A circuit logic for sexually shared and dimorphic aggressive behaviors in Drosophila.

Aggression involves both sexually monomorphic and dimorphic actions. How the brain implements these two types of actions is poorly understood. We have identified three cell types that regulate aggression in Drosophila: one type is sexually shared, and the other two are sex specific. Shared common aggression-promoting (CAP) neurons mediate aggressive approach in both sexes, whereas functionally downstream dimorphic but homologous cell types, called male-specific aggression-promoting (MAP) neurons in males and fpC1 in females, control dimorphic attack. These symmetric circuits underlie the divergence of male and female aggressive behaviors, from their monomorphic appetitive/motivational to their dimorphic consummatory phases. The strength of the monomorphic → dimorphic functional connection is increased by social isolation in both sexes, suggesting that it may be a locus for isolation-dependent enhancement of aggression. Together, these findings reveal a circuit logic for the neural control of behaviors that include both sexually monomorphic and dimorphic actions, which may generalize to other organisms.

Molecular Mechanisms of Sexually Dimorphic Nervous System Patterning in Flies and Worms.

Male and female brains display anatomical and functional differences. Such differences are observed in species across the animal kingdom, including humans, but have been particularly well-studied in two classic animal model systems, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Here we summarize recent advances in understanding how the worm and fly brain acquire sexually dimorphic features during development. We highlight the advantages of each system, illustrating how the precise anatomical delineation of sexual dimorphisms in worms has enabled recent analysis into how these dimorphisms become specified during development, and how focusing on sexually dimorphic neurons in the fly has enabled an increasingly detailed understanding of sex-specific behaviors.

Fly courtship song: triggering the light fantastic.

In a study in this issue, Clyne and Miesenböck (2008) apply an ingenious optogenetic technology to activate neurons that generate male-specific courtship song in flies. This work sheds new light on the neural circuitry underlying sexually dimorphic behaviors in Drosophila.

Generation and characterization of fruitless P1 promoter mutant in Drosophila melanogaster.

The identification of mutations in the gene fruitless (fru) paved the way for understanding the genetic basis of male sexual behavior in the vinegar fly Drosophila melanogaster. D. melanogaster males perform an elaborate courtship display to the female, ultimately leading to copulation. Mutations in fru have been shown to disrupt most aspects of the male's behavioral display, rendering males behaviorally sterile. The fru genomic locus encodes for multiple transcription factor isoforms from several promoters; only those under the regulation of the most distal P1 promoter are under the control of the sex determination hierarchy and play a role in male-specific behaviors. In this study, we used CRISPR/Cas9-based targeted genome editing of the fru gene, to remove the P1 promoter region. We have shown that removal of the P1 promoter leads to a dramatic decrease in male courtship displays towards females and male-specific sterility. We have expanded the analysis of fru P1-dependent behaviors, examining male's response to courtship song and general activity levels during12-hour light: dark cycles. Our novel allele expands the mutant repertoire available for future studies of fru P1-derived function in D. melanogaster. Our fruΔP1 mutant will be useful for future studies of fru P1-derived function, as it can be homozygosed without disrupting additional downstream promoter function and can be utilized in heterozygous combinations with other extant fru alleles.

Interactions between the sexual identity of the nervous system and the social environment mediate lifespan in Drosophila melanogaster.

Sex differences in lifespan are ubiquitous, but the underlying causal factors remain poorly understood. Inter- and intrasexual social interactions are well known to influence lifespan in many taxa, but it has proved challenging to separate the role of sex-specific behaviours from wider physiological differences between the sexes. To address this problem, we genetically manipulated the sexual identity of the nervous system-and hence sexual behaviour-in Drosophila melanogaster, and measured lifespan under varying social conditions. Consistent with previous studies, masculinization of the nervous system in females induced male-specific courtship behaviour and aggression, while nervous system feminization in males induced male-male courtship and reduced aggression. Control females outlived males, but masculinized female groups displayed male-like lifespans and male-like costs of group living. By varying the mixture of control and masculinized females within social groups, we show that male-specific behaviours are costly to recipients, even when received from females. However, consistent with recent findings, our data suggest courtship expression to be surprisingly low cost. Overall, our study indicates that nervous system-mediated expression of sex-specific behaviour per se-independent of wider physiological differences between the sexes, or the receipt of aggression or courtship-plays a limited role in mediating sex differences in lifespan.

Sex-specific responses to sexual familiarity, and the role of olfaction in Drosophila.

Studies of mating preferences have largely neglected the potential effects of individuals encountering their previous mates ('directly sexually familiar'), or new mates that share similarities to previous mates, e.g. from the same family and/or environment ('phenotypically sexually familiar'). Here, we show that male and female Drosophila melanogaster respond to the direct and phenotypic sexual familiarity of potential mates in fundamentally different ways. We exposed a single focal male or female to two potential partners. In the first experiment, one potential partner was novel (not previously encountered) and one was directly familiar (their previous mate); in the second experiment, one potential partner was novel (unrelated, and from a different environment from the previous mate) and one was phenotypically familiar (from the same family and rearing environment as the previous mate). We found that males preferentially courted novel females over directly or phenotypically familiar females. By contrast, females displayed a weak preference for directly and phenotypically familiar males over novel males. Sex-specific responses to the familiarity of potential mates were significantly weaker or absent in Orco(1) mutants, which lack a co-receptor essential for olfaction, indicating a role for olfactory cues in mate choice over novelty. Collectively, our results show that direct and phenotypic sexual familiarity is detected through olfactory cues and play an important role in sex-specific sexual behaviour.

Low-level repressive histone marks fine-tune gene transcription in neural stem cells.

Coordinated regulation of gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of gene transcription is lacking due to the compensatory effect of translational control. We used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). We demonstrate that the transcription factor FruitlessC (FruC) binds cis-regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fruC function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. FruC negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis-regulatory regions. Identical to fruC loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. We propose low-level H3K27me3 enrichment fine-tunes gene transcription in stem cells, a mechanism likely conserved from flies to humans.

From neurons to sperm, our bodies are formed of a range of cells tailored to perform a unique role. However, organisms also host small reservoirs of unspecialized 'stem cells' that retain the ability to become different kinds of cells. When these stem cells divide, one daughter cell remains a stem cell while the other undergoes a series of changes that allows it to mature into a specific cell type. This 'differentiation' process involves quickly switching off the stem cell programme, the set of genes that give a cell the ability to keep dividing while maintaining an unspecialized state. Failure to do so can result in the differentiating cell reverting towards its initial state and multiplying uncontrollably, which can lead to tumours and other health problems. While scientists have a good understanding of how the stem cell programme is turned off during differentiation, controlling these genes is a balancing act that starts even before division: if the program is over-active in the 'mother' stem cell, for instance, the systems that switch it off in its daughter can become overwhelmed. The mechanisms presiding over these steps are less well-understood. To address this knowledge gap, Rajan, Anhezini et al. set out to determine how stem cells present in the brains of fruit flies could control the level of activity of their own stem cell programme. RNA sequencing and other genetic analyses revealed that a protein unique to these cells, called Fruitless, was responsible for decreasing the activity of the programme. Biochemical experiments then showed that Fruitless performed this role by attaching a small amount of chemical modifications (called methyl groups) to the proteins that 'package' the DNA near genes involved in the stem cell programme. High levels of methyl groups present near a gene will switch off this sequence completely; however, the amount of methyl groups that Fruitless helped to deposit is multiple folds lower. Consequently, Fruitless 'fine-tunes' the activity of the stem cell programme instead, dampening it just enough to stop it from overpowering the 'off' mechanism that would take place later in the daughter cell. These results shed new light on how stem cells behave ­ and how our bodies stop them from proliferating uncontrollably. In the future, Rajan, Anhezini et al. hope that this work will help to understand and treat diseases caused by defective stem cell differentiation.

Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly.

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

Sexual dimorphism: can you smell the difference?

A powerful new technique for visualizing neurons in the fly brain has uncovered fine neuroanatomical differences between the olfactory circuitries of male and female Drosophila.

A sex-specific switch between visual and olfactory inputs underlies adaptive sex differences in behavior.

Although males and females largely share the same genome and nervous system, they differ profoundly in reproductive investments and require distinct behavioral, morphological, and physiological adaptations. How can the nervous system, while bound by both developmental and biophysical constraints, produce these sex differences in behavior? Here, we uncover a novel dimorphism in Drosophila melanogaster that allows deployment of completely different behavioral repertoires in males and females with minimum changes to circuit architecture. Sexual differentiation of only a small number of higher order neurons in the brain leads to a change in connectivity related to the primary reproductive needs of both sexes-courtship pursuit in males and communal oviposition in females. This study explains how an apparently similar brain generates distinct behavioral repertoires in the two sexes and presents a fundamental principle of neural circuit organization that may be extended to other species.

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 [1-6]. Sex mosaic studies have shown that the sex of certain regions of the central nervous system (CNS) is critical to song production [7]. Our examination of one of these regions, the mesothoracic ganglion (Msg), revealed the coexpression of two sex-determination genes, fruitless (fru) and doublesex (dsx). Because both genes are involved in creating a sexually dimorphic CNS [8, 9] and are necessary for song production [10-13], we investigated the individual contributions of fru and dsx to the specification of a male CNS and song production. We show a novel requirement for dsx in specifying a sexually dimorphic population of fru-expressing neurons in the Msg. Moreover, by using females constitutively expressing the male-specific isoforms of fru (Fru(M)), we show a critical requirement for the male isoform of dsx (Dsx(M)), alongside Fru(M), in the specification of courtship song. Therefore, although Fru(M) expression is sufficient for the performance of many male-specific behaviors [14], we have shown that without Dsx(M), the determination of a male-specific CNS and thus a full complement of male behaviors are not realized.

Distinct Roles and Synergistic Function of FruM Isoforms in Drosophila Olfactory Receptor Neurons.

Sexual dimorphism in Drosophila courtship circuits requires the male-specific transcription factor fruM, which is alternatively spliced to encode the FruMA, FruMB, and FruMC isoforms. Most fruM-positive neurons express multiple variants; however, the functional significance of their co-expression remains undetermined. Do co-expressed isoforms each play unique roles to jointly regulate dimorphism? By focusing on fruM-positive olfactory receptor neurons (ORNs), here, we show that FruMB and FruMC are both required for males' age-dependent sensitization to aphrodisiac olfactory cues in a cell-autonomous manner. Interestingly, FruMB expression is upregulated with age in Or47b and Ir84a ORNs, and its overexpression mimics the effect of age in elevating olfactory responses. Mechanistically, FruMB and FruMC synergistically mediate response sensitization through cooperation of their respective downstream effectors, namely, PPK25 and PPK23, which are both required for forming a functional amplification channel in ORNs. Together, these results provide critical mechanistic insight into how co-expressed FruM isoforms jointly coordinate dimorphic neurophysiology.

A single-cell transcriptomic atlas of the adult Drosophila ventral nerve cord.

The Drosophila ventral nerve cord (VNC) receives and processes descending signals from the brain to produce a variety of coordinated locomotor outputs. It also integrates sensory information from the periphery and sends ascending signals to the brain. We used single-cell transcriptomics to generate an unbiased classification of cellular diversity in the VNC of five-day old adult flies. We produced an atlas of 26,000 high-quality cells, representing more than 100 transcriptionally distinct cell types. The predominant gene signatures defining neuronal cell types reflect shared developmental histories based on the neuroblast from which cells were derived, as well as their birth order. The relative position of cells along the anterior-posterior axis could also be assigned using adult Hox gene expression. This single-cell transcriptional atlas of the adult fly VNC will be a valuable resource for future studies of neurodevelopment and behavior.

Ovipositor Extrusion Promotes the Transition from Courtship to Copulation and Signals Female Acceptance in Drosophila melanogaster.

Communication between male and female fruit flies during courtship is essential for successful mating, but, as with many other species, it is the female who decides whether to mate. Here, we show a novel role for ovipositor extrusion in promoting male copulation attempts in virgin and mated females and signaling acceptance in virgins. We first show that ovipositor extrusion is only displayed by sexually mature females, exclusively during courtship and in response to the male song. We identified a pair of descending neurons that controls ovipositor extrusion in mated females. Genetic silencing of the descending neurons shows that ovipositor extrusion stimulates the male to attempt copulation. A detailed behavioral analysis revealed that during courtship, the male repeatedly licks the female genitalia, independently of ovipositor extrusion, and that licking an extruded ovipositor prompts a copulation attempt. However, if the ovipositor is not subsequently retracted, copulation is prevented, as it happens with mated females. In this study, we reveal a dual function of the ovipositor: while its extrusion is necessary for initiating copulation by the male, its retraction signals female acceptance. We thus uncover the significance of the communication between male and female that initiates the transition from courtship to copulation.

Control of male sexual behavior in Drosophila by the sex determination pathway.

Understanding how genes influence behavior, including sexuality, is one of biology's greatest challenges. Much of the recent progress in understanding how single genes can influence behavior has come from the study of innate behaviors in the fruit fly Drosophila melanogaster. In particular, the elaborate courtship ritual performed by the male fly has provided remarkable insights into how the neural circuitry underlying sexual behavior--which is largely innate in flies--is built into the nervous system during development, and how this circuitry functions in the adult. In this review we will discuss how genes of the sex determination pathway in Drosophila orchestrate the developmental events necessary for sex-specific behaviors and physiology, and the broader lessons this can teach us about the mechanisms underlying the development of sex-specific neural circuitry.

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

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

To court or not to court - a multimodal sensory decision in Drosophila males.

When Drosophila males encounter another fly, they have to make a rapid assessment to ensure the appropriate response: should they court, fight or pursue a different action entirely? Previous work has focused on the significance of sensory cues detected by the male during these encounters; however, recent evidence highlights the importance of the male's own internal state in shaping his responses. Additionally, once triggered, courtship is not a rigid sequence of motor actions, but rather a finely tuned behavioural display that must continually update in response to sensory feedback. Here, we review recent findings highlighting how sensory information and internal states are integrated ensuring appropriate action selection, and how they sustain and fine-tune motor output. We further discuss recent advances in our understanding of species differences in sensory processing that may contribute to reproductive isolation.

Drosophila Courtship: Love Is Not Blind.

Animals rely on sensory cues to help them find suitable mates. Visual cues are particularly useful for locating mates during the day. A new study has revealed key visual neurons in male Drosophila used to identify and pursue potential mates.

Female Drosophila melanogaster respond to song-amplitude modulations.

Males in numerous animal species use mating songs to attract females and intimidate competitors. We demonstrate that modulations in song amplitude are behaviourally relevant in the fruit fly Drosophila We show that Drosophilamelanogaster females prefer amplitude modulations that are typical of melanogaster song over other modulations, which suggests that amplitude modulations are processed auditorily by D. melanogaster Our work demonstrates that receivers can decode messages in amplitude modulations, complementing the recent finding that male flies actively control song amplitude. To describe amplitude modulations, we propose the concept of song amplitude structure (SAS) and discuss similarities and differences to amplitude modulation with distance (AMD).This article has an associated First Person interview with the first author of the paper.