Dissertation
Novel molecular mechanisms governing cleft palate and ankyloglossia
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2020
DOI: 10.17077/etd.005398
Abstract
In humans, ankyloglossia and cleft palate are common congenital craniofacial anomalies, which are regulated by a complex gene regulatory network. The treatment of ankyloglossia is relatively easier than cleft palate. However, either of these conditions can result in speech and social deficiencies which are costly to treat. For this reason, understanding the genetic underpinnings of clefting and ankyloglossia are important first steps towards the rational treatment of these defects.
Using SYSFACE, we identified genes enriched for expression in the palate, and Six2 was the top candidate. The expression of Six2 was confirmed in palatogenesis and staining revealed high levels of Six2 protein in the palatal mesenchyme. Using mouse models due to the conservation of the palatogenesis processes with humans, Six2 null embryos were sectioned and we determined that the loss of Six2 prevented palate shelf elevation. Furthermore, the deletion of Six2 resulted in decreased cell proliferation and malformation of the maxillary process and palatine bone. Interestingly, we identified Pax9 as a novel downstream target of Six2. Previous work has demonstrated Pax9 is involved in palatogenesis and we demonstrate that Six2 binds directly to the Pax9 promoter to regulate its expression. Additionally, we identified a human mutation SIX2(G264E) that resulted in decreased protein stability compared with the WT SIX2 protein, and a decreased ability to activate PAX9 expression in vitro. Overall, we demonstrate how Six2 contributes to palatogenesis by regulating Pax9 and these findings support including the SIX2 allele in genetic counseling when determining the clefting risk in newborns.
In addition to the palatal mesenchyme, the epithelium also plays an essential role in palatogenesis. Immunofluorescence staining demonstrated Sox2 is highly expressed in the palatal epithelia, and Pitx2Cre used to delete Sox2 resulted in cleft palate. In this mouse model, we found defects in rugae formation, signaling centers controlling palatogenesis, which likely explain the clefting phenotype. In Sox2 deleted embryos, we also found ankyloglossia, a fusion of the tongue to the mandible occurring at E14.5 and later stages. Close examination revealed this fusion resulted from the lack of periderm formation, a transient layer that prevents the adhesion of opposing epithelial tissues. Further examination revealed that the adhesion of the tongue is more proliferative and less differentiated than control matched oral epithelial tissue. The expanded epithelia not only express E-cad and P63, but are also Sox9, Pitx2 and Tbx1 positive cells, which are markers of the dental epithelium, and thus the dental epithelia contributes to the development of oral adhesions.
In conclusion, Six2 and Sox2 are involve in different gene regulatory networks to control palatogenesis. Sox2 is also essential for the formation of periderm to prevent abnormal adhesion during craniofacial development.
Details
- Title: Subtitle
- Novel molecular mechanisms governing cleft palate and ankyloglossia
- Creators
- Yan Yan Sweat
- Contributors
- Brad A Amendt (Advisor)John F Engelhardt (Committee Member)Robert A Cornell (Committee Member)Huojun Cao (Committee Member)Michael D Henry (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Anatomy and Cell Biology
- Date degree season
- Spring 2020
- DOI
- 10.17077/etd.005398
- Publisher
- University of Iowa
- Number of pages
- xvi, 145 pages
- Copyright
- Copyright 2020 Yan Yan Sweat
- Comment
- This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 137-145).
- Public Abstract (ETD)
Cleft lip and palate patients have openings on one side or both sides of the upper lip, as well as a cleft, or split on the roof of the mouth. Cleft lip and palate occur in one out of 700 newborns in the U.S. It is the most common congenital defect treated by pediatric plastic surgeons. Cleft lip and palate commonly occur as isolated birth defects, however, sometimes can be associated with other genetic conditions. Patients with cleft lip and palate often require speech, social and hearing therapies, which can cost up to 200,000 dollars. Therefore, it is important to understand the genetic reasons causing cleft lip and palate.
Ankyloglossia is also called tongue-tie. It is due to the abnormal formation of the lingual frenulum, which ties the tongue to the floor of the mouth. Patients with ankyloglossia have difficulties in moving their tongue and breast-feeding.
In my study, we use mouse models to identify the function of Six2 and Sox2 in craniofacial development. Loss of Six2 or Sox2 results in cleft palate. In the Six2 null embryos, the palate shelves are much shorter than the wild-type embryos, which is due to decreased cell proliferation and premature bone formation. However, in the Sox2 conditional deletion embryos, they lack palatal rugae, which could be the reason causing cleft palate. Additionally, deletion of Sox2 in the oral epithelia can also cause tooth defects and ankyloglossia. The ankyloglossia phenotype is related with the lack of periderm layer, a transient epithelial layer in oral mucosa to prevent abnormal adhesion during development.
Tracing these genetic origins will help us understand the underpinnings of these genetic anomalies; more importantly, it will be used in genetic counseling and future gene therapies.
- Academic Unit
- Anatomy and Cell Biology; Craniofacial Anomalies Research Center
- Record Identifier
- 9983949695602771
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