In the developing vertebrate embryo, segmentation initiates very early and in a species-specific manner through the formation of somites, which later give rise to the vertebral column, most skeletal musculature and dermis. Somites are gradually and periodically pinched off from the anterior end of the presomitic mesoderm (PSM). The rhythmicity of somitogenesis is regulated by a molecular clock called the segmentation clock that drives the periodic expression of a number of “clock” genes in the PSM, with a periodicity that matches somite formation. Clock genes belong to the Notch, Wnt and FGF pathways, and Notch has been shown to be essential in mouse and chick for somite formation and for dynamic expression of clock genes. Since Notch signalling does not use second messengers, the pathway activity is exclusively driven by concentration of Notch intracellular domain (NICD). NICD itself is unstable and NICD production appears as pulsatile, spatiotemporal waves that traverse the rostrocaudal axis of the PSM in the manner of a clock gene. In a recent study from the Dale lab, using computational modelling together with experimental manipulation involving pharmacological inhibitors, it was shown that increasing the half-life of NICD and thus increasing its turnover time increases the period of the somitogenesis clock. However, that study did not determine the mechanism of action of the inhibitors used. NICD stability is a key requisite for its activity and to date the regulation of stability has been attributed to phosphorylation of the PEST domain by two kinases namely cyclin-dependent kinase-8 (CDK8) and GSK-3β, which then recruits the SCF Sel10/FBXW7 E3 ubiquitin ligase complex, that targets NICD for degradation by the proteasome. However, many of these studies have been conducted through overexpression assays in vitro and none of this regulation has been verified in the presomitic mesoderm. In this thesis, we demonstrated, for the first time, the interaction between NICD and FBXW7 at endogenous levels. We further identified phosphorylated residues within human NICD and we showed that a highly conserved site is crucial for NICD recognition by FBXW7. We also demonstrate for the first time that two new kinases can phosphorylate hNICD1 in the domain where this crucial residue lies Lastly, we demonstrate, using a variety of assays, that inhibition of these two kinases leads to increased levels of hNICD1 in vitro and mNICD1 in vivo and that this treatment also delays both the mouse somitogenesis clock and somite formation in the mouse PSM.
|Date of Award||2018|
|Supervisor||Kim Dale (Supervisor)|