Gene Regulatory Mechanisms Driving Temporal Dynamics of Lineage Specific Differentiation in the Cranial Neural Crest

Abstract

Genetic and environmental perturbations impacting neural crest (NC) development can result in pleiotropic structural and functional birth defects, many of which are associated with pediatric syndromes. As developmental precursors, the NC has the unique capacity to give rise to a diverse array of ectodermal and mesoectodermal cell types, from neurons and glia of the peripheral nervous system to the cartilage and bone of the face. In order to transition from a multipotent progenitor to a specific cell type, NC cells must undergo a series of dynamic morphological and behavioral transformations that gradually unfold over time. However, the NC is rare and transient cell population, and the genetic programming that governs the transition through the intermediate stages of differentiation toward a specific cell fate remain poorly understood. In order to investigate the temporal regulation of this process we established a robust in vitro model system of mammalian cranial and trunk NC cell differentiation. Systematic characterization of directed differentiation along the neural and chondrogenic lineages revealed reproducible, temporal benchmarks indicative of the intermediate stages underlying the progression, many of which mimic those described in vivo. Using this culture system, we explicitly interrogated cranial NC cells to uncover the dynamic gene expression changes that occur as the multipotent progenitors transition over time to become peripheral neurons or cartilage matrix-producing chondrocytes. Our analysis revealed that cranial NC cell diversification toward either cell type is mediated through gradual coordination of both common and lineage-specific programming, as well as concurrent suppression of competing cell fates. We further identified distinct transcriptional signatures corresponding to the intermediate state, as well as putative regulators that govern the overall progression in a stage-specific and time-sensitive manner. These data serve as a powerful tool for discovering previously unappreciated mechanisms contributing to cell fate acquisition in the NC, and novel molecular targets of genetic and environmental factors that contribute to NC-related birth defects and disorders.