Characterization of Treacher Collins Syndrome in Mice and Zebrafish

Objectives: This project compares two model organisms and their phenotypic expression of Treacher Collins Syndrome. Using a conditional mouse model, we compare levels of craniofacial apoptosis and cell proliferation between wildtype and conditional knockout mice of the mouse ortholog of the Tcof1 gene. Secondly, we analyzed the mutation and associated phenotype in a knockout zebrafish model with alcian blue, acridine orange and in situ hibridizations.
Methods: To assess craniofacial apoptosis levels and cell proliferation levels, we conducted TUNEL and brdU assays on conditional knockout mouse embryos (e9) which had their Tcof1 gene excised from neural crest cells through Cre mediated recombination. To assess cartilage development in the zebrafish we mated heterozygous fish with a mutation in the Nocl1 gene which is the ortholog of Tcof1 (Weiner et al., 2012). Using alciain blue staining to stain cartilage, whole-mount in situ hybridization of genes GSC and GBX2, and acridine orange to track levels of apoptosis, we tracked the craniofacial development of the embryos from 1-7dpf.
Results: The experiments involving the conditional Tcof1 KO mice show higher levels of apoptosis and less cell proliferation in the craniofacial region, specifically in the prefusion neural folds and first pharyngeal arch in the affected embryos. These regions of the developing embryo are the sites of the highest levels of Tcof1 expression in the knockout animals when compared to controls. In situ hybridization of GSC and GBX2, two genes associated with neural crest cell gene expression, showed altered levels of expression between the control and mutant zebrafish. Cartilage staining at days 5, 6, and 7 post fertilization reveals a consistently underdeveloped mandible in the mutant fish. Acridine oranges levels remain the same between the mutants and controls.
Conclusions: Based on our mouse model, the hypoplastic craniofacial structures associated with Treacher Collins syndrome are a result of decreased neural crest cell proliferation and increased apoptosis in the 1st pharyngeal arch and frontal facial regions. Zebrafish analysis allowed us to develop a phenotypic developmental timeline which highlights the morphological differences in the mutants from 1-7 dpf.