Deciphering the role of p63 in secondary palate development using systems biology

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Development of the palate, which ultimately separates the mouth from the nasal passages, involves a complex series of inter-dependent events that requires close co-ordination of cell growth, cell adhesion, cell migration, cellular differentiation, and cell death. Failure of these processes to occur correctly results in the birth defect cleft palate. Cleft palate, which has an incidence of approximately 1 in 2500 live births, results in considerable problems to affected individuals as they may experience problems with eating, speaking and psychosocial adjustment due to the altered facial appearance. The frequent occurrence and major healthcare burden imposed by cleft palate highlight the need to dissect the fundamental mechanisms that underlie normal development of the palate and how these are disturbed in cleft palate. As a result of ethical concerns, these studies cannot be performed in humans. In mice palate development mirrors that occurring in humans and the mouse is, therefore, the major animal model used in the study of palatogenesis. Although the descriptive embryology of mouse palate development is well-established, the underlying genetic events are poorly characterised.Recently, we have generated compelling evidence that the transcription factor p63, which is a master control gene, plays a central role in the genetic events driving palatal development. We have generated a complete profile of the genes expressed in the developing palate at the critical time-points in its development by using high-throughput sequencing of RNA (RNA-seq) isolated from carefully dissected mouse palates. In parallel, we have identified all the regions of the genome to which p63 binds in the developing palate using the ChIP-seq technique. Despite the generation of these genome-wide datasets and the confirmation of a large number of p63 transcriptional targets in the secondary palate, the gene regulatory networks controlled by p63 have not been elucidated. The behaviour of these complex networks cannot be predicted by simple intuitive approaches but requires sophisticated computational methods. In this project, we will firstly apply time-course methods to analyse RNA-seq data generated from cells and from palatal shelves dissected from mice in which the function of p63 has been disrupted. By combining the results from these two distinct but complementary systems, we will identify the direct and biologically-relevant early regulatory links in the p63-controlled developmental regulatory network which will facilitate our understanding of the role of p63 in secondary palate development. Secondly, we will use the data generated on the gene regulatory networks controlled by p63 to analyse a mouse which exhibits cleft palate as the result of increased p63 signalling. These experiments will allow us to determine why it is essential that the level of p63 signalling is reduced in a specific subset of cells during normal development of the palate. Finally, to facilitate collaborative working, we will ensure that all the data generated during the project are freely available via the World-Wide Web.Ultimately, the project will provide crucial information about a developmental process of major significance and serve as a test case for using systems-level developmental genetics to dissect the gene regulatory networks that are disrupted in congenital human malformations more widely.