Variation of TMDs by Transcript Variation and Alternative Splicing

<p>Variation of TMDs by Transcript Variation and Alternative Splicing</p

Variation of Signal Peptide by Transcript Variation and Alternative Splicing

<p>Sixteen TUs are represented in both categories. These TUs contain multiple transcripts using signal peptide coding regions generated from distinct regions of the genome while alternative transcripts within the same TU exclude these signal peptide coding regions altogether. Thin green and red bars across exons represent the location of the start and stop codons respectively. An orange dot following the start codon represents the presence of N-terminal signal peptide, while green blocks show the genomic localization of the predicted transmembrane domain features within exons.</p

Categories of Membrane Organization Observed in the 782 High-Confidence Variable TUs

<p>In total, 753 TUs occurred in two different membrane organization classes, while 29 TU's occurred in more than two membrane organization classes, and are present in a number of variation categories.</p

Systematic analysis of transcription start sites in avian development

<div><p>Cap Analysis of Gene Expression (CAGE) in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs) and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM]) were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using <i>Brachyury</i> as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals). By facilitating promoter-based molecular analysis and genetic manipulation, our work also underscores the value of avian models in unravelling the complex regulatory mechanism of cell lineage specification during amniote development.</p></div

Transcriptome Tomography for Brain Analysis in the Web-Accessible Anatomical Space

<div><p>Increased information on the encoded mammalian genome is expected to facilitate an integrated understanding of complex anatomical structure and function based on the knowledge of gene products. Determination of gene expression-anatomy associations is crucial for this understanding. To elicit the association in the three-dimensional (3D) space, we introduce a novel technique for comprehensive mapping of endogenous gene expression into a web-accessible standard space: Transcriptome Tomography. The technique is based on conjugation of sequential tissue-block sectioning, all fractions of which are used for molecular measurements of gene expression densities, and the block- face imaging, which are used for 3D reconstruction of the fractions. To generate a 3D map, tissues are serially sectioned in each of three orthogonal planes and the expression density data are mapped using a tomographic technique. This rapid and unbiased mapping technique using a relatively small number of original data points allows researchers to create their own expression maps in the broad anatomical context of the space. In the first instance we generated a dataset of 36,000 maps, reconstructed from data of 61 fractions measured with microarray, covering the whole mouse brain (ViBrism: <a href="http://vibrism.riken.jp/3dviewer/ex/index.html" target="_blank">http://vibrism.riken.jp/3dviewer/ex/index.html</a>) in one month. After computational estimation of the mapping accuracy we validated the dataset against existing data with respect to the expression location and density. To demonstrate the relevance of the framework, we showed disease related expression of Huntington’s disease gene and <i>Bdnf</i>. Our tomographic approach is applicable to analysis of any biological molecules derived from frozen tissues, organs and whole embryos, and the maps are spatially isotropic and well suited to the analysis in the standard space (e.g. Waxholm Space for brain-atlas databases). This will facilitate research creating and using open-standards for a molecular-based understanding of complex structures; and will contribute to new insights into a broad range of biological and medical questions.</p></div

Spatial integration of 3D expression maps into the WHS MRI digital atlas.

<p>(<b>A</b>) <b>A schematic for the integration.</b> E80, the brain volume common to all expression maps in the ViBrism space, (colored in green) was transformed (shown with an arrow) into the brain volume in the Waxholm Space (WHS in gray). (<b>B–D</b>) <b>Integration of Huntington’s disease-related maps.</b> Areas defined by the gene expression of a pathogenetic combination deduced from the previous knowledge, Htt(+)/Bdnf(−), are colored with the Htt expression density in the ViBrism space (a right small panel in B). After the transformation into WHS, the areas are highlighted and colored with anatomical labels based on MRI data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373-Johnson1" target="_blank">[26]</a> (a large panel in B). The areas contain vulnerable regions in this disease as follows: Label 1; Cerebral cortex (anterior rather than posterior, 22% of the volume with label 1), 9; Ventral thalamic nuclei (100%) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373-Dom1" target="_blank">[28]</a>, 15; Globus pallidus (100%) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373-Han1" target="_blank">[17]</a> shown in the panel D, 23; CPu (99%) also seen in the panel C, 24; hippocampus (77%), 37; cerebellum (21%). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s004" target="_blank">Table S3</a> shows % of volumes overlapped with the Htt(+)Bdnf(−) areas in each of 37 anatomical regions labeled in WHS. The MRI T1 and T2* atlases are shown in the rectangular vertical and horizontal planes, respectively.</p

Results for the computational experiment of reconstruction using 1,366 test spheres.

<p>Gene expression that was evenly distributed in one of the test spheres located randomly in the virtual brain of ViBrism was computationally reconstructed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045373#pone.0045373.s006" target="_blank">Text S2</a> for Supporting Methods). A histogram for the number of test spheres with true positive rates (% of TP: percentages of test sphere volumes overlapped with the reconstructed area) is shown. Maps of the reconstructed results (shown in yellow) with the test spheres (in red) are attached. In 2D maps, the 80% cutoff filter was applied to the results of left-upper S panels; otherwise, the reconstructed densities are shown in gray scales. 3D maps are shown with the filter. Approximately one fifth (20.4%) of the test spheres had more than 95% of TP, which is the mode in the histogram, and 94.7% in total had at least 5% of TP as indicated. One of the mode results, the median result (TP = 80%) and one of the poorly reconstructed results (TP<5%) are shown. Only 0.8% of the test spheres resulted in no TP, which was mainly due to the peripheral location of the test spheres in the virtual brain (data not shown).</p

Induction of endogenous Brachyury expression mediated by Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-on.

<p>(A) Chicken-ZENBU view of a 6-kb window on chromosome 3, showing the transcription start site (TSS) location for the <i>Brachyury</i> gene. Small rectangle represents the region shown in (B). (B) Sequences of 4 single guide RNAs (sgRNAs; red underline) located within 300 bp upstream of the <i>Brachyury</i> TSS. Blue: protospacer-adjacent motif (PAM). (C) Electroporated embryos were grown to Hamburger and Hamilton stage 10 (HH10), and sgRNA-expressing territories were assessed by co-electroporated GFP signal. Embryos were then processed for <i>Brachyury</i> in situ hybridization (shown here). (D) Magnified view of mid-region of the embryo, showing ectopic <i>Brachyury</i>+ cells in somites and neural tube.</p

List of rat Rescued CAGE peaks

Coordinates, associated Ensembl genes and human projected CAGE peaks are recorded

Chicken-ZENBU views of transcription start site (TSS) peaks and expression levels.

<p>(A) A robust single TSS peak (arrow) is correctly mapped to the known Ensembl and RefSeq 5′ end of <i>GAPDH</i>. Right panel: zoomed-in view of left panel. (B) Ambiguity in <i>ACTB</i> gene annotation can be resolved using Cap Analysis of Gene Expression (CAGE). (C) The TSS representative of <i>RPL32</i> gene does not confirm either available annotation, suggesting the incorrectness of both gene models. (D) Bar graph of expression values shows <i>NANOG</i> pluripotency gene present at early stages, then down-regulated at later stages. (E) Late stage–specific expression of <i>GFAP</i> gene (Hamburger and Hamilton stage 41 [HH41] and HH45). Samples in the bar graphs are sorted by developmental stage.</p