Craniofacial Defects in Embryos with Homozygous Deletion of Eftud2 in Their Neural Crest Cells Are Not Rescued by Trp53 Deletion.

Embryos with homozygous mutation of Eftud2 in their neural crest cells (Eftud2ncc-/-) have brain and craniofacial malformations, hyperactivation of the P53-pathway and die before birth. Treatment of Eftud2ncc-/- embryos with pifithrin-α, a P53-inhibitor, partly improved brain and craniofacial development. To uncover if craniofacial malformations and death were indeed due to P53 hyperactivation we generated embryos with homozygous loss of function mutations in both Eftud2 and Trp53 in the neural crest cells. We evaluated the molecular mechanism underlying craniofacial development in pifithrin-α-treated embryos and in Eftud2; Trp53 double homozygous (Eftud2ncc-/-; Trp53ncc-/-) mutant embryos. Eftud2ncc-/- embryos that were treated with pifithrin-α or homozygous mutant for Trp53 in their neural crest cells showed reduced apoptosis in their neural tube and reduced P53-target activity. Furthermore, although the number of SOX10 positive cranial neural crest cells was increased in embryonic day (E) 9.0 Eftud2ncc-/-; Trp53ncc-/- embryos compared to Eftud2ncc-/- mutants, brain and craniofacial development, and survival were not improved in double mutant embryos. Furthermore, mis-splicing of both P53-regulated transcripts, Mdm2 and Foxm1, and a P53-independent transcript, Synj2bp, was increased in the head of Eftud2ncc-/-; Trp53ncc-/- embryos. While levels of Zmat3, a P53- regulated splicing factor, was similar to those of wild-type. Altogether, our data indicate that both P53-regulated and P53-independent pathways contribute to craniofacial malformations and death of Eftud2ncc-/- embryos.

Snrpb is required in murine neural crest cells for proper splicing and craniofacial morphogenesis.

Heterozygous mutations in SNRPB, an essential core component of the five small ribonucleoprotein particles of the spliceosome, are responsible for cerebrocostomandibular syndrome (CCMS). We show that Snrpb heterozygous mouse embryos arrest shortly after implantation. Additionally, heterozygous deletion of Snrpb in the developing brain and neural crest cells models craniofacial malformations found in CCMS, and results in death shortly after birth. RNAseq analysis of mutant heads prior to morphological defects revealed increased exon skipping and intron retention in association with increased 5' splice site strength. We found increased exon skipping in negative regulators of the P53 pathway, along with increased levels of nuclear P53 and P53 target genes. However, removing Trp53 in Snrpb heterozygous mutant neural crest cells did not completely rescue craniofacial development. We also found a small but significant increase in exon skipping of several transcripts required for head and midface development, including Smad2 and Rere. Furthermore, mutant embryos exhibited ectopic or missing expression of Fgf8 and Shh, which are required to coordinate face and brain development. Thus, we propose that mis-splicing of transcripts that regulate P53 activity and craniofacial-specific genes contributes to craniofacial malformations. This article has an associated First Person interview with the first author of the paper.