Abstract
The mammalian palate is the physical and functional barrier between the oral and nasal cavities. It is also a common locus for human birth defects, with incidence of cleft lip/palate at 1:700 live births worldwide. Dysmorphias may arise at any stage of palatogenesis, from the initial ventral outgrowth of the paired palatal shelves (PSs), via their elevation above the tongue to their eventual fusion horizontally at the midline. Elevation is poorly understood, with no universally agreed-upon mechanism of action. It is thought to be driven by a force internal to the PSs in a heterogeneous manner, with the anterior 'flipping-up', and the posterior actively remodelling around the tongue. The cellular mechanisms of growth generating the internal shelf force are not wellunderstood, and little research has been carried out into both the biomechanics of this process and the material properties of the PSs and how they might facilitate elevation.Initially, I set out to investigate the cellular mechanisms of growth underlying force generation in the PSs using an established hyperoxic ex vivo rolling culture system at E12.5 and E13.5. Surprisingly, it appeared that elevation was not occurring during culture, but rather via elastic recoil immediately on the removal of the tongue and mandible. However, 50% of cultured explants still displayed fused PSs, which posed the question as to how PS approximation can occur in the absence of growth ex vivo.
To interrogate this, I carried-out a 2D morphometric analysis of explanted maxillaecontaining PSs, ‘maxillary explants’, during culture, which revealed that growth was poor and inconsistent across different metrics, and that the explants sometimes shrank. Increasing the gas change frequency marginally improved growth, though it remained inconsistent. On further morphometric analyses, I identified that PS fusion in ex vivo rolling culture is caused by a non-physiological deformation of the tissue, which narrows the space in the midline, causing the PSs to approximate.
PS elevation is a mammalian novelty, and non-mammalian PSs grow horizontally by default. This appeared to be the case in the anterior mouse PSs at E12.5, prior to contact and presumably downward deflection by the tongue. This led to the hypothesis that PS elevation evolved as a consequence of the mammalian suckling requirement and large tongue, which I tested by producing a time course of palate development in a previously undescribed non-suckling mammal, the short-beaked echidna. The echidna shelves deflected just as in other mammals, disproving my hypothesis.
Finally, the ex vivo culture data suggested that elevation was an elastic, and potentially viscoelastic, event. Therefore, I carried out a series of experiments investigating the generation and mediation of the internal shelf force and the material properties of the PSs. Nuclear morphometrics studies revealed a complex picture of nuclear deformation and internuclear spacing across the PSs, but there were technical limitations in the segmentation model used and it was difficult to separate heterogeneity from noise. Further investigation into the presence of elastic fibres and collagens was carried out via histological staining and second harmonic imaging, which suggested that there are no mature elastic fibres or bundles of collagen present in the PSs at this time. Atomic force microscopy was used to measure the stiffness of the PSs. This suggested that the PSs comprise a relatively soft mesenchymal core, which was softer anteriorly and stiffer posteriorly, with a stiffer outer epithelial layer. This might suggest a more significant role for the epithelium during elevation than previously thought.
This work highlights new directions for the study of the biomechanics underlying PS elevation. It also provides the first early description of the material properties of the PSs at E13.5 and demonstrates that ex vivo rolling culture is an inappropriate system for the study of both PS elevation and growth. The limitations and future directions of the project are discussed, including directions for modelling PS elevation in silico.
| Date of Award | 1 May 2024 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Jeremy Green (Supervisor) & Martyn Cobourne (Supervisor) |