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Title: Clusters of Temporal Discordances Reveal Distinct Embryonic Patterning Mechanisms in Drosophila and Anopheles}
Article:
Abstract:
Evolutionary innovations can be driven by spatial and temporal changes in gene expression. Several such differences have been documented in the embryos of lower and higher Diptera. One example is the reduction of the ancient extraembryonic envelope composed of amnion and serosa as seen in mosquitoes to the single amnioserosa of fruit flies. We used transcriptional datasets collected during the embryonic development of the fruit fly, Drosophila melanogaster, and the malaria mosquito, Anopheles gambiae, to search for whole-genome changes in gene expression underlying differences in their respective embryonic morphologies. We found that many orthologous gene pairs could be clustered based on the presence of coincident discordances in their temporal expression profiles. One such cluster contained genes expressed specifically in the mosquito serosa. As shown previously, this cluster is redeployed later in development at the time of cuticle synthesis. In addition, there is a striking difference in the temporal expression of a subset of maternal genes. Specifically, maternal transcripts that exhibit a sharp reduction at the time of the maternal-zygotic transition in Drosophila display sustained expression in the Anopheles embryo. We propose that gene clustering by local temporal discordance can be used for the de novo identification of the gene batteries underlying morphological diversity.
Introduction:
During the 1980s and 1990s methods of molecular genetics were used to determine the contributions of individual genes to different developmental processes, such as the segmentation of the Drosophila embryo[1]. However, during the past decade post-genome technologies have opened the door to identifying all of the genes engaged in such processes. In particular, collections of microarray data accumulated in public databases now cover a variety of different conditions and sometimes even the full life cycles for a range of evolutionarily distant species. These data provide new opportunities to identify complete ensembles of genes engaged in the specification of body plans and morphological diversification. Recently a number of such studies in different model systems have been conducted[2]–[9]. The initial reports as exemplified by[7], where the comparative analysis was extended across different phyla, provide evidence for the existence of deeply conserved co-regulated gene sets( kernels[10]) responsible for fundamental cellular functions. More recent studies focused on a set of fairly proximal yeast species have revealed that the regulation of even the most essential processes such as the cell cycle may not be conserved[2]–[4]. It was found instead that temporarily similar engagement of multi-component biological machines could be achieved by the species-specific regulation of different subunits within these complexes[11],[12]. Also, the regulation of genes, which are differentially regulated between species, was shown to be primarily driven by TATA-box containing promoters[5]. The aforementioned studies were done by “co-expression meta-analysis” extensively reviewed in[13] and in[14]. This method employs gene lists independently precompiled by condition-dependent clustering in individual species. The benefit of this approach is its ability to compare gene expression independently of species-specific experimental conditions. In contrast, “expression meta-analysis”[13] employs direct comparisons of the expression profiles of orthologue pairs. This approach can specifically pick up a condition or a time point when orthologous genes are differentially regulated between species. However, its use is restricted to a set of similar conditions, such as a time-series. This method requires data preprocessing, or in the case of temporal data, matching the corresponding time points, which due to differences in metabolism may not relate directly between the species. The computational frameworks as well as data sources for direct cross-species gene expression studies have only recently become available. In a recent study we used datasets spanning several cell cycles of synchronized culture of fission and baker's yeast as a comparative training data source to develop a computational platform for expression meta-analysis[15]. Using so-called “time warping”[16]–[20] enhanced by noise suppression[15], we created alignment paths and successfully predicted the comparative duration of the cell cycle phases in baker's and fission yeast. In the present study we have employed the time warping method to compare mosquito and fruitfly embryos. Thus far, the phylogenetic comparisons of gene expression have identified differential expression of orthologous genes implicated in similar processes. However, most such comparisons have been restricted to unicellular organisms[2]–[4] or to closely related multicellular species that manifest relatively few phenotypic differences. Moreover, such studies have examined excessively divergent species( yeast versus humans)[7], thereby complicating the association of discordant gene expression profiles with phenotypic variation. We have selected divergent species that are nonetheless members of a common order of insects, the Diptera. We reasoned that mosquitoes and fruitflies possess a number of distinctive phenotypic traits, but are sufficiently similar to link such differences with orthologous clusters of discordantly expressed genes. These two flies belong to separate suborders( Nematocera and Brachycera) of Diptera and are thought to be separated by an evolutionary distance of ∼200 million years[21]. Mosquito and fruitfly larvae possess a number of striking differences, such as the representation of larval head, which is involuted in Drosophila but fully extended in mosquitoes. Another prominent feature, which was recently examined at the level of gene expression by our group[22],[23], is the presence in the mosquito embryo of a double-layered extraembryonic envelope( amnion and serosa), which is substituted by single amnioserosa in fruitflies( reviewed in[24]). We were interested in whether these and additional features could be detected by the comparative analysis of gene expression datasets. The duration of embryogenesis in flies and mosquitoes differs significantly. At 25°C it takes ∼22 h for the fruit fly embryo and ∼50 h for the mosquito embryo to fully develop. For this reason, the temporal gene expression datasets for fly[25] and mosquito embryogenesis[22] were first aligned using time warping[15] and then analyzed for discordant gene expression. Since all the distinct developmental events taking place in the course of embryogenesis are expected to have specific timing and duration, the aim of the analysis was to cluster the orthologous gene pairs based on timing and duration of local discordances in their temporal expression profiles. These studies reveal that a major gene cluster matches those known to be involved in the function of the mosquito serosa. This cluster is shown to be re-engaged later in development during cuticle synthesis. Significant discordances were also observed for a number of maternally expressed genes in flies and mosquitoes, consistent with the evolution of a sharp maternal-to-zygotic transition in gene expression higher Diptera. These studies provide a framework for the identification of the genetic circuits underlying embryonic diversity.
Discussion:
This study provides a conceptual and computational framework for cross-organismal temporal alignments and comparisons of species-specific transcriptional datasets. The resulting heatmaps of discordant gene clusters identified distinctive patterning properties in Dipteran embryos, including the organization of extra-embryonic tissues and the maternal-zygotic transition( see Figure 2B and UCB web resource). There is surprising concordance in the development of Drosophila and Anopheles embryos, despite different rates of growth and distinctive patterning features. The heatmaps and alignments of gene expression datasets( representing more than 4, 000 orthologous gene pairs) obtained with the time warping algorithm suggest a near linear relationship in their embryonic development. Interestingly the alignment path manifested a subtle deviation from linearity at stage 9, presumably shortly after serosa completion in Anopheles( Figure 1B). The removal of serosal genes from the datasets did not result in change in the automatic alignment path, suggesting that this time point may indeed represent temporarily local slow down of gene expression programs in Anopheles embryo proper compared to Drosophila. The heatmaps obtained with the discordance mining algorithms implemented in Peak-mapper and Glob-mapper identify clusters of discordant genes that represent either unique tissue types( such as serosa) or changes in regulatory pathways such as that governing the turnover of maternal transcripts. The percentage of false positive genes( as judged by the analysis of the serosa gene cluster) was expected to be relatively high. One approach to this problem is the application of annotation methods such as GO-term enrichment or controlled vocabulary annotations( see UCB web resource). “Gene sharing” or “co-option” manifested in redeployment of gene batteries in a diverse set of tissues is a recurrent theme in the evolution of animal embryos[26]. One of the mechanisms for gene co-option, specifically when it happens in the absence of gene duplication, is the diversification of regulatory regions resulting in acquisition of new territories of expression. For example, the skeletogenic mesoderm of the sea urchin embryo employs a conserved gene battery that is used for the secretion of the adult exoskeleton[27],[28]. Previous studies showed that the serosa gene battery is reengaged later in development, during the time of embryonic cuticle production[22]. The fact that an identical gene set is used twice during development at different time points or in different tissues suggests that the genes don't fall within this set due to random co-occurrence. In fact this reengagement is observed due to similarities of the cuticle secreted by the serosa at early stages[29]–[32] and the embryonic cuticle produced at the end of embryogenesis. We propose that the incidence of gene battery reengagement might be used as a further indicator of functional relevance of the gene within the battery or otherwise a filter that could remove the false positives from the genes extracted from the discordance cluster. Indeed, serosa genes can be identified by their biphasic expression in the mosquito as compared with the single late peak of expression in the fruitfly embryo( Figure 4B). Perhaps the low threshold discordance clustering in combination with verification of the reengagement patterns can be used as an alternative strategy for the extraction of functional gene sets from comparative datasets. Previous whole-genome comparisons focused on ancestral gene networks based on phylogenetic conservation of gene expression patterns. Here, evidence was presented that discordant clusters provide a means for identifying gene batteries involved in evolutionary diversification and novelty. This approach was used to identify de novo the changes in the turnover of maternal transcripts in the Drosophila and Anopheles embryos and in temporal expression of a subset of yolk genes. The rapid turnover of maternal genes correlates with increased rates of embryogenesis in higher Dipterans.
Linking genotype to phenotype is a major undertaking in modern biological research . A variety of strategies are used but have generally failed to explain the maintenance and acquisition of new phenotypic traits in changing populations . We propose that whole-genome cross-species comparisons can be used to identify gene clusters underlying phenotypic variation . In the present study we used gene expression datasets collected during fruit fly and mosquito embryogenesis to identify temporal changes in gene expression . We found that differentially represented tissue types ( such as extraembryonic serosa ) were clearly manifested by clusters of local discordances in gene expression . Discordances were also observed for a suite of maternally expressed genes , consistent with the notion that the abrupt maternal-zygotic transition seen in Drosophila is an evolutionary innovation of higher Diptera . We propose that gene clustering by expression discordance can be used to determine the genetic basis of phenotypic variation .