Regulatory mutations in Shh cause brain and craniofacial malformations

Abstract

Craniofacial malformations are the most common birth defect found in humans, encompassing defects such as microsomia, hemangioma and the holoprosencephaly (HPE) spectrum of malformations. The most commonly mutated gene in HPE is Sonic hedgehog (Shh). Shh is a key morphogen in development which is essential throughout varied embryonic structures, including the brain, mandible and other craniofacial elements. The first aim of my thesis addresses how Shh is able to regulate mandibular formation. To assess this issue, I used a Shh regulatory region deletion, which removes Shh signalling in the pharyngeal endoderm, which is of particular interest as these mice display defects in the maxilla and mandible. By dissecting the mandibular prominences of these mutant embryos and control counterparts at key developmental stages, I was able to create RNA-seq and ATAC-seq libraries. My analyses of the RNA-seq, expressed genes, and ATAC-seq, accessible DNA, data revealed that Shh has a dual role in mandibular development. At early mandibular development stages Shh promotes cell proliferation whilst at later stages it promotes cell fate specification. Furthermore, I have implicated the Fox gene family as key downstream targets of Shh that are responsible for orchestrating multiple aspects of mandibular development. Shh null mice display lack of a brain and a complete absence of craniofacial elements. Whether Shh is needed for craniofacial element formation or the loss of craniofacial elements is secondary due to collapse of the brain is unclear. The second aim of my thesis addresses this question. To do so, I generated a Shh regulatory region mutant which specifically removes Shh signalling directed by the sonic brain enhancer 2 (SBE2). Phenotypic analysis of these mice during embryonic development revealed that they present brain malformations, including of the pituitary lobes. These deformities can be linked to issues in signalling found within the brain tissue. Interestingly, I have shown that the neural tube patterning pathway, which involves the cross-repressive activities of Shh and Bmp, has been co-opted, at least in part in murine hypothalamic development. In addition to brain defects, these mice also display defects in the craniofacial elements, including the nasal cavities and bones of the midline. These craniofacial defects likely occur directly due to loss of Shh signalling to the craniofacial condensations and also secondarily due to disruption of the basisphenoid bone, as a result of the pituitary malformations. These results indicate that Shh signalling, directed by SBE2, is required not only for appropriate brain development but also for normal craniofacial structure development.