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

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.

During embryonic development TGFbeta signalling is essential for gastrulation and patterning, including specification of body axes. In the adult it plays roles in tissue homeostasis by promoting differentiation and counteracting proliferation, it also exhibits tumour-suppressing functions in several tissues. Deregulation of signalling is implicated in various diseases, including vascular disorders, fibrosis and cancer. TGFbeta regulates epithelial to mesenchymal transition (EMT), a migratory cellular behaviour required during gastrulation and organogenesis, while within tumours EMT promotes metastasis.
The TGFbeta ligand Nodal is essential for gastrulation and anterior-posterior (A-P) patterning of the vertebrate embryo. A-P axis specification is the best-characterised dose-dependent function of this pathway, with high levels required for anterior and lower for more posterior structures. However, it is not known how this is established.
Several regulatory factors have been identified suggesting that TGFb/Nodal signalling levels are tightly regulated. In mice, loss of function mutations in single intracellular negative regulators of the pathway, such as SnoN, Ski, Smad7 and Smad6, failed to impair A-P patterning raising concerns about their normal function in vivo. On the contrary, loss of Arkadia/RNF111, which has been shown to degrade all the above negative factors to enhance intracellular Nodal signalling, causes severe anterior truncation of the A-P embryonic axis. This implies that in its absence there is hyperactivity of negative regulators that reduces signalling. We propose to examine this using mouse genetics and A-P patterning in the embryo, along with the differentiation of embryonic stem cells towards anterior endoderm-foregut cell fates in culture. We expect our results to shed light on the dose-dependent functions of Nodal by revealing the importance of intracellular regulation in establishing the different responses and the underlying mechanisms.

During embryonic development TGFbeta signalling is essential for gastrulation and patterning, including the specification of body axes (head-tail, left-right). In the adult it plays a role in tissue homeostasis by promoting differentiation and counteracting proliferation, it also exhibits tumour-suppressing functions in several tissues. Deregulation of signalling is implicated in various diseases, including vascular disorders, fibrosis, and cancer. TGFbeta regulates epithelial to mesenchymal transition (EMT), a migratory cellular behaviour required during gastrulation and organogenesis, while within tumours EMT promotes metastasis.

Shedding light onto the mechanisms that regulate the dose-dependent functions of TGFbeta signalling will lead to better understanding of normal development and the molecular mechanisms that underlie the multiple functionality of the pathway, but also would provide the foundations for understanding its role in cancer and other diseases. Importantly, such knowledge would improve prognosis and enable precise therapeutic manipulations targeted to a subset of TGFbeta functions.

The ability to control signalling levels will also allow precise and efficient generation of cell fates during differentiation of stem cells in culture, or in vivo, facilitating and improving their use in regenerative medicine. Specifically from this research we will identify molecular components for the efficient differentiation of ESC towards anterior definitive endoderm precursors, which give rise to foregut and associated organs (liver, pancreas, lung, oesophagus and pharynx).