Search

The generation of acoustic communication signals is widespread across the animal kingdom, and males of many species, including Drosophilidae, produce patterned courtship songs to increase their chance of success with a female. For some animals, song structure can vary considerably from one rendition to the next; neural noise within pattern generating circuits is widely assumed to be the primary source of such variability, and statistical models that incorporate neural noise are successful at reproducing the full variation present in natural songs. In direct contrast, here we demonstrate that much of the pattern variability in Drosophila courtship song can be explained by taking into account the dynamic sensory experience of the male. In particular, using a quantitative behavioural assay combined with computational modelling, we find that males use fast modulations in visual and self-motion signals to pattern their songs, a relationship that we show is evolutionarily conserved. Using neural circuit manipulations, we also identify the pathways involved in song patterning choices and show that females are sensitive to song features. Our data not only demonstrate that Drosophila song production is not a fixed action pattern, but establish Drosophila as a valuable new model for studies of rapid decision-making under both social and naturalistic conditions.

Motion vision is a major function of all visual systems, yet the underlying neural mechanisms and circuits are still elusive. In the lamina, the first optic neuropile of Drosophila melanogaster, photoreceptor signals split into five parallel pathways, L1-L5. Here we examine how these pathways contribute to visual motion detection by combining genetic block and reconstitution of neural activity in different lamina cell types with whole-cell recordings from downstream motion-sensitive neurons. We find reduced responses to moving gratings if L1 or L2 is blocked; however, reconstitution of photoreceptor input to only L1 or L2 results in wild-type responses. Thus, the first experiment indicates the necessity of both pathways, whereas the second indicates sufficiency of each single pathway. This contradiction can be explained by electrical coupling between L1 and L2, allowing for activation of both pathways even when only one of them receives photoreceptor input. A fundamental difference between the L1 pathway and the L2 pathway is uncovered when blocking L1 or L2 output while presenting moving edges of positive (ON) or negative (OFF) contrast polarity: blocking L1 eliminates the response to moving ON edges, whereas blocking L2 eliminates the response to moving OFF edges. Thus, similar to the segregation of photoreceptor signals in ON and OFF bipolar cell pathways in the vertebrate retina, photoreceptor signals segregate into ON-L1 and OFF-L2 channels in the lamina of Drosophila.

The complex, geometric colour patterns of many animal bodies have important roles in behaviour and ecology. The generation of certain patterns has been the subject of considerable theoretical exploration, however, very little is known about the actual mechanisms underlying colour pattern formation or evolution. Here we have investigated the generation and evolution of the complex, spotted wing pattern of Drosophila guttifera. We show that wing spots are induced by the Wingless morphogen, which is expressed at many discrete sites that are specified by pre-existing positional information that governs the development of wing structures. Furthermore, we demonstrate that the elaborate spot pattern evolved from simpler schemes by co-option of Wingless expression at new sites. This example of a complex design developing and evolving by the layering of new patterns on pre-patterns is likely to be a general theme in other animals.

Just how animals are able to use the Earth's magnetic field for navigation is the subject of much current interest. It is known that the UV-A/blue light photoreceptor cryptochrome (Cry) mediates the light-dependent magnetic sense in Drosophila. Steven Reppert and colleagues now use genetic manipulation to show that two cryptochromes from the monarch butterfly, Drosophila-like Cry1 and vertebrate-like Cry2, can restore magnetic sensation in cry-deficient Drosophila. This suggests that both types of Cry have the capacity to sense magnetic fields in the migratory monarch butterfly via a mechanism that remains unclear — and that light sensitivity is involved in some way. Animals use the Earth's magnetic field for orientation but the biophysical basis of this is unclear. The light-dependent magnetic sense of Drosophila melanogaster was recently shown to be mediated by the cryptochrome (Cry) photoreceptor; here, using a transgenic approach, the type 1 and 2 Cry of the monarch butterfly are shown to both function in the magnetoreception system of Drosophila, and probably use an unconventional photochemical mechanism. Understanding the biophysical basis of animal magnetoreception has been one of the greatest challenges in sensory biology. Recently it was discovered that the light-dependent magnetic sense of Drosophila melanogaster is mediated by the ultraviolet (UV)-A/blue light photoreceptor cryptochrome (Cry)1. Here we show, using a transgenic approach, that the photoreceptive, Drosophila-like type 1 Cry and the transcriptionally repressive, vertebrate-like type 2 Cry of the monarch butterfly (Danaus plexippus) can both function in the magnetoreception system of Drosophila and require UV-A/blue light (wavelength below 420 nm) to do so. The lack of magnetic responses for both Cry types at wavelengths above 420 nm does not fit the widely held view that tryptophan triad-generated radical pairs mediate the ability of Cry to sense a magnetic field. We bolster this assessment by using a mutant form of Drosophila and monarch type 1 Cry and confirm that the tryptophan triad pathway is not crucial in magnetic transduction. Together, these results suggest that animal Crys mediate light-dependent magnetoreception through an unconventional photochemical mechanism. This work emphasizes the utility of Drosophila transgenesis for elucidating the precise mechanisms of Cry-mediated magnetosensitivity in insects and also in vertebrates such as migrating birds.

A genome-wide RNAi screen has been used to define the genetic basis for the formation and function of Drosophila muscle. A total of 2,785 genes were recognized as having a role in muscle, many of then assigned to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, and the list of conserved and functionally validated muscle genes includes a strong enrichment for genes implicated in mammalian sarcomere organization and human muscle diseases. A genome-wide RNA interference screen to systematically test the genetic basis for formation and function of the Drosophila muscle is described. A role in muscle for 2,785 genes is identified; many of these genes are phylogenetically conserved. Systematic genetic approaches have provided deep insight into the molecular and cellular mechanisms that operate in simple unicellular organisms. For multicellular organisms, however, the pleiotropy of gene function has largely restricted such approaches to the study of early embryogenesis. With the availability of genome-wide transgenic RNA interference (RNAi) libraries in Drosophila1,2, it is now possible to perform a systematic genetic dissection of any cell or tissue type at any stage of the lifespan. Here we apply these methods to define the genetic basis for formation and function of the Drosophila muscle. We identify a role in muscle for 2,785 genes, many of which we assign to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, including genes implicated in mammalian sarcomere organization and human muscle diseases.

Aggression is regulated by pheromones in many animal species1, 2, 3. However, in no system have aggression pheromones, their cognate receptors and corresponding sensory neurons been identified. Here we show that 11-cis-vaccenyl acetate (cVA), a male-specific volatile pheromone, robustly promotes male–male aggression in the vinegar fly Drosophila melanogaster. The aggression-promoting effect of synthetic cVA requires olfactory sensory neurons (OSNs) expressing the receptor Or67d4, 5, 6, as well as the receptor itself. Activation of Or67d-expressing OSNs, either by genetic manipulation of their excitability or by exposure to male pheromones in the absence of other classes of OSNs, is sufficient to promote aggression. High densities of male flies can promote aggression by the release of volatile cVA. In turn, cVA-promoted aggression can promote male fly dispersal from a food resource, in a manner dependent on Or67d-expressing OSNs. These data indicate that cVA may mediate negative-feedback control of male population density, through its effect on aggression. Identification of a pheromone–OSN pair controlling aggression in a genetic organism opens the way to unravelling the neurobiology of this evolutionarily conserved behaviour.

The primary objective of the Human Genome Project was to produce high-quality sequences not just for the human genome but also for those of the chief model organisms: Escherichia coli, yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans), fly (Drosophila melanogaster) and mouse (Mus musculus). Free access to the resultant data has prompted much biological research, including development of a map of common human genetic variants (the International HapMap Project)1, expression profiling of healthy and diseased cells2 and in-depth studies of many individual genes. These genome sequences have enabled researchers to carry out genetic and functional genomic studies not previously possible, revealing new biological insights with broad relevance across the animal kingdom 3, 4.

The integrity of polarized epithelia is critical for development and human health. Many questions remain concerning the full complement and the function of the proteins that regulate cell polarity. Here we report that the Drosophila FERM proteins Yurt (Yrt) and Coracle (Cora) and the membrane proteins Neurexin IV (Nrx-IV) and Na(+),K(+)-ATPase are a new group of functionally cooperating epithelial polarity proteins. This 'Yrt/Cora group' promotes basolateral membrane stability and shows negative regulatory interactions with the apical determinant Crumbs (Crb). Genetic analyses indicate that Nrx-IV and Na(+),K(+)-ATPase act together with Cora in one pathway, whereas Yrt acts in a second redundant pathway. Moreover, we show that the Yrt/Cora group is essential for epithelial polarity during organogenesis but not when epithelial polarity is first established or during terminal differentiation. This property of Yrt/Cora group proteins explains the recovery of polarity in embryos lacking the function of the Lethal giant larvae (Lgl) group of basolateral polarity proteins. We also find that the mammalian Yrt orthologue EPB41L5 (also known as YMO1 and Limulus) is required for lateral membrane formation, indicating a conserved function of Yrt proteins in epithelial polarity.

Fruit flies exhibit robust attraction to food odors, which usually excite multiple glomeruli. To understand how the representation of such odors leads to behavior, we used genetic tools to dissect the contribution of each activated glomerulus. Apple cider vinegar triggers robust innate attraction at a relatively low concentration, which activates six glomeruli. By silencing individual glomeruli, we found that the absence of activity in two glomeruli, DM1 and VA2, markedly reduced attraction. Conversely, when each of these two glomeruli was selectively activated, flies exhibited as robust an attraction to vinegar as wild type flies. Notably, a higher concentration of vinegar excites an additional glomerulus and is less attractive to flies. Here we show that the activation of the additional glomerulus is necessary and sufficient to mediate the behavioral switch. Together, these results indicate that individual glomeruli, rather than the entire pattern of active glomeruli, mediate innate behavioral output.

The Drosophila protein Argos is an antagonist of epidermal growth factor receptor (EGFR) signalling that functions by binding and sequestering EGFR ligands. Here the structure of Argos bound to the EGFR ligand Spitz reveals that Argos engulfs the ligand using three related domains with structural resemblance to receptors for TGF-β and urokinase plasminogen activator. These results suggest that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anticancer therapeutics. Drosophila Argos is an antagonist of EGF receptor signalling that functions by binding and sequestering EGFR ligands. The structure of Argos bound to the EGFR ligand Spitz reveals that Argos engulfs the ligand using three related domains with structural resemblance to receptors for TGF-β and urokinase plasminogen activator. Members of the epidermal growth factor receptor (EGFR) or ErbB/HER family and their activating ligands are essential regulators of diverse developmental processes1,2. Inappropriate activation of these receptors is a key feature of many human cancers3, and its reversal is an important clinical goal. A natural secreted antagonist of EGFR signalling, called Argos, was identified in Drosophila4. We showed previously that Argos functions by directly binding (and sequestering) growth factor ligands that activate EGFR5. Here we describe the 1.6-A resolution crystal structure of Argos bound to an EGFR ligand. Contrary to expectations4,6, Argos contains no EGF-like domain. Instead, a trio of closely related domains (resembling a three-finger toxin fold7) form a clamp-like structure around the bound EGF ligand. Although structurally unrelated to the receptor, Argos mimics EGFR by using a bipartite binding surface to entrap EGF. The individual Argos domains share unexpected structural similarities with the extracellular ligand-binding regions of transforming growth factor-β family receptors8. The three-domain clamp of Argos also resembles the urokinase-type plasminogen activator (uPA) receptor, which uses a similar mechanism to engulf the EGF-like module of uPA9. Our results indicate that undiscovered mammalian counterparts of Argos may exist among other poorly characterized structural homologues. In addition, the structures presented here define requirements for the design of artificial EGF-sequestering proteins that would be valuable anti-cancer therapeutics.

This issue includes a landmark collection of papers on the stalwart of the genetics lab, the Drosophila fruit fly. The centrepiece is the publication by the Drosophila 12 Genomes Consortium of the genomic sequence for ten Drosophila species. The paper compares the newly sequenced genomes (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi species), with the two previously known sequences for D. melanogaster and D. pseudoobscura. The resulting database of genetic variation will be invaluable for the study of the forces of evolutionary change. A second major collaboration has mined the dozen Drosophila genome sequences for conserved elements, and reports the relationship between conservation and function for many specific sequence motifs. A detailed regulatory network emerges, identifying protein-coding genes and exons, RNA genes, microRNAs and their targets. These papers are discussed in News and Views. Two further research papers use the new genomic data to study gene expression, first for genes with male-biased expression and those unique to each species and second, to track the evolution of gene dosage compensation on Drosophila sex chromosomes. Four new reviews focus on how the latest work on Drosophila is taking this genetically pliant lab model into exciting new fields. Pierre Leopold and Norbert Perrimon review advances in the study of endocrinology and homeostasis that are establishing Drosophila as a model for mammalian physiology. Drosophila has proved a powerful system in which to study the pathways controlling cell shape in growing tissue, as reported by Thomas Lecuit and Loic Le Goff. Leslie Vosshall reviews the remarkable work linking neural circuits and behaviour and John Lis reviews work on Drosophila that has rewritten the textbook view of gene transcription. The cover shows anaesthetized individuals of all twelve Drosophila species. The evolution of dosage compensation on Drosophila sex chromosomes is examined by using microarrays to determine relative gene expression of sex-linked genes in a number of fly species. How this expression changes over time is discussed, particularly in species with a neo-X chromosome, to determine why genes might have male-biased expression. X chromosomes evolve differently from autosomes, but general governing principles have not emerged1. For example, genes with male-biased expression are under-represented on the X chromosome of D. melanogaster2, but are randomly distributed in the genome of Anopheles gambiae3. In direct global profiling experiments using species-specific microarrays, we find a nearly identical paucity of genes with male-biased expression on D. melanogaster, D. simulans, D. yakuba, D. ananassae, D. virilis and D. mojavensis X chromosomes. We observe the same under-representation on the neo-X of D. pseudoobscura. It has been suggested that precocious meiotic silencing of the X chromosome accounts for reduced X chromosome male-biased expression in nematodes4, mammals5 and Drosophila6. We show that X chromosome genes with male-biased expression are under-represented in somatic cells and in mitotic male germ cells. These data are incompatible with simple X chromosome inactivation models. Using expression profiling and comparative sequence analysis, we show that selective gene extinction on the X chromosome, creation of new genes on autosomes and changed genomic location of existing genes contribute to the unusual X chromosome gene content.

Since the early 1960s, imaging studies of Drosophila sp. polytene chromosomes have provided unique views of gene transcription in vivo. The dramatic changes in chromatin structure that accompany gene activation can be visualized as chromosome puffs. Now, live-cell imaging techniques coupled with protein-DNA crosslinking assays on a genome-wide scale allow more detailed mechanistic questions to be addressed and are prompting the re-evaluation of models of transcription regulation in both Drosophila and mammals.

Both genome content and deployment contribute to phenotypic differences between species1-5. Sex is the most important difference between individuals in a species and has long been posited to be rapidly evolving. Indeed, in the Drosophila genus, traits such as sperm length, genitalia, and gonad size are the most obvious differences between species6. Comparative analysis of sex-biased expression should deepen our understanding of the relationship between genome content and deployment during evolution. Using existing7,8 and newly assembled genomes9, we designed species-specific microarrays to examine sex-biased expression of orthologues and species-restricted genes in D. melanogaster, D. simulans, D. yakuba, D. ananassae, D. pseudoobscura, D. virilis and D. mojavensis. We show that averaged sex-biased expression changes accumulate monotonically over time within the genus. However, different genes contribute to expression variance within species groups compared to between groups. We observed greater turnover of species-restricted genes with male-biased expression, indicating that gene formation and extinction may play a significant part in species differences. Genes with male-biased expression also show the greatest expression and DNA sequence divergence. This higher divergence and turnover of genes with male-biased expression may be due to high transcription rates in the male germline, greater functional pleiotropy of genes expressed in females, and/or sexual competition.

The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of 'off-target effects' (OTEs). However, this study shows that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21–23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities1,2. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function3,4,5,6,7,8,9. However, owing to a tolerance for mismatches and gaps in base-pairing with targets10,11,12, small RNAs could have up to hundreds of potential target sequences in a genome13,14, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets15,16. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such ‘off-target effects’ (OTEs)5. Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction17 in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.

The internal organs of animals often have left-right asymmetry. Although the formation of the anterior-posterior and dorsal-ventral axes in Drosophila is well understood, left-right asymmetry has not been extensively studied. Here we find that the handedness of the embryonic gut and the adult gut and testes is reversed (not randomized) in viable and fertile homozygous Myo31DF mutants. Myo31DF encodes an unconventional myosin, Drosophila MyoIA (also referred to as MyoID in mammals; refs 3, 4), and is the first actin-based motor protein to be implicated in left-right patterning. We find that Myo31DF is required in the hindgut epithelium for normal embryonic handedness. Disruption of actin filaments in the hindgut epithelium randomizes the handedness of the embryonic gut, suggesting that Myo31DF function requires the actin cytoskeleton. Consistent with this, we find that Myo31DF colocalizes with the cytoskeleton. Overexpression of Myo61F, another myosin I (ref. 4), reverses the handedness of the embryonic gut, and its knockdown also causes a left-right patterning defect. These two unconventional myosin I proteins may have antagonistic functions in left-right patterning. We suggest that the actin cytoskeleton and myosin I proteins may be crucial for generating left-right asymmetry in invertebrates.

From flies to humans, the left and right side of the body plan differs. Exactly how symmetry is broken in the early embryo is a mystery. But now two groups working independently report a genetic defect in the fly that may help uncover the mechanism. Both groups studied a mutant with reversed looping of the viscera, and discovered that the mutation lies in an unconventional myosin. Myosin directs right-handed looping and represses the default left-handed fate. This discovery now links actin-based molecular motors and the actin cytoskeleton to left–right patterning in vertebrates. One of a pair of papers separately showing that an unconventional myosin directs handedness of the viscera of Drosophila. Breaking left–right symmetry in Bilateria embryos is a major event in body plan organization that leads to polarized adult morphology, directional organ looping, and heart and brain function1,2,3,4. However, the molecular nature of the determinant(s) responsible for the invariant orientation of the left–right axis (situs choice) remains largely unknown. Mutations producing a complete reversal of left–right asymmetry (situs inversus) are instrumental for identifying mechanisms controlling handedness, yet only one such mutation has been found in mice (inversin)5 and snails6,7. Here we identify the conserved type ID unconventional myosin 31DF gene (Myo31DF) as a unique situs inversus locus in Drosophila. Myo31DF mutations reverse the dextral looping of genitalia, a prominent left–right marker in adult flies. Genetic mosaic analysis pinpoints the A8 segment of the genital disc as a left–right organizer and reveals an anterior–posterior compartmentalization of Myo31DF function that directs dextral development and represses a sinistral default state. As expected of a determinant, Myo31DF has a trigger-like function and is expressed symmetrically in the organizer, and its symmetrical overexpression does not impair left–right asymmetry. Thus Myo31DF is a dextral gene with actin-based motor activity controlling situs choice. Like mouse inversin8, Myo31DF interacts and colocalizes with β-catenin, suggesting that situs inversus genes can direct left–right development through the adherens junction.

The independent evolution of morphological similarities is widespread. For simple traits, such as overall body colour, repeated transitions by means of mutations in the same gene may be common. However, for more complex traits, the possible genetic paths may be more numerous; the molecular mechanisms underlying their independent origins and the extent to which they are constrained to follow certain genetic paths are largely unknown. Here we show that a male wing pigmentation pattern involved in courtship display has been gained and lost multiple times in a Drosophila clade. Each of the cases we have analysed (two gains and two losses) involved regulatory changes at the pleiotropic pigmentation gene yellow. Losses involved the parallel inactivation of the same cis-regulatory element (CRE), with changes at a few nucleotides sufficient to account for the functional divergence of one element between two sibling species. Surprisingly, two independent gains of wing spots resulted from the co-option of distinct ancestral CREs. These results demonstrate how the functional diversification of the modular CREs of pleiotropic genes contributes to evolutionary novelty and the independent evolution of morphological similarities.

The Drosophila fruitless (fru) gene product Fru has been postulated to be a neural sex determination factor that directs development of the central nervous system (CNS), thereby producing male-typical courtship behaviour and inducing male-specific muscle. Male-specific Fru protein is expressed in small groups of neurons scattered throughout the CNS of male, but not female, Drosophila. Collectively, these observations suggest that Fru 'masculinizes' certain neurons, thereby establishing neural substrates for male-typical behaviour. However, specific differences between neurons resulting from the presence or absence of Fru are unknown. Previous studies have suggested that Fru might result in sexual differences in the CNS at the functional level, as no overt sexual dimorphism in CNS structure was discernible. Here we identify a subset of fru-expressing interneurons in the brain that show marked sexual dimorphism in their number and projection pattern. We also demonstrate that Fru supports the development of neurons with male-specific dendritic fields, which are programmed to die during female development as a result of the absence of Fru. Thus, Fru expression can produce a male-specific neural circuit, probably used during heterosexual courtship, by preventing cell death in identifiable neurons.

Most animal cells, even in tissue cultures, can develop the molecular oscillations underlying circadian rhythms. To harness this property into the complex time-related behavioural patterns seen in whole organisms requires the intervention of a series of individual brain oscillators. Drosophila is proving to be a good model in which to study this system. The flies manifest characteristic morning and evening locomotor activity, each controlled by a different group of adult brain clock neurons. Now, by generating transgenic animals with different circadian periods in these morning and evening cells, the brain clock cells are shown to be organized into two separate neuronal circuits. One circuit includes the morning and evening cells and drives circadian locomotor activity. The timing of the evening cells is determined by the morning cells as a result of a daily resetting signal from the morning to the evening cells, which then run at their genetically programmed pace between signals. The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells1,2,3. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour4,5,6,7. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: we and others have recently shown that a group of adult brain clock neurons expresses the neuropeptide PDF8 and controls morning activity (small LNv cells; M-cells), whereas another group of clock neurons controls evening activity (CRY+, PDF- cells; E-cells)6,9. We have generated transgenic mosaic animals with different circadian periods in morning and evening cells. Here we show, by behavioural and molecular assays, that the six canonical groups of clock neurons10 are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity.

The elastic properties of the protein called resilin were discovered about 40 years ago during studies of the flight systems of locusts and dragonflies. It is used in repetitive tasks by most insects, including jumping fleas and chirping cicadas. Resilin is formed by crosslinking of a precursor protein, pro-resilin. The elastic region of pro-resilin has now been isolated in pure form in large quantities following expression of its gene in Escherichia coli. The recombinant pro-resilin can be photochemically crosslinked into a rubber-like material with many of the properties of natural resilin. The synthetic material can be cast into useful shapes, and its capacity to recover after deformation exceeds that of high-resilience rubber, making it a promising candidate for industrial and in situ biomedical applications. Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage1,2; it is best known for its roles in insect flight3,4 and the remarkable jumping ability of fleas5,6 and spittle bugs7. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein8,9 (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications.

During courtship, male Drosophila fruitflies perform a series of innate stereotyped behaviours in response to specific sensory cues. Now the set of nerve cells that govern this behaviour has been identified. Inactivation of these neurons is sufficient to make male flies lose interest in mating, and altering female brains to produce the same proteins produced by these cells causes the females to display male courtship routines. The neurons produce a set of proteins called FruM, encoded by the fruitless (fru) gene, which has previously been implicated in courtship in male fruitflies. Inactivation of FruM-producing nerve cells inhibited courtship without changing other behaviours. And manipulating females' nerve cells so as to produce FruM was enough to cause them to view other females as potential mates. Robust innate behaviours are attractive systems for genetically dissecting how environmental cues are perceived and integrated to generate complex behaviours. During courtship, Drosophila males engage in a series of innate, stereotyped behaviours that are coordinated by specific sensory cues. However, little is known about the specific neural substrates mediating this complex behavioural programme1. Genetic, developmental and behavioural studies have shown that the fruitless (fru) gene encodes a set of male-specific transcription factors (FruM) that act to establish the potential for courtship in Drosophila2. FruM proteins are expressed in ∼2% of central nervous system neurons, at least one subset of which coordinates the component behaviours of courtship3,4. Here we have inserted the yeast GAL4 gene into the fru locus by homologous recombination and show that (1) FruM is expressed in subsets of all peripheral sensory systems previously implicated in courtship, (2) inhibition of FruM function in olfactory system components reduces olfactory-dependent changes in courtship behaviour, (3) transient inactivation of all FruM-expressing neurons abolishes courtship behaviour, with no other gross changes in general behaviour, and (4) ‘masculinization’ of FruM-expressing neurons in females is largely sufficient to confer male courtship behaviour. Together, these data demonstrate that FruM proteins specify the neural substrates of male courtship.

Wnt and Hedgehog family proteins are secreted signalling molecules (morphogens) that act at both long and short range to control growth and patterning during development. Both proteins are covalently modified by lipid, and the mechanism by which such hydrophobic molecules might spread over long distances is unknown. Here we show that Wingless, Hedgehog and glycophosphatidylinositol-linked proteins copurify with lipoprotein particles, and co-localize with them in the developing wing epithelium of Drosophila. In larvae with reduced lipoprotein levels, Hedgehog accumulates near its site of production, and fails to signal over its normal range. Similarly, the range of Wingless signalling is narrowed. We propose a novel function for lipoprotein particles, in which they act as vehicles for the movement of lipid-linked morphogens and glycophosphatidylinositol-linked proteins.

Most of us sleep 7-8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.

The gain, loss or modification of morphological traits is generally associated with changes in gene regulation during development. However, the molecular bases underlying these evolutionary changes have remained elusive. Here we identify one of the molecular mechanisms that contributes to the evolutionary gain of a male-specific wing pigmentation spot in Drosophila biarmipes, a species closely related to Drosophila melanogaster. We show that the evolution of this spot involved modifications of an ancestral cis-regulatory element of the yellow pigmentation gene. This element has gained multiple binding sites for transcription factors that are deeply conserved components of the regulatory landscape controlling wing development, including the selector protein Engrailed. The evolutionary stability of components of regulatory landscapes, which can be co-opted by chance mutations in cis-regulatory elements, might explain the repeated evolution of similar morphological patterns, such as wing pigmentation patterns in flies.

The epidermal growth factor receptor (EGFR) has critical functions in development and in many human cancers1,2,3. During development, the spatial extent of EGFR signalling is regulated by feedback loops comprising both well-understood activators and less well-characterized inhibitors3,4. In Drosophila melanogaster the secreted protein Argos functions as the only known extracellular inhibitor of EGFR5, with clearly identified roles in multiple stages of development3. Argos is only expressed when the Drosophila EGFR (DER) is activated at high levels6, and downregulates further DER signalling. Although there is ample genetic evidence that Argos inhibits DER activation, the biochemical mechanism has not been established. Here we show that Argos inhibits DER signalling without interacting directly with the receptor, but instead by sequestering the DER-activating ligand Spitz. Argos binds tightly to the EGF motif of Spitz and forms a 1:1 (Spitz:Argos) complex that does not bind DER in vitro or at the cell surface. Our results provide an insight into the mechanism of Argos function, and suggest new strategies for EGFR inhibitor design.

In Drosophila melanogaster, ageing is slowed when insulin-like signalling is reduced: life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (chico) are mutated, or when insulin-producing cells are ablated. But we have yet to resolve when insulin affects ageing, or whether insulin signals regulate ageing directly or indirectly through secondary hormones. Caenorhabditis elegans lifespan is also extended when insulin signalling is inhibited in certain tissues, or when repressed in adult worms, and this requires the forkhead transcription factor (FOXO) encoded by daf-16 (ref. 6). The D. melanogaster insulin-like receptor mediates phosphorylation of dFOXO, the equivalent of nematode daf-16 and mammalian FOXO3a. We demonstrate here that dFOXO regulates D. melanogaster ageing when activated in the adult pericerebral fat body. We further show that this limited activation of dFOXO reduces expression of the Drosophila insulin-like peptide dilp-2 synthesized in neurons, and represses endogenous insulin-dependent signalling in peripheral fat body. These findings suggest that autonomous and non-autonomous roles of insulin signalling combine to control ageing.

Gradients of regulatory factors are essential for establishing precise patterns of gene expression during development1,2,3; however, it is not clear how patterning information in multiple gradients is integrated to generate complex body plans. Here we show that opposing gradients of two Drosophila transcriptional repressors, Hunchback (Hb) and Knirps (Kni), position several segments by differentially repressing two distinct regulatory regions (enhancers) of the pair-rule gene even-skipped (eve). Computational and in vivo analyses suggest that enhancer sensitivity to repression is controlled by the number and affinity of repressor-binding sites. Because the kni expression domain is positioned between two gradients of Hb, each enhancer directs expression of a pair of symmetrical stripes, one on each side of the kni domain. Thus, only two enhancers are required for the precise positioning of eight stripe borders (four stripes), or more than half of the whole eve pattern. Our results show that complex developmental expression patterns can be generated by simple repressor gradients. They also support the utility of computational analyses for defining and deciphering regulatory information contained in genomic DNA.

Some morphological traits differ greatly between related species, but it is not clear whether diversity evolves through changes in the same genes and whether similar, independent (that is, convergent) changes occur by the same mechanism1,2. Pigmentation in fruitflies presents an attractive opportunity to explore these issues because pigmentation patterns are diverse, similar patterns have arisen in independent clades, and numerous genes governing their formation have been identified3,4,5 in Drosophila melanogaster. Here we show that both evolutionary diversification and convergence can be due to evolution at the same locus, by comparing abdominal pigmentation and trichome patterns and the expression of Bric-a-brac2 (Bab2), which regulates both traits in D. melanogaster3,6, in 13 species representing the major clades7,8 of the subfamily Drosophilinae. Modifications of Bab2 expression are frequently correlated with diverse pigmentation and trichome patterns that evolved independently in multiple lineages. In a few species, Bab2 expression is not correlated with changes in pigmentation but is correlated with a conserved pattern of trichomes, indicating that this locus can be circumvented to evolve new patterns when a correlated trait is under different constraints.

The PAR-4 and PAR-1 kinases are necessary for the formation of the anterior-posterior (A-P) axis in Caenorhabditis elegans. PAR-1 is also required for A-P axis determination in Drosophila. Here we show that the Drosophila par-4 homologue, lkb1, is required for the early A-P polarity of the oocyte, and for the repolarization of the oocyte cytoskeleton that defines the embryonic A-P axis. LKB1 is phosphorylated by PAR-1 in vitro, and overexpression of LKB1 partially rescues the par-1 phenotype. These two kinases therefore function in a conserved pathway for axis formation in flies and worms. lkb1 mutant clones also disrupt apical-basal epithelial polarity, suggesting a general role in cell polarization. The human homologue, LKB1, is mutated in Peutz-Jeghers syndrome and is regulated by prenylation and by phosphorylation by protein kinase A. We show that protein kinase A phosphorylates Drosophila LKB1 on a conserved site that is important for its activity. Thus, Drosophila and human LKB1 may be functional homologues, suggesting that loss of cell polarity may contribute to tumour formation in individuals with Peutz-Jeghers syndrome.

The cytoplasmically inherited bacterium Wolbachia pipientis is a widespread parasite of arthropods that manipulates the reproductive biology of its hosts, often to their detriment, in order to foster its own transmission through egg cytoplasm. Here we report that infection by Wolbachia restores fertility to Drosophila melanogaster mutant females prevented from making eggs by protein-coding lesions in Sex-lethal (Sxl), the master regulator of sex determination. Suppression of sterility by Wolbachia discriminates markedly among similar germline-specific Sxl alleles, and is not observed for mutations in other genes that produce similar 'tumorous ovary' phenotypes, including one that blocks Sxl germline expression. This allele and gene specificity indicates that suppression probably results from a specific interaction with Sxl protein, rather than from a bypass of the normal germline requirement for this developmental regulator or from an effect on Sxl expression. The Sxl-Wolbachia interaction provides a rare opportunity to explore host-parasite relationships at the molecular level in a model insect. Furthermore, demonstration that a parasite infection can counteract the deleterious effects of mutations in host genes illustrates how hosts might become dependent on parasites.

Sleep is controlled by two processes: a homeostatic drive that increases during waking and dissipates during sleep, and a circadian pacemaker that controls its timing. Although these two systems can operate independently, recent studies indicate a more intimate relationship. To study the interaction between homeostatic and circadian processes in Drosophila, we examined homeostasis in the canonical loss-of-function clock mutants period (per(01)), timeless (tim(01)), clock (Clk(jrk)) and cycle (cyc(01)). cyc(01) mutants showed a disproportionately large sleep rebound and died after 10 hours of sleep deprivation, although they were more resistant than other clock mutants to various stressors. Unlike other clock mutants, cyc(01) flies showed a reduced expression of heat-shock genes after sleep loss. However, activating heat-shock genes before sleep deprivation rescued cyc(01) flies from its lethal effects. Consistent with the protective effect of heat-shock genes, was the observation that flies carrying a mutation for the heat-shock protein Hsp83 (Hsp83(08445)) showed exaggerated homeostatic response and died after sleep deprivation. These data represent the first step in identifying the molecular mechanisms that constitute the sleep homeostat.

During the growth of Drosophila imaginal discs a process called ‘cell competition’1 eliminates slow-proliferating but otherwise viable cells. We report here that cell competition requires the function of the brinker (brk) gene, whose expression is normally repressed by Decapentaplegic (Dpp) signalling2,3,4 but is upregulated in slow-growing Minute/+ cells. Excess brk expression activates the c-Jun amino-terminal kinase pathway, which in turn triggers apoptosis in these cells. We propose that slow-proliferating cells upregulate Brk levels owing to a disadvantage in competing for, or in transducing, the Dpp survival signal. This sequence of events might represent a general mechanism by which weaker cells are eliminated from a growing population, and might serve as a method of controlling cell number and optimizing tissue fitness and hence organ function.

A fascinating question in biology is how molecular changes in developmental pathways lead to macroevolutionary changes in morphology. Mutations in homeotic (Hox) genes have long been suggested as potential causes of morphological evolution1,2, and there is abundant evidence that some changes in Hox expression patterns correlate with transitions in animal axial pattern3. A major morphological transition in metazoans occurred about 400 million years ago, when six-legged insects diverged from crustacean-like arthropod ancestors with multiple limbs4,5,6,7. In Drosophila melanogaster and other insects, the Ultrabithorax (Ubx) and abdominal A (AbdA, also abd-A) Hox proteins are expressed largely in the abdominal segments, where they can suppress thoracic leg development during embryogenesis3. In a branchiopod crustacean, Ubx/AbdA proteins are expressed in both thorax and abdomen, including the limb primordia, but do not repress limbs8,9,10,11. Previous studies led us to propose that gain and loss of transcriptional activation and repression functions in Hox proteins was a plausible mechanism to diversify morphology during animal evolution12. Here we show that naturally selected alteration of the Ubx protein is linked to the evolutionary transition to hexapod limb pattern.

The mechanisms that establish and sharpen pattern across epithelia are poorly understood. In the developing nervous system, the first pattern elements appear as ‘proneural clusters’. In the morphogenetic furrow of the immature Drosophila retina proneural clusters emerge in a wave as a patterned array of 6–10-cell groups, which are recognizable by expression of Atonal, a basic helix–loop–helix transcription factor that is required to establish and pattern the first cell fate1,2,3. The establishment and subsequent patterning of Atonal expression requires activity of the signalling transmembrane receptor Notch2,4. Here we present in vivo and biochemical evidence that the secreted protein Scabrous associates with Notch, and can stabilize Notch protein at the surface. The result is a regulation of Notch activity that sharpens proneural cluster boundaries and ensures establishment of single pioneer neurons.

Sexually dimorphic abdominal pigmentation and segment morphology evolved recently in the melanogaster species group of the fruitfly Drosophila. Here we show that these traits are controlled by the bric à brac [corrected] (bab) gene, which integrates regulatory inputs from the homeotic and sex-determination pathways. bab expression is modulated segment- and sex-specifically in sexually dimorphic species, but is uniform in sexually monomorphic species. We suggest that bab has an ancestral homeotic function, and that regulatory changes at the bab locus played a key role in the evolution of sexual dimorphism. Pigmentation patterns specified by bab affect mating preferences, suggesting that sexual selection has contributed to the evolution of bab regulation.

All insects possess homologous segments, but segment specification differs radically among insect orders. In Drosophila, maternal morphogens control the patterned activation of gap genes, which encode transcriptional regulators that shape the patterned expression of pair-rule genes. This patterning cascade takes place before cellularization. Pair-rule gene products subsequently 'imprint' segment polarity genes with reiterated patterns, thus defining the primordial segments. This mechanism must be greatly modified in insect groups in which many segments emerge only after cellularization. In beetles and parasitic wasps, for instance, pair-rule homologues are expressed in patterns consistent with roles during segmentation, but these patterns emerge within cellular fields. In contrast, although in locusts pair-rule homologues may not control segmentation, some segment polarity genes and their interactions are conserved. Perhaps segmentation is modular, with each module autonomously expressing a characteristic intrinsic behaviour in response to transient stimuli. If so, evolution could rearrange inputs to modules without changing their intrinsic behaviours. Here we suggest, using computer simulations, that the Drosophila segment polarity genes constitute such a module, and that this module is resistant to variations in the kinetic constants that govern its behaviour.

Metazoans use diverse and rapidly evolving mechanisms to determine sex. In Drosophila melanogaster an X-chromosome-counting mechanism determines the sex of an individual by regulating the master switch gene, Sex-lethal (Sxl)1. The X-chromosome dose is communicated to Sxl by a set of X-linked signal elements (XSEs), which activate transcription of Sxl through its ‘establishment’ promoter, SxlPe. Here we describe a new XSE called sisterlessC (sisC) whose mode of action differs from that of previously characterized XSEs, all of which encode transcription factors that activate Sxl Pe directly. In contrast, sisC encodes a secreted ligand for the Drosophila Janus kinase (JAK) and ‘signal transducer and activator of transcription’ (STAT) signal transduction pathway and is allelic to outstretched (os, also called unpaired). We conclude that sisC works indirectly on Sxl through this signalling pathway because mutations in sisC or in the genes encoding Drosophila JAK or STAT reduce expression of SxlPe similarly. The involvement of os in sex determination confirms that secreted ligands can function in cell-autonomous processes. Unlike sex signals for other organisms, sisC has acquired its sex-specific function while maintaining non-sex-specific roles in development, a characteristic that it shares with all other Drosophila XSEs2.

In nervous systems with symmetry about the midline, many neurons project axons from one side to the other. Although several of the components controlling midline crossing have been identified1,2,3,4, little is known about how axons choose the appropriate pathway when crossing. For example, in the Drosophila embryo axons cross the midline in one of two distinct tracts, the anterior or posterior commissure (AC or PC, respectively). Here we show that the Derailed (Drl) receptor tyrosine kinase is expressed by neurons that project in the AC, and that in the absence of Drl such neurons often project abnormally into the PC. Conversely, misexpression of Drl in PC neurons forces them to cross in the AC. The behaviour of Drl-misexpressing neurons and the in vivo binding pattern of a soluble Drl receptor probe indicate that Drl acts as a guidance receptor for a repellent ligand present in the PC. Our results show that Drl is a novel component in the control of midline crossing.

Vertebrate limbs grow out from the flanks of embryos, with their main axis extending proximodistally from the trunk. Distinct limb domains, each with specific traits, are generated in a proximal-to-distal sequence during development. Diffusible factors expressed from signalling centres promote the outgrowth of limbs and specify their dorsoventral and anteroposterior axes. However, the molecular mechanism by which limb cells acquire their proximodistal (P-D) identity is unknown. Here we describe the role of the homeobox genes Meis1/2 and Pbx1 in the development of mouse, chicken and Drosophila limbs. We find that Meis1/2 expression is restricted to a proximal domain, coincident with the previously reported domain in which Pbx1 is localized to the nucleus, and resembling the distribution of the Drosophila homologues homothorax (hth) and extradenticle (exd); that Meis1 regulates Pbx1 activity by promoting nuclear import of the Pbx1 protein; and that ectopic expression of Meis1 in chicken and hth in Drosophila disrupts distal limb development and induces distal-to-proximal transformations. We suggest that restriction of Meis1/Hth to proximal regions of the vertebrate and insect limb is essential to specify cell fates and differentiation patterns along the P-D axis of the limb.

The Drosophila wingless gene (wg) encodes a protein of the Wnt family and is a critical regulator in many developmental processes1. Biochemical studies have indicated that heparan sulphate proteoglycans, consisting of a protein core to which heparan sulphate glycosaminoglycans are attached2, are important for Wg function3. Here we show that, consistent with these findings, the Drosophila gene sulfateless (sfl), which encodes a homologue of vertebrate heparan sulphate N -deacetylase/N -sulphotransferase (an enzyme needed for the modification of heparan sulphate) is essential for Wg signalling. We have identified the product of division abnormally delayed (dally), a glycosyl-phosphatidyl inositol (GPI)-linked glypican, as a heparan sulphate proteoglycan molecule involved in Wg signalling. Our results indicate that Dally may act as a co-receptor for Wg, and that Dally, together with Drosophila Frizzled 2, modulates both short- and long-range activities of Wg.

Different classes of vertebrate motor neuron that innervate distinct muscle targets express unique combinations of LIM-homeodomain transcription factors, suggesting that a combinatorial code of LIM-homeodomain proteins may underlie the control of motor-neuron pathway selection. Studies of LIM-homeodomain genes in mouse, Drosophila melanogaster and Caenorhabditis elegans have revealed functions of these genes in neuronal survival, axon guidance, neurotransmitter expression and neuronal function, but, to our knowledge, none of these studies have addressed the issue of a functional code. Here we study two members of this gene family in Drosophila, namely lim3, the homologue of the vertebrate Lhx3 and Lhx4 genes, and islet, the homologue of the vertebrate Isl1 and Is12 genes. We show that Drosophila lim3 is expressed by a specific subset of islet-expressing motor neurons and that mutating or misexpressing lim3 switches motor-neuron projections predictably. Our results provide evidence that lim3 and islet constitute a combinatorial code that generates distinct motor-neuron identities.

The mechanisms underlying the evolution of morphology are poorly understood1,2. Distantly related taxa sometimes exhibit correlations between morphological differences and patterns of gene expression3,4,5,6,7,8, but such comparisons cannot establish how mechanisms evolve to generate diverse morphologies. Answers to these questions require resolution of the nature of developmental evolution within and between closely related species. Here I show how the detailed regulation of the Hox gene Ultrabithorax patterns trichomes on the posterior femur of the second leg in Drosophila melanogaster, and that evolution of Ultrabithorax has contributed to divergence of this feature among closely related species. The cis-regulatory regions of Ultrabithorax, and not the protein itself, appear to have evolved. This study provides experimental evidence that cis-regulatory evolution is one way in which conserved proteins have promoted morphological diversity1.

The pattern of genetic variation across the genome of Drosophila melanogaster is consistent with the occurrence of frequent ‘selective sweeps’, in which new favourable mutations become incorporated into the species so quickly that linked alleles can ‘hitchhike’ and also become fixed1. Because of the hitchhiking of linked genes, it is generally difficult to identify the target of any putative selective sweep. Here, however, we identify a new gene in D. melanogaster that codes for a sperm-specific axonemal dynein subunit. The gene has a new testes-specific promoter derived from a protein-coding region in a gene encoding the cell-adhesion protein annexin X (AnnX), and it contains a new protein-coding exon derived from an intron in a gene encoding a cytoplasmic dynein intermediate chain (Cdic). The new transcription unit, designated Sdic (for sperm-specific dynein intermediate chain), has been duplicated about tenfold in a tandem array. Consistent with the selective sweep of this gene, the level of genetic polymorphism near Sdic is unusually low. The discovery of this gene supports other results that point to the rapid molecular evolution of male reproductive functions2,3,4.

The subcellular compartmentalization of signalling molecules helps to ensure the selective activation of different signal-transduction cascades within a single cell. Although there are many examples of compartmentalized signalling molecules, there are few examples of entire signalling cascades being organized as distinct signalling complexes. In Drosophila photoreceptors, the InaD protein, which consists of five PDZ domains, functions as a multivalent adaptor that brings together several components of the phototransduction cascade into a macromolecular complex. Here we study single-photon responses in several photoreceptor mutant backgrounds, and show that the InaD macromolecular complex is the unit of signalling that underlies elementary responses. We show that the localized activity of this signalling unit promotes reliable single-photon responses as well as rapid activation and feedback regulation. Finally, we use genetic and electrophysiological tools to illustrate how the assembly of signalling molecules into a transduction complex limits signal amplification in vivo.