Despite a great degree of facial variability within vertebrates, and even within individual species, the initial outgrowth and formation of the face is a highly conserved process, particularly between mouse and man. This makes the mouse an excellent model to understand the basis of craniofacial development and the defects that commonly result in congenital disease.
The hedgehog (Hh) signalling pathway is instrumental in the development of most vertebrate organs and the face is no exception. In humans, craniofacial anomalies are amongst the most common and debilitating congenital defects. It is well established that inappropriate activation of the Hh pathway during development can dramatically impact on facial morphology. In humans, haploinsufficiency for the patched1 gene (PTCH1) causes Nevoid Basal Cell Carcinoma Syndrome, characterised by a number of craniofacial and other developmental defects and a predisposition towards a range of tumours. PTCH1 is the receptor of the Hh pathway and major negative regulator. In mouse, homozygous loss of Ptch1 leads to unrestrained Hh signalling, due to constitutive pathway activation through smoothened. This results in early embryonic lethality, precluding a detailed analysis of the face during development. As a result, a number of outstanding questions remain as to the role of Ptch1 during craniofacial development and congenital disease.
This thesis examines the importance of Ptch1-mediated inhibition of Hh signalling during craniofacial development. To this end, two Ptch1 conditional mouse mutants were analysed in this project, generated using Cre/LoxP technology. Both models inactivate Ptch1 in the facial mesenchyme by targeting the neural crest cells (NCCs), a highly migratory and multipotent population of cells, which contribute to the majority of cell types within the vertebrate face. To examine the importance of Ptch1 at different stages of craniofacial development, Ptch1 inactivation was targeted to NCCs before (using Wnt1 Cre) and after (using Tyrosinase Cre) their migration to the facial primordia. The removal of Ptch1 led to unrestrained Hh signalling throughout the facial primordia, and a number of craniofacial defects in both models.
In the first Ptch1 conditional mutant, using Wnt1 Cre, mutants displayed mid-facial widening, consistent with previous Hh gain-of-function models. In contrast to these other models, however, Wnt1Cre;Ptch1c/c mutants displayed facial clefting, a common craniofacial birth defect in humans. The clefting phenotype affected the lip and nose and as mouse mutants rarely display these types of clefts, this model provided an opportunity to better understand how these structures form and what role Ptch1 may play in this process. Work in this project identified a requirement for Ptch1 in the maintenance of FGF-dependent, epithelial-to-mesenchymal signalling interactions within the nasal pit that affected specification of the nasal processes. In order to analyse the growth and fusion of the nasal processes in more detail than has previously been possible, a technique was optimised to conduct time-lapse imaging of facial explant cultures. These analyses led to the identification of several, previously unknown cellular mechanisms important in the initiation of nasal pit invagination. These studies also suggested that several of these mechanisms may rely on Ptch1 function. More unexpectedly, Ptch1 was found to play an essential, non cell-autonomous role in the maintenance of epithelial cell shape during nasal pit morphogenesis. These findings begin to uncover the cell biological processes regulated downstream of Hh signalling, and the vital role these processes play in craniofacial morphogenesis.
In the second Ptch1 mouse mutant, using Tyrosinase Cre, clefting of both the face and palate was observed, reaffirming the requirement for Ptch1 in the prevention of orofacial clefts. As the TyrCre;Ptch1c/c mutant survived longer through embryogenesis than the Wnt1Cre;Ptch1c/c mutant, further requirements for Ptch1 were found in the survival and specification of the NCC-derived mesenchyme and in the patterning and differentiation of the craniofacial skeleton. These data provide the first in vivo support that Ptch1 may play a role in cranial NCC fate determination. Furthermore, these findings reiterate the context-dependent requirements for Ptch1 during craniofacial development.
It is clear that facial development is dependent on the strict regulation of Hh signalling, throughout many stages of embryogenesis. Work presented in this thesis demonstrates that Ptch1-mediated negative feedback, within cranial NCCs, plays a critical role in this process throughout facial development. These analyses extend our knowledge of the role of Hh signalling during craniofacial development and congenital disease. In particular, work in this project highlights some of the cell biological processes downstream of Hh signalling, which are essential for correctly sculpting the mammalian face.