Characterisation of the impact of NICD S2513 mutation on human segmentation using iPS derived somitoids

During early development cells differentiate into the different cell types required to form the tissues that make up the embryo. Research into human embryological development is challenging for practical and ethical reasons. This project will investigate the process of somitogenesis (the formation of segments that give rise to the bones and muscle of the skeleton) using human stem cell derived 3D structures called somitoids (also named segmentoids/axioloids) and 2D differentiation protocols. The 2D protocols are well established, however, the somitoid protocols have very recently been developed and represent state of the art tissue culture techniques and will require further development. Combined these two approaches allow for the use of human stem cells to study human development and disease and avoid the use of animal model embryos. 

One of the key signalling pathways regulating somitogenesis is the Notch pathway. Notch1 is a transmembrane protein that gets activated by signals from neighbouring cells. This results in the release of the intracellular domain (NICD) that translocates to the nucleus and forms a transcription complex which activates segmentation clock genes. The expression of these genes oscillates in time with somite formation. Therefore, regulation of activation and inactivation of NICD is of critical importance. Inactivation is mediated by phosphorylation of residue S2513. This recruits Fbxw7 and targets NICD to the ubiquitin pathway. 

This project aims to investigate the molecular mechanisms resulting in the phosphorylation of S2513 and the impact of mutagenesis of S2513 on somitogenesis in human iPSC derived somitoids. The results of this project are critical for the understanding of human somitogenesis as well as related birth defects. Moreover, T-ALL (T-cell acute lymphoblastic leukaemia) patients often have mutations in Notch1 (including S2513) and Fbxw7 that lead to high levels of NICD which correlate with poor prognosis. Therefore, this project will also improve our understanding of T-ALL and other cancers where Notch signalling is disturbed.

The project will incorporate a wide variety of techniques, such as stem cell maintenance, CRISPR, 2D and 3D differentiation, advanced microscopy including timelapse imaging, single cell sequencing, mass spectrometry and a variety of other biochemistry, molecular biology and cell biology approaches. The lab of Dr. Davies has longstanding collaborations with the labs of Prof. Dale (somitogenesis, developmental biology, Notch signalling) and Prof. Lowell (3D iPSC differentiation) who will be actively involved with this project. The PhD student will also be supported by the world class iPS facilities at Dundee University.

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