Extent and context dependence of pleiotropy revealed by high-throughput single-cell phenotyping.

Pleiotropy-when a single mutation affects multiple traits-is a controversial topic with far-reaching implications. Pleiotropy plays a central role in debates about how complex traits evolve and whether biological systems are modular or are organized such that every gene has the potential to affect many traits. Pleiotropy is also critical to initiatives in evolutionary medicine that seek to trap infectious microbes or tumors by selecting for mutations that encourage growth in some conditions at the expense of others. Research in these fields, and others, would benefit from understanding the extent to which pleiotropy reflects inherent relationships among phenotypes that correlate no matter the perturbation (vertical pleiotropy). Alternatively, pleiotropy may result from genetic changes that impose correlations between otherwise independent traits (horizontal pleiotropy). We distinguish these possibilities by using clonal populations of yeast cells to quantify the inherent relationships between single-cell morphological features. Then, we demonstrate how often these relationships underlie vertical pleiotropy and how often these relationships are modified by genetic variants (quantitative trait loci [QTL]) acting via horizontal pleiotropy. Our comprehensive screen measures thousands of pairwise trait correlations across hundreds of thousands of yeast cells and reveals ample evidence of both vertical and horizontal pleiotropy. Additionally, we observe that the correlations between traits can change with the environment, genetic background, and cell-cycle position. These changing dependencies suggest a nuanced view of pleiotropy: biological systems demonstrate limited pleiotropy in any given context, but across contexts (e.g., across diverse environments and genetic backgrounds) each genetic change has the potential to influence a larger number of traits. Our method suggests that exploiting pleiotropy for applications in evolutionary medicine would benefit from focusing on traits with correlations that are less dependent on context.

Analysis of Epistasis in Natural Traits Using Model Organisms.

The ability to detect and understand epistasis in natural populations is important for understanding how biological traits are influenced by genetic variation. However, identification and characterization of epistasis in natural populations remains difficult due to statistical issues that arise as a result of multiple comparisons, and the fact that most genetic variants segregate at low allele frequencies. In this review, we discuss how model organisms may be used to manipulate genotypic combinations to power the detection of epistasis as well as test interactions between specific genes. Findings from a number of species indicate that statistical epistasis is pervasive between natural genetic variants. However, the properties of experimental systems that enable analysis of epistasis also constrain extrapolation of these results back into natural populations.

High-Temporal-Resolution smFISH Method for Gene Expression Studies in Caenorhabditis elegans Embryos.

Recent development in fluorescence-based molecular tools has contributed significantly to developmental studies, including embryogenesis. Many of these tools rely on multiple steps of sample manipulation, so obtaining large sample sizes presents a major challenge as it can be labor-intensive and time-consuming. However, large sample sizes are required to uncover critical aspects of embryogenesis, for example, subtle phenotypic differences or gene expression dynamics. This problem is particularly relevant for single-molecule fluorescence in situ hybridization (smFISH) studies in Caenorhabditis elegans embryogenesis. Microfluidics can help address this issue by allowing a large number of samples and parallelization of experiments. However, performing efficient reagent exchange on chip for large numbers of embryos remains a bottleneck. Here, we present a microfluidic pipeline for large-scale smFISH imaging of C. elegans embryos with minimized labor. We designed embryo traps and engineered a protocol allowing for efficient chemical exchange for hundreds of C. elegans embryos simultaneously. Furthermore, the device design and small footprint optimize imaging throughput by facilitating spatial registration and enabling minimal user input. We conducted the smFISH protocol on chip and demonstrated that image quality is preserved. With one device replacing the equivalent of 10 glass slides of embryos mounted manually, our microfluidic approach greatly increases throughput. Finally, to highlight the capability of our platform to perform longitudinal studies with high temporal resolution, we conducted a temporal analysis of par-1 gene expression in early C. elegans embryos. The method demonstrated here paves the way for systematic high-temporal-resolution studies that will benefit large-scale RNAi and drug screens and in systems beyond C. elegans embryos.

Cryptic genetic variation: evolution's hidden substrate.

Cryptic genetic variation (CGV) is invisible under normal conditions, but it can fuel evolution when circumstances change. In theory, CGV can represent a massive cache of adaptive potential or a pool of deleterious alleles that are in need of constant suppression. CGV emerges from both neutral and selective processes, and it may inform about how human populations respond to change. CGV facilitates adaptation in experimental settings, but does it have an important role in the real world? Here, we review the empirical support for widespread CGV in natural populations, including its potential role in emerging human diseases and the growing evidence of its contribution to evolution.

The many faces of pleiotropy.

Pleiotropy is the well-established phenomenon of a single gene affecting multiple traits. It has long played a central role in theoretical, experimental, and clinical research in genetics, development, molecular biology, evolution, and medicine. In recent years, genomic techniques have brought data to bear on fundamental questions about the nature and extent of pleiotropy. However, these efforts are plagued by conceptual difficulties derived from disparate meanings and interpretations of pleiotropy. Here, we describe distinct uses of the pleiotropy concept and explain the pitfalls associated with applying empirical data to them. We conclude that, for any question about the nature or extent of pleiotropy, the appropriate answer is always 'What do you mean?'.

A web application for gene-based queries of CaeNDR RNA-seq data.

Variation in gene expression is a feature of all living systems and has recently been characterized extensively among wild strains of the model organism Caenorhabditis elegans. To enable researchers to query gene expression and gene expression variation at any gene of interest, we have created a user-friendly web application that shares RNA-seq transcription data for 208 wild C. elegans strains generated by the Caenorhabditis Natural Diversity Resource (CaeNDR). Here, we describe the features of the web application and the details of the data and data processing underlying it. We hope that this website, wildworm.biosci.gatech.edu/cendrexp/ , will help C. elegans researchers better understand their favorite genes and strains.

Diversification of small RNA pathways underlies germline RNA interference incompetence in wild Caenorhabditis elegans strains.

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, the efficacy of exogenous RNA interference (RNAi) varies dramatically within the Caenorhabditis elegans natural population, raising questions about our understanding of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some wild strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others, the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains but dampens it further in others. Among wild strains, genes known to mediate RNAi exhibited very high expression variation relative to other genes in the genome as well as allelic divergence and strain-specific instances of pseudogenization at the sequence level. Our results demonstrate functional diversification in the small RNA pathways in C. elegans and suggest that RNAi processes are evolving rapidly and dynamically in nature.

Beyond the reference: gene expression variation and transcriptional response to RNAi in C. elegans.

A universal feature of living systems is that natural variation in genotype underpins variation in phenotype. Yet, research in model organisms is often constrained to a single genetic background, the reference strain. Further, genomic studies that do evaluate wild strains typically rely on the reference strain genome for read alignment, leading to the possibility of biased inferences based on incomplete or inaccurate mapping; the extent of reference bias can be difficult to quantify. As an intermediary between genome and organismal traits, gene expression is well positioned to describe natural variability across genotypes generally and in the context of environmental responses, which can represent complex adaptive phenotypes. C. elegans sits at the forefront of investigation into small-RNA gene regulatory mechanisms, or RNA interference (RNAi), and wild strains exhibit natural variation in RNAi competency following environmental triggers. Here, we examine how genetic differences among five wild strains affect the C. elegans transcriptome in general and after inducing RNAi responses to two germline target genes. Approximately 34% of genes were differentially expressed across strains; 411 genes were not expressed at all in at least one strain despite robust expression in others, including 49 genes not expressed in reference strain N2. Despite the presence of hyper-diverse hotspots throughout the C. elegans genome, reference mapping bias was of limited concern: over 92% of variably expressed genes were robust to mapping issues. Overall, the transcriptional response to RNAi was strongly strain-specific and highly specific to the target gene, and the laboratory strain N2 was not representative of the other strains. Moreover, the transcriptional response to RNAi was not correlated with RNAi phenotypic penetrance; the two germline RNAi incompetent strains exhibited substantial differential gene expression following RNAi treatment, indicating an RNAi response despite failure to reduce expression of the target gene. We conclude that gene expression, both generally and in response to RNAi, differs across C. elegans strains such that choice of strain may meaningfully influence scientific conclusions. To provide a public, easily accessible resource for querying gene expression variation in this dataset, we introduce an interactive website at https://wildworm.biosci.gatech.edu/rnai/ .

Beyond the reference: gene expression variation and transcriptional response to RNA interference in Caenorhabditis elegans.

Though natural systems harbor genetic and phenotypic variation, research in model organisms is often restricted to a reference strain. Focusing on a reference strain yields a great depth of knowledge but potentially at the cost of breadth of understanding. Furthermore, tools developed in the reference context may introduce bias when applied to other strains, posing challenges to defining the scope of variation within model systems. Here, we evaluate how genetic differences among 5 wild Caenorhabditis elegans strains affect gene expression and its quantification, in general and after induction of the RNA interference (RNAi) response. Across strains, 34% of genes were differentially expressed in the control condition, including 411 genes that were not expressed at all in at least 1 strain; 49 of these were unexpressed in reference strain N2. Reference genome mapping bias caused limited concern: despite hyperdiverse hotspots throughout the genome, 92% of variably expressed genes were robust to mapping issues. The transcriptional response to RNAi was highly strain- and target-gene-specific and did not correlate with RNAi efficiency, as the 2 RNAi-insensitive strains showed more differentially expressed genes following RNAi treatment than the RNAi-sensitive reference strain. We conclude that gene expression, generally and in response to RNAi, differs across C. elegans strains such that the choice of strain may meaningfully influence scientific inferences. Finally, we introduce a resource for querying gene expression variation in this dataset at https://wildworm.biosci.gatech.edu/rnai/.

A toxin-antidote selfish element increases fitness of its host.

Selfish genetic elements can promote their transmission at the expense of individual survival, creating conflict between the element and the rest of the genome. Recently, a large number of toxin-antidote (TA) post-segregation distorters have been identified in non-obligate outcrossing nematodes. Their origin and the evolutionary forces that keep them at intermediate population frequencies are poorly understood. Here, we study a TA element in Caenorhabditis elegans called zeel-1;peel-1. Two major haplotypes of this locus, with and without the selfish element, segregate in C. elegans. We evaluate the fitness consequences of the zeel-1;peel-1 element outside of its role in gene drive in non-outcrossing animals and demonstrate that loss of the toxin peel-1 decreased fitness of hermaphrodites and resulted in reductions in fecundity and body size. These findings suggest a biological role for peel-1 beyond toxin lethality. This work demonstrates that a TA element can provide a fitness benefit to its hosts either during their initial evolution or by being co-opted by the animals following their selfish spread. These findings guide our understanding on how TA elements can remain in a population where gene drive is minimized, helping resolve the mystery of prevalent TA elements in selfing animals.

Single-molecule FISH in C. elegans embryos reveals early embryonic expression dynamics of par-2 , lgl-1 and chin-1 and possible differences between hyper-diverged strains.

Wild C. elegans strains harbor natural variation in developmental pathways, but investigating these differences requires precise and well-powered phenotyping methods. Here we employ a microfluidics platform for single-molecule FISH to simultaneously visualize the transcripts of three genes in embryos of two distinct strains. We capture transcripts at high resolution by developmental stage in over one hundred embryos of each strain and observe wide-scale conservation of expression, but subtle differences in par-2 and chin-1 abundance and rate of change. As both genes reside in a genomic interval of hyper-divergence, these results may reflect consequences of pathway evolution over long timescales.

Caenorhabditis nematodes colonize ephemeral resource patches in neotropical forests.

Factors shaping the distribution and abundance of species include life-history traits, population structure, and stochastic colonization-extinction dynamics. Field studies of model species groups help reveal the roles of these factors. Species of Caenorhabditis nematodes are highly divergent at the sequence level but exhibit highly conserved morphology, and many of these species live in sympatry on microbe-rich patches of rotten material. Here, we use field experiments and large-scale opportunistic collections to investigate species composition, abundance, and colonization efficiency of Caenorhabditis species in two of the world's best-studied lowland tropical field sites: Barro Colorado Island in Panamá and La Selva in Sarapiquí, Costa Rica. We observed seven species of Caenorhabditis, four of them known only from these collections. We formally describe two species and place them within the Caenorhabditis phylogeny. While these localities contain species from many parts of the phylogeny, both localities were dominated by globally distributed androdiecious species. We found that Caenorhabditis individuals were able to colonize baits accessible only through phoresy and preferentially colonized baits that were in direct contact with the ground. We estimate the number of colonization events per patch to be low.

Identification of a candidate adaptive polymorphism for Drosophila life history by parallel independent clines on two continents.

Life history traits are critical components of fitness and frequently reflect adaptive responses to environmental pressures. However, few genes that contribute to natural life history variation have been identified. Insulin signalling mediates the determination of life history traits in many organisms, and single gene manipulation in Drosophila melanogaster suggests that individual genes in the pathway have the potential to produce major effects on these quantitative traits. We evaluated allelic variation at two insulin signalling genes, the Insulin-like Receptor (InR) and its substrate, chico, in natural populations of D. melanogaster. We found different patterns of variation: InR shows evidence of positive selection and clines in allele frequency across latitude; chico exhibits neutral patterns of evolution. The clinal patterns at InR are replicated between North America and Australia, showing striking similarity in the distribution of specific alleles and the rate at which allele frequencies change across latitude. Moreover, we identified a polymorphism at InR that appears to be functionally significant and consistent with hypothetical patterns of selection across geography. This polymorphism provides new characterization of genic regions of functionality within InR, and is likely a component in a suite of genes and traits that respond adaptively to climatic variation.

A portable, low-cost device for precise control of specimen temperature under stereomicroscopes.

To facilitate precise and convenient control of biological sample temperature, we developed a low-cost device that can be used independently or with any stereomicroscope. The purpose of the device is to control the thermal environment during experimental intervals in which a specimen must be manipulated outside of an incubator, e.g. for dissection or slide-mounting in preparation for imaging. Sample temperatures can be both cooled to below and heated to above room temperatures, and stably maintained at a precision of +/- 0.1˚C. To demonstrate the utility of this device, we report improved characterization of the penetrance of a short-acting temperature-sensitive allele in C. elegans embryos, and identification of the upper temperature threshold for embryonic viability for six Caenorhabditis species. By controlling the temperature environment even as a specimen is manipulated, this device offers consistency and flexibility, reduces environmental noise, and enables precision timing in experiments requiring temperature shifts.

Reproductive diapause and life-history clines in North American populations of Drosophila melanogaster.

Latitudinal clines are widespread in Drosophila melanogaster, and many have been interpreted as adaptive responses to climatic variation. However, the selective mechanisms generating many such patterns remain unresolved, and there is relatively little information regarding how basic life-history components such as fecundity, life span and mortality rates vary across environmental gradients. Here, it is shown that four life-history traits vary predictably with geographic origin of populations sampled along the latitudinal gradient in the eastern United States. Although such patterns are indicative of selection, they cannot distinguish between the direct action of selection on the traits in question or indirect selection by means of underlying genetic correlations. When independent suites of traits covary with geography, it is therefore critical to separate the widespread effects of population source from variation specifically for the traits under investigation. One trait that is associated with variation in life histories and also varies with latitude is the propensity to express reproductive diapause; diapause expression has been hypothesized as a mechanism by which D. melanogaster adults overwinter, and as such may be subject to strong selection in temperate habitats. In this study, recently derived isofemale lines were used to assess the relative contributions of population source and diapause genotype in generating the observed variance for life histories. It is shown that although life span, fecundity and mortality rates varied predictably with geography, diapause genotype explained the majority of the variance for these traits in the sampled populations. Both heat and cold shock resistance were also observed to vary predictably with latitude for the sampled populations. Cold shock tolerance varied between diapause genotypes and the magnitude of this difference varied with geography, whereas heat shock tolerance was affected solely by geographic origin of the populations. These data suggest that a subset of life-history parameters is significantly influenced by the genetic variance for diapause expression in natural populations, and that the observed variance for longevity and fecundity profiles may reflect indirect action of selection on diapause and other correlated traits.

Cryptic Genetic Variation in Evolutionary Developmental Genetics.

Evolutionary developmental genetics has traditionally been conducted by two groups: Molecular evolutionists who emphasize divergence between species or higher taxa, and quantitative geneticists who study variation within species. Neither approach really comes to grips with the complexities of evolutionary transitions, particularly in light of the realization from genome-wide association studies that most complex traits fit an infinitesimal architecture, being influenced by thousands of loci. This paper discusses robustness, plasticity and lability, phenomena that we argue potentiate major evolutionary changes and provide a bridge between the conceptual treatments of macro- and micro-evolution. We offer cryptic genetic variation and conditional neutrality as mechanisms by which standing genetic variation can lead to developmental system drift and, sheltered within canalized processes, may facilitate developmental transitions and the evolution of novelty. Synthesis of the two dominant perspectives will require recognition that adaptation, divergence, drift and stability all depend on similar underlying quantitative genetic processes-processes that cannot be fully observed in continuously varying visible traits.

Genetic variance for diapause expression and associated life histories in Drosophila melanogaster.

The dipteran Drosophila melanogaster can express a form of reproductive quiescence or diapause when exposed to low temperature and shortened photoperiod. Among natural populations in the eastern United States, the frequency of lines that express reproductive diapause in the laboratory varies substantially and predictably with latitudinal origin. The goals of the present study were twofold: (1) to examine the impact of genetic variance for diapause expression on multiple traits associated with organismal fitness; and (2) to evaluate the potential for fitness trade-offs between diapause and nondiapause phenotypes that may result in the observed cline. Even prior to diapause entry or expression, inbred lines that express and do not express reproductive diapause in laboratory assays were constitutively distinct for life span, age-specific mortality rates, fecundity profiles, resistance to cold and starvation stress, lipid content, development time, and egg-to-adult viability. Furthermore, estimates of genetic correlations based on line means revealed significant differentiation for genetic variance/covariance matrices between diapause and nondiapause lines. The data indicate the potential for life-history trade-offs associated with variation for the diapause phenotype. The observed cline in diapause incidence in the eastern United States may be generated by these tradeoffs and the associated spatial and/or temporal variation in relative fitness of these two phenotypes in natural populations.

Wild worm embryogenesis harbors ubiquitous polygenic modifier variation.

Embryogenesis is an essential and stereotypic process that nevertheless evolves among species. Its essentiality may favor the accumulation of cryptic genetic variation (CGV) that has no effect in the wild-type but that enhances or suppresses the effects of rare disruptions to gene function. Here, we adapted a classical modifier screen to interrogate the alleles segregating in natural populations of Caenorhabditis elegans: we induced gene knockdowns and used quantitative genetic methodology to examine how segregating variants modify the penetrance of embryonic lethality. Each perturbation revealed CGV, indicating that wild-type genomes harbor myriad genetic modifiers that may have little effect individually but which in aggregate can dramatically influence penetrance. Phenotypes were mediated by many modifiers, indicating high polygenicity, but the alleles tend to act very specifically, indicating low pleiotropy. Our findings demonstrate the extent of conditional functionality in complex trait architecture.

A highly pleiotropic amino acid polymorphism in the Drosophila insulin receptor contributes to life-history adaptation.

Finding the specific nucleotides that underlie adaptive variation is a major goal in evolutionary biology, but polygenic traits pose a challenge because the complex genotype-phenotype relationship can obscure the effects of individual alleles. However, natural selection working in large wild populations can shift allele frequencies and indicate functional regions of the genome. Previously, we showed that the two most common alleles of a complex amino acid insertion-deletion polymorphism in the Drosophila insulin receptor show independent, parallel clines in frequency across the North American and Australian continents. Here, we report that the cline is stable over at least a five-year period and that the polymorphism also demonstrates temporal shifts in allele frequency concurrent with seasonal change. We tested the alleles for effects on levels of insulin signaling, fecundity, development time, body size, stress tolerance, and life span. We find that the alleles are associated with predictable differences in these traits, consistent with patterns of Drosophila life-history variation across geography that likely reflect adaptation to the heterogeneous climatic environment. These results implicate insulin signaling as a major mediator of life-history adaptation in Drosophila, and suggest that life-history trade-offs can be explained by extensive pleiotropy at a single locus.