Investigation into the regulation of NICD stability and its role in determining the periodicity of the vertebrate segmentation clock (360G-Wellcome-105306_Z_14_A)

Notch is one of the five major signalling pathways that regulate vertebrate and invertebrate development. Notch signalling relies on cell-cell contact as both ligand and receptor are transmembrane proteins. Upon ligand activation the intracellular domain of the receptor (NICD) is cleaved and translocates to the nucleus to activate gene transcription. NICD is extremely labile and phosphorylation allows this target to be recognised by the F-box protein Fbxw7 which then recruits the Skp1-Cui1-F-box protein (SCF) ubiquitin ligase complex that targets NICD for degradation. Thus, phosphorylation is a key event in NICD turnover but the details of this process remain obscure. Recent work in the lab has shown that a number of different small molecule kinase inhibitors, which delay the vertebrate segmentation clock, also lead to elevated NICD levels and prolonged NICD half-life. This research project aims to determine whether these inhibitors increase levels/half-life of NICD by changing NICD phosphorylation status and whether they affect the interaction of NICD with Fbxw7. We will employ a transgenic mouse that allows real time imaging of clock gene oscillations in tissue explant assays and will use CRISPR/Cas9 knock-in technology to generate NICD phospho-mutants to determine if this alters the periodicity of clock gene oscillations.