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.

Antennae sense heat stress to inhibit mating and promote escaping in Drosophila females.

Environmental stress is a major factor that affects courtship behavior and evolutionary fitness. Although mature virgin females of Drosophila melanogaster usually accept a courting male to mate, they may not mate under stressful conditions. Above the temperature optimal for mating (20-25 °C), copulation success of D. melanogaster declines with increasing temperature although we observed vigorous courtship attempts by males, and no copulation takes place at temperatures over 36 °C. We attempted to identify the sensory pathway for detecting heat threat that drives a female to escape rather than to engage in mating that detects hot temperature and suppresses courtship behavior. We found that the artificial activation of warmth-sensitive neurons ('hot cells') in the antennal arista of females completely abrogates female copulation success even at permissive temperatures below 32 °C. Moreover, mutational loss of the GR28b.d thermoreceptor protein caused females to copulate even at 36 °C. These results indicate that antennal hot cells provide the input channel for detecting the high ambient temperature in the control of virgin female mating under stressful conditions.

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.

The Neural Circuitry that Functions as a Switch for Courtship versus Aggression in Drosophila Males.

Courtship and aggression are induced in a mutually exclusive manner in male Drosophila melanogaster, which quickly chooses one of these behavioral repertoires to run depending on whether the encountered conspecific is a female or male, yet the neural mechanism underlying this decision making remains obscure. By targeted excitation and synaptic blockage in a subset of brain neurons, we demonstrate here that the fruitless (fru)-negative subfraction (∼20 cells) of a doublesex-positive neural cluster, pC1, acts as the aggression-triggering center whereas the fru-positive subfraction (∼20 cells) of pC1 acts as the courtship-triggering center, and that the mutually exclusive activation of these two centers is attained by a double-layered inhibitory switch composed of two fru single-positive clusters, LC1 and mAL. To our knowledge, this is the first report to unravel the cellular identity of the neural switch that governs the alternative activation of aggression and courtship in the animal kingdom.

REQUIREMENT OF THE TEC FAMILY TYROSINE KINASE BTK29A FOR COURTSHIP MEMORY IN Drosophila MALES.

A male Drosophila that is not successful in courtship will reduce his courtship efforts in the next encounter with a female. This courtship suppression persists for more than 1 h in wild-type males. The Btk29A(ficP) mutant males null for the Btk29A type 2 isoform, a fly homolog of the nonreceptor tyrosine kinase Btk, show no courtship suppression, while Btk29A hypomorphic males exhibit a rapid decline in courtship suppression, leading to its complete loss within 30 min. The males of a revertant stock or Btk29A(ficP) males that are also mutant for parkas, a gene encoding the presumptive negative regulator of Btk29A, exhibit normal courtship suppression. Since another behavioral assay has shown that Btk29A(ficP) mutants are sensitization-defective, we hypothesize that the mutant flies are unable to maintain the neural excitation state acquired by experience, resulting in the rapid loss of courtship suppression.

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.

The Dmca1D channel mediates Ca(2+) inward currents in Drosophila embryonic muscles.

We studied, in a genetic model organism, Drosophila melanogaster, the channel mechanisms underlying membrane excitation in the embryonic body wall muscle whose biophysical properties have been poorly characterized. The inward current underlying the action potential was solely mediated by a high-threshold class of voltage-gated Ca(2+) channels, which exhibited slow inactivation, Ca(2+) permeability with saturation at high [Ca(2+)]OUT, and sensitivity to a Ca(2+) channel blocker, Cd(2+). The Ca(2+) current in the embryonic muscle was completely eliminated in Dmca1D mutants, indicating that the Dmca1D-encoded Ca(2+) channel is the major mediator of inward currents in the body wall muscles throughout the embryonic and larval stages.

Drosophila ovipositor extension in mating behavior and egg deposition involves distinct sets of brain interneurons.

Oviposition is a female-specific behavior that directly affects fecundity, and therefore fitness. If a fertilized female encounters another male that she has evaluated to be of better quality than her previous mate, it would be beneficial for her to remate with this male rather than depositing her eggs. Females who decided not to remate exhibited rejection behavior toward a courting male and engaged in oviposition. Although recent studies of Drosophila melanogaster identified sensory neurons and putative second-order ascending interneurons that mediate uterine afferents affecting female reproductive behavior, little is known about the brain circuitry that selectively activates rejection versus oviposition behaviors. We identified the sexually dimorphic pC2l and female-specific pMN2 neurons, two distinct classes of doublesex (dsx)-expressing neurons that can initiate ovipositor extension associated with rejection and oviposition behavior, respectively. pC2l interneurons, which induce ovipositor extrusion for rejection in females, have homologues that control courtship behavior in males. Activation of these two classes of neurons appears to be mutually exclusive and each governs hierarchical control of the motor program in the VNC either for rejection or oviposition, contributing centrally to the switching on or off of the alternative motor programs.

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.

Neuroethology of male courtship in Drosophila: from the gene to behavior.

Neurogenetic analyses in the fruit fly Drosophila melanogaster revealed that gendered behaviors, including courtship, are underpinned by sexually dimorphic neural circuitries, whose development is directed in a sex-specific manner by transcription factor genes, fruitless (fru) and doublesex (dsx), two core members composing the sex-determination cascade. Via chromatin modification the Fru proteins translated specifically in the male nervous system lead the fru-expressing neurons to take on the male fate, as manifested by their male-specific survival or male-specific neurite formations. One such male-specific neuron group, P1, was shown to be activated when the male taps the female abdomen. Moreover, when artificially activated, P1 neurons are sufficient to induce the entire repertoire of the male courtship ritual. These studies provide a conceptual framework for understanding how the genetic code for innate behavior can be embodied in the neuronal substrate.

Insect pheromone behavior: fruit fly.

The amenability to genetics of Drosophila melanogaster has made this organism one of the best-suited models for studying the neurobiology of pheromone-guided behavior. Single-male assays use the minigene encoding the thermosensitive channel dTrpA1 to activate neurons expressing fruitless (fru), a major courtship regulator gene, and thereby induce most of the elementary courtship acts in a solitary male exposed to temperature increase. Tethered male assays allow Ca(2+)-imaging of neuronal activities of a male fly displaying courtship behavior on a treadmill when stimulated with a female or pheromones. Here we describe technical details of these assays.

Genes and circuits of courtship behaviour in Drosophila males.

In Drosophila melanogaster, the causal links among a complex behaviour, single neurons and single genes can be demonstrated through experimental manipulations. A key player in establishing the male courtship circuitry is the fruitless (fru) gene, the expression of which yields the FruM proteins in a subset of male but not female neurons. FruM probably regulates chromatin states, leading to single-neuron sex differences and, consequently, a sexually dimorphic circuitry. The mutual connections among fru-expressing neurons--including primary sensory afferents, central interneurons such as the P1 neuron cluster that triggers courtship, and courtship motor pattern generators--probably form the core portion of the male courtship circuitry.

Select interneuron clusters determine female sexual receptivity in Drosophila.

Female Drosophila with the spinster mutation repel courting males and rarely mate. Here we show that the non-copulating phenotype can be recapitulated by the elimination of spinster functions from either spin-A or spin-D neuronal clusters, in the otherwise wild-type (spinster heterozygous) female brain. Spin-D corresponds to the olfactory projection neurons with dendrites in the antennal lobe VA1v glomerulus that is fruitless-positive, sexually dimorphic and responsive to fly odour. Spin-A is a novel local neuron cluster in the suboesophageal ganglion, which is known to process contact chemical pheromone information and copulation-related signals. A slight reduction in spinster expression to a level with a minimal effect is sufficient to shut off female sexual receptivity if the dominant-negative mechanistic target of rapamycin is simultaneously expressed, although the latter manipulation alone has only a marginal effect. We propose that spin-mediated mechanistic target of rapamycin signal transduction in these neurons is essential for females to accept the courting male.

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

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

Sexually dimorphic shaping of interneuron dendrites involves the hunchback transcription factor.

Sexual dimorphism of the brain has been well characterized anatomically in Drosophila melanogaster at the single neuron level, yet little is known about the molecular mechanism whereby cellular sex differences are generated except that the neural sex determination gene fruitless (fru) plays a key role. The fru-expressing mAL interneuron cluster is sexually dimorphic in three aspects: the number of cells composing the cluster is 5 in females and 30 in males; the ipsilateral neurite is absent in females and present in males; the contralateral neurite forms Y-shaped branches in the subesophageal ganglion in females while it ends with a simple horsetail-like structure in males. By screens in the compound eye for modifiers of roughness induced by fru(+) overexpression, we identified a loss-of-function allele of hunchback (hb) to be a suppressor of this phenotype. Hb was expressed in most of the fru-expressing neurons in the pupal and adult stages. Knocking down hb in mAL MARCM (Mosaic Analysis with a Repressible Cell Marker) clones in the male brain resulted in partial demasculinization of the branching pattern of the contralateral neurites without affecting the cell number and the ipsilateral neurite formation. The present results suggest that Hb is essential for male-typical shaping of the contralateral neurites by Fru.

Gr39a, a highly diversified gustatory receptor in Drosophila, has a role in sexual behavior.

Sexual recognition among individuals is crucial for the reproduction of animals. In Drosophila, like in many other animals, pheromones are suggested to play an important role in conveying information about an individual, such as sex, maturity and mating status. Sex-specific cuticular hydrocarbon components are thought to be major sex pheromones in Drosophila, and are postulated to act through the gustatory system, since they are mostly non-volatile chemicals. However, very little is known about the molecular and neural bases of gustatory pheromone reception. So far, a few putative gustatory receptors, including Gr32a and Gr68a, have been implicated in courtship behavior. Here, we examine another putative gustatory receptor, Gr39a, which shares a cluster with both Gr32a and Gr68a in a molecular phylogeny of the gustatory receptor family, for its potential role in courtship behavior. The Gr39a gene produces four isoforms through alternative splicing of different 5'-most exons. A quantitative real-time PCR analysis showed that the expression levels of all four splice variants of Gr39a were reduced in a fly line in which a P element was inserted into the Gr39a locus. Homozygous and hemizygous males for the P-element insertion, as well as males in which Gr39a was knocked down by RNAi, all showed reduced courtship levels toward females. The courtship levels returned to normal when the P element was excised out. A close analysis of courtship behavior of the mutant males revealed that the average duration of a continuous courtship bout was significantly shorter in the mutants than in the wild type. The results suggest that Gr39a has a role in sustaining courtship behavior in males, possibly through the reception of a stimulating arrestant pheromone.

Female contact activates male-specific interneurons that trigger stereotypic courtship behavior in Drosophila.

We determined the cellular substrate for male courtship behavior by quasinatural and artificial stimulation of brain neurons. Activation of fruitless (fru)-expressing neurons via stimulation of thermosensitive dTrpA1 channels induced an entire series of courtship acts in male Drosophila placed alone without any courting target. By reducing the number of neurons expressing dTrpA1 by MARCM, we demonstrated that the initiation of courtship behavior is significantly correlated with the activation of the transmidline P1 interneurons, the descending P2b interneurons, or both, indicating that these interneurons trigger courtship. Using an experimental paradigm in which a tethered male can be stimulated to initiate courtship by touching his foreleg tarsus to a female's abdomen, we found that P1 neurites of tethered males showed a transient Ca(2+) rise after tarsal stimulation with the female-associated sensory cues. These observations strongly suggest that P1 neurons are the prime components of the neural circuitry that initiates male courtship.

Neuronal synaptic outputs determine the sexual fate of postsynaptic targets.

Synapses mediate inductive interactions for the proper development of pre- and postsynaptic cells: presynaptic electrical activities and synaptic transmission ensure the organization of postsynaptic structures, whereas neurotrophins produced in postsynaptic cells support the survival and enlargement of presynaptic partners. In Drosophila, a motor nerve has been implicated in the induction of the muscle of Lawrence (MOL), the formation of which is male specific and depends on the neural expression of fruitless (fru), a neural sex-determinant gene. Here we report the identification of a single motoneuron essential for inducing the MOL, which we call the MOL-inducing (Mind) motoneuron. The MOL is restored in fru mutant males, which otherwise lack the MOL, if the fru(+) transgene is selectively expressed in the Mind motoneuron by mosaic analysis with a repressible cell marker. We further demonstrate that synaptic outputs from the Mind motoneuron are indispensable to MOL induction, because the blockage of synaptic transmission by shibire(ts) (shi(ts)) during the critical period in development abolished the MOL formation in males. Our finding that sex-specific neurons instruct sexually dimorphic development of their innervating targets through synaptic interactions points to the novel mechanism whereby the pre- and postsynaptic partners coordinately establish their sexual identity.

Phenotypic interactions of spinster with the genes encoding proteins for cell death control in Drosophila melanogaster.

The spin gene was first identified by its mutant phenotype, which is characterized by extremely strong mate refusal by females in response to male courtship in Drosophila. Spin mutants are also known to be accompanied by a remarkable reduction in programmed cell death in the reproductive and nervous systems. To better understand the molecular functions of spin, we searched for its genetic modifiers. Forced expression of spin(+) in somatic cells as driven by ptc-Gal4 in the testis resulted in the invasion of mature sperm into the anterior testes tip, which is otherwise occupied only by immature germ cells. To obtain genes that modulate spin's effect, the gain-of-function spin phenotype was observed in the presence of a chromosome harboring an EP or GS P-element insertion, which initiates transcription of the genomic sequence neighboring the insertion site. We isolated th and emc as suppressors of spin and atg8a as a gene that reproduces the spin phenotype on its own. th encodes Inhibitor of apoptosis-1, and mammalian Id genes homologous to emc are known to inhibit apoptosis. atg8a encodes a protein essential for autophagy. These results suggest that spin promotes cell death mechanisms that are regulated negatively by th and emc and positively by atg8a.

The shaping of male courtship posture by lateralized gustatory inputs to male-specific interneurons.

BACKGROUND: Unilateral wing vibration to generate "love songs" is a hallmark of male courtship posture in Drosophila melanogaster. In contrast, males of some other Drosophila species extend both wings simultaneously during courtship. Thus, the patterns of wing movement vary among species and are under stringent genetic control, although there are few variations among individuals within a single species. These observations prompted the postulation that the proper wing display by courting males of D. melanogaster does not require sensory inputs. RESULTS: Here we show that when males of D. melanogaster are deprived of gustatory inputs mediated by the sensory neurons expressing the taste receptor gene Gr32a, a close relative to the pheromone receptor gene Gr68a, they often fail to perform unilateral wing extension during courtship because they become unable to keep a wing in the resting position while extending another wing. The tarsal amputation of a foreleg, but not other legs, increased the occurrence of simultaneous wing extension, indicating that Gr32a-expressing cells in this structure are involved in the regulation of courting posture. A similar simultaneous wing extension was also observed in males in which the putative pheromone-binding protein gene Obp57d was inactivated. The axons of Gr32a-expressing cells project to the subesophageal ganglion, where their terminals unilaterally contact mAL, which are male-specific fruitless (fru)-expressing interneurons that have bilateral branches. CONCLUSIONS: Our observations strongly suggest that gustatory pheromone inputs ensure the correct laterality of wing vibration that conforms to the species-specific behavioral pattern.