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

Abstract

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.

The −216‐ to −100‐bp Sequence in the 5′‐Flanking Region of the Oxytocin Gene Contains a Cell‐Type Specific Regulatory Element for its Selective Expression in Oxytocin Magnocellular Neurones

“…There was no inhibition of the EGFP expression ( Fig. 3), and the specificity of expression of this construct for AVP-MCNs was maintained, as reported previously (4). Therefore, the repressor hypothesis can be rejected.…”

“…The 2.0kVPI.EGFP plasmid described by Ponzio et al . was digested with Psh AI and the Psh AI digested OXT PCR product was ligated into it. This places the 216–100‐bp OXT derived sequence at position −1936 of the VP 5′ UTR in the AVP p2 kb plus OXT p‐216–100‐bp construct shown in Fig a .…”

“…A second test of the hypothesis that the −216‐ to −100‐bp domain contains a regulatory region responsible for the repression of expression of the oxytocin (OXT) gene in vasopressin (AVP)‐magnocellular neurones (MCNs) in the supraoptic nucleus (SON). ( a ) The construct in which the −216‐ to −100‐bp domain from the OXT p563 transgene was added to the 2‐kbp 5′ untranslated region ( UTR) of an AVP transgene that produces robust cell‐type expression of enhanced green fluorescent protein (EGFP) in AVP‐ MCN s in the SON . ( b ).…”

“…Taken together, these results are consistent with a conclusion that the −216‐ to −100‐bp sequence in the 5′‐flanking region of the oxytocin gene contains only an activator of transcription operating in the OXT‐MCNs. In the second experiment, we directly test the repressor hypothesis by injecting an AVP gene promoter construct known to be robustly expressed in AVP‐MCNs but which had the putative −216‐ to −100‐bp sequence repressor attached to its 5′ end. In this case, the repressor hypothesis would predict that the expression of the latter construct would be significantly decreased or eliminated.…”

MicroRNA profiling in the mouse hypothalamus reveals oxytocin‐regulating microRNA

“…However, it is largely unclear what mechanisms are responsible for the highly selective regulation of the cell‐type specific expression of Oxt and Avp (Ponzio et al . ). Therefore, it is important to understand how exactly the neurohypophysial hormones and neurotransmitters are regulated in the hypothalamus.…”