Studies on the dose-dependent functions of TGFbeta-Smad2/3 signalling

点击翻译按钮获取中文摘要

The body of an embryo forms an elongated structure with the head at the anterior end followed by the trunk and then the tail at the posterior end. This constitutes the basic body Anterior-Posterior (A-P) axis. Mutation of the gene named Nodal and its associated gene network causes a reduction in, or loss of, the anterior/head of the embryo. Such malformations are rarely viable and these embryos are aborted. Interestingly it has become apparent that reduction of Nodal gene activity causes head truncation, while an embryo without any Nodal does not form any A-P axis structures at all and dies very early after implantation. Therefore, high Nodal levels/activity are required for the formation of the head/anterior and lower for the trunk and even less for the tail/posterior. The focus of our study is to understand the dose-dependent effects of Nodal by analysing factors that are responsible for regulating the levels of Nodal activity within the cells. Nodal is produced by certain cells and secreted into the extracellular space where it diffuses and affects the fate and behaviour of cells that are exposed to it. The diffusion of Nodal creates a gradient with high close to the source and low further away. The cells exposed to Nodal receive it with specific proteins located at their cell surface (receptors). Then this signal travels through the cytoplasm as a relay of protein interactions leading to the modification of specific proteins that enter the nucleus of the cell and bind to a set of target genes on the chromosomes to modulate their expression. This gene activation (and sometimes repression) changes cell fate and behaviour. However, this "signal transduction" pathway is itself regulated in order to control more precisely the extent to which the target genes are affected, e.g. the proportion that are activated, and consequently the fate of the cell receiving the Nodal signal. This regulation is dependent on the balance between a few negative regulatory proteins and a factor that we identified and named Arkadia, which destroys these negative regulatory factors to enhance Nodal signalling. Arkadia therefore makes cells respond as if it they have been exposed to high levels of extracellular Nodal. But we do not yet know whether these factors are actually responsible for head/anterior-tail/posterior axis formation in the whole embryo and if they do indeed change the interpretation of the absolute concentration of Nodal to which cells are exposed.Here we propose to test how the combination of mutations in Nodal regulatory factors including Arkadia affects the development of the head/anterior versus tail/posterior in mouse embryos. Furthermore, we will also create these mutations in mouse embryonic stem cells (ESC) in culture and examine whether these affect their differentiation towards tissues that are associated with the head/anterior and depend on high Nodal signalling. One such tissue is the foregut (anterior gut) which gives rise to the oesophagus, larynx, lung, liver and pancreas. Therefore, the ability to manipulate Nodal activity levels in ESC will facilitate and improve the generation of the above tissues and organs that can be use in regenerative medicine.