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.

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 InformationCanadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Splicing, the removal of introns from pre‐messenger RNA is an essential step for expression of most genes in multicellular organisms and for expanding the number of proteins coded by genomes. Recently, exome sequencing revealed that mutations in splicing factors and small nuclear ribonucleoprotein particles (snRNPs) which form core components of the spliceosome, are responsible for craniofacial malformations. Our group used in situ hybridization to examine expression of three core components of the spliceosome: Eftud2 – mutated in patients with mandibulofacial dysostosis, Guion‐Almeida type (MFDGA); SnrpB – responsible for Cerebrocostomandibular Syndrome (CMS); and Sf3b4 which is responsible for Nager syndrome. Herein, we report expression of Eftud2, Sf3b4 and SnrpB during craniofacial development. In addition, we used CRISPR/Cas9 to generate mice with deletion (Eftud2 del) and conditional mutation of exon 2 of Eftud2 (Eftud2 flox). At embryonic days (E) 7.5 and 8.5, Eftud2 was highly expressed in ectodermal and mesodermal components of the future craniofacial region, by E9.5 expression was also found in the body wall and developing heart. Eftud2 del heterozygous mice were viable and fertile, though these embryos showed reduced levels of Eftud2 mRNA and protein. In contrast, Eftud2 del homozygous mutant embryos arrest at E3.5 and failed to grow and hatch ex vivo. To examine the requirement for Eftud2 in neural crest cells, the Wnt1‐Cre2 transgenic line was used to delete exon 2 of Eftud2 specifically in that lineage. Eftud2 flox homozygous mutant embryos carrying the Wnt1‐Cre2 transgene displayed hypoplasia of the midbrain and pharyngeal arches starting at E9.5. By E11.5, most embryos also had an open neural tube and all embryos showed exencephaly at E14.5. Cartilage preparations revealed an absence of cartilage in the head, reduction/or absence of Meckel's cartilage, and abnormal inner ear development. Since deletion of exon 2 is predicted to generate a truncated protein with partial function, our data suggest that normal levels of Eftud2 is crucial in neural crest cells for normal craniofacial development. Future studies are focused on elucidating the molecular and transcriptional basis of MFDM using this mouse model.Support or Funding InformationCanadian Institute of Health Research (MOP#142452)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.