Graduation Date

Fall 12-19-2025

Document Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Ali Nawshad

Second Advisor

Po-Jung Chen

Third Advisor

Peter Giannini

Abstract

Introduction: Failure in palatal fusion during palate development results in cleft palate, one of the most common birth defects in the US. During the final phases of palatogenesis, the palatal periderm (a protective cell layer that prevents premature fusion and adhesion with oral epithelium) must be removed, also known as desquamation. This process is a prerequisite for establishing the midline seam in the fused palate. Previous studies have shown that transforming growth factor-β3 (TGFβ3) plays an irreplaceable role in palate development in mice. Our previously published mechanistic models outline the role of TGFβ3 and its downstream signaling cascade involving proteins (e.g. Irf6 and dNp63) in periderm desquamation. Our previously published data have shown a regulatory feedback loop between ΔNp63 and Irf6 in differentiating oral/palatal epithelial cells that is potentially dependent on functional TGFβ3 signal. Our goal in this research is to explore how TGFβ3 fits into regulating Irf6 and ΔNp63 in periderm desquamation, hence, we ask the question, “How can TGFβ3 -/- palatal fusion defects be corrected through the timely induction of periderm desquamation?”. Objectives: In this aim, we investigate mechanisms by which TGFβ3 -/- cleft palate can be rescued in vivo by modulating ΔNp63 and Irf6 genes in triple transgenic TGFβ3, IRF6, and ΔNp63 mouse model systems. As per our published and preliminary data introducing a novel role for TGFβ3 in periderm desquamation, we will test the hypothesis that the timely removal of the 4 periderm or the key peridermal regulator ΔNp63 in Tgfb3-/- mice will rescue the defective palatal shelf adhesion seen in Tgfb3 null mutants. Our hypothesis is that the timely removal of the periderm or the key peridermal regulator ΔNp63 and Irf6 in Tgfb3-/- mice will rescue the defective palatal shelf adhesion seen in Tgfb3 null mutants. Results: Using Cre-loxp murine transgenic experimental models and innovative imaging of newborn palate to document the dynamic cellular changes that are controlled by TGFβ signaling during palatogenesis, such as micro-CT, palatal size’s shape and volume measurements, Hematoxylin-Eosin (H/E) and Safranin O staining. Our results demonstrate that while induction of ΔNp63 in Tgfb3-/- mice rescued cleft palate in 40% of mice, and as expected, induction of IRF6 in Tgfb3-/- mice did not rescue palatal cleft. This unique observation agrees with our hypothesis that TGFβ3 and ΔNp63 play crucial roles in periderm morphogenesis and desquamation. However, while IRF6 is necessary for palatal fusion, it is not dependent on functional TGFβ3 signaling. And we propose that IRF6 has its independent pathways in palate development. Conclusion: Using both in vitro model and in vivo genetic models in newborn palates to analyze their structural, functional, and biochemical properties places us in an advantageous position to be able to interpret the regulatory feedback loop among TGFβ3-ΔNp63-Irf6 signaling in greater detail, to improve our understanding of how the cells process vast information. And thus, our study presents a new paradigm with significant conceptual advances in understanding the detailed mechanisms, by which periderm desquamation takes place in the pathogenesis of cleft palate. The hierarchical order TGFβ3, Irf6, and ΔNp63 is critical in the development of approaches to prevent cleft palate as well as in the development of therapeutic strategies.

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2025 Copyright, the authors

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