Dissecting the role of sonic hedgehog signalling during development of the secondary palate: a systems-level approach

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

The palate (the roof of the mouth) develops from two halves that fuse together during embryonic development. 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 improved 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 Sonic Hedgehog protein is a key molecule in ensuring that the palate develops normally; however, only a small fraction of proteins acting in response to Sonic Hedgehog has been identified. Recently, methods have been developed that will allow us to identify the complete set of proteins controlled by Sonic Hedgehog. We will use these techniques to dissect the role of Sonic Hedgehog signalling in ensuring that the palate develops correctly thereby preventing cleft palate. To ensure that this research is completed ethically, we will perform these experiments in mice, in which the palate develops in exactly the same way as in humans. 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 will help us to provide improved diagnosis, genetic counselling and treatment to patients and their families who are affected by this distressing condition.

Technical Summary

Cleft palate is a common congenital malformation that affects up to 1 in 2000 live births resulting in approximately 320 new cases each year in the UK alone. Cleft palate leads to distressing problems with obstructive apnoea, feeding, speaking, hearing and social adjustment. Treatment of cleft palate constitutes a heavy burden to affected individuals and their next-of-kin as well as to society more generally as the therapy requires several years and the input of multidisciplinary teams including surgeons, orthodontists, speech therapists and psychologists. Identification of the molecules that are crucial for development of the secondary palate and knowledge of their downstream cascades is imperative to improve diagnosis, prophylaxis, genetic counselling and care for affected individuals and their families.

Appropriate specification, growth, elevation, adhesion and fusion of the palatal shelves are essential mechanisms involved in formation of the secondary palate and failure of these processes leads to cleft palate. Recent research has indicated that Sonic Hedgehog (Shh)-mediated signalling plays a fundamental role in ensuring appropriate palatal development; however, the downstream regulatory networks remain uncharacterised. The recent development of resources for defining the complete set of binding sites for Gli transcription factors, the effectors of Shh signalling, has provided a unique opportunity to dissect the Shh signalling cascade during development of the secondary palate

In this project, we will use a systems-level approach to dissect the cis-regulatory circuitry underlying Shh-mediated palatal development. The first aim will utilise expression profiling of the palatal shelves of mice carrying loss-of-function or gain-of-function mutations in Smoothened, an obligatory component of Shh signal transduction in responding tissues, to identify genes downstream of Shh signalling. In the second aim, the resulting dataset will be filtered by comparison with a palate-specific database of Gli binding sites generated using ChIP-seq thereby selecting for genes directly regulated by Shh signalling.

To prioritise these targets for subsequent analyses, we will filter our data further by intersecting them with a ChIP-seq dataset generated using antibodies specific for the transcriptional co-activator protein p300 to identify a genome-wide catalogue of enhancers active in the developing secondary palate. The presumed direct targets of Shh signalling will then be verified using expression analyses, bio-informatics, independent ChIP-qPCR, and luciferase reporter assays. This analysis will provide crucial insights into the fundamental molecular events driving palatal development and result in the identification of genes that can be screened for mutations in families with a history of cleft palate.

Publications

10 25 50
 
Description Collaboration with University of Bonn 
Organisation University of Bonn
Country Germany 
Sector Academic/University 
PI Contribution We are collaborating with Drs Elisabeth Mangold and Kerstin Ludwig on the possibility of using the data we have generated on non-coding regulatory elements to interpret genome-wide association signals
Collaborator Contribution The group in Bonn will sequence non-coding regulatory elements in their cleft lip/palate cohorts
Impact None as yet
Start Year 2014
 
Description FaceBase consortium 
Organisation National Institutes of Health (NIH)
Department National Institute of Dental and Craniofacial Research (NIDCR)
Country United States 
Sector Public 
PI Contribution We are members of the FacebAse co-orination hub. FaceBase is funded by NIDCR and has been constituted to advance understanding of craniofacial development/disorders world-wide.
Collaborator Contribution FaceBase is a consortium of USA (predominantly) and Uk laboratories. I coordinate the mouse sub-committee
Impact Paper in preparation
Start Year 2009
 
Description Zebrafish collaboration 
Organisation University of Texas at Austin
Department Molecular Cell and Developmental Biology Track
Country United States 
Sector Academic/University 
PI Contribution We are collaborating with Dr. Johann Eberhart to understand the molecular drivers of palatal development using zebrafish as a model organism. We are identifying targets of Shh signalling.
Collaborator Contribution We are collaborating with Dr. Johann Eberhart to understand the molecular drivers of palatal development using zebrafish as a model organism. Dr. Eberhart's laboratory is using morpholino technology to ablate the function of the genes that we are identifying
Impact Papers published
Start Year 2011
 
Description Support for school students on Nuffield Bursary 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact Two school students spent four weeks in my laboratory researching cleft lip and palate. The students were supported by a Nuffield Bursary

The students will present the results of their research at a regional event.
Year(s) Of Engagement Activity 2008,2009