Genome-wide association in Drosophila identifies a role for Piezo and Proc-R in sleep latency.

Sleep latency, the amount of time that it takes an individual to fall asleep, is a key indicator of sleep need. Sleep latency varies considerably both among and within species and is heritable, but lacks a comprehensive description of its underlying genetic network. Here we conduct a genome-wide association study of sleep latency. Using previously collected sleep and activity data on a wild-derived population of flies, we calculate sleep latency, confirming significant, heritable genetic variation for this complex trait. We identify 520 polymorphisms in 248 genes contributing to variability in sleep latency. Tests of mutations in 23 candidate genes and additional putative pan-neuronal knockdown of 9 of them implicated CG44153, Piezo, Proc-R and Rbp6 in sleep latency. Two large-effect mutations in the genes Proc-R and Piezo were further confirmed via genetic rescue. This work greatly enhances our understanding of the genetic factors that influence variation in sleep latency.

A conserved role for frizzled in sleep architecture.

Previous studies of natural variants in Drosophila melanogaster implicated the Wnt signaling receptor frizzled in sleep. Given that the Wnt signaling pathway is highly conserved across species, we hypothesized that frizzled class receptor 1 (Fzd1), the murine homolog of frizzled, would also have a role in sleep. Using a CRISPR transgenic approach, we removed most of the Fzd1 coding region from C57BL/6N mice. We used a video assay to measure sleep characteristics in Fzd1-deficient mice. As Wnt signaling is known to affect visuospatial memory, we also examined the impact of the deletion on learning and memory using the novel object recognition (NOR) paradigm. Fzd1-deficient mice had altered sleep compared to littermate controls. The mice did not respond differently to the NOR paradigm compared to controls but did display anxiety-like behavior. Our strategy demonstrates that the study of natural variation in Drosophila sleep translates into candidate genes for sleep in vertebrate species such as the mouse.

Nonlinear expression patterns and multiple shifts in gene network interactions underlie robust phenotypic change in Drosophila melanogaster selected for night sleep duration.

All but the simplest phenotypes are believed to result from interactions between two or more genes forming complex networks of gene regulation. Sleep is a complex trait known to depend on the system of feedback loops of the circadian clock, and on many other genes; however, the main components regulating the phenotype and how they interact remain an unsolved puzzle. Genomic and transcriptomic data may well provide part of the answer, but a full account requires a suitable quantitative framework. Here we conducted an artificial selection experiment for sleep duration with RNA-seq data acquired each generation. The phenotypic results are robust across replicates and previous experiments, and the transcription data provides a high-resolution, time-course data set for the evolution of sleep-related gene expression. In addition to a Hierarchical Generalized Linear Model analysis of differential expression that accounts for experimental replicates we develop a flexible Gaussian Process model that estimates interactions between genes. 145 gene pairs are found to have interactions that are different from controls. Our method appears to be not only more specific than standard correlation metrics but also more sensitive, finding correlations not significant by other methods. Statistical predictions were compared to experimental data from public databases on gene interactions. Mutations of candidate genes implicated by our results affected night sleep, and gene expression profiles largely met predicted gene-gene interactions.

Natural genetic variation in a dopamine receptor is associated with variation in female fertility in Drosophila melanogaster.

Fertility is a major component of fitness but its genetic architecture remains poorly understood. Using a full diallel cross of 50 Drosophila Genetic Reference Panel inbred lines with whole genome sequences, we found substantial genetic variation in fertility largely attributable to females. We mapped genes associated with variation in female fertility by genome-wide association analysis of common variants in the fly genome. Validation of candidate genes by RNAi knockdown confirmed the role of the dopamine 2-like receptor (Dop2R) in promoting egg laying. We replicated the Dop2R effect in an independently collected productivity dataset and showed that the effect of the Dop2R variant was mediated in part by regulatory gene expression variation. This study demonstrates the strong potential of genome-wide association analysis in this diverse panel of inbred strains and subsequent functional analyses for understanding the genetic architecture of fitness traits.

What have we learned about sleep from selective breeding strategies?

Selective breeding is a classic technique that enables an experimenter to modify a heritable target trait as desired. Direct selective breeding for extreme sleep and circadian phenotypes in flies successfully alters these behaviors, and sleep and circadian perturbations emerge as correlated responses to selection for other traits in mice, rats, and dogs. The application of sequencing technologies to the process of selective breeding identifies the genetic network impacting the selected trait in a holistic way. Breeding techniques preserve the extreme phenotypes generated during selective breeding, generating community resources for further functional testing. Selective breeding is thus a unique strategy that can explore the phenotypic limits of sleep and circadian behavior, discover correlated responses of traits having shared genetic architecture with the target trait, identify naturally-occurring genomic variants and gene expression changes that affect trait variability, and pinpoint genes with conserved roles.

Identification of Genes Contributing to a Long Circadian Period in Drosophila Melanogaster.

The endogenous circadian period of animals and humans is typically very close to 24 h. Individuals with much longer circadian periods have been observed, however, and in the case of humans, these deviations have health implications. Previously, we observed a line of Drosophila with a very long average period of 31.3 h for locomotor activity behavior. Preliminary mapping indicated that the long period did not map to known canonical clock genes but instead mapped to multiple chromosomes. Using RNA-Seq, we surveyed the whole transcriptome of fly heads from this line across time and compared it with a wild-type control. A three-way generalized linear model revealed that approximately two-thirds of the genes were expressed differentially among the two genotypes, while only one quarter of the genes varied across time. Using these results, we applied algorithms to search for genes that oscillated over 24 h, identifying genes not previously known to cycle. We identified 166 differentially expressed genes that overlapped with a previous Genome-wide Association Study (GWAS) of circadian behavior, strongly implicating them in the long-period phenotype. We tested mutations in 45 of these genes for their effect on the circadian period. Mutations in Alk, alph, CG10089, CG42540, CG6034, Kairos (CG6123), CG8768, klg, Lar, sick, and tinc had significant effects on the circadian period, with seven of these mutations increasing the circadian period of locomotor activity behavior. Genetic rescue of mutant Kairos restored the circadian period to wild-type levels, suggesting it has a critical role in determining period length in constant darkness.

Natural selection on sleep duration in Drosophila melanogaster.

Sleep is ubiquitous across animal species, but why it persists is not well understood. Here we observe natural selection act on Drosophila sleep by relaxing bi-directional artificial selection for extreme sleep duration for 62 generations. When artificial selection was suspended, sleep increased in populations previously selected for short sleep. Likewise, sleep decreased in populations previously selected for long sleep when artificial selection was relaxed. We measured the corresponding changes in the allele frequencies of genomic variants responding to artificial selection. The allele frequencies of these variants reversed course in response to relaxed selection, and for short sleepers, the changes exceeded allele frequency changes that would be expected under random genetic drift. These observations suggest that the variants are causal polymorphisms for sleep duration responding to natural selection pressure. These polymorphisms may therefore pinpoint the most important regions of the genome maintaining variation in sleep duration.

Evolution of Reproductive Behavior.

Behaviors associated with reproduction are major contributors to the evolutionary success of organisms and are subject to many evolutionary forces, including natural and sexual selection, and sexual conflict. Successful reproduction involves a range of behaviors, from finding an appropriate mate, courting, and copulation, to the successful production and (in oviparous animals) deposition of eggs following mating. As a consequence, behaviors and genes associated with reproduction are often under strong selection and evolve rapidly. Courtship rituals in flies follow a multimodal pattern, mediated through visual, chemical, tactile, and auditory signals. Premating behaviors allow males and females to assess the species identity, reproductive state, and condition of their partners. Conflicts between the "interests" of individual males, and/or between the reproductive strategies of males and females, often drive the evolution of reproductive behaviors. For example, seminal proteins transmitted by males often show evidence of rapid evolution, mediated by positive selection. Postmating behaviors, including the selection of oviposition sites, are highly variable and Drosophila species span the spectrum from generalists to obligate specialists. Chemical recognition features prominently in adaptation to host plants for feeding and oviposition. Selection acting on variation in pre-, peri-, and postmating behaviors can lead to reproductive isolation and incipient speciation. Response to selection at the genetic level can include the expansion of gene families, such as those for detecting pheromonal cues for mating, or changes in the expression of genes leading to visual cues such as wing spots that are assessed during mating. Here, we consider the evolution of reproductive behavior in Drosophila at two distinct, yet complementary, scales. Some studies take a microevolutionary approach, identifying genes and networks involved in reproduction, and then dissecting the genetics underlying complex behaviors in D. melanogaster Other studies take a macroevolutionary approach, comparing reproductive behaviors across the genus Drosophila and how these might correlate with environmental cues. A full synthesis of this field will require unification across these levels.

Short-Term Memory Deficits in the SLEEP Inbred Panel.

Although sleep is heritable and conserved across species, sleep duration varies from individual to individual. A shared genetic architecture between sleep duration and other evolutionarily important traits could explain this variability. Learning and memory are critical traits sharing a genetic architecture with sleep. We wanted to know whether learning and memory would be altered in extreme long or short sleepers. We therefore assessed the short-term learning and memory ability of flies from the Sleep Inbred Panel (SIP), a collection of 39 extreme long- and short-sleeping inbred lines of Drosophila. Neither long nor short sleepers had appreciable learning, in contrast to a moderate-sleeping control. We also examined the response of long and short sleepers to enriched social conditions, a paradigm previously shown to induce morphological changes in the brain. While moderate-sleeping control flies had increased daytime sleep and quantifiable increases in brain structures under enriched social conditions, flies of the Sleep Inbred Panel did not display these changes. The SIP thus emerges as an important model for the relationship between sleep and learning and memory.

Genome-Wide Association Study of Circadian Behavior in Drosophila melanogaster.

Circadian rhythms influence physiological processes from sleep-wake cycles to body temperature and are controlled by highly conserved cycling molecules. Although the mechanistic basis of the circadian clock has been known for decades, the extent to which circadian rhythms vary in nature and the underlying genetic basis for that variation is not well understood. We measured circadian period (Æ®) and rhythmicity index in the Drosophila Genetic Reference Panel (DGRP) and observed extensive genetic variation in both. Seven DGRP lines had sexually dimorphic arrhythmicity and one line had an exceptionally long Æ®. Genome-wide analyses identified 584 polymorphisms in 268 genes. We observed differences among transcripts for nine genes predicted to interact among themselves and canonical clock genes in the long period line and a control. Mutations/RNAi knockdown targeting these genes also affected circadian behavior. Our observations reveal that complex genetic interactions influence high levels of variation in circadian phenotypes.

The Sleep Inbred Panel, a Collection of Inbred Drosophila melanogaster with Extreme Long and Short Sleep Duration.

Understanding how genomic variation causes differences in observable phenotypes remains a major challenge in biology. It is difficult to trace the sequence of events originating from genomic variants to changes in transcriptional responses or protein modifications. Ideally, one would conduct experiments with individuals that are at either extreme of the trait of interest, but such resources are often not available. Further, advances in genome editing will enable testing of candidate polymorphisms individually and in combination. Here we have created a resource for the study of sleep with 39 inbred lines of Drosophila-the Sleep Inbred Panel (SIP). SIP lines have stable long- and short-sleeping phenotypes developed from naturally occurring polymorphisms. These lines are fully sequenced, enabling more accurate targeting for genome editing and transgenic constructs. This panel facilitates the study of intermediate transcriptional and proteomic correlates of sleep, and supports genome editing studies to verify polymorphisms associated with sleep duration.

Dosage-Dependent Expression Variation Suppressed on the Drosophila Male X Chromosome.

DNA copy number variation is associated with many high phenotypic heterogeneity disorders. We systematically examined the impact of Drosophila melanogaster deletions on gene expression profiles to ask whether increased expression variability owing to reduced gene dose might underlie this phenotypic heterogeneity. Indeed, we found that one-dose genes have higher gene expression variability relative to two-dose genes. We then asked whether this increase in variability could be explained by intrinsic noise within cells due to stochastic biochemical events, or whether expression variability is due to extrinsic noise arising from more complex interactions. Our modeling showed that intrinsic gene expression noise averages at the organism level and thus cannot explain increased variation in one-dose gene expression. Interestingly, expression variability was related to the magnitude of expression compensation, suggesting that regulation, induced by gene dose reduction, is noisy. In a remarkable exception to this rule, the single X chromosome of males showed reduced expression variability, even compared with two-dose genes. Analysis of sex-transformed flies indicates that X expression variability is independent of the male differentiation program. Instead, we uncovered a correlation between occupancy of the chromatin-modifying protein encoded by males absent on the first (mof) and expression variability, linking noise suppression to the specialized X chromosome dosage compensation system. MOF occupancy on autosomes in both sexes also lowered transcriptional noise. Our results demonstrate that gene dose reduction can lead to heterogeneous responses, which are often noisy. This has implications for understanding gene network regulatory interactions and phenotypic heterogeneity. Additionally, chromatin modification appears to play a role in dampening transcriptional noise.

Genotype Influences Day-to-Day Variability in Sleep in Drosophila melanogaster.

Patterns of sleep often vary among individuals. But sleep and activity may also vary within an individual, fluctuating in pattern across time. One possibility is that these daily fluctuations in sleep are caused by the underlying genotype of the individual. However, differences attributable to genetic causes are difficult to distinguish from environmental factors in outbred populations such as humans. We therefore employed Drosophila as a model of intra-individual variability in sleep using previously collected sleep and activity data from the Drosophila Genetic Reference Panel, a collection of wild-derived inbred lines. Individual flies had significant daily fluctuations in their sleep patterns, and these fluctuations were heritable. Using the standard deviation of sleep parameters as a metric, we conducted a genome-wide association study. We found 663 polymorphisms in 104 genes associated with daily fluctuations in sleep. We confirmed the effects of 12 candidate genes on the standard deviation of sleep parameters. Our results suggest that daily fluctuations in sleep patterns are due in part to gene activity.

Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep.

Why do some individuals need more sleep than others? Forward mutagenesis screens in flies using engineered mutations have established a clear genetic component to sleep duration, revealing mutants that convey very long or short sleep. Whether such extreme long or short sleep could exist in natural populations was unknown. We applied artificial selection for high and low night sleep duration to an outbred population of Drosophila melanogaster for 13 generations. At the end of the selection procedure, night sleep duration diverged by 9.97 hours in the long and short sleeper populations, and 24-hour sleep was reduced to 3.3 hours in the short sleepers. Neither long nor short sleeper lifespan differed appreciably from controls, suggesting little physiological consequences to being an extreme long or short sleeper. Whole genome sequence data from seven generations of selection revealed several hundred thousand changes in allele frequencies at polymorphic loci across the genome. Combining the data from long and short sleeper populations across generations in a logistic regression implicated 126 polymorphisms in 80 candidate genes, and we confirmed three of these genes and a larger genomic region with mutant and chromosomal deficiency tests, respectively. Many of these genes could be connected in a single network based on previously known physical and genetic interactions. Candidate genes have known roles in several classic, highly conserved developmental and signaling pathways-EGFR, Wnt, Hippo, and MAPK. The involvement of highly pleiotropic pathway genes suggests that sleep duration in natural populations can be influenced by a wide variety of biological processes, which may be why the purpose of sleep has been so elusive.

The Genetic Architecture of Ovariole Number in Drosophila melanogaster: Genes with Major, Quantitative, and Pleiotropic Effects.

Ovariole number has a direct role in the number of eggs produced by an insect, suggesting that it is a key morphological fitness trait. Many studies have documented the variability of ovariole number and its relationship to other fitness and life-history traits in natural populations of Drosophila However, the genes contributing to this variability are largely unknown. Here, we conducted a genome-wide association study of ovariole number in a natural population of flies. Using mutations and RNAi-mediated knockdown, we confirmed the effects of 24 candidate genes on ovariole number, including a novel gene, anneboleyn (formerly CG32000), that impacts both ovariole morphology and numbers of offspring produced. We also identified pleiotropic genes between ovariole number traits and sleep and activity behavior. While few polymorphisms overlapped between sleep parameters and ovariole number, 39 candidate genes were nevertheless in common. We verified the effects of seven genes on both ovariole number and sleep: bin3, blot, CG42389, kirre, slim, VAChT, and zfh1 Linkage disequilibrium among the polymorphisms in these common genes was low, suggesting that these polymorphisms may evolve independently.

Microenvironmental Gene Expression Plasticity Among Individual Drosophila melanogaster.

Differences in phenotype among genetically identical individuals exposed to the same environmental condition are often noted in genetic studies. Despite this commonplace observation, little is known about the causes of this variability, which has been termed microenvironmental plasticity. One possibility is that stochastic or technical sources of variance produce these differences. A second possibility is that this variation has a genetic component. We have explored gene expression robustness in the transcriptomes of 730 individual Drosophila melanogaster of 16 fixed genotypes, nine of which are infected with Wolbachia Three replicates of flies were grown, controlling for food, day/night cycles, humidity, temperature, sex, mating status, social exposure, and circadian timing of RNA extraction. Despite the use of inbred genotypes, and carefully controlled experimental conditions, thousands of genes were differentially expressed, revealing a unique and dynamic transcriptional signature for each individual fly. We found that 23% of the transcriptome was differentially expressed among individuals, and that the variability in gene expression among individuals is influenced by genotype. This transcriptional variation originated from specific gene pathways, suggesting a plastic response to the microenvironment; but there was also evidence of gene expression differences due to stochastic fluctuations. These observations reveal previously unappreciated genetic sources of variability in gene expression among individuals, which has implications for complex trait genetics and precision medicine.

Knockdown expression of Syndecan in the fat body impacts nutrient metabolism and the organismal response to environmental stresses in Drosophila melanogaster.

The heparan sulfate proteoglycan syndecans are transmembrane proteins involved in multiple physiological processes, including cell-matrix adhesion and inflammation. Recent evidence from model systems and humans suggest that syndecans have a role in energy balance and nutrient metabolism regulation. However, much remains to be learned about the mechanisms through which syndecans influence these phenotypes. Previously, we reported that Drosophila melanogaster Syndecan (Sdc) mutants had reduced metabolic activity compared to controls. Here, we knocked down endogenous Sdc expression in the fat body (the functional equivalent of mammalian adipose tissue and liver) to investigate whether the effects on metabolism originate from this tissue. We found that knocking down Sdc in the fat body leads to flies with higher levels of glycogen and fat and that survive longer during starvation, likely due to their extra energy reserves and an increase in gluconeogenesis. However, compared to control flies, they are also more sensitive to environmental stresses (e.g. bacterial infection and cold) and have reduced metabolic activity under normal feeding conditions. Under the same conditions, fat-body Sdc reduction enhances expression of genes involved in glyceroneogenesis and gluconeogenesis and induces a drastic decrease in phosphorylation levels of AKT and extracellular signal regulated kinase 1/2 (ERK1/2). Altogether, these findings strongly suggest that Drosophila fat body Sdc is involved in a mechanism that shifts resources to different physiological functions according to nutritional status.