During early embryogenesis, segments (somites) are formed during a process called somitogenesis. These somites will go on to form the bones and muscles of the skeleton. The timing of the segmentation process is regulated by a molecular oscillator, the segmentation clock, that drives cyclic gene expression with a periodicity that matches somite formation (1). This process is tightly controlled, and dysregulation of the segmentation clock results in diseases such as congenital scoliosis (2). For the segmentation clock several levels of regulation of clock gene expression are important: transcriptional activation and negative feedback loops, post-transcriptional regulation (from splicing to RNA stability) and protein degradation (3).
This project will investigate how post-transcriptional regulators control the segmentation clock in human induced pluripotent stem cell (hiPSC) derived presomitic mesoderm (PSM) cells. The project will also use somitoids, hiPSC derived 3D structures that will enable investigation of the role of post-transcriptional regulators during human somite formation, something that would be extremely challenging in human embryo’s for practical and ethical reasons. The project will provide insights into the mechanisms required for accurate segmentation clock gene expression in embryonic development as well as diseases associated with misregulation of the segmentation clock or the signaling pathways involved such as congenital scoliosis and T-cell acute lymphoblastic leukaemia (T-ALL).
Aims of the project:
- Identify the regulatory sequence elements in clock gene mRNAs and their interacting factors
- Determine the post-transcriptional mechanisms that regulate clock gene mRNAs
- Establish how post-transcriptional mechanisms control somite formation
Examples of techniques expected to be used during the project: maintenance of hiPSC, CRISPR modification of hiPSC, differentiation of hiPSC into PSM cells, generation of hiPSC derived somitoids, immuno fluorescence, in situ hybridisation, microscopy (e.g. time lapse imaging, confocal), purification of DNA, RNA and protein, RT-qPCR, Next Generation Sequencing (e.g. RNAseq, RIPseq, miRNASeq, RIBOseq, TAILseq, scSeq), immuno precipitation, western blotting, mass spectrometry, analysis of large data sets.