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, 7 (2), e32085

Cell-type Specific Oxytocin Gene Expression From AAV Delivered Promoter Deletion Constructs Into the Rat Supraoptic Nucleus in Vivo

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Cell-type Specific Oxytocin Gene Expression From AAV Delivered Promoter Deletion Constructs Into the Rat Supraoptic Nucleus in Vivo

Raymond L Fields et al. PLoS One.

Erratum in

  • PLoS One. 2012;7(6). doi:10.1371/annotation/2d183615-8b34-4ea4-ae9b-9833d6079d11

Abstract

The magnocellular neurons (MCNs) in the hypothalamus selectively express either oxytocin (OXT) or vasopressin (AVP) neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV) vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON). Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Description of AAVs containing OXT promoter deletion constructs used in this study.
A. Shows plasmid pFBGR, which was used for making all constructs, this plasmid contains the AAV left and right inverted terminal repeats (ITR). B. shows diagrams of constructs made containing sequences 568, 440, 325, 216, 100, and 50 bp upstream of the transcription start site of the OXT gene. These constructs contained all the three exons and two introns in the OXT gene and with the EGFP reporter inserted at the end of the coding region of exon III, followed by 768 bp downstream of OXT exon III. All constructs were placed into pFBGR between the XbaI sites after removal of the 2291 bp XbaI band (see panel A).
Figure 2
Figure 2. Efficacy of targeting both OXT and AVP magnocellular neurons (MCNs) in rat supraoptic nucleus (SON) by stereotaxic injections of AAVs.
The AAVs used were chimeric AAV6 serotypes with an AAV2 ITR and an AAV6 capsid (see Methods). A. Illustrates a rat brain coronal section showing the placement of 30 gauge needles used for bilateral injections of AAVs to target the SON. Stereotaxic coordinates are presented in Methods. Panels B–D show the results of injecting an AAV containing the CMV promoter fused to an EGFP reporter into the rat SON. B. Illustrates EGFP fluorescence in the MCNs of the SON found two weeks after injection. C. shows OXT MCNs in the SON after staining with PS 38 antibody (an OXT MCN marker). D. Shows merged EGFP fluorescence and PS 38 staining. White Arrows in panels B–D show MCNs in the SON that express EGFP co-localized with PS 38-immunoreactivity. Yellow arrow in panels B–D show MCNs that express EGFP which is not co-localized with PS 38-immunoreactivity, and hence are presumptive AVP MCNs. Abbreviations in A: cc, corpus callosum; LV, lateral ventricle; f, fornix; 3 V, third ventricle; ic, internal capsule; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; OC, optic chiasm. Scale line in D is the same for B and C.
Figure 3
Figure 3. Specificity of EGFP expression from the p563 AAV construct.
Immunohistochemistry was performed two weeks after injection of the AAVs into the rat SON. Brain sections in A and D show EGFP fluorescence, in B, PS38 antibody staining, in C, merged EGFP fluorescence and PS38 staining, in E, PS41 antibody staining, and in F, merged EGFP fluorescence and PS41 staining. Note that while the EGFP is abundantly expressed in the OXT MCNs (panel C), there is virtually no expression of the EGFP in the AVP MCNs (panel F). Abbreviations: OC, optic chiasm. Scale line in F is the same for all panels.
Figure 4
Figure 4. Analysis of the specificity of expression of EGFP from AAVs containing various promoter deletion constructs (shown in Fig. 1B ) two weeks after their injection into rat SONs.
The specific promoter lengths that were injected into the SONs in each of the experiments is shown in the lower left of the panels. Immunohistochemical analyses show that each of the oxytocin promoter AAV constructs was selectively expressed in oxytocin cells. Each panel illustrates merged images of AVP-neurophysin (PS 41) immunoreactivity shown as red, and the EGFF expression shown as green. Note that while the EGFP is clearly not expressed in the AVP MCNs in panels A–D where the promoter constructs contain 216 bp or more of the upstream region, the p100 construct (panel E) and p50 construct (Panel F) shows EGFP expression in both OXT. Hence the key elements regulating of the selectivity of OXT gene expression must reside between in the −216 to −100 bp domain in the promoter. Abbreviations: OC, optic chiasm. The 100 µm scale line in lower right is the same for all panels.
Figure 5
Figure 5. Removal of exons 2 and 3, and both introns does not alter the specificity of expression properties of the OXT promoter.
An AAV was made containing the p563 OXT promoter- EGFP reporter construct (pOTI) shown at the bottom of the figure. The construct contains 568 bp of the OXT upstream promoter region followed by OXT exon I with the EGFP reporter gene fused to the end of exon I followed by 768 bp of the region downstream of OXT exon III. The pOXTI AAV was injected into the SON of a salt loaded rat. A. shows EGFP Fluorescence, B. shows a merge of EGFP fluorescence and OXT antibody staining in the same section, C. shows EGFP fluorescence and AVP-NP antibody staining in a different section from the same rat SON. OC = optic chiasm. The scale line in A is the same for B and C.
Figure 6
Figure 6. Effects of systemic salt loading on the expression of EGFP in MCNs after injection of various OXT promoter deletion constructs into the rat SON.
After injection of the AAVs into the SONs, the control rats were given water to drink for two weeks, whereas the salt loaded rats were were given water for one week, followed by a second week of access only to water containing 2% NaCl (see Methods). Panels show EGFP fluorescence in SONs of control (left column) and salt loaded (right column) rats for each of the p563, 440 bp, p100, and p50 OXT promoter-containing AAVs. Control and salt loaded image capture times are identical for each construct. Note that for each deletion construct the SONs fom salt loaded rats express much more EGFP than the control SONs, this is also the case for the pOXTI promoter construct. OC = optic chiasm. Scale bar in bottom left panel is same for all panels.
Figure 7
Figure 7. Diagram summarizing the cis-regulatory domains in the oxytocin gene promoter as suggested by the data in this study.
The −50 bp and −100 bp OXT promoter domains are sufficient to produce expression of an EGFP reporter in both OXT and AVP MCNs in the SON, and in a population of smaller neurons just dorsal to the SON. The −100 to −216 bp domain appears to contain a repressor element that inhibits expression specifically in AVP MCNs. The −216 to −325 domain appears to repress expression in an unidentified dorsal neuron population above the SON, and the −216 to −440 bp domain may have enhancer elements which are operative in the OXT MCNs.

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