In Vivo Gene Transfer Studies on the Regulation and Function of the Vasopressin and Oxytocin Genes

Abstract

Novel genes can be introduced into the germline of rats and mice by microinjecting fertilized one-cell eggs with fragments of cloned DNA. A gene sequence can thus be studied within the physiological integrity of the resulting transgenic animals, without any prior knowledge of its regulation and function. These technologies have been used to elucidate the mechanisms by which the expression of the two genes in the locus that codes for the neuropeptides vasopressin and oxytocin is confined to, and regulated physiologically within, specific groups of neurones in the hypothalamus. A number of groups have described transgenes, derived from racine, murine and bovine sources, in both rat and mouse hosts, that mimic the appropriate expression of the endogenous vasopressin and genes in magnocellular neurones (MCNs) of the supraoptic and paraventricular nuclei. However, despite considerable effort, a full description of the cis-acting sequences mediating the regulation of the vasopressin-oxytocin locus remains elusive. Two general conclusions have nonetheless been reached. First, that the proximal promoters of both genes are unable to confer any cell-specific regulatory controls. Second, that sequences downstream of the promoter, within the structural gene and/or the intergenic region that separates the two genes, are crucial for appropriate expression. Despite these limitations, sufficient knowledge has been garnered to specifically direct the expression of reporter genes to vasopressin and oxytocin MCNs. Further, it has been shown that reporter proteins can be directed to the regulated secretory pathway, from where they are subject to appropriate physiological release. The use of MCN expression vectors will thus enable the study of the physiology of these neurones through the targeted expression of biologically active molecules. However, the germline transgenic approach has a number of limitations involving the interpretation of phenotypes, as well as the large cost, labour and time demands. High-throughput somatic gene transfer techniques, principally involving the stereotaxic injection of hypothalamic neuronal groups with replication-deficient adenoviral vectors, are now being developed that obviate these difficulties, and which enable the robust, long-lasting expression of biologically active proteins in vasopressin and oxytocin MCNs.'Neurobiology still has a long way to go to contend with consciousness. Perhaps we should be content to work instead on unconsciousness for a while, and find out about all those processes going on in our brains that we don't know about directly and need science to tell us the answers' (Sydney Brenner) (1).In vivo gene transfer (transgenic) techniques enable the study of any gene sequence within the physiological and developmental integrity of the whole animal. In this review, we describe the work of a number of laboratories, including our own, on the application of transgenesis to the study of the regulation and function of the neurones of the hypothalamic-neurohypophysial system ...

Neurohormones: oxytocin, vasopressin and related peptides – structure, genes, receptors, and evolution

“…Vasopressin mRNAs were identified in a subset of pituitary cells (Murphy et al, 1993), which is in concordance with the presence of their translation proteins in these cells (Boersma et al, 1993;Pu et al, 1995;Murphy and Wells, 2003).…”

“…The identification of specific mRNAs for oxytocin and vasopressin in the neural lobe (NL) of the rat pituitary gland (Murphy et al, 1989) stimulated much interest, research, and opinions concerning both their origin, regulation, and expected physiological role (Levy et al, 1990;Mohr et al, 1991;Murphy et al, 1993;Trembleau et al, 1994Trembleau et al, , 1996Murphy and Wells, 2003). The mRNA for VP was also found in the neural lobe of mice (Murphy et al, 1989) and in the bovine neurointermediate lobe of the pituitary (Chaturvedi et al, 1996).…”

“…Similarly, osmotic signals, such as salt-loading, resulting in functional demand for vasopressin, increase vasopressin gene transcription Herman et al, 1991) and mRNA abundance (Burbach et al, 1984;Zingg et al, 1986), and also result in stimulatory changes in oxytocin expression gene expression . In the parvocellular PVN, vasopressin gene expression is suppressed by adrenal glucocorticoids as part of a negative-feedback loop, and is upregulated by stress (Ma et al, 1997), while diurnal cues modulate vasopressin gene expression in the suprachiasmatic nucleus (SCN) (Carter and Murphy, 1991;Murphy and Wells, 2003). Abundant and detailed information on the subject can be found in important and prestigious publications (Gainer and Wray, 1994;Acher, 1996;Hoyle, 1999;Gimpl and Fahreholtz, 2001;Gwee et al, 2009).…”

Autosomal recessive familial neurohypophyseal diabetes insipidus: onset in early infancy

“…Biosynthesis of AVP starts with tightly regulated transcription of the AVP gene (13), continues with regulated translation and enzymatic cleavage of the AVP-neurophysin II (AVP-NPII) prohormone (14)(15)(16)(17), and ends with active AVP secretion into the circulation upon an appropriate stimulus (i.e. hyperosmolality and hypovolemia).…”

Transgenesis and the Study of Expression, Cellular Targeting and Function of Oxytocin, Vasopressin and Their Receptors

“…Finally, since most endocrinological and physiological data have been obtained using rats, it would be highly desirable to perform such experiments on transgenic rats. In fact, significant efforts towards this goal have been made in studies [26, 27, 28]on the rat hypothalamoneurohypophysial system (HNS). However, at this time, studies using transgenic rats still lag behind those with transgenic mice, and we are not aware of any studies using homologous recombination to study the rat nervous system.…”