Post transcriptional Regulation of Oscillatory clock gene expression during somitogenesis

该项目旨在研究负责发育中胚胎分割的调节机制。这些片段继续形成骨骼和肌肉，增加对这一过程的了解对于了解发育疾病(如脊柱侧弯和某些癌症)至关重要。在胚胎发育过程中，细胞需要分化，以产生胚胎所需的所有不同类型的细胞。这是通过一系列非常复杂的过程实现的，这些过程依赖于不同细胞之间的高度协调。一个这样的过程是分割，它导致被称为体节的片段的形成，体节是形成骨骼和肌肉的不同细胞组。这个过程是由一个被称为分段时钟的分子时钟驱动的，它以循环的方式激活一组基因的表达。这些基因需要被及时地开启和关闭，以产生正确的时钟基因表达的定时波。这些因素共同调节了这些片段的形成时间。如果分段时钟基因没有在正确的时间开启和关闭，这会导致更大或更小的片段，这可能会导致出生缺陷，如脊柱侧弯。基因表达的调节发生在几个不同的水平。当基因被激活时，所需基因的一个副本就产生了：信使RNA，然后它被翻译成所需的蛋白质。对于分段时钟，我们知道很多关于基因是如何激活的，但我们对这些信使的活动水平和稳定性如何调节的了解非常有限。对信使活性和稳定性的准确调控对于确定将被制造的蛋白质的时间和数量至关重要，并且对于成功的分割和胚胎存活是必不可少的。这一过程已被证明对其他振荡过程至关重要，如昼夜节律时钟，但遗憾的是，对分段时钟的研究不足。这项拟议的研究为我们对分段时钟的知识增加了一个新的、至关重要的和创新的方面。我们将使用两个干细胞衍生的模型系统来促进人类胚胎发育的研究，而不是使用人类或动物胚胎。1)人诱导的多能干细胞将被分化为形成体节的细胞：卵子前中胚层。这些通过荧光蛋白显示时钟基因的表达。2)我们将使用相同的细胞并产生AXILOID：形成体节状细胞块的3D结构。利用这些模型系统，我们可以分析扰动候选调节子的功能对时钟基因振荡和体节形成的影响。当对细胞产生的关键调控蛋白的时间和数量的调控被解除时，这是包括许多癌症在内的许多疾病的特征。时钟基因被信号通路激活，这些信号通路都与癌症密切相关，如果它们被异常调控的话。因此，更好地了解时钟基因所产生的信使和蛋白质的正确数量的调节机制，将有助于我们理解这些过程的错误调节如何可能导致某些癌症。这个项目的目的是发现我们可以检测到的所有时钟基因信使的调控机制，并将其与控制分段过程的信号通路联系起来。它将提供时钟基因调控的非常详细的分析，这将使我们更好地了解分段过程，并将有助于我们了解发育障碍，如脊柱侧弯以及与参与分段的基因的错误表达有关的几种癌症。

This project aims to investigate the regulatory mechanisms responsible for the segmentation of the developing embryo. These segments go on to form the bones and muscles of the skeleton and increased knowledge of this process is essential for the understanding of developmental diseases such as scoliosis as well as certain cancers.During embryonic development cells need to differentiate to generate all the different cell types that the embryo requires. This is achieved by a set of very complicated processes which are dependent on a high level of co-ordination between the different cells. One such process is segmentation which results in formation of segments called somites, distinct groups of cells that will form the bones and muscles of the skeleton. This process is driven by a molecular clock termed the segmentation clock which activates expression of a set of genes in a cyclic manner. These genes need to be switched on and off in a timely fashion to generate the correctly timed waves of clock gene expression. Together, these regulate the timing of the formation of the segments. If the segmentation clock genes are not switched on and off at the correct times this results in bigger or smaller segments which can lead to birth defects such as scoliosis.Regulation of gene expression occurs at several different levels. When genes are activated a copy is made of the desired gene: the messenger RNA, which then subsequently gets translated into the protein that is required. For the segmentation clock a lot is known about how genes are activated but our knowledge on how activity levels and stability of these messengers are regulated is very limited. Accurate regulation of messenger activity and stability are critical to determine the timing and the amount of the proteins that will be made and are essential for successful segmentation and embryonic survival. This process has been shown to be of critical importance for other oscillatory processes such as the circadian clock but has been woefully understudied for the segmentation clock. The proposed research adds a new, critically important and innovative aspect to our knowledge of the segmentation clock. It takes an unbiassed holistic approach to identify the regulatory processes governing regulation of messenger activity and stability of these critical genes.We will use two stem cell derived model systems that facilitate research into the development of human embryogenesis without the use of human or animal embryos. 1) Human induced pluripotent stem cells will be differentiated into the cells that form somites: presomitic mesoderm. These show clock gene expression via a fluorescent protein. 2) We will use the same cells and produce axioloids: 3D structures that form somite-like cell blocks. Using these model systems, we can analysis the effects of perturbing the function of candidate regulators on clock gene oscillations and somite formation. When regulation of the timing and the amount of key regulatory proteins produced by a cell is deregulated this is a hallmark of many diseases including many cancers. Clock genes are activated by signalling pathways which are all tightly associated with cancers if they are aberrantly regulated. Thus, a greater understanding of the mechanisms regulating the correct amount of the messengers and proteins made by clock genes will inform our understanding of how misregulation of those processes may contribute to certain cancers. The aim of this project is to discover the regulatory mechanism for all clock gene messengers that we can detect and to link that to the signalling pathways that control the segmentation process. It will provide a very detailed analysis of clock gene regulation which will provide a much better understanding of the segmentation process and will contribute to our knowledge regarding developmental disorders such as scoliosis as well as several cancers that are linked with misexpression of the genes involved in segmentation.