Bone Abstracts

Objectives: Craniosynostosis is a skeletal birth defect, resulting in premature fusion of cranial sutures. The patency of the sutures is essential to enable the growth of the skull in compliance to the developing brain. Mutations in TWIST1 and TCF12 have been identified in patients with Saethre-Chotzen syndrome, which is typically associated with coronal synostosis. Studies in mouse models suggest that the quantity of TCF12-TWIST1 heterodimers is one critical factor for the patency of coronal sutures. We present zebrafish (Danio rerio) as supportive disease model to further visualize the specific function of TCF12 and TWIST1 during suture development and emergence of craniosynostosis.

Methods: To characterize the dynamic expression patterns of tcf12 during development, we generated transgenic zebrafish, in which the green fluorescent protein (GFP) is expressed under the control of the tcf12 zebrafish promoter. Using customized bone staining methods we perform in vivo imaging of developing bones and are able to correlate tcf12 expression to skull development. By use of CRISPR/Cas9–mediated genome editing we additionally established a number of loss-of-function mutations in conserved regions of tcf12 and twist1.

Results: The GFP-transgenic zebrafish reveal a broad range of expression patterns of tcf12 in developing embryos with high levels of expression in tissues like the developing heart and in neurons of the epiphysis, the otic vesicle, and the spinal cord. More importantly tcf12 expressing cells are localized at the edges of the skull bones during development and could also be identified inside all cranial sutures in adult zebrafish. Our loss-of-function experiments further reveal that heterozygous mutations in tcf12 can lead to local fusions of the cranial sutures in zebrafish, whereas individuals with mutations in twist1 do not exhibit suture fusions.

Conclusion: We present zebrafish as a valuable disease model to gain deeper insight into the function and interaction of craniosynostosis genes. By generating transgenic individuals for tcf12 expression, we could detect tcf12 expressing cells in cranial sutures, skull bones, and neural tissues of zebrafish. With the establishment of tcf12 and twist1 loss-of-function fish we clarify that even heterozygous mutations can have minor effects on the maintenance of suture patency. Further experiments aim for the molecular characterization of tcf12 expressing cells detected in the sutures and for the generation of double knockout mutants.

Disclosure: The authors declared no competing interests.