Mice with neural crest cells lacking splicing factor Rbfox2 developed a cleft palate and defects in the bones of their head and face, new Singapore study shows.
Head and face abnormalities, such as cleft lip or palate, are among the most common birth defects, contributing to over a third of congenital diseases. Researchers at Duke-NUS Medical School and the National Heart Research Institute, Singapore, have uncovered a key mechanism underlying these conditions in a mouse model, which may be instrumental in the human disease and suggest new therapeutic strategies.
In Singapore, cleft lip and/or palate, characterised by an opening or split in the upper lip, the roof of the mouth (palate) or both, occurs in approximately two out of every 1,000 live births. These structures are formed from cells in the embryo known as neural crest cells. Most cases of cleft lip or palate link back to genes implicated in the impaired development of neural crest cells. These code for proteins involved in a wide range of different activities, such as switching genes on and off, packing DNA inside cells, or splicing different pieces of genetic code to build other proteins.
"Genetic defects that affect neural crest cell generation, migration, proliferation, or differentiation result in cardio-craniofacial malformations, including cleft lip and cleft palate," explained Assistant Professor Manvendra Singh, the study's corresponding author from the Cardiovascular and Metabolic Disorders Programme at Duke-NUS Medical School, Singapore. "However, the role of splicing factors during neural crest cell development remained unclear. Our study set out to investigate this so as to better understand the process that leads to these birth defects."
Splicing factors are important protein components of the gene proofreading or 'splicing' mechanism that is found in all cells. The researchers showed that neural crest cells from mice create a splicing factor called Rbfox2, which regulates the expression of numerous genes essential for neural crest and craniofacial development. They found that deleting the gene for the expression of this protein from the neural crest cells resulted in mice with a cleft palate and defects in the bones of their head and face.
Further analysis helped to identify the genetic code spliced by Rbfox2, and the effects that these splicing events have on gene activity in mouse tissues that develop from cranial neural crest cells. The research team went on to discover that a signalling pathway, associated with a cell-signalling protein called transforming growth factor-beta (TGF-b) – which plays a vital role in regulating cell growth, differentiation, and development in a wide array of biological systems – was impaired in the cells lacking Rbfox2. Forcing these cells to over-produce Tak1, one of the proteins involved in the TGF-b pathway, compensated for some of the defects caused by the lack of Rbfox2, suggesting it acts downstream of the splicing regulator.
Finally, the researchers showed that another protein in the TGF-b signalling pathway acted to increase the amount of Rbfox2 produced by the neural crest cells, creating a positive feedback loop. Given that altered TGF-b signalling is a common feature in many birth defects seen in humans, these findings may be relevant in human disease studies.
"Fundamental research is a vital component of new discoveries in medicine and healthcare. Understanding the molecular mechanism underlying craniofacial and cardiovascular abnormalities helps us to better understand the aetiology of these congenital diseases, with a view to discovering new treatment possibilities," remarked Professor Patrick Casey, Senior Vice Dean for Research at Duke-NUS.
"The neural crest is an interesting embryonic cell population as they contribute to a wide variety of derivatives, including the formation and septation of the cardiac outflow tract, as well as patterning and remodelling of aortic arch arteries," said Asst Prof Singh. "Our future work in this direction will help to understand the transcriptional network underlying the congenital craniofacial and cardiovascular defects." Palates from control (left) and neural crest-specific Rbfox2 knockout (right) embryos. Cleft Palate is observed in Rbfox2 mutant embryos. (Photo credit: Cibi Dasan Mary)
Reference: Cibi, D.M., Mia, M.M., Shekeran, S.G., et al. Neural crest-specific deletion of Rbfox2 in mice leads to craniofacial abnormalities including cleft palate. eLife 2019;8:e45418. doi: 10.7554/eLife.45418
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