Comparative analysis of the transcriptome across distant species

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters

Isolation of Specific Neurons from <i>C. elegans</i> Larvae for Gene Expression Profiling

<div><p>Background</p><p>The simple and well-described structure of the <i>C. elegans</i> nervous system offers an unprecedented opportunity to identify the genetic programs that define the connectivity and function of individual neurons and their circuits. A correspondingly precise gene expression map of <i>C. elegans</i> neurons would facilitate the application of genetic methods toward this goal. Here we describe a powerful new approach, SeqCeL (RNA-Seq of <i>C. elegans</i> cells) for producing gene expression profiles of specific larval <i>C. elegans</i> neurons.</p><p>Methods and Results</p><p>We have exploited available GFP reporter lines for FACS isolation of specific larval <i>C. elegans</i> neurons for RNA-Seq analysis. Our analysis showed that diverse classes of neurons are accessible to this approach. To demonstrate the applicability of this strategy to rare neuron types, we generated RNA-Seq profiles of the NSM serotonergic neurons that occur as a single bilateral pair of cells in the <i>C. elegans</i> pharynx. These data detected >1,000 NSM enriched transcripts, including the majority of previously known NSM-expressed genes.</p><p>Significance</p><p>This work offers a simple and robust protocol for expression profiling studies of post-embryonic <i>C. elegans</i> neurons and thus provides an important new method for identifying candidate genes for key roles in neuron-specific development and function.</p></div

GFP reporters validate NSM-enriched RNA-Seq data set.

<p>Transgenic animals expressing promoter::GFP reporter genes for NSM-enriched transcripts. Confocal images of adult hermaphrodites, anterior to left, ventral down. A–B, Bright <i>tph-1::</i>GFP (<i>vsIs45</i>) expression is limited to NSML and NSMR; note dim <i>tph-1::</i>GFP signal in ADF neurons (arrows). C, Cartoon depicting NSM neurons marked with <i>tph-1</i>::GFP in the anterior pharyngeal bulb. <i>Y39B6A.19</i>::GFP (D, E) and <i>F41E7.3</i>::GFP (F, G) are specifically expressed in neurons, including NSM, head neurons and ventral cord motor neurons (asterisks). <i>Y39B6A.19</i>::GFP is also expressed in sensory neuron, PDE (E). Scale bar is 25 microns.</p

Isolation of NSM serotonergic neurons by FACS.

<p>(A) Head region of an adult hermaphrodite depicting the NSML neuron (orange) in the pharynx (green) (WormAtlas). Anterior is to left. (B) Confocal image of NSML in L1 larval stage. Anterior is to left. (C) FACS profile of cells dissociated from L1 stage larvae expressing <i>tph-1::GFP</i> in NSM neurons. Propidium Iodide (PI) marks dead cells (blue). Viable GFP-labeled cells (green) are outlined with the box. Images of FACS-isolated NSM neurons (D) 24 hr, (E), 48 hr and (F) 72 hr after plating. Scale bars are 10 microns.</p

Specific sensory and motor neurons are accessible to isolation by FACS from multiple larval stages.

<p>Primary cultures of L1 stage cells 24 hr after dissociating from transgenic lines expressing (A) <i>mec-4::</i>mCherry to mark ALM and PLM neurons (red) and (B) <i>srh-142p::</i>dsRed to label ADF sensory neurons (red). (C) GABA motor neuron marked with <i>unc-47::</i>mCherry (red) and cultured for 24 hour after dissociating from L4 larval animals. (D) Primary culture of A-class motor neurons marked with <i>unc-4::</i>GFP (green) and isolated by FACS from L2 stage larvae. (E) <i>del-1::</i>GFP labels VB motor neurons (green) in the ventral nerve cord of an L2 stage larva. Anterior to left. (F) FACS profile of cells dissociated from <i>del-1::</i>GFP L2 larvae. Propidium Iodide (PI) marks dead cells (blue). Viable GFP-labeled cells (green) are outlined with the box. Scale bars are 10 microns.</p

Expression of promoter-GFP reporters for transcripts enriched in the NSM RNA-Seq data set.

<p>Genes are ranked according to statistical significance. VNC (Ventral Nerve Cord), DNC (Dorsal Nerve Cord).</p><p>Expression of promoter-GFP reporters for transcripts enriched in the NSM RNA-Seq data set.</p

Isolation of <i>C. elegans</i> neurons from larval animals by Fluorescence Activated Cell Sorting (FACS).

<p>(A) Expression of the pan-neural GFP marker, <i>evIs111</i>, in an L2 stage larval animal. Anterior is to left. (B) Procedure for generating dissociated suspensions of larval cells for primary culture and for isolation of GFP-marked cells (green) by FACS. (C) Primary culture of L1-stage cells 36 hr after plating. Arrows point to elongated processes extending from GFP-labeled neurons. Scatterplot of FACS profile for cells dissociated from the wild-type (N2) reference strain (D) and from the pan-neural <i>evIs111</i> line (E). Propidium Iodide (PI) marks dead cells (blue). GFP-labeled cells (green) were isolated by FACS (outlined with box). The majority (∼90%) of FACS-derived cells (F) are marked with GFP (G) in primary cultures examined within 2 hr of FACS isolation. Note that neurons are loosely attached which likely accounts for the displacement of DIC vs GFP images of individual cells in these micrographs. Scale bars = 10 micron.</p

5′-to-3′ coverage.

<p>(A) The average relative coverage for each library is shown for normalized transcript length. The Y-axis denotes the average coverage of each position in the transcript normalized to the total number of mapped reads. Coverage for coding sequence genes (B) <i>tph-1</i> and (C) <i>bas-1</i> and for (D) a small nuclear RNA (snoRNA) gene, <i>ZK643.9</i>. Lighter vs darker shades of color depict results for independent NSM (blue) and reference (green) samples.</p