InteractiveFly: GeneBrief

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

BIOLOGICAL OVERVIEW

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 (shi^TS). These manipulations have been extended to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shi^TS 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/tra^F 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 FRU^M protein is regulated by Tra, it was hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fru^MIR) would mimic the effects of tra^F; but C309/fru^MIR 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 FRU^M in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of tra^F or fru^MIR effects on courtship song, because C309 was found to be coexpressed with FRU^M within the same 10 neuronal clusters in pupae. Thus, it is hypothesized that elimination of singing behavior by the C309/shi^TS combination involves neurons acting downstream of FRU^M 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 (shi^TS), 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 FRU^M pattern (Villella, 2005).

The possibility was also entertained that the C309/shi^TS 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 FRU^M protein is not produced in females. If C309 and tra^F 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-shi^TS 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 FRU^M protein. One element of the courtship effects of C309/UAS-shi^TS involves subnormal interactions between males and females. From correlating C309/FRU^M coexpression with the fact that fruitless mutations lead to lower-than-normal male–female courtship, it is speculated that FRU^M brain regions 2, 8, 13, and 14 are connected with the deleterious effects of shi^TS. As to why two additional neuronal groups that coexpress FRU^M 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 FRU^M 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/FRU^M coexpressing cells led to no decrements in male-female interactions, despite the 7- to 10-fold coexpression reductions caused by XY/C309/UAS-tra^F 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 tra^F-affected courtship decrement were to be realized. An alternative to viewing this matter in the context of C309/FRU^M 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-shi^TS males. Once again, UAS-tra^F 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-shi^TS neurons in the thoracic ganglia could be the etiology of heat-induced songlessness exhibited by these doubly transgenic males. Regarding the absence of a tra^F effect, what if C309 was not expressed in any FRU^M-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 shi^TS 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 tra^F would occur too late to affect singing ability. However, substantial coexpression of FRU^M 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 shi^TS vs. tra^F, 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: FRU^M 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-tra^F combination knocked down driver/FRU^M 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-shi^TS 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 FRU^M's spatial domain for shi^TS- or tra^F-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-tra^F flies elicit fairly high levels of courtship. Thus, groups of C309/UAS-tra^F 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-tra^F fly is feminized could include self-stimulation that might contribute to intermale courting. However, recall the case of C309/UAS-tra^F/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-tra^F/Cha-gal80 males vigorously to court other ones, is minimally operating to induce the homosexual courtships performed by XY/C309/UAS-tra^F flies. Males carrying C309 and UAS-fru^MIR 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 tra^For fru^MIR? 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 tra^F could lead to the female (F) form of DSX (thus, no DSX^M, 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-fru^MIR 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-tra^F combination. It is as if the broad neural effects of a genetic abnormality such as a fruitless mutation, or combining C309 with UAS-tra^F, 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-fru^MIR flies were substantially less affected in terms of numbers of brain neurons within which FRU^M became undetectable, compared with the effect of the same driver combined with UAS-tra^F. 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-shi^TS 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 shi^TS-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-fru^MIR combination was found not to mimic the effects on courtship among males of combining the driver with UAS-tra^F. Thus, the broader pattern of FRU^M expression, unaffected by the IR compared with the substantial decrement caused by tra^F, 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 FRU^M 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 FRU^M 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-shi^TS. 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 (Fru^M), a critical requirement is shown for the male isoform of dsx (Dsx^M), alongside Fru^M, in the specification of courtship song. Therefore, although Fru^M expression is sufficient for the performance of many male-specific behaviors, this study has shown that without Dsx^M, 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 (Fru^M 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 Fru^M 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: Dsx^M and Dsx^F, 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).

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

Song analysis was based on 29 fru^M and fru^Δtra females because most fru^M and fru^Δtra females did not perform sufficient courtship behavior or song for analysis. The wing-extension indices (WEIs) of these 29 fru^M and fru^Δtra females were not significantly different from wild-type and control fru^M and fru^Δtra males; however, a significant decrease was foudn in the song index (SI) (the percentage of time spent singing during wing extension). Also, the fru^M and fru^Δtra 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 fru^M and fru^Δtra females were capable of wild-type wing extension, they spent significantly less time singing during courtship and produced song of poor quality. Thus, Fru^M expression alone cannot specify wild-type song production (Rideout, 2007).

To dissect the individual contributions of both Fru^M and Dsx to the specification of courtship song, males were analyzed lacking Fru^M 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. Fru^M expression in females is not sufficient for courtship song. Likewise, the expression of Dsx^M 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 Fru^M and Dsx^M, as in transformer (tra) mutant females, renders females capable of wild-type courtship song. Together, these results demonstrate a previously unrecognized requirement for Dsx^M, in conjunction with Fru^M, 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, Fru^M 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 Fru^M 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 Fru^M 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 Fru^M 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 Fru^M 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 fru^GAL4, 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 fru^GAL4 positive, and thus is a fru neuron. This neuron was fru^GAL4 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 Fru^M expression prevented reaper-mediated programmed cell death in a cluster of cells, resulting in more neurons in this cluster in males. Dsx^M, 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 Fru^M colocalize, as suggested by the ability of Fru^M 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 fru^GAL4, 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 Fru^M and Dsx in the creation of this difference was examined in fru^GAL4-positive neuron number in the Msg (Rideout, 2007).

A sexually dimorphic number of fru^GAL4-expressing neurons was found in the Msg, a region of the CNS central to song production and in which Fru^M and Dsx colocalize. To determine the individual contributions of dsx and fru in the creation of this sexually dimorphic number of neurons, fru^M and fru^Δtra females were examined to see if Fru^M expression alone abolishes the observed difference in neuronal number in the Msg between the sexes. It was found that the number of Fru^M-expressing neurons in the Msg of these females was significantly reduced in comparison to wild-type and control males. Furthermore, this decrease in Fru^M-expressing neurons was comparable to the difference in neuron number observed in the Msg of fru^GAL4 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 Fru^M-expressing neurons, and that Fru^M expression alone cannot eliminate this difference. Thus Fru^M 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 Fru^M in the Msg, plays a role in the specification of this sexually dimorphic population of neurons, helping to determine the full complement of Fru^M 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 Fru^M-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 Fru^M-expressing neurons in the Msg than did wild-type and control males, demonstrating that Dsx is indeed required to obtain a full complement of Fru^M-expressing neurons. Because fru^M and fru^Δtra females (who express the female-specific isoform of dsx, Dsx^F) do not have a full complement of Fru^M-expressing neurons in the Msg, this study has demonstrated a critical role for Dsx^M in the creation of a sexually dimorphic Msg. In fact, only when both Fru^M and Dsx^M are present, as in tra mutant females, can a full complement of Fru^M-expressing neurons in the Msg be obtained. Thus, this study has demonstrated a previously unrecognized requirement for Dsx^M in the specification of a population of Fru^M-expressing neurons in the Msg (Rideout, 2007).

Dsx^M prolongs the division of neuroblasts in the Abg of males, resulting in more neurons in the male Abg. Also in the Abg, Dsx^M plays a critical role alongside Fru^M in the differentiation of a male-specific serotonergic population of neurons. The current findings suggest that Dsx^M 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 Dsx^M generates a sexually dimorphic population of neurons, which is exploited by Fru^M to fashion a male-specific behavioral neural network (Rideout, 2007).

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

Thus, although Fru^M expression can specify many male-specific behaviors, this study showd that without Dsx^M, 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).

Male-specific fruitless isoforms target neurodevelopmental genes to specify a sexually dimorphic nervous system

In Drosophila, male courtship behavior is regulated in large part by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation or the role that isoform diversity plays in the formation of a male-specific nervous system. To characterize the roles of sex-specific Fru isoforms in specifying male behavior, this study generated novel isoform-specific mutants and used a genomic approach to identify direct Fru isoform targets during development. All Fru isoforms were shown to directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. fru behavioral phenotypes are specified by either a single isoform or a combination of isoforms. Finally, the utility of these data for the identification of novel sexually dimorphic genomic enhancers and novel downstream regulators of male sexual behavior is illustrated in this study. These findings suggest that Fru isoform diversity facilitates both redundancy and specificity in gene expression, and that the regulation of neuronal developmental genes may be the most ancient and conserved role of fru in the specification of a male-specific nervous system (Neville, 2014).

This study of fru isoform function exemplifies how complex behaviors involved in courtship can be controlled by a single locus. Differential expression of multiple isoforms with different binding specificities produces a 'neural code' of downstream gene expression, in which phenotypes can be specified by either a single isoform or a combination of isoforms. The DamID approach allowed identification of the association of Fru^M proteins to specific regions of the genome and relate this binding with downstream target genes. Genes known to play a role in the development of the nervous system are significantly overrepresented within these identified Fru^M target genes. This is certainly consistent with the established role that fru plays in the development of a number of neuronal structures. However, until this study, the identity of fru-regulated genes had not been determined. The identification of putative Fru^M binding motifs, the strategy for identifying and characterizing Fru-regulated genomic enhancers, and the production of a comprehensive set of fru^M isoform-specific mutant flies facilitates an unprecedented leap forward in the ability to study Fru^M transcriptional regulation (Neville, 2014).

For a more in-depth analysis, concentration was placed on a subset of putative Fru^MB target genes. The identification of a putative Fru^MB DNA-binding motif allowed this study to show that the majority of genomic enhancers containing this motif exhibit sexually dimorphic expression in fru neurons. Among the genes associated with these enhancers are the related BTB-Zn-finger genes lola and chinmo, both key neuronal morphogenesis genes. In addition, decreased expression of lola, specifically in fru neurons, led to dramatically reduced levels of male sexual behavior, establishing the necessity of this protein in fru neurons. Since Fru targets other BTB-Zn-finger genes, it is speculated that regulatory diversity of these transcription factors contributes to a neuron-specific transcriptional code leading to specific developmental outcomes. Future examination of these and other Fru^M target genes will allow deciphering of this code and connect specific dimorphic neural cell fates with behavioral outputs (Neville, 2014).

It was determined that there is a great deal of overlap in the genomic loci targeted by all of the Fru^M isoforms when it comes to genes involved in the development of the nervous system. Since each Fru isoform appears to have unique binding specificity, it follows that Fru^M isoforms could act independently on the same genes, either cooperatively or redundantly. Fru^MB and Fru^MC isoforms can associate with the same genomic regions containing the putative Fru^MB motif (see Motif Analysis of Fru^M Data Sets). Fru^MB exhibits the most consistent binding specificity, which this study determined to be dependent on amino acid residues that are required for DNA binding. In contrast, although the Fru^MC isoform is enriched for the Fru^MB motif, it appears to have a unique DNA binding specificity. Previous analysis of serotonergic neurons in the abdominal ganglion that innervate the male reproductive organs showed evidence of cooperative function between the Fru^MB and Fru^MC isoforms, as both were required for the development of these neurons. However, other functions appeared to be isoform specific: for example, only Fru^MC controls the innervation and formation of the male-specific muscle of Lawrence. Although the elimination of individual Fru^M isoforms generated overt behavioral deficits, they were not sufficient to abrogate courtship behaviors completely, suggesting some degree of redundancy in the determination of the neural networks directing these behaviors (Neville, 2014).

Alternatively spliced isoforms, like gene duplications, enable a diversification of gene function, by allowing essential (often ancestral) functions to be maintained while others are able to diverge and take on new roles. Evolutionary analysis of the fru C2H2 Zn-finger domains in various insect species shows the appearance and disappearance of these domains throughout evolution. However, the high conservation between all fru C2H2 Zn fingers supports the idea that they all originated from one or a few ancestral sequences and retained a common function (Neville, 2014).

The results support this scenario of evolution in fru, as evidence has been found for both conservation and divergence in function of the different isoforms. Loss of either Fru^MB or Fru^MC expression significantly disrupts the male's ability to perform courtship behavior, whereas loss of the Fru^MA isoform has no obvious consequence. Expression analysis of the individual isoforms in the CNS also mirrors these relationships. Fru^MB and Fru^MC isoforms are broadly expressed in most Fru^M-positive neurons, whereas Fru^MA expression is restricted to only a subset of Fru^M-positive neurons. The lack of an overt phenotype associated with the fruΔA mutant may be a reflection of the relative involvement of this subset of fru-positive neurons, or may indicate that the Fru^MA isoform fulfills more specialized nonessential functions. Evidence has been recently found of positive selection acting on fru exon A across Drosophila species, whereas exons B and C were found to be conserved, supporting the involvement of transcripts containing the A exon in nonessential functions, which may contribute to phenotypic differences between species (Neville, 2014).

Previous microarray experiments showed that genes regulated downstream of Fru^M (either directly or indirectly) appear to also be regulated by ecdysone. In addition, the ecdysone receptor was shown to act in fru neurons to mediate male courtship behavior. More recently, it was shown that females depleted in ecdysone display male-like courtship behaviors, and it was proposed that distinct ecdysone peaks might regulate the formation of distinct Fru^M-containing chromatin regulatory complexes. Although the developmental time course did not detect a dramatic shift in Fru^M DNA-binding specificity as a result of ecdysone pulses, there are more subtle dynamic shifts in binding throughout development that might result from these pulses. A small but significant enrichment was found of known ecdysone-responsive genes in all Fru^M data sets. Interestingly, this included the cell death gene reaper (rpr), which was identified as a putative target of all Fru^M isoforms throughout development. fru has been shown to be essential for the suppression of cell death in the male mAL neural cluster, potentially by downregulating key cell death genes. Direct targeting of rpr by Fru^M isoforms would support this mechanism. This study also identified the ecdysone-responsive transcription factor crooked legs (crol) as a putative target of both Fru^MB and Fru^MC isoforms in pupal and adult stages. A previous microarray analysis reported crol as being upregulated in the CNS of fru^M mutant males. Deficiency combinations resulting in complete loss of the fru locus (along with a small number of neighboring genes) result in early pupal developmental arrest, around the time of pupal ecdysis. It was noted that the phenotype of fru-deficient flies was similar to that of flies mutant for the ecdysone receptor and the crol gene. Therefore, some of these targets may link the sex-specific and common isoform functions of Fru in response to ecdysone (Neville, 2014).

The identified Fru^M targets overlap with those of the other key sex-determination protein, Doublesex (Dsx). The male-specific form of dsx (dsx^M) is expressed in far fewer cells in the adult CNS than fru, but almost all dsx^M cells coexpress fru. Dsx and Fru are the only identified factors at the bottom of the sex-determination hierarchy, and both of these transcriptional regulators act in the same neurons to bring about male-specific neuronal wiring and male-specific behavioral patterns. Given the overrepresentation of identified Dsx target genes in the Fru^M data sets, it is speculated that Fru^M and dsx^M act together, either in a physical complex or through coregulation of genomic targets, to determine the male-specific nervous system (Neville, 2014).

Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons

For successful mating, a male animal must execute effective courtship behaviors toward a receptive target sex, which is female. Whether the courtship execution capability and upregulation of courtship toward females are specified through separable sex-determining genetic pathways remains uncharacterized. This study found that one of the two Drosophila sex-determining genes, doublesex (dsx), specifies a male-specific neuronal component that serves as an execution mechanism for courtship behavior, whereas fruitless (fru) is required for enhancement of courtship behavior toward females. The dsx-dependent courtship execution mechanism includes a specific subclass within a neuronal cluster that co-express dsx and fru. This cluster contains at least another subclass that is specified cooperatively by both dsx and fru. Although these neuronal populations can also promote aggressive behavior toward male flies, this capacity requires fru-dependent mechanisms. These results uncover how sex-determining genes specify execution capability and female-specific enhancement of courtship behavior through separable yet cooperative neurogenetic mechanisms (Ishii, 2020).

This study has uncovered distinct yet cooperative roles of dsx and fru on male-type social behaviors through a specific subset of P1/pC1 neurons. For courtship behaviors, this study found that NP2631 ∩ dsx^FLP neurons are specified in a fru-independent manner, and in males, their capacity to generate courtship behaviors does not require fruM. However, activation of NP2631 ∩ dsx^FLP neurons in fruF males failed to increase courtship selectively toward female targets. These results suggest that dsx plays a major role in establishing a neuronal circuit that enables the male flies to execute courtship behavior, whereas fru is critical for enhancing courtship behavior toward females, likely through proper recognition of target sex. The fact that the sex of the target flies influences the function of P1/pC1 subsets implies that information about target sex can modulate the neural circuit units downstream of these neurons, and encourages revision of the linear circuit model for sexually dimorphic social behaviors. In contrast, the complete specification and courtship-promoting functions of P1^a neurons require both dsx and fru, revealing genetic and functional heterogeneity within P1/pC1 neurons. Lastly, NP2631 ∩ dsx^FLP and P1^a neurons require a fruM-dependent mechanism to promote male-type aggressive behavior. This suggests that neither of these neurons are part of the execution mechanism for male-type aggressive behavior, and that the genetic mechanisms specifying execution components for courtship and aggressive behaviors are different (Ishii, 2020).

Electrical stimulation of various parts of the brain has been known to elicit complex behaviors, including social behaviors, for almost a century. Recent technological advances have allowed researchers to identify specific, genetically labeled populations of neurons that can induce mating and aggressive behaviors upon acute optogenetic stimulation in both mice and in flies, even toward suboptimal targets (such as inanimate objects). These findings seem consistent with the idea that neuronal activation can override most contexts and generate specific behaviors depending on the identity of stimulated cells. However, interactions with a target animal can transmit important information which a tester animal may use to choose appropriate behaviors. In fact, attacks triggered by optogenetic stimulation of ventrolateral hypothalamus (VMH) in male mice tend to last longer toward castrated males than toward female targets, and chemogenetic activation of progesterone receptor-expressing VMH neurons appears to induce more attacks toward male than toward female targets. While effects on target sex are not consistently documented, the current results and previous observations in mice show that the target sex has a significant impact on behavioral choice even for optogenetically induced social behaviors. These results suggest that sensory or behavioral feedback from target animals can impact the operation of what may appear to be an 'execution mechanism' for a given behavior (Ishii, 2020).

Identification of neural sites where the information about the target sex is integrated with the activity of both NP2631 ∩ dsx^FLP and P1^a neurons will be an important step in understanding how such context cues modulate ongoing neural activity and, ultimately, behavioral outcome. While a 'command'-like center that irreversibly executes courtship or aggressive behaviors, like recently characterized egg-laying controlling neurons, may exist, it is also possible that information about target sex (and its behavioral response) can be injected at multiple levels of a neural circuit, thereby ensuring the target sex-specific execution of sexually dimorphic social behaviors. This is conceptually analogous to the neural control of fine motions, which can be constantly adjusted by sensory feedback and efference copies all the way down to the motoneuron level (Ishii, 2020).

Recently, the importance of addressing sex as a biological variable has been widely recognized. In the context of social behaviors, this variable in the tester animals can be critical for uncovering the underlying neural mechanisms (Ishii, 2020).

The functional segregation of dsx and fru that was observed in this study can be considered analogous to the organizational and activation functions of sex hormones in mammals. Differential exposure to gonadal steroid hormones, mostly through estrogen receptors, specifies neural circuits that are necessary for sex-specific reproductive behaviors, whereas hormonal surges in the adult stage (such as testosterone or progesterone) orchestrate activation of sex-specific behaviors. It is postulated that dsx has an organizational function for the courtship execution circuit, whereas fru is important for the appropriate activation of the circuit (Ishii, 2020).

The results do not mean that fru is not necessary for the establishment of all neuronal components involved in courtship. Nonetheless, the result suggests that the wing extension execution circuit that connects NP2631 ∩ dsx^FLP neurons and relevant motoneurons is specified even in the absence of fruM, which is consistent with previous observations that fruF males are capable of expressing at least a part of courtship behavior. While a specification role for dsx on P1/pC1 neurons has been previously reported, the current study showed for the first time the behavioral role of a specific P1/pC1 subset (NP2631 ∩ dsx^FLP neurons) in fruF males. dsx is important for the specification of a few other behaviorally relevant sexual dimorphisms in the Drosophila nervous system. For instance, the sexually dimorphic axon development of leg gustatory receptor neurons, which includes aphrodisiac pheromone sensors, requires dsx function. The neural connectivity and function of TN1 neurons, which are pre-motor neurons important for the production of pulse song, are also specified by dsx. Several classes of abdominal ganglia neurons involved in male copulation also express dsx. Although relatively few in number, these examples display the importance of dsx in key neuronal populations for organizing circuit components that are essential for the execution of courtship behaviors. It is noteworthy that dsx is involved in sex-determination across a variety of animal phyla, whereas fru's role in sex-determination seems confined to insects. This suggests that dsx may be evolutionarily more ancient in the context of sex-determination than fru, which can account for its dominance over fru when specifying sexually dimorphic neurons that co-express dsx and fru (Ishii, 2020).

The proposal that fruM may be important for enhancing courtship behavior specifically towards females is consistent with the fact that many characterized fru-expressing neurons are involved in processing sex- and species-specific sensory cues. Namely, P1^a neurons, as well as more broadly defined P1/pC1 neurons accessed by different genetic reagents, are known to respond to sex-specific chemical cues, underscoring their critical role in sensory integration for courtship. fruM can play the 'activation' role for courtship by establishing sensory circuits that transmit sex-specific sensory information to P1/pC1 neurons, or by enabling P1/pC1 neurons to properly integrate and transform such neural inputs. Neuroanatomical defects of P1^a neurons in fruF males could disrupt either process (Ishii, 2020).

Gain control of sex-specific sensory cues can be one neuronal mechanism for the 'activation' function, but courtship behavior can be enhanced in other ways as well. For instance, behavioral persistence or context-dependent intensity adjustment can result in an increase of the overall courtship vigor. Recently, a new class of fru-expressing neurons downstream of P1^a neurons has been found to mediate the persistence of courtship behavior triggered by P1^a neuronal activation (Jung, 2020). Even if fruM is not absolutely necessary for the formation of the minimal wing extension execution circuit, it can have a significant impact on the generation of effective wing extension toward female target flies (Ishii, 2020).

While it is concluded that the role of fru is not necessarily to specify the execution mechanism for courtship behavior, fruM females can still perform wing extensions or dsx^- expressing neurons in females can elicit wing extensions, suggesting that the residual execution mechanism for at least a part of courtship behavior may be specified in a sex-invariant manner. The presence of a latent mating execution circuit in female brains is also suggested in mice. Because the courtship songs produced by females or fruM females are defective, male-type splicing of dsx nonetheless seems to be instrumental in organizing the proper execution mechanism for Drosophila courtship behavior (Ishii, 2020).

In striking contrast to wing extensions, this study found that activation of neither NP2631 ∩ dsx^FLP nor P1^a neurons in fruF males induced lunges. This result points to the existence of a fruM-dependent execution mechanism for male-type aggressive behaviors, likely downstream of these neurons. In Wohl (2020), it was found that at least one group of fruM-dependent neurons can promote male-type aggressive behaviors independent of dsx. Therefore, a separation of the courtship execution mechanism and the aggression execution mechanism by two sex-determining genes is likely accomplished by a partial separation of underlying neural circuits. The aggression-promoting function of NP2631 ∩ dsx^FLP and P1^a neurons likely reflects their roles to coordinate aggression and courtship depending on internal and external conditions, instead of a simple decision switch that triggers fixed types of behavior (Ishii, 2020).

Both dsx and fru encode transcription factors. The sexually dimorphic morphology and wiring specificity of many fru-expressing neurons are determined in a cell-autonomous manner, suggesting that dsx and fru define Drosophila sex at a cellular level through regulation of a specific set of target genes. Mammalian sex hormones ultimately exert their effects through nuclear steroid receptors, which serve as transcription factors. Thus, both in flies and in mammals, organismal sex can be regarded as a collective phenotype of genetic 'sexes' that can be reduced down to the cellular leve. To understand how sex at the neuronal level influences sexually dimorphic behaviors, cell-type specific manipulation of sex-determining genes is required. The current study focused on neural functions in a whole animal mutant, which prevents addressing the role of either dsx or fru specifically within NP2631 ∩ dsx^FLP or P1^a neurons. For example, it is ot known whether NP2631 ∩ dsx^FLP neurons in fruF males failed to enhance courtship behavior toward female targets because of the absence of fruM within this population, or because of the lack of fruM in other neuronal populations, or both. In addition, the current approach does not address if it is the presence of dsxM or the absence of dsxF that is important for the specification of male-type NP2631 ∩ dsx^FLP neurons or P1^a neurons (Ishii, 2020).

It is important to note that sex specification is a developmental process of transformation. Both at genetic and organismal levels, one sex is not a loss-of-function mutant of the other. Loss-of-function manipulations at the cellular level, by cell type-specific RNA interference or CRISPR interference -based approaches, may show that either dsx or fru is necessary for the proper development or function of the given neurons, but may be insufficient to illuminate the genetic origin of the sex-specific transformation at the cellular level. In addition, temporally and spatially precise manipulation of genes during development remains difficult. This can create a difficulty interpreting the effects of either knock-down or over-expression of sex determining genes, which are dynamically regulated from early developmental stages. Creation of neuronal mutant clones may circumvent this problem, but the tra mutation, which has been previously used to convert a 'neuronal sex', cannot dissociate the roles of dsx and fru (Ishii, 2020).

Faced with these often overlooked limitations of cell-type specific gene manipulations, it would be informative to characterize what types of transformations are observed in mutants of sex-specific splicing at an organismal level, as in this and other studies. Although a constitutive mutants have above-mentioned limitations, they nonetheless establish fundamental functional differences among sex-determining genes, as well as benchmarks for the efficacy for cell-specific manipulations techniques. Although clearly out of the scope of the current study, electron microscopy-based connectome reconstructions of fruF male and fruM female brains could provide useful information for understanding the transformative nature of sex specification in the brain (Ishii, 2020).

Lastly, the serendipitous finding that NP2631 ∩ dsx^FLP and P1^a neurons contain genetically and functionally distinct populations underscores the importance of characterizing neuronal cell types in greater detail. How to determine cell types remains a challenge in neuroscience, but genetic access to a finely defined population of neurons even within what is considered as a single class of neurons can be the key to understand how a neural circuit generates complex behaviors such as social behaviors (Ishii, 2020).

In the posterior part of male brains, 'P1' neurons, as defined by fru-expressing cluster, and pC1 neurons, as defined by dsx-expressing cluster, extensively overlap. This raises a question about the distinction between 'P1' and 'pC1' neurons. Furthermore, recent single cell level analyses of the neurons that belong to the male 'P1' cluster or 'pC1' cluster revealed surprising neuroanatomical and functional diversity, raising a possibility that P1/pC1 neurons may be functionally heterogeneous as well (Ishii, 2020).

Surprisingly, this study found that behaviorally relevant NP2631 ∩ dsx^FLP and P1^a neurons, as well as NP2631 ∩ fruFLP and P1^a neurons, seldom overlap. Optogenetic stimulation of NP2631 ∩ dsx^FLP and P1^a neurons triggers social behaviors in temporally distinct manners. Moreover, fru has a different impact on the specification and function of these two neuron groups, suggesting that little overlap of NP2631 ∩ dsx^FLP and P1^a neurons does not necessarily reflect arbitrary labeling bias within a single homogeneous neuronal population by different genetic reagents. Instead, these observations support the idea that of P1/pC1 neurons consist of functionally diverse subtypes (Ishii, 2020).

It is acknowledged that the genetic reagents used in this study are likely insufficient to resolve the possible heterogeneity within either NP2631 ∩ dsx^FLP or P1^a neurons. Differential expression patterns of FruM proteins within both clusters alone suggest that such heterogeneity almost certainly exists. Recent advances in whole-brain neural reconstruction using electron microscopy images will provide a foundation for precise characterization of Drosophila neurons, as has been recently used for the female-type 'pC1' cluster. A large number of 'split-GAL4' collections will allow universal access to the specific subpopulations. These types of tools will facilitate cross-study comparisons of neuroanatomical and behavioral data, and will serve as a catalyst to understand the logic of neural control of behavior in general. With the advance of single cell-level genetic and epigenetic profiling techniques, the importance of precisely characterizing the targeted neuronal types will only grow not only in Drosophila, but in every model organism. Reproducible access to each neuronal type can uncover functional units for a given behavior at even finer detail, which will be fundamental for deconstructing the dynamics of neural circuits that are responsible for generating social behaviors in a context-dependent manner. Such knowledge will be also critical for establishing theoretical models that account for brain operations and population-level dynamics of animals engaging in social interactions (Ishii, 2020).

teiresias, a Fruitless target gene encoding an immunoglobulin-superfamily transmembrane protein, is required for neuronal feminization in Drosophila

This study aims at identifying transcriptional targets of FruitlessBM (FruBM), which represents the major isoform of male-specific FruM transcription factors that induce neural sexual dimorphisms. A promoter of the axon-guidance factor gene robo1 carries the 16-bp palindrome motif Pal1, to which FruM binds. A genome-wide search for Pal1-homologous sequences yielded ~200 candidate genes. Among these, CG17716 potentially encodes a transmembrane protein with extracellular immunoglobulin (Ig)-like domains similar to Robo1. Indeed, FruBM overexpression reduced CG17716 mRNA and protein expression. In the fru-expressing mAL neuron cluster exhibiting sexual dimorphism, it was found that CG17716 knockdown in female neurons completely transformed all neurites to the male-type. Conversely, CG17716 overexpression suppressed male-specific midline crossing of fru-expressing sensory axons. CG17716 was renamed teiresias (tei) based on this feminizing function. It is hypothesized that Tei interacts with other Ig superfamily transmembrane proteins, including Robo1, to feminize the neurite patterns in females, whereas FruBM represses tei transcription in males (Sato, 2020).

Reproductive success in many male animals relies on their behavioral performance during mating attempts with a female1. The neural circuitry that controls mating behavior has thus evolved under the strong pressure of sexual selection. Such an evolutionary drive led to the development of sexual dimorphisms in the neural circuitry and neurons that compose this circuitry1. Drosophila melanogaster is an outstanding model organism highly amenable to genetic dissection of complex traits, including mating behavior2. In this organism, the transcription factor gene fruitless (fru) plays a key role in organizing the sexually dimorphic circuitry for mating behavior by specifying sex-specific neuronal structures during development (Sato, 2020).

Among the four promoters identified in the fru gene, the most distal one (the P1 promoter) is dedicated to sex-specific functions ascribed to the male-specific translation of P1-derived mRNAs. As a result of sex-specific splicing of the fru-P1 primary transcript, only male mRNAs have a long ORF encoding male-specific functional fru proteins, called FruM. These proteins are translated as five isoforms, FruAM, FruBM, FruCM, FruDM, and FruEM, which share a common BTB domain in their N-terminus, but which each have a unique C-terminus. The most prevalent isoform is FruBM, which has two zinc-finger motifs in the C-terminus, and forms a complex with chromatin regulators such as the TIF1 homolog Bonus (Bon), histone deacetylase 1 (HDAC1), and heterochromatin protein 1a (HP1a), to bind to more than 100 target sites on the genome for transcriptional regulation of downstream genes. The best-characterized FruBM target is robo1, which encodes a transmembrane protein belonging to the immunoglobulin superfamily with an axon-guidance role in the developing nervous system. FruBM binds to a 42-bp promoter segment of the robo1 gene named the FruBM Response Obligatory Sequence (FROS), which contains a characteristic palindrome sequence of 16 nucleotides (Pal1). Partial deletion of Pal1 in the robo1 gene impairs neural sexual differentiation and mating behavior in male flies, indicating that Pal1 is pivotal for FruBM in order to execute its sexual functions (Sato, 2020).

Many of the fru-expressing neurons are sexually dimorphic, including neurons composing the mAL cluster. The mAL cluster displays sexual dimorphisms in the following four respects: (i) the number of neurons in the cluster (~5 in females vs. ~30 in males); (ii) the absence (females) or presence (males) of a neuronal subset carrying the ipsilateral neurite; (iii) the branched (females) vs. unbranched (males) tip structure of the descending contralateral neurite; and (iv) the focal (females) or expanded (males) terminal arbor distribution of the ascending contralateral neurite. While these four types of sexual dimorphisms of mAL neurons are established in a fru-dependent manner, they are also regulated separately and independently from each other. For example, Hunchback knockdown in male mAL neurons feminizes only the descending contralateral neurite, whereas robo1 knockdown feminizes only the ipsilateral neurite. On the other hand, loss of the cell-death genes, reaper, grim and head involution defective, increases the number of cells composing the mAL cluster from ~5 to ~30 in females, but has no effect in males. Therefore, FruBM likely interacts with different cofactors for transcription regulation, and/or FruBM acts on different targets to establish each of the four sexually dimorphic features of mAL neurons. With the aim of obtaining additional FruBM transcriptional targets for the establishment of neural sexual dimorphisms, this study examined the effects of knocking down the genes harboring a Pal1-homologous motif on mAL neuron structures. teiresias (tei), encoding another immunoglobulin-superfamily member with a transmembrane domain, was identified. Remarkably, knockdown of tei in female mAL neurons completely masculinized all three neurite characteristics, leaving the cell number unaffected. tei knockdown also masculinized fru-expressing sensory neurons and mcALa interneurons in females. It is proposed that the Tei protein functions as a common receptor and interacts with a second receptor, e.g., robo1, which confers the ligand specificity on the heteromeric receptor complex, producing distinct neuronal populations that are diversified in their sex-specific structures (Sato, 2020).

tei, a new transcriptional target of FruBM, was identified based on the in silico search for genes harboring the FruBM-binding consensus sequence and subsequent analysis of gene knockdown effects on sexually dimorphic neurite structures. Tei is a putative transmembrane protein carrying multiple immunoglobulin-like repeats in the extracellular portion. This structure is very much alike that of Robo1, the product of the best-characterized FruBM target gene. Indeed, tei knockdown in female mAL neurons induced the male-specific ipsilateral neurite as did robo1 knockdown, revealing a functional similarity between tei and robo1 in terms of the feminizing effect on this particular structure. Nonetheless, tei was distinctly different from robo1 in that tei is also required for the female-typical shaping of ascending as well as descending contralateral neurites, in which robo1 has no role. Robo1 has been shown to operate as a receptor for the secreted ligand Slit, activating several signal transduction pathways depending on the developmental context in both invertebrates and vertebrates. Promiscuous interactions of Robo1 with other transmembrane receptors in cis have been implicated as a basis for the versatile performances of Robo1 in multiple developmental contexts. The lack of the cytoplasmic portion in Tei might suggest that Tei contributes to the ligand specificity whereas the specificity of intracellular signal transduction following heteromeric receptor activation is determined by Robo1 or another Tei partner that carries the C-terminal cytoplasmic domain. It is therefore plausible that Tei is one of these transmembrane receptors that cooperate with Robo1, and likely initiates together an intracellular biochemical cascade which ultimately inhibits the male-specific ipsilateral neurite from forming in an mAL neuron. Perhaps Tei associates with transmembrane receptors other than Robo1 in specifying the sex-specific structure in the ascending and descending contralateral neurites of an mAL neuron. Systematic knockdown in mAL neurons of genes encoding putative transmembrane receptors with immunoglobulin-like domains other than Robo protein members might lead to the identification of additional Tei partner proteins that are involved in the sex-type specification of the contralateral neurites (Sato, 2020).

Despite its striking effects on neurite structures, tei knockdown had no effect on the sexually dimorphism in the number of mAL neurons. This contrasts with the effects of fru loss-of-function mutation, which reduces the number of cells composing the mAL cluster in males from ~30 to ~5, the cell number typical of the mAL cluster in a wild-type female. Removal of three major cell-death genes, hid, grim and rpr, in female mAL neurons increased the number of cells composing the female mAL cluster up to 29 (the mean cell number was 19). This observation led to the proposition that cell death is the primary cause of the smaller number of cells composing the mAL cluster in females. Moreover, the sex difference in the cell number of fru-expressing neurons has recently been attributed to a difference in proliferation in addition to a difference in cell death: male neuroblasts produce more cells than female neuroblasts, so that males have a larger number of cells even after blocking cell death. These considerations lead to the supposition that FruM proteins separately control the neurite sex-type and the cell number sex-type. In this study it was noted that the Pal1-homologous motif was not found in or around any of the hid, grim, and rpr loci, suggesting they are not direct transcriptional targets of FruM proteins. Alternatively, transcription of these genes might be regulated by a FruM isoform other than FruBM. In fact, the mAL cell number was shown to be affected by the fruB2 (null for FruEM) as well as the fruC1 (null for FruBM) mutation (Sato, 2020).

The 16-bp Pal1 core motif is located within the 42-bp FruBM-binding site, FROS. This motif was first identified in the robo1 gene by a combination of reporter assays, electromobility-shift assays, and CRISPR-Cas9-mediated in vivo mutagenesis in conjunction with phenotypic characterizations of single-cell clones of mAL neurons and mutant-fly behavior. Using different strategies, two other research groups determined the binding consensus sequences for FruAM and FruEM in addition to FruBM. The DNA adenosine methyltransferase identification (DamID) method was used, in which a Fru-fused bacterial methylase methylates the DNA around the genomic region to which the fru moiety of the fusion protein binds. One of the reported FruBM-binding motifs in the DamID-based search revealed a 4/6 match when compared with Pal1, yet robo1 was not obtained as a putative FruBM target in that study. Another study used in vitro screening of oligo-DNAs with random sequences for binding to fru zinc-finger motifs fused to glutathione S-transferase, revealing a binding consensus sequence for each isoform, but none of these had a similarity to Pal1. Of note, another study included CG17716 (tei), together with another ~1400 genes, in a list of potential FruBM targets having a deduced FruBM-binding sequence. Reliable determination of consensus sequences for the binding of fru isoforms and rigorous identification of fru transcriptional targets are indispensable for obtaining a more complete picture of the molecular mechanisms underlying sex-specific circuit formation (Sato, 2020).

Doublesex regulates fruitless expression to promote sexual dimorphism of the gonad stem cell niche

Doublesex (Dsx) and Fruitless (Fru) are the two downstream transcription factors that actuate Drosophila sex determination. While Dsx assists Fru to regulate sex-specific behavior, whether Fru collaborates with Dsx in regulating other aspects of sexual dimorphism remains unknown. One important aspect of sexual dimorphism is found in the gonad stem cell (GSC) niches, where male and female GSCs are regulated to create large numbers of sperm and eggs. This study reports that Fru is expressed male-specifically in the GSC niche and plays important roles in the development and maintenance of these cells. Unlike previously-studied aspects of sex-specific Fru expression, which is regulated by Transformer (Tra)-mediated alternative splicing, this study shows that male-specific expression of fru in the gonad is regulated downstream of dsx, and is independent of tra. fru genetically interacts with dsx to support maintenance of the niche throughout development. Ectopic expression of fru inhibited female niche formation and partially masculinized the ovary. fru is also required autonomously for cyst stem cell maintenance and cyst cell survival. Finally, this study identified a conserved Dsx binding site upstream of fru promoter P4 that regulates fru expression in the niche, indicating that fru is likely a direct target for transcriptional regulation by Dsx. These findings demonstrate that fru acts outside the nervous system to influence sexual dimorphism and reveal a new mechanism for regulating sex-specific expression of fru that is regulated at the transcriptional level by Dsx, rather than by alternative splicing by Tra (Zhou, 2021).

In sexually reproducing animals, the proper production of gametes and successful copulation are equally critical for reproductive success. It is therefore important that both the gonad and the brain know their sexual identity. The Doublesex/Mab-3 Related Transcription Factors act downstream of sex determination and play an evolutionarily conserved role to establish and maintain sexual dimorphism in the gonad. Meanwhile, sexual dimorphism in other tissues such as the brain is controlled, to varying degrees in different animals, through autonomous control by the sex determination and non-autonomous signaling from the gonads. In many invertebrate species, another sex-determination gene fruitless (fru), which encodes multiple BTB-Zinc finger transcription factors, plays a central role in controlling mate choice, courtship behavior and aggression. How sex determination in the gonad and the nervous system are related and coordinated in these species remains unclear (Zhou, 2021).

The founding member of the DMRT family is Drosophila doublesex (dsx). dsx and fru undergo sex-specific alternative mRNA splicing by the sex determination factor Transformer (Tra), together with its co-factor Transformer-2 (Tra-2), to produce transcripts encoding sex-specific protein isoforms. It was once thought that dsx controls sexual dimorphism outside the nervous system while fru regulates sex-specific nervous system development and behavior. But more recent evidence shows that dsx cooperates with fru to specify sex-specific neural circuitry and regulate courtship behaviors. However, whether fru acts along with dsx to control sexual dimorphism outside the nervous system remains unknown (Zhou, 2021).

The fru gene locus contains a complex transcription unit with multiple promoters and alternative splice forms (see Fruitless is expressed male-specifically in the germline stem cell niche and is independent of Fru^M). Sex-specific regulation of fru was only known to occur through alternative splicing of transcripts produced from the P1 promoter, which produces the FruM isoforms. The downstream promoters (P2-P4) produce Fru isoforms (collectively named FruCom) encoded by transcripts that are common to both sexes and are required for viability in both males and females. fru P1 transcripts have only been detected in the nervous system, suggesting that sex-specific functions of fru are limited to neural tissue. However, FruCom is expressed in several non-neural tissues, including sex-specific cell types of the reproductive system. Further, from a recent genome-wide search for putative Dsx targets, fru was identified as a candidate for transcriptional regulation by Dsx. These data raise the possibility that fru functions cooperatively with dsx to regulate gonad development (Zhou, 2021).

Over the past decades, much effort has been focused on understanding the functions of fru in regulating sex-specific behaviors, yet it remained unclear whether fru plays a role in regulating sexual dimorphism outside the nervous system. The work presented in this study demonstrates that Fru is expressed male-specifically in the gonad stem cell niche, and is required for CySC maintenance, cyst cell survival, and for the maintenance of the hub during larval development. Further, male-specific expression of Fru in the gonad is independent of the previously described mechanism of sex-specific alternative splicing by Tra, and is instead dependent on dsx. fru appears to be a direct target for transcriptional regulation by Dsx. This work provides evidence that fru regulates sex-specific development outside the nervous system and alters traditional thinking about the structure of the Drosophila sex determination pathway (Zhou, 2021).

While it was previously reported that fru is expressed in tissues other than the nervous system, including in the gonad, a function for fru outside the nervous system was previously unknown. This study found that Fru is expressed in the developing and adult testis in the hub, the CySC, and the early developing cyst cells. Importantly, it was found that fru is important for the proper function of these cells (Zhou, 2021).

Fru is not expressed at the time of hub formation during embryogenesis, but expression is initiated during the L2/L3 larval stage. This correlates with a time period when the hub must be maintained and resist transforming into female niche structures: in dsx mutants, all gonads in XX and XY animals develop hubs, but in half of each, hubs transform into terminal filament cells and cap cells. fru is not required for initial hub formation, consistent with it not being expressed at that time. fru is also not, by itself, required for hub maintenance under the conditions that were possible to assay (prior to the pupal lethality of fru null mutant animals). However, under conditions where hub maintenance is compromised by loss of dsx function, fru clearly plays a role in influencing whether a gonad will retain a hub, or transform into TF. Fru expression in dsx mutant gonads correlates with whether they formed male or female niche structures, and removing even a single allele of fru is sufficient to induce more hubs to transform into TFs. Finally, ectopic expression of Fru in females is sufficient to inhibit TF formation and partially masculinize the gonad, but does not induce hub formation. Thus, it is proposed that fru is one factor acting downstream of dsx in the maintenance of the male gonad stem cell niche, but that it acts in combination with other factors that also regulate this process (Zhou, 2021).

This study also demonstrated that fru is required for CySC maintenance and for the survival of differentiating cyst cells. Loss of fru from the CySC lineage led to rapid loss of these CySCs from the testis niche. Since precocious differentiation of CySCs or an increase in their apoptosis was not observed, these mechanisms do not appear to contribute to CySC loss. One possibility is that fru is needed for CySCs to have normal expression of adhesion proteins and compete with other stem cells for niche occupancy. It has been shown that fru regulates the Slit-robo pathway and that robo1 is a direct target of fru in the CNS. Interestingly, the Slit-Robo pathway also functions in the CySCs to modulate E-cadherin levels and control the ability of CySCs to compete for occupancy in the niche. Therefore, fru may use similar mechanisms to maintain CySC attachment to the hub. fru also influences survival in the differentiating cyst cells, as an increase in cell death was observed in these cells in fru mutants. Several reports have demonstrated that fru represses programmed cell death in the nervous system. It was further indicated that the cell death gene reaper is a putative target of Fru. Thus, fru may play a role in repressing the apoptosis of cyst cells (Zhou, 2021).

In summary, fru function is clearly important for male niche maintenance and the function of the CySCs and their differentiating progeny. This provides clear evidence that fru regulates sex-specific development in tissues other than the nervous system. Whether additional tissues are also regulated by fru remains to be determined (Zhou, 2021).

Previously, it was thought that the only mechanism by which sex-specific functions of fru were regulated was through Tra-dependent alternative splicing of the P1 transcripts. fru null alleles are lethal in both sexes and Fru proteins derived from non-P1 promoters were thought to be sex-nonspecific and not to contribute to sex determination. Thus, fru and dsx were considered as parallel branches of the sex determination pathway, each independently regulated by Tra. This study demonstrates that fru can also be regulated in a manner independent of tra and dependent on dsx, and provides evidence that fru is a direct target for transcriptional regulation by Dsx (see Proposed model of the Drosophila sex determination pathway. First, Fru expression in the testis is independent of the P1 transcript that is regulated by Tra. A P1 Gal4 reporter is not expressed in the testis and a mutation that prevents FruM expression from P1 does not affect Fru immunoreactivity in the testis. Second, in animals that simultaneously express the female form of tra (Tra on) and the male form of Dsx [XX; dsxD/Df(3R)dsx3], Fru is expressed in the male mode in the testis, demonstrating that it is regulated by dsx and not tra. Finally, an evolutionarily conserved Dsx consensus binding site upstream of the P4 promoter is required for proper expression levels of a fru P4 reporter in the testis. Together, these data demonstrate a novel mode for fru regulation by the sex determination pathway, where sex-specific expression of fru is regulated by dsx. It also means that the large number of fru transcripts that do not arise from the P1 promoter can be expressed in a sex-specific manner to contribute to sexual dimorphism (Zhou, 2021).

The male and female forms of Dsx contain the same DNA binding domain and can regulate the same target genes, but often have opposite effects on gene expression. Prior to this study, the documented Dsx targets (Yolk proteins 1 and 2, bric-a-brac and desatF), along with other proposed targets, were all expressed at higher levels in females than males. Thus, for these targets, DsxF acts as an activator and DsxM acts as a repressor (or DsxM has no role). Interestingly, fru is the first identified Dsx target that is expressed in a male-biased manner. Thus, for direct regulation of fru, DsxM would activate expression while DsxF represses. Mechanistically for Dsx, this implies that the male and female isoforms are not dedicated repressors and activators, respectively, but may be able to switch their mode of regulation in a tissue-specific or target-specific manner. Mouse DMRT1 has also been shown to regulate gene expression both as transcriptional activator and repressor. Thus, it is quite possible that bifunctional transcriptional regulation is a conserved characteristic of DMRTs (Zhou, 2021).

It is possible that dsx regulation of fru occurs in the nervous system as well, where it co-exists with direct regulation of fru alternative splicing by Tra. It was originally thought that alternative splicing of the fru P1 transcript by tra was essential for male courtship behavior. However, more recently it was found that these animals could exhibit male courtship behavior if they were simply stimulated by other flies prior to testing. Interestingly, the courtship behavior exhibited by these males was dependent on dsx. It is proposed that fru might still be essential for male courtship in these fru P1-mutants, but that sex-specific fru expression is dependent on transcriptional regulation of other fru promoters by Dsx (Zhou, 2021).

If sex-specific fru function can be regulated both through alternative splicing by Tra and through transcriptional regulation by Dsx, it raises the question of what is the relationship between these two modes of regulation? It is proposed that regulation of fru by Dsx is the more ancient version of the sex determination pathway and that additional regulation of fru by Tra evolved subsequently, through the acquisition of regulatory RNA elements in the fru P1 transcript. This model is supported by studies of fru gene structures in distantly related Dipteran species, and species of other insect orders, that illustrate the considerable variability in the organization of sequences controlling fru splicing. Further, in some insects, no evidence for alternative splicing of fru has been found, yet fru still plays an important role in males to control courtship behaviors. Finally, in the Hawaiian picture-winged group of subgenus Drosophila, the fru orthologues lack the P1 promoter, and non-P1 fru transcripts exhibit male-specific expression, similar to what is proposed for non-P1 fru transcripts in D. melanogaster. Thus, it appears that regulation of fru by dsx may be the evolutionarily more ancient mechanism for sex-specific control of fru, while Tra-dependent splicing of P1 transcripts is a more recent adaptation. More broadly, tra is not conserved in the sex determination pathway in the majority of animal groups, while homologs of Dsx, the DMRTs, are virtually universal in animal sex determination. Thus, if Fru orthologs are involved in the creation of sexual dimorphism in the body or the brain in other animals, they cannot be regulated by Tra but may be regulated by DMRTs (Zhou, 2021).

REGULATION

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 fru^M (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. retn^z2-428/retn^dri8 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/retn^Z2-428 larvae and pupae; retn^dri8/retn^Z2-428, and retn^Ro44/retn^RO44 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 retn^dri8/retn^Z2-428 and retn^R044/retn^R044 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 retn^dri 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).

The fruitless gene affects female receptivity and species isolation

Female mate rejection acts as a major selective force within species, and can serve as a reproductive barrier between species. In spite of its critical role in fitness and reproduction, surprisingly little is known about the genetic or neural basis of variation in female mate choice. This study identified fruitless as a gene affecting female receptivity within Drosophila melanogaster, as well as female Drosophila simulans rejection of male D. melanogaster. Of the multiple transcripts this gene produces, by far the most widely studied is the sex-specifically spliced transcript involved in the sex determination pathway. However, this study found that female rejection behaviour is affected by a non-sex-specifically spliced fruitless transcript. This is the first implication of fruitless in female behaviour, and the first behavioural role identified for a fruitless non-sex-specifically spliced transcript. It was found that this locus does not influence preferences via a single sensory modality, examining courtship song, antennal pheromone perception, or perception of substrate vibrations, and it is concluded that fruitless influences mate choice via the integration of multiple signals or through another sensory modality (Chowdhury, 2020).

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).

Fruitless recruits two antagonistic chromatin factors to establish single-neuron sexual dimorphism

The Drosophila fruitless (fru) gene encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the mechanism whereby fru establishes the sexual fate of neurons remains enigmatic. This study shows that Fru forms a complex with the transcriptional cofactor Bonus (Bon), which, in turn, recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1), which masculinizes individual sexually dimorphic neurons, or Heterochromatin protein 1a (HP1a), which demasculinizes them. Manipulations of HDAC1 or HP1a expression change the proportion of male-typical neurons and female-typical neurons rather than producing neurons with intersexual characteristics, indicating that on a single neuron level, this sexual switch operates in an all-or-none manner (Ito, 2012).

Sex-switching of the Drosophila brain by two antagonistic chromatin factors

In Drosophila melanogaster, the fruitless (fru) gene encoding BTB-Zn-finger transcription factors organizes male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the molecular mechanism by which fru controls the sexual fate of neurons has been unknown. A recent study represents a first step toward clarification of this mechanism. It was shown that: (1) Fru forms a complex with the transcriptional cofactor Bonus (Bon), which recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1) or Heterochromatin protein 1a (HP1a), to Fru-target sites; (2) the Fru-Bon complex has a masculinizing effect on single sexually-dimorphic neurons when it recruits HDAC1, whereas it has a demasculinizing effect when it recruits HP1a; (3) HDAC1 or HP1a thus recruited to Fru-target sites determines the sexual fate of single neurons in an all-or-none manner, as manipulations of HDAC1 or HP1a expression levels affect the proportion of male-typical neurons and female-typical neurons without producing neurons of intersexual characteristics. It is hypothesized that chromatin landscape changes induced by ecdysone surges direct the HDAC1- or HP1a-containing Fru complex to distinct targets, thereby allowing them to switch the neuronal sexual fate in the brain (Ito, 2013).

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 tra^F-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 Clock^Jrk 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 FRU^M protein is a BTB-Zn-finger transcription factor, FRU^M likely regulates expression of an array of genes to establish neural substrates controlling male behavior. However, such downstream targets of the FRU^M 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 FRU^M, suggesting that NPF is a neurochemical factor mediating FRU^M 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 FRU^M 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 FRU^M 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 LN_d 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-LN_vs and the LN_ds. 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 LN_ds are required for anticipation of lights-off. The data implicate NPF as a neuromodulatory substance within a subset of the LN_ds, involved in this 'late-day' component of the locomotor cycle in males (Lee, 2006).

Although the biological meaning of LN_d-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 LN_ds 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 fru^M and fru^Δtra females is a remarkable mimic of courtship by wild-type or control fru^C males. Some courtship steps, such as initiation, orientation, following, and wing extension, are indistinguishable in fru^M (and fru^Δtra) females and fru^C males. Other steps are clearly abnormal. fru^M 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 fru^M females resemble dsx males in that they lack male-specific Dsx isoforms (Dsx^M) and hence are anatomically female, yet they express the male-specific Fru isoforms (Fru^M) (Demir, 2005).

The distinct roles of fru and dsx in sexual development are clearly illustrated by the differences between animals that produce either only Fru^M or only Dsx^M. Animals that express Dsx^M but not Fru^M (either fru^F males or dsx^M females) resemble normal males but do not court. Conversely, animals that express Fru^M but not Dsx^M (either fru^M females or dsx males) do court, even though they resemble normal females. Thus, Fru^M is both necessary and sufficient for male courtship, whereas Dsx^M is neither necessary nor sufficient. The role of Dsx^M in courtship may simply be to provide the gross male anatomy needed for its optimal execution. This anatomical contribution of Dsx^M 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 Fru^M most likely exerts its effect by modulating the function rather than the assembly of this circuit. Nevertheless, the critical period for Fru^M 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 Fru^M 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, Fru^M 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 Fru^M proteins includes isoforms with at least four different DNA binding domains, which are likely to homo- and hetero-dimerize through their common BTB domain. Fru^M 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, Fru^M 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 fru^GAL4 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 fru^Δtra 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 Fru^M 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 Fru^M 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 Fru^M 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).

The core-promoter factor TRF2 mediates a Fruitless action to masculinize neurobehavioral traits in Drosophila

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 (Chowdhury, 2017).

EFFECTS OF MUTATION

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 fru^sat 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 fru^sat 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 (FRU^M) involved in the control of courtship. One approach toward understanding how fru regulates male courtship is to compare patterns of FRU^M 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 FRU^M is expressed in the CNS (or not expressed, as the case may be) in a given mutant. FRU^M spatial and temporal patterns were examined in fru mutants that exhibit aberrant male courtship. Chromosome breakpoints at the locus eliminate FRU^M. Homozygous viable mutants exhibit an intriguing array of defects. In fru¹ males, there are absences of FRU^M-expressing neuronal clusters or stained cells within certain clusters, reductions of signal intensities in others, and ectopic FRU^M expression in novel cells. fru² males exhibit an overall decrement of FRU^M expression in all neurons normally expressing the gene. fru⁴ and fru^sat mutants produce FRU^M in only a small number of neurons at extremely low levels, and no FRU^M 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 FRU^M and 5-HT in brain neurons. Yet, a newly identified set of sexually dimorphic FRU^M/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 FRU^M 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 FRU^M 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 FRU^M 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 FRU^M 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 FRU^M 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-FRU^M 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 FRU^M 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 FRU^M 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 fru^sat 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 FRU^M 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 FRU^M-less fru³ mutant (Lee, 2001).

Innervation by 5-HT/FRU^M 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 FRU^M 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 FRU^M 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)fru^4-40 and In(3R)fru^w27, 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)fru^w27 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 CI_m-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 fru^sat 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 fru^w12 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 fru^w12 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., fru^4-40/fru^sat15. 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 fru^w12 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 fru^w12/fru^sat15 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 fru^sat15/fru^AJ96u3 and fru^w12/fru^AJ96u3 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).

fru^ΔC, 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 Fru^ComC, whereas reduced male fertility and lack of the MOL are caused by the absence of Fru^MC. 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 Fru^MC, focus was placed on the behavioral function of the male-specific Fru^MC isoform. Male sexual behavior consists of a series of independent but interlinked steps. Fru^MC 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, Fru^MC 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 Fru^M isoforms specify enough neuronal substrates for male sexual behavior. Fru^M 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 Fru^MC has on courtship and its specific effect at the neuronal level. Fru^MC 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 Fru^MA or Fru^MB cannot rescue Fru^MC-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. Fru^MC is expressed in all Fru^M neurons, whereas Fru^MA 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 Fru^M 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 Fru^M 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. Fru^MC mutants develop four smaller muscles, each innervated by the motor neuron that would normally innervate the MOL. Thus expression of Fru^MC 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 Fru^MC'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 Fru^M neurons to induce sex-specific changes in other neurons (Billeter, 2006).

Understanding the neuronal substrates for male sexual behavior requires defining how groups of Fru^M neurons control distinct modalities of this behavior. The fact that only one isoform is missing in fru^ΔC mutants, resulting in hypomorphic behavioral phenotypes, allows the behavioral function of Fru^M neurons to be tested. Expression of Fru^MC in a subset of its normal expression pattern in a Fru^MC-null background rescues mating and fertility but not overall courtship behavior. This uncouples mating from courtship and shows that different populations of Fru^M neurons are responsible for distinct steps of male sexual behavior. This behavioral rescue coincides with rescue in defined Fru^M 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 Fru^M 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 Fru^M expression pattern and/or of its isoforms (Billeter, 2006).

Fru^M 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. Fru^MC-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 Fru^M 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 Fru^M 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. Dsx^M 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 Dsx^M-dependent proliferation of neurons appears to offer a substrate for Fru^M to induce serotonergic differentiation. A number of ectopic serotonergic neurons form in dsx-null females. Given that these females do not express Fru^M, the development of these neurons would appear normally to be inhibited by dsx (Billeter, 2006).

These experiments reveal a new feature of Fru^M 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 Fru^M, given that females missing Tra develop a complete organized set of male serotonergic neurons. Constitutive expression of Fru^M in females also induces two clusters of serotonergic neurons, though with fewer neurons than wild-type males. This reinforces the contention that Fru^M 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 Dsx^M and Fru^M, the other just Fru^M. Two lines of evidence support the idea that Fru^M acts directly on the neurogenesis of these cells: Their projections are greatly reduced in Fru^M-null males, suggesting that some of these neurons are absent, and the number of neurons expressing Fru^M in Fru^MC-null males is reduced. fru has been shown to prevent cell death in neurons in the male brain. Fru^M could use this mechanism to control the number of male serotonergic neurons during development (Billeter, 2006).

Although Fru^M 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 Dsx^M/Fru^M cooperation extends beyond the formation of the male serotonergic neurons because Fru^M 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 (Fru^M). A single Fru^M-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 Fru^M 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 (Fru^M) 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 Fru^M-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 Fru^M 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 Fru^M-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 Fru^M-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/Fru^M SOG neurons could also be linked to higher-order processing centers through synaptic contacts with the male-specific SOG projections of Fru^M-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 Fru^M-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, Fru^M proteins are expressed in small groups of neurons throughout the CNS, and eliminating Fru^M expression in a neuronal subset has profound effects on the progression of male courtship behaviors. At the gross level almost all of the Fru^M-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 fru^M-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 Fru^M-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/Fru^M 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 fru^M females are less active than males in performing male-type courtship behavior. fru^M 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 (Fru^M) acts in foreleg GRNs to promote midline crossing by their axons, whereas midline crossing is repressed in females by female-specific Dsx (Dsx^F). In addition, midline crossing by these neurons might be promoted in males by male-specific Dsx (Dsx^M). The roundabout (robo) paralogs also regulate midline crossing by these neurons, and evidence is provided that Fru^M 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 Fru^M function. Specifically, Fru^MC 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 Dsx^M have somewhat fewer contralateral GRN projections, and (2) Dsx^F prevents the appearance of contralateral GRN axons in females (Mellert, 2010).

The finding that Fru^M regulates GRN axon midline crossing is consistent with previous findings that, in some neurons, Fru^M regulates axonal morphology. Regulation of axonal morphology is likely to alter synaptic connectivity, suggesting that one of the roles of Fru^M 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 Fru^M 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 Fru^MC in female GRNs using poxn-Gal4 is sufficient to induce midline crossing, suggesting that the sex-nonspecific GRNs are not intrinsically nonresponsive to Fru^M (Mellert, 2010).

With respect to the latter result, it is worth considering the contrast between females that are masculinized with fru^δtra, where no contralateral GRN projections are observed, and females in which poxn-Gal4 is used to drive the expression of UAS-fru^MC::AU1 (AU epitope tagged Fruitless) in females, where GRN midline crossing is observed. In the case of females masculinized by fru^δtra it was shown that the absence of contralateral GRN projections was due to Dsx^F functioning to prevent midline crossing in a manner that was epistatic to fru^M function. One attractive explanation for the difference between these two situations is based on the fact that masculinization by fru^δtra occurs via Fru^M produced from the endogenous fruitless locus, whereas masculinization by UAS-fru^MC::AU1, occurs via fru^MC expressed from a UAS construct that contains none of the untranslated sequences present in endogenous fru^M transcripts. Thus, it might be that the difference in midline crossing seen in these two situations is due to Dsx^F directly regulating fru^M expression through noncoding fru sequences that are present in the endogenous fru gene, but absent in the fru cDNA expressed from UAS-fru^MC::AU1. It is not likely that Dsx^M represses fru^M transcription, fru^P1.LexA was seen to be expressed in GRNs in both males and females. Thus, if fru^M is downstream of dsx in these cells, Dsx^F probably affects the processing or translation of fru^M transcripts through sequences not present in the UAS-fru^MC::AU1 construct. Alternatively, differences between these two situations in expression levels or patterns of expression might result in differences in the ability of Fru^M versus Fru^MC to overcome a parallel repressive effect of Dsx^F (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 fru^M-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 fru^M regulate midline crossing? The data indicate that robo lies genetically downstream of fru^M. The most straightforward mechanistic explanation is that Fru^M suppresses the activity of the Robo signaling pathway. Several ways that this might occur can be envisioned. First, fru^M 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 fru^M regulates comm, it does so subtly. It is more probable that fru^M regulates the expression of either other regulators of robo signaling, robo itself, or robo effectors. Strategies are being pursued to identify candidate Fru^M 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 fru^M, and that fru^M 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 fru^M-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, fru^M, 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 fru^C 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 Fru^C rather than other single Fru isoforms. Removal of Fru^C 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).

Fru^A, Fru^B, and Fru^C 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. fru^C mutants were the most affected, and fru^A mutants the least. fru^C 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 fru^C 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 fru^C mutants suggest that Fru^C 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 Fru^C 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 Fru^C 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 Fru^C-dependent lateral arborizations that overlap with the processes of vAB3. Depleting Fru^C from aSP4 or vAB3 recapitulates one aspect of the courtship defect observed in mutants that lack Fru^C 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 (fru^M) gene; males without fru^M do not court females when held in isolation. Such fru^M 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 dsx^M is necessary and sufficient for the acquisition of the potential for such experience-dependent courtship. These results reveal a process that builds, via dsx^M 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 (fru^M) 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 fru^M 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 fru^M null males with either male or female flies for 1 or more days prior to testing. It was further demonstrated that such fru^M-independent, experience-dependent courtship is genetically specified by the dsx gene, whose expression significantly overlaps that of fru^M 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 fru^M as a dedicated regulatory gene that specifies the neural substrates of D. melanogaster male courtship and showing that fru^M 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 fru^M, in male courtship. Of particular relevance, in the light of the discovery of a fru^M-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 fru^M. 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 fru^M and dsx^M, is a particularly attractive candidate for having a significant role in experience-dependent courtship, based on its key role in initiating fru^M-dependent courtship (Pan, 2014).

These findings add significantly to understanding the role of dsx^M in specifying male courtship behavior. Previous studies showed that in males that are wild-type for fru^M one specific aspect of male courtship (e.g., sine song) is dependent on dsx^M function. Thus, it is likely that the potential for sine song is innately established in CNS by dsx^M in a manner analogous to how the potential for fru^M-dependent aspects of courtship are innately established. Additionally, in dsx null males that are wild-type for fru^M 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 dsx^M in the acquisition of the potential for courtship in the absence of fru^M function. This reasoning suggests that dsx^M functions both to facilitate acquisition of the potential for many aspects of courtship (in the absence of fru^M) and to (in the presence of fru^M) innately determine at least one aspect of courtship—sine song (Pan, 2014).

As noted above, the sex determination genes fru^M and dsx^M 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 fru^M 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 fru^M is not expressed. Thus social experience acting via dsx^M-mediated processes somehow compensates for many aspects of fru^M function. It is noted that other aspects of courtship behavior (e.g., attempted copulation) are not observed in fru^M null males, even after they have been group-housed, suggesting that the latter aspects of courtship are solely fru^M-dependent (Pan, 2014).

How does social experience change the courtship circuitry in the absence of fru^M? 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 fru^M 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 fru^M null males. Rather, it is suggested that social experience induces courtship in fru^M 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 dsx^M during adulthood and fru^M 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).

Optogenetic activation of the fruitless-labeled circuitry in Drosophila subobscura males induces mating motor acts

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. fru^soChrimV 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 fru^soChrimV 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 (Tanaka, 2017).

Function of Fruitless Neurons

Pheromones mediating copulation and attraction in Drosophila

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 (Dweck, 2015).

SIFamide acts on Fruitless neurons to modulate sexual behavior in Drosophila melanogaster

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 (Sellami, 2015).

Neurons that underlie Drosophila melanogaster reproductive behaviors: detection of a large male-bias in gene expression in fruitless-expressing neurons

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 (Mewell, 2016). s

Activation of latent courtship circuitry in the brain of Drosophila females induces male-like behaviors

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 (Rezaval, 2016).

Genetic and neuronal mechanisms governing the sex-specific interaction between sleep and sexual behaviors in Drosophila

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 fru^M 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 fru^M -positive sleep-controlling DN1 neurons. In addition, FRU^M 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 (Chen, 2018).

Characterization of the sexually dimorphic fruitless neurons that regulate copulation duration

Male courtship in Drosophila melanogaster is a sexually dimorphic innate behavior that is hardwired in the nervous system. Understanding the neural mechanism of courtship behavior requires the anatomical and functional characterization of all the neurons involved. Courtship involves a series of distinctive behavioral patterns, culminating in the final copulation step, where sperms from the male are transferred to the female. The duration of this process is tightly controlled by multiple genes. The fruitless (fru) gene is one of the factors that regulate the duration of copulation. Using several intersectional genetic combinations to restrict the labeling of GAL4 lines, this study found that a subset of a serotonergic cluster of fru neurons co-express the dopamine-synthesizing enzyme, tyrosine hydroxylase, and provide behavioral and immunological evidence that these neurons are involved in the regulation of copulation duration (Jois, 2018).

Partial proteasomal degradation of Lola triggers the male-to-female switch of a dimorphic courtship circuit

In Drosophila, some neurons develop sex-specific neurites that contribute to dimorphic circuits for sex-specific behavior. As opposed to the idea that the sexual dichotomy in transcriptional profiles produced by a sex-specific factor underlies such sex differences, the sex-specific cleavage was found to confer the activity as a sexual-fate inducer on the pleiotropic transcription factor Longitudinals lacking (Lola). Surprisingly, Fruitless, another transcription factor with a master regulator role for courtship circuitry formation, directly binds to Lola to protect its cleavage in males. Lola cleavage involves E3 ubiquitin ligase Cullin1 and 26S proteasome. This work adds a new dimension to the study of sex-specific behavior and its circuit basis by unveiling a mechanistic link between proteolysis and the sexually dimorphic patterning of circuits. These findings may also provide new insights into potential causes of the sex-biased incidence of some neuropsychiatric diseases and inspire novel therapeutic approaches to such disorders (Sato, 2019).

EVOLUTIONARY HOMOLOGS

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 FRU^MC 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).

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Search PubMed for articles about Drosophila fruitless

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