fruitless

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).

Targets of Activity

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

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

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

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

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

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

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

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

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

Drosophila neuropeptide F (NPF), a homolog of vertebrate neuropeptide Y, functions in feeding and coordination of behavioral changes in larvae and in modulation of alcohol sensitivity in adults, suggesting diverse roles for this peptide. To gain more insight into adult-specific NPF neuronal functions, how npf expression is regulated in the adult brain was studied. npf expression is regulated in both sex-nonspecific and male-specific manners. The data show that male-specific npf (ms-npf) expression is controlled by the transformer (tra)-dependent sex-determination pathway. Furthermore, fruitless, one of the major genes functioning downstream of tra, is apparently an upstream regulator of ms-npf transcription. Males lacking ms-npf expression (through 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).

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