Inferring evolutionary histories of pathway regulation from transcriptional profiling data

One of the outstanding challenges in comparative genomics is to interpret the evolutionary importance of regulatory variation between species. Rigorous molecular evolution-based methods to infer evidence for natural selection from expression data are at a premium in the field, and to date, phylogenetic approaches have not been well-suited to address the question in the small sets of taxa profiled in standard surveys of gene expression. We have developed a strategy to infer evolutionary histories from expression profiles by analyzing suites of genes of common function. In a manner conceptually similar to molecular evolution models in which the evolutionary rates of DNA sequence at multiple loci follow a gamma distribution, we modeled expression of the genes of an \emph{a priori}-defined pathway with rates drawn from an inverse gamma distribution. We then developed a fitting strategy to infer the parameters of this distribution from expression measurements, and to identify gene groups whose expression patterns were consistent with evolutionary constraint or rapid evolution in particular species. Simulations confirmed the power and accuracy of our inference method. As an experimental testbed for our approach, we generated and analyzed transcriptional profiles of four \emph{Saccharomyces} yeasts. The results revealed pathways with signatures of constrained and accelerated regulatory evolution in individual yeasts and across the phylogeny, highlighting the prevalence of pathway-level expression change during the divergence of yeast species. We anticipate that our pathway-based phylogenetic approach will be of broad utility in the search to understand the evolutionary relevance of regulatory change.Comment: 30 pages, 12 figures, 2 tables, contact authors for supplementary table

CHARACTERIZING ADAPTIVE NON-CODING CHANGES IN THE REGULATION OF HUMAN GENE EXPRESSION

Differential patterns of gene expression contribute to phenotypic differences between species. Understanding evolutionary changes in gene regulatory elements can help explain traits that separate humans from closely related species. Here, in two separate studies, we investigate gene expression and gene regulatory differences between humans our closest living evolutionary relatives, chimpanzees, in the context of uniquely human traits: increased susceptibility to epithelial cancers and neural developmental and functional processes that underlie our increased cognitive capacity. Using genomic methods to study gene expression and open chromatin, we compare human and chimpanzee responses to a serum challenge, an assay that that mimics patterns of gene expression that occur during cancer progression, and in another approach, we investigate the functional consequences of evolutionary changes in non-coding regulatory elements in neural progenitor cells and neurons. These studies identify recently evolved changes in physiological stress responses in humans, and patterns of adaptive changes in regulatory elements around highly conserved developmental pathways. Together, using these comparative genomic studies in relevant physiological contexts, we can thus further define the molecular basis for uniquely human phenotypes

Regulatory Pathway Analysis by High-Throughput In Situ Hybridization

Automated in situ hybridization enables the construction of comprehensive atlases of gene expression patterns in mammals. Such atlases can become Web-searchable digital expression maps of individual genes and thus offer an entryway to elucidate genetic interactions and signaling pathways. Towards this end, an atlas housing ∼1,000 spatial gene expression patterns of the midgestation mouse embryo was generated. Patterns were textually annotated using a controlled vocabulary comprising >90 anatomical features. Hierarchical clustering of annotations was carried out using distance scores calculated from the similarity between pairs of patterns across all anatomical structures. This process ordered hundreds of complex expression patterns into a matrix that reflects the embryonic architecture and the relatedness of patterns of expression. Clustering yielded 12 distinct groups of expression patterns. Because of the similarity of expression patterns within a group, members of each group may be components of regulatory cascades. We focused on the group containing Pax6, an evolutionary conserved transcriptional master mediator of development. Seventeen of the 82 genes in this group showed a change of expression in the developing neocortex of Pax6-deficient embryos. Electromobility shift assays were used to test for the presence of Pax6-paired domain binding sites. This led to the identification of 12 genes not previously known as potential targets of Pax6 regulation. These findings suggest that cluster analysis of annotated gene expression patterns obtained by automated in situ hybridization is a novel approach for identifying components of signaling cascades

Patterns and Processes of Speciation and Phenotypic Diversification in Palm-Pitvipers (Viperidae: \u3ci\u3eBothriechis\u3c/i\u3e)

Diversification is primarily a function of two processes—speciation and adaptation—that shape the history and trajectory of evolutionary lineages. These processes are often interdependent and are shaped by biotic and abiotic factors including existing and evolvable genetic variation, selection, and genetic connectivity among lineages. Genomic datasets present a means of disentangling complex evolutionary signals by sampling hundreds or thousands of loci to interrogate lineage and/or trait diversification. Palm-pitvipers (Bothriechis) are well suited for testing evolutionary hypotheses related to speciation and adaption. This group of arboreal vipers is well-recognized as monophyletic with a contentious phylogeographic history. Moreover, these species’ venoms are an ideal adaptive trait for examining genotype-phenotype interactions. To understand the processes affecting speciation in palm-pitvipers, I estimated the group’s phylogeny using an anchored phylogenomics approach and tested for evidence of reticulate evolution (i.e., ancient gene-flow) in the group\u27s history. The recovered phylogeny conflicted with key relationships inferred by mitochondrial genes and tests for reticulate evolution revealed strong support for historic gene flow among geographically proximate lineages. To examine the mechanisms promoting macroevolutionary divergence of venom phenotypes, I first focused on two sister taxa with strongly divergent venom types: The Black-Speckled (B. nigroviridis) and Talamancan (B. nubestris) Palm-Pitvipers. I tested whether toxins underlying venom differentiation would be associated with modular transcript expression. I found that toxins responsible for specific phenotypes segregate into distinct co-expression modules, which may permit rapid differentiation of venoms. To expand my investigation, I assembled venom gland transcriptomes from all recognized species of palm-pitvipers and assessed how gene family evolution affected patterns of toxin expression. Toxin expression was highly variable within toxin families but more variable following speciation events than gene duplications. Additionally, I identified multiple lineage specific regimes of expression for specific PLA2 and SVMP genes. Despite the broad expression variation characteristic of toxin genes, lineage-specific patterns of toxin expression emphasize the effect of shared evolutionary history on underlying genetic architecture. More broadly, the complexities of speciation and venom phenotype evolution observed in palm-pitvipers demonstrate the variety of processes that can influence evolutionary trajectories

Integrating genomic, transcriptomic and developmental approaches to investigate coloniality and life cycle evolution in the Hydractiniidae (Hydrozoa: Cnidaria)

Integrative approaches to evolutionary biology yield rich data through which we can truly begin to understand the marvels of life. This dissertation integrates genomic, transcriptomic, and developmental approaches to understand the evolution of prominent life history characters of the cnidarian class Hydrozoa, including the transition from solitary to colonial forms, an elaboration of coloniality known as polyp polymorphism, and medusae (jellyfish) evolution and loss. While these characters have been repeatedly explored phylogenetically, recognizing interesting and complex evolutionary patterns of character transitions, understanding of these complex patterns of character evolution will ultimately come from insight into their development. In this dissertation, I have developed workflows for analyzing RNA-Seq data in both an intra- and interspecific comparative context. Using next-generation sequencing I not only characterize entire transcriptomic expression profiles in various tissues of two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea, but also assess and accurately characterize intra- and interspecific changes in gene expression. Using these unbiased differential expression analyses, I identify correlated changes in expression and propose candidate genes and gene pathways that are potentially involved in these key transitions. Furthermore, using whole mount in situ hybridization to characterize the spatial expression of various candidates genes, I validated each approach showing expression consistent with their role in the development of a particular tissue or life cycle stage. Results presented in this dissertation suggest that the differential regulation of gene expression, as well as novel gene gain and loss appear to have played an important role in hydrozoan life cycle transitions. Moreover, these results reveal the power of these unbiased genomic/transcriptomic methods over traditional comparative candidate gene approaches to address longstanding questions of hydrozoan morphology and evolution

Gene expression in a paleopolyploid: a transcriptome resource for the ciliate Paramecium tetraurelia

International audienceBACKGROUND: The genome of Paramecium tetraurelia, a unicellular model that belongs to the ciliate phylum, has been shaped by at least 3 successive whole genome duplications (WGD). These dramatic events, which have also been documented in plants, animals and fungi, are resolved over evolutionary time by the loss of one duplicate for the majority of genes. Thanks to a low rate of large scale genome rearrangement in Paramecium, an unprecedented large number of gene duplicates of different ages have been identified, making this organism an outstanding model to investigate the evolutionary consequences of polyploidization. The most recent WGD, with 51% of pre-duplication genes still in 2 copies, provides a snapshot of a phase of rapid gene loss that is not accessible in more ancient polyploids such as yeast. RESULTS: We designed a custom oligonucleotide microarray platform for P. tetraurelia genome-wide expression profiling and used the platform to measure gene expression during 1) the sexual cycle of autogamy, 2) growth of new cilia in response to deciliation and 3) biogenesis of secretory granules after massive exocytosis. Genes that are differentially expressed during these time course experiments have expression patterns consistent with a very low rate of subfunctionalization (partition of ancestral functions between duplicated genes) in particular since the most recent polyploidization event. CONCLUSIONS: A public transcriptome resource is now available for Paramecium tetraurelia. The resource has been integrated into the ParameciumDB model organism database, providing searchable access to the data. The microarray platform, freely available through NimbleGen Systems, provides a robust, cost-effective approach for genome-wide expression profiling in P. tetraurelia. The expression data support previous studies showing that at short evolutionary times after a whole genome duplication, gene dosage balance constraints and not functional change are the major determinants of gene retention

Evolution of gene expression between closely related taxa of Mus

Regulatory changes in gene expression are thought to play an important role in evolutionary divergence. It has been suggested that a large proportion of the changes relevant to the process of differential adaptation and species formation can be attributed to changes in gene regulation. Moreover, differences in gene expression are heritable and thus provide a target for selection. To infer genome-wide evolutionary patterns of species divergence, I studied the evolution of gene expression in a comparison of Mus musculus with its closest relative Mus spretus, and among subspecies of Mus musculus (Mus musculus domesticus, Mus musculus musculus, Mus musculus castaneus, Mus musculus ssp.). RNA of individuals from different wild populations and wild-derived populations at and below the species level were screened for expression differences in three tissues (brain, pooled liver/kidney, testis) with a microarray approach. Six male individuals from M. m. domesticus, M. m. musculus and M. m. ssp. and three male individuals from M. m. castaneus and M. spretus were compared. A common reference design was employed, and all samples were hybridized against labeled cDNA from laboratory mice (C57BL/6). Using multiple animals from each population allowed to differentiate between the fraction of variation stemming from within- and that stemming from between-terms of population differences. Gene expression measures served as a common currency to compare evolutionary divergence across different tissues. A statistical analysis based on the identification of differentially expressed genes shows that the number of genes that changed expression between taxa depends on divergence time and the tissue under study. Across subspecies most expression changes are identified in the liver/kidney and almost none in the testis, whereas across species the highest number of differentially expressed genes is identified in the testis. Comparative Genomic Hybridizations (CGHs) ruled out hybridization differences as a cause for the observed pattern. Mitochondrial D-loop sequencing shows that Mus spretus is separated from the Mus musculus subspecies and that Mus musculus ssp. is not resolved as a phylogenetic entity. Functional annotation analysis of the differentially expressed genes shows that a wide variety of genes change expression, that transcription factors are a major group in all three tissues, and that functional classification categorization is able to reflect the tissues identity. Quantitative real-time PCR was used to confirm chosen target loci. In another approach, genome-wide patterns of evolution of gene expression were investigated. A study of the overall rates of divergence of gene expression shows the same tendency as the study based on gene counts: across subspecies, divergence is highest in the liver/kidney and across species, it is highest in the testis. In addition, it was tested whether a correlation between sequence divergence and gene expression divergence exists. Across subspecies there is a negative correlation between variation in gene expression and dN/dS ratios for genes that changed expression, whereas across species this correlation is positive. The observation of divergent gene expression in metabolic organs among incipient subspecies of the house mouse suggests a pervasive role of ecological and physiological adaptations in the early stage of divergence while late divergence seems to be primarily driven by non-ecological factors. The dN/dS analysis points to a role of positive selection for the genes that changed expression between subspecies. Whether or not gene expression divergence in later stages of divergence is a cause or a consequence of speciation and whether sexual selection or genetic drift is the major driving force behind this divergence remains open. Additional experiments are necessary to answer these questions

Gene circuit analysis of the terminal gap gene <i>huckebein</i>

The early embryo of Drosophila melanogaster provides a powerful model system to study the role of genes in pattern formation. The gap gene network constitutes the first zygotic regulatory tier in the hierarchy of the segmentation genes involved in specifying the position of body segments. Here, we use an integrative, systems-level approach to investigate the regulatory effect of the terminal gap gene huckebein (hkb) on gap gene expression. We present quantitative expression data for the Hkb protein, which enable us to include hkb in gap gene circuit models. Gap gene circuits are mathematical models of gene networks used as computational tools to extract regulatory information from spatial expression data. This is achieved by fitting the model to gap gene expression patterns, in order to obtain estimates for regulatory parameters which predict a specific network topology. We show how considering variability in the data combined with analysis of parameter determinability significantly improves the biological relevance and consistency of the approach. Our models are in agreement with earlier results, which they extend in two important respects: First, we show that Hkb is involved in the regulation of the posterior hunchback (hb) domain, but does not have any other essential function. Specifically, Hkb is required for the anterior shift in the posterior border of this domain, which is now reproduced correctly in our models. Second, gap gene circuits presented here are able to reproduce mutants of terminal gap genes, while previously published models were unable to reproduce any null mutants correctly. As a consequence, our models now capture the expression dynamics of all posterior gap genes and some variational properties of the system correctly. This is an important step towards a better, quantitative understanding of the developmental and evolutionary dynamics of the gap gene network

Gene Circuit Analysis of the Terminal Gap Gene huckebein

The early embryo of Drosophila melanogaster provides a powerful model system to study the role of genes in pattern formation. The gap gene network constitutes the first zygotic regulatory tier in the hierarchy of the segmentation genes involved in specifying the position of body segments. Here, we use an integrative, systems-level approach to investigate the regulatory effect of the terminal gap gene huckebein (hkb) on gap gene expression. We present quantitative expression data for the Hkb protein, which enable us to include hkb in gap gene circuit models. Gap gene circuits are mathematical models of gene networks used as computational tools to extract regulatory information from spatial expression data. This is achieved by fitting the model to gap gene expression patterns, in order to obtain estimates for regulatory parameters which predict a specific network topology. We show how considering variability in the data combined with analysis of parameter determinability significantly improves the biological relevance and consistency of the approach. Our models are in agreement with earlier results, which they extend in two important respects: First, we show that Hkb is involved in the regulation of the posterior hunchback (hb) domain, but does not have any other essential function. Specifically, Hkb is required for the anterior shift in the posterior border of this domain, which is now reproduced correctly in our models. Second, gap gene circuits presented here are able to reproduce mutants of terminal gap genes, while previously published models were unable to reproduce any null mutants correctly. As a consequence, our models now capture the expression dynamics of all posterior gap genes and some variational properties of the system correctly. This is an important step towards a better, quantitative understanding of the developmental and evolutionary dynamics of the gap gene network