Evita Mohr

Expression of the vasopressin and oxytocin genes in rats occurs in mutually exclusive sets of hypothalamic neurons

The genes for the hypothalamic hormones vasopressin and oxytocin are located in close proximity to each other within the rat genome. They are separated by only approx. 11 kbp of DNA sequence and oriented in such a way that their transcription occurs on opposite DNA strands. Although the two genes are structurally very similar including common potential regulatory elements in their putative promotor regions, they are expressed in discrete populations of magnocellular neurons of the hypothalamus. In rats placed under osmotic stress, the vasopressin gene is unregulated; concomitantly transcription of the oxytocin gene is also stimulated. To address the question of whether this coordinated rise in oxytocin‐encoding mRNA is the result of switching on oxytocin gene transcription in vasopressinergic neurons, in situ hybridization with double labelled cRNA probes was carried out. Biotinylated and [α‐35S]CTP labelled antisense cRNA probes specific for either vasopressin or oxytocin mRNA were constructed and hybridized to hypothalamic sections from salt‐loaded rats. The results demonstrate that upregulation of oxytocin gene transcription is restricted solely to oxytocinergic cells; no oxytocin gene transcripts can be detected in vasopressinergic neurons.

Sequence analysis of the promoter region of the rat vasopressin gene

The vasopressin gene is highly transcribed in magnocellular neurons of the supraoptic (SON) and paraventricular nucleus (PVN) in the rat hypothalamus. In order to identify cis‐acting elements involved in the expression of the vasopressin gene, approximately 1 kb upstream of the transcription start site has been sequenced. Several putative regulatory elements have been detected, including a glucocorticoid response element (GRE), a cAMP response element (CRE), and four AP2 binding sites. In gel shift assays performed with a labelled DNA fragment corresponding to nucleotide residues −214 to −36 and nuclear proteins extracted from SON‐derived tissue enriched in magnocellular neurons, three specific protein‐DNA complexes have been detected. Complex formation is effectively competed by addition of an excess of unlabelled fragment.

Functional characterization of estrogen and glucocorticoid responsive elements in the rat oxytocin gene

Expression of the gene encoding the oxytocin precursor occurs in the hypothalamus and, to a lesser extent, in a number of peripheral organs, the tissue-specific regulatory mechanisms of which are largely unknown. By DNA sequence analysis several elements upstream of the transcriptional start point of the rat oxytocin gene were identified matching the consensus sequence of enhancers inducible by estrogen or glucocorticoids, respectively. Their general transactivating capacities were investigated using heterologous gene constructs and revealed that the rat oxytocin gene harbours two functional estrogen responsive elements near the transcription initiation site, one of which is conserved in the respective human gene. In addition, one enhancer conferring glucocorticoid responsiveness to a reporter gene is located at nucleotide residues -2449 to -2464. These data might indicate a steroid hormone-mediated influence on oxytocin gene expression in the central nervous system and/or the periphery.

Subcellular RNA compartmentalization.

The phenomenon of mRNA sorting to defined subcellular domains is observed in diverse organisms such as yeast and man. It is now becoming increasingly clear that specific transport of mRNAs to extrasomal locations in nerve cells of the central and peripheral nervous system may play an important role in nerve cell development and synaptic plasticity. Although the majority of mRNAs that are expressed in a given neuron are confined to the cell somata, some transcript species are specifically delivered to dendrites and/or, albeit less frequently, to the axonal domain. The physiological role and the molecular mechanisms of mRNA compartmentalization is now being investigated extensively. Even though most of the fundamental aspects await to be fully characterized, a few interesting data are emerging. In particular, there are a number of different subcellular distribution patterns of different RNA species in a given neuronal cell type and RNA compartmentalization may differ depending on the electrical activity of nerve cells. Furthermore, RNA transport is different in neurons of different developmental stages. Considerable evidence is now accumulating that mRNA sorting, at least to dendrites and the initial axonal segment, enables local synthesis of key proteins that are detrimental for synaptic function, nerve cell development and the establishment and maintenance of nerve cell polarity. The molecular determinants specifying mRNA compartmentalization to defined microdomains of nerve cells are just beginning to be unravelled. Targeting appears to be determined by sequence elements residing in the mRNA molecule to which proteins bind in a manner to direct these transcripts along cytoskeletal components to their site of function where they may be anchored to await transcriptional activation upon demand.

Diversity of mRNAs in the Axonal Compartment of Peptidergic Neurons in the Rat.

Vasopressin and oxytocin mRNAs, which are normally translated in the perikarya of magnocellular neurons, have recently been demonstrated to be also present in axons and nerve terminals which are located in the posterior pituitary. The physiological significance of this observation has not yet been resolved. In order to gain further insight into the function and plasticity of the peptidergic neuron the question was addressed whether axonal localization is a unique feature of the above‐mentioned transcripts. Biochemical evidence is presented that magnocellular axons and nerve terminals also contain mRNA species encoding a member of the neurofilament protein family and the prodynorphin precursor. These data imply that axons may harbour a variety of additional protein‐encoding transcripts. Furthermore, it is shown that in the mutant (Brattleboro) rat, which lacks detectable levels of vasopressin but which still transcribes the corresponding gene, axonal vasopressin but not oxytocin mRNA contents are dramatically reduced. Most likely, vasopressin transcripts are absent from the nerve terminals as a consequence of the impaired precursor biosynthesis in the cytoplasm of the mutant rat.

Dendritic Localization of Rat Vasopressin mRNA: Ultrastructural Analysis and Mapping of Targeting Elements

Transcripts encoding the vasopressin precursor are located in axons and dendrites of rat hypothalamic magnocellular neurons. While the axonal vasopressin mRNA has been extensively characterized both at the biochemical and morphological level, little is known about those transcripts residing in dendrites of magnocellular neurons. As revealed by in situ hybridization at the electron microscopic level, the mRNA is located in proximal and distal dendritic segments and is exclusively confined to regions containing rough endoplasmic reticulum. These results suggest that dendrites of hypothalamic neurons may be capable of local precursor synthesis independent of that occurring in the cell somata. A heterologous system has been employed to define cis‐acting elements within the vasopressin mRNA which may be involved in dendritic compartmentalization. Expression vector constructs consisting of the cytomegalovirus promoter coupled to the rat vasopressin cDNA have been injected into the cell nuclei of cultured neurons derived from embryonic rat superior cervical ganglia. Vector‐encoded vasopressin transcripts were also sorted to dendrites of these neurons indicating that the molecular determinants of dendritic mRNA transport are not cell specific. Mapping of the targeting elements revealed two segments within the vasopressin mRNA that are able to confer dendritic compartmentalization to α‐tubulin mRNA which is normally confined to the cell body.

Rats with physically disconnected hypothalamo-pituitary tracts no longer contain vasopressin-oxytocin gene transcripts in the posterior pituitary lobe.

In rats, vasopressin‐ and oxytocin‐encoding mRNAs are present in the posterior but absent in the anterior lobe of the pituitary gland. RNase protection experiments indicate that in the posterior pituitary and hypothalamus identical transcriptional start points are used. Furthermore, the two transcripts from posterior pituitary and hypothalamus show identical nucleotide sequences. Animals operated by paired electrical lesions in such a way that connections between the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus and the posterior pituitary lobeare destroyed continue to express the vasopressin and oxytocin gene in the hypothalamus but not in the posterior pituitary. Operated animals subjected to chronic intermittent salt loading for 6 days similarly contain vasopressin and oxytocin encoding transcripts in the hypothalamus but not in the posterior pituitary.

A single rat genomic DNA fragment encodes both the oxytocin and vasopressin genes separated by 11 kilobases and oriented in opposite transcriptional directions

An 18 kb DNA fragment, containing the genes encoding both the vasopressin and oxytocin polyprotein precursors, has been isolated from a rat genomic library. The two genes are linked by approximately 11 kb of intervening sequence and transcribed from opposite DNA strands.

Axonal mRNAs: functional significance in vertebrates and invertebrates

Sorting of defined mRNA species to distinct cytoplasmic regions is observed throughout the animal kingdom in many cell types, including neurons. During the past years, mRNA localization to dendrites of nerve cells has been characterized in detail. The functional role of these transcripts appears to be obvious: Since dendrites are equipped with the basic translational machinery, certain proteins are likely to be synthesized on-site. Targeting of mRNAs to the axon of vertebrate neurons is less well understood. Even though some vertebrate nerve cells such as goldfish Mauthner neurons seem to have ribosomes within the axonal compartment, evidence for ongoing local translation is still preliminary. In most differentiated mammalian neurons the axon is thought to lack mRNAs and a protein synthesizing machinery. Although a few nerve cell types harbour substantial amounts of distinct mRNA species within the axonal domain, their functional significance has remained elusive until today. Recent evidence suggests that mRNA transport to neurites including the future axon and local translation might play a role in nerve cell maturation. mRNA targeting to the axon of young neurons is strictly correlated with differentiation. It is no longer observed in fully matured neurons. Finally, for many years invertebrate neurons have served as model systems to investigate axonal mRNA transport and its physiological relevance. There is no doubt that protein synthesis does take place in the axonal domain. However, it has to be considered that invertebrate neurons develop only one type of neurite, referred to as the axon. These axons are different when compared with those of vertebrate nerve cells since they combine characteristics of dendrites and axons. In fact, current evidence supports the view that the axonal mRNAs in invertebrate nerve cells have functions comparable to those of transcripts residing in the dendrites of vertebrate neurons.

Expression of the vasopressin and oxytocin genes in human hypothalami

Poly(A)+ RNA isolated from post‐mortem human hypothalami has been used to characterize the polyprotein precursors to vasopressin and oxytocin. Translation in a cell‐free system and subsequent immunoprecipitation with antibodies raised against either vasopressin or neurophysin identified a product of M r 19000 (prepro‐vasopressin). A second less intense product of M r 16500 was tentatively identified as prepro‐oxytocin. A cDNA library derived from the human hypothalamic poly(A)+ RNA was screened for vasopressin and oxytocin‐encoding cDNA using heterologous probes; clones encoding the two precursors were identified and found to be organized as their rat and bovine counterparts. Northern blot analysis shows that the mRNAs for the two prepro‐hormones consist of ~ 840 (AVP) and ~ 700 (OT) nucleotides.