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

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.

While the descriptive embryology of secondary palate development is well-established, the underlying molecular mechanisms are poorly characterised. Although the transcription factor p63 plays a central role in driving palate development, the gene regulatory networks in which p63 functions remain unknown. The behaviour of these complex networks cannot be predicted by simple intuitive approaches but requires sophisticated inference methods applied at a systems-level to informative datasets. This project will be completed under three objectives. In Objective 1, we will infer the direct, secondary, and downstream targets of p63 activation genome-wide by applying a model-based inference approach to ChIP-seq and RNA-seq time-course datasets. The resulting in silico network predictions will generate experimentally testable hypotheses that will be dissected using a combination of ChIP-qPCR, Nanostring technology, RT-qPCR, in situ hybridisation, and luciferase reporter assays. We will use network predictions to prioritise these experiments with the goal of validating the network links and accelerating the generation of experimental evidence for new genes and pathways involved in palatal development. In an iterative process, these experimental data will feed back into the models to refine the network predictions further. In Objective 2, we will determine the role of p63 during palatal fusion. We will analyse an existing transgenic mouse model which exhibits cleft palate as a result of over-expression of p63 in the palatal epithelia using a combination of RNA-seq, Nanostring technology, and in situ hybridisation. These experiments will be informed by the networks defined in Objective 1. In Objective 3, we will ensure that the data are fully reproducible, improve collaborative working, and make our new methodology widely available, by publishing the full data analysis pipeline as a Galaxy workflow, from raw data processing through to network inference and visualisation.

Cleft palate is a congenital disorder that occurs with a prevalence of 1 in 2500 live births. Although the morbidity resulting from cleft palate can be ameliorated by a variety of treatment interventions, the resulting costs to the NHS are significant. The frequent occurrence and major healthcare burden imposed by cleft palate highlight the need to dissect the fundamental mechanisms that underlie development of the secondary palate.

Who might benefit from this research? Potential non-academic beneficiaries of the research include: individuals affected by cleft palate; geneticists; genetic counsellors; industry; and prospective employers.

How might they benefit from the research? Dissecting the gene regulatory networks driving development of the palate and ultimately applying it to interpret human genetic studies of cleft palate is imperative to improve diagnosis, prophylaxis, genetic counselling and, ultimately, care for affected individuals and their families. Importantly, these areas have been identified as research priorities by a representative group of patients, carers and clinicians convened under the auspices of the James Lind Alliance.

While the immediate benefits will be realised in understanding the fundamental events driving development of the secondary palate, delineation of the underlying gene regulatory networks will facilitate interpretation of human genetic studies of cleft palate. While exome sequencing is a highly effective method for mutation identification, considerable challenges remain in interpreting the sequence data generated. Functional data provide a useful filter to reduce the complexity of analysis. Our research will therefore provide a rationale basis for studies of this type (Beneficiaries: geneticists, genetic counsellors, patients and their families; time-frame: 2+ years).

Although the impact will be longer-term, identification of non-coding, gene regulatory elements during this project will provide a rich resource of sequences that can be analysed in families with a history of non-syndromic clefting thereby facilitating the move from genome-wide association to causative variant(s). Such elements are also targets for understanding the interaction between (epi)genetic and environmental contributors to cleft palate. (Beneficiaries: geneticists, genetic counsellors, patients and their families; time-frame: 5+ years).

As outlined in the 'Pathways to Impact' section of the application, the significance and implication of our results will be communicated to patients and their families through the Cleft Lip and Palate Association and other relevant patient support groups (beneficiaries: patients and their families; time-frame: ongoing).

In terms of impact on industry, a diverse range of molecules has been associated with an increase in the incidence of cleft palate in animal models used to evaluate developmental toxicity. The data from these models are used as the basis for human risk assessment during the development of New Chemical Entities (for example, agrochemicals). A better understanding of the underlying perturbations to embryogenesis, with particular reference to cleft palate, would help to refine the utility of these animal models in terms of assessing risks to human health (beneficiaries: industry; time-frame: 3+ years).

Research staff employed on the project will be beneficiaries as they will be provided with comprehensive research training. As detailed in 'Pathways to Impact', the researchers will benefit from professional and career development opportunities at the University of Manchester. Past experience from the applicants' laboratories has indicated that the skills acquired by the researchers have been, and will continue to be, transferrable to careers in research, industry, the NHS, medical writing, education and funding agencies (beneficiaries: researchers employed on the programme; time-frame: ongoing; prospective employers; time-frame: 3-5 years).