A RNA-Seq Analysis of the Rat Supraoptic Nucleus Transcriptome: Effects of Salt Loading on Gene Expression

<div><p>Magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) are highly specialized to release large amounts of arginine vasopressin (Avp) or oxytocin (Oxt) into the blood stream and play critical roles in the regulation of body fluid homeostasis. The MCNs are osmosensory neurons and are excited by exposure to hypertonic solutions and inhibited by hypotonic solutions. The MCNs respond to systemic hypertonic and hypotonic stimulation with large changes in the expression of their Avp and Oxt genes, and microarray studies have shown that these osmotic perturbations also cause large changes in global gene expression in the HNS. In this paper, we examine gene expression in the rat supraoptic nucleus (SON) under normosmotic and chronic salt-loading SL) conditions by the first time using “new-generation”, RNA sequencing (RNA-Seq) methods. We reliably detect 9,709 genes as present in the SON by RNA-Seq, and 552 of these genes were changed in expression as a result of chronic SL. These genes reflect diverse functions, and 42 of these are involved in either transcriptional or translational processes. In addition, we compare the SON transcriptomes resolved by RNA-Seq methods with the SON transcriptomes determined by Affymetrix microarray methods in rats under the same osmotic conditions, and find that there are 6,466 genes present in the SON that are represented in both data sets, although 1,040 of the expressed genes were found only in the microarray data, and 2,762 of the expressed genes are selectively found in the RNA-Seq data and not the microarray data. These data provide the research community a comprehensive view of the transcriptome in the SON under normosmotic conditions and the changes in specific gene expression evoked by salt loading.</p></div

Transcriptional and Translational Regulator mRNAs increased in expression by SL.

<p>^aMean Control and Mean SL data are averages of three samples each and are expressed in quantile (log2 (RPKM+2)) units.</p><p>Transcriptional and Translational Regulator mRNAs increased in expression by SL.</p

Transcriptional and Translational Regulator mRNAs decreased in expression by SL.

<p>^aMean Control and Mean SL data are averages of three samples each and are expressed in quantile (log2 (RPKM+2)) units.</p><p>Transcriptional and Translational Regulator mRNAs decreased in expression by SL.</p

Network Analysis depicting gene products and known relationships between them for the second-ranked scoring network by Ingenuity (http://www.ingenuity.com/) when provided list of differentially expressed genes observed between salt-loaded and control by RNA-Seq (based on data in S9 Table).

<p>Gene products are represented using circle-shaped symbols with connected edges drawn between them to describe interactions (solid edge = direct interaction, dashed edge = indirect interaction). Color-filled shapes indicate the direction of differential expression observed between salt-loaded and control (green = up, red = down). Circle-shaped symbols not color-filled represent gene products not observed differentially expressed between salt-loaded and control.</p

Venn diagram of intersections of RNA-Seq and microarray data after correcting for noise threshold and statistical analyses (see S2 Fig, Steps 11 and 12, and S4 Fig steps 6 and 7 for microarray).

<p>There are 552 genes selected from the RNA-Seq data (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124523#pone.0124523.s018" target="_blank">S9 Table</a>) and 1030 unique genes selected from the microarray analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124523#pone.0124523.s022" target="_blank">S13 Table</a>). Note that 146 unique genes are found from both platforms to be unequivocally expressed and altered by salt-loading (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124523#pone.0124523.s023" target="_blank">S14</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124523#pone.0124523.s024" target="_blank">S15</a> Tables). Lower panel: Shows X-Y scatter plot of the fold changes in the 146 genes observed between Con & SL samples that were found in both the Microarray (X-axis) and RNA-Seq (Y-axis) data.</p

Pie Chart showing Locations and Types of 552 selected, expressed genes (See columns N and O in S9 Table).

<p>Pie Chart showing Locations and Types of 552 selected, expressed genes (See columns N and O in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124523#pone.0124523.s018" target="_blank">S9 Table</a>).</p