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The critical role of p63 during palatal shelf fusion

Richardson, Rose

[Thesis]. Manchester, UK: The University of Manchester; 2015.

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Abstract

Cleft palate affects approximately 1 in 2000 live births resulting in considerable morbidity to affected individuals and their families. Evidence that the p63 gene is mutated in at least seven human developmental syndromes which are each characterised by varying extents of orofacial clefting, coupled to the severe facial dysmorphism displayed by the p63 mutant mouse, highlight the need to elucidate the role of the p63 during normal and aberrant palatogenesis.In mice, secondary palate development closely mirrors that occurring in humans; consequently, the mouse is a pre-eminent model organism for studying palatogenesis. In mice, the palatal shelves initiate from the maxillary processes and grow vertically, lateral to the tongue. The shelves re-orientate and make contact above the tongue. The medial edge epithelia (MEE) of the apposed palatal shelves adhere to form a midline epithelial seam (MES). Subsequent degeneration of the MES allows mesenchymal confluence across the palate, at which point palatogenesis is considered complete. The mechanisms underlying degeneration of the MES remain contentious; however, in vivo studies suggest that cessation of proliferation, induction of apoptosis and periderm migration are essential to ensure removal of the midline seam. The data presented in this thesis uncover a key role for p63 in controlling these aspects of cell behaviour during palatal shelf fusion. Tgfb3-/- mice exhibit cleft palate with maintained expression of p63 in the MEE. This thesis reveals that epistatically lowering the dosage of p63 in Tgfb3-/- mice rescues this fusion defect, facilitating periderm cell migration out of the MEE and subsequent MES degradation. Recent research suggests that p63 orchestrates a cell adhesion network in the palate. In this context, this thesis suggests the importance of p63 down-regulation in the MES in compromising adhesion at the basal-periderm border, thereby allowing periderm cell migration out from the midline and subsequent MES degradation.To test the hypothesis that down-regulation of p63 is essential for palatal fusion, tetracycline-inducible transgenic animals in which ΔNp63α is targeted to the MEE of the developing palate have been engineered. ΔNp63α bi-transgenic mice presented with cleft palate in which the MES failed to degenerate. An observed lack of apoptotic activity in the MEE of ΔNp63α bi-transgenic mice suggested a role for p63-mediated apoptosis during MES degradation. Gene ontology analysis of a complete range of ΔNp63α transcriptional targets which have been identified in the secondary palate by ChIP-seq, lent support to this hypothesis. The data indicate that p63 down-regulation in the MES is essential to ensure complete removal of the MES and implicate p63 as a key regulator of apoptosis during this process; thereby building on work which suggests that cell death is the major fate of the MEE. In addition to dissecting a pathway of fundamental importance in secondary palate development, this research provides insights into ectodermal development more generally and has wider significance for the study of many congenital malformations.

Additional content not available electronically

Time-lapse confocal imaging of periderm cell migration in the medial edge epithelia of [mK17 5’]-GFP transgenic mice during palatal shelf fusion. Palatal shelves from wild-type, Tgfb3-/- and Tgfb3-/-; p63+/- mice were cultured and imaged over a 24 hour period.

Bibliographic metadata

Type of resource:
Content type:
Form of thesis:
Type of submission:
Degree programme:
PhD Basic Dental Sciences (Molecular Genetics) 4 Years Full-time
Publication date:
Location:
Manchester, UK
Total pages:
269
Abstract:
Cleft palate affects approximately 1 in 2000 live births resulting in considerable morbidity to affected individuals and their families. Evidence that the p63 gene is mutated in at least seven human developmental syndromes which are each characterised by varying extents of orofacial clefting, coupled to the severe facial dysmorphism displayed by the p63 mutant mouse, highlight the need to elucidate the role of the p63 during normal and aberrant palatogenesis.In mice, secondary palate development closely mirrors that occurring in humans; consequently, the mouse is a pre-eminent model organism for studying palatogenesis. In mice, the palatal shelves initiate from the maxillary processes and grow vertically, lateral to the tongue. The shelves re-orientate and make contact above the tongue. The medial edge epithelia (MEE) of the apposed palatal shelves adhere to form a midline epithelial seam (MES). Subsequent degeneration of the MES allows mesenchymal confluence across the palate, at which point palatogenesis is considered complete. The mechanisms underlying degeneration of the MES remain contentious; however, in vivo studies suggest that cessation of proliferation, induction of apoptosis and periderm migration are essential to ensure removal of the midline seam. The data presented in this thesis uncover a key role for p63 in controlling these aspects of cell behaviour during palatal shelf fusion. Tgfb3-/- mice exhibit cleft palate with maintained expression of p63 in the MEE. This thesis reveals that epistatically lowering the dosage of p63 in Tgfb3-/- mice rescues this fusion defect, facilitating periderm cell migration out of the MEE and subsequent MES degradation. Recent research suggests that p63 orchestrates a cell adhesion network in the palate. In this context, this thesis suggests the importance of p63 down-regulation in the MES in compromising adhesion at the basal-periderm border, thereby allowing periderm cell migration out from the midline and subsequent MES degradation.To test the hypothesis that down-regulation of p63 is essential for palatal fusion, tetracycline-inducible transgenic animals in which ΔNp63α is targeted to the MEE of the developing palate have been engineered. ΔNp63α bi-transgenic mice presented with cleft palate in which the MES failed to degenerate. An observed lack of apoptotic activity in the MEE of ΔNp63α bi-transgenic mice suggested a role for p63-mediated apoptosis during MES degradation. Gene ontology analysis of a complete range of ΔNp63α transcriptional targets which have been identified in the secondary palate by ChIP-seq, lent support to this hypothesis. The data indicate that p63 down-regulation in the MES is essential to ensure complete removal of the MES and implicate p63 as a key regulator of apoptosis during this process; thereby building on work which suggests that cell death is the major fate of the MEE. In addition to dissecting a pathway of fundamental importance in secondary palate development, this research provides insights into ectodermal development more generally and has wider significance for the study of many congenital malformations.
Additional digital content not deposited electronically:
Time-lapse confocal imaging of periderm cell migration in the medial edge epithelia of [mK17 5’]-GFP transgenic mice during palatal shelf fusion. Palatal shelves from wild-type, Tgfb3-/- and Tgfb3-/-; p63+/- mice were cultured and imaged over a 24 hour period.
Thesis main supervisor(s):
Thesis co-supervisor(s):
Language:
en

Institutional metadata

University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:247702
Created by:
Richardson, Rose
Created:
20th January, 2015, 22:17:53
Last modified by:
Richardson, Rose
Last modified:
6th February, 2020, 10:40:37

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