Mathematical models predict the period of clock gene oscillations can be approximated as a sum of the delays involved in transcription, splicing, translation and transport of clock gene products, and in particular through the regulation of the half-lives of both mRNA and protein of unstable regulators 14- 17. Synchronization of clock gene oscillations between neighbouring cells is reliant on Notch signalling 10- 13. On a single cell level in the PSM, oscillatory clock gene expression is established through positive and negative feedback loops of unstable clock gene products, which potentiate or inhibit the pathway that activates them. Notch is crucial to the segmentation process in mice, since in the absence of Notch signalling, the segmentation clock stops and no somites form 9. Significantly, four of these are components of the Notch pathway (DL元, MESP2, LFNG, HES7), which plays multiple roles during segmentation. For CS, while the aetiology is unclear, linkage analyses have shown mutations in six genes (DL元, MESP2, LFNG, HES7, TBX6 and RIPPLY2) lead to familial forms of SCD 8. In humans, defects in segmentation lead to congenital scoliosis (CS), with an infant mortality rate of 50% that comprises many vertebral skeletal and muscular pathologies, including the family of spondylocostal dysostoses (SCD). Aberrant somitogenesis leads to severe segmentation and skeletal defects 7. The genes that exhibit oscillatory PSM expression are termed clock genes and are targets of the Notch, Wnt and FGF pathways 5, 6. The periodicity of segment formation is regulated by a molecular oscillator, known as the somitogenesis clock, which drives oscillatory gene expression within the PSM tissue from which somites are derived 2- 4. Segmentation, a process which occurs early during vertebrate body plan formation, generates repeated segments (or somites) that later give rise to the vertebral column, most skeletal musculature and dermis 1, 2.ĭuring somitogenesis, pairs of somites bud off the rostral end of the unsegmented presomitic mesoderm (PSM) with a periodicity that is species specific. Inhibiting CDK1 or CDK2 activity increases NICD levels both in vitro and in vivo, leading to a delay of clock gene oscillations and an increase in somite size. We demonstrate both CDK1 and CDK2 can phosphorylate NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle-dependent manner. We identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. However, the mechanism by which NICD stability is regulated in this context is unclear. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. Notch signalling is crucial to this process. The segmentation clock is a molecular oscillator that exhibits dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Segments form from the rostral end of the presomitic mesoderm (PSM) with a periodicity that is regulated by the segmentation clock. All vertebrates share a segmented body axis.

Imagej western blot quantification