Experimental Evaluation of a Direct Fitness Effect of the De Novo Evolved Mouse Gene Pldi.

De novo evolved genes emerge from random parts of noncoding sequences and have, therefore, no homologs from which a function could be inferred. While expression analysis and knockout experiments can provide insights into the function, they do not directly test whether the gene is beneficial for its carrier. Here, we have used a seminatural environment experiment to test the fitness of the previously identified de novo evolved mouse gene Pldi, which has been implicated to have a role in sperm differentiation. We used a knockout mouse strain for this gene and competed it against its parental wildtype strain for several generations of free reproduction. We found that the knockout (ko) allele frequency decreased consistently across three replicates of the experiment. Using an approximate Bayesian computation framework that simulated the data under a demographic scenario mimicking the experiment's demography, we could estimate a selection coefficient ranging between 0.21 and 0.61 for the wildtype allele compared to the ko allele in males, under various models. This implies a relatively strong selective advantage, which would fix the new gene in less than hundred generations after its emergence.

Testing the accuracy of 3D automatic landmarking via genome-wide association studies.

Various advances in 3D automatic phenotyping and landmark-based geometric morphometric methods have been made. While it is generally accepted that automatic landmarking compromises the capture of the biological variation, no studies have directly tested the actual impact of such landmarking approaches in analyses requiring a large number of specimens and for which the precision of phenotyping is crucial to extract an actual biological signal adequately. Here, we use a recently developed 3D atlas-based automatic landmarking method to test its accuracy in detecting QTLs associated with craniofacial development of the house mouse skull and lower jaws for a large number of specimens (circa 700) that were previously phenotyped via a semiautomatic landmarking method complemented with manual adjustment. We compare both landmarking methods with univariate and multivariate mapping of the skull and the lower jaws. We find that most significant SNPs and QTLs are not recovered based on the data derived from the automatic landmarking method. Our results thus confirm the notion that information is lost in the automated landmarking procedure although somewhat dependent on the analyzed structure. The automatic method seems to capture certain types of structures slightly better, such as lower jaws whose shape is almost entirely summarized by its outline and could be assimilated as a 2D flat object. By contrast, the more apparent 3D features exhibited by a structure such as the skull are not adequately captured by the automatic method. We conclude that using 3D atlas-based automatic landmarking methods requires careful consideration of the experimental question.

Tracing the Origin and Evolutionary Fate of Recent Gene Retrocopies in Natural Populations of the House Mouse.

Although the contribution of retrogenes to the evolution of genes and genomes has long been recognized, the evolutionary patterns of very recently derived retrocopies that are still polymorphic within natural populations have not been much studied so far. We use here a set of 2,025 such retrocopies in nine house mouse populations from three subspecies (Mus musculus domesticus, M. m. musculus, and M. m. castaneus) to trace their origin and evolutionary fate. We find that ancient house-keeping genes are significantly more likely to generate retrocopies than younger genes and that the propensity to generate a retrocopy depends on its level of expression in the germline. Although most retrocopies are detrimental and quickly purged, we focus here on the subset that appears to be neutral or even adaptive. We show that retrocopies from X-chromosomal parental genes have a higher likelihood to reach elevated frequencies in the populations, confirming the notion of adaptive effects for "out-of-X" retrogenes. Also, retrocopies in intergenic regions are more likely to reach higher population frequencies than those in introns of genes, implying a more detrimental effect when they land within transcribed regions. For a small subset of retrocopies, we find signatures of positive selection, indicating they were involved in a recent adaptation process. We show that the population-specific distribution pattern of retrocopies is phylogenetically informative and can be used to infer population history with a better resolution than with SNP markers.

The Effects of Sequence Length and Composition of Random Sequence Peptides on the Growth of E. coli Cells.

We study the potential for the de novo evolution of genes from random nucleotide sequences using libraries of E. coli expressing random sequence peptides. We assess the effects of such peptides on cell growth by monitoring frequency changes in individual clones in a complex library through four serial passages. Using a new analysis pipeline that allows the tracing of peptides of all lengths, we find that over half of the peptides have consistent effects on cell growth. Across nine different experiments, around 16% of clones increase in frequency and 36% decrease, with some variation between individual experiments. Shorter peptides (8-20 residues), are more likely to increase in frequency, longer ones are more likely to decrease. GC content, amino acid composition, intrinsic disorder, and aggregation propensity show slightly different patterns between peptide groups. Sequences that increase in frequency tend to be more disordered with lower aggregation propensity. This coincides with the observation that young genes with more disordered structures are better tolerated in genomes. Our data indicate that random sequences can be a source of evolutionary innovation, since a large fraction of them are well tolerated by the cells or can provide a growth advantage.

Frequent lineage-specific substitution rate changes support an episodic model for protein evolution.

Since the inception of the molecular clock model for sequence evolution, the investigation of protein divergence has revolved around the question of a more or less constant change of amino acid sequences, with specific overall rates for each family. Although anomalies in clock-like divergence are well known, the assumption of a constant decay rate for a given protein family is usually taken as the null model for protein evolution. However, systematic tests of this null model at a genome-wide scale have lagged behind, despite the databases' enormous growth. We focus here on divergence rate comparisons between very closely related lineages since this allows clear orthology assignments by synteny and reliable alignments, which are crucial for determining substitution rate changes. We generated a high-confidence dataset of syntenic orthologs from four ape species, including humans. We find that despite the appearance of an overall clock-like substitution pattern, several hundred protein families show lineage-specific acceleration and deceleration in divergence rates, or combinations of both in different lineages. Hence, our analysis uncovers a rather dynamic history of substitution rate changes, even between these closely related lineages, implying that one should expect that a large fraction of proteins will have had a history of episodic rate changes in deeper phylogenies. Furthermore, each of the lineages has a separate set of particularly fast diverging proteins. The genes with the highest percentage of branch-specific substitutions are ADCYAP1 in the human lineage (9.7%), CALU in chimpanzees (7.1%), SLC39A14 in the internal branch leading to humans and chimpanzees (4.1%), RNF128 in gorillas (9%), and S100Z in gibbons (15.2%). The mutational pattern in ADCYAP1 suggests a biased mutation process, possibly through asymmetric gene conversion effects. We conclude that a null model of constant change can be problematic for predicting the evolutionary trajectories of individual proteins.

A humanized version of Foxp2 affects ultrasonic vocalization in adult female and male mice.

The transcription factor FoxP2 is involved in setting up the neuronal circuitry for vocal learning in mammals and birds and is thought to have played a special role in the evolution of human speech and language. It has been shown that an allele with a humanized version of the murine Foxp2 gene changes the ultrasonic vocalization of mouse pups compared to pups of the wild-type inbred strain. Here we tested if this humanized allele would also affect the ultrasonic vocalization of adult female and male mice. In a previous study, in which only male vocalization was considered and the mice were recorded under a restricted spatial and temporal regime, no difference in adult vocalization between genotypes was found. Here, we use a different test paradigm in which both female and male vocalizations are recorded in extended social contact. We found differences in temporal, spectral and syntactical parameters between the genotypes in both sexes, and between sexes. Mice carrying the humanized Foxp2 allele were using higher frequencies and more complex syllable types than mice of the corresponding wildtype inbred strain. Our results support the notion that the humanized Foxp2 allele has a differential effect on mouse ultrasonic vocalization. As mice carrying the humanized version of the Foxp2 gene show effects opposite to those of mice carrying disrupted or mutated alleles of this gene, we conclude that this mouse line represents an important model for the study of human speech and language evolution.

Natural copy number variation of tandemly repeated regulatory SNORD RNAs leads to individual phenotypic differences in mice.

Genic copy number differences can have phenotypic consequences, but so far this has not been studied in detail in natural populations. Here, we analysed the natural variation of two families of tandemly repeated regulatory small nucleolar RNAs (SNORD115 and SNORD116) in the house mouse (Mus musculus). They are encoded within the Prader-Willi Syndrome gene region, known to be involved in behavioural, metabolic, and osteogenic functions in mammals. We determined that the copy numbers of these SNORD RNAs show substantial natural variation, both in wild-derived mice as well as in an inbred mouse strain (C57BL/6J). We show that copy number differences are subject to change across generations, making them highly variable and resulting in individual differences. In transcriptome data from brain samples, we found SNORD copy-number correlated regulation of possible target genes, including Htr2c, a predicted target gene of SNORD115, as well as Ankrd11, a predicted target gene of SNORD116. Ankrd11 is a chromatin regulator, which has previously been implicated in regulating the development of the skull. Based on morphometric shape analysis of the skulls of individual mice of the inbred strain, we show that shape measures correlate with SNORD116 copy numbers in the respective individuals. Our results suggest that the variable dosage of regulatory RNAs can lead to phenotypic variation between individuals that would typically have been ascribed to environmentally induced variation, while it is actually encoded in individual differences of copy numbers of regulatory molecules.

The imprinted lncRNA Peg13 regulates sexual preference and the sex-specific brain transcriptome in mice.

Mammalian genomes include many maternally and paternally imprinted genes. Most of these are also expressed in the brain, and several have been implicated in regulating specific behavioral traits. Here, we have used a knockout approach to study the function of Peg13, a gene that codes for a fast-evolving lncRNA (long noncoding RNA) and is part of a complex of imprinted genes on chromosome 15 in mice and chromosome 8 in humans. Mice lacking the 3' half of the transcript look morphologically wild-type but show distinct behavioral differences. They lose interest in the opposite sex, instead displaying a preference for wild-type animals of the same sex. Further, they show a higher level of anxiety, lowered activity and curiosity, and a deficiency in pup retrieval behavior. Brain RNA expression analysis reveals that genes involved in the serotonergic system, formation of glutamatergic synapses, olfactory processing, and estrogen signaling-as well as more than half of the other known imprinted genes-show significant expression changes in Peg13-deficient mice. Intriguingly, these pathways are differentially affected in the sexes, resulting in male and female brains of Peg13-deficient mice differing more from each other than those of wild-type mice. We conclude that Peg13 is part of a developmental pathway that regulates the neurobiology of social and sexual interactions.

The mutational load in natural populations is significantly affected by high primary rates of retroposition.

Gene retroposition is known to contribute to patterns of gene evolution and adaptations. However, possible negative effects of gene retroposition remain largely unexplored since most previous studies have focused on between-species comparisons where negatively selected copies are mostly not observed, as they are quickly lost from populations. Here, we show for natural house mouse populations that the primary rate of retroposition is orders of magnitude higher than the long-term rate. Comparisons with single-nucleotide polymorphism distribution patterns in the same populations show that most retroposition events are deleterious. Transcriptomic profiling analysis shows that new retroposed copies become easily subject to transcription and have an influence on the expression levels of their parental genes, especially when transcribed in the antisense direction. Our results imply that the impact of retroposition on the mutational load has been highly underestimated in natural populations. This has additional implications for strategies of disease allele detection in humans.

Independent evolution toward larger body size in the distinctive Faroe Island mice.

Most phenotypic traits in nature involve the collective action of many genes. Traits that evolve repeatedly are particularly useful for understanding how selection may act on changing trait values. In mice, large body size has evolved repeatedly on islands and under artificial selection in the laboratory. Identifying the loci and genes involved in this process may shed light on the evolution of complex, polygenic traits. Here, we have mapped the genetic basis of body size variation by making a genetic cross between mice from the Faroe Islands, which are among the largest and most distinctive natural populations of mice in the world, and a laboratory mouse strain selected for small body size, SM/J. Using this F2 intercross of 841 animals, we have identified 111 loci controlling various aspects of body size, weight and growth hormone levels. By comparing against other studies, including the use of a joint meta-analysis, we found that the loci involved in the evolution of large size in the Faroese mice were largely independent from those of a different island population or other laboratory strains. We hypothesize that colonization bottleneck, historical hybridization, or the redundancy between multiple loci have resulted in the Faroese mice achieving an outwardly similar phenotype through a distinct evolutionary path.

Testing Implications of the Omnigenic Model for the Genetic Analysis of Loci Identified through Genome-wide Association.

Organismal phenotypes usually have a quantitative distribution, and their genetic architecture can be studied by genome-wide association (GWA) mapping approaches. In most of such studies, it has become clear that many genes of moderate or small effects contribute to the phenotype.1-4 Hence, the attention has turned toward the loci falling below the GWA cut-off, which may contribute to the phenotype through modifier interactions with a set of core genes, as proposed in the omnigenic model.5 One can thus predict that both moderate effect GWA-derived candidate genes and randomly chosen genes should have a similar likelihood to affect a given phenotype when they are analyzed via gene disruption assays. We have tested this hypothesis by using an automated phenotyping system for Drosophila pupal phenotypes.6,7 We first identified candidate genes for pupal length in a GWA based on the Drosophila Genetic Reference Panel (DGRP)8,9 and showed that most of these candidate genes are indeed involved in the phenotype. We then randomly chose genes below a GWA significance threshold and found that three-quarters of them had also an effect on the trait with comparable effect sizes as the GWA candidate genes. We further tested the effects of these knockout lines on an independent behavioral pupal trait (pupation site choice) and found that a similar fraction had a significant effect as well. Our data thus confirm the implication that a large number of genes can influence independent quantitative traits.

Effects of the Expression of Random Sequence Clones on Growth and Transcriptome Regulation in Escherichia coli.

Comparative genomic analyses have provided evidence that new genetic functions can emerge out of random nucleotide sequences. Here, we apply a direct experimental approach to study the effects of plasmids harboring random sequence inserts under the control of an inducible promoter. Based on data from previously described experiments dealing with the growth of clones within whole libraries, we extracted specific clones that had shown either negative, neutral or positive effects on relative cell growth. We analyzed these individually with respect to growth characteristics and the impact on the transcriptome. We find that candidate clones for negative peptides lead to growth arrest by eliciting a general stress response. Overexpression of positive clones, on the other hand, does not change the exponential growth rates of hosts, and they show a growth advantage over a neutral clone when tested in direct competition experiments. Transcriptomic changes in positive clones are relatively moderate and specific to each clone. We conclude from our experiments that random sequence peptides are indeed a suitable source for the de novo evolution of genetic functions.

Evolution of a New Testis-Specific Functional Promoter Within the Highly Conserved Map2k7 Gene of the Mouse.

Map2k7 (synonym Mkk7) is a conserved regulatory kinase gene and a central component of the JNK signaling cascade with key functions during cellular differentiation. It shows complex transcription patterns, and different transcript isoforms are known in the mouse (Mus musculus). We have previously identified a newly evolved testis-specific transcript for the Map2k7 gene in the subspecies M. m. domesticus. Here, we identify the new promoter that drives this transcript and find that it codes for an open reading frame (ORF) of 50 amino acids. The new promoter was gained in the stem lineage of closely related mouse species but was secondarily lost in the subspecies M. m. musculus and M. m. castaneus. A single mutation can be correlated with its transcriptional activity in M. m. domesticus, and cell culture assays demonstrate the capability of this mutation to drive expression. A mouse knockout line in which the promoter region of the new transcript is deleted reveals a functional contribution of the newly evolved promoter to sperm motility and the spermatid transcriptome. Our data show that a new functional transcript (and possibly protein) can evolve within an otherwise highly conserved gene, supporting the notion of regulatory changes contributing to the emergence of evolutionary novelties.

Inbred lab mice are not isogenic: genetic variation within inbred strains used to infer the mutation rate per nucleotide site.

For over a century, inbred mice have been used in many areas of genetics research to gain insight into the genetic variation underlying traits of interest. The generalizability of any genetic research study in inbred mice is dependent upon all individual mice being genetically identical, which in turn is dependent on the breeding designs of companies that supply inbred mice to researchers. Here, we compare whole-genome sequences from individuals of four commonly used inbred strains that were procured from either the colony nucleus or from a production colony (which can be as many as ten generations removed from the nucleus) of a large commercial breeder, in order to investigate the extent and nature of genetic variation within and between individuals. We found that individuals within strains are not isogenic, and there are differences in the levels of genetic variation that are explained by differences in the genetic distance from the colony nucleus. In addition, we employ a novel approach to mutation rate estimation based on the observed genetic variation and the expected site frequency spectrum at equilibrium, given a fully inbred breeding design. We find that it provides a reasonable per nucleotide mutation rate estimate when mice come from the colony nucleus (~7.9 × 10-9 in C3H/HeN), but substantially inflated estimates when mice come from production colonies.

Dedicated transcriptomics combined with power analysis lead to functional understanding of genes with weak phenotypic changes in knockout lines.

Systematic knockout studies in mice have shown that a large fraction of the gene replacements show no lethal or other overt phenotypes. This has led to the development of more refined analysis schemes, including physiological, behavioral, developmental and cytological tests. However, transcriptomic analyses have not yet been systematically evaluated for non-lethal knockouts. We conducted a power analysis to determine the experimental conditions under which even small changes in transcript levels can be reliably traced. We have applied this to two gene disruption lines of genes for which no function was known so far. Dedicated phenotyping tests informed by the tissues and stages of highest expression of the two genes show small effects on the tested phenotypes. For the transcriptome analysis of these stages and tissues, we used a prior power analysis to determine the number of biological replicates and the sequencing depth. We find that under these conditions, the knockouts have a significant impact on the transcriptional networks, with thousands of genes showing small transcriptional changes. GO analysis suggests that A930004D18Rik is involved in developmental processes through contributing to protein complexes, and A830005F24Rik in extracellular matrix functions. Subsampling analysis of the data reveals that the increase in the number of biological replicates was more important that increasing the sequencing depth to arrive at these results. Hence, our proof-of-principle experiment suggests that transcriptomic analysis is indeed an option to study gene functions of genes with weak or no traceable phenotypic effects and it provides the boundary conditions under which this is possible.

The amylase gene cluster in house mice (Mus musculus) was subject to repeated introgression including the rescue of a pseudogene.

BACKGROUND: Amylase gene clusters have been implicated in adaptive copy number changes in response to the amount of starch in the diet of humans and mammals. However, this interpretation has been questioned for humans and for mammals there is a paucity of information from natural populations. RESULTS: Using optical mapping and genome read information, we show here that the amylase cluster in natural house mouse populations is indeed copy-number variable for Amy2b paralogous gene copies (called Amy2a1 - Amy2a5), but a direct connection to starch diet is not evident. However, we find that the amylase cluster was subject to introgression of haplotypes between Mus musculus sub-species. A very recent introgression can be traced in the Western European populations and this leads also to the rescue of an Amy2b pseudogene. Some populations and inbred lines derived from the Western house mouse (Mus musculus domesticus) harbor a copy of the pancreatic amylase (Amy2b) with a stop codon in the first exon, making it non-functional. But populations in France harbor a haplotype introgressed from the Eastern house mouse (M. m. musculus) with an intact reading frame. Detailed analysis of phylogenetic patterns along the amylase cluster suggest an additional history of previous introgressions. CONCLUSIONS: Our results show that the amylase gene cluster is a hotspot of introgression in the mouse genome, making it an evolutionary active region beyond the previously observed copy number changes.

Population Genomics of the House Mouse and the Brown Rat.

Mice (Mus musculus) and rats (Rattus norvegicus) have long served as model systems for biomedical research. However, they are also excellent models for studying the evolution of populations, subspecies, and species. Within the past million years, they have spread in various waves across large parts of the globe, with the most recent spread in the wake of human civilization. They have developed into commensal species, but have also been able to colonize extreme environments on islands free of human civilization. Given that ample genomic and genetic resources are available for these species, they have thus also become ideal mammalian systems for evolutionary studies on adaptation and speciation, particularly in the combination with the rapid developments in population genomics. The chapter provides an overview of the systems and their history, as well as of available resources.

Identification of a genetic network for an ecologically relevant behavioural phenotype in Drosophila melanogaster.

Pupation site choice of Drosophila third-instar larvae is critical for the survival of individuals, as pupae are exposed to various biotic and abiotic dangers while immobilized during the 3-4 days of metamorphosis. This singular behavioural choice is sensitive to both environmental and genetic factors. Here, we developed a high-throughput phenotyping approach to assay the variation in pupation height in Drosophila melanogaster, while controlling for possibly confounding factors. We find substantial variation of mean pupation height among sampled natural stocks and we show that the Drosophila Genetic Reference Panel (DGRP) reflects this variation. Using the DGRP stocks for genome-wide association (GWA) mapping, 16 loci involved in determining pupation height could be resolved. The candidate genes in these loci are enriched for high expression in the larval central nervous system. A genetic network could be constructed from the candidate loci, which places scribble (scrib) at the centre, plus other genes known to be involved in nervous system development, such as Epidermal growth factor receptor (Egfr) and p53. Using gene disruption lines, we could functionally validate several of the initially identified loci, as well as additional loci predicted from network analysis. Our study shows that the combination of high-throughput phenotyping with a genetic analysis of variation captured from the wild can be used to approach the genetic dissection of an environmentally relevant behavioural phenotype.

Effects of a male meiotic driver on male and female transcriptomes in the house mouse.

Not all genetic loci follow Mendel's rules, and the evolutionary consequences of this are not yet fully known. Genomic conflict involving multiple loci is a likely outcome, as restoration of Mendelian inheritance patterns will be selected for, and sexual conflict may also arise when sexes are differentially affected. Here, we investigate effects of the t haplotype, an autosomal male meiotic driver in house mice, on genome-wide gene expression patterns in males and females. We analysed gonads, liver and brain in adult same-sex sibling pairs differing in genotype, allowing us to identify t-associated differences in gene regulation. In testes, only 40% of differentially expressed genes mapped to the approximately 708 annotated genes comprising the t haplotype. Thus, much of the activity of the t haplotype occurs in trans, and as upregulation. Sperm maturation functions were enriched among both cis and trans acting t haplotype genes. Within the t haplotype, we observed more downregulation and differential exon usage. In ovaries, liver and brain, the majority of expression differences mapped to the t haplotype, and were largely independent of the differences seen in the testis. Overall, we found widespread transcriptional effects of this male meiotic driver in the house mouse genome.

Human core duplicon gene families: game changers or game players?

Illuminating the role of specific gene duplications within the human lineage can provide insights into human-specific adaptations. The so-called human core duplicon gene families have received particular attention in this respect, due to special features, such as expansion along single chromosomes, newly acquired protein domains and signatures of positive selection. Here, we summarize the data available for 10 such families and include some new analyses. A picture emerges that suggests broad functions for these protein families, possibly through modification of core cellular pathways. Still, more dedicated studies are required to elucidate the function of core-duplicons gene families and how they have shaped adaptations and evolution of humans.