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Using Small Molecule Screens to Identify Novel IRF6 Gene Regulatory Pathways in Orofacial Cleft Pathogenesis
Edward Li, BA, Dawn Truong, BA, Brittany Garrity, BS, Christina Nguyen, BS, Kusumika Mukherjee, PhD, Eric C. Liao, MD-PhD.
Massachusetts General Hospital, Boston, MA, USA.

PURPOSE:
Mutations in the transcription factor IRF6 represent the most common genetic determinant of both syndromic and nonsyndromic cleft lip and/or palate (CL/P). We hypothesized that the IRF6 gene regulatory network contains pharmacological targets that could prevent CL/P in utero, much like the dramatic effect of prenatal folate supplementation on the incidence of spina bifida. CRISPR genome editing was used to disrupt irf6 in zebrafish. All mutant embryos displayed an embryonic epithelium (periderm) rupture phenotype, which represents a sensitive platform for high-throughput chemical screening identifying small molecules that could modulate the irf6 pathways to prevent periderm rupture and potentially CL/P pathogenesis. Using our irf6 mutant model, here we present a small molecule screen of known bioactives for modulation of Irf6 activity in zebrafish, and identification of molecular pathways that could play a role in palate development.
METHODS:
Mutant irf6-/- embryos were dispensed 10 embryos per well in 96-well plates and incubated in media containing propidium iodide (renders ruptured embryos fluorescent). The ICCB known-bioactives library containing FDA-approved drugs with well-characterized biological targets was screened. Mutant embryos treated with DMSO were used as solvent controls. Wildtype embryos treated with drugs were used as toxicity controls. Time-lapse images of the wells were captured by automated bright-field and fluorescence microscopy and analyzed with ImageJ. Molecular pathways associated with the positive hits were identified through the library index and analyzed using computational modeling programs.
RESULTS:
65 of the 480 small molecules screened reached statistical significance in delaying periderm rupture compared to DMSO-treated controls without causing developmental delays in wildtype embryos. The molecular targets of the small molecule hits were analyzed by Gene Ontology and revealed not only molecular pathways previously known to play important roles in palate development such as PDGF and FGF, but also novel pathway connections between IRF6 and the retinoic acid, aryl hydrocarbon, and adenosine pathways among others. Furthermore, when these pathways were aberrantly modulated in wildtype zebrafish embryos, craniofacial defects were observed.
CONCLUSION:
Zebrafish irf6-/- embryos represent a robust platform for high-throughput small molecule screens to identify modulators of IRF6 capable of mitigating cleft pathogenesis. The results identified many critical developmental pathways, some of which have been previously reported as essential in palate development while others have not yet been extensively characterized. These pathways could represent unexplored regulatory mechanisms of palate development and novel nodes of pharmacological intervention for orofacial clefting.


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