Determination of tissue-specific functions of Gnasxl derived proteins from the imprinted Gnas gene cluster.

Lead Research Organisation: University of Liverpool
Department Name: Biomedical Sciences

Abstract

This research project aims to analyse the functions of a gene in mice, termed Gnasxl, which is important for normal growth, food intake and the balancing of energy reserves. It is especially required at the postnatal stage of development, but has continued effects on metabolism and adiposity throughout adulthood. Mice that lack Gnasxl are very lean and use up their energy resources quickly.
Genetic abnormalities of the corresponding human gene cause similar disease symptoms, although these occur rarely and not all parallels have been fully investigated yet.
The Gnasxl gene is not active in all tissues, but functions only in a few areas of the brain and in some peripheral tissues that have a role in balancing energy resources (e.g. adipose tissue, pancreas. With this project we want to find out, which tissues are the most important sites of Gnasxl function. We will approach this question by generating mice that lack Gnasxl in specific tissues only, for example in brain or in adipose tissue, and analyse the physiological effects of these disturbances.
Results from our experiments should clarify, in which regulatory pathways Gnasxl acts and improve our understanding of control mechanisms for energy balance and obesity in mouse and human.

Technical Summary

Genetically modified mice are contributing substantially to our understanding of obesity, metabolism and the regulation of energy homeostasis. Recently, I generated a novel mouse model (Gnasxl ?knock-out?), which shows postnatal feeding difficulties, severe lack of adiposity and increased metabolism in adulthood. To understand this complex and often lethal phenotype, tissue-specific functions of Gnasxl need to be determined through restricted ablations.
The Gnasxl transcript is part of the composite Gnas locus, which comprises several alternatively spliced transcripts derived from different promoters. They are regulated by genomic imprinting, which results in monoallelic, parental-origin-dependent expression. Gnas encodes the Gsalpha subunit of trimeric G-proteins involved in cAMP signalling. The Gnasxl transcript produces XLalphas, an amino-terminally extended variant of Gsalpha, and a truncated neural protein XLN1. Gnasxl is only expressed from the paternal chromosome and the proteins XLalphas/XLN1 are required for postnatal growth, suckling and energy homeostasis. Similar symptoms have been described in human patients, who lack GNASXL expression due to genetic defects. GNAS1 itself is implicated in the human neuroendocrine disorders Albright?s Hereditary Osteodystrophy and Pseudohypoparathyroidism.
Gnasxl has a defined expression pattern in postnatal brain areas implicated in feeding (orofacial motornuclei), energy homeostasis (hypothalamus) and sympathetic nervous system (SNS) activity (brainstem). It is also detected in some peripheral tissues (adipose tissue, pancreas). To what extent is the complex phenotype of Gnasxl deficient mice due to dysfunctions in central and/or peripheral tissues? Both, SNS hyperactivity and adipose tissue autonomous functions have been suggested to cause the severe lack of lipid reserves.
This research proposal aims at defining the roles of XLalphas/XLN1 in individual tissues by creating a conditionally targeted mouse strain for cell-type specific ablations. The organisation of the locus demands an unconventional targeting approach and I have designed a conditional gene-trap strategy. In crosses with transgenic mice expressing Cre-recombinase from tissue-specific promoters, e.g. Nestin-Cre (brain), aP2-Cre (adipose tissue), restricted deficiencies of XLalphas/XLN1 can be evaluated. The severity of the phenotypes will be compared to the general Gnasxl ?knock-out? (postnatal survival, growth, suckling activity, SNS activity, adipose tissue histology, cAMP signalling). Complementary studies include the determination of the Gnasxl expression pattern in adult brain and its co-localisation with other neural markers. Potential changes in expression levels of candidate genes involved in the regulation of energy homeostasis and SNS outflow will be examined by q-PCR and protein analysis. These approaches will provide new insights into the mechanisms, by which XLalphas/XLN1 regulate metabolism, feeding and energy balance.
 
Description Centre for Integrative Mammalian Biology of the Universities of Manchester and Liverpool (funded by BBSRC, MRC, industrial partners), PhD Studentship
Amount £84,850 (GBP)
Organisation University of Liverpool 
Sector Academic/University
Country United Kingdom
Start 10/2008 
End 09/2012
 
Title Conditional Gnasxl LacZ gene trap 
Description Conditionally targeted gene trap mouse line for the Gnasxl transcript of the Gnas locus. Expresses LacZ from the Gnasxl promoter after Cre recombination. Strain name XLlacZGT (or Gnasxltm2Pla). 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Publication: Krechowec SO, Burton KL, Newlaczyl AU, Nunn N, Vlatkovic N, Plagge A, 'Postnatal changes in the expression pattern of the imprinted signalling protein XLas underlie the changing phenotype of deficient mice.' PLoS ONE 7(1): e29753 (2012) 
URL http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0029753
 
Title pCLacZGT 
Description Conditional gene targeting / gene trap vector. Generally useful plasmid for conditional gene trap targeting with a lacZ reporter gene. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Vector sequence has been deposited to Genbank with acc. no.: JN159854 Vector will be made available upon request. 
URL http://www.ncbi.nlm.nih.gov/nuccore/JN159854
 
Description Analysis of circadian rhythms, sleep, wakefulness and behaviour in Gnasxl KO mice. 
Organisation Italian Institute of Technology (Istituto Italiano di Tecnologia IIT)
Country Italy 
Sector Academic/University 
PI Contribution I provided the Gnasxl KO mouse line to Dr Valter Tucci for neurobiological experiments on circadian rhythm analysis, sleep, wakefulness and related behaviours.
Collaborator Contribution Dr Valter Tucci's laboratory is currently analysing circadian rhythmicity, sleep (REM, NREM) patterns and wakefulness as well as associated behaviours in the Gnasxl KO mice.
Impact Initial data have been obtained by Dr Tucci and they have been included in a grant applicaton to the European Research Council - Consolidator grant. I am a named collaborator on this grant application, to carry out a specific type of experiment in my laboratory. This collaboration is not multi-disciplinary.
Start Year 2013
 
Description Investigation of sympathetic nervous system (SNS) activity in mice deficient for the signalling protein XLas. 
Organisation University of Liverpool
Department School of Veterinary Science Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint supervision of a 4-year PhD student in molecular biological and physiological experiments. Contribution of genetically modified mouse models to the research.
Collaborator Contribution Joint supervision of MRes/PhD student in physiological / cardiovascular experiments.
Impact PhD student training in Integrative Mammalian Biology within the 'Centre for Integrative Mammalian Biology' of the Universities of Manchester and Liverpool (Co-funded by BBSRC, MRC, industrial partners and others). Poster presentation at the following conferences: The 7th James Black Conference, Joint Meeting of the British Pharmacological Society and the Physiological Society, King's College, London, UK, 01. - 03.09.2009. 'Metabolism & Endocrinology, Themed Meeting of The Physiological Society, AstraZeneca, Macclesfield, UK, 24. - 26.03.2010. Main Meeting of the Physiological Society, Oxford, UK, 11.-14.07.2011 20th Northern Cardiovascular Research Group Meeting, Hull, UK, 19.04.2011 22nd Mammalian Genetics and Development Workshop (The Genetics Society), London, 17. 11. 2011 Annual Meeting of the Physiological Society, Edinburgh, UK, 02. - 05.07.2012 EMBO Conference 'Obesity and Diabetes', Heidelberg, Germany, 13. - 16. 09. 2012 Biochemical Society Annual Symposium: 'Epigenetic Mechanisms in Development and Disease'; Leeds, UK, 11. - 13.12. 2012 Paper publication: Nunn N, Feetham CH, Martin J, Barrett-Jolley R, Plagge A, 'Elevated blood pressure, heart rate and body temperature in mice lacking the XLas protein of the Gnas locus is due to increased sympathetic tone.' Exp. Physiol. 98: 1432 - 1445 (2013)
Start Year 2008
 
Description RNAseq analysis of Gnasxl KO hypothalami 
Organisation University of Liverpool
Department Department of Functional and Comparative Genomics
Country United Kingdom 
Sector Academic/University 
PI Contribution I provided RNA from hypothalamus tissues of Gnasxl WT and KO mice for RNAseq analysis. We helped with the analysis of the data on differences in gene expression levels. I taught a collaborating PhD student how to confirm data using additional, different techniques, i.e. Western blots and immunohistochemistry. Additional members of my lab have added further data to this project. I have written a paper describing the results, the manuscript has been submitted to a journal and is currently under review for publication.
Collaborator Contribution They provided access to next-generation sequencing technology (Illumina) and carried out the RNAseq experiment and the associated bioinformatics analyses of the RNAseq data.
Impact Poster at Conference: British Neuroscience Association, 2015 Festival of Neuroscience Edinburgh, UK, 12. - 15.04.2015 Poster at conference: Obesity: A Physiological Perspective Newcastle, UK, 10. - 12.09.2014 Talk at conference: Programming Obesity: Central and Peripheral Contributors; Cambridge, UK, 14. - 16.04.2013 Poster at conference: Programming Obesity: Central and Peripheral Contributors Cambridge, UK, 14. - 16.04.2013 Invited talk: Nottingham Trent University; School of Biomedical and Natural Sciences Nottingham, UK, 14.03.2014
Start Year 2011