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

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.

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.