The detachment of neural crest cells occurs via a process called epithelial to mesenchymal transition—where static cells change their shape and acquire migratory properties—the subject of a recent review paper from the Trainor Lab. Once situated in the facial region, neural crest cells differentiate, or decide their cell fate, ultimately generating nearly all the of the craniofacial cartilage, bone and connective tissues.

The rapid proliferation and growth of neural crest cells in concert with epithelial to mesenchymal transition requires a disproportionate quantity of ribosomes and hence proteins compared to surrounding non-neural crest cells and tissues. One of the key steps of ribosome biogenesis—RNA Polymerase I-mediated rRNA transcription—is a rate-limiting step in the formation of ribosomes, and is highly active within neural crest cells.

“Large quantities of protein translation are needed for neural crest cell proliferation and survival,” said Dash.

By turning off genes responsible for rRNA transcription and ribosome formation in mice, the researchers uncovered a cellular imbalance between rRNA transcription and ribosomal proteins that greatly impacted neural crest cells.

This imbalance promotes neural crest cell death via increased concentrations of the tumor suppressor protein, p53, which causes the embryo to develop malformations of the head and face.

In normal neural crest cells, p53 levels are tightly controlled by the protein, Mdm2. However, when rRNA transcription is reduced, an imbalance arises where ribosomal proteins that normally form part of the ribosome, become free and instead bind to Mdm2 preventing its control of p53. This leads to increased levels of p53 that cause the newly formed neural crest cells to die.

“We have known for many years that problems in craniofacial development are often the result of neural crest cells dying before they can form the cartilage and bone structures of the head and face,” said Falcon.

“Our previous studies have shown that inhibiting p53 in Treacher-Collins Syndrome animal models can stop neural crest cells from dying and thus prevent craniofacial anomalies from occurring, but this made the animals much more susceptible to cancer,” said Watt.

“The significance of this study is that at the genetic, cellular, and biochemical level, we can now connect rRNA transcription and the ribosome biogenesis pathway in neural crest cells, to proper craniofacial development and to the pathogenesis of birth defects,” said Trainor. “We understand how each step in the pathway links to p53-dependent apoptosis.”

Coauthors include Ruonan Zhao, Daisuke Sakai PhD, Emma L. Moore, Sharien Fitriasari, Melissa Childers, Mihaela E. Sardia, Selene Swanson, Dai Tsuchiya, Jay Unruh, PhD, George Bugarinovic, Lin Li, Rita Shiang, PhD, Annita Achilleos, PhD, Jill Dixon, PhD, and Michael Dixon, PhD.

Studying multiple research organisms is key

The study of rare diseases is challenging but deserves no less attention than common diseases. However, a universal mechanism to explain rare birth defects simply doesn’t exist and developing preventative therapies therefore requires a thorough understanding of the cellular and genetic mechanisms responsible for each individual disorder, which is derived from our knowledge of normal craniofacial development in many different animal species.

For example, in another recent study published in Development on June 28, 2022, Dash in the Trainor Lab investigated the function of Nucleolin in neural crest cells and craniofacial development in zebrafish. Mutations in Nucleolin disrupt the differentiation of neural crest cells resulting in craniofacial anomalies resembling Treacher-Collins Syndrome. Dash discovered that Nucleolin, among its many functions, regulates signaling pathways that are important for neural crest cell differentiation into bone and cartilage.

“By identifying the biochemical pathway that was involved, we were able to bypass the problem by providing the zebrafish embryos with the specific proteins they needed for proper differentiation,” said Dash.

Using research organisms such as mice and zebrafish to understand the basis for birth defect disorders can lead to discoveries on how to prevent them. “If it’s successful in animal models, it may give people hope that something can be done in the future,” said Trainor.

This work was funded by the Kirschstein-NRSA F31 predoctoral fellowship (awards DE027860, DE023017) from the National Institute for Dental and Craniofacial Research of the National Institutes of Health, K99 Pathway to independence awards (DE030971 and DE030972), the American Association for Anatomy Post-Doctoral Fellowship, and from institution support from the Stowers Institute for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.