Reactivation of the pluripotency program precedes formation of the cranial neural crest

Reactivating neural crest pluripotency

Cranial neural crest cells (CNCCs) are a transient cell group with an extraordinary differentiation potential that extends beyond its ectodermal lineage to form the majority of facial mesenchyme. Zalc et al. identified a neuroepithelial precursor population that transiently reactivates pluripotency factors to generate CNCCs. The pluripotency factor Oct4 is required for the expansion of CNCC developmental potential to form facial mesenchyme. Analysis of the chromatin landscape of Oct4+ CNCC precursors showed that these cells resemble those of epiblast stem cells, with additional features suggestive of future priming for neural crest programs. Thus, to expand their cellular potency, CNCC precursors undergo a natural in vivo reprogramming event.

Science, this issue p. eabb4776

Structured Abstract


Cell differentiation is classically described as a unidirectional process that progresses through a series of lineage restriction events, with cellular potential being increasingly reduced as the embryo develops, a concept famously illustrated by Conrad Waddington in his epigenetic landscape. However, the vertebrate-specific transient cell population called cranial neural crest cells (CNCCs) challenges this paradigm. Although they originate in the ectoderm and are capable of differentiating into cell types typical of this germ layer, CNCCs can also give rise to mesenchymal cell types canonically associated with the mesoderm lineage, such as bone, cartilage, and smooth muscle. How CNCCs expand their differentiation potential beyond their germ layer of origin remains unresolved.


We hypothesized that unbiased analysis of transcriptional heterogeneity during the early stages of mammalian CNCC development may identify a precursor population and provide clues as to how these specialized cells gain their extraordinary differentiation potential. To test this, we combined single-cell RNA-sequencing analysis of murine CNCCs from staged mouse embryos with follow-up lineage-tracing, loss-of-function, and epigenomic-profiling experiments.


We found that premigratory CNCCs are heterogeneous and carry positional information reflective of their origin in the neuroepithelium, but this early positional information is subsequently erased, with delaminating CNCCs showing a relatively uniform transcriptional signature that later rediversifies as CNCCs undergo first commitment events. We identify an early precursor population that expresses canonical pluripotency transcription factors and gives rise to CNCCs and craniofacial structures. Rather than being maintained from the epiblast, pluripotency factor Oct4 is transiently reactivated in the prospective CNCCs after head-fold formation, and its expression shifts from the most anterior to the more posterior part of the cranial domain as development progresses. Oct4 is not required for the induction of CNCCs in the neuroepithelium, but instead is important for the specification and survival of facial mesenchyme, thus directly linking this pluripotency factor with the expansion of CNCC cellular potential. Open chromatin landscapes of Oct4+ CNCC precursors are consistent with their neuroepithelial origin while also broadly resembling those of pluripotent epiblast stem cells. In addition, we saw priming of distal regulatory regions at a subset of loci associated with future neural crest migration and mesenchyme formation.


Our results show that premigratory CNCCs first form as a heterogeneous population that rapidly changes its transcriptional identity during delamination, resulting in the formation of a transcriptionally (and likely also functionally) equivalent cell group capable of adapting to future locations during and after migration. Such functional equivalency and plasticity of CNCCs is consistent with previous embryological studies. Furthermore, the demonstration that CNCC precursors transiently reactivate pluripotency factors suggests that these cells undergo a natural in vivo reprogramming event that allows them to climb uphill on Waddington’s epigenetic landscape. Indeed, our results show that at least one of the pluripotency factors, Oct4, is required for the expansion of CNCC developmental potential to include formation of facial mesenchyme. Whether this mechanism is specific to CNCCs and if such expansion of cellular plasticity could be harnessed for regenerative medicine purposes remain interesting questions for future investigations.

CNCCs expand their developmental potential through transient reactivation of a pluripotency program.

(A) Single-cell RNA (scRNA) sequencing of genetically labeled murine CNCCs over 14 hours of development revealed rapid transcriptional changes and identified a precursor population expressing pluripotency factors. (B) Uphill on Waddington’s epigenetic landscape, reactivation of Oct4 endows CNCC precursors with the ability to form derivatives typical of mesoderm, such as mesenchyme.


During development, cells progress from a pluripotent state to a more restricted fate within a particular germ layer. However, cranial neural crest cells (CNCCs), a transient cell population that generates most of the craniofacial skeleton, have much broader differentiation potential than their ectodermal lineage of origin. Here, we identify a neuroepithelial precursor population characterized by expression of canonical pluripotency transcription factors that gives rise to CNCCs and is essential for craniofacial development. Pluripotency factor Oct4 is transiently reactivated in CNCCs and is required for the subsequent formation of ectomesenchyme. Furthermore, open chromatin landscapes of Oct4+ CNCC precursors resemble those of epiblast stem cells, with additional features suggestive of priming for mesenchymal programs. We propose that CNCCs expand their developmental potential through a transient reacquisition of molecular signatures of pluripotency.

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