Maintenance of pluripotency in the entire ectoderm enables neural crest formation

Background

The vertebrate body plan, like that of its invertebrate counterparts, is built from a single, fertilized egg cell. As development proceeds, cells gradually lose potency as the set of possible cell types they can give rise to shrinks. While cells at the blastula stage of an embryo exhibit pluripotency, the ability to give rise to all cell types, it is thought that pluripotency is lost during gastrulation when cells are specified to become one of the three germ layers: endoderm, mesoderm, or ectoderm. Further fate restriction is observed as the embryo passes from gastrulation into neurulation.

The neural crest, a vertebrate-specific cell population, provides an exception to this rule. Neural crest cells are formed at the border of neural and non-neural ectoderm during gastrulation and begin to migrate at the end of neurulation. These cells ultimately give rise to cell types that are traditionally thought to be both ectodermal and mesodermal, including the bones, cartilage, smooth muscle and sensory organs found in the head that serve as some of the defining features of the vertebrate clade [1].

In recent years, debates surrounding the potency of the neural crest have heated up thanks to experiments applying different techniques across different organisms. Experiments in Xenopus embryos and explanted pluripotent cells identified a shared transcriptional regulatory network between pluripotent cells of the blastula and neural crest cells [2], suggesting that pluripotency may be retained in the neural crest of the early embryo. Meanwhile, experiments in mice showed reactivation of the pluripotency factor Oct4 immediately preceding formation of the cranial neural crest [3].

In this preprint, Pajanoja and colleagues use Multiplex Single Cell Spatial Transcriptomics (scMST), a technique used previously to identify a neural crest stem cell niche [4], to understand the transition from pluripotency to fate commitment in the neural crest and surrounding ectoderm.

Main Findings

A pluripotent transcriptional signature is observed throughout the entire ectoderm at early neurula stages
The authors performed scMST on sectioned tissue from the midbrain level of chick embryos at four different developmental stages, ranging from the end of gastrulation to the end of neurulation (HH5 to 7SS). The set of 30 genes chosen for imaging represented pluripotency and stem cell markers, as well as differentiation markers for neural, neural crest, epidermal, and mesodermal fates. Hierarchical clustering of spatial transcriptomic data grouped cells into subpopulations that were manually annotated based on their gene expression, allowing the authors to pseudo-color images of the midbrain sections and observe spatial distributions of these subpopulations.

 

Individual cells expressing high levels of pluripotency markers, annotated as uncommitted stem cells, were observed broadly throughout the ectoderm at the early neurula stage, contrasting with a model in which pluripotency is lost by the end of gastrulation. The authors also identified transitioning stem cell domains marked by co-expression of pluripotency and differentiation markers that were spatially distinct from the committed cell populations. This observation raises the possibility that ectodermal cells maintain developmental plasticity, and potentially pluripotency, beyond gastrulation and into neurulation.

Figure 1: (A) Pseudocoloring of cells identified by scMST with Z-scores greater than the mean for all three pluripotency markers. The presence of these uncommitted cells is observed throughout neurulation, and is gradually restricted from the whole ectoderm to the dorsal neural tube where neural crest cells are specified. (B) The expression of pluripotency markers overlapped with a pan-ectodermal stem cell signature, suggesting that developmental plasticity in the ectoderm is essential for patterning.

Pluripotency marker expression is maintained in the neural crest through end of neurulation

The authors observed that the shared expression of pluripotency factors was maintained through the end of neurulation, with gradual spatial restriction to the presumptive neural crest in the dorsal neural tube (Fig. 1 a). Expression of these pluripotency factors also overlapped with expression of a pan-ectoderm stem cell module, reflecting a transcriptional state where the cells remain undecided regarding the future ectodermal domains including epidermis, central nervous system, and the neural crest (Fig. 1b). Bulk RNA-seq data from dissected neural folds, which include presumptive neural crest domains, also showed that the expression of multiple pluripotency factors was maintained until the end of neurulation, even as the expression of neural crest markers increased. Using single-cell RNA-seq data to observe transcriptional dynamics in the entire ectoderm, the authors confirmed that expression of the pan-ectoderm pluripotency module maintained co-expression of markers for all ectodermal domains throughout neurulation. suggesting a role in the ectodermal patterning process. At the end of neurulation the pan-ectodermal cells overlapped with the neural crest module, suggesting a mechanisms by which the neural crest obtains its exceptionally high stem cell potential. When the authors used whole-mount fluorescent in situ hybridization (FISH) to visualize the spatial distribution of pluripotency markers, they observed co-expression throughout the neural crest at all axial levels, suggesting the broad ectodermal pluripotency is required for development of all neural crest cells despite their future lineage later in the embryo.

Why I chose this preprint

In many developmental contexts, there is not yet a perfect technique for interrogating the developmental potential of populations of cells. Even when we make inferences about pluripotency from gene expression data, tradeoffs are made – sequencing experiments sacrifice spatial information for large numbers of genes, while imaging-based studies can examine the spatial distributions of a smaller set of genes. The scMST technique used in this preprint nicely bridges the gap between these two approaches and allows the authors to visualize how entire networks of transcription factors are distributed within the embryo. The results were striking, providing evidence of a pluripotency network that persists much longer in development than previously thought, proving once again that there is a lot to learn from simply looking at gene expression in the embryo.

Questions for the Authors

1. Is it possible to perform scMST in sections from blastula or early gastrula embryos? If so, would you expect to see major differences in the transcriptome of pluripotent blastula cells versus the cells at later stages that still express the pluripotency module? In other words, do you think you might get distinct clusters representing “Blastula pluripotency stem cells” and “Undecided neurula stage stem cells” if you compared across those stages?
2. Since many transcription factors get redeployed in different roles throughout development, there’s often overlap in the transcription factors that make up different regulatory networks. You alluded to the roles that Sox2 plays in both pluripotency and neural induction, and c-Myc is another example of a transcription factor with roles in both pluripotency and neural crest development. In cases like those where transcription factors don’t have just one clear cut module that they belong to, how were decisions made on which module to include them in?

References

[1]      C. Gans and R. G. Northcutt, “Neural Crest and the Origin of Vertebrates: A New Head,” Science (1979), vol. 220, no. 4594, pp. 268–273, Apr. 1983, doi: 10.1126/science.220.4594.268.

[2]      E. Buitrago-Delgado, K. Nordin, A. Rao, L. Geary, and C. LaBonne, “Shared regulatory programs suggest retention of blastula-stage potential in neural crest cells,” Science (1979), vol. 348, no. 6241, pp. 1332–1335, Jun. 2015, doi: 10.1126/science.aaa3655.

[3]      A. Zalc et al., “Reactivation of the pluripotency program precedes formation of the cranial neural crest,” Science (1979), vol. 371, no. 6529, Feb. 2021, doi: 10.1126/science.abb4776.

[4]      A. Lignell, L. Kerosuo, S. J. Streichan, L. Cai, and M. E. Bronner, “Identification of a neural crest stem cell niche by Spatial Genomic Analysis,” Nat Commun, vol. 8, no. 1, p. 1830, Nov. 2017, doi: 10.1038/s41467-017-01561-w.

 

Tags: microscopy, neural crest, pluripotency, single cell, smfish, waddington's landscape

doi: https://doi.org/10.1242/prelights.34411

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