Abstract

Binary decisions refine fate decisions Neural crest cells develop into tissues ranging from craniofacial bones to peripheral neurons. Combining single-cell RNA sequencing with spatial transcriptomics, Soldatov et al. analyzed how neural crest cells in mouse embryos decide among the various fates available to them (see the Perspective by Mayor). These multipotent cells become biased toward a given fate early on and step through a progression of binary decisions as their fate is refined. Competing fate programs coexist until increased synchronization favors one and repression disfavors the other. Science, this issue p. eaas9536; see also p. 937 Differentiation of cells of the neural crest proceeds through binary decisions that restrict and refine developmental paths. INTRODUCTION Multipotent progenitors must choose among multiple downstream fates. In developing embryos, progenitor cells exhibit transcriptional or epigenetic heterogeneity that is related to early biases in cell fate choices, and can be externally induced or stochastic in nature. Molecular assessment of the transient states assumed by cells during these developmental progressions has the potential to illuminate how such fate-specific biases emerge and unfold to ensure fate commitment. With this aim, we examine multipotent neural crest cells—transient embryonic progenitors unique to vertebrates that build the head, teeth, neuroendocrine tissue, and autonomic and sensory nervous systems. Cranial neural crest preferentially gives rise to a multitude of mesenchymal types of facial cartilage and bones, in addition to neuronal, glial, and pigment cell–type progeny. By contrast, trunk neural crest does not form bone or cartilage derivatives in vivo. The logic and molecular mechanisms that allow neural crest to resolve multiple potential cell fates at each axial level remain poorly understood. RATIONALE Here we used single-cell and spatial transcriptomics with statistical analysis of branching trajectories to investigate lineage relationships in mouse neural crest. Combined with lineage tracing and functional perturbations, we addressed spatiotemporal dynamics associated with early cell fate decisions in mouse trunk and cranial neural crest cells with different fate potential. RESULTS We find that up to early migration, neural crest cells progress through a sequence of common transcriptional states, followed by fate bifurcations during migration that can be formalized as a series of sequential binary decisions. The first decision separates sensory neuro-glial fate from all other fates, whereas the second decision occurs between autonomic and mesenchymal lineages and reveals a bipotent Phox2b+/Prrx1+ subpopulation. Decision points uncover distinct roles of neural crest regulators: Neurog2 is involved in early repression of melanocytes and activation of sensory fate at later steps. Each decision consists of initial coactivation, gradual biasing, and commitment phases. Early genes of competing cell fate programs coactivate in the same cells, starting from premigratory stage. As cells approach cell fate bifurcation points, increased synchronization of fate-specific programs and repulsion of competing fate programs lead to gradual appearance of cell fate bias, which becomes pronounced upon neural crest migration. Cell fate commitment culminates with activation of mutually exclusive, fate-specific gene expression programs. Early transcriptional patterns reveal that fate biasing of neural crest is already detectable when neural crest cells delaminate from the neural tube. In particular, the neuronal bias of trunk and mesenchymal bias of cranial neural crest emerge during delamination, indicating that this might be the time when the mesenchymal potential, distinct between cranial and trunk neural crest, is installed. In support to this hypothesis, we find that sustained overexpression of a single gene, Twist1, normally activated upon delamination only in the cranial compartment, is sufficient to reverse the trunk crest developmental program to a mesenchymal route. CONCLUSION Our analysis resolved a branching transcriptional trajectory of the differentiating neural crest, illustrating transcriptional implementation of major cell fate decisions and pinpointing the key differences defining cranial versus trunk neural crest potential. Our results show that neural crest cells differentiate through a series of stereotypical lineage-restriction events that involve coexpression and competition of genes driving alternative fate programs. Cell fate acquisition in neural crest. (A) Stepwise model of neural crest developmental dynamics. EMT, epithelial-to-mesenchymal transition. (B) Key phases of fate decision in neural crest cells. (C) Different fate potential of cranial versus trunk neural crest is encoded by the mesenchymogenic genes Twist1 and Prrx2. A single factor, Twist1, confers mesenchymal potential to trunk neural crest. Neural crest cells are embryonic progenitors that generate numerous cell types in vertebrates. With single-cell analysis, we show that mouse trunk neural crest cells become biased toward neuronal lineages when they delaminate from the neural tube, whereas cranial neural crest cells acquire ectomesenchyme potential dependent on activation of the transcription factor Twist1. The choices that neural crest cells make to become sensory, glial, autonomic, or mesenchymal cells can be formalized as a series of sequential binary decisions. Each branch of the decision tree involves initial coactivation of bipotential properties followed by gradual shifts toward commitment. Competing fate programs are coactivated before cells acquire fate-specific phenotypic traits. Determination of a specific fate is achieved by increased synchronization of relevant programs and concurrent repression of competing fate programs.