Molecular and cellular origins of behavioral sex differences: a tiny little fly tells a lot.

Behavioral sex differences primarily derive from the sexually dimorphic organization of neural circuits that direct the behavior. In Drosophila melanogaster, the sex-determination genes fruitless (fru) and doublesex (dsx) play pivotal roles in producing the sexual dimorphism of neural circuits for behavior. Here we examine three neural groups expressing fru and/or dsx, i.e., the P1 cluster, aSP-f and aSP-g cluster pairs and aDN cluster, in which causal relationships between the dimorphic behavior and dimorphic neural characteristics are best illustrated. aSP-f, aSP-g and aDN clusters represent examples where fru or dsx switches cell-autonomously their neurite structures between the female-type and male-type. Processed sensory inputs impinging on these neurons may result in outputs that encode different valences, which culminate in the execution of distinct behavior according to the sex. In contrast, the P1 cluster is male-specific as its female counterpart undergoes dsx-driven cell death, which lowers the threshold for the induction of male-specific behaviors. We propose that the products of fru and dsx genes, as terminal selectors in sexually dimorphic neuronal wiring, induce and maintain the sex-typical chromatin state at postembryonic stages, orchestrating the transcription of effector genes that shape single neuron structures and govern cell survival and death.

Mutually exclusive expression of sex-specific and non-sex-specific fruitless gene products in the Drosophila central nervous system.

The fruitless gene of Drosophila produces multiple protein isoforms, which are classified into two major classes, sex-specific Fru proteins (FruM) and non-sex specific proteins (FruCOM). Whereas FruM proteins are expressed in ∼2000 neurons to masculinize their structure and function, little is known about FruCOM's roles. As an attempt to obtain clues to the roles of FruCOM, we compared expression patterns of FruCOM and FruM in the central nervous system at the late larval stage. We found that nearly all neuroblasts express FruCOM but not FruM, whereas a subset of ganglion mother cells and differentiated neurons express FruM but not FruCOM. It is inferred that FruCOM proteins support fundamental stem cell functions, contrasting to FruM proteins, which play major roles in sex-specific differentiation of neurons.

Evolution of a neuromuscular sexual dimorphism in the Drosophila montium species group.

While epigamic traits likely evolve via sexual selection, the mechanism whereby internal sexual dimorphism arises remains less well understood. Seeking clues as to how the internal sexual dimorphism evolved, we compared the abdominal musculature of 41 Drosophila montium group species, to determine whether any of these species carry a male-specific muscle of Lawrence (MOL). Our quantitative analysis revealed that the size of a sexually dimorphic MOL analog found in 19 montium group species varied widely from species to species, suggesting the gradual evolution of this sexually dimorphic neuromuscular trait. We attempted the ancestral state reconstitution for the presence or absence of the neuromuscular sexual dimorphism in the A5 segment; the neuromuscular sexual dimorphism existed in an old ancestor of the montium group, which was lost in some of the most recent common ancestors of derived lineages, and subsequently some species regained it. This loss-and-gain history was not shared by evolutionary changes in the courtship song pattern, even though both traits were commonly regulated by the master regulator male-determinant protein FruM. It is envisaged that different sets of FruM target genes may serve for shaping the song and MOL characteristics, respectively, and, as a consequence, each phenotypic trait underwent a distinct evolutionary path.

Contact-Chemosensory Evolution Underlying Reproductive Isolation in Drosophila Species.

The main theme of the review is how changes in pheromone biochemistry and the sensory circuits underlying pheromone detection contribute to mate choice and reproductive isolation. The review focuses primarily on gustatory and non-volatile signals in Drosophila. Premating isolation is prevalent among closely related species. In Drosophila, preference for conspecifics against other species in mate choice underlies premating isolation, and such preference relies on contact chemosensory communications between a female and male along with other biological factors. For example, although D. simulans and D. melanogaster are sibling species that yield hybrids, their premating isolation is maintained primarily by the contrasting effects of 7,11-heptacosadiene (7,11-HD), a predominant female pheromone in D. melanogaster, on males of the two species: it attracts D. melanogaster males and repels D. simulans males. The contrasting preference for 7,11-HD in males of these two species is mainly ascribed to opposite effects of 7,11-HD on neural activities in the courtship decision-making neurons in the male brain: 7,11-HD provokes both excitatory and inhibitory inputs in these neurons and differences in the balance between the two counteracting inputs result in the contrasting preference for 7,11-HD, i.e., attraction in D. melanogaster and repulsion in D. simulans. Introduction of two double bonds is a key step in 7,11-HD biosynthesis and is mediated by the desaturase desatF, which is active in D. melanogaster females but transcriptionally inactivated in D. simulans females. Thus, 7,11-HD biosynthesis diversified in females and 7,11-HD perception diversified in males, yet it remains elusive how concordance of the changes in the two sexes was attained in evolution.

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

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

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Behavioral Evolution of Drosophila: Unraveling the Circuit Basis.

Behavior is a readout of neural function. Therefore, any difference in behavior among different species is, in theory, an outcome of interspecies diversification in the structure and/or function of the nervous system. However, the neural diversity underlying the species-specificity in behavioral traits and its genetic basis have been poorly understood. In this article, we discuss potential neural substrates for species differences in the courtship pulse song frequency and mating partner choice in the Drosophila melanogaster subgroup. We also discuss possible neurogenetic mechanisms whereby a novel behavioral repertoire emerges based on the study of nuptial gift transfer, a trait unique to D. subobscura in the genus Drosophila. We found that the conserved central circuit composed primarily of fruitless-expressing neurons (the fru-circuit) serves for the execution of courtship behavior, whereas the sensory pathways impinging onto the fru-circuit or the motor pathways downstream of the fru-circuit are susceptible to changes associated with behavioral species differences.

The mode of action of Fruitless: Is it an easy matter to switch the sex?

The fruitless (fru) locus was originally defined by a male sterile mutation that promotes male-to-male courtship while suppressing male-to-female courtship in Drosophila melanogaster. The fru promoter-1 pre-RNA generates a set of BTB-zinc finger family FruM proteins expressed exclusively in the male neurons, leading to the formation of sexual dimorphisms in neurons via male-specific neuroblast proliferation, male-specific neural survival, male-specific neuritegenesis or male-specific arbor patterning. Such a wide spectrum of phenotypic effects seems to result from chromatin modifications, in which FruBM recruits Bonus, Histone deacetylase 1 (HDAC1) and/or Heterochromatin protein 1a (HP1a) to ~130 target sites. One established FruBM transcriptional target is the axon guidance protein gene robo1. Multiple transcriptional regulator-binding sites are nested around the FruBM-binding site, and mediate sophisticated modulation of the repressor activity of FruBM. FruBM also binds to the Lola-Q transcriptional repressor to protect it from proteasome-dependent degradation in male but not female neurons as FruBM exists only in male neurons, leading to the formation of sexually dimorphic neural structures. These findings shed light on the multilayered network of transcription regulation orchestrated by the master regulator FruBM.

Sex Mysteries of the Fly Courtship Master Regulator Fruitless.

The fruitless (fru) gene of Drosophila melanogaster generates two groups of protein products, the male-specific FruM proteins and non-sex-specific FruCOM proteins. The FruM proteins have a 101 amino acids (a.a.)-long extension at the N-terminus which is absent from FruCOM. We suggest that this N-terminal extension might confer male-specific roles on FruM interaction partner proteins such as Lola, which otherwise operates as a transcription factor common to both sexes. FruM-expressing neurons are known to connect with other neurons to form a sexually dimorphic circuit for male mating behavior. We propose that FruM proteins expressed in two synaptic partners specify, at the transcriptional level, signaling pathways through which select pre- and post-synaptic partners communicate, and thereby pleiotropic ligand-receptor pairs for cell-cell interactions acquire the high specificity for mutual connections between two FruM-positive cells. We further discuss the possibility that synaptic connections made by FruM-positive neurons are regulated by neural activities, which in turn upregulate Fru expression in active cells, resulting in feedforward enhancement of courtship activities of the male fly.

Calmodulin-binding transcription factor shapes the male courtship song in Drosophila.

Males of the Drosophila melanogaster mutant croaker (cro) generate a polycyclic pulse song dissimilar to the monocyclic songs typical of wild-type males during courtship. However, cro has not been molecularly mapped to any gene in the genome. We demonstrate that cro is a mutation in the gene encoding the Calmodulin-binding transcription factor (Camta) by genetic complementation tests with chromosomal deficiencies, molecular cloning of genomic fragments that flank the cro-mutagenic P-insertion, and phenotypic rescue of the cro mutant phenotype by Camta+-encoding cDNA as well as a BAC clone containing the gene for Camta. We further show that knockdown of the Camta-encoding gene phenocopies cro mutant songs when targeted to a subset of fruitless-positive neurons that include the mcALa and AL1 clusters in the brain. cro-GAL4 and an anti-Camta antibody labeled a large number of brain neurons including mcALa. We conclude that the Camta-encoding gene represents the cro locus, which has been implicated in a species-specific difference in courtship songs between D. sechellia and simulans.

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

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

Ecdysone signaling regulates specification of neurons with a male-specific neurite in Drosophila.

Some mAL neurons in the male brain form the ipsilateral neurite (ILN[+]) in a manner dependent on FruBM, a male-specific transcription factor. FruBM represses robo1 transcription, allowing the ILN to form. We found that the proportion of ILN[+]-mALs in all observed single cell clones dropped from ∼90% to ∼30% by changing the heat-shock timing for clone induction from 4-5 days after egg laying (AEL) to 6-7 days AEL, suggesting that the ILN[+]-mALs are produced predominantly by young neuroblasts. Upon EcR-A knockdown, ILN[+]-mALs were produced at a high rate (∼60%), even when heat shocked at 6-7 days AEL, yet EcR-B1 knockdown reduced the proportion of ILN[+]-mALs to ∼30%. Immunoprecipitation assays in S2 cells demonstrated that EcR-A and EcR-B1 form a complex with FruBM. robo1 reporter transcription was repressed by FruBM and ecdysone counteracted FruBM. We suggest that ecdysone signaling modulates the FruBM action to produce an appropriate number of male-type neurons.

Processing and Thermal Response of Temperature-Sensitive-Gel(TSG)/Polymer Composites.

Temperature-sensitive gels (TSGs) are generally used in the fields of medical, robotics, MEMS, and also in daily life. In this paper, we synthesized a novel TSG with good thermal durability and a lower melting temperature below 60 °C. We discussed the physical properties of he TSG and found it provided excellent thermal expansion. Therefore, we proposed the usage of TSG to develop a strategic breathable film with controllable gas permeability. The TSG particles were prepared firstly and then blended with linear low-density polyethylene/calcium carbonate (LLDPE/CaCO3) composite to develop microporous TSG/LLDPE/CaCO3 films. We investigated the morphology, thermal, and mechanical properties of TSG/LLDPE/CaCO3 composite films. The film characterization was conducted by gas permeability testing and demonstration temperature control experiments. The uniformly porous structure and the pore size in the range of 5⁻40 µm for the TSG/LLDPE/CaCO3 composite films were indicated by SEM micrographs. The demonstration temperature control experiments clearly proved the effect of the controllable gas permeability of the TSG and, more promisingly, the great practical value and application prospects of this strategic effect for the temperature-sensitive breathable film was proved.

Optogenetic Activation of the fruitless-Labeled Circuitry in Drosophila subobscura Males Induces Mating Motor Acts.

It remains an enigma how the nervous system of different animal species produces different behaviors. We studied the neural circuitry for mating behavior in Drosophila subobscura, a species that displays unique courtship actions not shared by other members of the genera including the genetic model D. melanogaster, in which the core courtship circuitry has been identified. We disrupted the D. subobscura fruitless (fru) gene, a master regulator for the courtship circuitry formation in D. melanogaster, resulting in complete loss of mating behavior. We also generated frusoChrimV , which expresses the optogenetic activator Chrimson fused with a fluorescent marker under the native fru promoter. The fru-labeled circuitry in D. subobscura visualized by frusoChrimV revealed differences between females and males, optogenetic activation of which in males induced mating behavior including attempted copulation. These findings provide a substrate for neurogenetic dissection and manipulation of behavior in non-model animals, and will help to elucidate the neural basis for behavioral diversification.SIGNIFICANCE STATEMENT How did behavioral specificity arise during evolution? Here we attempted to address this question by comparing the parallel genetically definable neural circuits controlling the courtship behavior of Drosophila melanogaster, a genetic model, and its relative, D. subobscura, which exhibits a courtship behavioral pattern unique to it, including nuptial gift transfer. We found that the subobscura fruitless circuit, which is required for male courtship behavior, was slightly but clearly different from its melanogaster counterpart, and that optogenetic activation of this circuit induced subobscura-specific behavior, i.e., regurgitating crop contents, a key element of transfer of nuptial gift. Our study will pave the way for determining how and which distinctive cellular elements within the fruitless circuit determine the species-specific differences in courtship behavior.

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

In fruit flies, the male-specific fruitless (fru) gene product FruBM plays a central role in establishing the neural circuitry for male courtship behavior by orchestrating the transcription of genes required for the male-type specification of individual neurons. We herein identify the core promoter recognition factor gene Trf2 as a dominant modifier of fru actions. Trf2 knockdown in the sexually dimorphic mAL neurons leads to the loss of a male-specific neurite and a reduction in male courtship vigor. TRF2 forms a repressor complex with FruBM, strongly enhancing the repressor activity of FruBM at the promoter region of the robo1 gene, whose function is required for inhibiting the male-specific neurite formation. In females that lack FruBM, TRF2 stimulates robo1 transcription. Our results suggest that TRF2 switches its own role from an activator to a repressor of transcription upon binding to FruBM, thereby enabling the ipsilateral neurite formation only in males.

Serotonergic neuronal death and concomitant serotonin deficiency curb copulation ability of Drosophila platonic mutants.

Drosophila platonic (plt) males court females, but fail to copulate. Here we show that plt is an allele of scribbler (sbb), a BMP signalling component. sbb knockdown in larvae leads to the loss of approximately eight serotonergic neurons, which express the sex-determinant protein Doublesex (Dsx). Genetic deprivation of serotonin (5-HT) from dsx-expressing neurons results in copulation defects. Thus, sbb+ is developmentally required for the survival of a specific subset of dsx-expressing neurons, which support the normal execution of copulation in adults by providing 5-HT. Our study highlights the conserved involvement of serotonergic neurons in the control of copulatory mechanisms and the key role of BMP signalling in the formation of a sex-specific circuitry.

Fruitless Represses robo1 Transcription to Shape Male-Specific Neural Morphology and Behavior in Drosophila.

The Drosophila fruitless (fru) gene is regarded as a master regulator of the formation of male courtship circuitry, yet little is known about its molecular basis of action. We show that roundabout 1 (robo1) knockdown in females promotes formation of the male-specific neurite in sexually dimorphic mAL interneurons and that overexpression of the male-specific Fru(BM) diminishes the expression of Robo1 in the fly brain. Our electrophoretic mobility shift and reporter assays identify the 42-bp segment encompassing the palindrome sequence T T C G C T G C G C C G T G A A in the 5' UTR of robo1 exon1 as the Fru(BM)-responsive element. We find that ∼10-bp deletions in the palindrome sequence induce a loss of the male-specific neurite and disrupt male courtship patterns. This study paves the way for a thorough understanding of the mechanism whereby Fru proteins orchestrate transcription for the formation of courtship circuitry.

Zeste tunes the timing of ecdysone actions in triggering programmed tissue degeneration in Drosophila.

In the pupal stage, the fly body undergoes extensive metamorphic remodeling, in which programmed cell death plays a critical role. We studied two of the constituent processes in this remodeling, salivary gland degeneration and breakdown of the eclosion muscle, which are triggered by an increase and a decrease in the circulating steroid hormone ecdysone at the start and end of metamorphosis, respectively. We found that knockdown of zeste (z), a gene encoding a sequence-specific DNA-binding protein implicated in transvection, in salivary gland cells advances the initiation of their degeneration, whereas z knockdown in neurons delays muscle breakdown. We further showed that knockdown of an ecdysone-inducible gene, E74, retards salivary gland degeneration with little effect on eclosion muscle breakdown. We propose that Z tunes the sensitivity of ecdysone targets to this hormone in order to ensure a high safety margin so that the cell death program will be activated when the ecdysone titer is at a sufficiently high level that is reached only at a defined stage during metamorphosis.

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

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

The Drosophila lingerer protein cooperates with Orb2 in long-term memory formation.

Recently mated Drosophila females were shown to be reluctant to copulate and to exhibit rejecting behavior when courted by a male. Males that experience mate refusal by a mated female subsequently attenuate their courtship effort toward not only mated females but also virgin females. This courtship suppression persists for more than a day, and thus represents long-term memory. The courtship long-term memory has been shown to be impaired in heterozygotes as well as homozygotes of mutants in orb2, a locus encoding a set of CPEB RNA-binding proteins. We show that the impaired courtship long-term memory in orb2-mutant heterozygotes is restored by reducing the activity of lig, another putative RNA-binding protein gene, yet on its own the loss-of-function lig mutation is without effect. We further show that Lig forms a complex with Orb2. We infer that a reduction in the Lig levels compensates the Orb2 deficiency by mitigating the negative feedback for Orb2 expression and thereby alleviating defects in long-term memory.