Neural crest-specific deletion of Rbfox2 in mice leads to craniofacial abnormalities including cleft palate

Abstract

Alternative splicing (AS) creates proteomic diversity from a limited size genome by generating numerous transcripts from a single protein-coding gene. Tissue-specific regulators of AS are essential components of the gene regulatory network, required for normal cellular function, tissue patterning, and embryonic development. However, their cell-autonomous function in neural crest development has not been explored. Here, we demonstrate that splicing factor Rbfox2 is expressed in the neural crest cells (NCCs), and deletion of Rbfox2 in NCCs leads to cleft palate and defects in craniofacial bone development. RNA-Seq analysis revealed that Rbfox2 regulates splicing and expression of numerous genes essential for neural crest/craniofacial development. We demonstrate that Rbfox2-TGF-β-Tak1 signaling axis is deregulated by Rbfox2 deletion. Furthermore, restoration of TGF-β signaling by Tak1 overexpression can rescue the proliferation defect seen in Rbfox2 mutants. We also identified a positive feedback loop in which TGF-β signaling promotes expression of Rbfox2 in NCCs.

eLife digest

Abnormalities affecting the head and face – such as a cleft lip or palate – are among the most common of all birth defects. These tissues normally develop from cells in the embryo known as the neural crest cells, and specifically a subset of these cells called the cranial neural crest cells. Most cases of cleft lip or palate are linked back to genes that affect the biology of this group of cells.

The list of genes implicated in the impaired development of cranial neural crest cells code for proteins with a wide range of different activities. Some encode transcription factors – proteins that switch genes on or off. Others code for chromatin remodeling factors, which control how the DNA is packed inside cells. However, the role of another group of proteins – the splicing factors – remains unclear and warrants further investigation.

When a gene is switched on its genetic code is first copied into a short-lived molecule called a transcript. These transcripts are then edited to form templates to build proteins. Splicing is one way that a transcript can be edited, which involves different pieces of the transcript being cut out and the remaining pieces being pasted together to form alternative versions of the final template. Splicing factors control this process.

Cibi et al. now show that neural crest cells from mice make a splicing factor called Rbfox2 and that deleting this gene for this protein from only these cells leads to mice with a cleft palate and defects in the bones of their head and face.

Further analysis helped to identify the transcripts that are spliced by Rbfox2, and the effects that these splicing events have on gene activity in mouse tissues that develop from cranial neural crest cells. Cibi et al. went on to find a signaling pathway that was impaired in the mutant cells that lacked Rbfox2. Forcing the mutant cells to over-produce one of the proteins involved in this signaling pathway (a protein named Tak1) was enough to compensate for the some of the defects caused by a lack of Rbfox2, suggesting it acts downstream of the splicing regulator.

Lastly, Cibi et al. showed that another protein in this signaling pathway, called TGF-β, acted to increase how much Rbfox2 was made by neural crest cells. Together these findings may be relevant in human disease studies, given that altered TGF-β signaling is a common feature in many birth defects seen in humans.