Student thesis: Doctoral ThesisDoctor of Philosophy


    Approximately 3-6% of babies worldwide are born with serious birth defects each year. Among these, craniofacial malformations collectively represent about one-third of all congenital anomalies. The clinical conditions have lifelong consequences for the patients, however, the mechanisms of pathology and etiology associated with most craniofacial birth defects are unknown. My PhD project seeks to provide an in-depth analysis focusing on an important but understudied embryonic craniofacial structure the vestibular lamina (VL). Defects in the VL are present in a number of craniofacial syndromes and are associated with the formation of dental tumours (odontomas) in later life.

    The VL is an embryonic structure that forms the oral vestibule. It shares a common origin with the neighbouring dental lamina (DL). The development of the VL is disrupted with a high incidence in Ellis-van Creveld (EvC) syndrome. In this syndrome, patients display multiple oral frenula physically linking the lips and teeth. EvC syndrome is a recessive disorder considered as a ciliopathy, which is caused by a mutation in EVC or EVC2. EVC and EVC2 proteins localize at the base of the primary cilia, and function to regulate hedgehog signalling. To understand the underlying mechanisms linking mutations in EVC genes to the physical abnormalities, my PhD project aims to: 1) To examine the early development of VL in mouse and human, and assess the mechanisms underlying furrow formation. 2) To identify the molecular signature for the VL and to explore the differences in signalling pathway expression in VL and DL. 3) To investigate the development of the VL in Gas1 and compound mutants and explore the role of Shh signalling in the VL and DL. 4) To compare the VL defects in Gas1 and Evc mice, looking for common underlying mechanisms. The key methods include organ culture, RNA-Scope, DIG in situ hybridization, bioinformatic analysis, immunofluorescence, statistical analysis, and histological staining.

    I first assessed the VL development in human and mouse, and revealed the mechanisms of furrow formation during normal development. The VL shows a close relationship with DL from initiation to differentiation, with fissure formation caused by terminal differentiation without an obvious contribution from apoptosis. Extension of the VL is driven by localized proliferation at the edges of the VL in the human and mouse. Bulk RNA-Seq was performed to explore the molecules involved during early development of the VL and DL. Novel gene expression data and potential signalling pathways were identified. The highlighted differences of Shh pathway components in the VL and DL drove me to concentrate on the Shh signalling. I have identified that the VL development rely on the Shh signals from the adjacent tooth germ using the Shh pathway mutants (Gas1 and compound mutants). A deeper understanding of the mechanisms that lead to EvC syndrome defects was achieved through a further comparative analysis of Evc mutants and Gas1 mutants. Despite sharing a similar truncated VL phenotype, Gas1 and Evc mutants exhibit different pathological mechanisms. Both Gas1 and Evc mutants displayed a reduction in proliferation within the truncated VL. However, the Gas1 mutants showed decreased expression of Gli1, whereas Evc mutants exhibited an upregulation of Gli1. Additionally, in Evc mutants, I have identified other VL and DL defects mimicking the phenotypes seen in EvC patients, such as multiple VL, conical teeth, fused teeth, and supernumerary tooth originated from VL. Moreover, the opening of the short VL occurred as normal and the basic cilia structure appeared unaffected in the Evc mutants. These findings will help our understanding of the oral problems in syndromes such as EvC syndrome and other ciliopathies.

    Date of Award1 Jul 2023
    Original languageEnglish
    Awarding Institution
    • King's College London
    SupervisorAbigail Tucker (Supervisor) & Isabelle Miletich (Supervisor)

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