A segmented body plan is a conserved feature of all vertebrate species. It is established early in embryonic development by the formation of somites, epithelised balls of tissue from which many of the axial tissues of the adult organism are derived. Produced during the process of somitogenesis, somites periodically bud off in pairs with strict, species-specific periodicity from an area of unsegmented tissue in the caudal end of the embryo termed the pre-somitic mesoderm (PSM). This periodicity is regulated by a molecular oscillator underlain by negative feedback loops termed the ‘segmentation clock,’ which manifests as a dynamic pattern of gene expression that sweeps the PSM in a caudo-rostral direction. Key components of the segmentation clock machinery are intracellular constituents of the Notch, Wnt and FGF signalling pathways. However, Notch signalling has been shown to be crucially important in establishing and regulating the segmentation clock oscillator and is essential for somitogenesis in higher vertebrates. In this thesis, a potential mechanism for the coordination of intracellular clock oscillations across the PSM is revealed. The expression levels of the obligate Notch signalling ligand and receptor in the PSM, Delta-like 1 (Dll1) and Notch1, are shown to vary dynamically across the PSM of both chick and mouse. This proposes that the signalling components mediating intercellular coupling of pulsatile gene expression themselves exhibit oscillatory behaviour. Additionally,
Notch1 is found to be a target of Notch signalling, whereas
Dll1 is demonstrated to Wnt regulated. The differential regulation of
Notch1 and
Dll1 expression thus links the activity of Wnt and Notch signalling, two of the main signalling pathways driving the segmentation clock. I also go on to explore transcriptionally independent mechanisms by which the dynamic Notch activity in the clock oscillator is reinforced.
The latter half of this thesis examines the temporal delays associated with the life cycle of clock components, which are proposed to dictate the periodicity of the segmentation clock oscillator. Recent research has employed the use of small molecule inhibitors to elicit changes in the stability of the intracellular portion of the Notch receptor and effector of Notch signalling, NICD. As judged by expression pattern changes in fixed PSM tissue, NICD stability was proposed to be tightly correlated to the periodicity of segmentation clock oscillations. Through further development of a published
ex vivo model of vertebrate segmentation, I build on this work by examining the effect of these same pharmacological inhibitors on the periodicity of segmentation clock oscillations in real-time using live-imaging approaches. Additionally, I uncover interesting physiological and molecular properties not previously observed in this
ex vivo model system that provide that a potential insight into the origin and initiation of segmentation clock oscillations. Expanding on current published literature, I examine the temporal delays associated with transcription, splicing and mRNA nuclear export of
Hes7, Dll1 and
Notch1 expression in the PSM. Given the differences in the regulation and size of the
Dll1 and
Notch1 genes, novel questions could be asked about the contribution of these temporal delays to the periodicity of the molecular oscillator underlying vertebrate segmentation.
| Date of Award | 2015 |
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| Original language | English |
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| Awarding Institution | |
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| Sponsors | Medical Research Council |
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| Supervisor | Kim Dale (Supervisor) |
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