Significant insights into the development of the vertebrate head

Cranial neural crest-derived cells (CNCCs) are a vertebrate-specific population, often referred to as the fourth germ layer. They have extraordinary potential to form diverse cell types. In addition to pigment cells and the peripheral nervous system, CNCCs form the ectomesenchyme that populates the pharyngeal arches and gives rise to much of the skeleton and connective tissue of the jaws and face.

How neural crest cells acquire such extraordinary lineage potential such as to generate a huge diversity of derivatives, including the bulk of connective and skeletal tissues of the vertebrate head, remains unresolved.

Now, scientists from the lab of Gage Crump created a series of atlases over time to understand the molecular decisions by which CNCCs commit to forming specific tissues in developing zebrafish. Their findings, published in Nature Communications, may provide new insights into normal head development, as well as craniofacial birth defects.

“CNCCs have long fascinated biologists by the incredible diversity of cell types they can generate. By studying this process in the genetically tractable zebrafish, we have identified many of the potential switches that allow CNCCs to form these very different cell types,” said Gage Crump, professor of stem cell biology and regenerative medicine at the Keck School of Medicine of USC.

Led by postdoc Peter Fabian and Ph.D. students Kuo-Chang Tseng, Mathi Thiruppathy, and Claire Arata, the scientists’ team permanently labeled CNCCs with a red fluorescent protein to keep track of which cell types came from CNCCs throughout the lifetime of zebrafish.

To understand the emergence and diversification of CNCC lineages across the lifetime of a vertebrate, researchers constructed a longitudinal single-cell atlas of gene expression and chromatin accessibility of zebrafish CNCC derivatives.

The massive quantity of data generated required the scientists to develop a new computational tool to make sense of it.

“We created a type of computational analysis that we called ‘Constellations,’ because the final visual output of the technique is reminiscent of constellations of stars in the sky,” said Fabian. “In contrast to astrology, our Constellations algorithm really can predict the future of cells and reveal the key genes that likely control their development.”

Through this new bioinformatic approach, the team discovered that CNCCs do not start out with all the information required to make the huge diversity of cell types. Instead, only after they disperse throughout the embryo do CNCCs begin reorganizing their genetic material in preparation for becoming specific tissues. Constellations accurately identified genetic signs that point to these specific destinies for CNCCs. Real-life experiments confirmed that Constellations correctly pinpointed the role of a family of “FOX” genes in facial cartilage formation, and a previously unappreciated function for “GATA” genes in the formation of gill respiratory cell types that allow fish to breathe.

“By conducting one of the most comprehensive single-cell studies of a vertebrate cell population to date, we not only gained significant insights into the development of the vertebrate head, but also created a broadly useful computational tool for studying the development and regeneration of organ systems throughout the body,” said Crump.

Journal Reference

1. Fabian, P., Tseng, KC., Thiruppathy, M. et al. Lifelong single-cell profiling of cranial neural crest diversification in zebrafish. Nat Commun 13, 13 (2022). DOI: 10.1038/s41467-021-27594-w