Single-cell atlas of early chick development reveals gradual segregation of neural crest lineage from the neural plate border during neurulation

  1. Ruth M Williams
  2. Martyna Lukoseviciute
  3. Tatjana Sauka-Spengler
  4. Marianne E Bronner

  • Curated by eLife

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    Public Evaluation Summary:

    Determining when cells acquire their individual identity is critical for understanding the patterning and growth of embryos. The authors use state-of-the-art methods to map the lineages of cells that emerge during the earliest stages of development in chick embryos and that contribute to the central and peripheral nervous system. The authors have characterised the gene signature of ectoderm sub-clusters and used algorithms to infer lineage trajectories using the dataset. However, the analysis of placode and neural crest emergence is not clear-cut or well supported by in vivo experiments in the embryo. The work overall will be of broad interest to developmental and stem cell biologists, as well as neurobiologists interested in the understanding of the neural and neural crest gene regulatory networks.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)

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Abstract

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harboring neural crest and cranial placode progenitors. Here, we a generate single-cell atlas of the developing chick epiblast from late gastrulation through early neurulation stages to define transcriptional changes in the emerging ‘neural plate border’ as well as other regions of the epiblast. Focusing on the border territory, the results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridization. Developmental trajectory analysis infers that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined neural crest and placode lineages. The data also can be mined to reveal changes throughout the developing epiblast.

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  1. eLife

    Author Response

    Reviewer #1 (Public Review):

    A discussion of how the current results fit into the larger framework of GRNs (both for known and novel genes) would provide a more complete context for the work. A schematic figure that maps these genes onto a GRN could be quite informative and clarifying.

    We thank the reviewer for this insightful comment. To address this, we have added discussion of how some of the genes identified here fit into the broader neural crest gene regulatory network using previously published data (Williams et al., 2019) (lines 384-386 and 392-394). While the idea of putting some of the novel genes described here into a GRN is very attractive, it is premature in the absence of functional data since it is not possible to construct a GRN on expression data alone; this would require epistatic/regulatory data which is outside the scope of the current work.

  2. eLife

    Public Evaluation Summary:

    Determining when cells acquire their individual identity is critical for understanding the patterning and growth of embryos. The authors use state-of-the-art methods to map the lineages of cells that emerge during the earliest stages of development in chick embryos and that contribute to the central and peripheral nervous system. The authors have characterised the gene signature of ectoderm sub-clusters and used algorithms to infer lineage trajectories using the dataset. However, the analysis of placode and neural crest emergence is not clear-cut or well supported by in vivo experiments in the embryo. The work overall will be of broad interest to developmental and stem cell biologists, as well as neurobiologists interested in the understanding of the neural and neural crest gene regulatory networks.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript.The reviewers remained anonymous to the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    The work by Williams et al. represents a significant effort to understand the lineages that emerge from the epiblast, which is the layer of cells that will give rise to the chick embryo, during the period from gastrulation through neurulation. Previously published work has addressed the question of when and where cells in the epiblast at the neural plate border become specified, but the authors strive to refine such data further and more precisely by using single-cell RNA-sequencing to characterize the signatures of these lineages in vivo. The authors focus on identifying cells that generate the placodes, neural crest, and neural tissues. Studying these populations of cells is well justified because of their relevance to human disease phenotypes particularly in the craniofacial complex, heart, gut, and malignancies more broadly. Another important contribution of the work is the descriptions of novel markers, which may ultimately result in identifying new genes associated with human disease.

    A strength is that the manuscript is concise, clear, and well-written. Additionally, the figures are nicely organized and easy to follow. The images of embryonic gene expression (both known markers and validation of novel genes) are excellent in terms of tissue quality and confocal microscopy, and they provide robust support for the conclusions. The strategies employed for single-cell RNA-seq analysis represent a technical advance, and the results generated by this study will be of broad interest to the community. The use Hybridization Chain Reaction (HCR) with multiplexed probes to validate the RNA-seq dataset adds significant depth to the analysis.

    A weakness is that the study is primarily descriptive with no experimental test of a hypothesis. For example, the work could benefit from some loss-of-function analyses for any of the novel genes identified. On this point, the authors have an excellent track record using high-throughput screening with chick embryos and morpholinos either in terms of characterizing gene regulatory networks (GRNs) or assaying for developmental phenotypes related to the neural crest. Moreover, work from the Bronner and Sauka-Spengler labs has previously contributed much in the way of our understanding of the GRNs that direct the delineation of the neural and non-neural lineages in the ectoderm. A discussion of how the current results fit into the larger framework of GRNs (both for known and novel genes) would provide a more complete context for the work. A schematic figure that maps these genes onto a GRN could be quite informative and clarifying.

    Nonetheless, the results are significant in that they advance our understanding of the spatial and temporal expression of genes that are associated with cell specification and lineage restriction during the embryonic time course from gastrulation to neurulation. The conclusions are further supported by a modified RNA velocity analysis that reconstructs the temporal sequence of transcriptional steps and resolves gene-specific transcriptional dynamics over time. Importantly, based on this analysis, the authors find that cells from the emerging neural plate border are more heterogeneous than previously believed and they do not see the emergence of neural crest cells as a distinct lineage until later than what has been suggested by other studies.

  4. eLife

    Reviewer #2 (Public Review):

    This study performs single-cell transcriptome analysis of chicken embryos from gastrulation to neurulation stages (HH4-HH7) with the goal of understanding the emergence and transition of the neural plate border domain to placodal and neural crest lineages. The main impact of the study is the first detailed single-cell analysis of early chick embryos, which should complement similar studies in human, mouse, and other vertebrates. Major cell types are described at each stage, and this is coupled with in situ hybridization for a set of known and novel genes, as well as RNA velocity to deduce cell trajectories (based on the ratio of old cytoplasmic to new nuclear transcripts). Based on the absence of a distinct Pax7/Tfap2a cluster during gastrulation stages, the authors make an argument that the neural plate border and placode/neural crest does not emerge until later stages (i.e. neurulation), later than previously reported. However, the study largely falls short of making significant new insights into the timing and lineage trajectories of placodes and neural crest. The manuscript is very dense with descriptive data of gene expression in numerous clusters, which makes it difficult to extract big picture messages about lineage emergence. There are also numerous typos and figure errors that make the reading difficult. The RNA velocity analysis is not particularly convincing as to lineage emergence, and there are numerous overstatements about the ability of such single-cell genomics approaches to define in vivo lineages in the absence of experimental confirmation. While some new genes with neural plate border expression are presented, others are already well known or are shown to have very broad expression, thus limiting their utility in defining domains. There is a feeling that this work is quite preliminary in terms of its analysis of neural plate border lineages, though it is clearly an important resource for the chick embryology community.

  5. eLife

    Reviewer #3 (Public Review):

    In this study, Williams and colleagues use single cell transcriptomics to describe the cell fates and lineages in the dorsal ectoderm of chick embryos taken from gastrula to early neurula stage. Specifically, they provide an atlas of whole embryo epiblast at stages 4,HH, 5HH, 6HH and 7H. They further subcluster the ectoderm cells and use cVelo algorithm (based on velocity of RNA splicing) to infer trajectories and lineages in this dataset.

    Description of each stage and each cluster of cells is carefully done and retrieves the canonical markers for each territory. The neural plate border is found expressing pax7 early on, as previously described in chick embryos (Basch et al., 2006) but a specific clustering of these cells is not found, suggesting that the identity of the neural plate border is rather the overlap of gene signatures from adjacent tissues and that the neural crest and the placode signatures arise at the early neurula stage, in the elevating neural folds of the embryo.

    From previous lineage tracing studies done at the gastrula stage, it was shown that the border between the prospective neural plate and the future non-neural ectoderm gives rise to four main cell fates: dorsal neural tube cells, neural crest cells, posterior placodal cells and non-neural ectoderm cells (e.g. Steventon et al., 2009). Genetically, this territory has previously been defined by the overlap between gene expressions defining the non-neural ectoderm and the neural ectoderm (e.g. De Crozé et al., 2011, Grooves and Labonne 2014)

    Previous transcriptomic studies have pointed out the high similarity of the neural plate border territory compared to adjacent ectoderm regions, the failure of "classical" biostatistics tools to evidence a specific signature and the need for tailored strategies to do so (Plouhinec et al., 2017). Using those, the neural plate border could be defined from gastrula stage by its expression of pax3/pax7 ortholog and a couple of other genes, then by a more extended signature at neurulation stage.

    In conclusion, although this study explores the same question with the latest tools of single cell transcriptomics, it is mostly descriptive and brings little novel insight into the biology of neural and neural plate border induction. However, it highlights a series of additional genes that could be of interest for further functional study.

  6. Version published to 10.7554/elife.74464 on eLife
  7. Version published to 10.1101/2021.08.08.455573v1 on bioRxiv