Cell biological determinants underlying phenotypic severity of holoprosencephaly



 DESCRIPTION (provided by applicant): Many structural diseases of the craniofacial complex are characterized by a high degree of phenotypic variation, but the underlying causation of such variation is largely unknown. A key example of such a disease is holoprosencephaly (HPE), which can produce phenotypes in humans ranging from minor midfacial narrowing to cyclopia. Mutations in the Sonic hedgehog (SHH) pathway are a predominant cause of familial forms of HPE. Family members often exhibit phenotypes of greatly varying severity despite possessing identical mutations; it remains unclear what produces such heterogeneous phenotypes. HPE occurs 1 in 250 conceptions, but only 1 in 10,000 live births due to intrauterine lethality. Determining factors that impact the severity of HPE would thus have a major impact on public health. The objective of this research application is to elucidate mechanisms by which variation in midline patterning is produced in the HPE population by modulating SHH pathway activation experimentally. Previous research has demonstrated that decreased SHH-signaling in the avian brain correlates with a continuous distribution of craniofacial phenotypes mimicking the HPE spectrum. Significantly, the relationship between SHH-signaling and facial morphology appears to be non-linear, suggesting that a very slight range of SHH molecule concentrations can underlie the production of highly dissimilar phenotypes. The SHH signaling pathway itself, however, appears to be a binary switch, wherein very low or high ligand concentrations produce little phenotypic variation, while concentrations very close to the middle produce greater phenotypic heterogeneity. Although this model could account for the phenotypic spectrum found in HPE patients, it has not been demonstrated in a genetic in vivo model to date. This application aims to test the hypothesis that modifications in SHH pathway activation can produce a continuous spectrum of midfacial phenotypes mimicking the HPE patient population. In Aim 1, Subaim 1, I will determine whether an HPE-like spectrum can be produced in a chick population by the presence of a HPE- patient derived mutant SHH ligand shown to act in a dominant-negative manner in vitro. In Aim 1, Subaim 2, I will determine whether five HPE patient-derived SHH N-terminal mutations produce heterogeneous outcomes by replacing the endogenous SHH locus of cells with each mutation using CRISPR/Cas technology and assessing subsequent SHH activity levels using qPCR. In Aim 2, I will test whether modulating the cellular response to SHH in an allelic series of mouse embryos contributes to the production of variable HPE phenotypes. Results from this study may provide the preliminary tools and/or information necessary for the development of new and innovative approaches for treating craniofacial defects.