Genetic variation drives cancer cell adaptation to ECM stiffness.

The progression of many solid tumors is accompanied by temporal and spatial changes in the stiffness of the extracellular matrix (ECM). Cancer cells adapt to soft and stiff ECM through mechanisms that are not fully understood. It is well known that there is significant genetic heterogeneity from cell to cell in tumors, but how ECM stiffness as a parameter might interact with that genetic variation is not known. Here, we employed experimental evolution to study the response of genetically variable and clonal populations of tumor cells to variable ECM stiffness. Proliferation rates of genetically variable populations cultured on soft ECM increased over a period of several weeks, whereas clonal populations did not evolve. Tracking of DNA barcoded cell lineages revealed that soft ECM consistently selected for the same few variants. These data provide evidence that ECM stiffness exerts natural selection on genetically variable tumor populations. Soft-selected cells were highly migratory, with enriched oncogenic signatures and unusual behaviors such as spreading and traction force generation on ECMs with stiffness as low as 1 kPa. Rho-regulated cell spreading was found to be the directly selected trait, with yes-associated protein 1 translocation to the nucleus mediating fitness on soft ECM. Overall, these data show that genetic variation can drive cancer cell adaptation to ECM stiffness.

Direct inference of the distribution of fitness effects of spontaneous mutations from recombinant inbred Caenorhabditis elegans mutation accumulation lines.

The distribution of fitness effects of new mutations plays a central role in evolutionary biology. Estimates of the distribution of fitness effect from experimental mutation accumulation lines are compromised by the complete linkage disequilibrium between mutations in different lines. To reduce the linkage disequilibrium, we constructed 2 sets of recombinant inbred lines from a cross of 2 Caenorhabditis elegans mutation accumulation lines. One set of lines ("RIAILs") was intercrossed for 10 generations prior to 10 generations of selfing; the second set of lines ("RILs") omitted the intercrossing. Residual linkage disequilibrium in the RIAILs is much less than in the RILs, which affects the inferred distribution of fitness effect when the sets of lines are analyzed separately. The best-fit model estimated from all lines (RIAILs + RILs) infers a large fraction of mutations with positive effects (∼40%); models that constrain mutations to have negative effects fit much worse. The conclusion is the same using only the RILs. For the RIAILs, however, models that constrain mutations to have negative effects fit nearly as well as models that allow positive effects. When mutations in high linkage disequilibrium are pooled into haplotypes, the inferred distribution of fitness effect becomes increasingly negative-skewed and leptokurtic. We conclude that the conventional wisdom-most mutations have effects near 0, a handful of mutations have effects that are substantially negative, and mutations with positive effects are very rare-is likely correct, and that unless it can be shown otherwise, estimates of the distribution of fitness effect that infer a substantial fraction of mutations with positive effects are likely confounded by linkage disequilibrium.

Pervasive conservation of intron number and other genetic elements revealed by a chromosome-level genomic assembly of the hyper-polymorphic nematode Caenorhabditis brenneri.

With within-species genetic diversity estimates that span the gambit of that seen across the entirety of animals, the Caenorhabditis genus of nematodes holds unique potential to provide insights into how population size and reproductive strategies influence gene and genome organization and evolution. Our study focuses on Caenorhabditis brenneri, currently known as one of the most genetically diverse nematodes within its genus and metazoan phyla. Here, we present a high-quality gapless genome assembly and annotation for C. brenneri, revealing a common nematode chromosome arrangement characterized by gene-dense central regions and repeat rich peripheral parts. Comparison of C. brenneri with other nematodes from the 'Elegans' group revealed conserved macrosynteny but a lack of microsynteny, characterized by frequent rearrangements and low correlation iof orthogroup sizes, indicative of high rates of gene turnover. We also assessed genome organization within corresponding syntenic blocks in selfing and outcrossing species, affirming that selfing species predominantly experience loss of both genes and intergenic DNA. Comparison of gene structures revealed strikingly small number of shared introns across species, yet consistent distributions of intron number and length, regardless of population size or reproductive mode, suggesting that their evolutionary dynamics are primarily reflective of functional constraints. Our study provides valuable insights into genome evolution and expands the nematode genome resources with the highly genetically diverse C. brenneri, facilitating research into various aspects of nematode biology and evolutionary processes.

Variation in mutational (co)variances.

Because of pleiotropy, mutations affect the expression and inheritance of multiple traits and, together with selection, are expected to shape standing genetic covariances between traits and eventual phenotypic divergence between populations. It is therefore important to find if the M matrix, describing mutational variances of each trait and covariances between traits, varies between genotypes. We here estimate the M matrix for six locomotion behavior traits in lines of two genotypes of the nematode Caenorhabditis elegans that accumulated mutations in a nearly neutral manner for 250 generations. We find significant mutational variance along at least one phenotypic dimension of the M matrices, but neither their size nor their orientation had detectable differences between genotypes. The number of generations of mutation accumulation, or the number of MA lines measured, was likely insufficient to sample enough mutations and detect potentially small differences between the two M matrices. We then tested if the M matrices were similar to one G matrix describing the standing genetic (co)variances of a population derived by the hybridization of several genotypes, including the two measured for M, and domesticated to a lab-defined environment for 140 generations. We found that the M and G were different because the genetic covariances caused by mutational pleiotropy in the two genotypes are smaller than those caused by linkage disequilibrium in the lab population. We further show that M matrices differed in their alignment with the lab population G matrix. If generalized to other founder genotypes of the lab population, these observations indicate that selection does not shape the evolution of the M matrix for locomotion behavior in the short-term of a few tens to hundreds of generations and suggests that the hybridization of C. elegans genotypes allows selection on new phenotypic dimensions of locomotion behavior.

Phenotypic stasis with genetic divergence.

Whether or not genetic divergence in the short-term of tens to hundreds of generations is compatible with phenotypic stasis remains a relatively unexplored problem. We evolved predominantly outcrossing, genetically diverse populations of the nematode Caenorhabditis elegans under a constant and homogeneous environment for 240 generations and followed individual locomotion behavior. Although founders of lab populations show highly diverse locomotion behavior, during lab evolution, the component traits of locomotion behavior - defined as the transition rates in activity and direction - did not show divergence from the ancestral population. In contrast, transition rates' genetic (co)variance structure showed a marked divergence from the ancestral state and differentiation among replicate populations during the final 100 generations and after most adaptation had been achieved. We observe that genetic differentiation is a transient pattern during the loss of genetic variance along phenotypic dimensions under drift during the last 100 generations of lab evolution. These results suggest that short-term stasis of locomotion behavior is maintained because of stabilizing selection, while the genetic structuring of component traits is contingent upon drift history.

Mutation, selection, and the prevalence of the Caenorhabditis elegans heat-sensitive mortal germline phenotype.

Caenorhabditis elegans strains with the heat-sensitive mortal germline phenotype become progressively sterile over the course of a few tens of generations when maintained at temperatures near the upper range of C. elegans' tolerance. Mortal germline is transgenerationally heritable, and proximately under epigenetic control. Previous studies have suggested that mortal germline presents a relatively large mutational target and that mortal germline is not uncommon in natural populations of C. elegans. The mortal germline phenotype is not monolithic. Some strains exhibit a strong mortal germline phenotype, in which individuals invariably become sterile over a few generations, whereas other strains show a weaker (less penetrant) phenotype in which the onset of sterility is slower and more stochastic. We present results in which we (1) quantify the rate of mutation to the mortal germline phenotype and (2) quantify the frequency of mortal germline in a collection of 95 wild isolates. Over the course of ∼16,000 meioses, we detected one mutation to a strong mortal germline phenotype, resulting in a point estimate of the mutation rate UMrt≈ 6×10-5/genome/generation. We detected no mutations to a weak mortal germline phenotype. Six out of 95 wild isolates have a strong mortal germline phenotype, and although quantification of the weak mortal germline phenotype is inexact, the weak mortal germline phenotype is not rare in nature. We estimate a strength of selection against mutations conferring the strong mortal germline phenotype s¯≈0.1%, similar to selection against mutations affecting competitive fitness. The appreciable frequency of weak mortal germline variants in nature combined with the low mutation rate suggests that mortal germline may be maintained by balancing selection.

The distribution of mutational effects on fitness in Caenorhabditis elegans inferred from standing genetic variation.

The distribution of fitness effects (DFE) for new mutations is one of the most theoretically important but difficult to estimate properties in population genetics. A crucial challenge to inferring the DFE from natural genetic variation is the sensitivity of the site frequency spectrum to factors like population size change, population substructure, genome structure, and nonrandom mating. Although inference methods aim to control for population size changes, the influence of nonrandom mating remains incompletely understood, despite being a common feature of many species. We report the DFE estimated from 326 genomes of Caenorhabditis elegans, a nematode roundworm with a high rate of self-fertilization. We evaluate the robustness of DFE inferences using simulated data that mimics the genomic structure and reproductive life history of C. elegans. Our observations demonstrate how the combined influence of self-fertilization, genome structure, and natural selection on linked sites can conspire to compromise estimates of the DFE from extant polymorphisms with existing methods. These factors together tend to bias inferences toward weakly deleterious mutations, making it challenging to have full confidence in the inferred DFE of new mutations as deduced from standing genetic variation in species like C. elegans. Improved methods for inferring the DFE are needed to appropriately handle strong linked selection and selfing. These results highlight the importance of understanding the combined effects of processes that can bias our interpretations of evolution in natural populations.

Mutability of mononucleotide repeats, not oxidative stress, explains the discrepancy between laboratory-accumulated mutations and the natural allele-frequency spectrum in C. elegans.

Important clues about natural selection can be gleaned from discrepancies between the properties of segregating genetic variants and of mutations accumulated experimentally under minimal selection, provided the mutational process is the same in the laboratory as in nature. The base-substitution spectrum differs between C. elegans laboratory mutation accumulation (MA) experiments and the standing site-frequency spectrum, which has been argued to be in part owing to increased oxidative stress in the laboratory environment. Using genome sequence data from C. elegans MA lines carrying a mutation (mev-1) that increases the cellular titer of reactive oxygen species (ROS), leading to increased oxidative stress, we find the base-substitution spectrum is similar between mev-1, its wild-type progenitor (N2), and another set of MA lines derived from a different wild strain (PB306). Conversely, the rate of short insertions is greater in mev-1, consistent with studies in other organisms in which environmental stress increased the rate of insertion-deletion mutations. Further, the mutational properties of mononucleotide repeats in all strains are different from those of nonmononucleotide sequence, both for indels and base-substitutions, and whereas the nonmononucleotide spectra are fairly similar between MA lines and wild isolates, the mononucleotide spectra are very different, with a greater frequency of A:T → T:A transversions and an increased proportion of ±1-bp indels. The discrepancy in mutational spectra between laboratory MA experiments and natural variation is likely owing to a consistent (but unknown) effect of the laboratory environment that manifests itself via different modes of mutability and/or repair at mononucleotide loci.

Reverse Plasticity Underlies Rapid Evolution by Clonal Selection within Populations of Fibroblasts Propagated on a Novel Soft Substrate.

Mechanical properties such as substrate stiffness are a ubiquitous feature of a cell's environment. Many types of animal cells exhibit canonical phenotypic plasticity when grown on substrates of differing stiffness, in vitro and in vivo. Whether such plasticity is a multivariate optimum due to hundreds of millions of years of animal evolution, or instead is a compromise between conflicting selective demands, is unknown. We addressed these questions by means of experimental evolution of populations of mouse fibroblasts propagated for approximately 90 cell generations on soft or stiff substrates. The ancestral cells grow twice as fast on stiff substrate as on soft substrate and exhibit the canonical phenotypic plasticity. Soft-selected lines derived from a genetically diverse ancestral population increased growth rate on soft substrate to the ancestral level on stiff substrate and evolved the same multivariate phenotype. The pattern of plasticity in the soft-selected lines was opposite of the ancestral pattern, suggesting that reverse plasticity underlies the observed rapid evolution. Conversely, growth rate and phenotypes did not change in selected lines derived from clonal cells. Overall, our results suggest that the changes were the result of genetic evolution and not phenotypic plasticity per se. Whole-transcriptome analysis revealed consistent differentiation between ancestral and soft-selected populations, and that both emergent phenotypes and gene expression tended to revert in the soft-selected lines. However, the selected populations appear to have achieved the same phenotypic outcome by means of at least two distinct transcriptional architectures related to mechanotransduction and proliferation.

Short-term heritable variation overwhelms 200 generations of mutational variance for metabolic traits in Caenorhabditis elegans.

Metabolic disorders have a large heritable component, and have increased markedly in human populations over the past few generations. Genome-wide association studies of metabolic traits typically find a substantial unexplained fraction of total heritability, suggesting an important role of spontaneous mutation. An alternative explanation is that epigenetic effects contribute significantly to the heritable variation. Here, we report a study designed to quantify the cumulative effects of spontaneous mutation on adenosine metabolism in the nematode Caenorhabditis elegans, including both the activity and concentration of two metabolic enzymes and the standing pools of their associated metabolites. The only prior studies on the effects of mutation on metabolic enzyme activity, in Drosophila melanogaster, found that total enzyme activity presents a mutational target similar to that of morphological and life-history traits. However, those studies were not designed to account for short-term heritable effects. We find that the short-term heritable variance for most traits is of similar magnitude as the variance among MA lines. This result suggests that the potential heritable effects of epigenetic variation in metabolic disease warrant additional scrutiny.

Evolution: Environmental Dependence of the Mutational Process.

Environmental dependence of mutation in microbes is well-known, but most experiments have investigated contexts in which growth rate is greatly reduced below optimum. A new experiment shows mutational variability extends to contexts in which growth is near optimum.

Evolution of the Mutational Process under Relaxed Selection in Caenorhabditis elegans.

The mutational process varies at many levels, from within genomes to among taxa. Many mechanisms have been linked to variation in mutation, but understanding of the evolution of the mutational process is rudimentary. Physiological condition is often implicated as a source of variation in microbial mutation rate and may contribute to mutation rate variation in multicellular organisms.Deleterious mutations are an ubiquitous source of variation in condition. We test the hypothesis that the mutational process depends on the underlying mutation load in two groups of Caenorhabditis elegans mutation accumulation (MA) lines that differ in their starting mutation loads. "First-order MA" (O1MA) lines maintained under minimal selection for ∼250 generations were divided into high-fitness and low-fitness groups and sets of "second-order MA" (O2MA) lines derived from each O1MA line were maintained for ∼150 additional generations. Genomes of 48 O2MA lines and their progenitors were sequenced. There is significant variation among O2MA lines in base-substitution rate (µbs), but no effect of initial fitness; the indel rate is greater in high-fitness O2MA lines. Overall, µbs is positively correlated with recombination and proximity to short tandem repeats and negatively correlated with 10 bp and 1 kb GC content. However, probability of mutation is sufficiently predicted by the three-nucleotide motif alone. Approximately 90% of the variance in standing nucleotide variation is explained by mutability. Total mutation rate increased in the O2MA lines, as predicted by the "drift barrier" model of mutation rate evolution. These data, combined with experimental estimates of fitness, suggest that epistasis is synergistic.

Head-to-head comparison of three experimental methods of quantifying competitive fitness in C. elegans.

Organismal fitness is relevant in many contexts in biology. The most meaningful experimental measure of fitness is competitive fitness, when two or more entities (e.g., genotypes) are allowed to compete directly. In theory, competitive fitness is simple to measure: an experimental population is initiated with the different types in known proportions and allowed to evolve under experimental conditions to a predefined endpoint. In practice, there are several obstacles to obtaining robust estimates of competitive fitness in multicellular organisms, the most pervasive of which is simply the time it takes to count many individuals of different types from many replicate populations. Methods by which counting can be automated in high throughput are desirable, but for automated methods to be useful, the bias and technical variance associated with the method must be (a) known, and (b) sufficiently small relative to other sources of bias and variance to make the effort worthwhile. The nematode Caenorhabditis elegans is an important model organism, and the fitness effects of genotype and environmental conditions are often of interest. We report a comparison of three experimental methods of quantifying competitive fitness, in which wild-type strains are competed against GFP-marked competitors under standard laboratory conditions. Population samples were split into three replicates and counted (1) "by eye" from a saved image, (2) from the same image using CellProfiler image analysis software, and (3) with a large particle flow cytometer (a "worm sorter"). From 720 replicate samples, neither the frequency of wild-type worms nor the among-sample variance differed significantly between the three methods. CellProfiler and the worm sorter provide at least a tenfold increase in sample handling speed with little (if any) bias or increase in variance.

Network Architecture and Mutational Sensitivity of the C. elegans Metabolome.

A fundamental issue in evolutionary systems biology is understanding the relationship between the topological architecture of a biological network, such as a metabolic network, and the evolution of the network. The rate at which an element in a metabolic network accumulates genetic variation via new mutations depends on both the size of the mutational target it presents and its robustness to mutational perturbation. Quantifying the relationship between topological properties of network elements and the mutability of those elements will facilitate understanding the variation in and evolution of networks at the level of populations and higher taxa. We report an investigation into the relationship between two topological properties of 29 metabolites in the C. elegans metabolic network and the sensitivity of those metabolites to the cumulative effects of spontaneous mutation. The correlations between measures of network centrality and mutability are not statistically significant, but several trends point toward a weak positive association between network centrality and mutational sensitivity. There is a small but significant negative association between the mutational correlation of a pair of metabolites (rM ) and the shortest path length between those metabolites. Positive association between the centrality of a metabolite and its mutational heritability is consistent with centrally-positioned metabolites presenting a larger mutational target than peripheral ones, and is inconsistent with centrality conferring mutational robustness, at least in toto. The weakness of the correlation between rM and the shortest path length between pairs of metabolites suggests that network locality is an important but not overwhelming factor governing mutational pleiotropy. These findings provide necessary background against which the effects of other evolutionary forces, most importantly natural selection, can be interpreted.

The mutational decay of male-male and hermaphrodite-hermaphrodite competitive fitness in the androdioecious nematode C. elegans.

Androdioecious Caenorhabditis have a high frequency of self-compatible hermaphrodites and a low frequency of males. The effects of mutations on male fitness are of interest for two reasons. First, when males are rare, selection on male-specific mutations is less efficient than in hermaphrodites. Second, males may present a larger mutational target than hermaphrodites because of the different ways in which fitness accrues in the two sexes. We report the first estimates of male-specific mutational effects in an androdioecious organism. The rate of male-specific inviable or sterile mutations is ⩽5 × 10-4/generation, below the rate at which males would be lost solely due to those kinds of mutations. The rate of mutational decay of male competitive fitness is ~ 0.17%/generation; that of hermaphrodite competitive fitness is ~ 0.11%/generation. The point estimate of ~ 1.5X faster rate of mutational decay of male fitness is nearly identical to the same ratio in Drosophila. Estimates of mutational variance (VM) for male mating success and competitive fitness are not significantly different from zero, whereas VM for hermaphrodite competitive fitness is similar to that of non-competitive fitness. Two independent estimates of the average selection coefficient against mutations affecting hermaphrodite competitive fitness agree to within two-fold, 0.33-0.5%.

Experimental Evolution with Caenorhabditis Nematodes.

The hermaphroditic nematode Caenorhabditis elegans has been one of the primary model systems in biology since the 1970s, but only within the last two decades has this nematode also become a useful model for experimental evolution. Here, we outline the goals and major foci of experimental evolution with C. elegans and related species, such as C. briggsae and C. remanei, by discussing the principles of experimental design, and highlighting the strengths and limitations of Caenorhabditis as model systems. We then review three exemplars of Caenorhabditis experimental evolution studies, underlining representative evolution experiments that have addressed the: (1) maintenance of genetic variation; (2) role of natural selection during transitions from outcrossing to selfing, as well as the maintenance of mixed breeding modes during evolution; and (3) evolution of phenotypic plasticity and its role in adaptation to variable environments, including host-pathogen coevolution. We conclude by suggesting some future directions for which experimental evolution with Caenorhabditis would be particularly informative.

Considerations when choosing a genetic model organism for metabolomics studies.

Model organisms are important in many areas of chemical biology. In metabolomics, model organisms can provide excellent samples for methods development as well as the foundation of comparative phylometabolomics, which will become possible as metabolomics applications expand. Comparative studies of conserved and unique metabolic pathways will help in the annotation of metabolites as well as provide important new targets of investigation in biology and biomedicine. However, most chemical biologists are not familiar with genetics, which needs to be considered when choosing a model organism. In this review we summarize the strengths and weaknesses of several genetic systems, including natural isolates, recombinant inbred lines, and genetic mutations. We also discuss methods to detect targets of selection on the metabolome.

The mutational structure of metabolism in Caenorhabditis elegans.

A properly functioning organism must maintain metabolic homeostasis. Deleterious mutations degrade organismal function, presumably at least in part via effects on metabolic function. Here we present an initial investigation into the mutational structure of the Caenorhabditis elegans metabolome by means of a mutation accumulation experiment. We find that pool sizes of 29 metabolites vary greatly in their vulnerability to mutation, both in terms of the rate of accumulation of genetic variance (the mutational variance, VM) and the rate of change of the trait mean (the mutational bias, ΔM). Strikingly, some metabolites are much more vulnerable to mutation than any other trait previously studied in the same way. Although we cannot statistically assess the strength of mutational correlations between individual metabolites, principal component analysis provides strong evidence that some metabolite pools are genetically correlated, but also that there is substantial scope for independent evolution of different groups of metabolites. Averaged over mutation accumulation lines, PC3 is positively correlated with relative fitness, but a model in which metabolites are uncorrelated with fitness is nearly as good by Akaike's Information Criterion.

Mutation Is a Sufficient and Robust Predictor of Genetic Variation for Mitotic Spindle Traits in Caenorhabditis elegans.

Different types of phenotypic traits consistently exhibit different levels of genetic variation in natural populations. There are two potential explanations: Either mutation produces genetic variation at different rates or natural selection removes or promotes genetic variation at different rates. Whether mutation or selection is of greater general importance is a longstanding unresolved question in evolutionary genetics. We report mutational variances (VM) for 19 traits related to the first mitotic cell division in Caenorhabditis elegans and compare them to the standing genetic variances (VG) for the same suite of traits in a worldwide collection C. elegans Two robust conclusions emerge. First, the mutational process is highly repeatable: The correlation between VM in two independent sets of mutation accumulation lines is ∼0.9. Second, VM for a trait is a good predictor of VG for that trait: The correlation between VM and VG is ∼0.9. This result is predicted for a population at mutation-selection balance; it is not predicted if balancing selection plays a primary role in maintaining genetic variation.

Scaling, selection, and evolutionary dynamics of the mitotic spindle.

BACKGROUND: Cellular structures such as the nucleus, Golgi, centrioles, and spindle show remarkable diversity between species, but the mechanisms that produce these variations in cell biology are not known. RESULTS: Here we investigate the mechanisms that contribute to variations in morphology and dynamics of the mitotic spindle, which orchestrates chromosome segregation in all Eukaryotes and positions the division plane in many organisms. We use high-throughput imaging of the first division in nematodes to demonstrate that the measured effects of spontaneous mutations, combined with stabilizing selection on cell size, are sufficient to quantitatively explain both the levels of within-species variation in the spindle and its diversity over ∼100 million years of evolution. Furthermore, our finding of extensive within-species variation for the spindle demonstrates that there is not just one "wild-type" form, rather that cellular structures can exhibit a surprisingly broad diversity of naturally occurring behaviors. CONCLUSIONS: Our results argue that natural selection acts predominantly on cell size and indirectly influences the spindle through the scaling of the spindle with cell size. Previous studies have shown that the spindle also scales with cell size during early development. Thus, the scaling of the spindle with cell size controls its variation over both ontogeny and phylogeny.