Mutation of Eftud2 in Mouse Neural Crest Cells Models MFDM and Reveals Contribution of P53 in Abnormal Craniofacial Development

Abstract

Haploinsufficiency of EFTUD2 is associated with MFDM (mandibulofacial dysostosis with microcephaly), but the etiology of this syndrome remains unknown. Our goal is to determine the tissue and temporal requirement for Eftud2 during craniofacial development. We generated a mouse with a conditional mutation of exon 2 of Eftud2 (Eftud2 flox) using the CRISPR/Cas9 editing system. We used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Embryos carrying one mutated allele of Eftud2 were normal. However, when both alleles were mutated, all embryos displayed hypoplasia of the midbrain and pharyngeal arches at E9.5. By E11.5, most of these embryos showed an open neural tube and all live embryos exhibited exencephaly at E14.5. At this stage, only 3 out of 12 embryos were still alive. Cartilage preparations revealed an absence of cartilage in the head, reduced/or absence of Meckel’s cartilage, and abnormal inner ear development. Crosses with the Rosa26RlacZ mice reporter line revealed a reduced number of neural crest cells in the pharyngeal region at E9.5 and E10.5, but not at E9.0. We next performed RNA sequencing analysis from the head of Eftud2 neural crest cells mutant embryos at E9.0, before any phenotype was observed. The P53 pathway was the most significantly upregulated in the mutant embryos, compared to their wild‐type littermates. Overexpression of P53 target genes identified in the RNAseq (Ccng1, Phlad3 and Trp53inp1) was validated by qRT‐PCR. Additionally, 373 genes showed increased skipped exons in the mutant embryos. Among them, only 2 genes were also overexpressed, including Mdm2, a major regulator of P53. We found that mutant embryos had significantly increased levels of the Mdm2 transcript lacking exon 3, which was previously reported to produce a truncated MDM2 protein, allowing stabilization of P53. Finally, we evaluated the contribution of P53 in craniofacial malformation of Eftud2 mutants by treating pregnant females with daily i.p. injection of pifithrinα, a P53 inhibitor, from E6.5 to E8.5. 37% of pifithrinα‐treated mutant embryos had normal midbrain development at E9.5. Also, the area perimeter of the first pharyngeal arch of pifithrinα‐treated mutant embryos was significantly higher than the vehicle‐treated ones. Altogether, these data suggest that Eftud2 specifically regulates Mdm2 splicing and leads to neural crest cell death due to overactivation of the P53 pathway. We propose that cranial neural crest cell death is responsible for craniofacial malformations in MFDM patients.Support or Funding InformationCanadian Institutes of Health Research