Eye-Transcriptome and Genome-Wide Sequencing for Scolecophidia: Implications for Inferring the Visual System of the Ancestral Snake.

Molecular genetic data have recently been incorporated in attempts to reconstruct the ecology of the ancestral snake, though this has been limited by a paucity of data for one of the two main extant snake taxa, the highly fossorial Scolecophidia. Here we present and analyze vision genes from the first eye-transcriptomic and genome-wide data for Scolecophidia, for Anilios bicolor, and A. bituberculatus, respectively. We also present immunohistochemistry data for retinal anatomy and visual opsin-gene expression in Anilios. Analyzed in the context of 19 lepidosaurian genomes and 12 eye transcriptomes, the new genome-wide and transcriptomic data provide evidence for a much more reduced visual system in Anilios than in non-scolecophidian (=alethinophidian) snakes and in lizards. In Anilios, there is no evidence of the presence of 7 of the 12 genes associated with alethinophidian photopic (cone) phototransduction. This indicates extensive gene loss and many of these candidate gene losses occur also in highly fossorial mammals with reduced vision. Although recent phylogenetic studies have found evidence for scolecophidian paraphyly, the loss in Anilios of visual genes that are present in alethinophidians implies that the ancestral snake had a better-developed visual system than is known for any extant scolecophidian.

Characterization of hormone-producing cell types in the teleost pituitary gland using single-cell RNA-seq.

The pituitary is the vertebrate endocrine gland responsible for the production and secretion of several essential peptide hormones. These, in turn, control many aspects of an animal's physiology and development, including growth, reproduction, homeostasis, metabolism, and stress responses. In teleost fish, each hormone is presumably produced by a specific cell type. However, key details on the regulation of, and communication between these cell types remain to be resolved. We have therefore used single-cell sequencing to generate gene expression profiles for 2592 and 3804 individual cells from the pituitaries of female and male adult medaka (Oryzias latipes), respectively. Based on expression profile clustering, we define 15 and 16 distinct cell types in the female and male pituitary, respectively, of which ten are involved in the production of a single peptide hormone. Collectively, our data provide a high-quality reference for studies on pituitary biology and the regulation of hormone production, both in fish and in vertebrates in general.

3D Atlas of the Pituitary Gland of the Model Fish Medaka (Oryzias latipes).

In vertebrates, the anterior pituitary plays a crucial role in regulating several essential physiological processes via the secretion of at least seven peptide hormones by different endocrine cell types. Comparative and comprehensive knowledge of the spatial distribution of those endocrine cell types is required to better understand their physiological functions. Using medaka as a model and several combinations of multi-color fluorescence in situ hybridization, we present the first 3D atlas revealing the gland-wide distribution of seven endocrine cell populations: lactotropes, thyrotropes, Lh and Fsh gonadotropes, somatotropes, and pomca-expressing cells (corticotropes and melanotropes) in the anterior pituitary of a teleost fish. By combining in situ hybridization and immunofluorescence techniques, we deciphered the location of corticotropes and melanotropes within the pomca-expressing cell population. The 3D localization approach reveals sexual dimorphism of tshba-, pomca-, and lhb-expressing cells in the adult medaka pituitary. Finally, we show the existence of bi-hormonal cells co-expressing lhb-fshb, fshb-tshba and lhb-sl using single-cell transcriptomics analysis and in situ hybridization. This study offers a solid basis for future comparative studies of the teleost pituitary and its functional plasticity.

Swimming exercise enhances brain plasticity in fish.

It is well-established that sustained exercise training can enhance brain plasticity and boost cognitive performance in mammals, but this phenomenon has not received much attention in fish. The aim of this study was to determine whether sustained swimming exercise can enhance brain plasticity in juvenile Atlantic salmon. Brain plasticity was assessed by both mapping the whole telencephalon transcriptome and conducting telencephalic region-specific microdissections on target genes. We found that 1772 transcripts were differentially expressed between the exercise and control groups. Gene ontology (GO) analysis identified 195 and 272 GO categories with a significant overrepresentation of up- or downregulated transcripts, respectively. A multitude of these GO categories was associated with neuronal excitability, neuronal signalling, cell proliferation and neurite outgrowth (i.e. cognition-related neuronal markers). Additionally, we found an increase in proliferating cell nuclear antigen (pcna) after both three and eight weeks of exercise in the equivalent to the hippocampus in fish. Furthermore, the expression of the neural plasticity markers synaptotagmin (syt) and brain-derived neurotrophic factor (bdnf) were also increased due to exercise in the equivalent to the lateral septum in fish. In conclusion, this is the first time that swimming exercise has been directly linked to increased telencephalic neurogenesis and neural plasticity in a teleost, and our results pave the way for future studies on exercise-induced neuroplasticity in fish.

Author Correction: Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Rapid de novo assembly of the European eel genome from nanopore sequencing reads.

We have sequenced the genome of the endangered European eel using the MinION by Oxford Nanopore, and assembled these data using a novel algorithm specifically designed for large eukaryotic genomes. For this 860 Mbp genome, the entire computational process takes two days on a single CPU. The resulting genome assembly significantly improves on a previous draft based on short reads only, both in terms of contiguity (N50 1.2 Mbp) and structural quality. This combination of affordable nanopore sequencing and light weight assembly promises to make high-quality genomic resources accessible for many non-model plants and animals.

Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors.

The rapidly growing world population has a greatly increasing demand for plant biomass, thus creating a great interest in the development of methods to enhance the growth and biomass accumulation of crop species. In this study, we used zinc finger artificial transcription factor (ZF-ATF)-mediated genome interrogation to manipulate the growth characteristics and biomass of Arabidopsis plants. We describe the construction of two collections of Arabidopsis lines expressing fusions of three zinc fingers (3F) to the transcriptional repressor motif EAR (3F-EAR) or the transcriptional activator VP16 (3F-VP16), and the characterization of their growth characteristics. In total, six different 3F-ATF lines with a consistent increase in rosette surface area (RSA) of up to 55% were isolated. For two lines we demonstrated that 3F-ATF constructs function as dominant in trans acting causative agents for an increase in RSA and biomass, and for five larger plant lines we have investigated 3F-ATF induced transcriptomic changes. Our results indicate that genome interrogation can be used as a powerful tool for the manipulation of plant growth and biomass and that it might supply novel cues for the discovery of genes and pathways involved in these properties.

De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing.

Background: The introduction of the MinION TM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing. It has been shown that the nanopore sequence data, in combination with other sequencing technologies, is highly useful for accurate annotation of all genes in the genome. However, it also offers great potential for de novo assembly of complex genomes without using other technologies. In this manuscript we used nanopore sequencing as a tool to classify yeast strains. Methods: We compared various technical and software developments for the nanopore sequencing protocol, showing that the R9 chemistry is, as predicted, higher in quality than R7.3 chemistry. The R9 chemistry is an essential improvement for assembly of the extremely AT-rich mitochondrial genome. Results: In this study, we used this new technology to sequence and de novo assemble the genome of a recently isolated ethanologenic yeast strain, and compared the results with those obtained by classical Illumina short read sequencing. This strain was originally named Candida vartiovaarae ( Torulopsis vartiovaarae) based on ribosomal RNA sequencing. We show that the assembly using nanopore data is much more contiguous than the assembly using short read data. Conclusions: The mitochondrial and chromosomal genome sequences showed that our strain is clearly distinct from other yeast taxons and most closely related to published Cyberlindnera species. In conclusion, MinION-mediated long read sequencing can be used for high quality de novo assembly of new eukaryotic microbial genomes.

Snake Genome Sequencing: Results and Future Prospects.

Snake genome sequencing is in its infancy-very much behind the progress made in sequencing the genomes of humans, model organisms and pathogens relevant to biomedical research, and agricultural species. We provide here an overview of some of the snake genome projects in progress, and discuss the biological findings, with special emphasis on toxinology, from the small number of draft snake genomes already published. We discuss the future of snake genomics, pointing out that new sequencing technologies will help overcome the problem of repetitive sequences in assembling snake genomes. Genome sequences are also likely to be valuable in examining the clustering of toxin genes on the chromosomes, in designing recombinant antivenoms and in studying the epigenetic regulation of toxin gene expression.

Changes in ovarian gene expression profiles and plasma hormone levels in maturing European eel (Anguilla anguilla); Biomarkers for broodstock selection.

Complete sexual maturation of European eels (Anguilla anguilla) in captivity can only be achieved via injections with gonadotropins. For female eels this procedure takes 4-6months and the response ranges from "unresponsive" to final maturation and ovulation. Reproductive success could be significantly increased via early selection of responders based on predictive markers and minimally invasive sampling methods. To get a better understanding of the genetic background of ovarian maturation of the European eel we performed a pilot deep-sequencing transcriptome analysis of ovarian tissue derived from a yellow eel, a prepubertal silver eel and a post-spawning matured eel. Two key players in steroidogenesis were strongly correlated with advanced sexual maturation, namely P450c17 and liver receptor homolog-1, suggesting that blood plasma steroids might qualify as minimally invasive markers for early detection of responders. Since the predictive value of plasma sex steroid levels for final maturation of the European eel had not yet been carefully examined, we performed an extensive artificial maturation trial. Farmed silver eels were treated with pituitary extracts and sampled at multiple time intervals. Expression of steroidogenesis-related genes in ovarian tissue of responding and non-responding eels after four weekly injections with pituitary extract was compared using a custom-built microarray and RNAseq. Increased expression of 17ß-hsd1 was strongly linked to sexual maturation. Blood plasma levels of sex steroids were measured using ELISAs. We show that a 2.5-fold increase in blood-plasma estradiol level after 4 weekly pituitary extract injections is a strong predictor of final sexual maturation of female European eel.

The Agrobacterium tumefaciens virulence protein VirE3 is a transcriptional activator of the F-box gene VBF.

During Agrobacterium tumefaciens-mediated transformation of plant cells a part of the tumour-inducing plasmid, T-DNA, is integrated into the host genome. In addition, a number of virulence proteins are translocated into the host cell. The virulence protein VirE3 binds to the Arabidopsis thaliana pBrp protein, a plant-specific general transcription factor of the TFIIB family. To study a possible role for VirE3 in transcriptional regulation, we stably expressed virE3 in A. thaliana under control of a tamoxifen-inducible promoter. By RNA sequencing we showed that upon expression of virE3 the RNA levels of 607 genes were increased more than three-fold and those of 132 genes decreased more than three-fold. One of the strongly activated genes was that encoding VBF (At1G56250), an F-box protein that may affect the levels of the VirE2 and VIP1 proteins. Using Arabidopsis cell suspension protoplasts we showed that VirE3 stimulates the VBF promoter, especially when co-expressed with pBrp. Although pBrp is localized at the external surface of plastids, co-expression of VirE3 and pBrp in Arabidopsis cell suspension protoplasts resulted in the accumulation of pBrp in the nucleus. Our results suggest that VirE3 affects the transcriptional machinery of the host cell to favour the transformation process.

Using normalization to resolve RNA-Seq biases caused by amplification from minimal input.

RNA-Seq has become a widely used method to study transcriptomes, and it is now possible to perform RNA-Seq on almost any sample. Nevertheless, samples obtained from small cell populations are particularly challenging, as biases associated with low amounts of input RNA can have strong and detrimental effects on downstream analyses. Here we compare different methods to normalize RNA-Seq data obtained from minimal input material. Using RNA from isolated medaka pituitary cells, we have amplified material from six samples before sequencing. Both synthetic and real data are used to evaluate different normalization methods to obtain a robust and reliable pipeline for analysis of RNA-Seq data from samples with very limited input material. The analysis outlined here shows that quantile normalization outperforms other more commonly used normalization procedures when using amplified RNA as input and will benefit researchers employing low amounts of RNA in similar experiments.

Genome Sequence of the Octopine-Type Agrobacterium tumefaciens Strain Ach5.

We have sequenced the complete genome of the plant pathogen Agrobacterium tumefaciens strain LBA4213, a derivative of the wild-type strain A. tumefaciens Ach5 and the ancestor of A. tumefaciens strain LBA4404 used in genetic engineering. The genome consists of a circular chromosome and a linear chromosome, as well as a megaplasmid and a tumor-inducing plasmid.

The king cobra genome reveals dynamic gene evolution and adaptation in the snake venom system.

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue-specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection.

The Burmese python genome reveals the molecular basis for extreme adaptation in snakes.

Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

The pituitary gland of the European eel reveals massive expression of genes involved in the melanocortin system.

Hormones secreted from the pituitary gland regulate important processes such as development, growth and metabolism, reproduction, water balance, and body pigmentation. Synthesis and secretion of pituitary hormones are regulated by different factors from the hypothalamus, but also through feedback mechanisms from peripheral organs, and from the pituitary itself. In the European eel extensive attention has been directed towards understanding the different components of the brain-pituitary-gonad axis, but little is known about the regulation of upstream processes in the pituitary gland. In order to gain a broader mechanistic understanding of the eel pituitary gland, we have performed RNA-seq transcriptome profiling of the pituitary of prepubertal female silver eels. RNA-seq reads generated on the Illumina platform were mapped to the recently assembled European eel genome. The most abundant transcript in the eel pituitary codes for pro-opiomelanocortin, the precursor for hormones of the melanocortin system. Several genes putatively involved in downstream processing of pro-opiomelanocortin were manually annotated, and were found to be highly expressed, both by RNA-seq and by qPCR. The melanocortin system, which affects skin color, energy homeostasis and in other teleosts interacts with the reproductive system, has so far received limited attention in eels. However, since up to one third of the silver eel pituitary's mRNA pool encodes pro-opiomelanocortin, our results indicate that control of the melanocortin system is a major function of the eel pituitary.

Parallel deep transcriptome and proteome analysis of zebrafish larvae.

BACKGROUND: Sensitivity and throughput of transcriptomic and proteomic technologies have advanced tremendously in recent years. With the use of deep sequencing of RNA samples (RNA-seq) and mass spectrometry technology for protein identification and quantitation, it is now feasible to compare gene and protein expression on a massive scale and for any organism for which genomic data is available. Although these technologies are currently applied to many research questions in various model systems ranging from cell cultures to the entire organism level, there are few comparative studies of these technologies in the same system, let alone on the same samples. Here we present a comparison between gene and protein expression in embryos of zebrafish, which is an upcoming model in disease studies. RESULTS: We compared Agilent custom made expression microarrays with Illumina deep sequencing for RNA analysis, showing as expected a high degree of correlation of expression of a common set of 18,230 genes. Gene expression was also found to correlate with the abundance of 963 distinct proteins, with several categories of genes as exceptions. These exceptions include ribosomal proteins, histones and vitellogenins, for which biological and technical explanations are discussed. CONCLUSIONS: By comparing state of the art transcriptomic and proteomic technologies on samples derived from the same group of organisms we have for the first time benchmarked the differences in these technologies with regard to sensitivity and bias towards detection of particular gene categories in zebrafish. Our datasets submitted to public repositories are a good starting point for researchers interested in disease progression in zebrafish at a stage of development highly suited for high throughput screening technologies.

Deep RNA sequencing of the skeletal muscle transcriptome in swimming fish.

Deep RNA sequencing (RNA-seq) was performed to provide an in-depth view of the transcriptome of red and white skeletal muscle of exercised and non-exercised rainbow trout (Oncorhynchus mykiss) with the specific objective to identify expressed genes and quantify the transcriptomic effects of swimming-induced exercise. Pubertal autumn-spawning seawater-raised female rainbow trout were rested (n = 10) or swum (n = 10) for 1176 km at 0.75 body-lengths per second in a 6,000-L swim-flume under reproductive conditions for 40 days. Red and white muscle RNA of exercised and non-exercised fish (4 lanes) was sequenced and resulted in 15-17 million reads per lane that, after de novo assembly, yielded 149,159 red and 118,572 white muscle contigs. Most contigs were annotated using an iterative homology search strategy against salmonid ESTs, the zebrafish Danio rerio genome and general Metazoan genes. When selecting for large contigs (>500 nucleotides), a number of novel rainbow trout gene sequences were identified in this study: 1,085 and 1,228 novel gene sequences for red and white muscle, respectively, which included a number of important molecules for skeletal muscle function. Transcriptomic analysis revealed that sustained swimming increased transcriptional activity in skeletal muscle and specifically an up-regulation of genes involved in muscle growth and developmental processes in white muscle. The unique collection of transcripts will contribute to our understanding of red and white muscle physiology, specifically during the long-term reproductive migration of salmonids.

First draft genome sequence of the Japanese eel, Anguilla japonica.

The Japanese eel is a much appreciated research object and very important for Asian aquaculture; however, its genomic resources are still limited. We have used a streamlined bioinformatics pipeline for the de novo assembly of the genome sequence of the Japanese eel from raw Illumina sequence reads. The total assembled genome has a size of 1.15 Gbp, which is divided over 323,776 scaffolds with an N50 of 52,849 bp, a minimum scaffold size of 200 bp and a maximum scaffold size of 1.14 Mbp. Direct comparison of a representative set of scaffolds revealed that all the Hox genes and their intergenic distances are almost perfectly conserved between the European and the Japanese eel. The first draft genome sequence of an organism strongly catalyzes research progress in multiple fields. Therefore, the Japanese eel genome sequence will provide a rich resource of data for all scientists working on this important fish species.

Conserved expression signatures between medaka and human pigment cell tumors.

Aberrations in gene expression are a hallmark of cancer cells. Differential tumor-specific transcript levels of single genes or whole sets of genes may be critical for the neoplastic phenotype and important for therapeutic considerations or useful as biomarkers. As an approach to filter out such relevant expression differences from the plethora of changes noted in global expression profiling studies, we searched for changes of gene expression levels that are conserved. Transcriptomes from massive parallel sequencing of different types of melanoma from medaka were generated and compared to microarray datasets from zebrafish and human melanoma. This revealed molecular conservation at various levels between fish models and human tumors providing a useful strategy for identifying expression signatures strongly associated with disease phenotypes and uncovering new melanoma molecules.