Brian D. Eads

The developmental transcriptome of Drosophila melanogaster

Drosophila melanogaster is one of the most well studied genetic model organisms, nonetheless its genome still contains unannotated coding and non-coding genes, transcripts, exons, and RNA editing sites. Full discovery and annotation are prerequisites for understanding how the regulation of transcription, splicing, and RNA editing directs development of this complex organism. We used RNA-Seq, tiling microarrays, and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. Together, these data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

The ecoresponsive genome of Daphnia pulex

The Daphnia genome reveals a multitude of genes and shows adaptation through gene family expansions. We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia’s genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.

Diversity and dynamics of the Drosophila transcriptome

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Population-genomic insights into the evolutionary origin and fate of obligately asexual Daphnia pulex

Significance Drawing from whole-genome sequences of multiple genotypes, this study documents the molecular-genetic consequences of the loss of recombination in a microcrustacean, demonstrating that loss of heterozygosity via gene conversion-like processes is a dramatically more powerful force than accumulation of new mutations. Despite much theoretical work, the molecular-genetic causes and evolutionary consequences of asexuality remain largely undetermined. Asexual animal species are rare, evolutionarily short-lived, and thought to suffer mutational meltdown as a result of lack of recombination. Whole-genome analysis of 11 sexual and 11 asexual genotypes of Daphnia pulex indicates that current asexual lineages are in fact very young, exhibit no signs of purifying selection against accumulating mutations, and have extremely high rates of gene conversion and deletion. The reconstruction of chromosomal haplotypes in regions containing SNP markers associated with asexuality (chromosomes VIII and IX) indicates that introgression from a sister species, Daphnia pulicaria, underlies the origin of the asexual phenotype. Silent-site divergence of the shared chromosomal haplotypes of asexuals indicates that the spread of asexuality is as recent as 1,250 y, although the origin of the meiosis-suppressing element or elements could be substantially older. In addition, using previous estimates of the gene conversion rate from Daphnia mutation accumulation lines, we are able to age each asexual lineage. Although asexual lineages originate from wide crosses that introduce elevated individual heterozygosities on clone foundation, they also appear to be constrained by the inbreeding-like effect of loss of heterozygosity that accrues as gene conversion and hemizygous deletion expose preexisting recessive deleterious alleles of asexuals, limiting their evolutionary longevity. Our study implies that the buildup of newly introduced deleterious mutations (i.e., Muller’s ratchet) may not be the dominant force imperiling nonrecombining populations of D. pulex, as previously proposed.

PUMPING IONS: RAPID PARALLEL EVOLUTION OF IONIC REGULATION FOLLOWING HABITAT INVASIONS

Marine to freshwater colonizations constitute among the most dramatic evolutionary transitions in the history of life. This study examined evolution of ionic regulation following saline‐to‐freshwater transitions in an invasive species. In recent years, the copepod Eurytemora affinis has invaded freshwater habitats multiple times independently. We found parallel evolutionary shifts in ion‐motive enzyme activity (V‐type H+ ATPase, Na+/K+‐ATPase) across independent invasions and in replicate laboratory selection experiments. Freshwater populations exhibited increased V‐type H+ ATPase activity in fresh water (0 PSU) and declines at higher salinity (15 PSU) relative to saline populations. This shift represented marked evolutionary increases in plasticity. In contrast, freshwater populations displayed reduced Na+/K+‐ATPase activity across all salinities. Most notably, modifying salinity alone during laboratory selection experiments recapitulated the evolutionary shifts in V‐type H+ ATPase activity observed in nature. Maternal and embryonic acclimation could not account for the observed shifts in enzyme activity. V‐type H+ ATPase function has been hypothesized to be critical for freshwater and terrestrial adaptations, but evolution of this enzyme function had not been previously demonstrated in the context of habitat transitions. Moreover, the speed of these evolutionary shifts was remarkable, within a few generations in the laboratory and a few decades in the wild.

Ecological genomics in Daphnia : stress responses and environmental sex determination

Ecological genomics is the study of adaptation of natural populations to their environment, and therefore seeks to link organism and population level processes through an understanding of genome organization and function. The planktonic microcrustacean Daphnia, which has long been an important system for ecology, is now being used as a genomic model as well. Here we review recent progress in selected areas of Daphnia genomics research. Production of parthenogenetic male offspring occurs through environmental cues, which clearly involves endocrine regulation and has also been studied as a toxicological response to juvenoid hormone analog insecticides. Recent progress has uncovered a putative juvenoid cis-response element, which together with microarray analysis will stimulate further research into nuclear hormone receptors and their associated transcriptional regulatory networks. Ecotoxicological studies indicate that mRNA profiling is a sensitive and specific research tool with promising applications in environmental monitoring and for uncovering conserved cellular processes. Rapid progress is expected to continue in these and other areas, as genomic tools for Daphnia become widely available to investigators.

Daphnia as an emerging model for toxicological genomics

Daphnia are already an established model species in toxicology. This freshwater crustacean is used commonly for environmental monitoring of pollutants around the globe and plays an important role in establishing regulatory criteria by government agencies (e.g., US EPA, Environment Canada organization for Economic Cooperation and Development, Environment Agency of Japan). Consequently, daphniids represent 8% of all experimental data for aquatic animals within the toxicological databases (Denslow et al., 2007). As such, their incorporation within the new field of toxicological genomics is limited only by the advancement of genomic resources. Because the development of these technologies requires the input and feedback of a large research community that extends far beyond the boundaries of any one discipline, the Daphnia Genomics Consortium (DGC) was formed in 2001 to: (i) provide the organizational framework to coordinate efforts at developing the Daphnia genomic toolbox; (ii) facilitate collaborative research and (iii) develop bioinformatics strategies for organizing the rapidly growing database. This chapter reviews the progress in establishing Daphnia as model species for genomic studies, with emphasis on toxicological applications. As the goals of the DGC are defined largely by extending the boundaries of current biological research in light of genomic information, this chapter first reviews Daphnias unique biological attributes that make it ideal for such an expansion of research efforts. These attributes include a long tradition of ecological, evolutionary and toxicological study, culminating in the benefits provided by emerging genomic tools.

Localization of the Genetic Determinants of Meiosis Suppression in Daphnia pulex

Although ∼1 in 10,000 animal species is capable of parthenogenetic reproduction, the evolutionary causes and consequences of such transitions remain uncertain. The microcrustacean Daphnia pulex provides a potentially powerful tool for investigating these issues because lineages that are obligately asexual in terms of female function can nevertheless transmit meiosis-suppressing genes to sexual populations via haploid sperm produced by environmentally induced males. The application of association mapping to a wide geographic collection of D. pulex clones suggests that sex-limited meiosis suppression in D. pulex has spread westward from a northeastern glacial refugium, conveyed by a dominant epistatic interaction among the products of at least four unlinked loci, with one entire chromosome being inherited through males in a nearly nonrecombining fashion. With the enormous set of genomic tools now available for D. pulex, these results set the stage for the determination of the functional underpinnings of the conversion of meiosis to a mitotic-like mode of inheritance.

Meiosis genes in Daphnia pulex and the role of parthenogenesis in genome evolution

BackgroundThousands of parthenogenetic animal species have been described and cytogenetic manifestations of this reproductive mode are well known. However, little is understood about the molecular determinants of parthenogenesis. The Daphnia pulex genome must contain the molecular machinery for different reproductive modes: sexual (both male and female meiosis) and parthenogenetic (which is either cyclical or obligate). This feature makes D. pulex an ideal model to investigate the genetic basis of parthenogenesis and its consequences for gene and genome evolution. Here we describe the inventory of meiotic genes and their expression patterns during meiotic and parthenogenetic reproduction to help address whether parthenogenesis uses existing meiotic and mitotic machinery, or whether novel processes may be involved.ResultsWe report an inventory of 130 homologs representing over 40 genes encoding proteins with diverse roles in meiotic processes in the genome of D. pulex. Many genes involved in cell cycle regulation and sister chromatid cohesion are characterized by expansions in copy number. In contrast, most genes involved in DNA replication and homologous recombination are present as single copies. Notably, RECQ2 (which suppresses homologous recombination) is present in multiple copies while DMC1 is the only gene in our inventory that is absent in the Daphnia genome. Expression patterns for 44 gene copies were similar during meiosis versus parthenogenesis, although several genes displayed marked differences in expression level in germline and somatic tissues.ConclusionWe propose that expansions in meiotic gene families in D. pulex may be associated with parthenogenesis. Taking into account our findings, we provide a mechanistic model of parthenogenesis, highlighting steps that must differ from meiosis including sister chromatid cohesion and kinetochore attachment.

Profiling sex-biased gene expression during parthenogenetic reproduction in Daphnia pulex

BackgroundSexual reproduction is a core biological function that is conserved throughout eukaryotic evolution, yet breeding systems are extremely variable. Genome-wide comparative studies can be effectively used to identify genes and regulatory patterns that are constrained to preserve core functions from those that may help to account for the diversity of animal reproductive strategies. We use a custom microarray to investigate gene expression in males and two reproductive stages of females in the crustacean Daphnia pulex. Most Daphnia species reproduce by cyclical parthenogenesis, alternating between sexual and clonal reproduction. Both sex determination and the switch in their mode of reproduction is environmentally induced, making Daphnia an interesting comparative system for the study of sex-biased and reproductive genes.ResultsPatterns of gene expression in females and males reveal that 50% of assayed transcripts show some degree of sex-bias. Female-biased transcription is enriched for translation, metabolic and regulatory genes associated with development. Male-biased expression is enriched for cuticle and protease function. Comparison with well studied arthropods such as Drosophila melanogaster and Anopheles gambiae suggests that female-biased patterns tend to be conserved, whereas male-biased genes are evolving faster in D. pulex. These findings are based on the proportion of female-biased, male-biased, and unbiased genes that share sequence similarity with proteins in other animal genomes.ConclusionSome transcriptional differences between males and females appear to be conserved across Arthropoda, including the rapid evolution of male-biased genes which is observed in insects and now in a crustacean. Yet, novel patterns of male-biased gene expression are also uncovered. This study is an important first step towards a detailed understanding of the genetic basis and evolution of parthenogenesis, environmental sex determination, and adaptation to aquatic environments.