How do cranial sutures work?

Supervisors: Prof Andrew Wilkie and Dr Steve Twigg  

Brief description
The Clinical Genetics Group is one the world’s leading laboratories working on craniosynostosis, the premature closure of one or more of the cranial sutures of the skull. This is a relatively common condition affecting 1 in 2,250 babies, which requires major operations to rearrange the skull bones to avoid problems like increased pressure on the brain1. Whilst human molecular genetics has been very successful at identifying many of the common causes of craniosynostosis, we still know very little about what happens biologically in the cranial sutures themselves2. These structures, narrow gaps between the skull bones filled with cells and fibrous tissue, must achieve a delicate balancing act of enabling growth of new bone at the margins of the suture, whilst also ensuring that the mid-part of the suture remains open along its entire length. This project represents an exciting opportunity to explore how the suture works by mapping out the cellular hierarchy of activities from undifferentiated stem cell to fully formed osteoblast3. You will exploit the state-of-the-art facilities available in the WIMM for fluorescence-activated cell sorting (FACS), single cell transcriptomics and microscopy, and apply your discoveries to understand the mechanisms of craniosynostosis in two mouse models, caused by mutations in Zic1 and Erf, available in our laboratory4,5.

Aim
Following techniques established in the laboratory, you will isolate single cells from mouse and human cranial sutures at different developmental stages, and carry out single cell RNA sequencing. There will be an opportunity to learn the bioinformatic tools used to cluster cells with similar transcriptomic profiles. This approach will identify specific markers (genes) that define cell types and the initial characterisation will involve visualising these populations within intact sutures using in situ hybridisation analysis and microscopy. To enable an in depth molecular analysis of the chromatin landscape and gene expression patterns, these markers will be utilised to isolate cell populations by FACS. This will require tagging these genes with fluorescent flags using CRISPR-Cas9 targeting – a method used routinely in the laboratory.

In the course of the work you will analyse the roles of Zic1 and Erf in the development of cranial sutures. Working with both embryonic and postnatal mutant and WT mice you will learn the histological and immunohistochemical approaches used to gain insight into pathological mechanisms. CRISPR-Cas9 targeting will be used to develop cell or mouse lines as an aid for visualising cellular and developmental pathology. These studies could be complemented by in vitro analysis of mesenchymal stem cells (MSC) isolated from the sutures of mutant and WT mice. Such studies will involve analysing the capacity of MSC to differentiate to bone, RNAseq to detect altered gene expression patterns, and ChIPseq to profile the DNA binding pattern of specific transcription factors.

Training opportunities
Training in molecular and developmental biology (in situ hybridisation of whole or sectioned embryos, analysis of proliferation, differentiation and bone formation) as well as state of the art technologies such as CRISPR-Cas9 genome editing and single cell analysis.

References:

  1. Johnson and Wilke (2011) Craniosynostosis. Eur J Hum Genet 19:369-376.
  2. Twigg and Wilkie (2015) A genetic-pathophysiological framework for craniosynostosis. AJHG 97: 359-377.
  3. Zhao et al. (2015) The suture provides a niche for mesenchymal stem cells of craniofacial bones. Nat Cell Biol 17:386-96
  4. Twigg and Wilkie (2015) Gain-of-function mutations in ZIC1 are associated with coronal craniosynostosis and learning disability. AJHG 97: 378-388.
  5. Twigg et al. (2013) Reduced dosage of ERF causes complex craniosynostosis in humans and mice and links ERK1/2 signaling to regulation of osteogenesis. Nat Genet 45: 308-313.

For further information, please contact Prof Andrew Wilkie or Dr Steve Twigg