Prevention of cleft palate: the critical role of p63 and IRF6 signalling

The palate (the roof of the mouth) develops from two distinct parts that fuse together during development of the baby. Currently, we do not completely understand what controls these events, but we do know that the common and distressing birth defect cleft palate results when fusion of the two halves of the palate fails to occur. Patients with cleft palate experience difficulties with eating and speaking, which can be corrected to some degree by long-term surgery, dental treatment, and speech therapy; it is therefore essential that we have more information on how genes work together during normal development and how these are affected in cleft palate. The aim of this project is to study the way in which two genes, designated p63 and IRF6, function together during development of the palate. So far, we have discovered that p63 switches on the function of IRF6 and that this, in turn, causes the levels of p63 to drop just before fusion of the two halves of the palate. In our initial studies, we will determine whether or not cleft palate results when p63 is maintained at an artificially high level in the palate of developing mice. Subsequently, we will obtain a more complete picture of the ways in which the palate develops by discovering the target genes controlled by p63 and IRF6. In the short-term, this research will help us to understand the processes that underlie normal development of the palate and how these are disrupted in cleft palate. In the longer term, this information may help us to provide improved genetic diagnosis and counselling to patients and their families who are affected by this distressing condition.

Cleft palate is a common congenital disorder that affects up to 1 in 2500 live births; consequently, in the UK approximately 1000 babies are born with a cleft each year. Cleft palate results in considerable morbidity to affected families as individuals exhibiting this condition experience problems with eating, speaking and hearing which can be corrected to varying degrees by surgery, dental treatment, speech therapy and psychosocial intervention.

Although mutations that cause cleft palate have been identified at several loci, we have little knowledge of how the proteins encoded by these genes integrate into the signalling hierarchies guiding palatal development. Recently, we have demonstrated that the transcription factor p63 binds directly to an enhancer to transactivate Interferon Regulatory Factor 6 (IRF6) and that induction of IRF6 ultimately results in down-regulation of p63. These data lead us to hypothesise that integration of p63 and IRF6 signalling is central to the control of palatal development. The overall objective of this proposal is to dissect the role of this signalling cascade in palatal shelf fusion. Specific aims are: (1) to analyse whether down-regulation of p63 is a pre-requisite for palatal fusion; (2) to identify the transcriptional targets of p63; and (3) to identify the downstream targets of IRF6 during palatogenesis.

Firstly, we will test the hypothesis that down-regulation of p63 is essential for palatal fusion by mis-expressing DeltaNp63alpha in the medial edge epithelia of the pre-fusion palatal shelves using transgenic mice. The resulting mice will be used to investigate the consequences of altered p63 signalling using histology, electron microscopy, in situ hybridization, immunofluorescence, and analysis of cell behaviour. Secondly, we will define a more complete range of DeltaNp63alpha targets in the secondary palate using an existing ChIP-seq dataset. To prioritise these targets for subsequent analysis, we will use micro-array analysis of the developing palate and hypothesis-driven approaches based on published data and our own preliminary results. Thirdly, we will identify transcriptional targets of IRF6 using ChIP-seq analysis. Target genes that are identified in the second and third phases of the project will be verified using a combination of expression analyses, independent ChIP-qPCR, and luciferase reporter assays.

In addition to dissecting a key molecular pathway underlying palatal development, this project will provide crucial insights into ectodermal development more generally and will simultaneously result in the identification of candidate genes that can be screened for mutations in congenital malformations encompassing cleft palate and ectodermal dysplasia.