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.
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.
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.
Organisations
People |
ORCID iD |
| Michael Dixon (Principal Investigator) |