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

胚胎的身体形成一个细长的结构，头部在前端，随后是躯干，然后是尾部在后端。这构成了基本的身体前后（A-P）轴。名为Nodal的基因及其相关基因网络的突变会导致胚胎前部/头部的减少或丢失。这样的畸形很少是可行的，这些胚胎被流产。有趣的是，Nodal基因活性的降低会导致头部截短，而没有任何Nodal的胚胎根本不会形成任何A-P轴结构，并且在植入后很早就死亡。因此，头部/前部的形成需要高的节点水平/活动，躯干的形成需要低的节点水平/活动，尾部/后部的形成甚至更少。我们研究的重点是通过分析负责调节细胞内Nodal活性水平的因素来了解Nodal的剂量依赖性效应。Nodal由某些细胞产生并分泌到细胞外空间，在那里它扩散并影响暴露于它的细胞的命运和行为。Nodal的扩散产生了一个梯度，靠近源高，远离源低。暴露于Nodal的细胞通过位于其细胞表面的特定蛋白质（受体）接受它。然后，该信号作为蛋白质相互作用的中继穿过细胞质，导致特定蛋白质的修饰，这些蛋白质进入细胞核并与染色体上的一组靶基因结合以调节其表达。这种基因激活（有时是抑制）改变了细胞的命运和行为。然而，这种“信号转导”途径本身受到调节，以便更精确地控制靶基因受影响的程度，例如被激活的比例，以及因此接收Nodal信号的细胞的命运。这种调节依赖于一些负调节蛋白和我们鉴定并命名为Arkadia的因子之间的平衡，该因子破坏这些负调节因子以增强Nodal信号传导。因此，Arkadia使细胞的反应就像它们暴露在高水平的细胞外Nodal中一样。但我们还不知道这些因素是否真的是负责头/前后/后轴形成在整个胚胎，如果他们确实改变了Nodal的绝对浓度的解释，其中细胞exposed.Here我们建议测试如何结合突变的Nodal调节因子，包括Arkadia影响小鼠胚胎的头/前部与尾部/后部的发展。此外，我们还将在培养的小鼠胚胎干细胞（ESC）中产生这些突变，并检查这些突变是否影响它们向与头部/前部相关的组织分化，并依赖于高Nodal信号。一种这样的组织是前肠（前肠），其产生食道、喉、肺、肝和胰腺。因此，在ESC中操纵Nodal活性水平的能力将促进和改善可用于再生医学的上述组织和器官的产生。

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.