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

Gene name - fruitless

Synonyms -

Cytological map position - 91B2--91B3

Function - transcription factor

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

Symbol - fru

FlyBase ID:FBgn0004652

Genetic map position - 3-[64]

Classification - C2H2 zinc finger - BTB domain

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene

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

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

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

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

Rezaval, C., Pattnaik, S., Pavlou, H. J., Nojima, T., Bruggemeier, B., D'Souza, L. A., Dweck, H. K. and Goodwin, S. F. (2016). Activation of latent courtship circuitry in the brain of Drosophila females induces male-like behaviors. Curr Biol 26: 2508-2515. PubMed ID: 27568592
Summary:
Courtship in Drosophila melanogaster offers a powerful experimental paradigm for the study of innate sexually dimorphic behaviors. Fruit fly males exhibit an elaborate courtship display toward a potential mate. Females never actively court males, but their response to the male's display determines whether mating will actually occur. Sex-specific behaviors are hardwired into the nervous system via the actions of the sex determination genes doublesex (dsx) and fruitless (fru). Activation of male-specific dsx/fru+ P1 neurons in the brain initiates the male's courtship display, suggesting that neurons unique to males trigger this sex-specific behavior. In females, dsx+ neurons play a pivotal role in sexual receptivity and post-mating behaviors. This study manipulated the function of dsx+ neurons in the female brain to investigate higher-order neurons that drive female behaviors. Surprisingly, it was found that activation of female dsx+ neurons in the brain induces females to behave like males by promoting male-typical courtship behaviors. Activated females display courtship toward conspecific males or females, as well other Drosophila species. Specific dsx+ neurons critical for driving male courtship were uncovered and pheromones were identified that trigger such behaviors in activated females. While male courtship behavior was thought to arise from male-specific central neurons, this study shows that the female brain is equipped with latent courtship circuitry capable of inducing this male-specific behavioral program.
Chen, D., Sitaraman, D., Chen, N., Jin, X., Han, C., Chen, J., Sun, M., Baker, B. S., Nitabach, M. N. and Pan, Y. (2017). Genetic and neuronal mechanisms governing the sex-specific interaction between sleep and sexual behaviors in Drosophila. Nat Commun 8(1): 154. PubMed ID: 28754889
Summary:
Animals execute one particular behavior among many others in a context-dependent manner, yet the mechanisms underlying such behavioral choice remain poorly understood. This research studied how two fundamental behaviors, sex and sleep, interact at genetic and neuronal levels in Drosophila. An increased need for sleep was shown to inhibits male sexual behavior by decreasing the activity of the male-specific P1 neurons that coexpress the sex determination genes fruM and dsx, but does not affect female sexual behavior. Further, a sex-specific neuronal circuit was delineated wherein the P1 neurons encoding increased courtship drive suppressed male sleep by forming mutually excitatory connections with the fruM -positive sleep-controlling DN1 neurons. In addition, FRUM was found to regulates male courtship and sleep through distinct neural substrates. These studies reveal the genetic and neuronal basis underlying the sex-specific interaction between sleep and sexual behaviors in Drosophila, and provide insights into how competing behaviors are co-regulated. Genes and circuits involved in sleep and sexual arousal have been extensively studied in Drosophila. This study has identified the sex determination genes fruitless and doublesex, and a sex-specific P1-DN1 neuronal feedback that governs the interaction between these competing behaviors.
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 (shiTS). These manipulations have been extended to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shiTS at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, C309/traF combination was also found to induce high levels of courtship between pairs of males and 'chaining' behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRUM protein is regulated by Tra, it was hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fruMIR) would mimic the effects of traF; but C309/fruMIR males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRUM in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of traF or fruMIR effects on courtship song, because C309 was found to be coexpressed with FRUM within the same 10 neuronal clusters in pupae. Thus, it is hypothesized that elimination of singing behavior by the C309/shiTS combination involves neurons acting downstream of FRUM cells (Villella, 2005).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Courtship song is a critical component of male courtship behavior in Drosophila, making the female more receptive to copulation and communicating species-specific information. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production. Examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS and are necessary for song production, the individual contributions of fru and dsx to the specification of a male CNS and song production was investigated. A novel requirement is shown for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (FruM), a critical requirement is shown for the male isoform of dsx (DsxM), alongside FruM, in the specification of courtship song. Therefore, although FruM expression is sufficient for the performance of many male-specific behaviors, this study has shown that without DsxM, the determination of a male-specific CNS and thus a full complement of male behaviors are not realized (Rideout, 2007).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


GENE STRUCTURE

The fruitless transcription unit produces at least seven overlapping classes of transcription. The presence of three different 5' ends suggests that there are at least three promoters. Most transcripts contain a common middle region. There are at least four alternative 3' ends. It is not known whether transcripts with a particular 5' end can have all the possible 3' ends and vice versa. Transcripts generated from the most distal promoter appear to be alternatively spliced near three 13 nucleotide repeats known to be recognized by the the Tra and Tra-2 proteins, which regulate pre-mRNA splicing of Doublesex mRNA. Similar 13 nucleotide repeats are repeate six times in Doublesex. Different transcripts are female-specific (9.0, 8.0, and 7.4 kb), male-specific (7.9, 6.4 and 5.4 kb), and one sex-nonspecific (4.4 kb) (Ryner, 1996).

Genomic length - At least 140 kb

cDNA clone length - 2876 bases (Ito, 1996)


PROTEIN STRUCTURE

One cDNA codes for a protein of 855 amino acids (Ito, 1996). Another codes for a male-specific form of FRU transcript class I and contains a long open reading frame that starts upstream of the male-specific 5' splice site and encodes a polypeptide of 776 amino acids. In females, use of a start codon downstream of the female-specific splice site results in a polypeptide of 675 amino acids missing the first 101 amino acids (Ryner, 1996). The 101 amino acids specific to the male form contain a stretch of 12 histidines, alternating with neutral residues, followed by a proline rich stretch (Ryner, 1996).

Both the male and long female open reading frames encode proteins containing a BTB domain (also called a POX domain, a domain of approximately 115 amino acids, suggested to function in dimerization (Albagli, 1995). The FRU BTB domain is followed by stretches of repeated amino acids. The carboxy-terminus of the proteins contains a zinc finger pair related to the C2H2 class. At least two other alternative FRU transcripts encode proteins with different zinc finger pairs at their carboxy-termini.

Proteins with the same overall structure as FRU have been termed the BTB-ZF protein family or Ttk subgroup. A set of two family members, Drosophila proteins Tramtrack and Broad-complex, are sequence specific transcription factors. Both contain alternative carboxy-terminal zinc finger pairs that confer distinct DNA binding specificities, and are expressed in overlapping, but different, cell-specific patterns. Other Drosophila transcription factors with BTB domains include Abrupt, Trithorax-like and Suppressor of Hairy wing (Ryner, 1996 and Ito, 1996)

The BTB/POZ domain defines a conserved region of about 120 residues; it has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. The BTB/POZ domain from PLZF (PLZF-BTB/POZ) has been overexpressed, purified, characterized, and crystallized. Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a Kd below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. Crystals of the PLZF-BTB/POZ have been prepared that are suitable for a high resolution structure determination using x-ray crystallography. The data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function (Li, 1997).

A novel zinc finger protein, ZID (standing for zinc finger protein with interaction domain) was isolated from humans. ZID has four zinc finger domains and a BTB domain, also know ans a POZ (standing for poxvirus and zinc finger) domain. At its amino terminus, ZID contains the conserved POZ or BTB motif present in a large family of proteins that include otherwise unrelated zinc fingers, such as Drosophila Abrupt, Bric-a-brac, Broad complex, Fruitless, Longitudinals lacking, Pipsqueak, Tramtrack, and Trithorax-like (GAGA). The POZ domains of ZID, TTK and TRL act to inhibit the interaction of their associated finger regions with DNA. This inhibitory effect is not dependent on interactions with other proteins and does not appear dependent on specific interactions between the POZ domain and the zinc finger region. The POZ domain acts as a specific protein-protein interaction domain: The POZ domains of ZID and TTK can interact with themselves but not with each other, or POZ domains from ZF5, or the viral protein SalF17R. However, the POZ domain of TRL can interact efficiently with the POZ domain of TTK. In transfection experiments, the ZID POZ domain inhibits DNA binding in NIH-3T3 cells and appears to localize the protein to discrete regions of the nucleus (Bardwell, 1994).


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 FRUMC in fruit flies; this results in MOL development in both fru-mutant males and fru+ females who otherwise develop no MOL. In parallel, evidence is provided of a unique feature of muscle differentiation within the fifth abdominal segment of male mosquitoes that strongly resembles the fruit fly MOL. Given these conserved features within the context of 250 Myr of evolutionary divergence between Drosophila and Anopheles, it is hypothesized that fru is the prototypic gene of male sexual behavior among dipteran insects (Gailey, 2006).

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

In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. This study reports the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). It was found to be sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. It is proposed that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects, and the potential use of fru and dsx for developing new genetic strategies in vector control is discussed (Salvemini, 2013).


fruitless: Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 January 2007  

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