Plastic Surgery Research Council
Members Only  |  Contact  |  PSRC on Facebook
PSRC 60th Annual Meeting
Program and Abstracts

Back to 2015 Annual Meeting Program


Generating an irf6 Mutant Model for Rapid Analysis of Human Genetic Variants, Elucidate Gene Regulatory Network, and Drug Discovery to Mitigate Cleft Lip and Palate
Christina A. Nguyen, BS, Edward B. Li, BA, Kusumika Mukherjee, PhD, Lisa Gfrerer, MD, Eric C. Liao, MD, PhD.
Massachusetts General Hospital, Boston, MA, USA.

PURPOSE:
Interferon regulatory factor 6 (IRF6) is a transcription factor known to regulate epithelial proliferation and differentiation. Mutations in IRF6 are associated with Van der Woude syndrome, the most common form of syndromic cleft lip and palate (CLP), and nonsyndromic CLP identified through genome wide association studies. Previous work demonstrated that mice deficient in Irf6 exhibited cleft palates, skin and limb malformations, while transgenic expression of dominant-negative irf6 in zebrafish also produced cleft palates. Moreover, studies in zebrafish periderm identified irf6 and downstream effectors as regulators of epithelial development. Here, we generated irf6 mutant zebrafish lines by targeted mutagenesis as a model to further elucidate the role of irf6 in CLP pathogenesis and to utilize this platform for future drug discovery.
METHODS:
The CRISPR-Cas9 system was used to generate targeted mutations in irf6. P0 zebrafish with mutations in irf6 were identified by PCR and DNA fragment length analysis. Genotype-directed breeding produced irf6+/- heterozygotes that were then intercrossed to generate irf6-/- homozygotes. Cartilage structures were visualized by Alcian blue staining. Spatiotemporal gene expression was detected and visualized by whole-mount in situ hybridization with anti-sense RNA probes. Embryonic development was monitored with time-lapse compound microscopy. Human variants of IRF6 mRNA transcripts were used to rescue zebrafish mutants by microinjection.
RESULTS:
Zebrafish carrying an 8-bp deletion in the region encoding the protein-binding domain of irf6 were isolated. The mutant protein is predicted to be truncated to 257 amino acids compared to wild type (492 aa). While irf6+/- heterozygous and irf6-/- homozygous zebrafish were viable and did not demonstrate mutant phenotypes, the progeny of irf6-/- females crossed with irf6+/- males exhibited embryonic lethality during mid-epiboly. In contrast, the progeny of irf6-/- males crossed with irf6+/- females progressed normally through embryonic development. Time-lapse microscopy of embryos from irf6-/- mothers revealed mid-epiboly arrest and yolk rupture through an unstable blastoderm and/or periderm.
CONCLUSION:
The results indicate that disruptions in irf6 function could compromise the structural integrity of the zebrafish blastoderm and/or periderm during early embryogenesis. The results also reveal a novel critical role for maternal irf6 transcripts in embryonic development and potentially CLP pathogenesis. This preliminary study offers insights on the pattern of irf6 transmission and effect on embryogenesis when the irf6 protein-binding domain is truncated. Study of downstream molecular pathways using this irf6 mutant model will further elucidate the gene regulatory network governing Irf6 function. Importantly, this study generated a platform for the rapid assessment of irf6 gene function that can be applied to the study of human IRF6 variants and the discovery of small molecules that can mitigate the effects of IRF6 mutations and modify CLP pathogenesis in utero.


Back to 2015 Annual Meeting Program