Abstract
The mammalian tooth, specifically the molar, is a highly specialised and intricate organ. The molar’s cusps, ridges and sulci generate an occlusal surface, interfacing with its counterpart in the opposite jaw to chew and grind food. Due to its importance in evolutionary history and ease of study, the development of the molar has been extensively explored. However, the cellular mechanisms orchestrating its morphogenesis have been historically overlooked. The predominant hypothesis of molar morphogenesis is by buckling due to proliferation under confinement (BPUC), and differential proliferation has been modelled as the sole requirement for shape creation, whilst other cellular behaviours have received minimal attention.In this thesis, I set out to test the model of BPUC and explore other cellular mechanisms involved in molar bell stage epithelial folding. Contrary to in-silco models (but in line with findings at earlier stages), I found that proliferation was not essential to generate the characteristic bends of the sulcus and cusps. Tooth explants with and without growth generated indistinguishable shapes when analysed with landmark morphometrics. While some local sharpening of the sulcus required proliferation, it became evident that other mechanisms are involved in the shaping. Furthermore, as well as not requiring proliferation, I observed that cusp development proceeded without confinement from the surrounding mesenchyme and that compaction of the dental papilla, specifically below the cusps, is likely more necessary for shaping than global external force.
Given the non-essential role of proliferation, I then investigated the contribution of the epithelium and found that localised cell constriction correlated with curvature. Basal constriction (BC), by a reduction in the basal width, was seen in the concave curves of the cusps whereas apical constriction (with basal expansion) was seen in the convex curves of the tips of the cervical loops. Additionally, there was some evidence of apical constriction in the bending of the sulcus. Staining for F-Actin revealed basal or apical enrichment corresponding to this polarised reduction in width, implying the cell shape change is an active process, rather than a passive result of bending by buckling.
Inhibition of F-Actin and Focal Adhesion Kinase disrupted tooth morphology supporting the idea that cell shape control is required for proper bending. However, global inhibition in explant culture is not sufficient to localise the regions of force to the epithelium. To address this, teeth with epithelium-specific knockouts of Myosin were analysed, and I found some evidence that the molar shape was disrupted. The effect depended on the amount of knock- out, implying an essential role for the epithelium.
Interestingly, BC cells at earlier (cap) stages flank the primary enamel knot (PEK). The BC cells I identified at the bell stage also seemed to neighbour the secondary enamel knots (SEKs), leading to a hypothesis that these so-called juxta-knot cells are responsible for shaping the epithelium. To capture the identity of these cells, as well as any markers for the sulcus, I used Laser Capture Microdissection (LCM) and RNA sequencing. I found that the BC cells at cap stage represent an outer ring of the enamel knot gene nested expression, promoting a reimaging of the structure of the knot which will require further investigation. Unfortunately, the results at later stages were highly variable and so inconclusive, although some sulcus markers are suggested.
I investigated genes potentially involved in constriction and identified several shared actin- binding genes in both the BC and AC cells. Additionally, each cell type expressed unique genes in each that may be associated with distinct mechanisms. Further, investigation of the genes unique to BC cells revealed an enrichment of genes involved in microtubule organization. Considering the increased nuclear height in these cells, I propose a hypothesis suggesting a potential role for interkinetic nuclear movement in shaping the cells.
Overall, this work proposes a previously overlooked role for the epithelium in shaping the bell stage germ, with specific cell behaviours potentially relating to the locations of the enamel knots. Although modelling proliferation in-silco can generate realistic tooth germs, this thesis highlights the importance of functionally testing models and considering complex cell behaviours. Several hypotheses are proposed for mechanisms of epithelial bending, which provide future directions for this project and the limitations are discussed.
| Date of Award | 1 Jun 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Jeremy Green (Supervisor) & Martyn Cobourne (Supervisor) |