Mouse vasopressin deletion constructs and plasmid that were used to make the recombinant AAV (rAAV) vectors.

<p>A. All vasopressin constructs contained an EGFP reporter fused to exon I, as well as the endogenous poly-A signal found at the untranslated region of exon III, and an additional 173 bases that follow exon III. The vasopressin promoter region was systematically reduced such that promoter lengths tested were: 2.0 kbp, 1.5 kbp, 950 bp, 543 bp, 421 bp, 288 bp, 116 bp and 50 bp upstream of the transcription start site. B. Illustration of the AAV plasmid into which the assorted sequences described in A were inserted is shown. Abbreviations: ITR, inverted terminal repeat; 3′ UTR, 173 untranslated bases that follow exon III.</p

Diagram illustrating the cis-regulatory domains in the vasopressin gene promoter as suggested by the data in this study.

<p>Since the 288 VPI.EGFP construct still maintains the vasopressin MCN specific expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g006" target="_blank">Fig. 6F</a>) we hypothesize that there is an activator of expression in the Avp MCNs and a possible repressor element with an upper boundary of −288 kbp that inhibits expression selectively in the Oxt MCNs. We further propose that there are at least three enhancer domains upstream of the transcription start site of the Avp gene that are involved in expression in the Avp MCNs, which we designate enhancer 3 located between −2 kbp to −1.5 kbp, enhancer 2 located between −1.5 kbp to −950 bp, and enhancer 1 located between −543 bp to −288 bp (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g004" target="_blank">Fig. 4</a>). The −288 to 116 bp region appears to contain the cell-type specific element(s) (see text), and an osmotically-responsive element also appears to be present within the 288 bp construct (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone.0048860.s003" target="_blank">Fig. S3</a>).</p

Cell-Type Specific Expression of the Vasopressin Gene Analyzed by AAV Mediated Gene Delivery of Promoter Deletion Constructs into the Rat SON In Vivo

<div><p>The magnocellular neurons (MCNs) in the supraoptic nucleus (SON) of the hypothalamus selectively express either oxytocin (Oxt) or vasopressin (Avp) neuropeptide genes. In this paper we examine the cis-regulatory domains in the Avp gene promoter that are responsible for its cell-type specific expression. AAV vectors that contain various Avp gene promoter deletion constructs using EGFP as the reporter were stereotaxically injected into the rat SON. Two weeks following the injection immunohistochemical assays of EGFP expression from these constructs were done to determine whether the expressed EGFP reporter co-localizes with either the Oxt- or Avp-immunoreactivity in the MCNs. The results identify three major enhancer domains located at −2.0 to −1.5 kbp, −1.5 to −950 bp, and −950 to −543 bp in the Avp gene promoter that regulate the expression in Avp MCNs. The results also show that cell–type specific expression in Avp MCNs is maintained in constructs containing at least 288 bp of the promoter region upstream of the transcription start site, but this specificity is lost at 116 bp and below. Based on these data, we hypothesize that the −288 bp to −116 bp domain contains an Avp MCN specific activator and a possible repressor that inhibits expression in Oxt-MCNs, thereby leading to the cell-type specific expression of the Avp gene only in the Avp-MCNs.</p> </div

Analysis of the selectivity of expression of EGFP in MCNs in the SON after injections of rAAVs containing the 50 bp promoter deletion construct.

<p>Panels A and D illustrate the EGFP immunoreactivity (EGFP-ir) only, B shows the Avp marker (PS41-ir) only, E shows the Oxt marker (PS38) only. The merged views of the EGFP-ir with either the Avp marker (PS41-ir in C) or the Oxt marker (PS38-ir in F) are shown. Examples of MCNs with colocalized EGFP-ir and PS38-ir or PS41-ir are depicted by yellow arrows and the white arrows show cells containing EGFP-ir only. The EGFP-ir was detected using 33-second photographic exposures as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g005" target="_blank">Fig. 5</a>. Scale bar in panel D is 100 µm, and is the same for all the panels. Abbreviations: OC, optic chiasm.</p

Stereotaxic microinjection of AAV vectors into the supraoptic nuclei (SONs) of adult male rats.

<p>A. Illustrates the coordinates of the injections relative to bregma: −1.3 mm caudal, 1.8 mm lateral (left and right), and −8.8 mm ventral. A total volume of 3 µl of vector was injected at a convection-enhanced delivery rate of 0.3 µl/minute, which resulted in vector completely filling the SON (see inset in A). B. Shows a view of a unilateral SON in a coronal section of the rat hypothalamus after injection into the SON of a rAAV containing the pan-specific CMV promoter fused to an EGFP reporter. Note that the MCNs in the SON show intense EGFP fluorescence and that areas dorsal to the SON also show significant although much less dense cellular fluorescence. This illustrates the total area of transduction deriving from this AAV injection. Asterisk shows the estimated position of the tip of the injection needle. The white dotted line borders the dorsal side of the SON. Scale line represents 100 µm. Abbreviations: OC, optic chiasm; SON, supraoptic nucleus.</p

Efficacy of expression of the 116 bp (A–D) and 50 bp (E–H) VPI.EGFP promoter deletion constructs in the SON.

<p>In these experiments we found that both salt loading and IHC were required for any detection of the EGFP expression. In addition, exposure times needed to be increased three-fold from the 11.8 seconds used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g004" target="_blank">Figure 4</a> (panels B and F), to 33 seconds (panels C and G) in order to achieve a level of detection of expression that would allow for double label analyses of cell-type specific expression (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g007" target="_blank">Figs. 7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g008" target="_blank">8</a>). Panels D and H represent merges of panels A and C, and E and G, respectively. 100 µm scale bar in panel F is the same for all images. Abbreviation: OC, optic chiasm.</p

Efficacy of expression of the 2.0 kbp to 288 bp VPI.EGFP promoter deletion constructs (shown in <b>Fig. 1A</b>) in the SON.

<p>In these experiments after injection of the rAAVs into the SONs the control (normosmotic) rats were given water to drink for two weeks. In parallel experiments, the salt loaded rats were given water to drink for one week, followed by a second week of access only to water containing 2% NaCl (see Methods). The images in panels A1–6 and B1–6 present endogenous EGFP fluorescence (i.e. without the use of EGFP antibody in immunohistochemistry, −EGFP Ab). This was done in order to better assess quantitative differences between the constructs. In the third column (panels C1–6), IHC using EGFP antibodies together with salt loading was employed in order to maximize the detection of EGFP expression. Photographic image capture (exposure) times are identical for each panel. Under normosmotic conditions, clear expression of EGFP is obtained from injections of the 2.0 kVPI.EGFP (A1) and 1.5 kVPI.EGFP rAAV constructs (A2), and a much higher expression is seen for both under salt loading conditions (B1, B2). For the 950VPI.EGFP construct there was no detectable EGFP fluorescence under normosmotic conditions (A3), but salt loading was shown to significantly increase the EGFP expression in the SON (B3), and a greater increase in fluorescence was observed with both salt loading and IHC (C3). Neither normosmotic nor salt loading conditions produced detectable endogenous EGFP expression from the 543 bp to 288 bp VPI.EGFP constructs (A4–6, B4–6). However, application of both EGFP IHC (+EGFP Ab) and salt loading together clearly show that the EGFP was being expressed in the SON from the 543 bp to 288 bp VPI.EGFP constructs (C4–6). The red arrows denote three putative enhancer domains. Scale bar in panel A6 is 100 µm, and is the same for all the panels. Abbreviation: OC, optic chiasm.</p

Analysis of the selectivity of expression of EGFP in MCNs from rAAVs containing the 2.0 kbp to 288 bp promoter deletion constructs (see <b>Fig. 1A</b>) two weeks after their injection into rat SONs.

<p>For each of the experiments the specific promoter deletion construct that was injected into the SONs is shown in the lower right of each panel. Immunohistochemical analyses confirm that each of the vasopressin rAAV constructs was preferentially expressed in vasopressin cells. EGFP immunoreactivity (in green) and oxytocin (PS38) immunoreactivity in red are shown as merged images in the panels. As a rule, the EGFP is not expressed in oxytocin immunoreactive cells. Occasionally EGFP was found to colocalize with oxytocin immunoreactivity to the extent of colocalization found for the endogenous peptide genes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone.0048860-Kiyama1" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone.0048860-Mezey1" target="_blank">[10]</a>. While the data for the 2.0VPI.EGFP and 1.5VPI.EGFP constructs produce good endogenous EGFP fluorescence under normosmotic conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048860#pone-0048860-g004" target="_blank">Fig. 4</a>), both salt loading as well as immunohistochemistry with antibodies to EGFP were required for optimal analysis of the 950–288 bp VPI.EGFP constructs. Scale bar in panel A is 100 µm, and is the same for all the panels. Abbreviations: OC, optic chiasm.</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

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