Post transcriptional Regulation of Oscillatory clock gene expression during somitogenesis

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.

The segmentation clock is a molecular oscillator that drives the cyclic gene expression required for regulating the timing of somitogenesis in the presomitic mesoderm (PSM) during early embryogenesis. Functional disturbance of key genes required for segmentation clock gene expression leads to developmental diseases such as congenital scoliosis as well as several cancers. For dynamic clock gene expression three levels of regulation are relevant: transcriptional control, post transcriptional regulation (every step from splicing to RNA stability) and protein degradation. A complex system of transcriptional activation and negative feedback loops is emerging, however, current knowledge of the post transcriptional mechanisms that control oscillatory clock gene expression is limited. This multidisciplinary project will combine mathematical modelling, bioinformatics and functional assays, with both targeted and omics scale data collection, to systematically identify the post transcriptional mechanisms that regulate oscillatory clock gene expression, using iPS-derived PSM cells and axioloid models. We will analyse ribosomal association and poly(A) tail length changes to investigate translational efficiency and mRNA stability of the clock genes and will establish which sequence elements in clock gene mRNAs determine post transcriptional regulation and what factors are interacting with these elements during the clock cycle. Regulators at the post transcriptional level include RNA binding proteins, microRNAs and long noncoding RNAs. We will establish the function of the newly identified post transcriptional regulators in regulating clock gene expression and somitogenesis. The project will improve our understanding of developmental disorders such as scoliosis and cancers linked to misexpression of clock gene pathways.