Cellular development and evolution of the mammalian cerebellum.

The expansion of the neocortex, a hallmark of mammalian evolution1,2, was accompanied by an increase in cerebellar neuron numbers3. However, little is known about the evolution of the cellular programmes underlying the development of the cerebellum in mammals. In this study we generated single-nucleus RNA-sequencing data for around 400,000 cells to trace the development of the cerebellum from early neurogenesis to adulthood in human, mouse and the marsupial opossum. We established a consensus classification of the cellular diversity in the developing mammalian cerebellum and validated it by spatial mapping in the fetal human cerebellum. Our cross-species analyses revealed largely conserved developmental dynamics of cell-type generation, except for Purkinje cells, for which we observed an expansion of early-born subtypes in the human lineage. Global transcriptome profiles, conserved cell-state markers and gene-expression trajectories across neuronal differentiation show that cerebellar cell-type-defining programmes have been overall preserved for at least 160 million years. However, we also identified many orthologous genes that gained or lost expression in cerebellar neural cell types in one of the species or evolved new expression trajectories during neuronal differentiation, indicating widespread gene repurposing at the cell-type level. In sum, our study unveils shared and lineage-specific gene-expression programmes governing the development of cerebellar cells and expands our understanding of mammalian brain evolution.

Sex-biased gene expression across mammalian organ development and evolution.

Sexually dimorphic traits are common among mammals and are specified during development through the deployment of sex-specific genetic programs. Because little is known about these programs, we investigated them using a resource of gene expression profiles in males and females throughout the development of five organs in five mammals (human, mouse, rat, rabbit, and opossum) and a bird (chicken). We found that sex-biased gene expression varied considerably across organs and species and was often cell-type specific. Sex differences increased abruptly around sexual maturity instead of increasing gradually during organ development. Finally, sex-biased gene expression evolved rapidly at the gene level, with differences between organs in the evolutionary mechanisms used, but more slowly at the cellular level, with the same cell types being sexually dimorphic across species.

The molecular evolution of spermatogenesis across mammals.

The testis produces gametes through spermatogenesis and evolves rapidly at both the morphological and molecular level in mammals1-6, probably owing to the evolutionary pressure on males to be reproductively successful7. However, the molecular evolution of individual spermatogenic cell types across mammals remains largely uncharacterized. Here we report evolutionary analyses of single-nucleus transcriptome data for testes from 11 species that cover the three main mammalian lineages (eutherians, marsupials and monotremes) and birds (the evolutionary outgroup), and include seven primates. We find that the rapid evolution of the testis was driven by accelerated fixation rates of gene expression changes, amino acid substitutions and new genes in late spermatogenic stages, probably facilitated by reduced pleiotropic constraints, haploid selection and transcriptionally permissive chromatin. We identify temporal expression changes of individual genes across species and conserved expression programs controlling ancestral spermatogenic processes. Genes predominantly expressed in spermatogonia (germ cells fuelling spermatogenesis) and Sertoli (somatic support) cells accumulated on X chromosomes during evolution, presumably owing to male-beneficial selective forces. Further work identified transcriptomal differences between X- and Y-bearing spermatids and uncovered that meiotic sex-chromosome inactivation (MSCI) also occurs in monotremes and hence is common to mammalian sex-chromosome systems. Thus, the mechanism of meiotic silencing of unsynapsed chromatin, which underlies MSCI, is an ancestral mammalian feature. Our study illuminates the molecular evolution of spermatogenesis and associated selective forces, and provides a resource for investigating the biology of the testis across mammals.

NIMA-related kinase 9 regulates the phosphorylation of the essential myosin light chain in the heart.

To adapt to changing hemodynamic demands, regulatory mechanisms modulate actin-myosin-kinetics by calcium-dependent and -independent mechanisms. We investigate the posttranslational modification of human essential myosin light chain (ELC) and identify NIMA-related kinase 9 (NEK9) to interact with ELC. NEK9 is highly expressed in the heart and the interaction with ELC is calcium-dependent. Silencing of NEK9 results in blunting of calcium-dependent ELC-phosphorylation. CRISPR/Cas9-mediated disruption of NEK9 leads to cardiomyopathy in zebrafish. Binding to ELC is mediated via the protein kinase domain of NEK9. A causal relationship between NEK9 activity and ELC-phosphorylation is demonstrated by genetic sensitizing in-vivo. Finally, we observe significantly upregulated ELC-phosphorylation in dilated cardiomyopathy patients and provide a unique map of human ELC-phosphorylation-sites. In summary, NEK9-mediated ELC-phosphorylation is a calcium-dependent regulatory system mediating cardiac contraction and inotropy.

ExoPLOT: Representation of alternative splicing in human tissues and developmental stages with transposed element (TE) involvement.

Advances in RNA high-throughput sequencing and large-scale functional assays yield new insights into the multifaceted activities of transposed elements (TE) and many other previously undiscovered sequence elements. Currently, no tool for easy access, analysis, quantification, and visualization of alternatively spliced exons across multiple tissues or developmental stages is available. Also, analysis pipelines demand computational skills or hardware requirements, which often are hard to meet by wet-lab scientists. We developed ExoPLOT to enable simplified access to massive RNA high throughput sequencing datasets to facilitate the analysis of alternative splicing across many biological samples. To demonstrate the functonality of ExoPLOT, we analyzed the contributon of exonized TEs to human coding sequences (CDS). mRNA splice variants containing the TE-derived exon were quantified and compared to expression levels of TE-free splice variants. For analysis, we utilized 313 human cerebrum, cerebellum, heart, kidney, liver, ovary, and testis transcriptomes, representing various pre- and postnatal developmental stages. ExoPLOT visualizes the relative expression levels of alternative transcripts, e.g., caused by the insertion of new TE-derived exons, across different developmental stages of and among multiple tissues. This tool also provides a unique link between evolution and function during exonization (gain of a new exon) and exaptation (recruitment/co-optation) of a new exon. As input for analysis, we derived a database of 1151 repeat-masked, exonized TEs, representing all prominent families of transposons in the human genome and the collection of human consensus coding sequences (CCDS). ExoPLOT screened preprocessed RNA high-throughput sequencing datasets from seven human tissues to quantify and visualize the dynamics in RNA splicing for these 1151 TE-derived exons during the entire human organ development. In addition, we successfully mapped and analyzed 993 recently described exonized sequences from the human frontal cortex onto these 313 transcriptome libraries. ExoPLOT's approach to preprocessing RNA deep sequencing datasets facilitates alternative splicing analysis and significantly reduces processing times. In addition, ExoPLOT's design allows studying alternative RNA isoforms other than TE-derived in a customized - coordinate-based manner and is available at http://retrogenomics3.uni-muenster.de:3838/exz-plot-d/.

Developmental and evolutionary dynamics of cis-regulatory elements in mouse cerebellar cells.

Organ development is orchestrated by cell- and time-specific gene regulatory networks. In this study, we investigated the regulatory basis of mouse cerebellum development from early neurogenesis to adulthood. By acquiring snATAC-seq (single-nucleus assay for transposase accessible chromatin using sequencing) profiles for ~90,000 cells spanning 11 stages, we mapped cerebellar cell types and identified candidate cis-regulatory elements (CREs). We detected extensive spatiotemporal heterogeneity among progenitor cells and a gradual divergence in the regulatory programs of cerebellar neurons during differentiation. Comparisons to vertebrate genomes and snATAC-seq profiles for ∼20,000 cerebellar cells from the marsupial opossum revealed a shared decrease in CRE conservation during development and differentiation as well as differences in constraint between cell types. Our work delineates the developmental and evolutionary dynamics of gene regulation in cerebellar cells and provides insights into mammalian organ development.

Alternative splicing during mammalian organ development.

Alternative splicing (AS) is pervasive in mammalian genomes, yet cross-species comparisons have been largely restricted to adult tissues and the functionality of most AS events remains unclear. We assessed AS patterns across pre- and postnatal development of seven organs in six mammals and a bird. Our analyses revealed that developmentally dynamic AS events, which are especially prevalent in the brain, are substantially more conserved than nondynamic ones. Cassette exons with increasing inclusion frequencies during development show the strongest signals of conserved and regulated AS. Newly emerged cassette exons are typically incorporated late in testis development, but those retained during evolution are predominantly brain specific. Our work suggests that an intricate interplay of programs controlling gene expression levels and AS is fundamental to organ development, especially for the brain and heart. In these regulatory networks, AS affords substantial functional diversification of genes through the generation of tissue- and time-specific isoforms from broadly expressed genes.

Transcriptome and translatome co-evolution in mammals.

Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer1. Here we report an analysis of the co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.

Developmental Gene Expression Differences between Humans and Mammalian Models.

Identifying the molecular programs underlying human organ development and how they differ from model species is key for understanding human health and disease. Developmental gene expression profiles provide a window into the genes underlying organ development and a direct means to compare them across species. We use a transcriptomic resource covering the development of seven organs to characterize the temporal profiles of human genes associated with distinct disease classes and to determine, for each human gene, the similarity of its spatiotemporal expression with its orthologs in rhesus macaque, mouse, rat, and rabbit. We find clear associations between spatiotemporal profiles and the phenotypic manifestations of diseases. We also find that half of human genes differ from their mouse orthologs in their temporal trajectories in at least one of the organs. These include more than 200 genes associated with brain, heart, and liver disease for which mouse models should undergo extra scrutiny.

Developmental dynamics of lncRNAs across mammalian organs and species.

Although many long noncoding RNAs (lncRNAs) have been identified in human and other mammalian genomes, there has been limited systematic functional characterization of these elements. In particular, the contribution of lncRNAs to organ development remains largely unexplored. Here we analyse the expression patterns of lncRNAs across developmental time points in seven major organs, from early organogenesis to adulthood, in seven species (human, rhesus macaque, mouse, rat, rabbit, opossum and chicken). Our analyses identified approximately 15,000 to 35,000 candidate lncRNAs in each species, most of which show species specificity. We characterized the expression patterns of lncRNAs across developmental stages, and found many with dynamic expression patterns across time that show signatures of enrichment for functionality. During development, there is a transition from broadly expressed and conserved lncRNAs towards an increasing number of lineage- and organ-specific lncRNAs. Our study provides a resource of candidate lncRNAs and their patterns of expression and evolutionary conservation across mammalian organ development.

Gene expression across mammalian organ development.

The evolution of gene expression in mammalian organ development remains largely uncharacterized. Here we report the transcriptomes of seven organs (cerebrum, cerebellum, heart, kidney, liver, ovary and testis) across developmental time points from early organogenesis to adulthood for human, rhesus macaque, mouse, rat, rabbit, opossum and chicken. Comparisons of gene expression patterns identified correspondences of developmental stages across species, and differences in the timing of key events during the development of the gonads. We found that the breadth of gene expression and the extent of purifying selection gradually decrease during development, whereas the amount of positive selection and expression of new genes increase. We identified differences in the temporal trajectories of expression of individual genes across species, with brain tissues showing the smallest percentage of trajectory changes, and the liver and testis showing the largest. Our work provides a resource of developmental transcriptomes of seven organs across seven species, and comparative analyses that characterize the development and evolution of mammalian organs.

Investigating human placentation and pregnancy using first trimester chorionic villi.

Chorionic villus sampling (CVS), routinely used for prenatal diagnosis of cytogenetic disorders, also possesses great potential for the study of placentation. To better understand villus biology, human placentation, and how these relate to pregnancy outcomes, we examined the morphology and transcriptomes of villi obtained via CVS from 10 to 14 weeks of pregnancy and correlated these with pregnancy attributes and clinical outcomes. First, we established a morphological scoring system based on three main villus features: branching, budding and vascularization. We then tested whether morphology scores were predictive of pregnancy attributes and clinical outcomes. Finally, we used RNA sequencing to assess the transcriptional basis of villus morphology and tested the hypothesis that gene expression may predict pregnancy outcomes. We demonstrate that villus morphology varies tremendously between patients, irrespective of gestational age, and that transcriptional differences are highly predictive of villus morphology. We show that pre-eclampsia markers are associated with villi with low morphology scores. Additionally, we identify SVEP1 as a possible biomarker for defining gestational age. Overall, chorionic villi in the first trimester remain one of the few means to correlate placental function with pregnancy outcome and these samples are a valuable and increasingly rare resource.

Survey of Global Genetic Diversity Within the Drosophila Immune System.

Numerous studies across a wide range of taxa have demonstrated that immune genes are routinely among the most rapidly evolving genes in the genome. This observation, however, does not address what proportion of immune genes undergo strong selection during adaptation to novel environments. Here, we determine the extent of very recent divergence in genes with immune function across five populations of Drosophila melanogaster and find that immune genes do not show an overall trend of recent rapid adaptation. Our population-based approach uses a set of carefully matched control genes to account for the effects of demography and local recombination rate, allowing us to identify whether specific immune functions are putative targets of strong selection. We find evidence that viral-defense genes are rapidly evolving in Drosophila at multiple timescales. Local adaptation to bacteria and fungi is less extreme and primarily occurs through changes in recognition and effector genes rather than large-scale changes to the regulation of the immune response. Surprisingly, genes in the Toll pathway, which show a high rate of adaptive substitution between the D. melanogaster and D. simulans lineages, show little population differentiation. Quantifying the flies for resistance to a generalist Gram-positive bacterial pathogen, we found that this genetic pattern of low population differentiation was recapitulated at the phenotypic level. In sum, our results highlight the complexity of immune evolution and suggest that Drosophila immune genes do not follow a uniform trajectory of strong directional selection as flies encounter new environments.

LTR-mediated retroposition as a mechanism of RNA-based duplication in metazoans.

In a broad range of taxa, genes can duplicate through an RNA intermediate in a process mediated by retrotransposons (retroposition). In mammals, L1 retrotransposons drive retroposition, but the elements responsible for retroposition in other animals have yet to be identified. Here, we examined young retrocopies from various animals that still retain the sequence features indicative of the underlying retroposition mechanism. In Drosophila melanogaster, we identified and de novo assembled 15 polymorphic retrocopies and found that all retroposed loci are chimeras of internal retrocopies flanked by discontinuous LTR retrotransposons. At the fusion points between the mRNAs and the LTR retrotransposons, we identified shared short similar sequences that suggest the involvement of microsimilarity-dependent template switches. By expanding our approach to mosquito, zebrafish, chicken, and mammals, we identified in all these species recently originated retrocopies with a similar chimeric structure and shared microsimilarities at the fusion points. We also identified several retrocopies that combine the sequences of two or more parental genes, demonstrating LTR-retroposition as a novel mechanism of exon shuffling. Finally, we found that LTR-mediated retrocopies are immediately cotranscribed with their flanking LTR retrotransposons. Transcriptional profiling coupled with sequence analyses revealed that the sense-strand transcription of the retrocopies often lead to the origination of in-frame proteins relative to the parental genes. Overall, our data show that LTR-mediated retroposition is highly conserved across a wide range of animal taxa; combined with previous work from plants and yeast, it represents an ancient and ongoing mechanism continuously shaping gene content evolution in eukaryotes.

Extensive local adaptation within the chemosensory system following Drosophila melanogaster's global expansion.

How organisms adapt to new environments is of fundamental biological interest, but poorly understood at the genetic level. Chemosensory systems provide attractive models to address this problem, because they lie between external environmental signals and internal physiological responses. To investigate how selection has shaped the well-characterized chemosensory system of Drosophila melanogaster, we have analysed genome-wide data from five diverse populations. By couching population genomic analyses of chemosensory protein families within parallel analyses of other large families, we demonstrate that chemosensory proteins are not outliers for adaptive divergence between species. However, chemosensory families often display the strongest genome-wide signals of recent selection within D. melanogaster. We show that recent adaptation has operated almost exclusively on standing variation, and that patterns of adaptive mutations predict diverse effects on protein function. Finally, we provide evidence that chemosensory proteins have experienced relaxed constraint, and argue that this has been important for their rapid adaptation over short timescales.

Evidence for the fixation of gene duplications by positive selection in Drosophila.

Gene duplications play a key role in the emergence of novel traits and in adaptation. But despite their centrality to evolutionary processes, it is still largely unknown how new gene duplicates are initially fixed within populations and later maintained in genomes. Long-standing debates on the evolution of gene duplications could be settled by determining the relative importance of genetic drift vs. positive selection in the fixation of new gene duplicates. Using the Drosophila Global Diversity Lines (GDL), we have combined genome-wide SNP polymorphism data with a novel set of copy number variant calls and gene expression profiles to characterize the polymorphic phase of new genes. We found that approximately half of the roughly 500 new complete gene duplications segregating in the GDL lead to significant increases in the expression levels of the duplicated genes and that these duplications are more likely to be found at lower frequencies, suggesting a negative impact on fitness. However, we also found that six of the nine gene duplications that are fixed or close to fixation in at least one of the five populations in our study show signs of being under positive selection, and that these duplications are likely beneficial because of dosage effects, with a possible role for additional mutations in two duplications. Our work suggests that in Drosophila, theoretical models that posit that gene duplications are immediately beneficial and fixed by positive selection are most relevant to explain the long-term evolution of gene duplications in this species.

Mutation spectrum of Drosophila CNVs revealed by breakpoint sequencing.

BACKGROUND: The detailed study of breakpoints associated with copy number variants (CNVs) can elucidate the mutational mechanisms that generate them and the comparison of breakpoints across species can highlight differences in genomic architecture that may lead to lineage-specific differences in patterns of CNVs. Here, we provide a detailed analysis of Drosophila CNV breakpoints and contrast it with similar analyses recently carried out for the human genome. RESULTS: By applying split-read methods to a total of 10x coverage of 454 shotgun sequence across nine lines of D. melanogaster and by re-examining a previously published dataset of CNVs detected using tiling arrays, we identified the precise breakpoints of more than 600 insertions, deletions, and duplications. Contrasting these CNVs with those found in humans showed that in both taxa CNV breakpoints fall into three classes: blunt breakpoints; simple breakpoints associated with microhomology; and breakpoints with additional nucleotides inserted/deleted and no microhomology. In both taxa CNV breakpoints are enriched with non-B DNA sequence structures, which may impair DNA replication and/or repair. However, in contrast to human genomes, non-allelic homologous-recombination (NAHR) plays a negligible role in CNV formation in Drosophila. In flies, non-homologous repair mechanisms are responsible for simple, recurrent, and complex CNVs, including insertions of de novo sequence as large as 60 bp. CONCLUSIONS: Humans and Drosophila differ considerably in the importance of homology-based mechanisms for the formation of CNVs, likely as a consequence of the differences in the abundance and distribution of both segmental duplications and transposable elements between the two genomes.

The origin and evolution of new genes.

New genes are a major source of genetic innovation in genomes. However, until recently, understanding how new genes originate and how they evolve was hampered by the lack of appropriate genetic datasets. The advent of the genomic era brought about a revolution in the amount of data available to study new genes. For the first time, decades-old theoretical principles could be tested empirically and novel and unexpected avenues of research opened up. This chapter explores how genomic data can and is being used to study both the origin and evolution of new genes and the surprising discoveries made thus far.

Drosophila duplication hotspots are associated with late-replicating regions of the genome.

Duplications play a significant role in both extremes of the phenotypic spectrum of newly arising mutations: they can have severe deleterious effects (e.g. duplications underlie a variety of diseases) but can also be highly advantageous. The phenotypic potential of newly arisen duplications has stimulated wide interest in both the mutational and selective processes shaping these variants in the genome. Here we take advantage of the Drosophila simulans-Drosophila melanogaster genetic system to further our understanding of both processes. Regarding mutational processes, the study of two closely related species allows investigation of the potential existence of shared duplication hotspots, and the similarities and differences between the two genomes can be used to dissect its underlying causes. Regarding selection, the difference in the effective population size between the two species can be leveraged to ask questions about the strength of selection acting on different classes of duplications. In this study, we conducted a survey of duplication polymorphisms in 14 different lines of D. simulans using tiling microarrays and combined it with an analogous survey for the D. melanogaster genome. By integrating the two datasets, we identified duplication hotspots conserved between the two species. However, unlike the duplication hotspots identified in mammalian genomes, Drosophila duplication hotspots are not associated with sequences of high sequence identity capable of mediating non-allelic homologous recombination. Instead, Drosophila duplication hotspots are associated with late-replicating regions of the genome, suggesting a link between DNA replication and duplication rates. We also found evidence supporting a higher effectiveness of selection on duplications in D. simulans than in D. melanogaster. This is also true for duplications segregating at high frequency, where we find evidence in D. simulans that a sizeable fraction of these mutations is being driven to fixation by positive selection.

Mutational bias shaping fly copy number variation: implications for genome evolution.

Copy number variants (CNVs) underlie several genomic disorders and are a major source of genetic innovation. Consequently, any bias affecting their placement in the genome will impact our understanding of human disease and genome evolution. Here we report a mutational bias affecting CNVs that generates different probabilities of duplication and deletion across the genome in association with DNA replication time. We show that this mutational bias has important consequences for genome evolution by leading to different probabilities of gene duplication for different classes of genes and by linking the probability of gene duplication with the transcriptional activity of genes.