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Optogenetic Disruption Of Irf6 Leads To Cleft Palate Development In Zebrafish
Claudio Macias-Trevino, BS1,2, Edward B. Li, PhD1,2, Eric C. Liao, MD-PhD1,2.
1Massachusetts General Hospital, Boston, MA, USA, 2Shriners Hospital for Children, Boston, MA, USA.

PURPOSE: Mutations of the IRF6 transcription factor are strongly associated with the development of cleft lip and/or palate (CL/P). Germline mutagenesis of irf6 in zebrafish resulted in embryonic lethality, which precluded analysis of irf6 requirement in embryonic development. We utilized an optogenetic system to induce irf6 loss-of-function in developing zebrafish embryos to circumvent embryonic lethality and assess palate phenotype. This system confers precise temporal control of gene disruption using a light sensitive activation protein, particularly useful in zebrafish embryos that are transparent and develop ex vivo.
METHODS: Zebrafish embryos at the 1-cell stage were microinjected with mRNA encoding for VP16-EL222, a protein responsive to 465nm light, and a plasmid encoding for a dominant-negative form of the irf6 protein under control of the C120 promoter (C120-irf6-ENR). The embryos were grown in the dark to allow for normal development through epiboly (~10h), and subsequently exposed to 465nm light or grown in the dark for 72h. Palate phenotype was assessed by Alcian blue staining of zebrafish embryos.
RESULTS: Early exposure (0-4hpf) of zebrafish embryos co-injected with VP16-EL222 mRNA and C120-irf6-ENR plasmid to 465nm light resulted in development of embryos with a truncated body axis. When embryos were exposed to blue light post epiboly (~10h), zebrafish developed a hanging jaw phenotype and cleft palate.
CONCLUSION: Optogenetic activation of irf6 mutant protein was successfully implemented in zebrafish, enabling us to determine irf6 requirement in palate development. This system is critical in studying gene expression patterns of irf6 mutant fish, including downstream genes such as ghl3, as conventional zebrafish irf6 knockout models result in embryonic lethality. Since these fish develop cleft palate, we can utilize the model to carry additional experiments such as lineage tracing, gene expression analysis, or cell proliferation assays to understand the mechanism behind cleft palate development in the context of irf6 loss-of-function. Such analysis will be critical in understanding the function of irf6 in palatogenesis and how irf6 mutations lead to cleft palate in syndromes such as Van der Woude and Popliteal Pterygium Syndromes.


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