Gene networks involved in hypothalamic plasticity in response to dehydration; assessing the in vivo functions of candidate nodal genes.
Lead Research Organisation:
University of Bristol
Department Name: Henry Wellcome LINE
Abstract
The driving force behind this project is the need to rapidly exploit genomic information in order to obtain physiological understanding. We now know that mammals have approximately 30,000 genes. These data prompt two questions, firstly, where and when are these genes expressed, and secondly, what do these genes do? We have addressed these questions in a robust model system, namely the physiologically challenged vasopressin (VP) neurones of the hypothalamus. When an animal is dehydrated, the peptide hormone VP is released and travels through the blood stream to specific receptor targets located in the kidney, where it reduces the excretion of water, thus promoting water conservation. This is accompanied by a plethora of changes in the morphology, electrophysiological properties and biosynthetic and secretory activity of VP neurones. We wish to understand this functional plasticity and its physiological consequences in terms of the differential expression of genes. We have used microarray techniques that allow us to look at the expression of tens of thousands of genes in a single assay. We have thus compiled catalogues that represent comprehensive descriptions of the RNA populations expressed in different regions of the hypothalamus and pituitary. Further, we have identified transcripts that are either up- or down-regulated as a consequence of chronic dehydration. These catalogues are an important resource for researchers working on all aspects of VP physiology, particularly the central neuro-humoral control of cardiovascular homeostasis. Based on unbiased mathematical criteria, we have selected 4 genes for further study on the basis that they are key hubs, or nodes, in a gene network that, we hypothesise, might be involved in regulating and mediating hypothalamic functional plasticity. In order to test this hypothesis, we will: 1. check that the array data are correct using independent methods; 2. use gene transfer into the whole organism, coupled with the latest non-invasive monitoring technologies to determine the functional consequences of the increased or decreased activity of target gene products in terms of integrated cardiovascular control. This will be the first time that, based on a microarray output, a gene network will be studied functionally in the context of a whole animal physiological system. The data will undoubtedly lead to a better understanding of gene networks involved in the plasticity of a physiological system in health and disease states.
Technical Summary
We have used array technology to comprehensively describe the pattern of gene expression in the hypothalamus, and how this changes following the physiological challenge of dehydration. We now wish to study the functions of key differentially expressed genes in vivo. We have employed a rational and unbiased approach to gene selection. We have utilised machine-learning algorithms to describe a gene network that, we hypothesise, might be involved in regulating and mediating hypothalamic plasticity. Of particular interest are those genes with many connections. Such genes may represent crucial functional hubs, or nodes. We will now test this hypothesis in vivo, focusing on 4 genes with 4 or more connections. We will now determine the functional and regulatory roles of these four key signalling nodes within a hypothetical gene network activated in the SON as a consequence of dehydration. To test this hypothesis we will: - validate the transcriptome data by determining the expression patterns of our candidate genes in the brain, hypothalamus and HNS at both the RNA and protein levels in terms of both specific brain cell-types and responses to dehydration - assess the functions of these genes in basal hypothalamic activity and stress-induced remodelling using in vivo gene manipulation techniques. Three systems will be exploited - knockout' transgenic mice, transgenic rats and somatic gene delivery using viral vectors. Gene activity will be manipulated by over-expression of wild-type proteins, or inhibition using RNAi. This will be followed by expression analysis of putative interacting genes, and by robust, but wherever possible, non-invasive, quantification of water balance, vasopressin release, the electrical activity of hypothalamic neurons, and hypothalamic morphology.
People |
ORCID iD |
David Murphy (Principal Investigator) | |
Julian Paton (Co-Investigator) |
Publications
Hindmarch CC
(2013)
Whole transcriptome organisation in the dehydrated supraoptic nucleus.
in Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas
Qiu J
(2011)
Transcriptomic analysis of the osmotic and reproductive remodeling of the female rat supraoptic nucleus.
in Endocrinology
Greenwood M
(2014)
Transcription factor CREB3L1 regulates vasopressin gene expression in the rat hypothalamus.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Greenwood M
(2015)
Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus.
in Molecular brain
Hindmarch CC
(2011)
The transcriptome of the medullary area postrema: the thirsty rat, the hungry rat and the hypertensive rat.
in Experimental physiology
CC Hindmarch (Co-Author)
(2010)
The Transcriptome and the Hypothalamo-Neurohypophyseal System.
Greenwood MP
(2018)
The effects of aging on biosynthetic processes in the rat hypothalamic osmoregulatory neuroendocrine system.
in Neurobiology of aging
Yao ST
(2011)
Temporal profile of arginine vasopressin release from the neurohypophysis in response to hypertonic saline and hypotension measured using a fluorescent fusion protein.
in Journal of neuroscience methods
Colombari DS
(2011)
Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration.
in The Journal of physiology
Katoh A
(2010)
Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary.
in The Journal of endocrinology
Greenwood MP
(2014)
Salt appetite is reduced by a single experience of drinking hypertonic saline in the adult rat.
in PloS one
Konopacka A
(2015)
RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response.
in eLife
Greenwood MP
(2017)
Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1) Expression by Orphan Nuclear Receptor Nr4a1.
in Frontiers in molecular neuroscience
Greenwood MP
(2016)
Rasd1, a small G protein with a big role in the hypothalamic response to neuronal activation.
in Molecular brain
Konopacka A
(2015)
Osmoregulation requires brain expression of the renal Na-K-2Cl cotransporter NKCC2.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Stewart L
(2011)
Hypothalamic transcriptome plasticity in two rodent species reveals divergent differential gene expression but conserved pathways.
in Journal of neuroendocrinology
Hazell GG
(2012)
G protein-coupled receptors in the hypothalamic paraventricular and supraoptic nuclei--serpentine gateways to neuroendocrine homeostasis.
in Frontiers in neuroendocrinology
Waki H
(2013)
Excessive Leukotriene B4 in Nucleus Tractus Solitarii Is Prohypertensive in Spontaneously Hypertensive Rats
in Hypertension
Suzuki H
(2009)
Exaggerated response of a vasopressin-enhanced green fluorescent protein transgene to nociceptive stimulation in the rat.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Yoshimura M
(2013)
A c-fos-monomeric red fluorescent protein 1 fusion transgene is differentially expressed in rat forebrain and brainstem after chronic dehydration and rehydration.
in Journal of neuroendocrinology
Brunton PJ
(2015)
5a-Reduced neurosteroids sex-dependently reverse central prenatal programming of neuroendocrine stress responses in rats.
in The Journal of neuroscience : the official journal of the Society for Neuroscience
Description | The driving force behind this project is the need to rapidly exploit genomic information in order to obtain physiological understanding. We now know that mammals have approximately 30,000 genes. These data prompt two questions, firstly, where and when are these genes expressed, and secondly, what do these genes do? We have addressed these questions in a robust model system, namely the physiologically challenged vasopressin (VP) neurones of the hypothalamus. When an animal is dehydrated, the peptide hormone VP is released and travels through the blood stream to specific receptor targets located in the kidney, where it reduces the excretion of water, thus promoting water conservation. This is accompanied by a plethora of changes in the morphology, electrophysiological properties and biosynthetic and secretory activity of VP neurones. We have sought to understand this functional plasticity and its physiological consequences in terms of the differential expression of genes. We have used microarray techniques that allow us to look at the expression of tens of thousands of genes in a single assay. We have thus compiled catalogues that represent comprehensive descriptions of the RNA populations expressed in different regions of the hypothalamus and pituitary. Further, we have identified transcripts that are either up- or down-regulated as a consequence of chronic dehydration. These catalogues are an important resource for researchers working on all aspects of VP physiology, particularly the central neuro-humoral control of cardiovascular homeostasis. Based on unbiased mathematical criteria, we have selected genes for further study on the basis that they are key hubs, or nodes, in a gene network that, we hypothesise, might be involved in regulating and mediating hypothalamic functional plasticity. In order to test this hypothesis, we have: 1. check that the array data are correct using independent methods; 2. use gene transfer into the whole organism, coupled with the latest non-invasive monitoring technologies to determine the functional consequences of the increased or decreased activity of target gene products in terms of integrated cardiovascular control. Striking physiological data have been obtained. This has been the first time that, based on a microarray output, a gene network will be studied functionally in the context of a whole animal physiological system. Ongoing studies are seeking to understand molecular mechanisms. |
Exploitation Route | We have identified numerous candidate genes that might be important in the central control of salt and water homeostasis. We hope that these genes will be studied by other members of the research community. |
Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |
URL | http://www.vasopressin.org |
Description | Project Grant |
Amount | £1,270,000 (GBP) |
Funding ID | BB/J005452/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2012 |
End | 03/2016 |
Description | Project Grant |
Amount | £342,729 (GBP) |
Funding ID | EP/K008250/1 |
Organisation | Engineering and Physical Sciences Research Council (EPSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2013 |
End | 01/2015 |
Description | Project grant |
Amount | £1,700,000 (GBP) |
Funding ID | BB/J01515/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2013 |
End | 01/2016 |
Description | USA Partnering award |
Amount | £49,000 (GBP) |
Funding ID | BB/J01981X/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 06/2012 |
End | 07/2016 |
Description | Saudi Arabia collaboration |
Organisation | King Saud University |
Department | Department of Zoology |
Country | Saudi Arabia |
Sector | Academic/University |
PI Contribution | Transcriptomic analysis of Jerboa tissues. |
Collaborator Contribution | Privision of Jerboa tissues. |
Impact | Not applicable as yet/ |
Start Year | 2018 |