Inactivation of <i>Zeb1</i> in GRHL2-deficient mouse embryos rescues mid-gestation viability and secondary palate closure

Abstract

Cleft lip and palate are common birth defects resulting from failure of the facial processes to fuse during development. The mammalian grainyhead-like (<i>Grhl1-3</i>) genes play key roles in a number of tissue fusion processes including neurulation, epidermal wound healing and eyelid fusion. One family member, <i>Grhl2</i>, is expressed in the epithelial lining of the first pharyngeal arch in mice at embryonic day (E)10.5, prompting analysis of the role of this factor in palatogenesis. <i>Grhl2</i>-null mice die at E11.5 with neural tube defects and a cleft face phenotype, precluding analysis of palatal fusion at a later stage of development. However, in the first pharyngeal arch of <i>Grhl2</i>-null embryos, dysregulation of transcription factors that drive epithelial-mesenchymal transition (EMT) occurs. The aberrant expression of these genes is associated with a shift in RNA-splicing patterns that favours the generation of mesenchymal isoforms of numerous regulators. Driving the EMT perturbation is loss of expression of the EMT-suppressing transcription factors <i>Ovol1</i> and <i>Ovol2</i>, which are direct GRHL2 targets. The expression of the miR-200 family of microRNAs, also GRHL2 targets, is similarly reduced, resulting in a 56-fold upregulation of <i>Zeb1</i> expression, a major driver of mesenchymal cellular identity. The critical role of GRHL2 in mediating cleft palate in <i>Zeb1^-/-</i> mice is evident, with rescue of both palatal and facial fusion seen in <i>Grhl2^-/-;Zeb1^-/-</i> embryos. These findings highlight the delicate balance between GRHL2/ZEB1 and epithelial/mesenchymal cellular identity that is essential for normal closure of the palate and face. Perturbation of this pathway may underlie cleft palate in some patients.