Loss of function mutation of Eftud2, the gene responsible for mandibulofacial dysostosis with microcephaly (MFDM), leads to pre-implantation arrest in mouse

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 specific expression and requirement for Eftud2 during craniofacial development. We used RT‐PCR and in situ hybridization to examine expression of Eftud2 during embryogenesis. Using CRISPR/Cas9 we designed guide RNAs to generate mice with deletion (Eftud2 del) and conditional mutation of exon 2 of Eftud2 (Eftud2 flox). At embryonic day (E) 7.5 and 8.5 of mouse development, Eftud2 was highly expressed in both ectodermal and mesodermal components of the developing head and craniofacial region, by E9.5 Eftud2 was more broadly expressed, in the body wall and developing heart. Eftud2 del heterozygous mice and embryos were viable and fertile and showed a 38% and a 30% reduction of Eftud2 mRNA and protein expression, respectively. Before the onset of organogenesis, Eftud2 del heterozygous embryos had reduced number of somites when compared to their wild type litter mates, indicating a delay in development. Noticeably, RNA sequencing revealed that Eftud2 was the only transcript significantly affected in these heterozygous mice. Eftud2 del homozygous mutant embryos did not implant and failed to grow and hatch ex vivo. Next, we used the Wnt1‐Cre2 transgenic line to delete exon 2 of Eftud2 specifically in the neural crest cells. Eftud2 flox heterozygous embryos carrying the Wnt‐Cre2 transgene were normal. However, Eftud2 flox homozygous mutant embryos that also carry the Wnt1‐Cre2 transgene displayed hypoplasia of the midbrain and pharyngeal arches starting as early as E9.5. By E11.5, most of these embryos showed an open neural tube, all embryos showed exencephaly at E14.5. 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 Rosa26R mice reporter line, revealed reduced neural crest cell migration into the pharyngeal region at E10.5. Since the mutation designed is predicted to generate a truncated protein with partial function, our data suggest that normal levels of Eftud2 is crucial during embryogenesis. Future studies are focused on determining the molecular and transcriptional basis of MFDM using this mouse model. Support or Funding Information Canadian Institutes of Health Research This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mis-splicing ofMdm2leads to Increased P53-Activity and Craniofacial Defects in a MFDMEftud2Mutant Mouse Model

“…We reported a unique mouse model with heterozygous Eftud2 mutation in which exon 2, part of the N-terminal acidic region, was deleted. In an outbred genetic background, heterozygous mice were viable and fertile and showed no major phenotypic abnormalities whereas homozygous mutants arrested pre-implantation (Beauchamp et al, 2019). However, although heterozygous pups on the inbred C57Bl/6 genetic background showed no obvious craniofacial malformations, a small but significant number of these pups were lost between birth and weaning.…”

“…To characterize the role of Eftud2 in neural crest cells, we used Wnt1-Cre2 transgenic mice (Lewis et al, 2013). Since Eftud2 +/embryos were morphologically and molecularly indistinguishable from wild-type embryos (Beauchamp et al, 2019), matings of Eftud2 loxP/+ ; Wnt1-Cre2 tg/+ or Eftud2 +/-; Wnt1-Cre2 tg/+ mice to Eftud2 loxP/loxP mice were used to generate homozygous mutant embryos for analysis. From E8.5 to E17.5, embryos of all genotypes were found at the expected Mendelian ratio (Table S1).…”

“…A number of animal models carrying Eftud2 mutations have been reported in zebrafish and in mouse (Beauchamp et al, 2019;Deml et al, 2015;Lei et al, 2017;Wu et al, 2019). Three zebrafish models targeted eftud2 gene in different ways: one TALEN-derived deletion in the 5 th exon, c.378_385, disrupts part of the GTP binding domain (Deml et al, 2015), a 5 bp deletion in exon 2 also generated using TALEN mutates part of the N-acidic terminal domain (Wu et al, 2019), and an N-ethyl-Nnitrosourea (ENU) induced nonsense C-T mutation in exon 24 of eftud2 mutates part of the conserved EF 2 domain IV (Lei et al, 2017).…”

“…Our aim is to uncover the etiology of abnormal craniofacial malformations in MFDM using mutant mouse models of Eftud2. However, despite a 30% reduction of Eftud2 mRNA and protein levels, mice with constitutive heterozygous deletion of exon 2 of Eftud2 (Eftud2 +/-) are viable and fertile, and exhibit no craniofacial malformations (Beauchamp et al, 2019). Additionally, RNA sequencing revealed no significant splicing changes in Eftud2 +/embryos when compared to wild-type littermates.…”

EFTUD2 missense variants disrupt protein function and splicing in mandibulofacial dysostosis Guion‐Almeida type

“…However, recent cell and animal models of MFDGA have begun to analyze the consequences of reduced EFTUD2 expression (Beauchamp et al, 2019;Deml, Reis, Muheisen, Bick, & Semina, 2015;Lei et al, 2017;Wood et al, 2019).…”