Intron size minimisation in teleosts.

BACKGROUND: Spliceosomal introns are parts of primary transcripts that are removed by RNA splicing. Although introns apparently do not contribute to the function of the mature transcript, in vertebrates they comprise the majority of the transcribed region increasing the metabolic cost of transcription. The persistence of long introns across evolutionary time suggests functional roles that can offset this metabolic cost. The teleosts comprise one of the largest vertebrate clades. They have unusually compact and variable genome sizes and provide a suitable system for analysing intron evolution. RESULTS: We have analysed intron lengths in 172 vertebrate genomes and show that teleost intron lengths are relatively short, highly variable and bimodally distributed. Introns that were long in teleosts were also found to be long in mammals and were more likely to be found in regulatory genes and to contain conserved sequences. Our results argue that intron length has decreased in parallel in a non-random manner throughout teleost evolution and represent a deviation from the ancestral state. CONCLUSION: Our observations indicate an accelerated rate of intron size evolution in the teleosts and that teleost introns can be divided into two classes by their length. Teleost intron sizes have evolved primarily as a side-effect of genome size evolution and small genomes are dominated by short introns (<256 base pairs). However, a non-random subset of introns has resisted this process across the teleosts and these are more likely have functional roles in all vertebrate clades.

Complete loss of the MHC II pathway in an anglerfish, Lophius piscatorius.

Genome studies in fish provide evidence for the adaptability of the vertebrate immune system, revealing alternative immune strategies. The reported absence of the major compatibility complex (MHC) class II pathway components in certain species of pipefish (genus Syngnathus) and cod-like fishes (order Gadiformes) is of particular interest. The MHC II pathway is responsible for immunization and defence against extracellular threats through the presentation of exogenous peptides to T helper cells. Here, we demonstrate the absence of all genes encoding MHC II components (CD4, CD74 A/B, and both classical and non-classical MHC II α/ß) in the genome of an anglerfish, Lophius piscatorius, indicating loss of the MHC II pathway. By contrast, it has previously been reported that another anglerfish, Antennarius striatus, retains all MHC II genes, placing the loss of MHC II in the Lophius clade to their most recent common ancestor. In the three taxa where MHC II loss has occurred, the gene loss has been restricted to four or five core MHC II components, suggesting that, in teleosts, only these genes have functions that are restricted to the MHC II pathway.

Intrinsic transition of embryonic stem-cell differentiation into neural progenitors.

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

A continuum of transcriptional identities visualized by combinatorial fluorescent in situ hybridization.

Oligonucleotide-based fluorescent in situ hybridization (FISH) coupled with high-resolution high-sensitivity microscopy allows the visualization of single RNA molecules within fixed cells and tissues as distinct foci. We show here that combinatorial labeling of RNA molecules with several fluorescent dyes extends the number of genes that can be targeted simultaneously beyond the number of fluorophores used. This approach also inherently validates the identification of transcripts reducing false positive counts. We have used combinatorial FISH and image analysis to measure the transcript densities of six genes using three fluorophores. This has allowed us to visualize the endothelial maturation of lateral mesoderm in an in vitro ES differentiation assay from a single snapshot of molecular identities. Our observations show that, under these specific conditions, endothelial maturation follows a homogeneous course with a gradual increase in expression of Cdh5 and a concomitant loss of early transcription factors, arguing that maturation is governed in a generally deterministic manner. This methodology is limited by the number of fluorophores that can be used and by the available microscopic resolution, but currently available equipment should allow the visualization of transcripts from 10 or more genes simultaneously.

DNA methylation restricts lineage-specific functions of transcription factor Gata4 during embryonic stem cell differentiation.

DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.

A competitive protein interaction network buffers Oct4-mediated differentiation to promote pluripotency in embryonic stem cells.

Pluripotency in embryonic stem cells is maintained through the activity of a small set of transcription factors centred around Oct4 and Nanog, which control the expression of 'self-renewal' and 'differentiation' genes. Here, we combine single-cell quantitative immunofluorescence microscopy and gene expression analysis, together with theoretical modelling, to investigate how the activity of those factors is regulated. We uncover a key role for post-translational regulation in the maintenance of pluripotency, which complements the well-established transcriptional regulatory layer. Specifically, we find that the activity of a network of protein complexes involving Nanog, Oct4, Tcf3, and ß-catenin suffices to account for the behavior of ES cells under different conditions. Our results suggest that the function of the network is to buffer the transcriptional activity of Oct4, which appears to be the main determinant to exit pluripotency. The protein network explains the mechanisms underlying the gain and loss of function in different mutants, and brings us closer to a full understanding of the molecular basis of pluripotency.

Exploring the potential of mRNA for taxonomic delineation of marine benthic eukaryotes.

Direct sequencing of mRNA isolated from environmental samples has been commonly used to analyze the functional activity of ambient communities and, occasionally used for taxonomic identification. Here we assess the viability of using mRNA for investigating the species composition of marine benthic eukaryotes. Total RNA was extracted from sediments sampled close to fish farms and mRNA was sequenced after poly-A enrichment on an Illumina MiSeq sequencer. We investigated the origin of both raw reads and assembled contigs by aligning them to the NCBI non-redundant (nr/nt) nucleotide database. Although sequences were predominantly of eukaryotic origin, the analyses were complicated both by experimental and database artefacts. These issues were addressed by applying filtering procedures that removed the majority of ambiguous sequences from downstream analyses. These processes resulted in a set of 436 high-confidence contigs, the vast majority of which mapped to benthic organisms. Our alignments were dominated by annelids, consistent with burrowing groups found in a parallel morphological analysis. This study shows that it is possible to obtain adequate taxonomic information from the RNA of an eukaryotic community from a limited sample at a moderate cost, demonstrates how both laboratory and in silico artefacts can be overcome through appropriate bioinformatic procedures, and finally highlights some of the drawbacks and caveats of using NCBI as a reference database for such a dataset.

Necdin restricts proliferation of hematopoietic stem cells during hematopoietic regeneration.

Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndn as being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndn show accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.

Generation and Profiling of 2,135 Human ESC Lines for the Systematic Analyses of Cell States Perturbed by Inducing Single Transcription Factors.

Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.

The mitochondrial transcriptome of the anglerfish Lophius piscatorius.

OBJECTIVE: Analyze key features of the anglerfish Lophius piscatorius mitochondrial transcriptome based on high-throughput total RNA sequencing. RESULTS: We determined the complete mitochondrial DNA and corresponding transcriptome sequences of L. piscatorius. Key features include highly abundant mitochondrial ribosomal RNAs (10-100 times that of mRNAs), and that cytochrome oxidase mRNAs appeared > 5 times more abundant than both NADH dehydrogenase and ATPase mRNAs. Unusual for a vertebrate mitochondrial mRNA, the polyadenylated COI mRNA was found to harbor a 75 nucleotide 3' untranslated region. The mitochondrial genome expressed several non-canonical genes, including the long noncoding RNAs lncCR-H, lncCR-L and lncCOI. Whereas lncCR-H and lncCR-L mapped to opposite strands in a non-overlapping organization within the control region, lncCOI appeared novel among vertebrates. We found lncCOI to be a highly abundant mitochondrial RNA in antisense to the COI mRNA. Finally, we present the coding potential of a humanin-like peptide within the large subunit ribosomal RNA.

Profiling DNA methylation patterns of zebrafish liver associated with parental high dietary arachidonic acid.

Diet has been shown to influence epigenetic key players, such as DNA methylation, which can regulate the gene expression potential in both parents and offspring. Diets enriched in omega-6 and deficient in omega-3 PUFAs (low dietary omega-3/omega-6 PUFA ratio), have been associated with the promotion of pathogenesis of diseases in humans and other mammals. In this study, we investigated the impact of increased dietary intake of arachidonic acid (ARA), a physiologically important omega-6 PUFA, on 2 generations of zebrafish. Parental fish were fed either a low or a high ARA diet, while the progeny of both groups were fed the low ARA diet. We screened for DNA methylation on single base-pair resolution using reduced representation bisulfite sequencing (RRBS). The DNA methylation profiling revealed significant differences between the dietary groups in both parents and offspring. The majority of differentially methylated loci associated with high dietary ARA were found in introns and intergenic regions for both generations. Common loci between the identified differentially methylated loci in F0 and F1 livers were reported. We described overlapping gene annotations of identified methylation changes with differential expression, but based on a small number of overlaps. The present study describes the diet-associated methylation profiles across genomic regions, and it demonstrates that parental high dietary ARA modulates DNA methylation patterns in zebrafish liver.

A mitochondrial long noncoding RNA in atlantic cod harbors complex heteroplasmic tandem repeat motifs.

A heteroplasmic tandem repeat (HTR) array occupies 100 to 300 bp of the mitochondrial DNA control region in the Atlantic cod, and recently we noted that the repeat appeared integrated in a polyadenylated mitochondrial long noncoding RNA. Here we provide a more detailed analysis of the mitochondrial HTR in the mitochondrial genome of 134 Atlantic cod specimens. We report all specimens to harbor mitochondrial HTRs in the control region, and identified 26 distinct variants among the 402 repeat motifs assessed. Whereas most specimens contained HTR profiles of 2-5 copies consisting of the same 40-bp motif, 22 specimens showed compound HTR arrays of at least two types of motifs present in the same mitochondrial DNA molecule. We found HTR profiles to be highly conserved between different tissue types of a single individual, and strictly maternally inherited in a mating experiment between parental Atlantic cod expressing different HTR profiles and array motifs. We conclude that mitochondrial heteroplasmy in the control region is very common in Atlantic cod, and results in length heterogenity of the long noncoding RNA lncCR-H.

Array-based functional screening for genes that regulate vascular endothelial differentiation of Flk1-positive progenitors derived from embryonic stem cells.

Functional genomics is a central topic of current biological research, where a reverse genetic approach is one of the most promising strategies to discover functions of novel genes. Such an approach requires high-throughput methodologies to assess biological functions for a huge number of genes. We have developed a transfection array that permits parallel introduction of multiple plasmids separately into living cells. The feasibility of this array was examined in an assay system. Eleven genes were over-expressed alone, or in combination in vascular progenitors derived from embryonic stem cells. Endothelial differentiation of the cells was monitored through a stably transformed EGFP reporter construct that is expressed only in endothelial cells. Transcriptional activators that promote endothelial differentiation, such as Ets1 and Sox7, were identified. In addition, the assays also revealed an inhibitory effect on endothelial differentiation by several of the factors. These results demonstrate the feasibility of the transfection array for use in cell-based, high-throughput functional assays.

Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells.

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2R alpha (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+ Sox17+ definitive endoderm and Gsc- Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.

Early hematopoietic and vascular development in the chick.

The field of hematopoietic and vascular developmental research owes its origin to the chick embryo. Many key concepts, such as the hematopoietic stem cell, hemangioblast and hemogenic endothelium, were first proposed in this model organism. Genetically tractable models have gradually replaced the chick in the past two decades. However, advances in comparative genomics, transcriptomics and promoteromics promise a re-emergence of the chick embryo as a powerful model for hematopoietic/vascular research. This review summarizes the current status of our understanding of early blood/vascular development in the chick, focusing primarily on the processes of primitive hematopoiesis and early vascular network formation in the extraembryonic and lateral plate mesoderm territories. Emphasis is given to ontological and molecular association between the blood and endothelial cells and to the evolutionary relationship between the hemangioblasts, common precursors for the blood and endothelial lineages, and the coelomic epithelial lining cells. Links between early blood/vascular development and later definitive hematopoiesis are also discussed. Finally, potential applications of the chick model for comparative and omics-level studies of the blood/vascular system are highlighted.

Parental micronutrient deficiency distorts liver DNA methylation and expression of lipid genes associated with a fatty-liver-like phenotype in offspring.

Micronutrient status of parents can affect long term health of their progeny. Around 2 billion humans are affected by chronic micronutrient deficiency. In this study we use zebrafish as a model system to examine morphological, molecular and epigenetic changes in mature offspring of parents that experienced a one-carbon (1-C) micronutrient deficiency. Zebrafish were fed a diet sufficient, or marginally deficient in 1-C nutrients (folate, vitamin B12, vitamin B6, methionine, choline), and then mated. Offspring livers underwent histological examination, RNA sequencing and genome-wide DNA methylation analysis. Parental 1-C micronutrient deficiency resulted in increased lipid inclusion and we identified 686 differentially expressed genes in offspring liver, the majority of which were downregulated. Downregulated genes were enriched for functional categories related to sterol, steroid and lipid biosynthesis, as well as mitochondrial protein synthesis. Differential DNA methylation was found at 2869 CpG sites, enriched in promoter regions and permutation analyses confirmed the association with parental feed. Our data indicate that parental 1-C nutrient status can persist as locus specific DNA methylation marks in descendants and suggest an effect on lipid utilization and mitochondrial protein translation in F1 livers. This points toward parental micronutrients status as an important factor for offspring health and welfare.

Mitochondrial genome variation of Atlantic cod.

OBJECTIVE: The objective of this study was to analyse intraspecific sequence variation of Atlantic cod mitochondrial DNA, based on a comprehensive collection of completely sequenced mitochondrial genomes. RESULTS: We determined the complete mitochondrial DNA sequence of 124 cod specimens from the eastern and western part of the species' distribution range in the North Atlantic Ocean. All specimens harboured a unique mitochondrial DNA haplotype. Nine hundred and fifty-two polymorphic sites were identified, including 109 non-synonymous sites within protein coding regions. Eighteen variable sites were identified as indels, exclusively distributed in structural RNA genes and non-coding regions. Phylogeographic analyses based on 156 available cod mitochondrial genomes did not reveal a clear structure. There was a lack of mitochondrial genetic differentiation between two ecotypes of cod in the eastern North Atlantic, but eastern and western cod were differentiated and mitochondrial genome diversity was higher in the eastern than the western Atlantic, suggesting deviating population histories. The geographic distribution of mitochondrial genome variation seems to be governed by demographic processes and gene flow among ecotypes that are otherwise characterized by localized genomic divergence associated with chromosomal inversions.

ALCAM (CD166) is a surface marker for early murine cardiomyocytes.

ALCAM (activated leukocyte cell adhesion molecule, CD166) belongs to the immunoglobulin superfamily and is involved in axon guidance, hematopoiesis, immune response and tumor metastasis. During embryogenesis, mRNA encoding ALCAM was expressed in the cardiac crescent and the neural groove at embryonic day (E) 7.75 and predominately in the tubular heart at E8.5. A newly generated monoclonal antibody against the ALCAM molecule (ALC-48) exclusively stained cardiomyocytes at E8.25-10.5. However, ALCAM expression was lost by cardiomyocytes by E12.5 and its expression shifts to a variety of organs during later stages. ALCAM was found to be a prominent surface marker for cardiomyocytes in early embryonic hearts. The transient expression of ALCAM during early developmental stages marks specific developmental stages in cardiomyocyte differentiation.

Parental vitamin deficiency affects the embryonic gene expression of immune-, lipid transport- and apolipoprotein genes.

World Health Organization is concerned for parental vitamin deficiency and its effect on offspring health. This study examines the effect of a marginally dietary-induced parental one carbon (1-C) micronutrient deficiency on embryonic gene expression using zebrafish. Metabolic profiling revealed a reduced 1-C cycle efficiency in F0 generation. Parental deficiency reduced the fecundity and a total of 364 genes were differentially expressed in the F1 embryos. The upregulated genes (53%) in the deficient group were enriched in biological processes such as immune response and blood coagulation. Several genes encoding enzymes essential for the 1-C cycle and for lipid transport (especially apolipoproteins) were aberrantly expressed. We show that a parental diet deficient in micronutrients disturbs the expression in descendant embryos of genes associated with overall health, and result in inherited aberrations in the 1-C cycle and lipid metabolism. This emphasises the importance of parental micronutrient status for the health of the offspring.

Longitudinal Analysis of DNA Methylation in CD34+ Hematopoietic Progenitors in Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease progression and how DNA methylation contributes to MDS remain unclear. We analyzed global DNA methylation in purified CD34+ hematopoietic progenitors from MDS patients undergoing multiple rounds of AZA treatment. Differential methylation between MDS phenotypes was observed primarily at developmental regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high-risk MDS patients. Haploinsufficiency in mice of one of these genes (NR4A2) has been shown to lead to excessive HSC proliferation, and our data suggest that suppression of NR4A2 by DNA methylation may be involved in MDS progression.