-specific isoform of fruitless in about 2000 neurons appears to be necessary and sufficient for many aspects of male courtship behavior. Initial work found limited evidence for anatomical dimorphism in these fru+ neurons. Subsequently, three discrete anatomical differences in central brain fru+ neurons have been reported, but the global organization of sex differences in wiring is unclear. A global search for structural differences in the Drosophila brain identified large volumetric differences between males and females, mostly in higher brain centers. In parallel, saturating clonal analysis of fru+ neurons using mosaic analysis with a repressible cell marker identified 62 neuroblast lineages that generate fru+ neurons in the brain. Coregistering images from male and female brains identified 19 new dimorphisms in males; these are highly concentrated in male-enlarged higher brain centers. Seven dimorphic lineages also had female-specific arbors. In addition, at least 5 of 51 fru+ lineages in the nerve cord are dimorphic. These data were used to predict >700 potential sites of dimorphic neural connectivity. These are particularly enriched in third-order olfactory neurons of the lateral horn, where strong evidence is provided for dimorphic anatomical connections by labeling partner neurons in different colors in the same brain. This analysis reveals substantial differences in wiring and gross anatomy between male and female fly brains. Reciprocal connection differences in the lateral horn offer a plausible explanation for opposing responses to sex pheromones in male and female flies (Cachero, 2010).

This study constitutes the first global, voxelwise (voxel mean volumetric pixel) analysis of structural differences in an insect brain. This allowed identification of extensive sex-specific volume differences missed in earlier studies of Drosophila. These are so reliable that brain shape can identify the sex of individual animals, which can be of experimental benefit. The strong overlap of these volumetric differences with dimorphic arbors of fru+ neurons suggests a role in male behavior. Early studies of sex mosaics showed that the superior protocerebrum is a keyregulator of courtship behavior. These results strongly support that localization and identify specific regions in the superior protocerebrum likely to regulate both male and female behavior (Cachero, 2010).

At the circuit level, a 3D atlas was constructed containing 62 fru+ neuroblast lineages in the central brain and 51 in the VNC. These numbers reveal an impressive heterogeneity in the developmental origin of fru+ neurons. The central brain consists of the cerebrum (made by 100 neuroblasts, 57 of which are fru+) and the smaller subesophageal ganglion. Few of these lineages, or their component neurons, have previously been described in adult flies, making this study a substantial contribution to understanding the development and neuroanatomy of the fly brain (Cachero, 2010).

In contrast to the preliminary conclusion that the fruitless circuit is anatomically largely isomorphic, this study showed that one-third of fru+ neuronal clones are dimorphic. Previous work on sexual dimorphisms in insects has focused on changes that increase the sensitivity of sensory systems to visual or olfactory cues from conspecifics. However, this study found that most dimorphisms are in the arbors of fru+ higher interneurons of the protocerebrum. In those cases where arbors are denser in males than females but still similarly located, the effect could be to increase the strength of connections between the same groups of neurons; this could be analogous to the increases in sensory sensitivity mentioned above, increasing the gain for some signals without fundamentally altering circuit logic. However, all 21 dimorphic clones analyzed have arbors in locations where there are no processes in the opposite sex; in these cases, it seems very likely that different groups of neurons are connected in the two sexes. The sex-specific projection differences generated during development may therefore be somewhat analogous to the jumper switches in an electronic circuit—a few specific locations where connections can be made or altered in order to change the logic of a largely conserved circuit. Pursuing this analogy, engineers place jumper switches at key locations in order to efficiently alter circuit logic. Can any logic be proposed to rationalize the location of fru+ projection dimorphisms (Cachero, 2010)?

The olfactory system provides an excellent example to explore how these circuit level dimorphisms may regulate dimorphic behavior. In the periphery, fru+ olfactory sensory neurons are more numerous in males. This could enhance sensory sensitivity but cannot explain, for example, why the male pheromone cVA should be repulsive for males but stimulatory for females. It is proposed that changes in connectivity between second- and third-order olfactory neurons, largely because of shifts in the dendritic arbors of fru+ lateral horn neurons, result in the rewiring of sensory signals detected by both sexes. Several populations of lateral horn neurons were identified that show precise overlap with the axons of cVA-responsive PNs. This includes two populations with overlap only in males and another with female-specific overlap. It is therefore hypothesized that these lateral horn neurons are the first elements in the olfactory pathway that show different responses to the same stimulus in males and females (Cachero, 2010).

In flies, odors are represented in the first two stages of the olfactory system by the activity of about 50 channels (corresponding to the glomeruli of the antennal lobe). Odors usually activate many channels, and different odors can activate the same channel. PNs show little integration of information across olfactory channels, in clear contrast to third-order neurons. Although cVA is a rather specific signal known to activate only two channels, it occurs in a variety of behavioral contexts. Third-order neurons that integrate cVA signals with olfactory signals from other PNs may therefore respond with increased selectivity to behaviorally relevant odor objects such as male and female flies. It would make sense to restrict substantial anatomical dimorphism to such neurons capable of selective integration. More generally, it is hypothesized that it would be efficient to alter connections involving higher-order neurons that robustly represent a relevant feature of an external stimulus (e.g., the presence of a fly of the opposite sex) rather than more peripheral neurons, which can be activated by more diverse stimuli (Cachero, 2010).

Outside the olfactory system, a number of relatively discrete dimorphisms could alter the processing of external stimuli or the behaviors to which they are eventually connected. However, the analogy with an electronic circuit that can be (developmentally) reconfigured by altering some connections between the same set of elements is incomplete. There are some dimorphic clones that have many more neurons in males, most obviously pMP-e and aSP-a, which have extensive male-specific processes in the MER (Male-enlarged region). The MER is rich in dimorphic fru+ neurons and appears to receive diverse sensory information and to be connected to the VNC. Masculinization of pMP-e (P1) in otherwise female flies is strongly correlated with induction of male courtship behavior, so it is proposed to initiate male behavior. Although the circuit logic of the MER is not yet clear, two specific models are proposed for these highly dimorphic neurons. First, they may integrate multiple sensory streams and, when activated, directly excite descending neurons that control male motor programs. Alternatively, they may reflect the state of excitation of the male by generating a long-lasting potentiation of connections in the MER between sensory inputs specific to particular descending neurons (Cachero, 2010).

Alterations in the presence and wiring of specific circuit elements according to the logic that has been described in this study could result in sex-specific activation of motor programs that are largely conserved between the sexes. This is consistent with the finding that direct activation of fru+ neurons in the VNC of females can induce courtship song. However, song is abnormal unless those fru+ neurons are masculinized by fruM, so there may be similar developmental alterations in motor circuits of the VNC. The logic of sexually dimorphic behavior has strong parallels in mice, in which removal of the pheromone-sensing vomeronasal organ from females results in male behavior. The resultant hypothesis proposes that circuits for male and female behavior exist in females but that sensory information from the vomeronasal organ normally represses male behavior. This, then, begs the question of what circuit difference alters the behavioral significance of vomeronasal organ activation. Genetic approaches to identifying and characterizing circuit level differences in mice will find answers in the long run. However, w how dimorphic anatomy can alter neural processing and behavior in Drosophila can now be tested (Cachero, 2010).

Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain

The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. This study developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. The power of CLIn was demonstrated in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. 18 dopaminergic neurons from three distinct clusters were mapped to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. Type II lineages that produce doublesex- and fruitless-expressing neurons were identified, and whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain was examined. Blocking apoptosis in these lineages results in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism (Ren, 2016).

The relationship between neuron classes and lineages is complex in the Drosophila brain, where analogous neurons of a given class may arise from distinct lineages and a single lineage can yield multiple neuron classes. Therefore, a method was developed that would enable mapping and and analysis of neuron classes with respect to lineage identity using intersectional transgenic strategies. Specifically, the neuron class of interest expresses the GAL4 transcriptional activator from a class-specific transgene, while the lineage(s) of interest expresses the KD recombinase from a lineage-specific transgene. The KD recombinase activity triggers production of another recombinase, Cre, under the control of the deadpan (dpn) promoter, which is active in all NBs. Cre recombinase activity then triggers the simultaneous production of the LexA::p65 transcriptional activator and loss of the GAL4 inhibitor, GAL80, in all subsequently born progeny within the lineage(s). The LexA::p65 activates reporter-A expression within lineages of interest via lexAop. Because all other neurons outside lineage(s) of interest express GAL80, GAL4 is only active in neurons of the LexA::p65-expressing lineage(s) and thus can positively mark these neurons by activating expression of a reporter-B under UAS control. One can therefore subdivide any complex set of neurons that express a class-specific GAL4 transgene based on their developmental lineage(s). Consequently, CLIn enables the unambiguous determination of the lineage origins of particular neuron classes, which is essential for understanding the development and organization of the Drosophila brain (Ren, 2016).

The CLIn system unambiguously establishes the correspondence between cell classes and their lineage origins and enables the subdivision of a given neuronal class among certain NB lineages. It also allows interrogation of serially derived neuronal diversity. One can therefore map individual neurons of a given class with respect to their lineage and temporal origins in an effort to unravel the intricate neuron class-lineage relationships in the brain (Ren, 2016).

Revealing diverse cell classes of a lineage, by carefully choosing different GAL4 drivers that each distinguish a particular cell class, will allow better characterization of progeny heterogeneity within a lineage. It is therefore possible to explore how cellular diversity is generated during development. For example, it will be interesting to determine whether a specific cell class develops from one fixed temporal window. Moreover, comparing the cell-class diversity of different lineages will provide insight into the developmental heterogeneity of stem cells (Ren, 2016).

Conversely, for cell classes that originate from multiple lineages, CLIn analysis reveals the distribution of a cell class among different lineages. Vertebrate studies found that neurons of the same lineage origin, compared to neurons of the same class but different lineage origins, are more likely structurally connected via gap junction and have similar network functions. In Drosophila, lineage has been shown to be a developmental and a functional unit. Thus, assigning a cell class to different lineages may reveal the particular function of a neuronal subset within a cell class (Ren, 2016).

Moreover, the CLIn system permits incorporations of additional effectors driven by the GAL4-UAS system or the LexA system to manipulate cell class or lineage, respectively. The toolkit of effectors for different purposes is growing rapidly over recent years. Multiple reporter constructs are available to label specific sub-domains of the cell (dendrite, axon, or synapse). Effectors that affect cell viability could eliminate or immortalize specific neurons or glia. Effectors that alter membrane activity can be used to modulate neural activity. In addition, CLIn enables distinguishing gene’s functions in whole lineage including stem and progenitor cells versus only in a subset of lineage progeny by independent gene manipulations via lexAop versus UAS systems (Ren, 2016).

However, the CLIn system requires further improvement to reach its full potential. In particular, the drivers for targeting various NB subsets remain to be fully characterized. Moreover, their targeting efficiency and specificity could vary individually. Engineering drivers based on genes known to be expressed in defined subsets of embryonic NBs may provide an initial complete set of more reliable NB drivers. An additional challenge for the study of type II lineages is how to selectively target INP sublineages. Via the current dpn enhancer, the frequencies of INP1 sublineages are very low compared with that of NB lineages (Ren, 2016).

Type II NBs yield supernumerary neurons plus glia, which are expected to be highly diverse in cell classes. CLIn unambiguously assigned various neuronal classes to common type II lineages. In this study, the majority of progeny remained negative for the drivers employed. Revealing the full spectrum of neuronal heterogeneity within type II lineages requires characterization of additional cell-class drivers (Ren, 2016).

Diverse cell classes could arise from a single INP. Single-cell lineage analysis has shown that one INP can produce multiple morphological classes of neurons most likely pertaining to different functional classes. Temporal mapping by CLIn revealed the birth of both TH-GAL4 and dsxGAL4 neurons in early windows of larval type II lineages. This lends further support to the production of diverse neuronal classes by common INPs. Examining INP clones labeled by CLIn did validate that the first larval-born INP of the DM6 lineage makes one fruGAL4 neuron in addition to two TH-GAL4 neurons (Ren, 2016).

Per the limited cell-lineage analysis along the NB axis of type II lineages, sibling INPs produce morphologically similar series of neurons that differ in subsets of terminal neurite elaborations. These phenomena indicate expansion of related neurons across sibling INP sublineages. Assuming production of about 50 sibling INPs and in the absence of apoptosis, one would expect composition of 50 cell units for each neuronal class made by one type II NB. Notably, rescuing apoptotic dsx- or fru-expressing neurons throughout lineage development did restore complements of 50 or so cells in several, but not all, type II lineages. However, most type II lineages yield very few, if any, TH-GAL4 neurons. For instance, the DL1 lineage produces two TH-GAL4 neurons that innervate the upper FB layers. Temporal mapping of the DL1 lineage reveals the existence of multiple (n > 3) morphologically distinct INP clones that contain neurons projecting to the FB upper layers, similar to the DL1 TH-GAL4 neurons. Thus, morphologically similar neurons may belong to different functional classes, highlighting the challenges in sorting out neuronal classes and their interrelationships in the brain (Ren, 2016).

Pioneering studies in C. elegans showed that the acquisition of neurotransmitter identity could be achieved through distinct mechanisms. A shared regulatory signature consisting of three terminal-selector types of transcription factors regulates the terminal identity of all dopaminergic neurons. By contrast, different combinations of terminal selectors act in distinct subsets of glutamatergic neurons to initiate and maintain their glutamatergic identity. In the present study, it was observed that six type II lineages produce 18 dopaminergic neurons but all during early larval neurogenesis. The derivation of TH neurons from multiple neuronal lineages at similar temporal windows argues for their specification by combinations of different lineage-identity genes with common temporal factors (Ren, 2016).

Previous analysis of fruGAL4 neurons has uncovered 62 discrete MARCM clones in the fly central brain that might arise from an equal number of lineages. Ten clones show dimorphic cell numbers, and 22 clones exhibit dimorphic trajectories. Contrasting the stochastic clonal labeling of only fruGAL4 neurons, CLIn allows determination among a collection of lineages of whether a given lineage yields any fruGAL4 neurons. Based on the additional lineage information, two clones (pIP-j and pIP-h) were attributed as being partial clones of another two full-sized clones (pIP-g and pMP-f). Moreover, a more focused approach reveals sexual dimorphism of fru-expressing neurons in all type II NB lineages (Ren, 2016).

The presence of many more dsx- or fru-expressing neurons in male than female brains is proposed to result from selective loss of specific neurons in females through apoptosis. However, blocking apoptosis increased dsx- or fru-expressing neurons in both male and female lineages. This is consistent with a previous report showing that sex-independent apoptosis occur widely in type II lineages. Although the number of apoptosis-blocked female neurons was similar, if not identical, to that of the control male neurons, blocking apoptosis unexpectedly increased the number of male dsx- or fru-expressing neurons such that there were more neurons in the apoptosis-blocked male than female lineages. This unmasks the original potential of the male and female NBs to produce different numbers of dsx- or fru-expressing neurons in type II lineages (Ren, 2016).

Distinct fates have been reported for male and female NBs in the abdominal ganglion of Drosophila CNS. In this study, the male isoform of Dsx, DsxM, promotes additional NB divisions in males relative to females. Similarly, it has been reported that DsxM specifies additional cell divisions in the male, relative to female, central brain NBs that give rise to the pC1 and pC2 clusters. The proliferation of Drosophila intestinal stem cells is also determined by their sexual identity, although this is controlled by genes other than dsx and fru. Consistent with the notion that male and female NBs may possess distinct fates, this study found that male type II lineages contain more neurons committed to express dsx or fru, which possibly results from the greater number of NB divisions in males, as shown in the previous study. Elucidating the underlying molecular mechanisms of sex-specific neuron numbers in the central brain will require additional studies of the sex-dependent production and specification of different dsx- or fru-expressing neurons in the apoptosis-blocked type II NB lineages (Ren, 2016).

Lineage mapping based on morphology provides limited information about neuronal classes. Given the intricate relationship between neuronal classes and cell lineages, CLIn is needed to resolve the detail even in fly brains where invariant neuronal lineages exist. This is critical for fully understanding how cell lineages guide the formation of variant neural circuits with distinct combinations of neuronal classes and types (Ren, 2016).

In mammalian brains, extensive neuronal migration obscures the roles of cell lineages in the global organization of neural networks. However, clonally related neurons preferentially make local connections. Moreover, ample evidence exists for the heterogeneity of mammalian neural stem cells and the control of neuronal identity by spatiotemporal patterning of neural progenitors. Untangling of a further sophisticated brain and its development may absolutely require examination of cell lineages and neuronal classes at the same time. Systems like CLIn with its emphasis on the relationship between cell class and lineage potentially aid greatly in the study of mammalian brain development, anatomy, and function (Ren, 2016).


In both sexes, only about 500 of the roughly 100,000 neurons of the CNS label with an mRNA probe: there is no labeling in other tissues of the body. In both sexes, labeled neurons are found most commonly as small groups and less frequently as single cells; most labeled cells are distributed in similar areas of the brain and ventral nerve cord of males and females. A set of nine groups of labeled neurons, ranging from 10-30 cells is detected in the CNS of males in positions likely to be involved in particular male courtship behaviors. Comparably positioned cells are found in six of these locations in females.

A set of two groups of labeled neurons is detected in areas that must have a male identity for flies to perform particular steps in male courtship. One is a prominently labeled group of cells in the dorsal-posterior protocerebral region; early steps in courtship (following and wing extension) map to this region using gynandromorphs. The second is a group of cells located in the ventral mesothoracic ganglion in males (but not females), an area to which courtship song has been mapped. FRU positive neurons are also found in areas not known to be involved in sexual behavior, such as three groups of neurons in the anterior ventrolateral protocerebrum. These are found associated with higher order sensory processing neuropils of the CNS. There is also a male-specific neuronal cluster in the abdominal ganglion, a ganglion that controls abdominal movements during copulation (Ryner, 1996).

The small number and the locations of the neurons expressing male-specific transcripts suggest that fru is directly involved in only some of the sensory and motor systems necessary for male sexual behavior. For example, the sex-specific transcripts of FRU are abundantly expressed in a group of primary sensory interneurons in the antennal lobe involved in the processing of chemosensory information. These appear to be relay interneurons, known to project to higher brain centers such as the calyx of the mushroom body.

One unexpected finding is that some fru expressing cells are detected only in females (for example, in the subesophageal ganglion), yet no female behavioral phenotypes of extant fru mutations have been detected. Often the intensity of hybridization also differs between the labeled neurons in male versus female CNSs. Other tissues labeled by a common coding-region probe are a subset of direct flight muscle cells, gonadal nurse cells, spermatocytes, fat body cells, gut epithelium, and ectodermal epithelium, including the internal and external genitalia (Ryner, 1996).

The Drosophila fruitless gene product Fru has been postulated to be a neural sex-determination factor that directs the development of at least two male-specific characteristics, namely courtship behavior and formation of the muscle of Lawrence (MOL). The fru gene possesses consensus Tra-binding sequences, and encodes a putative transcription factor that has a BTB domain and two zinc-finger motifs. The binding of Tra to the Tra-binding sequences results in sex-specific alternative splicing of the FRU mRNA, leading to production of the 'male-type' or 'female-type' Fru protein. The Fru protein is not detected in the female central nervous system (CNS), despite the similar level of expression of FRU mRNA in both male and female CNS. Since ectopic expression of both the 'male-type' (with the sequence for the amino-terminal extension) and 'female-type' (without the sequence for the amino-terminal extension) fru cDNA can induce formation of the MOL in females, the presence or absence of the Fru protein, and not its sex-specific structure, seems to be responsible for the sexually dimorphic actions of the fru gene (Usui-Aoki, 2000).

The fru gene has been reported to have four promoters, two of which generate the 5' ends of the different transcripts identified in this study. The most distal promoter is required for the transcription of the exon that undergoes sex-dependent splicing. As a result of the sex-dependent splicing, the N terminus of the male-specific protein is expected to have a unique extension of 101 amino acids, followed by the BTB domain, which is shared by all Fru isoforms. This is because the female exon II has a stop codon that prematurely truncates the open reading frame (ORF), which begins near the 5' end of exon II, whereas this stop codon is spliced out in the male exon 2. There is no evidence for or against the possibility that the truncated ORF is transcribed to produce a peptide of 94 amino acids in females. The possibility cannot be excluded that translation starts from the second AUG that is present 113 base pairs (bp) downstream of the first AUG of exon 2, allowing the transcript to encode an ORF of 81 amino acids, with a one-base frameshift relative to the ORF defined by the first AUG. In addition to the N-terminal variants, five Fru isoforms with variations in their carboxy terminus have been identified. Three of these contain two zinc-finger motifs, each of which is encoded by a different exon. The remaining two do not have any zinc-finger motifs. Northern blot analysis of the wild-type head extracts with a DNA probe for the BTB domain detects at least three sets of transcripts differing in size between males and females, and two transcripts that show no such sex difference. In contrast, using the same DNA probe, this sexually dimorphic pattern of transcripts is not detected in fru-mutant homozygotes (Usui-Aoki, 2000).

frusat and fru3 represent strong viable alleles, which in males lead to flies showing male preference in courtship and complete lack of formation of the MOL. When the second exon sequence is used as a probe, a sex difference in the size of the transcripts has been demonstrated, even in frusat homozygotes. Since the frusat mutation was induced by a P-element insertion into the second large intron, the aberrant transcripts found in frusat homozygotes appear to represent chimaeric products of the fru gene and the P-element vector sequence; this was subsequently confirmed by reverse transcription polymerase chain reaction (RT-PCR). The second exon of the fru gene, which undergoes sexually dimorphic splicing, is spliced so as to fuse with the P-element sequence, resulting in the termination of transcription in the vector. The second aberrant mRNA in frusat is transcribed from the mini-white promoter in the vector, and spliced to the downstream fru exon that encodes the BTB domain. Due to the dicistronic organization, however, translation of functional proteins from this aberrant mRNA cannot be expected. Thus, P-element insertion into the second intron inactivates the sexually dimorphic transcripts in the frusat mutant (Usui-Aoki, 2000).

To determine the expression pattern of the fru gene product in the nervous system by immunohistochemical analysis, anti-Fru antibody was generated. Attention was focused on the expression of the Fru protein during the pupal stage, during which the protein is required for normal courtship after emergence, as well as for the development of the MOL. The antibody stains a proportion of the cellular nuclei in the brain and ventral ganglia in wild-type males. A significant proportion of the Fru-expressing cells in the CNS are neurons, since they were also immunopositive for the anti-Elav antibody, a neuron-specific marker. Fru-expressing neurons form discrete clusters in various regions of the brain. In the mid-pupal stage (72 h after puparium formation, APF), 59-69 cells per hemisphere are stained by the anti-Fru antibody in the posterior brain. These dorsal neurons do not contribute to the structure of the mushroom body, as revealed by a double-labelling experiment using the enhancer-trap reporter specific for the mushroom body, and the anti-Fru antibody. In the anterior brain, the largest cluster, composed of 43-44 cells per hemisphere, is found in the anterolateral protocerebrum. Another large cluster containing 34-39 cells per hemisphere is observed in the medio-dorso-anterior protocerebrum. The third largest cluster is formed by 19-20 cells per hemisphere, and is located in the medio-anterior protocerebrum. In addition, the s ganglion has a cluster composed of 16-20 cells per hemisphere. The localization of these immunoreactive cells coincides with that of cells expressing FRU mRNA. In addition to these areas, a few previously undescribed clusters of Fru-expressing cells were identified, including a cluster of 31-32 cells per hemisphere in the dorso-anterior-lateral protocerebrum at 15 h APF. The Fru immunoreactivity in this region decays thereafter, and becomes undetectable at 72 h APF. In the ventral ganglia, Fru-positive neurons are found in three thoracic segments as well as in all the abdominal segments. The same sets of neurons, with the exception of those in the dorso-anterior-lateral protocerebrum as mentioned above, continue to express the Fru protein throughout the pupal stage. After adult eclosion, the immunoreactivity of these neurons with the anti-Fru antibody decreases markedly (Usui-Aoki, 2000).

Surprisingly, the Fru antibody does not stain any neurons in the CNS of wild-type females, even though it stains the eye disc when the Fru protein is expressed ectopically in the disc in females. Furthermore, the antibody does not stain any neurons in the CNS of frusat homozygotes, regardless of sex, reflecting the absence of any functional Fru protein in this mutant. Lack of staining by the anti-Fru antibody in the wild-type female CNS indicates that the Fru protein is not present here. fru mRNA is, however, detected by in situ hybridization in the CNS of female wild-type pupae, at a level similar to that in the male wild-type pupae (Usui-Aoki, 2000).

These observations raise the intriguing possibility that although the fru gene is transcribed, the mRNA is not translated to produce the Fru protein in female neurons. Because the sex-dependent actions of the Fru protein are under the control of Tra, this sex-specific repression of the translation of fru mRNA might be mediated by the binding of Tra to the fru mRNA in females. Indeed, Tra binding appears to repress the translation of FRU mRNA in females. Significant reporter activity is, however, detected, even in the presence of Tra and Tra-2, unlike in the case of the female CNS, where the Fru protein is undetectable. This observation suggests that an additional mechanism is involved in the translation block in females in vivo (Usui-Aoki, 2000).

If Tra and Tra-2 function to directly or indirectly repress translation in females, then this repression should be alleviated or removed by tra or tra-2 mutations. In keeping with this expectation, anti-Fru antibody-reactive neurons are clearly observed in tra mutant females in a similar pattern to that in wild-type males. These observations collectively indicate that Tra represses the Fru protein expression in females in vivo (Usui-Aoki, 2000).

These observations suggest the interesting possibility that the presence or absence of Fru determines the sexual fate of certain neurons. This consideration led the authors to examine whether ectopic expression of fru in females can induce male-like characteristics, in particular, the formation of the MOL. It first had to be determined which isoform of the Fru protein is responsible for the formation of the MOL in males. To address this issue, the ability of heat-shock promoter-driven transgenes to rescue the MOL phenotype in frusat-homozygous males was evaluated. When either type A or type B transgene expression was induced during the third instar larval to pupal stage, the MOL is restored in a proportion of the fru sat mutant males, some of whom have one or two MOL(s), but only unilaterally. A few other rescued males exhibited one of the restored MOLs ectopically in the abdominal segment A4 rather than in segment A5 where it is normally formed. However, similar heat-shock treatment applied from the embryonic through the third instar larval stages has no effect. Types C, D and E transgenes are unable to rescue the MOL phenotype of frusat males, irrespective of heat-shock treatment. On the basis of these results, type A and type B transcripts appear to be essential for MOL formation in males. Another important finding is that the 'female-type' transcripts devoid of the male-specific 5' extension are able to produce the male-specific MOL in frusat males. Thus, the male-specific N-terminal polypeptide does not appear to be necessary to rescue the MOL phenotype of frusat males (Usui-Aoki, 2000).

In another series of rescue experiments, the GAL4-UAS system was used for induction instead of heat-shock promoter-mediated induction. As the Gal4 source, the D42-GAL4 strain which expresses Gal4 in almost all motor neurons but not in muscles was tested. Motor nerve endings on the MOL in the D42-GAL4 strain express Gal4. The basis for this experiment resides in the knowledge that the motor neurons innervating the MOL must be male, while the sex of the myocytes that form the MOL does not affect the muscle development. Using the D42-GAL4 line, it was confirmed that the type B transcript can rescue the MOL phenotype of frusat mutant males. The possibility, however, that the male-specific N-terminal extension enhances the activity of the Fru protein, even though it does appear to be unnecessary, cannot be excluded (Usui-Aoki, 2000).

To determine whether ectopic expression of the Fru protein leads to the formation of the MOL in females, UAS-fru+ type B was expressed, driven by D42-GAL4, in either the wild-type background or the frusat mutant background. In both cases, it was found that the MOL is formed ectopically in most of these female flies; 92% of wild-type females and 91% of frusat homozygous females with ectopic fru+ develop the MOL. Some of these females had one to three MOL(s) in the A4 (and A3) segments in addition to a pair of MOLs in the A5 segment of the abdomen. This is in contrast to the results obtained using hs-fru+ cDNA, which did not induce the formation of the MOL in any females. This result shows unequivocally that the absence or presence of the Fru protein determines whether the MOL is formed (Usui-Aoki, 2000).

Several possible mechanisms are proposed for the sex-specific expression of Fru. One possibility is that the Fru protein is translated only in males, even though its mRNA is transcribed in both males and females. The second is that the Fru protein is rapidly degraded in females whereas its durability is significantly higher in males. The third possibility is that the primary mRNA is not processed to give the mature fru transcript in the female, resulting in the absence of the Fru protein. This last possibility is the least likely, because sex-specific splicing by Tra and Tra-2 of the primary fru transcript has been shown both in vivo and in vitro. Sex-specific control of Fru protein synthesis is a more plausible hypothesis, in which Tra binding to the primary fru transcript in females is postulated to repress translation. Only female-type mature mRNA possesses the Tra-binding site, which lies 61 bp upstream of the translation start site in females. In fact, in transfection assays, the TraB sequence of the fru female transcript placed 5' to the translation start site of the luciferase gene inhibits translation from this reporter in the presence of Tra and Tra-2 (Usui-Aoki, 2000).

The translational regulation of sex-dependent processes in Drosophila is not without precedent. In Drosophila males, the activity of the X chromosome is doubled by a mechanism for gene dosage compensation, mediated by Male-specific lethal (Msl) proteins. Msl-2, one of the Msl proteins, is translated only in males because binding of the Sxl protein to the msl-2 mRNA represses its translation in females. The Sxl protein also functions as a splicing inhibitor when it binds to the same site. By analogy with Sxl, the Tra protein may act as both a translational repressor and a splicing regulator when bound to the fru mRNA. Note, however, that Tra and Tra-2 only partially repress translation from the reporter mRNA with TraB in transfected S2 cells, in contrast to the case in the female fly CNS, where the Fru protein is undetectable. A large amount of the reporter mRNA might have titrated Tra and Tra-2, resulting in unbound mRNA that contributes to residual translation. Alternatively, a separate mechanism could be involved in in vivo repression of fru translation. In this regard, the dicistronic organization of the female FRU mRNA merits discussion. The long ORF encoding the functional Fru protein is preceded by a short ORF for 94 amino acids in females. If this short ORF is translated, the second long ORF is likely not to be translated. A comparable mRNA organization has been found in the male form Sxl transcript, in which the first ORF yields only a non-functional protein because of the presence of a stop codon in male-specific exon III, yet the second ORF starting in exon IV is translated at extremely low levels and thus has no biological effect on sex determination (Usui-Aoki, 2000).

If the absence of Fru proteins in female CNS is entirely due to this mechanism, then the effects of tra mutation on the induction of Fru expression in females are solely due to male-type splicing of the FRU mRNA precursor in female flies. To evaluate this possibility, it is crucial to determine whether or not the 94-amino-acid peptide encoded by the short ORF is present in females (Usui-Aoki, 2000).

The prevailing thought behind the sexually dimorphic function of fru is that the male-type Fru protein and the female-type Fru protein, each derived from the sex-specific mRNA produced by Tra-mediated alternative splicing, probably have different target specificities, and thus activate distinct sets of genes required for either male or female development (Usui-Aoki, 2000).

The 'female-type' Fru protein can support the formation of the male-specific MOL, indicating that the male-specific function of the fru product cannot be ascribed to the 'male-specific' structure of the protein. It should be noted, however, that the sex-specific difference in the Fru protein structure deduced from the cDNA structure needs to be verified experimentally. The size difference of 101 amino acids could be easily resolved by Western blotting, if the antibody were reactive to denatured Fru protein. Whereas the male-type and female-type cDNAs are equally effective in rescuing the MOL phenotype when they are expressed by D42-GAL4 , their effects are different when driven by the hsp 70 promoter: the rescuing ability of the male-type cDNA can not be assessed, because of its lethal effect, whereas the female-type cDNA supports the formation of the MOL. Although the lethality seems to reflect the non-physiological interference of an unknown process by misexpression of the protein in inappropriate tissues, one might argue, on the basis of this observation, that the male-type protein has higher activity than the female-type protein. To better understand the functional significance of the sexual differences in the Fru protein structure, the female- and male-type proteins must be compared for their ability to bind to putative target genes and their transcriptional regulatory activity (Usui-Aoki, 2000).

The fruitless gene of Drosophila produces both sex-specifically and non-sex-specifically spliced transcripts. Male-specific fru products are believed to regulate male courtship. To further an understanding of this gene's behavioral role, the central nervous system (CNS) was examined for temporal, spatial, and sexually dimorphic expression patterns of sex-specific fru products by in situ hybridization and immunohistochemistry. For the latter, antibodies were designed to detect only male-specific forms of the protein (FRUM) or amino acid sequences that are in common among all translated products (FRUCOM). Sex-specific mRNAs and male-specific proteins are first observed in mature larvae and peak in their apparent abundances during the first half of the pupal period. At later stages and in adults, faint mRNA signals are seen in only a few neural clusters; in contrast, relatively strong FRUM signals persist into adulthood. Twenty neuronal groups composed of 1700 fru-expressing neurons were identified in the midpupal CNS. These groups overlap most of the neural sites known to be involved in male courtship. Staining with anti-FRUCOM leads to widespread labeling of neural and nonneural tissues in both sexes, but in the female CNS, this occurs only in developing ganglia, and in a pattern different from that of the male's FRUM cells. Expression of sex-specific fru mRNAs in the CNS of males analyzed from the earliest pupal stages indicates that sex-specific alternative splicing is not the exclusive mechanism regulating expression of fruitless transcripts (Lee, 2000a).

The spatial distribution of fru products overlaps with most brain regions inferred to be involved in male sexual behaviors from analysis of sex mosaics. The so-called foci underlying the sequential steps of male courtship include a dorsal-brain region associated (in genetically male form) with orientation, following, and wing extension. Five groups of FRUM neurons that have now been specified (fru-aSP1, fru-aSP2, fru-aSP3, fru-pSP1, fru-pSP2) are within dorsal brain regions determined from mosaics to be involved in early stages of the behavioral sequence. Additional brain regions, in the antennal lobe and within the mushroom body, are involved in sexual recognition; and clusters of FRUM neurons (fru-mAL and fru-AL) located near the antennal lobe might be involved in this process. This hypothesis is supported by the absence of sex-specific fru transcripts (or the cells that usually express them) in the antennal lobe of the fru1 mutant: fru1males exhibit dramatic defects in sex recognition (Lee, 2000a).

Given the courtship-song defects exhibited by these and other fru mutants and that a portion of the VNC -- possibly the mesothoracic ganglion -- needs to be genetically male for normal courtship song to be produced, it is interesting that FRUM-containing neurons (fru-PrMs, fru-MsMt) are found in this region of the CNS (Lee, 2000a).

Two sex-specific characteristics are associated with the abdominal ganglion, probably through the action of certain abdominal motorneurons: the formation of a male-specific abdominal muscle and the male's abdominal bending that occurs during copulation attempts, both of which are defective to varying degrees in fruitless mutants. Many FRUM neurons (fru-Ab) are present in this posteriormost ganglion. It is hypothesized that certain subsets of the fru-Ab cluster are involved in these two sexually dimorphic phenotypes (Lee, 2000a).

An unexpected result in this study was the presence of fru gene products in the optic lobes. Although vision is not essential for courtship and mating in this Drosophila species, visual cues are important for rapid recognition and sustained tracking of a sexual partner. However, no particular significance to fru expression in the visual system has yet been found. For example, it is not known whether male-specific female tracking behavior is impaired in these mutants. Yet it is interesting that fru3 and fru4 mutant males initiate subnormal frequencies of courtship following bouts and that the turning responses and locomotion necessary to sustain such bouts are relatively brief (Lee, 2000a).

The antibodies generated against a stretch of amino acids common to all forms of FRU (which lack the N-terminal, male-specific amino acids) led to immunostaining in the CNS of both males and females. It is inferred that anti-FRUCOM elicited staining of both non-sex-specific anti-FRUCOM proteins and FRUM in males and in females, only of anti-FRUCOM proteins produced under the control of promoters other than P1. Genetic-mosaic studies point to portions of the CNS involved in female-specific sexual behaviors, including an anterior brain region that perforce is included within the broad expression pattern of Fru in females. However, such female neural foci, including the anterior one that underlies receptivity to male mating attempts, may not require fruitless in order to form or function (Lee, 2000a).

In the CNS of females there should be no FRUM for anti-FRUM or anti-FRUCOM to detect, consistent with the patterns observed. Anti-FRUCOM was labeled by the latter reagent in many neural tissues of both sexes, but only during development. Here it is assumed, from the absence of anti-FRUM-like anti-FRUCOM immunostaining in the CNS of animals carrying fru deletions, that the neural signals labeled by anti-FRUCOM in adult males stem from FRUM alone. That protein type almost certainly is involved in regulating the behavior of adult males. However, the larval- and pupal-specific anti-FRUCOM proteins in the CNS could also have behavioral significance with respect to features of the CNS that have formed after either males or females have progressed through these developmental stages (Lee, 2000a).

This supposition implies that the reproductive behavior of females as well as males would be abnormal in fruitless mutants; however, no impairments of female courtship have been detectable, including that the mating receptivity of these mutants is normal. Yet, all the fru mutants tested as females can be inferred primarily to affect P1-promoted transcripts, either in terms of severe quantitative reductions (transposon mutants) or qualitative spatial-expression abnormalities (the effect of a chromosome breakpoint near the P1 promoter in the fru1 mutant). Those mRNAs encode FRUM, which is undetectable in females. At any developmental stage, no anti-FRUCOM-containing female counterparts could be detected of the CNS cells that express P1 transcripts with sharply defined, relatively high-level signals. Such mRNAs are translatable on paper, provided that the relatively downstream start codon is used in females. That this appears not to be the case (or that translated products of ORF-2 do not accumulate) suggests that P1-controlled female forms of Fru are not candidates for proteins that would be affected by the viable fru mutations in flies of this sex (Lee, 2000a).

Females are affected by certain genetic variations at the fru locus, in that the chromosomal breakpoints in question kill them, along with their brothers, during late developmental stages. These near-lethal effects are likely to be associated with the broad and non-sex-specific spatial patterns of anti-FRUCOM proteins. Indeed, the chromosomal lesions that cause severe decrements in viability eliminate all detectable Fru proteins, including the common forms produced under the control of three promoters located downstream of P1. Eliminating the P1 promoter from chromosomes [Df(3R)ChaM5 or Df(3R)4-40] that otherwise can encode some of the anti-FRUCOM proteins leads to the expected retention of such material. This rationalizes the viability of M5/P14 and 4-40/P14 flies, bearing in mind that the P1-retaining chromosome in these variants [Df(3R)P14] is missing most of fru's coding region. That these Df/Df males and females show much lower-than-normal levels of anti-FRUCOM-stained tissues is probably due to the fact that the M5 and 4-40 fru deletions are missing certain of the downstream promoters. In particular 4-40 is deleted of P2 and may possess a damaged P3; M5 could lack P2 or possess only a portion of that gene-regulatory region. At least one fru promoter (P4) is likely to be intact in both the M5 and 4-40 chromosomes (Lee, 2000a).

That regulatory region is far downstream of the P1 promoter, thus rather close to the bulk of fru's ORF. P4's action appears sufficient to allow for normal viability of the Df/Df flies, their reduced levels of anti-FRUCOM notwithstanding. Within the CNS of both sexes, the spatial distributions of fru transcripts are complex. There are so many ganglia and cells involved that is it difficult to determine whether the 'high-mRNA' cells in females have exact counterparts in males (Lee, 2000a).

Are the similarly labeled cells in males, which presumably correspond to FRUM-expressing neurons, present at all in females? In this regard, throughout the pupal period sexually dimorphic expression of mRNAs were detected whose production is controlled by the P1 promoter. These in situ hybridizations involved male-exclusive neural signals. Such sexually dimorphic features of the RNA spatial pattern arise just after the time when sex-specific forms of the gene products are first observed. These findings reveal that there is more to sex-specific expression of fruitless than the alternative RNA splicing of P1 transcripts that occurs under the control of the sex-determination hierarchy. One possibility is that the gene's transcriptional control is an additional component of its sex-specific regulation. For example, P1-promoted expression patterns could be different in males and females from the earliest stages of pupal development because of gene activation that would occur in the relevant CNS cells of males only. Alternatively, transcriptional activation of fru may be initiated identically in both sexes, with the sex-specific pattern being generated by repression of P1 transcription in certain cells of females or by selective cell death in the early-pupal CNS. These two possibilities are subsumed under the broader question of whether fruitless gene action is primarily involved in the structure of the CNS (including early aspects of sex-specific pattern formation) or the subcellular quality of its components (aspects of terminal cell differentiation). A further subsidiary question involves how the fruitless mutants might differ from wild type: are the former only devoid of the gene's expression in many or all of the normal CNS locations, or are they neuroanatomically defective as well? The inferred absence of P1-promoted, FRUM-encoding mRNA in some of the most severely affected but viable fru mutants (such as those carrying transposon inserts within the locus) makes an answer to this question by application of anti-FRU reagents problematical. If no FRUM protein are immunohistochemically detectable in a mutant such as fru3 (which is nearly null for P1-promoted mRNA), this could be because the relevant neurons did not form or underwent cell death during development. Alternatively, all the relevant neurons could be present in this mutant, but their connectivity or neurochemical qualities might be aberrant (Lee, 2000a).

Given the prominent expression of fruitless during the earliest stages of imaginal development, it is suspected that fruitless is crucially involved in 'early-formative' features of sex-specific neural development (whether or not the gene products also play a role at later stages). This conjecture includes the possibility that fru influences the presence versus absence of a CNS component in one sex or the other, as opposed to controlling only the fine features of neuronal differentiation. In either case, sex-specific anatomy in Drosophila needs to be understood at levels deeper than those involving certain gross ganglionic differences. How the reproductively relevant structures are formed and take on their detailed features must be apprehended by higher resolution examinations of females and males and of fru mutants (Lee, 2000a).

Neural circuitry that governs Drosophila male courtship behavior

Male-specific fruitless (fru) products (FruM) are both necessary and sufficient to 'hardwire' the potential for male courtship behavior into the Drosophila nervous system. FruM is expressed in ~2% of neurons in the male nervous system, but not in the female. The insertion of GAL4 was targeted into the fru locus, allowing visualization and manipulation of the FruM-expressing neurons in the male as well as their counterparts in the female. Evidence suggests that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent (Stockinger, 2005).

An important step toward understanding the neural basis of any behavior is to trace the neural circuits involved. This study characterizes at the level of single identifiable neurons, the circuitry that governs Drosophila male courtship behavior. These neurons are defined by their expression of fruGAL4, created by the targeted insertion of GAL4 into the fru locus. It is believed that fruGAL4 identifies most if not all of the neurons with sex-specific functions in courtship because male-specific FruM proteins are necessary and sufficient for courtship (Demir, 2005) and fruGAL4 includes all the neurons that express FruM. Synaptic silencing of these neurons impairs courtship behavior but leaves unrelated behaviors intact. Thus, the fruGAL4 neurons function directly in male courtship, and are largely dedicated to this behavior (Stockinger, 2005).

Of course, this is not to say that courtship only involves fruGAL4 neurons, nor that these neurons function only in courtship. Clearly, courtship also involves many neurons that do not express fruGAL4. However, these neurons most likely have more general functions, common to many behaviors and to both sexes. It is also possible that fruGAL4 neurons function in other behaviors that have not yet been examined, in particular other sex-specific behaviors such as aggression. Also, almost all of the fruGAL4 neurons have counterparts in females. The functions of these neurons in females are unknown (Stockinger, 2005).

How many of the fruGAL4 neurons are actually involved in male sexual behavior? At one extreme, just one or a few of the fruGAL4 neurons might be critical, with most fruGAL4 neurons having nothing to do with courtship. Alternatively, most or even all of the fruGAL4 neurons might be directly involved, each contributing in some way to the behavior. The latter scenario is favored. (1) There are no obvious examples of cells that express fruGAL4 but are clearly not involved in courtship. fruGAL4 is not detected at all in embryos nor during larval stages until shortly before pupariation, and even in adults it is confined to only a small fraction of neurons. (2) Distinct roles in sexual behavior have already been defined, or seem likely, for several subsets of these fruGAL4 neurons, such as the fruGAL4 ORNs and the fruGAL4 motor neurons that innervate the penis and ejaculatory bulb. It thus appears that male sexual behavior involves the contributions of many different fruGAL4 neurons, including neurons at each of the sensory, central, and motor levels (Stockinger, 2005).

Dissecting out the contributions of each of these neurons will require a means to manipulate defined subsets of fruGAL4 neurons, as was done in this study for the fruGAL4 ORNs by using antenna-specific FLP expression and FLP-dependent silencers. With the appropriate FLP reagents, it might be possible to extend this approach to other parts of the nervous system. Similarly, expression of GAL80, a GAL4 repressor, should allow the selective exclusion of specific subsets of fruGAL4 neurons. These approaches could also be combined, providing logical AND and NOT operations that might ultimately allow the operation of the entire fruGAL4 circuit to be examined, piece by piece (Stockinger, 2005).

fruGAL4 neurons are dispersed throughout the nervous system, generally comprising a small subset of neurons at each successive neural level. For example, fruGAL4 labels subsets of both first-order olfactory neurons (ORNs) and second-order olfactory neurons (probably PNs). Remarkably, the fruGAL4 ORNs innervate the same three glomeruli in the antennal lobe as the fruGAL4 PNs, indicating that they are most likely synaptic partners. Third-order olfactory neurons are located in the superior protocerebrum and mushroom bodies, and here too subsets of neurons express fruGAL4. Thus, it is possible that a “fruGAL4 connects to fruGAL4” principle might even extend into higher brain centers, and perhaps even continue through to the descending pathways and motor neurons that express fruGAL4. Clearly, fruGAL4 neurons also make synaptic contact with neurons that do not express fruGAL4, many of which will also have important (but general) roles in the neural processing that drives male courtship. It is speculated that many if not all of the neurons with sex-specific roles in courtship express fruGAL4 (and normally FruM), and that these neurons may be directly interconnected in a circuit that extends from sensory input through to motor output. A precedent for this organization, albeit for a much simpler behavior, is the connectivity of sensory and motor neurons that express the same Ets transcription factors in the vertebrate monosynaptic spinal reflex circuit (Stockinger, 2005).

In >Drosophila, as in many other animals, male mating behavior is triggered by sex pheromones emitted by the female. Drosophila females produce both volatile (long-range) and nonvolatile (contact) sex pheromones. Volatile pheromones are thought to stimulate courtship behavior, whereas nonvolatile pheromones may facilitate sex and species discrimination. The major nonvolatile female pheromones are 7,11-heptacosadiene and 7,11-nonacosadiene, for which Gr68a is a candidate receptor. The volatile pheromones have not yet been identified nor have the receptors or neurons that detect them. It is postulated that these pheromones are detected by the fruGAL4 ORNs. This conclusion rests on two main lines of evidence. (1) Sensory stimuli of particular significance often have enlarged representations in the brain, and so the fact that the fruGAL4 ORNs innervate glomeruli that are larger in males than in females suggests that the odors they detect are more important to males than to females. (2) Selective silencing of these fruGAL4 ORNs severely impairs courtship behavior, both in males exposed to normal females, and in fruM females exposed to males that emit female pheromones (Stockinger, 2005).

Just as the fruGAL4 ORNs appear to comprise a distinct class of 'specialist' ORNs involved in pheromone detection, the fruGAL4 PNs are also distinct from the 'generalist' PNs, many of which are labeled by GH146-GAL4. This suggests that the processing of pheromones and general odors is anatomically segregated in the Drosophila brain, just as it is for example in rodents and fish. This segregation may not be complete, however, as the glomeruli targeted by fruGAL4 ORNs and PNs are also innervated by GH146-GAL4 PNs, and silencing the GH146-GAL4 neurons also inhibits male courtship (as well as other olfactory behaviors) (Stockinger, 2005).

What is the role of fru in this olfactory circuit? fru is not required for the synaptic specificity of fruGAL4 ORNs and PNs, nor for the expression of putative pheromone receptors in the fruGAL4 ORNs. fru is however responsible for the sex differences in the size of each of the three glomeruli targeted by fruGAL4 ORNs and PNs. This sexual dimorphism is probably due to fru function in the ORNs rather than the PNs, since genetic feminization of ORNs reduces the size of at least two of these glomeruli in males. Similarly, for the sphinx moth Manduca sexta, the enlargement of pheromone-processing glomeruli in males also depends on the sex of the antenna, not of the brain (Stockinger, 2005).

Behavioral differences between males and females reflect sex differences in neural function. An important question, for any species, is whether the essential difference between the sexes lies primarily in their neuroanatomy or their neurophysiology. In some species, sexually dimorphic behaviors correlate with striking differences in neuroanatomy. For example, in some songbirds, such as the zebra finch, only males sing, and brain regions involved in the acquisition and performance of the song are much larger in males than females. However, in many other species, including humans and mice, sex differences in neuroanatomy are much more subtle, and their functional significance, if any, is still unknown (Stockinger, 2005).

For Drosophila sexual behavior, the reason why males court but females do not must reflect some sex-specific property of the fruGAL4 neurons. It is thought that it is not their gross anatomy. With the trivial exception of neurons innervating the reproductive organs, only subtle differences are detected in the numbers of these neurons and no differences at all in their morphologies or projections (see however Kimura, 2005). Pending further studies at higher resolution, it is tentatively concluded that sex differences in courtship behavior do not rest on differences in the production, survival, or connectivity of the neurons involved (Stockinger, 2005).

This conclusion offers a rather sobering perspective on the considerable effort that continues to be devoted to identifying and characterizing sexual dimorphisms in the mammalian brain. In Drosophila, the sexual behaviors of males and females are dramatically different and highly stereotyped; this difference can be attributed to a single splicing event in a single gene, and the neurons that express this gene can be examined at single-cell resolution. Yet even under these ideal circumstances, anatomical differences that might account for the dramatically different sexual behaviors of males and females have not yet been found. This suggests that differences in neural chemistry, rather than gross neuroanatomy, might underlie the profound differences in behavior between males and females in Drosophila, and surely in many other species as well (Stockinger, 2005).

If the essential difference between the sexes in Drosophila lies in the physiology of the fruGAL4 neurons, then it is necessary to begin to search for this difference. fruGAL4 itself will be a powerful tool in this endeavor. Coupled with optical indicators of neuronal activity and tools such as FLP and GAL80 to highlight specific subsets of fruGAL4 neurons, it should now be possible to look for sex differences in the patterns of neuronal activity elicited by sexual stimuli. Thus, by defining the neural circuit that governs male sexual behavior and providing a tool for its manipulation, this work paves the way for a mechanistic investigation of a complex innate behavior. It is now possible to begin to explore how this circuit operates, why it operates differently in males and females, and how this difference is programmed during development (Stockinger, 2005).

Obesity-blocking neurons in Drosophila

In mammals, fat store levels are communicated by leptin and insulin signaling to brain centers that regulate food intake and metabolism. By using transgenic manipulation of neural activity, the isolation is reported of two distinct neuronal populations in flies that perform a similar function, the c673a-Gal4 and fruitless-Gal4 neurons. When either of these neuronal groups is silenced, fat store levels increase. This change is mediated through an increase in food intake and altered metabolism in c673a-Gal4-silenced flies, while silencing fruitless-Gal4 neurons alters only metabolism. Hyperactivation of either neuronal group causes depletion of fat stores by increasing metabolic rate and decreasing fatty acid synthesis. Altering the activities of these neurons causes changes in expression of genes known to regulate fat utilization. These results show that the fly brain measures fat store levels and can induce changes in food intake and metabolism to maintain them within normal limits (Al-Anzi, 2009).

This paper describes the isolation of two distinct populations of Drosophila brain neurons that regulate fat deposition. These populations, denoted as c673a-Gal4 and Fru-Gal4, were identified by using Gal4 driver lines to express neuronal silencing or hyperactivating genes. For both neuronal populations, silencing produces obesity, defined as excess fat deposition, and hyperactivation produces leanness, defined as a reduction in fat store levels. Silencing and hyperactivation affect the expression of genes that are likely to be regulators of fat storage. However, the observed phenotypes are unlikely to be mediated by signaling through receptors for NPY-like or insulin-like peptides, which are important regulators of growth, feeding, and fat deposition (Al-Anzi, 2009).

The two populations have only a few neurons in common, and the analysis suggests that the shared neurons are not responsible for the observed phenotypes. Metabolic analysis shows that the two populations affect fat deposition by different mechanisms. The obesity phenotype produced by silencing is reversible (Al-Anzi, 2009).

Reduced use of fat stores and increases in de novo fatty acid synthesis correlate with the obesity phenotype when either Fru-Gal4 or c673a-Gal4 neurons are silenced, and c673a-Gal4-silenced animals also consume excess food. Conversely, the depletion of fat stores that occurs when either neuronal population is hyperactivated is likely to be caused by increased metabolism and decreases in de novo fatty acid synthesis. Interestingly, when Fru-Gal4 neurons, but not c673a-Gal4 neurons, are hyperactivated, the animals enter a state in which they use protein precursors to synthesize carbohydrates, and probably catabolize their own proteins via autophagy. This suggests that Fru-Gal4 hyperactivated animals are in a state of perceived starvation, despite the fact that they consume a normal amount of food (Al-Anzi, 2009).

Fru-Gal4 is expressed in a large number of brain neurons. Fru-Gal4-silenced flies accumulate excess fat despite consuming less food and are less obese than c673a-Gal4 silenced flies, which consume more food. These facts suggest that driving the silencing gene with Fru-Gal4 might have two opposing effects. Neurons that are positive regulators of feeding might be silenced as well as neurons that sense fat store levels. Because of this, the flies might reduce food intake, which would decrease the severity of the obesity phenotype that would have been produced by silencing only the fat-sensing subset of the Fru-Gal4 neurons (Al-Anzi, 2009).

Alternatively (or in addition), Fru-Gal4-silenced flies might still be able to detect an increase in their fat stores and respond to it by decreasing feeding. However, the decrease would be insufficient to prevent the accumulation of excess fat that is driven by the metabolic changes occurring when Fru-Gal4 neurons are silenced. c673a-Gal4-silenced flies probably cannot sense fat store levels at all, since they consume more food despite having an excess of energy reserves (Al-Anzi, 2009).

The different defects underlying the obesity phenotype when the two neuronal populations are silenced and the observation that there is very little overlap between these populations suggest that they are parts of two independent neural circuits. It is speculated that c673a-Gal4 and Fru-Gal4 neurons may have different roles in the wild, regulating fat stores in response to different environmental or internal stimuli. Since silencing of c673a-GAL4 neurons increases food intake, the activity of these neurons might be turned down under unfavorable environmental conditions in order to increase the ability of the flies to accumulate additional energy stores. For Fru-Gal4 neurons, whose hyperactivation induces an autophagic state, it is speculated that activity might be increased under severe starvation conditions to allow the utilization of cellular protein as an energy source (Al-Anzi, 2009).

Lipid metabolism is essential for generating much of the energy needed during periods of starvation. In Drosophila, stored fats are released from the fat body through the activity of lipases such as Bmm lipase. This is in turn causes the accumulation of fat molecules in the oenocytes, where they will be further metabolized through the activity of cytochrome P450 proteins such as Cyp4g1. It was observed that altering the activity of Fru-Gal4 neurons affected the expression levels of the cyp316a1, cyp4g1, and bmm lipase genes, while neural activity of c673a-Gal4 only affects cyp316a1 levels. Cyp316a1 is a cytochrome P450 that is closely related to Cyp4g1, and although its role in fat metabolism has not been studied, the fact that it belongs to the same cytochrome c family as Cyp4g1 indicates that it might have similar functions. Perturbation of both neuronal groups affects fatty acid synthesis by inducing changes in the expression of acetyl CoA-carboxylase, the main regulatory enzyme of the de novo fatty acid synthesis pathway (Al-Anzi, 2009).

In mammals, hypothalamic brain centers such as the ventromedial nuclei (VMN), paraventricular nuclei (PVN), and the lateral hypothalamic area (LHA) are informed about the status of body fat storage by the leptin and insulin pathways. These centers respond by inducing changes in food intake and metabolism that maintain constant body weight. Electrical stimulation of VMN or PVN neurons suppresses food intake, while bilateral lesions of VMN or PVN cause hyperphagia and obesity (Al-Anzi, 2009).

Leptin and insulin circulating in the bloodstream affect the activity of neurons in the arcuate nucleus of the hypothalamus (ARN). ARN is located in an area with a reduced blood-brain barrier, thus endowing it with the ability to sense leptin, insulin, and circulating nutrient levels. A subset of ARN neurons express the leptin receptor. ARN axons project to VMN, PVN, and LHA, and thereby communicate the status of fat stores to these feeding centers (Al-Anzi, 2009).

It is unknown whether the fly brain has feeding centers with equivalent roles to these mammalian hypothalamic nuclei. The 673a-Gal4 and Fru-Gal4 populations are dispersed throughout the brain, so the locations of neurons expressing these drivers do not indicate that any particular region of the brain is central to regulation of fat storage. However, the phenotypes produced by silencing and hyperactivation of these populations suggest that, like mammalian hypothalamic nuclei, they respond to humoral signals made by adipocytes that report on fat store levels. In particular, the fact that the obesity phenotype caused by silencing is reversible and that previously silenced flies dramatically reduce food consumption in order to reduce fat stores back to normal levels suggest that adipocytes alter release of a humoral factor when their fat content changes. The levels of this humoral factor are interpreted by the c673a-Gal4 and Fru-Gal4 neurons and used to control food consumption. Thus, flies that have accumulated excess fat stores during the silencing period communicate this fact to the brain, and brain neurons respond by reducing caloric intake when the activity block is released (Al-Anzi, 2009).

The isolation of c673a-Gal4 and Fru-Gal4 neurons in Drosophila should allow the future identification of genes involved in brain/fat store communication, possibly including those encoding the putative adipocyte humoral factor(s). This might be done by examining the consequences of transgenic expression of components of candidate signaling pathways in these neurons on flies' fat stores or by finding transcripts selectively expressed in them. The role of such genes in regulating fat storage could then be tested by RNAi or overexpression. The expression patterns of functionally validated genes could, in turn, more precisely identify which neurons within both populations are required for regulation of fat storage and what receptors they use to detect circulating humoral regulators that convey information about fat store levels (Al-Anzi, 2009).

Sensory neurons in the Drosophila genital tract regulate female reproductive behavior

Females of many animal species behave very differently before and after mating. In Drosophila, changes in female behavior upon mating are triggered by the sex peptide (SP), a small peptide present in the male's seminal fluid. SP activates a specific receptor, the sex peptide receptor (SPR), which is broadly expressed in the female reproductive tract and nervous system. This study pinpoints the action of SPR to a small subset of internal sensory neurons that innervate the female uterus and oviduct. These neurons express both fruitless (fru), a marker for neurons likely to have sex-specific functions, and pickpocket (ppk), a marker for proprioceptive neurons. SPR expression in these fru+ ppk+ neurons is both necessary and sufficient for behavioral changes induced by mating. These neurons project to regions of the central nervous system that have been implicated in the control of reproductive behaviors in Drosophila and other insects (Häsemeyer, 2009).

SPR was initially identified in a genome-wide pan-neuronal RNAi screen. In this screen, the panneuronal elav-GAL4 driver was crossed to a genome-wide collection of RNAi transgenes, and female progeny were scored for egg-laying defects. Mated elav-GAL4 UAS-SPR-IR females lay very few eggs and remain sexually receptive, and thus, like SPR null mutants, behave as though they were still virgins. To define the cellular requirement for SPR function, the logic of this screen was inverted, crossing the UAS-SPR-IR transgene to a collection of 998 GAL4 lines and scoring the female progeny for egg-laying defects in the same fashion. In each of these lines, the GAL4 transcriptional activator is expressed in a random but stereotyped subset of cells, in which SPR function should now be inhibited by the UAS-SPR-IR transgene (Häsemeyer, 2009).

Fifty-nine lines were identified that resulted in a strong and reproducible egg-laying defect. Many of these lines were found to be broadly expressed, as revealed with a UAS-mCD8-GFP reporter. These lines were not examined further. More restricted neuronal expression was observed in seven lines, and for each of these a series of secondary assays was performed to confirm the egg-laying defect and to assess the receptivity of both virgin and mated females. For all seven GAL4 lines, SPR knockdown resulted in reduced egg laying and increased remating of mated females, but little if any change in the receptivity of virgin females. These defects were indistinguishable from those observed upon panneuronal SPR knockdown with the elav-GAL4 driver, or in SPR null mutant females. For the most restricted of the positive GAL4 lines, ppk-GAL4, it was confirmed that these defects can indeed be attributed to a diminished response to SP. It was then determined that SPR is required in ppk+ sensory neurons in the female reproductive tract (Häsemeyer, 2009).

This study describes a set of internal ppk+ fru+ sensory neurons in the female reproductive tract and provides evidence that SPR functions in these neurons to trigger the behavioral changes induced by SP upon mating. This conclusion rests on two complementary sets of observations. First, SPR is required in both ppk+ and fru+ cells, because postmating responses are eliminated upon knockdown of SPR in either cell population. Second, SPR is sufficient in either ppk+ or fru+ cells alone, as expression in either restores the postmating response in SPR null mutant females. This forces the conclusion that SPR acts exclusively in cells that are both ppk+ and fru+. The sensory neurons innervating the uterus are the only cells that were identified that express both of these markers. There are typically four to six such cells, and it is not yet known if they are functionally equivalent, or if egg laying and receptivity are regulated by two distinct cell subtypes (Häsemeyer, 2009).

Silencing synaptic transmission of ppk+ fru+ neurons mimics the activity of SP, in that they both cause virgin females to become unreceptive and initiate egg laying. Thus, an attractive hypothesis is that activation of SPR by SP reduces the synaptic output of these neurons. Like other ppk+ neurons, the ppk+ fru+ uterus neurons are probably mechanosensory. They may therefore have an important function as uterus stretch receptors in the coordination of sperm transfer, fertilization, and egg release. They may have two distinct functional states, depending on the presence or absence of SP. Because receptivity can be genetically uncoupled from egg production and egg laying, it is inferred that SP can also act independently of any stretch signal in the uterus. Modulation of receptivity and egg laying might be mediated through either distinct ppk+ fru+ subtypes or distinct central synapses (Häsemeyer, 2009).

How might SP regulate these sensory neurons? Two possibilities are envisioned. First, the ppk+ fru+ neurons may detect SP in the reproductive tract and alter their firing rate accordingly. In this model, passage of SP into the hemolymph would not be required to induce the postmating response. A second possibility is that SP enters the circulatory system and acts presynaptically to modulate the release of these neurons at their central targets. The fact that SP can indeed be detected in the hemolymph of mated females does not in itself exclude the former possibility. At least some effects of SP, such as stimulating juvenile hormone synthesis in the corpus allatum, probably do require SP to enter the hemolymph. Similarly, the fact that SP triggers a postmating response even when injected directly into the hemolymph is also consistent with either model. The somata and some processes of the ppk+ fru+ neurons lie outside the uterus and would be readily accessible to factors in the hemolymph. A neural rather than a circulatory route has been proposed to mediate postmating responses in several species of moths. However, this conclusion is based upon the loss of this response upon nerve cord transection, a result predicted by both of these models. Thus, both models are consistent with currently available evidence from studies in Drosophila and other species, and distinguishing between them will require detailed studies of the physiological properties of the ppk+ fru+ neurons in response to SP (Häsemeyer, 2009).

The central targets of the ppk+ fru+ sensory neurons include the abdominal and/or subesophageal ganglia -- regions of the CNS likely to contain circuits that mediate behavioral responses to mating. The abdominal ganglion houses the octopaminergic neurons that are believed to regulate the release and passage of mature eggs from the ovary to the uterus. It is suspected that these neurons are direct or indirect targets of the ppk+ fru+ sensory neurons and that these circuits serve to ensure that ovulation and oviposition are coordinated with the presence of sperm (Häsemeyer, 2009).

Some ppk+ fibers project from the abdominal trunk nerve right through to the SOG, potentially forming a direct neural connection from the reproductive tract to the brain. It is suspected that these projections may feed into circuits that regulate female receptivity and other postmating behaviors. Virgin females are enticed to mate by the male's courtship song. Most auditory sensory neurons project to the mechanosensory neuropil in the lateral SOG, close to the terminal arborizations of the ppk+ neurons. The proximity of the auditory processing centers and the ascending ppk+ projections raises the attractive possibility that mating modulates an early step in song processing. The SOG also contains processes of the Ilp7 neurons, which function in egg-laying site selection after mating. Direct evidence for mating-induced changes in SOG circuit function is lacking in flies but has been obtained in other insects. In some species of moth, mating induces a long-term inhibition of the SOG neurosecretory cells that regulate female pheromone biosynthesis, making mated females less attractive to other males (Häsemeyer, 2009).

Having identified sensory neurons that detect SP in the reproductive tract, it will now be important to characterize the central pathways that process these signals to regulate female behavior. In the olfactory system, sensory neurons that detect pheromones are fru+, as are their postsynaptic partners in the brain. Given that the sensory neurons that detect SP are also fru+, and many fru+ neurons are also located in both the abdominal and subesophageal ganglia, it is enticing to think that a similar logic may apply in these pathways too. Elucidating the operation of these circuits should reveal how the female CNS integrates both external and internal information to switch between two very different behavioral patterns (Häsemeyer, 2009).

Neural circuitry underlying Drosophila female postmating behavioral responses

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. This study demonstrates an essential role played by neurons expressing the sex-determination gene doublesex (dsx) in regulating the female postmating response. Shared circuitry was uncovered between dsx and a subset of the previously described SP-responsive fru+/ppk+-expressing neurons in the reproductive system. In addition, sexually dimorphic dsx circuitry was identified within the abdominal ganglion (Abg) that was critical for mediating postmating responses. Some of these dsx neurons target posterior regions of the brain while others project onto the uterus. It is proposed 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 (Rezával, 2012).

These results show that in the female, dsx neurons associated with the internal genitalia not only form a component part of the previously described fru+/ppk+ network, but in fact define a more minimal SP-responsive neural circuit capable of inducing postmating changes, such as reduced receptivity, increased levels of rejection, and egg deposition (Rezával, 2012).

In addition to these 'classic' postmating behavioral responses, it was also noted that SP signaling to dsx neurons induces postmating changes in locomotor activity between unmated and mated females. Studies have shown that Drosophila males court immobilized females less than moving females; essentially, males react to changes in female locomotion, suggesting a causal link between female locomotion and increased courtship levels. It has been proposed that males are 'acoustically tuned' to signals generated by active females, stimulating increased courtship by changing the attention state of the male. Therefore, female mobility appears to contribute to her 'sex appeal' and decreased locomotion in mated females is likely to affect the male's willingness to copulate (Rezával, 2012).

The female's nervous system must have the capacity to receive, and interpret, postcopulatory signals derived from the male seminal package to direct physiological and behavioral responses required for successful deposition of fertilized eggs. It was demonstrated that two dsx clusters, composed of three bilateral neurons of the uterus, comprise a more defined component of the SP-responsive sensory circuit. In addition, the majority of other dsx neurons originating on the internal genitalia were shown to coexpress ppk. As ppk neurons are mechanosensory, these may be acting as uterine stretch receptors, facilitating sperm and egg transport, fertilization, and oviposition. Silencing neural function of ppk neurons appears to inhibit egg deposition, presumably by impeding egg transport along the oviducts. Similarly, in dsxGal4 females expressing TNT no egg deposition is ever observed, with unfertilized eggs atrophying in the lateral oviducts. In contrast, when fru+ neurons are silenced, deposition of successfully fertilized eggs is still observed, suggesting that different subsets of the dsx+/fru+/ppk+ SP-responsive sensory circuit may direct distinct postmating behavioral responses. As SP has been detected in the hemolymph of mated females, it has been suggested that this peptide could pass from the reproductive tract into the hemolymph to reach CNS targets. The fact that neither receptivity nor oviposition was restored to control levels when ppk-Gal80 (or Cha-Gal80) was expressed in dsxGal4/UAS-mSP flies opens the possibility that SP expression might affect additional dsx neurons in the CNS (Rezával, 2012).

Triggering of postmating responses via SP reception appears to occur via a small number of neurons expressing SPR on the female reproductive tract; however, SPR is also found on surface regions of the CNS as well as in endocrine glands and other reproductive tissues. Surprisingly, SPR may even be detected in the Drosophila male CNS, where no exposure to SP would be expected, and in insects that apparently lack SP-like. SPRs are therefore potentially responsive to other ligands, performing functions other than those associated with postmating responses in the diverse tissues in which SPRs are expressed (Rezával, 2012).

Extensive coexpression was found of dsx-expressing cells and SPR in the epithelium of the lower oviduct and spermathecae in females. However, mSP expression (or SPR downregulation) specifically in spermathecal secretory cells (SSC) or oviduct epithelium cells had no effect on receptivity or egg laying. In agreement with rescue experiments using neuronal Gal80 drivers to intersect Gal4-responsive UAS expression in dsx cells, this suggests that these cells are neither neuronal nor directly involved in SP-mediated postmating behaviors. SPR staining in the CNS was more difficult to determine given the limitations of the antibody; while no colocalization in the brain was observed, apparent coexpression was observed between SPR and a small subset of ventral (Rezával, 2012).

The results indicate that dsx-Abg neurons are required for the induction and regulation of specific components of the postmating response. It has been shown that inhibition of neurotransmission in apterous-expressing Abg neurons impairs SP-mediated postmating changes in receptivity and oviposition, emphasizing the importance of these neurons in the modulation of postmating responses (Rezával, 2012).

The level of dsx neuronal expression within the Abg and their associated fascicles projecting to the brain, where they form extensive presynaptic arborizations within the SOG, coupled with the effects that impairment of function in these neurons has on postmating responses, speaks to the involvement of these neurons in relaying information from the reproductive tract to the brain. That dsx-Abg neurons also project, and form presynaptic arborizations on the uterus, and that the effects on postmating responses when their function is impaired again argue that these neurons play a direct role in mediating processes such as egg fertilization and oviposition. Interestingly, most dsx intersecting neurons are specific to females. Sex-specific behaviors can arise from either shared circuits between males and females that operate differently and/or sex-specific circuits that result from the presence/absence of unique circuit components in one sex versus the other. The results support the latter (Rezával, 2012).

The VNC has been implicated in the modulation of postmating responses, with an identified focus specifically involved in ovulation and transfer of eggs into the uterus for fertilization. Octopaminergic modulatory neurons located at the distal tip of the VNC projecting to the reproductive tract are required for triggering ovulation, possibly by regulating muscle contractions in the ovaries and oviducts. Since earlier studies have shown that ablation of the pars intercerebralis revealed an additional focus for egg laying in the head, and the brain appears to be required for sexual behaviors, such that decapitated virgin females neither mate nor lay eggs, it seems likely that neurons in the Abg also require signals from the brain to regulate postmating responses such as egg transport, fertilization, and deposition (Rezával, 2012 and references therein).

Higher-order circuits in the female brain must be capable of integrating sensory inputs from the olfactory, auditory, and reproductive systems to decide between the alternative actions of acceptance or rejection of the male. Early gynandromorph studies mapped a region of the dorsal brain that must be female for an animal to be receptive; it has been recently shown that the majority of dsx neuronal clusters are located in this region. While neurons coexpressing dsx and fru in male brains define a more restricted circuitry for determining male mating decisions, in females no overlap between dsx+ and fru+ neurons is observable in the brain. It is also important to note that the sex-specific Fru isoform is absent in females; thus any circuits that are actively specified in the female are likely to depend on the female isoform DsxF. Most dsx neurons in the brain are found in the lateral protocerebrum, a region where multiple sensory inputs are thought to be integrated and discrete motor actions selected and coordinated. Further high-resolution functional and connectivity mapping will help to define which neurons participate in specific pre- and postmating behaviors in the female, allowing circuit architecture to be integrated with underlying cellular and synaptic properties. Future experiments will define what activity patterns trigger these behaviors and what activity patterns correlate with these behaviors (Rezával, 2012).

An olfactory receptor for food-derived odours promotes male courtship in Drosophila

Many animals attract mating partners through the release of volatile sex pheromones, which can convey information on the species, gender and receptivity of the sender to induce innate courtship and mating behaviours by the receiver. Male Drosophila melanogaster fruitflies display stereotyped reproductive behaviours towards females, and these behaviours are controlled by the neural circuitry expressing male-specific isoforms of the transcription factor Fruitless (FRUM). However, the volatile pheromone ligands, receptors and olfactory sensory neurons (OSNs) that promote male courtship have not been identified in this important model organism. This study describes a novel courtship function of Ionotropic receptor 84a (IR84a), a member of the chemosensory ionotropic glutamate receptor family, in a previously uncharacterized population of FRUM-positive OSNs. IR84a-expressing neurons are activated not by fly-derived chemicals but by the aromatic odours phenylacetic acid and phenylacetaldehyde, which are widely found in fruit and other plant tissues that serve as food sources and oviposition sites for drosophilid flies. Mutation of Ir84a abolishes both odour-evoked and spontaneous electrophysiological activity in these neurons and markedly reduces male courtship behaviour. Conversely, male courtship is increased -- in an IR84a-dependent manner -- in the presence of phenylacetic acid but not in the presence of another fruit odour that does not activate IR84a. Interneurons downstream of IR84a-expressing OSNs innervate a pheromone-processing centre in the brain. Whereas IR84a orthologues and phenylacetic-acid-responsive neurons are present in diverse drosophilid species, IR84a is absent from insects that rely on long-range sex pheromones. Our results suggest a model in which IR84a couples food presence to the activation of the fruM courtship circuitry in fruit flies. These findings reveal an unusual but effective evolutionary solution to coordinate feeding and oviposition site selection with reproductive behaviours through a specific sensory pathway (Grosjean, 2012).

While mapping the projections of Ionotropic receptor (IR)-expressing OSNs to the primary olfactory centre, the antennal lobe, it was observed that an Ir84a reporter was labelling neurons innervating the VL2a glomerulus. VL2a is one of only three glomeruli that are larger in males and whose OSN inputs and projection neuron outputs express male-specific isoforms of the behavioural sex determination gene fruitless (fruM). fruM-expressing OSNs have been implicated in promoting male sexual behaviours, because inhibition of synaptic transmission in all of these neurons simultaneously reduces male courtship of female. The expression of Ir84a in fruM-expressing neurons was confirmed by visualizing the co-expression of an Ir84a reporter, as well as endogenous Ir84a transcripts, with a fruM reporter. No sexual dimorphism was observed either in the number of Ir84a-expressing cells or in their targeting to VL2a, indicating that FRUM does not have an essential role in the development of these neurons, similar to other fruM-expressing OSNs (Grosjean, 2012).

A GAL4 knock-in null allele, Ir84aGAL4 was generated. Ir84aGAL4/+ heterozygotes expressed a GAL4-responsive, membrane-targeted, green fluorescent protein (GFP) transgene (UAS-mCD8:GFP) exclusively in Ir84a-expressing OSNs. In Ir84aGAL4 homozygotes, the endogenous expression of Ir84a was lost, but the distribution and dendritic projections of these neurons, as revealed by mCD8:GFP, was unaffected. The axons of Ir84a-expressing neurons in heterozygous and homozygous Ir84aGAL4 flies projected only to VL2a. Ir84a is therefore dispensable for the specification and wiring of the neurons in which it is expressed. An amino-terminal enhanced GFP (EGFP)-tagged version of this receptor (Abuin, 2011) localized to the cell bodies and the ciliated dendritic endings of these neurons but not to their axon termini, consistent with an exclusive role for IR84a as an olfactory receptor in the fruM circuitry (Grosjean, 2012).

The responses of IR84a-expressing neurons to chemicals produced by male or virgin female flies was tested, both by delivering headspaces of flies from a distance (simulating the action of volatile pheromones) and by presenting extracts from fly cuticles at close range (mimicking exposure to non-volatile hydrocarbons, such as contact pheromones. These stimuli produced no or extremely small responses, as detected by extracellular recordings in ac4 sensilla, which belong to the class of olfactory hair that houses IR84a-expressing neurons, as well as OSNs that express IR75d, or IR76a and IR76b. These observations suggest that IR84a is not tuned to fly-derived pheromones. Therefore 163 structurally diverse odours were tested. Only three of these gave responses of >50 spikes s−1 above basal activity: phenylacetaldehyde, phenylacetic acid and phenylethylamine. Dose response curves that revealed sensitivity to these ligands are similar in both sexes (Grosjean, 2012).

In Ir84aGAL4 homozygous mutants, the responses to phenylacetic acid and phenylacetaldehyde were completely abolished. Re-introduction of Ir84a function in these neurons, by using UAS-Ir84a or UAS-EGFP:Ir84a cDNA transgenes, rescued these phenotypes, indicating a cell-autonomous function for IR84a in mediating these odour responses. By contrast, responses to phenylethylamine were unaffected, corroborating the evidence that this chemical is detected by the neurons that express both IR76a and IR76b. Consistent with these loss-of-function data, misexpression of IR84a in Odorant receptor 35a (OR35a)-expressing neurons was sufficient to confer responsiveness to phenylacetic acid and phenylacetaldehyde. The basal activity in Ir84a mutant ac4 sensilla was also lower than that in the ac4 sensilla of wild-type and rescue genotypes, indicating that IR84a has a role in promoting spontaneous firing (Grosjean, 2012).

Phenylacetic acid and phenylacetaldehyde are aromatic compounds found in a diverse range of fruit and other plant tissues, as well as in their fermentation products, and they are used in human perfumes for their floral, honey-like, sweet smell. The presence of these chemicals in two host fruit for drosophilid flies, overripe bananas and the prickly-pear cactus Opuntia ficus-indica, as well as in laboratory Drosophila medium, was confirmed by using gas chromatography- mass spectrometry analysis. The ubiquity of phenylacetic acid in vegetal tissues may be linked with its activity as a growth-regulating auxin and/or its production by plant-associated microorganisms. Small, but reproducible, quantities of phenylacetic acid and phenylacetaldehyde were also detected in whole-body cuticular extracts of male and virgin female D. melanogaster. The similarity in the relative amounts of these chemicals in laboratory medium and fruitfly extracts suggested that these chemicals are transferred from food to flies during their culture. 'Clean' cuticular extracts from animals grown on a minimal medium containing only sucrose and agarose consistently contain no detectable phenylacetaldehyde or phenylacetic acid (Grosjean, 2012).

The expression of IR84a in fruM-expressing neurons implicates this receptor in the regulation of male courtship. Indeed, in single-pair courtship assays, Ir84aGAL4 mutant males court wild-type females significantly less than do wild-type males. This phenotype was observed using both decapitated virgin females (which do not produce feedback signals) and in more natural conditions, with intact females together with food. Most individual components of the courtship ritual were affected in Ir84a mutant flies. These defects were rescued with a UAS-Ir84a transgene, confirming that they result from the absence of IR84a in OSNs. The observed reduction in male heterosexual courtship index (~50%) is highly comparable to the phenotype of flies in which all FRUM-positive OSNs are silenced, suggesting that IR84a-expressing neurons are the major olfactory fruM channel contributing to this behaviour. Residual courtship is presumably stimulated by other sensory modalities, such as taste. Male wild-type D. melanogaster also show a low level of courtship towards other males, and this homosexual courtship was also markedly reduced in Ir84aGAL4 mutants. By contrast, Ir84aGAL4 mutant females did not show overt defects in reproductive behaviours, including copulation latency, success or duration (Grosjean, 2012).

In innate olfactory preference assays, Ir84aGAL4 mutant flies still show robust avoidance of acetic acid, indicating that they do not have a general impairment in sensory detection. By contrast, no obvious responses of flies to phenylacetic acid was observed, suggesting that this food-derived odour is not a volatile stimulus that attracts flies but is a salient cue at close range. Notably, phenylacetic acid has a low vapour pressure compared with other fruit volatiles (for example, ethyl butyrate). The observation that courtship is reduced in Ir84aGAL4 mutants in assays in which only small amounts of phenylacetic acid are present on fly cuticles raises the possibility that spontaneous activity of these neurons also contributes to establishing a basal courtship level, which is abolished in the absence of IR84a (Grosjean, 2012).

To test whether IR84a ligands are sufficient to promote courtship, the assay was adapted by using killed female objects (which males court at only low levels) and by replacing the base of the chamber with gauze, beneath which a filter paper treated with odour or solvent was placed. Perfuming with phenylacetic acid nearly doubled the courtship index of wild-type flies compared with a solvent control. This effect was abolished in Ir84aGAL4 mutants and could be restored, albeit not fully, by introducing a UAS-Ir84a transgene. By contrast, ethyl butyrate, which does not activate IR84a, did not increase courtship. The courtship chamber was also perfumed with Drosophila food—which contains phenylacetic acid, and this complex olfactory stimulus was observed to induced IR84a-dependent increases in male courtship behaviour (Grosjean, 2012).

The other fruM-expressing OSN populations express either OR67d, which is a receptor for the antiaphrodisiac male pheromone cis-vaccenyl acetate, or OR47b, which is activated by unidentified fly-derived odours from both sexes and may participate in mate localization. How IR84a sensory information is integrated with these pheromonal pathways was examined by visualizing the axons of projection neurons innervating the VL2a (IR84a), VA1lm (OR47b) and DA1 (OR67d) glomeruli, which carry sensory information to the mushroom body and lateral horn. Images of single-labelled projection neurons of different glomerular classes were registered onto a common reference brain. DA1 and VA1lm excitatory projection neurons target an anterior-ventral pheromone-processing region of the lateral horn, which is segregated from projection neurons that are responsive to general food odours. Importantly, it was found that VL2a projection neurons—and no other IR-expressing projection neuron class are highly interdigitated with pheromone pathways and not food pathways. Indeed, VL2a projection neuron axon terminals overlap more strongly with VA1lm projection neurons than any of the other 44 projection neuron classes, consistent with projection neurons of both of these classes transmitting courtship-promoting sensory signals. The VL2a, DA1 and VA1lm inhibitory projection neurons were observed to overlap to a similar extent. The anatomical convergence of combinations of excitatory and inhibitory inputs from VL2a, VA1lm and DA1 projection neurons may allow the integration of olfactory signals by fruM-expressing third-order neurons (Cachero, 2010; Yu, 2010) to control male courtship behaviour (Grosjean, 2012).

Many olfactory IRs are conserved in insectsand may detect odours that are important for all species. By contrast, although IR84a orthologues are present in ecologically diverse drosophilids, they are absent from other Diptera and more divergent insects. In the cactophilic species Drosophila mojavensis, coeloconic sensilla with neurons were identified that are responsive to phenylacetic acid and phenylacetaldehyde on their anterior antennal surface (similar to ac4 sensilla in D. melanogaster). Thus, IR84a may have a conserved, drosophilid-specific function (Grosjean, 2012).

Despite the widely held assumption of the existence of volatile chemicals that promote courtship in Drosophila, behavioural evidence for long-range pheromones is inconclusive, and no female-specific volatile compound that activates male OSNs has been identified. The characterization of IR84a identifies an olfactory receptor that is expressed in FRUM-positive neurons and is required to promote male courtship. Surprisingly, this receptor is not activated by fly-derived odours but rather by aromatic compounds that are present in the vegetal substrates in which fruitflies feed, breed and oviposit. Thus, the IR84a pathway may promote male courtship in the presence of food, complementing the functions of pheromone receptors in regulating mate choice. This model can account for the widespread observations that D. melanogaster and other drosophilids mate predominantly on their food substrates. Whereas many insects and other animal classes use long-range sex pheromones to attract potential mates, the evolution of IR84a in fruitflies has provided an alternative (although not necessarily exclusive) olfactory mechanism to unite males with females by integrating food-sensing neurons with the circuitry controlling sexual behaviour. Whether other animals have dedicated sensory pathways for environmental 'aphrodisiacs' remains an open question (Grosjean, 2012).

GABAergic projection neurons route selective olfactory inputs to specific higher-order neurons

This study characterizes an inhibitory circuit motif in the Drosophila olfactory system, parallel inhibition, which differs from feedforward or feedback inhibition. Excitatory and GABAergic inhibitory projection neurons (ePNs and iPNs) each receive input from antennal lobe glomeruli and send parallel output to the lateral horn, a higher center implicated in regulating innate olfactory behavior. Ca2+ imaging of specific lateral horn neurons as an olfactory readout revealed that iPNs selectively suppress food-related odor responses, but spare signal transmission from pheromone channels. Coapplying food odorant did not affect pheromone signal transmission, suggesting that the differential effects likely result from connection specificity of iPNs, rather than a generalized inhibitory tone. Ca2+ responses in the ePN axon terminals show no detectable suppression by iPNs, arguing against presynaptic inhibition as a primary mechanism. The parallel inhibition motif may provide specificity in inhibition to funnel specific olfactory information, such as food and pheromone, into distinct downstream circuits (Liang, 2013).

Two general circuit motifs involving inhibitory neurons are widely used in vertebrate and invertebrate nervous systems. In feedback inhibition, inhibitory neurons are locally activated by excitatory neurons. In turn, they inhibit a broad array of excitatory neurons, including those that excite them. In feedforward inhibition, excitatory input activates both excitatory and inhibitory target neurons, and the activated inhibitory target neurons further inhibit the excitatory target neurons. The mammalian olfactory bulb, for instance, provides examples of both motifs. As an example of feedback inhibition, granule cells are activated by mitral cells in response to odor stimuli. In turn, they inhibit the same and neighboring mitral cells. As an example of feedforward inhibition, ORN axons excite periglomerular cells and mitral cells in parallel; some periglomerular cells inhibit mitral cells in the same and adjacent glomeruli. Both granule cells and periglomerular cells contribute to the lateral inhibition and sharpening of the olfactory signals that mitral cells deliver to the olfactory cortex. Similarly, the fly antennal lobe, the equivalent of the mammalian olfactory bulb, has a diversity of GABAergic local interneurons (LNs). Some LNs are excited by ORNs and subsequently provide feedback inhibition onto ORN axon terminals for gain control. Other LNs may act on PN dendrites for feedforward inhibition. This study describes an inhibitory circuit motif that differs from classic feedforward and feedback inhibition, which is termed parallel inhibition, wherein excitatory and inhibitory projection neurons receive parallel input and send parallel output to a common target region (the lateral horn) (Liang, 2013).

What are the possible roles of iPNs, and what advantages might the parallel inhibition motif confer? By monitoring olfactory responses of a subset of putative third-order lateral horn neurons (the vlpr neurons) and by laser transecting the ascending mACT input from iPNs while sparing ePNs, this study has shown that iPNs selectively route olfactory input to vlpr neurons. Specifically, the vlpr responses to the food odors are inhibited by the iPNs, but the response to the cVA pheromone-processing channel is not subjected to this inhibition. Previous anatomical studies revealed highly stereotyped branching and terminal arborization patterns for uniglomerular ePNs and iPNs (Jefferis, 2007: Lai, 2008). Results in this study provide functional demonstration that GABAergic iPNs regulate olfactory inputs to the lateral horn neurons. Indeed, the fact that removing iPN inhibition allows isoamyl acetate and vinegar signals to activate vlpr neurons suggests that anatomical segregation of PN axon terminals representing food and pheromone (Jefferis, 2007) alone is not sufficient to prevent food odors to activate vlpr neurons, at least some of which are normally activated by pheromones. iPN inhibition provides another level of specificity of the higher-order neuronal responses to olfactory input (Liang, 2013).

This specificity of inhibition provides a special feature of parallel inhibition in comparison with feedforward and feedback inhibition. Feedforward and feedback inhibition tend to be nonspecific with respect to their target population within the same neuronal type, which is optimal for certain functions these motifs serve, such as lateral inhibition and gain control. In the Drosophila antennal lobe, for example, while exhibiting a large variety of arborization patterns, most local interneurons (LNs) innervate many to all glomeruli, where they both receive input and send output. By contrast, the specific dendritic glomerular innervation of individual iPNs in the antennal lobe, as well as their stereotyped axonal arborization patterns in the lateral horn, enable iPNs to selectively inhibit some olfactory-processing channels, but not others. It is speculated that food odors should activate other lateral horn higher-order neurons relevant to foraging and that such activation is not strongly inhibited by iPNs, perhaps also due to inhibition specificity (Liang, 2013).

Another interesting feature of parallel inhibition is the timing of inhibition. Inhibition from feedforward and certainly feedback motifs arrive later than excitation due to transmission through an extra synapse, which is used to confine the magnitude and/or duration of excitation. The parallel inhibition motif in principle allows for simultaneous arrival of excitation and inhibition at the postsynaptic neurons, potentially enabling inhibition to completely suppress excitation, and is ideally suited for information gating. This study provides evidence that the primary action of iPNs is unlikely through presynaptic inhibition of ePNs, as ePN presynaptic Ca2+ signals in response to olfactory stimuli were not elevated by middle antennocerebral tract (mACT) transection. A caveat of this interpretation is that some forms of presynaptic inhibition can bypass Ca2+ entry, for instance through Gβγ action on the release machinery; however, GABAergic inhibition that acts in this manner has not currently been identified. Thus, the idea is favored that iPNs act directly on postsynaptic third-order neurons under the experimental conditions. Due to the limited temporal resolution of Ca2+ imaging, the temporal property of parallel inhibition has not been explored in this study. It will be interesting for future research to measure the arrival time of both excitatory and inhibitory input directly with more sensitive and temporally precise electrophysiological methods (Liang, 2013).

This study describes the use of the parallel inhibition motif in sensory systems. Long-distance GABAergic projections are prevalent in the mammalian brain. Specifically, some GABAergic neurons in the hippocampus and cortex have recently been identified that send long-distance projections, sometimes to the same area as the glutamatergic projection neurons. Thus, parallel inhibition can potentially be a widely used mechanism in the nervous system (Liang, 2013).

This study has identified a unique class of higher-order neurons that respond to Or67d [and presumably the pheromone 11-cis-vaccenyl acetate (cVA)] activation. Or67d ORNs and their postsynaptic partner DA1 excitatory PNs express FruM, a male-specific transcription factor that is a key regulator of sexual behavior. A previous study identified a number of Fru+ higher-order cVA-responsive neurons whose cell bodies reside dorsal and lateral to the lateral horn (Ruta, 2010). Indeed, the analyses of Fru+ neurons have so far provided many examples where Fru+ neurons are connected with each other to regulate different aspects of sexual behavior. However, lateral horn-projecting Mz699+ vlpr neurons do not appear to express FruM, despite their robust activation by Fru+ Or67d ORNs. This may reflect a broad function of cVA as a pheromone that regulates not only mating but also aggression and social aggregation (Liang, 2013).

This study revealed a difference between food- and pheromone-processing channels in their susceptibility to inhibition by iPNs and suggests that pheromone channels may be insulated from general inhibition by iPNs. It is almost certain that iPNs play additional functions than reported in this study, as iPN function was studied only from the perspective of their effect on the olfactory response of a specific subset of higher-order neurons. Indeed, in a companion manuscript, Parnas (2013) showed that iPNs play an instrumental role in facilitating the discrimination of mostly food odors, as assayed by quantitative behavioral experiments. Taken together, these studies uncovered two distinct aspects of iPN function: increased discrimination of diverse food odors and information gating between qualitatively different olfactory stimuli (Liang, 2013).

Finally, it is notable that of the two major ePN targets, iPN axons only project to the lateral horn but spare the mushroom body. The mushroom body is a well-documented center for olfactory learning and memory, whereas PN projections to the lateral horn are implicated in regulating innate olfactory behavior (see Parnas, 2013). ePN axons exhibit striking stereotypy in their terminal arborization patterns in the lateral horn, but not in the mushroom body. Recent anatomical tracing in mice also revealed differential input organization in distinct olfactory cortical areas, suggesting a common principle in olfactory systems of insects and mammals. The selective innervation by iPNs of targeting neurons in the lateral horn suggests that regulation of innate olfactory behavior engages an additional level of specific inhibition to ensure that olfactory information carrying different biological values, such as food and pheromone, is funneled into distinct downstream circuits, resulting in the activation of distinct behavioral outputs (Liang, 2013).

Visualization of neural activity in insect brains using a conserved immediate early gene. Hr38

Many insects exhibit stereotypic instinctive behavior, but the underlying neural mechanisms are not well understood due to difficulties in detecting brain activity in freely moving animals. Immediate early genes (IEGs), such as c-fos, whose expression is transiently and rapidly upregulated upon neural activity, are powerful tools for detecting behavior-related neural activity in vertebrates. In insects, however, this powerful approach has not been realized because no conserved IEGs have been identified. This study identified Hr38 as a novel IEG that is transiently expressed in the male silkmoth Bombyx mori by female odor stimulation. Using Hr38 expression as an indicator of neural activity, comprehensive activity patterns of the silkmoth brain were mapped in response to female sex pheromones. Hr38 could also be used as a neural activity marker in the fly Drosophila melanogaster. Using Hr38, a neural activity map of the fly brain was mapped that partially overlaps with fruitless (fru)-expressing neurons in response to female stimulation. These findings indicate that Hr38 is a novel and conserved insect neural activity marker gene that will be useful for a wide variety of neuroethologic studies (Fujita, 2013).

Dhr38 expression was investigated in the brain of a naive male fly stimulated with a decapitated virgin female body. D. melanogaster males recognize conspecific females through visual, olfactory, and gustatory cues and show courtship behavior even to decapitated females. In response to female stimulation, Dhr38 was robustly expressed in various brain regions. In particular, strong signals were detected in the cells located dorsal to the AL (defined as area 1) and around the MBs (area 2). Signals were also reproducibly detected between the protocerebrum (PC) and OL (area 3), around the SOG (area 4), and around the lobula (area 5). The number of Dhr38-positive cells was comparable between male brains stimulated with one or three decapitated virgin females, suggesting that Dhr38 detection is sensitive enough to detect neural activity induced by a single virgin females (Fujita, 2013).

The gross expression pattern of Dhr38 was similar between the brains of males stimulated with a virgin and those stimulated with a mated female. Although mated females emit an antiaphrodisiac male pheromone, cis-vaccenyl acetate, they are still attractive to naive males and induce courtship behaviors (Fujita, 2013).

Next the contribution of female pheromones to Dhr38 expression in male brains was investigated. To examine the contribution of contact pheromone input, the Dhr38 expression pattern was examined in the brains of males with both foreleg tarsi surgically removed. In males with foreleg amputations, virgin female stimulation still induced a Dhr38 expression pattern similar to that in intact males. Then the contribution of olfactory input was evaluated using males whose antennae were surgically removed. Antennae amputation led to a significant decrease in the number of Dhr38-positive cells in all brain areas in response to female stimulation. The remaining Dhr38 expression completely disappeared when foreleg amputation was combined with antennae removal. In addition, in anosmic Orco mutants, female stimulation induced Dhr38 expression in a smaller number of cells than in wild-type, and Dh38 expression was decreased by foreleg amputation. The Dhr38 expression remaining after foreleg amputation might be derived from Orco-independent olfactory inputs. These findings indicate that Dhr38 expressed in response to female stimulation is derived from both olfactoryand contact-dependent neural pathways. Further, decapitated male stimulation induced Dhr38 expression in a moderate number of cells in areas 1, 2, and 5, indicating that these areas comprise heterologous neurons responsive to females and males. Dhr38 expression was examined in Or47b mutants, because Or47b is thought to be a female pheromone receptor (van der Goes van Naters, 2007). Double in situ hybridization of Dhr38 and Or47b confirmed that Or47b-expressing cells are responsive to virgin female stimulation. In Or47b mutants, female stimulation induced Dhr38 expression in a smaller number of cells than in wild-type, and this response was significantly decreased by foreleg amputation. These findings indicate that a large part of the neural activity induced by female stimulation is regulated by chemical inputs from contact pheromones and female odors (Fujita, 2013).

Male fly courtship behavior is regulated by neural circuits comprising fruitless (fru)-expressing neurons. Thus, it was asked whether virgin female-induced Dhr38 expression overlaps with fru-expressing neurons. To address this question, Dhr38 expression was examined in the brains of males whose fru-expressing neurons were visualized using an NP21-GAL4 strain, which covers 82% of Fru-expressing neurons. In areas 1, 4, and 5, Dhr38 was not expressed in fru-expressing cells. In contrast, in area 2, a small portion of Dhr38-positive cells was positive for GFP. Because the majority of Dhr38-positive cells were located dorsal to the MB calyces and fru-expressing cells were located ventral to the MB calyces, this area was analyzed in detail by focusing on each cell cluster. Dhr38-positive cells were detected in GFP-positive P1 and P4 cells (one or two double-positive cells per cluster) in three specimens. No Dhr38-positive cells were detected in P2 and P3 clusters. Interestingly, Dhr38-positive cells in area 3 frequently colocalized with fru-expressing neurons and are assumed to be Lv1+Ld and Lv2 cluster cells, which extend neurites to the OLs. P1 neurons are the master command neurons of male courtship behavior and are activated on contact with females through the foreleg tarsus (Kimura, 2008; Kohatsu, 2011). Consistent with this notion, no P1 neurons were positive for Dhr38 in males with both foreleg tarsi amputated. In contrast, the number of cells positive for both Dhr38 and GFP in area 3 was not affected by foreleg amputation, indicating that Dhr38-positive area 3 neurons are not involved in contact pheromone recognition. Taken together, these findings indicate that the neural circuit activated by virgin female stimulation partially overlaps with that comprising fru-expressing cells, supporting the notion that Dhr38 can be used to detect physiologically relevant neural activities (Fujita, 2013).

This study has identified Hr38 as a conserved IEG that can be used as a neural activity marker in insect brains. HR38 is the sole insect ortholog of the NR4A nuclear receptor family, which is highly conserved among metazoans and whose expression is increased by a variety of cellular signals. It is therefore reasonable to assume that neural activity-dependent Hr38 expression is widely conserved among insects. Because the DNA binding domain of NR4A family genes is highly conserved among species and in situ hybridization is feasible in nontransgenic animals, this study provides a powerful approach for neuroethologic studies in a wide variety of animalss (Fujita, 2013).

Hr38 was previously identified as an interaction partner of Ultraspiracle that binds to Ultraspiracle in competition with the ecdysone receptor, which is suggested to contribute to the fine-tuning of the ecdysone signaling pathway. Recently, ecdysone signaling was confirmed to be involved in memory formation in vinegar flies. Activity-dependent Hr38 expression suggests that ecdysone signaling may be modified in a neural activity-dependent manner, leading to the hypothesis that Hr38 has important roles in higher neural function, such as memory formation. Further studies are needed to elucidate the mechanism regulating activity-dependent Hr38 expression and its neural functions (Fujita, 2013).

A small subset of fruitless subesophageal neurons modulate early courtship in Drosophila

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. A fruM-dependent sexually dimorphic arborization was identified 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 (Tran, 2014).

Initiation of unilateral wing extension is heavily dependent on visual, olfactory, and gustatory cues. By forcing males to depend on non-visual pathways for courtship and co-expressing tissue-specific fruM RNAi, this study screened for fruM(+) neurons that likely regulate chemosensory-dependent processes in courtship, which manifested as infrared-specific courtship latency defects. The P[GawB]4-57 line driving UAS-fruMIR possessed normal courtship latency in ambient light and significant infrared-specific delays. Notably fruM overlap was strongest in the SOG, while lacking any detectable peripheral expression. Behavioral and anatomical studies using Cha-Gal80, to subdivide the P[GawB]4-57 expression pattern, highlighted a small subpopulation of fruM(+) neurons in the SOG, two-four anterior SG x 4-57 neurons (marked by the intersection of two drivers) and two medial SG x 4-57 neurons as responsible for the courtship defects (Tran, 2014).

Several lines of evidence suggest a direct role for the mSG x 4-57 neurons in regulating the initiation of wing extension and copulatory behaviors. First, expression of fluorescent markers was detected in the mSG x 4-57 neurons driven by P[GawB]4-57 in all brains, whereas fluorescence was only detected in a subset of animals for the other fruM x 4-57 subpopulations. The mSG x 4-57 neurons made sexually dimorphic arbors in the tritocerebrum, where male arbors were significantly larger than in wild type female and fru mutant male brains. The mSG x 4-57 neuronal tracts extended into the VNC where presynaptic innervation of the mesothoracic triangle was seen. The mesothoracic triangle is a target of descending command neurons that control wing song. Faint projections were detected in the posterior metathoracic/anterior abdominal ganglia, which suggest possible regulation of motor circuitry needed for abdominal curling during copulatory behaviors (Tran, 2014).

The sexually dimorphic projections of the mSG x 4-57 suggest sex-specific roles in receiving tritocerebral signals in males. In males, fruM knockdown and silencing of fruFLP x 4-57 neurons resulted in a failure to progress to copulation, a behavior that follows proboscis contact with a female ('licking'). The internal mouthparts house gustatory sensilla that likely detect contact female pheromones accessed via licking behavior (Tran, 2014).

Functions for the non-mSG x 4-57 neurons cannot be ruled out, particularly the DT6 x 4-57 (aSG) neurons in regulating courtship initiation, however. The current approach infers, but does not conclusively demonstrate that the mSG x 4-57 neurons are responsible for the courtship initiation and copulation defects. Further studies are required to conclusively identify the neurons responsible for each behavioral phenotype and their exact roles (Tran, 2014).

Several studies have examined the projections of fruM(+) neurons in the SOG. Antibody staining using anti-FruM identified 12±2 total FruM(+) nuclei in the SOG in the 2-day pupal brain. An intersectional study, using 131 Gal4 lines with sparse overlap with fruFLP, identified 8 fruFLP(+) SOG neuronal classes divided into six anterior, aSG1-6, and two posterior neuronal types, pSG1-2. At least one aSG x 4-57 neuron’s projection pattern, identified in this study, is consistent with the aSG5 class identified in that larger-scale study. Cachero (2010) used mosaic analyses of fruGal4 to identify larval neuroblast clonal populations of fruGal4 (+) neurons. Cachero identified six clones in SOG, however, none appear to correspond to neurons identified in this study. It appears that these broad mapping studies, while extensive, have not exhaustively identified fru-expressing neurons in the SOG (Tran, 2014).

Using tdc2-Gal4, three studies characterized three octopaminergic FruM(+) neurons in the SOG: designated VPM1 and VPM2 (ventral paired median) and one VUM1 (ventral unpaired median) neuron. Expression of tdc2-Gal4-driven UAS-fruMIR leads to courtship latency delays but no copulation defect. The VUM1 neuron tritocerebral projections appear similar to the mSG x 4-57 projections, however, no descending tracts to the VNC were reported. The VPM1 and VPM2 appear to correspond to the DT8 neurons Repression of fruM using tdc2-Gal4 appeared to primarily disrupt male-female discrimination, resulting in significant male-male courtship, whereas no significant male-male courtship was detected using P[GawB]4-57 (Tran, 2014).

Given the extensive projections of fruM(+) innervations, the tritocerebrum appears to be a site of gustatory integration with higher-order information in male courtship. The extensive, sexually dimorphic arbors from the mSG x 4-57 receive signals in the tritocerebrum that serve to regulate the progression to copulation in males and the performance of courtship. The tritocerebrum is targeted directly by gustatory afferents from the mouthparts via the pharyngeal nerves, indirectly via the SOG interneurons, which could relay signals from proboscis gustatory afferents entering via the labial nerve, and by descending tracts from the par interecerebralis of the superior medial protocerebrum (SMPR), which contains many neurosecretory cells. These mSG x 4-57 cells could then relay signals to circuitry controlling wing extension/song in the metathoracic triangle and copulation/abdominal curling in the anterior abdominal ganglia (Tran, 2014).

The decision to perform courtship by males likely weighs the receptivity of the female versus the cost of female rejection via escape, with greater costs associated with later steps in the ritual, i.e. copulation. In open environs, escape behaviors exhibited by rejecting females likely results in the cessation of the courtship unless the male correctly gauges receptivity. It is proposed that the fruM(+) SOG neurons identified in this study play a vital link between detection of female receptivity cues and integration of higher-order signals in order to appropriate initiate wing extension and copulatory behaviors (Tran, 2014).

Single serotonergic neurons that modulate aggression in Drosophila

Monoamine serotonin (5HT) has been linked to aggression for many years across species. However, elaboration of the neurochemical pathways that govern aggression has proven difficult because monoaminergic neurons also regulate other behaviors. There are approximately 100 serotonergic neurons in the Drosophila nervous system, and they influence sleep, circadian rhythms, memory, and courtship. In the Drosophila model of aggression, the acute shut down of the entire serotonergic system yields flies that fight less, whereas induced activation of 5HT neurons promotes aggression. Using intersectional genetics, the population of 5HT neurons that can be reproducibly manipulated were restricted to identify those that modulate aggression. Although similar approaches were used recently to find aggression-modulating dopaminergic and Fru(M)-positive peptidergic neurons, the downstream anatomical targets of the neurons that make up aggression-controlling circuits remain poorly understood. This study identified a symmetrical pair of serotonergic PLP neurons (5HT-PLP neurons) that are necessary for the proper escalation of aggression. Silencing these neurons reduced aggression in male flies, and activating them increased aggression in male flies. GFP reconstitution across synaptic partners (GRASP) analyses suggested that 5HT-PLP neurons form contacts with 5HT1A receptor-expressing neurons in two distinct anatomical regions of the brain. Activation of these 5HT1A receptor-expressing neurons, in turn, caused reductions in aggression. These studies, therefore, suggest that aggression may be held in check, at least in part, by inhibitory input from 5HT1A receptor-bearing neurons, which can be released by activation of the 5HT-PLP neurons (Alekseyenko, 2014).

Displays of appropriate levels of aggression rely on the ability of an animal to analyze many factors, including the following: the correct identification and evaluation of the abilities of potential competitors; the evaluation of the value of a territory and the likelihood of acquiring it; and the physiological state of the animal. Multiple sensory systems and circuits will be utilized in making such evaluations. The fixed number of neurons and neuronal circuits in nervous systems might limit the abilities of an animal to evaluate such a multiplicity of factors, but great flexibility is introduced into the system by the availability of neuromodulators. These have the capability of rapidly, efficiently, and reversibly reconfiguring the networks of neurons without changing the 'hardwiring.' The current studies illustrate the modulation by 1-2 pairs of serotonergic neurons that enhance aggression. Other modulatory neurons and systems that influence aggression have been identified previously in Drosophila, including dopaminergic neurons, FruM-positive octopamine neurons that influence the behavioral choice between courtship and aggression, FruM-positive tachykinin neurons that enhance aggression, and neuropeptide F circuits that decrease aggression. The arbors of processes of the 5HT-PLP neurons examined in this study densely innervate several integrative centers in the fly brain, but thus far, they do not seem to overlap with the processes of the other reported aggression-influencing neuromodulatory neurons. The 5HT-PLP neurons do not coexpress FruM or Dsx. Thus, the modulatory control of the male-specific higher-level aggression appears to involve both sex-specific regulatory factors and other as-yet-unidentified control elements. The current studies further suggest that going to higher-intensity levels in fights may be held in check by inhibition, which can be released by activation of the 5HT-PLP neurons. Learning more about the neurons and neuronal circuits involved with a suggested downstream aggression-suppressing system and with the sensory systems that trigger aggression in the first place will be essential steps in further unraveling the complex circuitry that controls the release of aggression in Drosophila (Alekseyenko, 2014).

In summary, using a Drosophila model system and an intersectional genetic strategy, this study identified a pair of serotonergic neurons in the PLP cluster that modulate aggressive behavior. These neurons arborize through several neuropil regions in the central brain, where they influence the escalation of aggression, at least in part, via 5HT1A receptor-bearing neurons and also independently influence locomotion and sleep. The single-cell resolution in identification of neuronal connections and explorations of their functions in behaving animals provides an entry point into unraveling the circuitry associated with complex behaviors like aggression (Alekseyenko, 2014).

Central neural circuitry mediating courtship song perception in male Drosophila

Animals use acoustic signals across a variety of social behaviors, particularly courtship. In Drosophila, song is detected by antennal mechanosensory neurons and further processed by second-order aPN1/aLN(al) neurons. However, little is known about the central pathways mediating courtship hearing. This study identified a male-specific pathway for courtship hearing via third-order ventrolateral protocerebrum Projection Neuron 1 (vPN1) neurons and fourth-order pC1 neurons. Genetic inactivation of vPN1 or pC1 disrupts song-induced male-chaining behavior. Calcium imaging reveals that vPN1 responds preferentially to pulse song with long inter-pulse intervals (IPIs), while pC1 responses to pulse song closely match the behavioral chaining responses at different IPIs. Moreover, genetic activation of either vPN1 or pC1 induced courtship chaining, mimicking the behavioral response to song. These results outline the aPN1-vPN1-pC1 pathway as a labeled line for the processing and transformation of courtship song in males (Zhou, 2015).

Courtship behavior of Drosophila males provides a fundamental model for understanding how species-specific courtship signals may be processed and integrated to drive stereotyped motor outputs. Using anatomical, behavioral, and physiological approaches, this study outlines a male-specific pathway for courtship hearing, which processes and transforms song stimuli to activate central fruM+ or dsx+ neurons that support multimodal integration and drive courtship behavior (Zhou, 2015).

fru and dsx are two key transcription factors with restricted expression patterns that specify the potential for sexual behaviors in Drosophila. fruM expression in primary auditory, tactile, gustatory, and visual neurons as well as the central brain may suggest that there are multiple fruM-labeled pathways conveying and integrating diverse sensory signals related to courtship, and an appealing hypothesis is that fruM labels interconnected neurons in a circuit that is dedicated to courtship. For example, the male-specific pheromone cVA is processed by a four-neuron pathway extending from sensory neurons through to the ventral nerve cord. This circuit appears to function as an olfactory labeled line, in that neurons in this circuit are functionally connected and selectively responsive to cVA (Zhou, 2015).

This study has focused on elucidating the auditory pathway underlying courtship song perception. The aPN1-vPN1-pC1 pathway is a labeled line for courtship hearing, by fulfilling four criteria: (1) these neurons are functionally connected; (2) these neurons respond preferentially to courtship song; (3) these neurons are necessary for the behavioral response to courtship song in male flies, and (4) activation of this labeled line provides a fictive stimulus, observable by the chaining response elicited upon CsChrimson activation. Strikingly, this labeled line appears to be specified by the expression of fruM or dsx (Zhou, 2015).

The auditory labeled line for courtship hearing begins with fruM-expressing JONs and second-order auditory neurons aPN1/aLN(al) in the antennal mechanosensory and motor center (AMMC). Silencing either fruM JONs or fruM aPN1 neurons reduced male song-induced responses. In addition, this study has also demonstrated the connectivity, response patterns, necessity, and sufficiency of fruM vPN1 and pC1 neurons in this pathway, thus, delineating a labeled line of fruM neurons leading directly from sensory neuron to multimodal integration (Zhou, 2015).

The inclusion of vPN1 in this pathway is supported by three lines of evidence. First, vPN1 projections extensively overlap with projections extended by aPN1 neurons within WED, and functional connectivity was observed between aPN1 and vPN1. Second, silencing vPN1 reduced pulse song-induced chaining in male flies, while optogenetic activation of vPN1 neurons mimicked a song signal to induce male chaining. Third, GCaMP recordings reveal that vPN1 responds strongly to both pulse song and sine song. It is therefore concluded that fruM+ vPN1 neurons are the third-order neurons mediating courtship hearing (Zhou, 2015).

vPN1 may provide its output via innervation of the lateral protocerebrum (LPC), a region receiving multimodal input that is likely to be a site for multi-sensory integration. This area is heavily innervated by dsx+ pC1 neurons, which include most of the male-specific fruM+ P1 neuron. While the broader pC1 population is important for both male courtship and female receptivity, the P1 neurons play a critical role in the initiation of male courtship and respond to both male and female pheromones. These neurons appear to be the downstream targets of vPN1, based on three lines of evidence. First, the arborizations of pC1 neurons match very closely with the projection of vPN1 neurons in the LPC, and optogenetic activation of vPN1 generates robust activity in pC1. Second, pC1 neurons show calcium responses to pulse song stimuli, with IPI tuning that matches that of the behavioral response. Third, silencing pC1 neurons in male flies almost completely abolishes song-induced chaining, while activation induces robust chaining in the absence of song. It is therefore concluded that vPN1 may carry song stimuli to activate pC1, where these stimuli are integrated with other sensory modalities such as pheromonal olfactory and gustatory cues to modulate the courtship level in males (Zhou, 2015).

Taken together, the neural circuit identified in this study suggests that song information flows via a labeled line of fruM neurons from the antenna to AMMC, to WED, and then to LPC, providing a functional explanation of how pulse song induces male courtship behavior (Zhou, 2015).

IPI is a key parameter of courtship song that exhibits great variation across Drosophila species. D. melanogaster not only produces song with a specific IPI, but also behaviorally recognizes song with that conspecific IPI in both males and females. This study also shows that song-induced male-chaining behavior is most responsive to a 35-ms IPI, although longer IPIs (35–65 ms) are still able to induce robust chaining behavior (Zhou, 2015).

While Drosophila has behavioral preferences toward the conspecific IPI, it has not been clear how IPIs are represented in the nervous system or how the fly discriminates specific IPIs. The results suggest there is a significant change in pulse song representation across the ascending aPN1-vPN1-pC1 pathway. For aPN1, the GCaMP ΔF/F responses in female flies reflect an integration of pulse rate at IPIs longer than 25 ms. In contrast, vPN1 responses observed here are low-passed and preferentially tuned to longer IPIs. Interestingly, the vPN1 response saturates above ∼35-ms IPI, consistent with the saturating response observed when comparing dendritic and axonal GCaMP signals in aPN1. Notably, however, neither the aPN1 nor vPN1 response corresponds well with the behavioral sensitivity to IPI observed in male or female flies (Zhou, 2015).

In contrast, the IPI sensitivity of pC1 reflects a band-pass response to IPI that closely matches the behavioral sensitivity of the chaining response. Indeed, the correlation between pC1 response and chaining behavior is significantly higher than the correlation observed for vPN1. Thus, while the mechanistic details remain unclear, the IPI sensitivity appropriate for species-appropriate responses is likely to be generated through a multi-stage transformation of song stimuli (Zhou, 2015).

Sexual dimorphism at multiple levels in the Drosophila brain may give rise to sex-specific differences in sensory processing and multimodal integration. The central integrators of courtship-related sensory cues in male and female flies, the pC1 neurons, are themselves sexually dimorphic in both cell number and morphology. pC1 neurons arborize within the triangular lateral junction of the LPC in both sexes, where integration of multiple sensory modalities may occur, but they also show male-specific innervation of the LPC arch and male-specific contralateral projections (Zhou, 2015).

For courtship hearing, pC1 neurons are stimulated by pulse song in both sexes, but are also stimulated by sine song in females. This result is consistent with the behavioral observation that both males and females are responsive to pulse song, while females are also responsive to sine song. However, the pC1 auditory response cannot be easily explained by the dimorphism of vPN1, which responds to pulse song in males but is absent in females. Moreover, the absence of vPN1 in females begs the question of how pC1 receives song information in females. One explanation comes from the observation that vPN1 is a subset of the fru+ aSP-k clone. aSP-k shows arborization in VLP and the LPC ring in both male and females, as well as male-specific innervation of the LPC arch that corresponds with vPN1 morphology. These neurons, including non-fru+ neurons in the same lineage, may compose a parallel pathway for female hearing (Zhou, 2015).

More generally, this study observed a gradient of sexual dimorphism across the ascending pathway for both olfaction and audition. In both cases, only limited sexual dimorphism was noticed in second-order neurons (DA1 and aPN1, respectively), but dramatic changes in third-order neurons (aSP-f/aSP-g and vPN1) and integrative neurons (pC1), which show significant dimorphisms in cell number and morphology. This gradient may reflect a general rule for the flexible assembly of sexually dimorphic circuits on an evolutionary timescale (Zhou, 2015).

These anatomical, behavioral, and physiological analyses have outlined the architecture of a system supporting species-specific courtship hearing, built upon genetically labeled lines expressing fruM or dsx within the fly. Although it is clear that courtship song representations are systematically transformed along the aPN1-vPN1-pC1 pathway, a circuit and synapse-level explanation for how this occurs, as well as an understanding of how pC1 activation gives rise to distinct and appropriate behavioral outputs in each sex, requires additional study (Zhou, 2015).


The fruitless (fru) gene functions in Drosophila males to establish the potential for male sexual behaviors. fru encodes a complex set of sex-specific and sex-nonspecific mRNAs through the use of multiple promoters and alternative pre-mRNA processing. The male-specific transcripts produced from the distal (P1) fru promoter are believed to be responsible for its role in specifying sexual behavior and are only expressed in a small fraction of central nervous system (CNS) cells (Goodwin, 2000).

The fru locus spans at least 140 kb and produces a complex array of transcripts due to the use of four promoters and alternative splicing. Transcripts from the distal promoter (P1) are alternatively spliced near their 5' termini to generate sex-specific transcripts. It is thought that the male-specific P1 transcripts encode fru's sex determination function, whereas the transcripts produced from the more proximal promoters encode fru's vital function(s). Alternative splicing at the extreme 3' end of the fru transcripts leads to the inclusion of one of three mutually exclusive exons that encode alternative pairs of zinc fingers. Fifteen transcript classes are possible if the five identified 5' ends (two are produced by sex-specific splicing from P1) are combined with all the identified 3' ends; cDNAs corresponding to seven of these classes have been identified. However, the potential transcript diversity may be more extensive, due to the discovery of additional fru transcripts containing one of several micro-exons. The transcripts from all fru promoters have open reading frames that encode proteins related to the BTB-ZF protein family (Goodwin, 2000 and references therein).

When a probe common to all known classes of fru cDNAs was hybridized to Northern blots of poly(A)+ RNA from sexed wild-type adult heads, three female-specific (9.0, 8.0, and 7.4 kb), three male-specific (7.9, 6.4, and 5.4 kb), and one common (4.4 kb) transcripts were detected. Sex-specific fru transcripts are generated by the sex-specific usage of alternative 5' splice sites that are 1590 nt apart in the P1-derived pre-mRNA (Ryner, 1996). It has been suggested that the three male-specific and three female-specific transcripts are generated from P1-derived transcripts by sex-specific splicing at their 5' ends and by sex-nonspecific alternative splicing to exons containing three alternative zinc-finger pairs at their 3' ends. This predicts that the three classes of female-specific transcripts would have common sequences at their 5' ends, as should the three male-specific forms. To test this, a fru probe from upstream of the female-specific 5' splice site was used to probe Northern blots of poly(A)+ RNA from sexed adult heads. Only the three female-specific (9.0, 8.0, and 7.4 kb) transcripts seen previously were detected; no signals were observed in male head RNA, even with long autoradiographic exposures. These results are consistent with the interpretation that all three female-specific fru transcripts share common 5' female-specific sequences (Goodwin, 2000).

The sequences of the sex-specific fru transcripts revealed that there are no sequences unique to the male transcripts: both male- and female-specific fru transcripts share common sequences from upstream of the male-specific 5' splice site (Ryner, 1996). Therefore a probe from this region was used on Northern blots of poly(A)+ RNA from sexed wild-type heads; only the three female sex-specific transcripts (9.0, 8.0, and 7.4 kb) and three male sex-specific transcripts (7.9, 6.4, and 5.4 kb) were observed. This set of results provides strong support for the conclusion that these six sex-specific fru transcripts arise from sex-specific splicing at the 5' end of transcripts produced from the distal (P1) fru promoter (Goodwin, 2000).

An important feature of P1-derived transcripts is that they are expressed in several hundred neurons in the CNS of adults. The spatial pattern of P1 expression was analyzed in wild type in more detail and, at this higher level of resolution, an examination was made to determine whether any of the viable fru alleles alter the gene's spatial expression pattern. Nine groups emerged from this analysis: six in the brain and three in the ventral nerve cord. In the brain, the locations of the six groups are as follows: (1) a large group in the dorsal posterior protocerebrum, medial and ventral to the mushroom bodies; (2) a lateral group in the protocerebrum, anterior to the medullary division of the optic lobes; (3-5) three anterior groups in the protocerebrum, subdivided into lateral (3), intermediate (4), and medial (5) sets; and (6) one anterior group near the mechanosensory part of the antennal lobe. In the ventral nerve cord, the three groups of male-specific fru P1-expressing neurons are as follows: (7) a lateral group between the prothoracic and mesothoracic neuromeres and ventral to the wing neuromere; (8) a ventro-medial group in the mesothoracic ganglion; and (9) a ventral group in the abdominal ganglion. In addition to these groups of cells, both sexes have a small population of labeled neurons that are found as singletons or are too widely separated from other labeled cells to recognize them as belonging to a group (Goodwin, 2000).

In contrast to the restricted pattern of expression of P1 fru transcripts, a probe (C1) that detects the protein-coding region common to all fru transcripts has revealed expression in virtually all neurons in the CNS and in several other tissues. Most cells in the CNS have a relatively low level of expression, whereas a small number of neurons display markedly higher levels of fru expression. By their location and number, the subsets of neurons labeled by the C1 probe that have relatively high levels of fru expression appear to correspond to the nine groups of neurons detected by the S1 probe (Goodwin, 2000).

To determine whether there were changes in the cellular pattern of fru expression in the five viable fruitless mutants, analogous in situ hybridizations were undertaken with both the S1 and C1 probes. Only the presence and relative size of the aforementioned nine groups of labeled neurons were analyzed, because these groups of fru-expressing cells are distinct and could be unambiguously identified in tissue sections (Goodwin, 2000).

Examination of the expression pattern of P1-derived transcripts in fru1 males revealed distinct groups of labeled neurons in only four of the nine regions where cells expressing these transcripts are found in wild-type males. The four groups of neurons detected were those in the dorsal posterior protocerebrum (1), the optic lobe (2), the antennal lobe, and the ventro-medial mesothoracic groups. The numbers of labeled neurons in the antennal lobe and dorsal posterior groups in fru1 males are not significantly different from wild type (Goodwin, 2000).

The other five groups of cells detected in wild-type males are very difficult to detect in fru1 males. In the anterior protocerebrum, the normal pattern for three groups of neurons (3-5) is not observed. In the dorsal anterior protocerebral region, only about one-quarter of the expected number of labeled neurons are detected. These labeled neurons are distributed throughout the medial, intermediate, and lateral subdivisions, suggesting that fru1 leads to a reduced number of cells expressing P1 transcripts in all three regions. It was not possible to identify definitively the remaining labeled neurons as belonging to one of the three anterior protocerebral groups. Likewise, labeled neurons are difficult to detect in two of the male-specific neuronal groups in the ventral nerve cord of fru1. A small cluster of neurons, ~15% of the expected number, is found in the ventral area, between the prothoracic and mesothoracic neuromeres. By contrast, almost no labeled cells are found in the ventral abdominal region, where group 9 neurons are observed in wild-type males. The reduced numbers of cells expressing P1 transcripts in fru1 males may be caused by a reduction in transcription from the P1 promoter, instability of these transcripts, or the loss of neurons that normally express these transcripts (Goodwin, 2000).

When a probe (C1) common to all fruitless transcripts is used, all neurons in fru1 male CNSs are labeled at a low level, comparable to what is seen in the wild-type CNS. In addition, subsets of neurons show relatively high levels of fru expression. Neurons with such heavy labeling are detected in the dorsal posterior protocerebrum (1), optic lobe (2), antennal lobe (6), and mesothoracic groups (8); these are the same regions in which the neurons expressing the sex-specific transcripts are abundant in fru1 males. In fru1, fewer-than-normal numbers of heavily labeled neurons are detected with the C1 probe in the anterior protocerebrum (3-5) as well as within the thoracic (7) and the abdominal ganglion (9) groups. These regions are the same as those in which fewer or no labeled neurons are found by in situ hybridization with the S1 probe (Goodwin, 2000).

in situ hybridization was used to determine whether the spatial expression of P1-derived transcripts was affected in the transposon mutants. In all four mutants, the S1 probe detects groups of labeled neurons in the nine locations where P1-expressing neurons are found in wild-type males. For example, labeled neurons were found in the antennal lobe, the dorsal anterior protocerebrum, the thoracic ganglion, and the abdominal ganglion. The numbers of neurons labeled in these groups are similar to those found in wild-type males. However, there was an overall increase in the number of heavily labeled neurons in the brains of frusat males compared to the other fru mutant or wild-type males. In wild-type males, cells labeled with the S1 probe often have darkly stained dots within the nucleus, as well as cytoplasmic staining. In a similar fashion, in all the P-element-mutant males, neurons labeled with the S1 probe often have very darkly stained dots in the nucleus in addition to weak cytoplasmic staining (Goodwin, 2000).

The loss of fruitless expression in the regions described likely accounts for the striking courtship abnormalities exhibited by fru1 males. Sequences in the transposons in the four P-element mutants lead to nonproductive splicing of fru transcripts. This fact offers a first-order explanation for how the insertions of these transposons into the large fru intron generate mutant phenotypes. However, what is not accounted for by these findings is that these four insertion mutations differ in the severity of their phenotypic effects. One possible explanation for these phenotypic differences is that low levels of normal FRU mRNAs are generated from the P1 promoter in these mutants. Indeed, RT-PCR experiments using poly(A)+ RNA from heads are able to detect normally spliced products from the P1 promoter in all four of the transposon mutants. These RT-PCR experiments are not quantitative, allowing for the possibility that there are different residual levels of normal, P1-derived mRNAs in the mutants; this could account for the different severities of these alleles. That some of the transposons are not only intrinsically different, but also are inserted at different intronic positions, could contribute to the probability of a given transcript being subject to aberrant splicing during its processing. Thus, it is suggested that the mutant phenotypes in fru2, fru3, fru4, and frusat animals are due to a failure to appropriately splice P1 transcripts, whereas the mutant phenotype of fru1 animals is due to the reduction or absence of P1 transcripts within specific regions of the CNS (Goodwin, 2000).

Various fru mutations have varying effects on male sexual behavior, Muscle of Lawrence development and locomotion. Many mutant alleles result in male flies that court indiscriminately, fail to copulate and have MOL defects. Some mutant males show very little wing extension; during wing displays, they generate no song pulse signals. This defect is specific to courtship, as these mutants are normal for flight and are able to flick their wings when rejecting advances made by another male. Further reductions in sexual behavior are seen with other alleles or combinations. More severly impaired males barely court, yet they exhibit essentially normal locomotor activity. When fru mutant males are grouped together, they form male-male courtship chains in which each male is simultaneously both courting and being courted. All mutant combinations show some male-male chaining. Thus, the early steps of courtship (orientation, following, and wing extension) as well as the later steps (courtship song and attempted copulation) are disrupted in various mutants and mutant combinations (Ryner, 1996).

A genetically defined element of the fruitless locus regulates the development of a male-specific muscle spanning the fifth abdominal segment in adult males, the "muscle of Lawrence" (MOL). The region is defined by two cytological deletions, each with a breakpoint that co-maps with previously described mutant courtship phenotypes at cytogenetic interval 91B on the third chromosome. Flies that carry both of these deletions are viable, and males express abnormalities of courtship similar to those caused by the fru inversion breakpoint at 91B. In addition, these double-deletion males show the complete absence of the MOL, suggesting that they have little or no gene expression of a postulated MOL determinant; the musculature in the fifth abdominal segment of these mutants to indistinguishable from that of a normal female. Other mutant combinations that produce fruitless courtship phenotypes--including deletion and inversion breakpoints, and a marked transposon inserted at 91B--produce intermediate forms of the MOL. A new genetic variant, induced by imprecise excision of the marked transposon, is homozygous lethal and disrupts fru functions related to courtship and the MOL. The MOL is shown to be dispensable for fertility and is therefore not the causative factor of fru-induced behavioral sterility (Gailey, 1991).

Muscle of Lawrence (MOL) development, which is induced by male-specific innervation, was studied in a variety of genotypes: in wild-type males, in males fed hydroxyurea to ablate the muscle precursors and in fruitless mutants, in which the MOL develops aberrantly. One striking feature of MOLs in wild-type males is the presence of additional muscle nuclei compared with neighboring muscles or MOL-homologs in females. Does muscle length and the sex-specific expression of a reporter gene depend critically on the number of nuclei present within a MOL fiber? MOL fibers developing from a reduced myoblast pool in hydroxyurea-affected hemisegments are recognizable by their attachment points and still contain more nuclei than do neighboring medial fibers, suggesting that these MOL fibers are able to actively recruit myoblasts nearly as well as wild-type MOLs. However, many of the hydroxyurea-affected MOL fibers are incapable of the normal male-specific expression of a muscle-specific reporter gene. It is suggested that early events in MOL development, such as finding the correct muscle attachment points, are relatively insensitive to the number of MOL nuclei, as compared with later events, such as the sex-specific expression of a reporter gene. In fruitless mutant males, MOL-position fibers are smaller and have substantially fewer nuclei, when compared to wild-type MOLs. Since the number and distribution of muscle precursors is the same in fruitless mutant and wild-type animals, it is proposed that one fru+ function is to direct the male-specific recruitment of myoblasts into MOL-myotubes. However, fruitless+ must have more than one role in MOL fiber development, since simple reduction in the number of muscle nuclei, as demonstrated by the hydroxyurea ablations, is insufficient to account for all of the MOL muscle phenotypes in fruitless mutant males (Taylor, 1995).

The muscle of Lawrence (MOL) is a bilaterally symmetrical muscle spanning the tergite of the fifth abdominal segment of adult male Drosophila melanogaster. It is not, however, a general feature of male-specific development within the subfamily Drosophilinae. Of 95 species surveyed within this subfamily, 67 exist with no MOL at all. By drawing comparisons with published cladograms of species relatedness, three conclusions have been reached regarding the evolutionary history of the MOL: (1) it predates the major radiations of the genus Drosophila, given its presence in earlier-branching Chymomyza and Scaptodrosophila; the MOL has been subsequently excluded in at least one present species of each of these two primitive genera. (2) Within the genus Drosophila the MOL is present sporadically in the radiation of the subgenus Sophophora, showing repetitive loss even in very close evolutionary lineages. (3) The MOL may have been entirely excluded from the prolific radiation of the subgenus Drosophila. Thus the MOL shows a uniquely incongruous pattern of presence or absence relative to accepted Drosophilid phylogeny (Gailey, 1997).

The fruitless mutants fru3 and fru4 were assessed for sex-specific reproductive-behavioral phenotypes and compared to the previously reported fru mutants. Among the several behavioral anomalies exhibited by males expressing these relatively new mutations, some are unique. fru3 and fru4 males are less stimulated to court females than fru1 and fru2. No courtship pulse song is generated by either fru3 or fru4 males, even though they perform brief wing extensions. fru3 and fru4 males display significantly less chaining behavior than do fru1 males. The hierarchy of courtship responses by fru males directed toward females vs. males, when presented with both sexes simultaneously, is that fru1 males perform vigorous and indiscriminant courtship directed at either sex; fru4 males are similarly indiscriminant, but courtship levels are lower than fru1; fru2 males prefer females and fru3 males show a courtship bias toward males. fru3 and fru4 males essentially lack the Muscle of Lawrence (MOL). On several reproductive criteria, there was no difference between fru- variant females and fru+ (Gailey, 1997).

There exists in Drosophila a tra-dependent, dsx-independent mechanism for the control of aspects of male and female sexual behavior and neural development. One dissatisfaction function, proper ventral A5 abdominal innervation in males, is downstream of tra but not downstream of dsx. These results lead to the inferrence that dsf functions in a tra-dependent and dsx-independent process. Another gene, fruitless (fru), has also been postulated to be part of a dsx-independent pathway controlling behavior and neuronal development. Males with strong fru mutations (1) fail to differentiate tissues known as the muscle of Lawrence (MOL); (2) show abnormal courtship partner choice, courting both mature males and females; (3) are sterile as a result of an inability to curl their abdomens into a copulatory position, and (4) generate an abnormal courtship song. Mutant phenotypes have not been reported for fru females (Finley, 1997 and references).

The identification of dsf as a second dsx-independent gene controlling sexual behavior and neural development raises questions about whether dsf and fru are part of a single regulatory pathway or are parts of two different regulatory pathways. There are both substantial similarities and substantial differences in phenotype between these two genes. dsf and fru are similar in that mutations in both genes lead to male by male courtship and to abnormality (dsf) or failure (fru) in abdominal curling during copulation. Yet, there are behavioral and neurological differences between dsf and fru mutants. The most notable among these is the lack of any reported abnormalities for fru females in either courtship or fertility relative to the substantial abnormalities exhibited by dsf females. There are also phenotypic differences between dsf and fru males. Males with strong fru alleles do not produce male-like MOLs, while dsf males produce normal MOLs with normal innervation. In addition, the failure of abdominal bending is absolute in males carrying strong fru alleles and only partial in dsf mutants, while ventral abdominal muscles are innervated normally in fru males and abnormally in dsf males. The multiple differences between the fru and dsf phenotypes lead to the conclusion that these genes act in separate regulatory pathways, each of which is required for appropriate sexual behavior (Finley, 1997 and references).

Male sexual behavior is regulated by the sex-determination hierarchy (SDH) in Drosophila melanogaster. The fruitless gene, one of the regulatory factors functioning downstream of other SDH factors, plays a prominent role in male sexual behavior. fru mutations cause a previously unappreciated behavioral anomaly: high levels of head-to-head interactions between mutant males. These apparent confrontations between males are exhibited by all of the homozygous-viable fru mutants (expressing the effects of a given allele, among the four tested). Mutant dissatisfaction (dsf) males also exhibit this behavior at higher-than-normal levels, but it was barely displayed by doublesex or intersex mutants. For fru, a social component is involved in the head-interaction phenotype, while increasing age is a modifying factor for the behavior of dsf males. It is suggested that head-to-head interactions, especially those performed by fru males, are instances of putative aggression analogous to those exhibited by wild-type males and that head interactions are, to an extent, operationally separable from courtship behavior (Lee, 2000b).

The fruitless gene produces male-specific protein (FRUM) involved in the control of courtship. One approach toward understanding how fru regulates male courtship is to compare patterns of FRUM expression in the CNS of various fruitless mutants that display behavioral phenotypes ranging from mildly to severely defective. The courtship subnormalities and bisexual behavior caused by fru mutations could be understood in terms of where FRUM is expressed in the CNS (or not expressed, as the case may be) in a given mutant. FRUM spatial and temporal patterns were examined in fru mutants that exhibit aberrant male courtship. Chromosome breakpoints at the locus eliminate FRUM. Homozygous viable mutants exhibit an intriguing array of defects. In fru1 males, there are absences of FRUM-expressing neuronal clusters or stained cells within certain clusters, reductions of signal intensities in others, and ectopic FRUM expression in novel cells. fru2 males exhibit an overall decrement of FRUM expression in all neurons normally expressing the gene. fru4 and frusat mutants produce FRUM in only a small number of neurons at extremely low levels, and no FRUM signals were detected in fru3 males. This array of abnormalities was inferred to correlate with the varying behavioral defects exhibited by these mutants. Such abnormalities include courtship among males, which has been hypothesized to involve anomalies of serotonin (5-HT) function in the brain. However, double-labeling uncovered no coexpression of FRUM and 5-HT in brain neurons. Yet, a newly identified set of sexually dimorphic FRUM/5-HT-positive neurons was identified in the abdominal ganglion of adult males. These sexually dimorphic neurons (s-Abg) project toward regions of the abdomen involved in male reproduction. The s-Abg neurons and the proximal extents of their axons were unstained or absent in wild-type females and exhibit subnormal or no 5-HT immunoreactivity in certain fru-mutant males, indicating that fruitless controls the formation of these cells or 5-HT production in them (Lee, 2001).

To examine the possible relationship between fruitless function and 5-HT, whole-mounted CNSs were double-labeled with antibodies against FRUM and the neuromodulator. 5-HT-immunoreactive neurons are broadly distributed throughout the brain, the thoracic ganglia, and the abdominal ganglion of adult males. Nine groups of serotonergic neurons have been reported in the adult brain and five groups in the ventral nervous system of Drosophila. A comparison was made of of serotonergic neurons of wild type and fru mutant adult flies (Lee, 2001).

It was assumed that fruitless mutations and ectopic expression of the white gene (Zhang, 1995) cause males to court other flies because of anomalous brain function (possible involvement of the VNC is counterintuitive). Ectopic expression (and probably overexpression) of w+ in the brain could deplete 5-HT levels in cells that normally express the fru gene, mutations of which can easily be found to cause a similar neurochemical deficit. Thus FRUM and 5-HT would be coexpressed in at least some of the neurons that normally contain these substances. However, within the brain of wild-type males, no FRUM neurons whatsoever were double-labeled with anti-5-HT. The usual locations of cells and processes immunoreactive for this substance were observed. The number of 5-HT neurons is not particularly large, reinforcing the possibility that global uptake of a serotonin precursor throughout the brain could deplete levels of this substance in their usual locations. However, if ectopic expression of w+ is mechanistically related to fru-mutational effects via 5-HT, the current results indicate that there is a need to formulate a hypothesis different from one involving direct intracellular effects of the latter genotypes. Perhaps white and the tryptophan transporter it encodes cause this neuromodulator to be anomalously present in FRUM cells or other neurons that directly interact with them; such effects might derange fru-controlled brain functions insofar as sex recognition is regulated. Another possibility, not mutually exclusive, is that ectopic w+ leads to anomalous 5-HT levels in cells that interact with FRUM neurons, deranging brain functions that are not directly controlled by fruitless but are components of the neural substrates for courtship. In any case, the lack of a simple relationship between fruitless gene products and serotonergic neurons, which would have bolstered the notion that both abnormal genotypes cause their courtship effects via 5-HT depletion in the same key brain cells, suggests that ectopic-white males are made to behave in a manner that caricatures the phenotype of fruitless mutants (Lee, 2001).

In the course of these double-labeling tests, signals elicited by anti-FRUM and anti-5-HT were scrutinized in all CNS ganglia. Within the male's ventral cord, the great majority of fru-expressing neurons in the four pairs of ganglia contain no detectable 5-HT. There was, however, an exception within one VNC region. It involves certain newly identified serotonergic cells in the abdominal ganglion. For these neurons, coexpression of FRUM and 5-HT was observed in a total of eight cells at the posterior tip of the VNC. These serotonergic-abdominal giant neurons (s-Abg) are located close to one another in a relatively dorsal side of the abdominal ganglion and have conspicuously large cell bodies. Larval serotonergic neurons in the developing nervous system are reorganized during metamorphosis. In this respect, putative precursors of the s-Abg neurons were not detected in the third-instar larval CNS or in the abdominal ganglion of 2-d-old male pupae. Therefore, these s-Abg neurons in Drosophila may form during metamorphosis, or they may have been born earlier and only take on their final neurochemical quality during late stages of development (Lee, 2001).

With regard to the projection patterns of the s-Abg cells that were revealed by 5-HT-immunostaining, each neuron appears to have more than one neurite. In most specimens, the s-Abg neurons are closely clumped together. A few preparations exhibited fairly clear bilaterality of these cell bodies and their posterior projections. These 5-HT-immunoreactive neurites also appear to be within the median trunk (which is known to innervate posterior abdominal segments), genital segments, and internal reproductive organs (Lee, 2001).

The putatively fru-related function of these cells and their processes would seem to involve aspects of male reproduction because the patterns of 5-HT immunoreactivity being described were not observed in or posterior to the abdominal ganglion of adult females. Whether these cells exist in females, as opposed to being present but devoid of 5-HT, is unknown. In this regard, bear in mind that there is no FRUM immunostaining anywhere in the CNS of females (Lee, 2001).

In fru-mutant males, anti-5-HT immunoreactivity in the s-Abg neurons as well as the axons projecting from them was either absent or defective. fru1 and frusat show low levels of transmitter staining in some of the s-Abg neurons and their processes (Lee, 2001).

In fru3, there is no detectable 5-HT immunoreactivity in s-Abg neurons or their axons. At best, fru4 mutant males present weakly detectable 5-HT immunoreactivity in these structures. fru2 males are normal with respect to numbers of s-Abg neurons and their projections (as stained by anti-5-HT), although the levels of staining intensity in both subcellular compartments of these neurons appears to be lower than in wild type (Lee, 2001).

For the most severely subnormal mutant (fru3) in terms of FRUM and 5-HT expression in the abdominal ganglion, it was not immediately possible to determine whether the general absence of both kinds of immunoreactivity is caused by an absence of s-Abg neurons or by the lack of serotonin production in these cells. To address this question, 5-HT-uptake experiments were performed. These were based on the fact that exogenously applied 5-HT was found to be absorbed selectively by serotonergic neurons in the CNSs of third-instar larvae that express late-developmentally lethal Dopa decarboxylase (Ddc) mutations; relatively severe (but viable) Ddc variants cause severe decrements in 5-HT synthesis. Ventral nerve cords from adult fru3 males were exposed to a series of 5-HT-creatinine concentrations. The resulting immunostaining led to the following patterns. In wild-type VNCs, lowered endogenous 5-HT levels were observed in the serotonergic neurons that are undisturbed by this fru mutation; it was inferred that the incubation procedure necessary for 5-HT uptake is the major cause of this depletion. In fru3 VNCs, at the lowest concentration applied to fru3 specimens, neither s-Abg-like cell bodies nor neurites could be recognized; but as incubations with increasing 5-HT concentrations were performed, there were increasing numbers of immunostained cells along with stronger signal strengths. At the highest concentration of 5-HT applied, a subset of these structures in the ganglion of the mutant exhibited what appeared to be the appropriate immunoreactivity. The signals associated with the VNC cell bodies and processes in question appeared similar to those of genetically normal s-Abgs in their size, shape, and intraganglionic location. Thus, it seems as if at least some of these VNC cells are retained in this mutant and able to take up serotonin. However, it was not possible to determine unambiguously whether the normal fru/5-HT-expressing cells and their projections were labeled in the fru3 ganglia. Therefore, it remains an open question as to whether these neurons are eliminated by a developmental effect of this mutation, or whether, if present, the cells are unable to absorb exogenously applied 5-HT under the conditions used (Lee, 2001).

The discovery of sexually dimorphic s-Abg neurons in the abdominal ganglion could provide an anatomical link to fru+-dependent sex-specific phenotypes not yet known to be influenced by this gene. The s-Abg neurite signals elicited by anti-5-HT also provide the first information on a projection pattern for fru-expressing cells. These findings indicate that the formation of the s-Abg neurons or production of 5-HT in them is male-specific and under fru control. The 5-HT-uptake results suggest that s-Abg cells are present but are unable to synthesize this transmitter in the FRUM-less fru3 mutant (Lee, 2001).

Innervation by 5-HT/FRUM neurons of abdominal muscles influenced by fru-gene action is unlikely. This is because glutamate is the canonical neuromuscular transmitter in Drosophila, although comprehensive information is understandably lacking as to whether this molecule is responsible for intercellular communication at all such synapses. In addition to the muscles in this body region, there are male-specific organs that have better-appreciated structures (compared with unknown muscles hypothetically devoted to attempted copulation) and functions (compared with the MOL) for which reproductive significance is unknown. In this regard, the s-Abg neurons seem to send their axonal projections into the median-trunk nerve, the terminal branches of which innervate the genital segments and internal reproductive organs as well as certain abdominal muscles (Lee, 2001 and references therein).

5-HT has been suggested to play a role in altered sexual orientation in Drosophila (Zhang, 1995). A potentially close connection between the action of FRUM and serotonin in terms of inter-male courtship would have been worthy of deeper consideration if these factors had been found to be coexpressed. But the current results uncovered no overt 5-HT link to fru-expressing brain neurons. The fact that there is a distinctly separate colocalization of FRUM and serotonin at the opposite end of the CNS suggests that regulation of the presence and action of this neurotransmitter can be a downstream target of fru function, an unexpected connection between the control of sexual differentiation and this piece of Drosophila neurochemistry. This bonus was but one of the results of being able to monitor the presence of fruitless gene products at high resolution (Lee, 2001).

A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here has shown that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. Eight new fru mutations, created by X-ray mutagenesis and P-element excision, have been analyzed to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers shows that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts; fru also functions sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes (Anand, 2001).

It has been proposed that fru's role in controlling male sexual behavior is through the products of the distal (P1) fru promoter. Briefly, the reasoning is as follows. Genetic analysis has shown that while all aspects of male somatic sexual differentiation are controlled by the transformer (tra) and transformer-2 (tra-2) sex determination genes, doublesex (dsx), the only known sex determination regulatory gene below tra and tra-2 in the sex determination hierarchy, does not control two aspects of male sexual differentiation (the MOL and male sexual behavior). These results indicated that there is a previously unrecognized branch of the sex determination hierarchy below tra and tra-2 and has lead to the molecular search for other genes that are direct targets of tra and tra-2. That search identified fru as a direct target of tra and tra-2 because the transcripts from the P1 fru promoter are sex-specifically alternatively spliced under the control of the TRA and TRA-2 proteins. fru function is known to be required for some aspects of male sexual behavior and the formation of the MOL, and the fru P1 transcripts are expressed in regions of the CNS previously implicated in certain aspects of male sexual behavior. This has provided a strong case for the products of the P1 fru promoter functioning to control aspects of sexual differentiation in the CNS needed for male sexual behavior and the MOL formation. However compelling that case may seem, it falls short of establishing that the products of the fru P1 promoter have the proposed functions, since there has no demonstration that mutations that impair P1 fru function alone have the expected phenotypes. Recent data consistent with the proposed function of P1 fru products have come from the following findings: (1) the fru1 allele, which affects male sexual behavior, is an inversion broken 5' to the P1 promoter and alters the spatial pattern of P1 expression in the CNS (and based on its location probably does not affect expression from the other fru promoters, and (2) the fru3 and fru4 alleles, which have strong effects on male sexual behavior, result in a substantial reduction in the amount of normal P1 and P2 (but not P3 and P4)-derived transcripts (Anand, 2001 and reference therein).

Further support for the proposed roles of the P1 fru transcripts is provided by various genotypes involving the new fru alleles reported here. The findings that males, which express P3 and P4 transcripts, but no P1 or P2 transcripts, are sterile and exhibit little or no male-specific reproductive behaviors in single-pair tests strongly support the hypothesis that the products encoded by fru's P1 (and possibly P2) transcripts control male sexual behavior. Flies of some of the these genotypes show reduced general activity and thus one might argue that this contributes to their reduced sexual behavior. However, as quantified in this study, there is in general only a weak correlation between the scores on courtship tests and the test of general activity. Moreover, for several genotypes, males had moderate levels of activity, indicating that for these genotypes the absence of male sexual behavior is unrelated to any general behavioral deficit (Anand, 2001).

Males having P1-encoded functions that are either lacking or seriously impaired show as severe decrements in their sexual behavior as males lacking both P1- and P2-encoded functions. This suggests that fru's role in sexual behavior may be entirely attributed to its P1-encoded functions. In single-pair tests of courtship behavior, males lacking or severely subnormal for P1 functions show essentially no sexual behavior. Moreover, all types of males show either normal or moderate levels of general activity. Since a genotype that lacks only P2-encoded functions is not available, the possibility that P2-encoded functions are also important for male sexual behavior cannot be excluded. However, all of the data currently available can be explained by the proposal that fru's P1-encoded functions are responsible for fru's role in male sexual behavior (Anand, 2001).

The new fru genotypes analyzed also provide some insight into which fru products are necessary for fru's vital function. With respect to whether P2 encodes a vital function, two aberrations, Df(3R)fru4-40 and In(3R)fruw27, both of which lack P1 and P2 transcripts and produce P3 and P4 transcripts, give completely discordant results when used in complementation tests with other fru alleles that lack fru's vital function. It is thought that this disparity is due to In(3R)fruw27 being defective in more than just fru's P1 and P2 functions (Anand, 2001).

In summary, these studies implicate the products of the P1 fru promoter as being responsible for fru's control of male sexual behavior and the products of the P3 fru promoter for carrying out fru's vital function. The functions of the products of the P2 and P4 promoters are currently unclear. However, it should be noted that genotypes that individually remove the functions encoded by P2-, P3-, or P4-derived transcripts are unavailable. Thus conclusions as to the functions of P1- and P3-derived transcripts are the simplest ones compatible with the data. Ghe possibilities that P2-, P3-, or P4-derived products may play some role in male sexual behavior or that P4-encoded products may also carry out a vital function cannot be excluded. Moreover, the findings that flies lacking P1 and P2 promoter-derived products are viable, whereas flies lacking P1-, P2-, and P3-derived products are lethal are most simply compatible with the proposal that P3 products carry out a vital function. These data do not preclude some models in which there is redundancy between the P1, P2, and P3 products in providing fru's vital function (Anand, 2001).

From a detailed analysis of the new fru alleles it has been shown that wild-type fru function is necessary for the production of nearly all aspects of male sexual behavior. fru mutant males lacking P1-encoded products no longer performs any courtship behavior with either single male or female partners in standard courtship tests. The observed failure of males of various fru genotypes to court might reflect a specific defect in sexual behavior, or alternatively it might be a by-product of a general reduction in activity. Although males of several fru genotypes did have reduced levels of short-term activity, regression analysis has shown that, overall, there is only a poor correlation between the level of activity and male courtship behavior; this indicates that low activity by these males could at most account for only a small part of the variation in male courtship behavior. Moreover, the reduction in courtship in single-pair tests occurs in some males that have moderate to high levels of activity. Further evidence indicating that reduced courtship with a single partner is a specific defect is shown by a number of fru genotypes in which males do not court a male or female partner but do engage in male-male group courtship as measured by the ChI. This dichotomy in CI vs. ChI shows that males are capable of generating at least some courtship behaviors in one situation but do not express that similar behavior in a different setting. Thus, the elimination of male courtship to both males and females in a subset of fru mutant genotypes reflects a sex-specific courtship effect of the loss of fru+ function (Anand, 2001).

There are two ways these results could be interpreted in terms of the wild-type function of fru. First, since male courtship is a dependent action pattern, with the occurrence of one step in the courtship sequence generally requiring the completion of preceding steps, these results could simply mean that these fru males are blocked in some way prior to the very earliest steps of courtship. Alternatively these results could mean that fru+ function is necessary for each step of courtship. The data support the latter alternative. Using several hypomorphic alleles, previous studies have shown that fru mutant males courted males and females but were blocked at certain individual steps occurring during courtship. For example, fru3 and fru4 males are blocked in the middle of the courtship behavioral sequence such that although they court they do not vibrate their wings to generate the courtship song and do not attempt copulation. By comparison, fru1 males are blocked only in the penultimate step of attempted copulation. These studies have provided strong evidence that male-specific fru+ function is involved with the specific neuronal circuitry required for the wing vibration needed for courtship song and the abdominal bending for copulation. Various heteroallelic combinations tested also show similar defects at intermediate to late stages of courtship. Thus many of the combinations of new fru alleles with fru1 or fru2 show significant levels of courtship activity as measured by either the CI or the WEI yet are sterile or have greatly reduced fertility due to failures to copulate. Similarly, a number of the fru genotypes tested fail to produce a courtship song during the reduced period that they are extending their wings. Taken together, these findings suggest that fru functions to establish the potential for essentially all aspects of male sexual behavior (Anand, 2001).

The exact molecular role that the P1 products play in controlling male-specific behaviors is not known, but they are likely to affect the development and differentiation of particular male-specific neurons and neuronal circuits. Of the clusters of P1-expressing neurons that have been identified, focus was placed on nine different clusters of P1-expressing neurons that have been identified by in situ hybridization in late stage pharate male brains and are prominent in sectioned preparations. Mutant males of a genotype that did not produce any courtship behavior appear to retain many or most of these neurons in the CNS even though these animals are not producing any functional P1 or P2 transcripts. This finding strongly suggests that the primary role of the P1 transcripts is to direct sex-specific differentiation and not direct the production or facilitate the survival of these neurons (Anand, 2001).

One aspect of the courtship phenotypes of the fru mutant combinations that requires special consideration is what might appear to be somewhat discordant results between single-pair courtship tests (both male-male and male-female) and the male-male group courtship (chaining) test. Of the 10 genotypes that were tested that lacked P1 function, all displayed little or no courtship in single-pair tests, but 6 of these genotypes displayed significant levels of male-male group courtship. In addition, of the 15 genotypes tested in which one of the new alleles was heterozygous with either fru2, fru3, or fru4, 12 genotypes displayed little or no courtship in single-pair tests, yet 13 of these genotypes had significant, often substantial, levels of male-male group courtship. In thinking about these data it is important to recall that although single-pair male-female courtship, single-pair male-male courtship, and male-male group courtship are all referred to as courtship, male-male group courtship is a mutant phenotype, whereas single-pair male-female courtship is the wild-type phenotype in D. melanogaster. Mature males courting each other in pairs essentially never occurs in a sustained manner in this species, so single-pair male-male courtship might also be viewed as a 'mutant' phenotype. Thus one might expect a priori that there would not be parallel dependencies of these three different 'courtship' phenotypes on fru function. In that regard it is worth recalling that the levels of courtship as measured by the CIs of males of a given genotype with single males or single females are highly correlated. Strikingly, a similar linear regression analysis of the mean CIm-m and the ChI across all fru genotypes shows a similarly strong positive correlation. These high degrees of concordance suggest that these three behavioral assays are measuring behaviors that are largely equivalent in their fru dependency. The difference between wild type and any of the fru mutants that have been tested to date would be that in the fru mutant situations males as well as females would be seen as appropriate partners for whatever level of courtship males of a particular genotype can attain (Anand, 2001).

As was just noted for a number of genotypes, males show little or no courtship in single-pair tests but are able to carry out the very first steps of courtship (orientation and following), but not later steps, of courtship in group tests. These differences within a genotype are likely related to the different ways single-pair and group tests are done. In particular, male-male group tests are carried out by placing a group of males together for 3-4 days prior to testing, whereas males used in single-pair courtship tests were stored individually until tested. The richer environment experienced by males used in male-male group courtship tests (more stimulation through dynamic interactions between animals and a longer time for association between animals) is likely the reason for the different levels of courtship seen in single-pair and group tests. The preceding interpretation is suggested by the prior observation that fru2, fru3, and fru4 homozygous males chained little, if at all, when initially placed together in groups, but that the level of chaining increased over the next couple of days. The finding that fru mutant animals perform some courtship steps under group conditions also shows that the appropriate parts of the brain for at least the very initial steps of courtship are still present and can be activated in these genotypes but that the conditions of the test for pairwise courtship do not lead to the activation of these same centers and the expression of orientation and following (Anand, 2001).

Another striking aspect of these data is that 6 of the 10 genotypes that completely lack P1 promoter-encoded functions showed significant levels of male-male group courtship. The finding that 4 of these 10 genotypes showed no courtship in either single pairs or as groups of males is consistent with the conclusion that it is the products of the P1 fru promoter that establish the potential for male sexual behavior. However, the results from the 6 genotypes that show significant male-male group courtship suggest that other factors are involved that are not understood. There does not seem to be an obvious genetic explanation for these data, since genotypes that should be identical in terms of their arrays of fru products do not have equivalent phenotypes (Anand, 2001).

It is suggested that the products of the P1 fru promoter are responsible for both MOL development and male sexual behavior. An obvious question is whether this fru-regulated male-specific muscle has any connection with fru-regulated male-specific courtship behavior. To date, the physiological function of the MOL is unknown. It has been demonstrated that the MOL is not necessary for copulation to occur in this species, and, in fact, the males of most Drosophila species do not even develop a MOL (Anand, 2001).

Given the data indicating that the P1-derived male-specific FRU proteins govern MOL development, a recent report comes as a surprise: it found that expression of a fru cDNA construct encoding a protein lacking the 101-amino-acid male-specific N terminus could partially rescue the MOL phenotype in frusat homozygous males and lead to MOL development in wild-type females was surprising. The fact that a Fre protein can induce MOL development in an otherwise wild-type female provides striking evidence that Fru function is sufficient to trigger MOL development. With respect to the question of which class of Fre proteins controls MOL development in wild type, the following is noted. The Fru protein shown to be capable of producing a MOL is identical to a P1-derived Fru protein, except for the absence of the male-specific 101-amino-acid N terminus. It would not be surprising if there were some overlap in the functionality of these two proteins. The heat-shock and GAL4 expression systems used for Fru expression are likely to produce Fru protein at significantly higher levels than are present for that protein in wild type. Thus the production of MOLs in their experiments may be being brought about by the overexpression of a protein whose function partially overlaps that of P1-encoded proteins. Alternatively, it could be that these proteins are functionally fully synonymous, and these functions are biologically determined by the locations and levels at which they are expressed (Anand, 2001).

Studies of the phenotypes of lethal fru mutant combinations provide insights into the nature of fru's essential function. Genotypes that lack P3 (and P1 and P2) promoter function have lethal phases in the mid- to late-pupal period, but in all cases a substantial fraction of these individuals are viable if assisted in emerging from the pupal case. Thus the vital function encoded by P3 is only essential very late in development (Anand, 2001).

P3-encoded functions appear to be necessary for the differentiation of imaginal-disc derivatives, such as legs and wings. The rare survivors with fru lethal genotypes show defects in their external morphology and have specific sensory bristle organs duplicated. Moreover, imaginal discs frequently failed to evert in adult escapers of genotypes that lacked P3 function. These observations suggest that there is a role for fru in imaginal disc development or differentiation. The gross morphological defects produced in adult derivatives of imaginal discs makes it likely that the nearly lethal phenotype of fru genotypes lacking P3 function arises from these defects (Anand, 2001).

In addition, many of the adult escapers that lack P3 function have neuroanatomical defects in the motor innervation to the abdominal muscles. These defects in the motorterminals may explain part of the inability of many of these fru mutant genotypes to successfully eclose from the pupal case. All abdominal muscles appear to be innervated, suggesting that the defects in these fru mutants are not due to abnormalities in neuronal pathfinding but rather some feature of synaptogenesis once the motorneuronal axons reached their target muscles. Changes in the branching of motorneurons at the neuromuscular junction in larval Drosophila have been described for various mutants that affect neuronal excitability. In other respects the development of the nervous system of fru mutant genotypes appears normal. No obvious change in the number or patterns of divisions of postembryonic neuroblasts was found in either a null fru genotype or a genotype lacking P1, P2, and P3 functions, suggesting that the normal complement of adult-specific neurons is likely produced in the complete absence of fru function. The apparently normal level of postembryonic divisions of neuroblasts in the larval CNSs of mutants which are destined to die within a day, makes it unlikely that the loss of adult neurons is responsible for the death of these animals (Anand, 2001).

While there is currently not a fru genotype that is null for all fru functions and wild type for neighboring genes, it was possible to place a limit on how extreme a completely null fru genotype might be by examining the lethal phase of mutant combinations that are null for fru as well as a small number of adjacent genes. The development of these individuals is arrested in early pupal development, around the time of pupal ecdysis. The morphology of the pupal case in these genotypes is similar to the phenotypes of animals mutant for the ecdysone receptor and the crooked legs gene. However, the fru genotypes lack not only all fru function but also the function of at least four, and possibly six, neighboring genes. Thus it is not clear if the earlier lethal phase caused by these genotypes -- as compared to fru genotypes that are lacking just P1, P2, and P3 function -- is a consequence of the absence of P4 function or the absence of one of the neighboring genes. In any case, these two null genotypes establish that a complete absence of fru function does not result in a lethal phase prior to the beginning of the pupal period (Anand, 2001).

In summary, these results establish that fru functions sex-specifically in the sex-determination regulatory hierarchy to control male sexual behavior and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during development. Perhaps most importantly these results show that just the absence of the products of the P1 fru promoter results in the loss of all normal male sexual behavior and thus provide strong support for the proposal that these proteins function to establish the potential for nearly all components of a Drosophila male's sexual behavior (Anand, 2001).

The fruitless (fru) gene acts sex-nonspecifically in the development of the embryonic central nervous system (CNS) and has sex as well as sex-nonspecific functions in the development of the adult CNS. In the embryo, sex-nonspecific fru mRNAs and proteins are widely expressed during neurogenesis and present in both neurons and glia. To assess whether the fru gene plays any role in fate determination of neuronal precursors and neurons, the development of Eve-positive [Eve(+)] GMCs and neurons was examined in fru mutants. In fru mutant embryos in which most or all fru transcripts are eliminated, the normal complement of Eve(+) neurons is present initially, but some neurons are unable to maintain their Eve-expression. Concomitantly, a subset of Eve(+) neurons also showed inappropriate expression of the glial marker, Reversed polarity. In addition, neurons that normally do not express Eve became Eve(+) in these fru mutants. These defects are rescued in fru mutant embryos expressing specific fru transgenes under the control of the sca-GAL4 and elav-GAL4 drivers. These phenotypic analyses and rescue experiments provide evidence that one of the sex-nonspecific functions of the fru gene is the maintenance of neuronal identity rather than establishment of a neuron's initial fate (Song, 2003)

fruitless is required for the proper formation of axonal tracts in the embryonic CNS

The fruitless gene in Drosophila is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. This study examines fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. These results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS (Song, 2002).

To determine the spatial and temporal distribution of embyonic fru transcripts, in situ hybridizations were carried out with antisense-BTB and antisense-Com riboprobes, which detect most or all fru transcripts. fru mRNAs are expressed in a dynamic temporal and spatial pattern from the beginning of embryogenesis until stage 16. fru transcripts are uniformly distributed in very early embryos and become incorporated into segregating pole cells. At the start of gastrulation (stage 6), heavily labeled cells are found in the ventral and cephalic furrows. In slightly older embryos (stages 7-9) the most prominent distribution of fru transcripts is found in the developing CNS within mesectodermal and ventral neuroectodermal cells. Transcripts become localized to delaminating neuroblasts and after stage 10, fru transcripts are detected in medial but not in lateral neuroblasts. Following expression in the progeny of neuroblasts, the level of fru expression in the CNS continues to decline until becoming undetectable at stage 16. Thus, fru transcripts are expressed throughout the development and early differentiation of the CNS but become undetectable at later stages. Cells in some non-CNS tissues, the amnioserosa and tracheal placodes, also expressed fru transcripts (stages 9-11), but no in situ hybridization signal was detected in other tissues, such as the PNS or body wall muscles (Song, 2002). The fru locus encodes a complex set of transcripts. To better understand the embryonic pattern of fru transcripts, in situ hybridization was performed with a set of 5' end riboprobes to distinguish transcripts made from different fru promoters. No P1 or P2 transcripts were detected at any embryonic stage using riboprobes specific to these transcripts. Thus, P1 transcripts, which encode the male-specific fru proteins, are not expressed in the embryo. Transcripts from P3 and P4 promoters were expressed during embryogenesis in a temporal and spatial pattern that mirrored that of fru transcripts detected by antisense-BTB or -Com riboprobes. In the developing CNS, both P3- and P4-specific riboprobes labeled mesectodermal and neuroectodermal cells followed by labeling of delaminating neuroblasts. At slightly later stages (stages 9-11), medial neuroblasts continued to express P3 and P4 transcripts, but lateral neuroblasts no longer had detectable transcripts. In stages 7-12 embryos, the in situ hybridization signal for P4 transcripts was less intense, suggesting that the level of P4 transcripts was lower than that of P3 transcripts in these stages. However, P3 transcript levels became undetectable in all tissues after stage 12, whereas P4 transcripts were still detectable up to stage 16. In summary, the in situ hybridization data show that both P3 and P4 fru transcripts are expressed in the developing CNS and, very likely, in the same cells. Furthermore, the higher level of fru transcripts detected with riboprobes to fru common sequences in stages 9-12 CNSs is likely due to the presence of both P3 and P4 transcripts whereas the lower level of fru transcripts in stages 12-16 CNSs reflects the presence of only P4 transcripts (Song, 2002).

An additional complexity in fru transcripts reflects alternative splicing at the 3' end, which generates transcripts containing one of three different pairs of Zn-finger domains. While it is known that P1 transcript isoforms are spliced to each of the three alternative 3' ends, the full complexity of the 3' alternative splicing of transcripts produced from the P2, P3, and P4 promoters is not known. Embryos were examined by in situ hybridization with three different 3' end riboprobes to detect transcripts having the A, B, or C 3' ends. Overall, the temporal and spatial expression pattern of transcripts containing the A, B, and C 3' ends was consistent with the pattern found for fru transcripts labeled with antisense-BTB and antisense-Com riboprobes. In the developing CNS, transcripts containing the C 3' end appeared to be more abundant than those containing the A or B 3' ends. In addition, some tissues, such as the tracheal placodes and amnioserosal cells, were labeled only when riboprobes to the C 3' end were used, suggesting that there is some tissue-specific regulation of the 3' alternative splicing of P3 and or P4 fru primary transcripts. The localization of Fru proteins during embryogenesis is largely consistent with the spatial and temporal RNA distribution. All Elav-positive cells in the CNS (neurons) express Fru proteins, even though there were variable levels of Fru protein expression in individual cells. Likewise, all Repo-positive cells in the CNS (glia) were also Fru positive. In summary, all neurons and glia appear to express Fru proteins. All neurons and some lateral glia contain Fru isoforms having at least two different carboxy termini, but some lateral glia and midline cells contain Fru proteins that may have only one type of carboxy terminus (Song, 2002).

Given the findings that the fru gene is widely expressed in the embryonic CNS, fru mutant embryos are likely to have defects in CNS development: two antibodies, anti-FasII and mAb BP102, were used to assay axonal projections within the CNS. FasII is a neural adhesion molecule expressed on the cell surface of axons forming specific longitudinal fascicles or tracts running throughout the entire ventral nerve cord and into the brain. In whole-mount or flat-dissected preparations of stage 16 and 17 wild-type embryos, three tracts, medial, intermediate, and lateral, are visible in the CNS (Song, 2002 and references therein).

FasII-positive tracts are abnormal in fru mutants lacking all or most fru transcripts. Between 12% and 25% of fru null mutant embryos have abnormal FasII-positive tracts. By comparison, <3% of wild-type embryos have any segments with disrupted FasII tracts. In all fru null mutant CNSs, FasII-positive axons no longer form distinct tracts in one or more adjacent hemisegment, suggesting that these axons have defasciculated from other axons within the tracts. In some cases, axons that have defasciculated cross and join an adjacent fascicle or approach and cross the midline. In other cases, the left and right medial tracts appear to merge along the midline (Song, 2002).

To determine whether many or most axonal tracts are disrupted in fru mutants, the longitudinal connectives and commissures were labelled with the BP102 antibody. Almost 20% of the fru null mutant embryos have defects in the pattern and distribution of BP102-positive axons in the connectives and commissures compared to only 1% of wild-type embryos. Most commonly, in these mutants, the commissures and connectives are not uniform but are either thicker, as though more axons are present, or thinner, as though fewer axons are present (Song, 2002).

To demonstrate that these FasII and BP102 axonal defects depend on the loss of fru function, the fruw12 allele, which has a chromosomal break within the fru locus, was used in combination with fru deletion mutations. 15%-23% of these mutant embryos have defects in their FasII and BP102 tracts; the frequency and the severity of the defects are similar to those found in fru null embryos. The chromosomal break in the fruw12 allele in the fru locus separates the P1, P2, and P3 promoters from the fru coding region but leaves the P4 fru transcription unit intact. This result suggests that fru transcripts from the P1, P2, or P3 promoters are important for wild-type axonal pathfinding (Song, 2002).

To further define which fru transcripts are required for the formation of FasII and BP102 tracts, fru mutants were examined in which P1 transcripts are affected or where P1 and P2 transcripts are eliminated (e.g., fru4-40/frusat15. These mutants have wild-type FasII and BP102 axonal tracts and produce P3 and P4 transcripts. The finding that P1 and P2 fru transcripts are not required for the formation of FasII and BP102 tracts is consistent with evidence by in situ hybridization that these transcripts are not present in embryos. By considering the different fru mutant genotypes examined, it is inferred that expression of P3 and, perhaps, P4 fru transcripts is sufficient for the development of wild-type FasII and BP102 tracts. The role of P4 transcripts in this process is inferred from data of mutants expressing the fruw12 allele and it is possible that P4 transcripts, while present, are not expressed as in wild-type animals (Song, 2002).

If the axonal pathfinding defects in fru mutants are due to the loss of fru function in neuronal precursors or in neurons themselves, then there are two likely explanations for the altered axonal trajectories in these mutants. One explanation is that neurons have not adopted, or only partially adopted, their wild-type identity in fru mutants and thus their axons fasciculate with different axonal partners as they grow in the CNS. An alternative explanation is that neurons in fru mutants adopt their wild-type identity but are unable to carry out their normal program of axonal pathfinding and differentiation. To distinguish between these two possibilities, neuronal and axonal markers were used to identify the earliest developmental abnormalities in fru mutants (Song, 2002).

To assess whether fru plays a role in neuroblast delamination and identity, developing neuroblasts were labelled with antibodies to the Hunchback (Hb) protein, which labels all delaminating neuroblasts by early stage 9 in wild-type embryos. Fewer neuroblasts were labeled with anti-Hb antibody in early stage 9 fruw12/frusat15 embryos than in wild type, but neuroblasts in all three rows did become Hb positive in late stage 9 embryos. Thus, the final pattern of Hb expression is wild type in these fru mutant embryos but there is a slight temporal delay in either neuroblast delamination itself or the onset of Hb expression in delaminating neuroblasts (Song, 2002).

Next, the development of neurons that pioneer the FasII fascicles was examined to better assess whether there were changes in neuronal identity or early defects in axonal differentiation in fru mutants. In the wild-type CNS, pCC, vMP2, dMP2, and MP1 axons initiate the formation of the medial and intermediate FasII tracts within each segment. The axonal process of the pioneer neurons vMP2 and pCC ascends while the processes of the MP1 and dMP2 axons fasciculate and extend posteriorly. These axons initially produce one fascicle at stage 13, which then splits into two fascicles, the pCC/vMP2 (medial) and dMP2/MP1 (intermediate) fascicles (Song, 2002).

The development of aCC, pCC, vMP2, and dMP2 neurons was examined in frusat15/fruAJ96u3 and fruw12/fruAJ96u3 embryos. Along the midline, the MP2 precursor expresses the Odd-skipped protein and upon division the dMP2 daughter cell maintains Odd-skipped expression while the vMP2 daughter downregulates Odd-skipped expression. In fru mutant embryos all segments have the expected complement of Odd-skipped neurons along the midline. The other pioneer neurons, aCC and pCC neurons, can be recognized by their location and FasII expression in stage 12 embryos. In fru mutant embryos, aCC and pCC neurons were present and beginning to extend their axonal processes. These results show that in fru mutant embryos, neurons pioneering the medial and intermediate FasII tracts express markers appropriate for their expected neuronal fate (Song, 2002).

fru mutants that lack most or all fru transcript classes form longitudinal and commissural axonal tracts in which axons do not coalesce into fascicles, fasciculate with inappropriate partners, or are unable to maintain proper fasciculation. In fru mutants where P1, P2, and P3 transcripts are disrupted, but P4 transcripts were present, the defects in FasII and BP102 axonal tracts are as severe as the defects in embryos completely lacking fru function. Consideration of the axonal phenotypes in these different fru mutant genotypes suggests that P3 fru transcripts are sufficient for the formation of wild-type FasII and BP102 tracts. Even though this explanation is the simplest that accounts for the data, it was not possible to assess the effects of the loss of transcripts from only the P2, P3, or P4 promoter. Thus, the possibility that elimination of other fru transcripts or combinations of transcripts might also result in defective axonal pathfinding cannot be ruled out (Song, 2002).

The embryonic phenotypes of complete loss of fru mutants are mild with only a fraction of mutant embryos showing defects in their axonal tracts. This relatively benign phenotype suggests that the activity of other genes may be able to compensate for the loss of fru function. Mutants in other genes, such as fasII, Dlar, and other receptor protein tyrosine phosphatases that encode fasciculation and guidance molecules, also exhibit weak phenotypes in single mutants but show much stronger phenotypes in double mutants or when heterozgyous with mutations that reduce the function of genes that operate in the same developmental pathway (Song, 2002).

The FasII and BP102 axonal pathfinding defects found in fru mutant CNSs might result from the failure of neurons to adopt their proper cell fate or their ability to differentiate according to their fate. Since P3 and P4 fru transcripts are strongly expressed during NB delamination and early neurogenesis, the time at which neuronal fate decisions are being made, it was considered possible that fru's main function would be in fate determination. Four neurons, dMP2, vMP2, aCC, and pCC, responsible for pioneering the medial and intermediate FasII tracts expressed the appropriate identity markers, Odd-skipped and FasII. These results suggest that in fru mutants these neurons have adopted, at least partially, their initial wild-type fate. In support of this finding, it was found that in fru mutants lacking all or most fru transcripts, all aCC and pCC neurons express Even-skipped. The loss of fru function, however, does affect the ability of some of these neurons to maintain Even-skipped expression at later embryonic stages. All possible markers for these neurons have not been examined, and it may be that some cell fate markers are not expressed appropriately in fru mutants. The delay in the onset of Hb expression in neuroblasts may also indicate that fru has a small early role in neurogenesis. In addition, neuronal identity was examined in a very small population of neurons that have a very specific pioneering function; it may very well be that the fru gene plays a role in establishing neuronal identity in other embryonic neurons (Song, 2002).

In fru mutants, the earliest defects observed in FasII pioneering neurons were in the orientation and outgrowth of their initial axonal projections. In some neurons, axonogenesis appeared to be delayed, whereas in other neurons the initial axonal process was oriented abnormally and/or did not appear to be fasciculating properly with other axons. If these pioneering axons are unable to form normal fascicles or are unable to coalesce into discrete fascicles, then other later developing neurons may also be expected to have difficulties in fasciculating along their normal pathways. These results suggest that the loss of fru function may very well affect the expression of the specific receptor systems on axons that are necessary to recognize their fasciculation partners. In the dMP2 and vMP2 neurons, the expression of the Futsch protein was also delayed, suggesting that this gene is a target of fru function. Similar weak labeling of neurons by mab22C10 has also been described in embryos mutant for the argos, pointed, and prospero genes; these genes are known to be important for establishing cell fate and in some cases are required for the formation of FasII axonal tracts. There is no direct evidence that the loss of futsch expression in fru mutants leads to abnormalities or delays in the outgrowth of dMP2/vMP2 axons. The UAS-fruA and UAS-fruC transgenes were able to rescue the FasII axonal pattern without rescuing the initial defects in mab22C10 expression in dMP2/vMP2. Thus, it is possible that the defects in axonal outgrowth growth by these neurons depend on alterations in other proteins involved in axonogenesis or axonal pathfinding (Song, 2002).

Glial cells have been implicated as important regulators of axonal pathfinding by neurons. Glial cells in the CNS can be grouped into two major categories, midline and the lateral glia, according to their position and gene expression profiles in wild-type embryos. Four segmental midline glial cells, closely associated with the developing commissures, are characterized by the expression of the epidermal growth factor receptor, argos, and pointedP2. Lateral glial cells consist of several functional subgroups and express the pointedP1, repo, and glial cell missing genes. Lateral glial cells, identified by their expression of Repo, express Fru proteins. Cell counts in fru mutant embryos revealed no change in the number of Repo-immunoreactive glial cells in these embryos compared with wild type. Likewise, no defects were found in the number of midline glial cells in fru mutant embryos (Song, 2002).

Glial cells of both subtypes are required for the formation of the axonal scaffold of the ventral nerve cord. The loss of lateral glial cells has been implicated in defasciculation phenotypes of tramtrack and glial cells missing mutant embryos. Defasciculation of FasII axons has also been found in repo mutants in which lateral glial cells are largely present, but are in some way unable to support axonal fasciculation. Other studies have identified mutations in genes involved in midline or glial development causing defects in FasII and BP102 CNS tracts similar to the phenotypes of fru mutants. The phenotypic similarity between these mutants and fru raises the possibility that fru acts in the same pathway as these other genes in glial cells (Song, 2002).

From these findings, it is concluded that the fru gene functions in the process of axonal pathfinding by neurons in the embryonic CNS. The earliest neuronal defect observed was in the initial outgrowth of axons, which suggests that the fru gene plays an important role in neurons during axonogenesis. Since fru is expressed in neuronal progenitors as well as in neurons and glia, fru may also have a role in cell fate acquisition or maintenance in these cell types (Song, 2002).

Isoform-specific control of male neuronal differentiation and behavior in Drosophila by the fruitless gene

How the central nervous system (CNS) develops to implement innate behaviors remains largely unknown. Drosophila male sexual behavior has long been used as a model to address this question. The male-specific products of fruitless (fru) are pivotal to the emergence of this behavior. These putative transcription factors, containing one of three alternative DNA binding domains, determine the neuronal substrates for sexual behavior in male CNS. This study reports on the isolation of he first fru coding mutation, resulting in complete loss of one isoform. At the neuronal level, this isoform alone controls differentiation of a male-specific muscle and its associated motorneuron. Conversely, a combination of isoforms is required for development of serotonergic neurons implicated in male copulatory behavior. Full development of these neurons requires the male-specific product of doublesex, a gene previously thought to act independently of fru. At the behavioral level, missing one isoform leads to diminished courtship behavior and infertility. This study achieved the first rescue of a distinct fru behavioral phenotype, expressing a wild-type isoform in a defined subset of its normal expression pattern. This study exemplifies how complex behaviors can be controlled by a single locus through multiple isoforms regulating both developmental and physiological pathways in different neuronal substrates (Billeter, 2006).

fruΔC, the first fru coding mutation, completely removes all type-C Zinc-finger Fru isoforms, yet is a hypomorphic mutation with separable phenotypes associated with the two classes of Fru proteins. Viability and morphological defects in both sexes are due to the lack of FruComC, whereas reduced male fertility and lack of the MOL are caused by the absence of FruMC. Phenotypes common to both sexes uncovered new fru requirements for adult morphology, as in the development of imaginal disc derivatives such as eyes, legs, and genitalia, as well as reduction in the number of fru neurons in the abdominal ganglion (Billeter, 2006).

However, using animals missing only FruMC, focus was placed on the behavioral function of the male-specific FruMC isoform. Male sexual behavior consists of a series of independent but interlinked steps. FruMC specifies a subset of these functions, because males lacking only this isoform qualitatively perform all courtship steps, but in a quantitatively subnormal manner, often failing to mate. Therefore, FruMC functions in the specification of all steps of sexual behavior, rather than one specific modality. Because flies lacking this isoform still exhibit courtship behaviors, and sometimes are even fertile, the remaining FruM isoforms specify enough neuronal substrates for male sexual behavior. FruM isoforms, therefore, have an additive, not cumulative, effect on the specification of a given behavior (Billeter, 2006).

Behavior not only emerges from the functionality of single cells, but also depends on a network of neuronal interactions. This is exemplified by the broad effect that FruMC has on courtship and its specific effect at the neuronal level. FruMC is the only isoform controlling the innervation and formation of the MOL, demonstrating that an individual isoform, through differentiation of a single neuron, can control a specific fru phenotype. However, a combination of isoforms is required for complete development of the male serotonergic neurons. Individual isoforms can induce formation of subsets of these neurons, but this is not functional redundancy given that FruMA or FruMB cannot rescue FruMC-null phenotypes. This illustrates how isoforms perform different functions in a single cell type to regulate a phenotypic outcome. fru exploits multiple isoforms to create a 'neural code' where each phenotype is specified by either a single isoform or a combination of isoforms (Billeter, 2006).

This study has elucidated some of the principles of this 'code'. The Zinc fingers determine the functional difference of each isoform, with selective use of only three of the four possible domains indicating a requirement for specific binding domains to control different genes. The isoforms also display differential spatial expression patterns. FruMC is expressed in all FruM neurons, whereas FruMA is more restricted and notably is not expressed in the male serotonergic neurons and does not appear to participate in their formation. Finally, it is shown that the BTB and Zinc-finger domains are necessary to confer functional activity and specificity to FruM isoforms. longitudinal lacking (lola), a member of the same family of transcription factors as fru, uses similar mechanisms to control a wide range of axonal-guidance decisions. Through alternative splicing, it generates multiple isoforms with unique Zinc-finger domains and expression patterns; moreover, mutants lacking single isoforms show that alternative isoforms have different functions. Alternative splicing and differential expression are not just central to the diversity of FruM function, but appear to be key principles for explaining how a single locus controls complex biological processes (Billeter, 2006).

The formation of the MOL is determined by its innervation, with fru directing the recruitment of myoblasts from a limited non-sex-specific pool into the larger MOL in males. FruMC mutants develop four smaller muscles, each innervated by the motor neuron that would normally innervate the MOL. Thus expression of FruMC in the MOL motor neuron regulates the patterning of myoblasts into one bigger muscle, and in its absence these myoblasts are partitioned into four fibers. This patterning effect of motor neurons has been documented in the development of the indirect flight muscles in Drosophila. The sexually dimorphic appearance of the MOL NMJ is likely a secondary effect of myoblast reorganization into the larger male structure. Indeed, as muscle size increases, a concomitant increase in synaptic efficacy, or number, is required to ensure appropriate muscle contraction. Understanding FruMC's role in orchestrating the differentiation of the MOL and its motor neuron may come from investigating the proteins controlling synaptogenesis and homeostasis during muscle growth. Perhaps this mechanism is exploited by subsets of FruM neurons to induce sex-specific changes in other neurons (Billeter, 2006).

Understanding the neuronal substrates for male sexual behavior requires defining how groups of FruM neurons control distinct modalities of this behavior. The fact that only one isoform is missing in fruΔC mutants, resulting in hypomorphic behavioral phenotypes, allows the behavioral function of FruM neurons to be tested. Expression of FruMC in a subset of its normal expression pattern in a FruMC-null background rescues mating and fertility but not overall courtship behavior. This uncouples mating from courtship and shows that different populations of FruM neurons are responsible for distinct steps of male sexual behavior. This behavioral rescue coincides with rescue in defined FruM neurons involved in mating behavior, including the male serotonergic neurons, and motor neurons innervating the MOL and abdominal muscles necessary for successful copulation (Billeter, 2006).

That subsets of FruM neurons control certain behavioral modalities, but not others, suggests that fru does not work strictly by establishing one main closed circuit but rather establishes, and/or links, many foci whose independent activities are recruited into a series of behavioral steps. These neurons are thus better described as forming a network rather than a circuit. This is in keeping with experiments on mosaic animals, whose brains are part male and part female, that showed that the ability to perform individual behaviors resides in different CNS regions. The concept that male sexual behavior is not strictly controlled by a closed circuit offers interesting possibilities to evolve subtle differences by adding, changing, or removing the functionality of cells among the network of fru neurons. This might affect quantitative parameters, such as the difference between the songs of the closely related D. simulans and D. melanogaster, or accumulate to give qualitatively different sexual behaviors, as between A. gambiae and D. melanogaster. Given that Fru proteins are functionally conserved between A. gambiae and D. melanogaster, these differences could be due to changes in FruM expression pattern and/or of its isoforms (Billeter, 2006).

FruM controls the development of male serotonergic neurons in the adult abdominal ganglion. A striking feature of these neurons is their organization into two opposing clusters that both innervate the same reproductive organs. That these projections are collateral suggests that they act to coordinate the function of related targets like the vas deferens (controlling sperm emission from the testes) and the accessory glands (producing seminal fluids). Such modulation is required for the synchronized emission of sperm, fertilizing the female, and seminal fluids, preventing remating. Defects in sperm and seminal-fluid transfer in certain fru allelic combinations may be due to subnormal production of serotonin in neurites innervating the reproductive organs. FruMC-null males exhibit similar defects, but they also have reduced numbers of male serotonergic neurons, which are not always properly organized into two clusters. This aberrant development and organization may be one reason for their sterility. It is hypothesized that FruM directs the development of two clusters of male serotonergic neurons to form a neuronal network controlling male reproductive physiology (Billeter, 2006).

Previously, fru and dsx have been described as acting independently in the sex-determination pathway, with FruM expressed specifically in the male CNS determining male sexual behavior and with dsx expressed in the soma determining the dimorphic morphology of the sexes. However, this study found that male serotonergic neurons exhibit abnormal differentiation in dsx-null animals and fail to differentiate in fru mutant males. These experiments evoke a mechanism for this apparent overlap in function. DsxM controls the formation of 20 neurons in the abdominal ganglion by prolonging neuroblast proliferation at the end of the larval stage. In dsx-null animals these neuroblasts completely fail to develop, and, notably, the number of male serotonergic neurons is reduced in mutant males. This DsxM-dependent proliferation of neurons appears to offer a substrate for FruM to induce serotonergic differentiation. A number of ectopic serotonergic neurons form in dsx-null females. Given that these females do not express FruM, the development of these neurons would appear normally to be inhibited by dsx (Billeter, 2006).

These experiments reveal a new feature of FruM function. Whereas dsx-null males exhibit less male serotonergic neurons, these retain their typical dorso-ventral patterning. Conversely, the ectopic serotonergic neurons in dsx-null females develop randomly either ventrally or dorsally. This lack of dorso-ventral patterning must be linked to the absence of FruM, given that females missing Tra develop a complete organized set of male serotonergic neurons. Constitutive expression of FruM in females also induces two clusters of serotonergic neurons, though with fewer neurons than wild-type males. This reinforces the contention that FruM is fundamental for controlling not only the serotonergic differentiation of these neurons but their structural organization into a functional circuit. These experiments also show that the male serotonergic neurons stem from at least two different populations of neurons, one requiring both DsxM and FruM, the other just FruM. Two lines of evidence support the idea that FruM acts directly on the neurogenesis of these cells: Their projections are greatly reduced in FruM-null males, suggesting that some of these neurons are absent, and the number of neurons expressing FruM in FruMC-null males is reduced. fru has been shown to prevent cell death in neurons in the male brain. FruM could use this mechanism to control the number of male serotonergic neurons during development (Billeter, 2006).

Although FruM is sufficient to determine most aspects of male sexual behavior, part of its function requires a male-specific neuronal substrate determined by dsx. It is predicted that DsxM/FruM cooperation extends beyond the formation of the male serotonergic neurons because FruM and Dsx colocalize in more than 100 neurons in the abdominal ganglion. Moreover, given that dsx is required for males to generate normal courtship song, it is anticipated their interaction to have a broader importance in the determination of male sexual behavior (Billeter, 2006).

Modulation of Drosophila male behavioral choice

The reproductive and defensive behaviors that are initiated in response to specific sensory cues can provide insight into how choices are made between different social behaviors. This study manipulated both the activity and sex of a subset of neurons and found significant changes in male social behavior. Results from aggression assays indicate that the neuromodulator octopamine (OCT; see Tyramine β hydroxylase) is necessary for Drosophila males to coordinate sensory cue information presented by a second male and respond with the appropriate behavior: aggression rather than courtship. In competitive male courtship assays, males with no OCT or with low OCT levels do not adapt to changing sensory cues and court both males and females. A small subset of neurons was identified in the s ganglion region of the adult male brain that coexpress OCT and male forms of the neural sex determination factor, Fruitless (FruM). A single FruM-positive OCT neuron sends extensive bilateral arborizations to the s ganglion, the lateral accessory lobe, and possibly the posterior antennal lobe, suggesting a mechanism for integrating multiple sensory modalities. Furthermore, eliminating the expression of FruM by transformer expression in OCT/tyramine neurons changes the aggression versus courtship response behavior. These results provide insight into how complex social behaviors are coordinated in the nervous system and suggest a role for neuromodulators in the functioning of male-specific circuitry relating to behavioral choice (Certel, 2007).

Modulation of classical neurotransmitter action on target neurons adds great flexibility to synaptic output between neurons and is suggested to be at the core of important behavioral processes like learning and memory. In vertebrates, amines like serotonin, dopamine, and norepinephrine; peptides like arginine vasopressin, and oxytocin; gonadal steroids; and various glucocorticoids serve as well known neuromodulatory substances. Through selective actions at individual synaptic sites, neuromodulators coordinate the output of neuronal ensembles to generate behavioral patterns of varying complexity (Certel, 2007).

An elegant example of coordinating network output comes from studies with the stomatogastric ganglion of crustaceans. In this small neuronal ensemble, neuromodulators function either singly or in various combinations at multiple sites in the ganglion to alter the patterned output of the ganglion and thereby the movement of food through the stomach. An example of changing network ensembles in vertebrates is seen in studies of vole social behavior. Here, the distribution of oxytocin, vasopressin, and dopamine receptors within different brain regions appears linked to the differences seen in social behavior between prairie voles and montane voles (Certel, 2007 and references therein).

This paper focuses on the roles of octopamine, a phenolamine structurally related to the catecholamine norepinephrine, in modulating the choice between courtship and aggression in male flies. Norepinephrine has been shown to be important in many aspects of vertebrate behavior, including arousal, anxiety, learning and memory, opiate reward, and aggression. Among invertebrates, OCT influences foraging behavior in honey bees; resets aggressive motivation in crickets; and functions in appetitive associative learning, ethanol tolerance development, and possibly aggression levels in Drosophila. Like their vertebrate amine neuron counterparts, OCT neurons in Drosophila (1) are few in number but have enormous fields of innervation covering essentially all neuropil areas in the fly brain and (2) function by activating multiple G protein-coupled receptors (Certel, 2007).

Aggression and courtship usually are mutually exclusive behaviors. By examining the choices made between these behaviors by male flies, a powerful approach is offered with which to study the genetic and neural basis of complex behaviors. Multiple decision-making actions are required for each of these behaviors, including the processing of chemosensory and visual information and deciding whether another fly is a potential opponent or a potential mate. Using aggression and competitive courtship assays, OCT was found to be necessary for pairs of Drosophila males to respond to the sensory cues presented and to coordinate expression of the appropriate response: aggression. Feminizing OCT/tyramine (TYR) neurons in males also changes the aggression vs. courtship response behavior. Because the gene fruitless directs both courtship and aggression in flies, the expression patterns of OCT and the male forms of Fruitless (FruM) was analyzed and the were found to be coexpressed in distinct s ganglion (SOG) neurons in the male brain. This region receives the contact gustatory pheromone information thought to facilitate sex and species discrimination. The arborizations of one of the FruM-octopaminergic neurons were found to project bilaterally and appear to ramify in the posterior antennal lobe, multiple SOG layers, as well as the lateral accessory lobe (ventral body). These results offer insight into how sensory cues are integrated and modulated in the nervous system to direct sex-specific complex behaviors and indicate a role for the neuromodulator OCT in the functioning of the male-specific circuitry relating to behavioral choice (Certel, 2007).

Males and females react to environmental cues with distinct sex-specific innate behaviors particularly in the areas of courtship/reproduction and aggression/defense. Results from a number of studies have demonstrated that functional and structural sex differences in the brain can influence and direct these behaviors, but how sensory cues contribute to the appropriate response of one of these two mutually exclusive behaviors remains unclear. This study presents evidence that the neuromodulator OCT functions within a defined circuit to provide at least one means of regulating the choice between courtship and aggression. The results of these aggression studies indicate that male flies require OCT to respond with an appropriate aggressive response to another male. The results of the male–female courtship assays suggest that normal OCT function provides increased behavioral response confidence about the sensory cues being presented (Certel, 2007).

Identifying a potential mate or opponent relies on discriminating specific stimuli from background and then integrating this information with other sensory modalities. Anatomically, the extensive arrays of OCT-immunoreactive processes that are found throughout the Drosophila brain offer one such overlying integration network that may fine-tune sensory input and activate sex-specific behavioral subcircuits. In Drosophila, male-specific behavioral circuits are specified by the male-specific products of the fruitless gene. In this study, it was demonstrated that three VUM neurons in the male SOG coexpress FruM and OCT. The SOG is the primary taste-processing center in the fly. The sensory information sent to this neuropil includes the female pheromone recognition cues necessary for male courtship behavior. Therefore, an intriguing possibility is that OCT is necessary in the subset of FruM-positive SOG neurons to accurately relay contact gustatory pheromone information (Certel, 2007).

Morphological results suggest that a single neuron can provide a simple integration network of multisensory cues. The arborizations of one of the VUM 1 FruM-positive OCT neurons extensively ramify throughout multiple neuropil regions, including the SOG, posterior antennal lobe, and the lateral accessory lobe (ventral body), suggesting a link between various sensory modalities. Gustatory information from OCT/FruM SOG neurons could also be linked to higher-order processing centers through synaptic contacts with the male-specific SOG projections of FruM-expressing mAL neurons identified. The superior lateral protocerebrum has been proposed to be the output site of these interneurons. Linkages of this type may be of particular significance because FruM-expressing neurons play critical roles in two sex-specific social behaviors: aggression and courtship. Thus, the same circuits may need to integrate the context-specific sensory information necessary to direct the output of appropriate behavioral patterns (Certel, 2007).

How might OCT modify distinct SOG neurons to regulate behavioral choice by males? In the spider, OCT increases the overall sensitivity of mechanosensory neurons by local release from efferent endings. This local release suggests that sensory input from specific sensilla relative to others can be emphasized depending on behavioral circumstances. In the silkworm moth, OCT specifically increases the sensitivity of male pheromone-sensitive receptor neurons but not general odorant-sensitive responses. Recent modeling studies in vertebrates suggest that neuromodulators can play a key role at specific times in decision-making tasks by regulating competition between populations of neurons that represent choices. This regulation may allow an organism to integrate noisy sensory information and past experience to make optimal decisions (Certel, 2007).

Although the mouse neural pathways that mediate the output of two sex-specific behaviors, reproduction and defense, are anatomically segregated, a recent study identified a hypothalamic point of convergence that may function as a choice selection mechanism for sensory activation of defensive responses over reproduction. The results suggest that whether an individual male mouse responds with the appropriate behavior depends on the coordinated activation of the appropriate subcircuits by amygdalo–hypothalamic projections. Likewise the different behavioral outputs of Drosophila males and females could be generated through the activation of sex-specific segregated neural ensembles. However, behavioral differences also could emerge through sex-specific modulation of circuits that are common to both sexes. In males, FruM proteins are expressed in small groups of neurons throughout the CNS, and eliminating FruM expression in a neuronal subset has profound effects on the progression of male courtship behaviors. At the gross level almost all of the FruM-producing neurons have counterparts in the female and in terms of function, a recent report indicates that the sex-specific reproductive behaviors of females and males involve shared neural circuits. The splicing of fruM-specific transcripts have been proposed to modify neurons common in both sexes for male-specific functions through differences in neuron morphology and/or physiology (Certel, 2007).

In addition to changing the activity of OCT neurons, OCT/TYR neurons were feminized in an otherwise masculine brain and altered male behavioral choice was demonstrated. The results from OCT immunostaining do not indicate any sex-dependent changes in SOG neuron number. The identification of a sex-independent marker for the FruM-positive OCT neurons should allow determination of whether feminizing these neurons changes either their branching patterns, their synaptic connections, or their OCT-related biochemical properties. Further examination of these OCT/FruM SOG neurons should offer a behaviorally relevant ensemble with which to address questions of sex-specific morphology and function-related physiology (Certel, 2007).

Fruitless and Doublesex coordinate to generate male-specific neurons that can initiate courtship

Biologists postulate that sexual dimorphism in the brain underlies gender differences in behavior, yet direct evidence for this has been sparse. A male-specific, fruitless (fru)/doublesex (dsx)-coexpressing neuronal cluster, P1, was identified in Drosophila. The artificial induction of a P1 clone in females effectively provokes male-typical behavior in such females even when the other parts of the brain are not masculinized. P1, located in the dorsal posterior brain near the mushroom body, is composed of 20 interneurons, each of which has a primary transversal neurite with extensive ramifications in the bilateral protocerebrum. P1 is fated to die in females through the action of a feminizing protein, DsxF. A masculinizing protein Fru is required in the male brain for correct positioning of the terminals of P1 neurites. Thus, the coordinated actions of two sex determination genes, dsx and fru, confer the unique ability to initiate male-typical sexual behavior on P1 neurons (Kimura, 2008).

An effort at identifying the neural centers for specific acts in courtship behavior was initiated by Hotta and Benzer (1976), who inferred the foci for a variety of behavioral acts in the brain by correlating the body surface markers or neural markers with the incidence of each act in sexually mosaic flies. Hall (1979) generated female-male (XX-XO) mosaic flies that were subjected to mating assays and then sacrificed, after which their brain sections were stained with the Acph-1 enzymatic marker to determine which parts of the brains of individual flies were female and which parts male. In this classic work with gynandromorphic flies, the conclusion was reached that the flies perform the early part of courtship behavior when a specific site located in the dorsal posterior brain is composed of male tissues, at least on one side of the brain. This conclusion was supported by a study in which part of a male brain was feminized with tra+. In addition, a cluster of cells in the posterior lateral protocerebrum has been suggested to mediate the initiation and early steps of male courtship by experiments in which transgenes that either activated or suppressed neural activities were focally expressed (Kimura, 2008 and references therein).

The present study succeeded in pinpointing a single neural cluster that plays an important role in initiating courtship by large-scale screens of tra1 mosaic females for the ability to perform male-typical behavior. The cluster is called P1, and is located in close proximity to the mushroom body. It is intriguing that P1 is located roughly in the area Hall (1979) assigned as the focus of male-courtship. He showed that 'SP3' ('supraoesophageal ganglion cortex site 3') is the 'domineering' focus for the early steps of male-type courtship behavior, which include tapping, following, and wing extension. To perform the late steps of male-type courtship behavior, flies need to have additional male tissues in other parts of the brain. Further study with gynandromorphs led to the identification of 'SP2' as a second focus for wing extension in male-type courtship behavior. Note that 'SP2' and 'SP3' represent the posterior brain sites distinct from current aSP2 and aSP3, which lie in the anterior brain (Kimura, 2008).

Female mosaic flies with the masculinized P1 cluster showed the early parts but not the late parts of male-type courtship, reminiscent of the behavior displayed by gynandromorphic flies with male 'SP3'. Thus, these two groups of flies share masculinized brain cells in a similar location and exhibit similar behavioral characteristics. P1 neurons have an input site and an output site in the lateral and medial protocerebra, to which sensory information on different modalities is conveyed by higher-order interneurons and from which descending interneurons extend their axons to motor centers in the brain and the ventral nerve cord. Although these structural features imply a role for P1 in the integration of multiple sensory inputs for the control of outputs for sexual behavior, this remains speculation, as the physiological properties of these neurons have not been characterized (Kimura, 2008).

Although the presence of P1 in the brain makes a fly more likely to engage in courtship, flies without P1 are able to perform male sexual behavior and other flies with P1 are not. This is not surprising in light of recent findings that the female flies with DsxF and devoid of Fru and DsxM perform male-type courtship behavior, provided that they are mutant for retained (Ditch, 2005; Shirangi, 2006; Shirangi, 2007). This may mean that entire motor patterns of male-type courtship behavior can be generated in the neural circuitry that is common to both sexes. A recent study using light-activated ion channels to stimulate fru-expressing neurons in the thoracic-abdominal ganglia reveals the presence of pattern-generating circuit for courtship song in both sexes (Clyne, 2008). P1 might function as a cluster of neurons that powerfully drive lower motor centers, which are not necessarily sexually dimorphic by themselves, to generate male-type courtship behavior. It is envisaged that male-sexual behavior is initiated by the activation of P1 under normal conditions in response to adequate key stimuli, yet can be commenced by the lower motor centers without any involvement of P1 neurons, when they are somehow sensitized or disinhibited under the influence of genetic or environmental factors. This may also explain why the fruM females are less active than males in performing male-type courtship behavior. fruM females lack P1 neurons so that they need additional drive to initiate male-type behavior (Kimura, 2008).

P1 neurons require functioning dsx expression to acquire sexual dimorphism: for example, DsxF contributes to the active elimination of P1 from the female brain, thereby preventing the females from behaving similarly to males in a sexual context. There are, however, precedent cases in which both dsx and fru are required for sexually dimorphic neural development, i.e., serotonergic neurons that innervate the seminal glands of males and a group of thoracic neurons that are suspected to function in specifying the courtship song (Kimura, 2008).

It is plausible that the neural circuit underlying sexual behavior is composed of three different neuronal species, namely, class 1 sex-specific or sexually dimorphic neurons whose sex specificity is determined by the coordinated actions of dsx and fru, class 2 sex-specific or sexually dimorphic neurons whose sex specificity is determined by either dsx or fru, and class 3 neurons that show no sex-related differences and do not express either dsx or fru. The class 3 neurons likely form a circuit common to both sexes, although this circuit is involved in the generation of male-specific motor outputs for male-type sexual behavior. Because of the presence of such a non-sex-specific circuit in the female brain, female flies are able to exhibit male sexual behavior after being genetically manipulated to possess male-specific P1 neurons. A recent study with mice deficient for pheromone sensation has led to a similar hypothesis, i.e., functional neural circuits underlying male-specific behavior exist in the normal female mouse brain (Kimura, 2008).

The three classes of neurons defined above may interconnect to form circuits that control different aspects of sexual behavior. In fact, some of the olfactory pheromone receptor neurons on the antenna express Fru. Male-specific expression of a gustatory pheromone receptor in the foreleg sensory hairs is dsx dependent but fru independent. The sexually dimorphic mAL interneurons suspected to be involved in the integration of pheromone inputs express Fru but not Dsx. The P1 cluster can initiate male-type courtship behavior and can thus be placed on the highest rung of the neural hierarchy. P1 is composed of class 1 neurons whose sex-specific differentiation is governed by both dsx and fru and thus are under the stringent control of the developmental program (Kimura, 2008).

Alternatively, P1 may contribute to one of several neural pathways, each with the potential to initiate male-type courtship behavior. It is worth examining whether or not this behavior can be elicited by the selective activation of individual neural clusters with the aid of photosensitive tools such as channelrhodopsins or the ionotropic purinoceptor P2X2, both of which are expressed only in a small MARCM clone in the brain (Kimura, 2008).

Midline crossing by gustatory receptor neuron axons is regulated by fruitless, doublesex and the Roundabout receptors

Although nervous system sexual dimorphisms are known in many species, relatively little is understood about the molecular mechanisms generating these dimorphisms. Recent findings in Drosophila provide the tools for dissecting how neurogenesis and neuronal differentiation are modulated by the Drosophila sex-determination regulatory genes to produce nervous system sexual dimorphisms. This paper reports studies aimed at illuminating the basis of the sexual dimorphic axonal projection patterns of foreleg gustatory receptor neurons (GRNs): only in males do GRN axons project across the midline of the ventral nerve cord. The sex determination genes fruitless (fru) and doublesex (dsx) both contribute to establishing this sexual dimorphism. Male-specific Fru (FruM) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (DsxF). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (DsxM). The roundabout (robo) paralogs also regulate midline crossing by these neurons, and evidence is provided that FruM exerts its effect on midline crossing by directly or indirectly regulating Robo signaling (Mellert, 2010).

This study shows that the male-specific presence of contralateral GRN projections is primarily due to FruM function. Specifically, FruMC acts in foreleg GRNs to promote the crossing of the VNC midline by their axons. A role for dsx was identified in this dimorphism since (1) males that lack DsxM have somewhat fewer contralateral GRN projections, and (2) DsxF prevents the appearance of contralateral GRN axons in females (Mellert, 2010).

The finding that FruM regulates GRN axon midline crossing is consistent with previous findings that, in some neurons, FruM regulates axonal morphology. Regulation of axonal morphology is likely to alter synaptic connectivity, suggesting that one of the roles of FruM is to support the formation of male-specific connections, and possibly prevent the formation of female-specific connections, between neurons that are present in both sexes. Determining how such changes alter information processing will contribute to understanding how the potential for male courtship behavior is established (Mellert, 2010).

It is also notable that dsx plays a role in regulating sexually dimorphic midline crossing, given that it also specifies the sexual dimorphism in gustatory sensilla number in the foreleg. It might be that dsx regulates gustatory sensilla development independently of its regulation of GRN axon morphology. That dsx can independently specify multiple sexual dimorphisms within particular cell lineages has been previously shown for the foreleg bristles that comprise the sex comb teeth of the male foreleg and their homologous bristles in the female. There, dsx was shown to function at one time to determine the sex-specific number of bristles that are formed and at another time to determine their sex-specific morphology. In support of a similar sequential role in the developing GRNs, dsx is expressed in the gustatory sense organ precursor cells and continues to be expressed in the terminally differentiated GRNs (Mellert, 2010).

It is also possible that the effect of dsx on the presence of contralateral GRN projections is indirect. The two pools of gustatory sensilla, those that are male-specific and those that are homologous between males and females, might differ in their competence for midline crossing (i.e. only the male-specific GRNs will cross the midline when FruM is expressed). This is thought not to be the case for two reasons. First, dsx is expressed in the GRNs throughout their development, consistent with a role in regulating axon guidance. Second, the expression of FruMC in female GRNs using poxn-Gal4 is sufficient to induce midline crossing, suggesting that the sex-nonspecific GRNs are not intrinsically nonresponsive to FruM (Mellert, 2010).

With respect to the latter result, it is worth considering the contrast between females that are masculinized with fruδtra, where no contralateral GRN projections are observed, and females in which poxn-Gal4 is used to drive the expression of UAS-fruMC::AU1 (AU epitope tagged Fruitless) in females, where GRN midline crossing is observed. In the case of females masculinized by fruδtra it was shown that the absence of contralateral GRN projections was due to DsxF functioning to prevent midline crossing in a manner that was epistatic to fruM function. One attractive explanation for the difference between these two situations is based on the fact that masculinization by fruδtra occurs via FruM produced from the endogenous fruitless locus, whereas masculinization by UAS-fruMC::AU1, occurs via fruMC expressed from a UAS construct that contains none of the untranslated sequences present in endogenous fruM transcripts. Thus, it might be that the difference in midline crossing seen in these two situations is due to DsxF directly regulating fruM expression through noncoding fru sequences that are present in the endogenous fru gene, but absent in the fru cDNA expressed from UAS-fruMC::AU1. It is not likely that DsxM represses fruM transcription, fruP1.LexA was seen to be expressed in GRNs in both males and females. Thus, if fruM is downstream of dsx in these cells, DsxF probably affects the processing or translation of fruM transcripts through sequences not present in the UAS-fruMC::AU1 construct. Alternatively, differences between these two situations in expression levels or patterns of expression might result in differences in the ability of FruM versus FruMC to overcome a parallel repressive effect of DsxF (Mellert, 2010).

robo, robo2 and robo3 are involved in GRN axon guidance. Of these three genes, robo appears to be most important in regulating GRN midline crossing because only reductions in levels of robo transcript result in midline crossing in females or fruM-null males. Reducing levels of robo2 and robo3 transcripts in addition to robo enhances the robo phenotype but individual reductions of robo2 or robo3 function have the opposite effect, a reduction in midline crossing, suggesting that these receptors function to promote crossing in the presence of wild-type levels of robo expression (Mellert, 2010).

It is not surprising that robo differs in function from robo2 and robo3 with respect to foreleg GRN development. robo2 and robo3 are more similar in sequence to each other than to robo, and robo contains two cytoplasmic motifs not found in its paralogs. Furthermore, functional differences have been recognized since the original reports of robo2 and robo3. Finally, robo2 might promote midline crossing if pan-neuronally overexpressed at low levels and yet repress midline crossing when overexpressed at high levels. This 'switch' in function might explain why reduced midline crossing is seen under conditions of both robo2 overexpression and reduction (Mellert, 2010).

Given that the Robo receptors play such an important role in GRN development, how might fruM regulate midline crossing? The data indicate that robo lies genetically downstream of fruM. The most straightforward mechanistic explanation is that FruM suppresses the activity of the Robo signaling pathway. Several ways that this might occur can be envisioned. First, fruM might regulate commissureless, which itself participates in the midline crossing decision by regulating the subcellular localization of Robo. No sexual dimorphism could be detected in the subcellular localization of a Robo::GFP fusion protein in GRNs in either the axons or cell body (UAS-robo::GFP), so if fruM regulates comm, it does so subtly. It is more probable that fruM regulates the expression of either other regulators of robo signaling, robo itself, or robo effectors. Strategies are being pursued to identify candidate FruM targets that might be involved in regulating midline crossing (Mellert, 2010).

How does midline crossing by GRN axons affect gustatory perception? Given that male-typical GRN morphology requires fruM, and that fruM has a major regulatory role for social behavior, one hypothesis is that the contralateral GRN projections in males play a role in mediating the processing of contact cues during male courtship and/or aggression. Previous reports have shown that fruM-masculinized females, which do not have contralateral GRN projections, readily perform tapping and proceed to subsequent steps in the male courtship ritual, and behave like males with respect to aggressive behaviors. Thus, contralateral GRN projections are not necessary for the initiation and execution of these male-specific behaviors. Nevertheless, midline crossing might still be important for mediating socially relevant gustatory information. For instance, amputation experiments suggest that the detection of contact stimuli is important for courtship initiation under conditions when the male cannot otherwise see or smell the female (Mellert, 2010).

It is possible that midline crossing by GRN axons facilitates the comparison of chemical contact cues between the two forelegs. Such a comparison might help the male to determine the orientation of another fly, which would be a useful adaptation for performing social behaviors in conditions of sensory deprivation, such as in the dark. Alternatively, midline crossing might simply be a mechanism to form additional neuronal connections that integrate gustatory information into circuits underlying male-specific behaviors. Armed with the results of the present study, fruM, dsx, and the robo genes can be used as handles for developing tools and strategies to specifically manipulate midline crossing in the foreleg GRNs, with the goal of understanding its importance with regard to male behavior (Mellert, 2010).

Cellular and behavioral functions of fruitless isoforms in Drosophila courtship

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. This study examined the expression and function of each of these isoforms. Most fru+ cells were shown to 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. 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 (von Philipsborn, 2014).

The primary goal of this study was to determine the expression pattern of each of the Fru isoforms (A-D) in the developing and adult male CNS, at cellular resolution, and to assess the contribution that each makes to both anatomical and behavioral dimorphisms. Such information is essential to the ultimate goal of understanding how fru sculpts the sex-specific neural circuitry, and hence the neural computations, that generate male courtship behavior. Previous reports were confirmed that the D isoform is not expressed in the developing or adult CNS and this isoform was not examined further. Each of the other three isoforms is expressed in the CNS and makes some contribution to male courtship behavior (von Philipsborn, 2014).

FruA, FruB, and FruC are coexpressed in many of the fru+ neurons, although several cell types express only one or two of these isoforms. A similar pattern of substantial but incomplete overlap of Fru isoforms has also been observed in the embryonic nervous system. Splicing at the 30 end of the fru transcripts thus appears to be regulated in a cell-specific manner, independent of the sex-specific splicing that some transcripts undergo at their 5' end (von Philipsborn, 2014).

Functionally, specific deficits were found in courtship behavior in flies lacking any one of the three isoforms. Thus, each isoform has some nonredundant contribution to courtship behavior and, presumably, the construction or function of the underlying circuitry. In general, however, the deficits observed upon eliminating just one isoform were relatively mild compared to those observed upon complete loss of the male-specific fru transcripts. All three isoform mutants performed male-female courtship, but with significantly reduced success. fruC mutants were the most affected, and fruA mutants the least. fruC mutants showed two striking defects in courtship behavior: the complete loss of sine song, and a dramatic reduction in pulse song in the absence of visual stimuli. The loss of sine song, which accounts for approximately half of the total song in control flies, is consistent with the significant reduction of wing extension observed previously in fruC mutants. Deprived of visual cues, a male presumably becomes more reliant on volatile or contact pheromones. Consistent with this, males unable to detect the female stimulatory pheromone 7,11-heptacosadiene show loss of courtship song in the dark, but not in the light. The similar defects in fruC mutants suggest that FruC might also be essential for detection or processing of this pheromone or other nonvisual stimuli from the female (von Philipsborn, 2014).

At the cellular level, this study found that almost all sexual dimorphisms within the fru circuit depend upon the function of fru itself. It has been shown previously that the absence of P1 neurons in females is due to dsx and not fru. On the other hand, dimorphisms in the number of mAL/aDT2 neurons, the size of the glomerular targets of Or67d+ OSNs, and the terminal arborizations of DA1 PNs had all been attributed to fru function. The current analysis thus confirms and extends the general observation that most cellular dimorphisms among the fru+ neurons are indeed dependent upon fru. Moreover, an interesting pattern was noticed in the requirement for specific isoforms for each of these dimorphisms. For those cell types that are present in both sexes but differ in their arborization patterns, the FruC isoform was strictly required. In contrast, for those cell types that differ in number, no single isoform was essential. This might reflect redundancy among the distinct target genes of each isoform, or among their distinct binding sites at common target genes. Alternatively, Fru might regulate cell birth or survival by a mechanism that is independent of its zinc-finger domain (von Philipsborn, 2014).

In addition to determining how fru establishes all of these cellular dimorphisms, the impact that each has on neural processing and behavior needs to be understood. A useful strategy here might be to individually feminize each cell type and ask how this perturbs behavior and, as the tools become available, the underlying physiological processes. Here, this study found that loss of FruC specifically in either aSP4 or vAB3 neurons feminizes their morphology and, it is inferred, changes their connectivity with pre- and postsynaptic partners. This might include connections between these two neurons, as aSP4 has FruC-dependent lateral arborizations that overlap with the processes of vAB3. Depleting FruC from aSP4 or vAB3 recapitulates one aspect of the courtship defect observed in mutants that lack FruC entirely: the pronounced loss of pulse song in the dark. The appropriate morphology and connectivity of aSP4 and vAB3 might therefore be essential for male-specific processing of pheromone signals. From their anatomy, both aSP4 and vAB3 appear to be candidate input neurons for P1, which is activated by the female gustatory pheromone (von Philipsborn, 2014).

As further cellular dimorphisms within the fru circuit are revealed, the tools generated here can be used to assess the genetic determinants and behavioral consequences of these dimorphisms. These analyses will guide the formulation of hypotheses about the role of specific cellular dimorphisms in the information processing that occurs within these circuits—hypotheses that can be readily tested as the physiological methods for circuit analysis advance. It may then ultimately be possible to establish mechanistic links from a single gene to the complex behavior that it specifies, encompassing the expression of the specific Fru isoforms and the target genes they regulate, the cellular properties these genes influence, the circuit-level information processing these properties enable, and ultimately the probabilistic mapping of sensory input to motor output that characterizes courtship behavior in the fly (von Philipsborn, 2014).

Genetic identification and separation of innate and experience-dependent courtship behaviors in Drosophila

Wild-type D. melanogaster males innately possess the ability to perform a multistep courtship ritual to conspecific females. The potential for this behavior is specified by the male-specific products of the fruitless (fruM) gene; males without fruM do not court females when held in isolation. Such fruM null males acquire the potential for courtship when grouped with other flies; they apparently learn to court flies with which they were grouped, irrespective of sex or species and retain this behavior for at least a week. The male-specific product of the doublesex gene dsxM is necessary and sufficient for the acquisition of the potential for such experience-dependent courtship. These results reveal a process that builds, via dsxM and social experience, the potential for a more flexible sexual behavior, which could be evolutionarily conserved as dsx-related genes that function in sexual development are found throughout the animal kingdom (Pan, 2014).

For nearly 100 years male courtship behavior in D. melanogaster has been recognized as a robust, complex, and largely innate behavior: a male fly is fully capable of performing all steps of courtship behavior when raised in complete isolation from egg to adulthood and then presented with a female fly as his first encounter with another creature. Thus male courtship has been used as a model system for the analysis of such topics as, how innate behaviors are elicited by specific environmental cues and how sequential motor programs are coordinated (Pan, 2014).

One of the most significant findings with respect to courtship behavior during the last decade is that a single gene (fruM) is both necessary and sufficient for building the potential of courtship behavior into a dedicated courtship circuitry. This study shows that while courtship behavior is abolished in fruM null males that are raised in isolation, a condition used by most studies in this field, many steps of courtship behavior can be alternatively established simply by group-housing fruM null males with either male or female flies for 1 or more days prior to testing. It was further demonstrated that such fruM-independent, experience-dependent courtship is genetically specified by the dsx gene, whose expression significantly overlaps that of fruM in the CNS. Finally, this study shows that the experience-dependent acquisition of the potential for courtship has properties indicative of learning and memory, but is independent of mushroom bodies. Integrating these results with previous findings deepens our understanding of both the genetic and neuronal underpinnings of courtship (Pan, 2014).

Numerous studies have contributed to identifying fruM as a dedicated regulatory gene that specifies the neural substrates of D. melanogaster male courtship and showing that fruM largely functions to regulate fine neural connectivity and/or neural physiology. Recent findings have highlighted the importance of dsx-expressing neurons, and in particular those that also express fruM, in male courtship. Of particular relevance, in the light of the discovery of a fruM-independent, dsx-dependent, and experience-dependent courtship pathway, is the finding that artificial activation of all dsx neurons elicits courtship by males independent of whether they had functional fruM. Approximately two-thirds of all dsx-expressing CNS neurons are found in the ventral nerve cord, and in particular the abdominal ganglion, where they likely function in the execution of sexual behaviors. This leaves five bilaterally present clusters of dsx-expressing neurons in the brain (300 neurons total counting both hemispheres) as likely containing the dsx neurons that mediate the acquisition of experience-dependent courtship. Of these five clusters of dsx-expressing neurons, the male-specific PC1 (also termed P1) cluster, which expresses both fruM and dsxM, is a particularly attractive candidate for having a significant role in experience-dependent courtship, based on its key role in initiating fruM-dependent courtship (Pan, 2014).

These findings add significantly to understanding the role of dsxM in specifying male courtship behavior. Previous studies showed that in males that are wild-type for fruM one specific aspect of male courtship (e.g., sine song) is dependent on dsxM function. Thus, it is likely that the potential for sine song is innately established in CNS by dsxM in a manner analogous to how the potential for fruM-dependent aspects of courtship are innately established. Additionally, in dsx null males that are wild-type for fruM there is a poorly understood deficit in the overall level of courtship (as measured by the CI), but all steps of courtship occur, except for sine song and copulation itself, which is mechanically not possible due to dsx-dependent defects in genital development. These results reveal additional roles of dsxM in the acquisition of the potential for courtship in the absence of fruM function. This reasoning suggests that dsxM functions both to facilitate acquisition of the potential for many aspects of courtship (in the absence of fruM) and to (in the presence of fruM) innately determine at least one aspect of courtship—sine song (Pan, 2014).

As noted above, the sex determination genes fruM and dsxM in males function developmentally to build some aspects of courtship behavior into the CNS. Although the majority of neurons comprising the courtship circuitry are still present in fruM null males, they do not function effectively in transducing sensory cues to motor centers that execute courtship behavior. Strikingly, group-housing experience allows efficient transduction from sensory cues to motor centers when fruM is not expressed. Thus social experience acting via dsxM-mediated processes somehow compensates for many aspects of fruM function. It is noted that other aspects of courtship behavior (e.g., attempted copulation) are not observed in fruM null males, even after they have been group-housed, suggesting that the latter aspects of courtship are solely fruM-dependent (Pan, 2014).

How does social experience change the courtship circuitry in the absence of fruM? It is noted that many recent studies on flies have found that social experience can change gene expression, synaptic connectivity, and/or pheromone profiles. As this study showed that when fruM null males that had been group housed were isolated and then singly housed for 7 days, they still courted fresh females intensively, it is unlikely that changed pheromone profiles, if any, play essential roles in establishing courtship behavior in fruM null males. Rather, it is suggested that social experience induces courtship in fruM null males by changing gene expression and/or neuronal connectivity to allow efficient transduction from sensory perception to motor output. Whether social experience functioning through dsxM during adulthood and fruM functioning during development, act through identical or synonymous mechanisms to specify the courtship circuitry is unknown and awaits further study. In this regard, it is noted that the experience- dependent acquisition of the potential for male courtship behavior during adulthood provides a robust single fly paradigm for learning that may facilitate studies of learning at a variety of levels (Pan, 2014).

Looking under the lamp post: neither fruitless nor doublesex has evolved to generate divergent male courtship in Drosophila

How do evolved genetic changes alter the nervous system to produce different patterns of behavior? This question was addressed 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. Their role in courtship evolution was tested by transferring the entire locus for each gene from divergent species to D. melanogaster. It was found that despite differences in Fru+ and Dsx+ cell numbers 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 (Cande, 2014).

Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila

Males of most species are more aggressive than females, but the neural mechanisms underlying this dimorphism are not clear. This study identified a neuron and a gene that control the higher level of aggression characteristic of Drosophila melanogaster males. Males, but not females, contain a small cluster of FruM+ neurons that express the neuropeptide tachykinin (Tk). Activation and silencing of these neurons increased and decreased, respectively, intermale aggression without affecting male-female courtship behavior. Mutations in both Tk and a candidate receptor, Takr86C, suppressed the effect of neuronal activation, whereas overexpression of Tk potentiated it. Tk neuron activation overcame reduced aggressiveness caused by eliminating a variety of sensory or contextual cues, suggesting that it promotes aggressive arousal or motivation. Tachykinin/Substance P has been implicated in aggression in mammals, including humans. Thus, the higher aggressiveness of Drosophila males reflects the sexually dimorphic expression of a neuropeptide that controls agonistic behaviors across phylogeny (Asahina, 2014).

Aggression is an innate, species-typical social behavior that is widespread in animal phylogeny. Expression of agonistic behavior is commonly observed between conspecific males in conflict over access to reproductively active females, food, territory, or other resources. In many animal species, aggression is often quantitatively higher in males than in females. In humans, violent aggression constitutes a major public health problem and its incidence is overwhelmingly higher among males than females. In addition, the behavioral expression of aggression is often qualitatively different between males and females, and may differ in the contexts in which it is exhibited (Asahina, 2014).

Despite recent progress, the neurobiological mechanisms underlying the evolutionarily conserved sexual dimorphism in aggressiveness remain poorly understood. Pheromones are known to play an important role in intermale aggression. However, in cases where the relevant receptors are known, dimorphic expression of these molecules does not appear to explain sex differences in aggressiveness. Studies in numerous vertebrate species have identified sexual dimorphisms in the size of brain nuclei or their constituent neuronal subpopulations that are controlled by gonadal steroid hormones in a manner that parallels the influence of these hormones on aggressive behavior. Recent studies have shown that genetic ablation of hypothalamic neurons expressing the progesterone receptor decreases both aggression and mounting in males, and mating behavior in females (Yang, 2013). These neurons display sexual dimorphisms in their projections, but whether this dimorphism is causally responsible for sex differences in levels of aggressiveness is not yet clear. As in other species, Drosophila males flies are more aggressive than females and also exhibit qualitative differences in agonistic behavior. These sex differences in aggression are known to be under the control of fruitless (fru), a master regulator of sexual differentiation of the brain. Although some efforts have been made to identify circuits through which fru exerts its influence on aggressive behavior, FruM+ neurons that are necessary, sufficient, and specific for male-type aggression have not yet been identified (Asahina, 2014).

This study has identified a small group of sexually dimorphic, FruM+ neurons that promote aggressiveness in Drosophila males but have no influence on male-female courtship behavior. These neurons enhance aggression, at least in part, through the release of a neuropeptide, Drosophila tachykinin (DTK). Tachykinin/Substance P has been implicated in certain forms of aggression in several mammalian species. Thus, the higher level of aggression that is characteristic of Drosophila males is promoted by sexually dimorphic neurons, which express a neuropeptide that regulates agonistic behavior across phylogeny (Asahina, 2014).

This study has identified a sexually dimorphic neuron and a gene that play a critical and specific role in the expression of intermale aggression in Drosophila. The gene encodes a neuropeptide homologous to mammalian Substance P, and its release from the identified neurons is important for aggression. Substance P has been implicated in aggression in several mammalian systems. Together, the data suggest that the higher level of aggressiveness in Drosophila males may be controlled by the expression in sexually dimorphic neurons of a neuropeptide that regulates forms of agonistic behavior across phylogeny (Asahina, 2014).

Previous studies have investigated the role of FruM+ neurons in aggression versus courtship. Selective masculinization of certain groups of neurons in females masculinized courtship behavior, but not aggression, suggesting that distinct subsets of FruM neurons may control these behaviors; however, a selective masculinization of aggression, but not courtship, was not observed. Feminization of most or all octopaminergic (OA) or cholinergic neurons, via expression of UAS-Tra, altered the balance between male-male courtship and aggression, or enhanced aggression, respectively. Feminization of a small subset of OA neurons increased male-male courtship, but not aggression. Specific OA and dopaminergic neurons that influence aggression have been identified, but these neurons are not sexually dimorphic. The present results identify sexually dimorphic Tk-GAL4FruM neurons that are necessary, sufficient, and specific for the quantitatively higher level of aggressiveness that is characteristic of Drosophila males. The neurons responsible for the qualitative sex-specific differences in the behavioral expression of aggression remain to be identified (Asahina, 2014).

Studies in mice have localized aggression-promoting neurons to the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl). Genetic ablation of anatomically dimorphic neurons within VMHvl that express the progesterone receptor (PR) was shown to partially reduce aggressive behavior. However, this effect of this ablation was not specific to aggression, since male mating behavior and female mating behavior were attenuated as well. In contrast, the Tk-GAL4FruM neurons identified in this study control aggression, but not mating behavior. Unlike PR+ neurons, moreover, these cells are not detectable in females (Asahina, 2014).

The fact that the Tk-GAL4FruM neurons were not observed in females suggests that either the developmental generation of these neurons and/or their expression of the neuropeptide is male specific. Whatever the case, the absence of these neural elements from the female brain is likely to contribute to their lower level of aggressive behavior. The data suggest that sex-typical features of some innate behaviors in Drosophila may be achieved, at least in part, by the sexually dimorphic expression in specific neurons of neuropeptides that coordinate males-pecific behavioral subprograms. Dimorphic populations of FruM-expressing neurons also regulate sexually dimorphic behaviors through the release of classical fast neurotransmitters that act on sexually dimorphic chemical synapses (Asahina, 2014).

Several lines of evidence presented in this study argue that Tk-GAL4FruM neurons influence aggressive arousal or motivation, rather than simply acting as 'command neurons' for aggressive actions. First, activation of these neurons did not trigger a single aggressive action, as would be expected for a command neuron, but rather increased the frequency of multiple agonistic behaviors, including wing-threat, lunging, and tussling. Second, thermogenetic activation of these neurons supervened the requirement for several aggression-permissive conditions and cues, some of which (such as male-specific pheromones) could be construed as 'releasing signals'. The activation of Tk-GAL41 neurons was even able to promote lunging toward a moving dummy fly (albeit in a minority of trials). To the extent that increased arousal decreases the requirement for specific releasing signals to evoke innate behaviors, activation of Tk-GAL4FruM neurons may generate an arousal-like state that is specific for aggression. Alternatively, Tk-GAL4FruM neurons may enhance behavioral sensitivity to multiple releasing signals that characterize an attackable object, either at the level of parallel sensory processing pathways or at a locus downstream of the integration of these multisensory cues, analogous to the neuropeptide regulation of feeding behavior in C. elegans (Asahina, 2014).

Several lines of evidence presented in this study suggest that the release of DTK peptides indeed contributes to the aggression-promoting function of Tk-GAL4FruM neurons. Nevertheless, the release of a classical neurotransmitter, probably acetylcholine, likely contributes to the behavioral influence of Tk-GAL4FruM neurons as well. Furthermore, while the data implicate Takr86C as a receptor for Tk in the control of aggression, they do not exclude a role for Takr99D. Among three species of vertebrate Tachykinin neuropeptides, Substance P has been implicated, directly or indirectly, in various forms of aggression, including defensive rage and predatory attack in cats, and intermale aggression in rats. Although not all functions of Substance P are necessarily conserved (such as nociception in mammals and olfactory modulation in the fly, these data suggest that this neuropeptide is broadly involved in the control of agonistic behavior in both vertebrates and invertebrates. They therefore add to the growing list of neuropeptide systems that show a remarkable evolutionary conservation of functions in the regulation of innate 'survival behaviors' such as feeding and mating. Biogenic amines also control aggression across phylogeny. However, in the case of serotonin, the directionality of its influence is opposite in flies and humans (Asahina, 2014).

The findings of this study indicate that studies of agonistic behavior in Drosophila can identify aggression-regulating genes with direct relevance to vertebrates. Interestingly, in humans, the concentration of Substance P-like immunoreactivity in cerebrospinal fluid has been positively correlated with aggressive tendencies in patients with personality disorders. Substance P antagonists have been tested in humans as anxiolytic and antidepressant agents, although they failed to show efficacy . The present findings, taken together with mammalian animal studies, suggest that it may be worthwhile to investigate the potential of these antagonists for reducing violent aggression in humans (Asahina, 2014).


Male sexual behavior in the fruit fly Drosophila melanogaster is regulated by fruitless (fru), a sex-determination gene specifying the synthesis of BTB-Zn finger proteins that likely function as male-specific transcriptional regulators. Expression of fru in the nervous system specifies male sexual behavior and the muscle of Lawrence (MOL), an abdominal muscle that develops in males but not in females. The fru ortholog was isoltated from the malaria mosquito Anopheles gambiae; the gene's conserved genomic structure is shown. Male-specific mosquito fru protein isoforms arise by conserved mechanisms of sex-specifically activated and alternative exon splicing. A male-determining function of mosquito fru is revealed by ectopic expression of the male mosquito isoform FRUMC in fruit flies; this results in MOL development in both fru-mutant males and fru+ females who otherwise develop no MOL. In parallel, evidence is provided of a unique feature of muscle differentiation within the fifth abdominal segment of male mosquitoes that strongly resembles the fruit fly MOL. Given these conserved features within the context of 250 Myr of evolutionary divergence between Drosophila and Anopheles, it is hypothesized that fru is the prototypic gene of male sexual behavior among dipteran insects (Gailey, 2006).

The orthologue of the fruitfly sex behaviour gene fruitless in the mosquito Aedes aegypti: evolution of genomic organisation and alternative splicing

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. This study reports the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). It was found to be 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. Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. It is proposed 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 the potential use of fru and dsx for developing new genetic strategies in vector control is discussed (Salvemini, 2013).


Search PubMed for articles about Drosophila fruitless

Al-Anzi, B., et al. (2009). Obesity-blocking neurons in Drosophila. Neuron 63(3): 329-41. PubMed Citation: 19679073

Albagli, O., et al. (1995). The BTB/POX domain: a new protein-protein interaction motif common to DNA- and actin-binding proteins. Cell Growth Differ. 6: 1193-98. PubMed Citation: 8519696

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