Nager syndrome is a poorly understood rare disorder that severely affects skeletal development, predominantly in the face and head. Approximately 60% of cases are caused by mutations to the gene encoding mRNA splicing factor 3b, subunit 4 (SF3B4), with the cause of the remaining cases unknown. Alternative splicing happens during mRNA processing before it is translated into protein, enabling a greater diversity of protein products from the same number of genes. Splicing disorders have been implicated in other diseases of face development, making understanding the pathogenic role of variant SF3B4 and disrupted splicing important to a range of conditions.
In this study, Griffin, Saint-Jeannet and colleagues modelled the loss of sf3b4 in the frog species Xenopus tropicalis to understand how mutations in this gene can impair facial development. They used CRISPR-Cas9 gene editing to remove part of the sf3b4 gene that corresponds to a region commonly mutated in Nager syndrome patients, leading to complete loss of the protein. While the human disease is caused by mutations in a single copy of the gene, frog embryos with one mutated copy of sf3b4 were broadly comparable to wild type, suggesting that humans are more reliant on SF3B4 for healthy development. Frogs lacking both copies of sf3b4 showed shortened facial features, recapitulating aspects of Nager disease; however, these frogs failed to survive past tadpole stage.
To understand why loss of sf3b4 disrupts facial development, the authors looked at the neural crest, the region of the embryo where the cartilage of the face and head originates. Tailbud-stage embryos completely lacking sf3b4 showed impaired migration of neural crest cells and increased cell death in this region, suggesting a likely cause of the shortened facial features. Bulk RNA sequencing showed that aberrant mRNA splicing is particularly pronounced at the tailbud stage in embryos completely lacking sf3b4, with considerably fewer splicing defects observed later in tadpoles. However, overall gene expression changes were most pronounced at the tadpole stage, probably driven by this mis-regulated splicing at the earlier stage.
Altogether, these results show successful characterisation of a novel frog model that recapitulates many aspects of Nager disease, and other splicing disorders that affect the face and head. Uncovering commonly disrupted pathways between these conditions – such as increased cell death in the neural crest – will strengthen our understanding of how these mutations contribute to rare disease.
