The role of mechanical cues in the regulation of post-migratory neural crest cell fate

Neural crest cells (NCC) are a multipotent cell population that contribute to the majority of the face and neck regions during formation of the embryo. Aberrant NCC regulation and differentiation can result in craniofacial disorders, which are associated with high infant mortality, therefore it is important to uncover the mechanisms that determine NCC fate and differentiation. Existing evidence suggests a role for mechanical cues in the cellular microenvironment in driving cell differentiation in adult cells such as mesenchymal stem cells. There is now evidence that mechanoregulation can control cell fate in the embryo, although this remains limited. Therefore, the aim of this project is to investigate whether mechanical forces also control NCC fate, specifically in the post-migratory NCC population.

A variety of in vitro and in vivo methods will be employed to answer this question. Mechanical cues can be attributed to stiffnesses encountered by cells, as well as cell density and cell shape. How stiffness influences NCC fate will be assessed by in vitro culture of post-migratory NCC on very soft and very stiff surfaces to assess their response to environmental mechanical cues. The role of the cellular cytoskeleton will be determined by modulation of the actin cytoskeletal tension through the use of specific inhibitors and activators. In addition, RNAseq will be used to globally assess the impact of mechanical cues on NCC differentiation at a molecular level.

In vivo, the contribution of cell shape to terminal differentiation will be assessed in various embryonic regions by analysis of mTmG mice, in which a subset of cells are labelled with GFP. Atomic Force Microscopy (AFM) will be used to quantify the relative stiffness present in various regions of tissue slices taken through the embryonic craniofacial region. Tissue stiffness will also be assessed at different stages of development (E10.5 and E15.5), to assess any changes in micro-environmental forces that NCC are likely to encounter as the embryo ages.