Single-cell RNA analysis identifies pre-migratory neural crest cells expressing markers of differentiated derivatives
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Evaluation Summary:
This manuscript is of interest to researchers interested in specification and differentiation of the neural crest. This work presents a small single-cell RNAseq dataset from zebrafish trunk neural crest cells during the early stages of migration that identifies the subpopulations of trunk neural crest cells, new genetic markers and a subset of Rohon-Beard neurons. The paper confirms and extends previous work and reports expression of differentiated pigment cell types in the pre-migratory neural crest populations.
(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 neural crest is a migratory population of stem-like cells that contribute to multiple traits including the bones of the skull, peripheral nervous system, and pigment. How neural crest cells differentiate into diverse cell types is a fundamental question in the study of vertebrate biology. Here, we use single-cell RNA sequencing to characterize transcriptional changes associated with neural crest cell development in the zebrafish trunk during the early stages of migration. We show that neural crest cells are transcriptionally diverse and identify pre-migratory populations already expressing genes associated with differentiated derivatives, specifically in the xanthophore lineage. Further, we identify a population of Rohon–Beard neurons in the data. The data presented identify novel genetic markers for multiple trunk neural crest cell populations and Rohon–Beard neurons providing insight into previously uncharacterized genes critical for vertebrate development.
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Evaluation Summary:
This manuscript is of interest to researchers interested in specification and differentiation of the neural crest. This work presents a small single-cell RNAseq dataset from zebrafish trunk neural crest cells during the early stages of migration that identifies the subpopulations of trunk neural crest cells, new genetic markers and a subset of Rohon-Beard neurons. The paper confirms and extends previous work and reports expression of differentiated pigment cell types in the pre-migratory neural crest populations.
(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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Reviewer #1 (Public Review):
In this study, Lentzer and colleagues perform single-cell sequencing of migratory trunk neural crest population in zebrafish trunk during the early stages of migration. The authors provide a characterisation of gene expression in those cells, identify new genetic markers and identify a subset of Rohon-Beard (RB) neurons as a source of Fgf signalling but remain very descriptive in their analysis. However, the dataset is rather limited, and there is no in-depth validation. The study mostly expands the list of markers (albeit previously known in the nervous and hematopoietic system) and reports on differentiated derivative markers' presence in the pre-migratory NC populations (that were previously reported).
Major concerns are listed below:
The authors argue that RB cells share a developmental origin path with …
Reviewer #1 (Public Review):
In this study, Lentzer and colleagues perform single-cell sequencing of migratory trunk neural crest population in zebrafish trunk during the early stages of migration. The authors provide a characterisation of gene expression in those cells, identify new genetic markers and identify a subset of Rohon-Beard (RB) neurons as a source of Fgf signalling but remain very descriptive in their analysis. However, the dataset is rather limited, and there is no in-depth validation. The study mostly expands the list of markers (albeit previously known in the nervous and hematopoietic system) and reports on differentiated derivative markers' presence in the pre-migratory NC populations (that were previously reported).
Major concerns are listed below:
The authors argue that RB cells share a developmental origin path with NCC based on scRNA-seq data of 607 cells at a single time point (20-24hpf). Cell number is quite low if one is attempting to resolve transcriptional dynamics underlying cellular behaviour over time. This finding essentially validates the past work from the same lab that demonstrated the presence of HNK1+ (NV migratory marker) RB neurons in a well-characterised pdrm1 mutant (Hernandez-Lagunas et al., 2014). Such analysis performed at a single stage and at a relatively late time point cannot be used to infer common developmental origin or path. Furthermore, the authors seem to "brush" over the finding that they identify both sox10+ and sox10- RB neurons - both of which are lost in pdrm1 mutant - this finding is not addressed appropriately. This indicates the limitations of single-stage analysis with a relatively limited dataset.
There seems to be a disconnect between the title and what forms the bulk of the discussion and figures in this paper.
The authors claim to have identified new markers for these groups of neurons. However, they do not attempt to compare/integrate their dataset with other more extensive existing datasets (Wagner et al.,2018) to investigate whether markers (fgf13a, cxcr4b) are expressed at earlier time points to help support their hypothesis. Similarly, the authors do not seek to utilise single-cell datasets at later time points obtained using the same transgenic line to verify whether these markers are expressed (Aubrey et al., 2021). Further mining and integration with available datasets would help strengthen the authors' point.
The authors also postulate that some premigratory cells already express differentiated genes as a novelty but fail to cite multiple studies that have shown this in the neural crest in zebrafish and other models (Soldatov et al., 2019, Ling et al., 2019).
The "unknown" cluster 7 described by the authors as a potential new NCC lineage cluster is most likely (authors should verify this) a previously reported mesenchymal cluster expressing a wealth of collagen genes a{section sign}nd this should be verified and rectified.
The claim 'Some of 156 cells (Cluster 5) are presumably neural tube tissue' is unclear, as sox10 found in Cluster 5 does not label neural tube. It is unclear what are the Cluster 5 marker genes. Cluster 5 also seems to be split into two subclusters. It is unclear whether different genes mark these regions. It would have been helpful if the authors elaborated on the differential split of sox10-expressing and non-expressing cells within the cluster (feature plots in 1F indicate that sox10 is downregulated in the top left portion of this cluster and the RB cluster).
One of the primary novelty points emphasized by the authors is the subset of RB neurons. If this is to be confirmed, the authors would need to perform KO of some of the known marker genes specific to this cell population to show their relevance to RB cell development and determine what role these subsets of NCC-RB cells play.
Important technical points:
The study lacks sufficient information to verify the data quality and level of rigour in the analysis. For instance, information such as the number of embryos used to get 607 cells would need to be provided (this is important for defining genetic heterogeneity). It would also be important to indicate what proportion of embryos were 20hpf and 24hpf, how many cells were loaded into the channel, and what were the mean number of genes and UMI's that were found per cell. It is alosounclear how the analysis was performed, what were the parameters/cut-offs used and how many cells are found within each cluster. Furthermore, the quality of the data and figure annotations could be improved.
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Reviewer #2 (Public Review):
This work presents a small single-cell RNAseq dataset from 20-24 hpf zebrafish trunk neural crest cells (FACS of sox10::GFP from dissected trunk/tails). Within this data, the authors identify trunk neural crest cells (both pre-migratory and migratory), otic/lateral line cells (also labeled by sox10::GFP), posterior arches, and Rohon-beard neurons. A small selection of novel markers of xanthoblasts and Rohon-Beard neurons are identified, validated by in situ hybridization, and also shown to lose expression in prdm1a mutants (which lack trunk neural crest cells). Moreover, neural crest cells were classified as pre-migratory or migratory. Surprisingly, markers of distinct pigment cell fates were expressed prior to the onset of migration, but markers of neural fates were not observed prior to the onset of …
Reviewer #2 (Public Review):
This work presents a small single-cell RNAseq dataset from 20-24 hpf zebrafish trunk neural crest cells (FACS of sox10::GFP from dissected trunk/tails). Within this data, the authors identify trunk neural crest cells (both pre-migratory and migratory), otic/lateral line cells (also labeled by sox10::GFP), posterior arches, and Rohon-beard neurons. A small selection of novel markers of xanthoblasts and Rohon-Beard neurons are identified, validated by in situ hybridization, and also shown to lose expression in prdm1a mutants (which lack trunk neural crest cells). Moreover, neural crest cells were classified as pre-migratory or migratory. Surprisingly, markers of distinct pigment cell fates were expressed prior to the onset of migration, but markers of neural fates were not observed prior to the onset of migration, indicating that some populations of NCCs express differentiation markers prior to migration (but that this is not universal to all neural crest derivatives). Additionally, this work highlights that a small percentage of Rohon-Beard neurons are marked by the sox10 transgene, suggesting that they have expressed sox10 at some point in their developmental history.
This is a compelling study and a pleasure to read. The data are of good quality (a sensible isolation protocol, quality metrics seem reasonable) and the processing steps used are well established standards in the field. The data annotation is well presented and convincing, and the novel xanthophore and RB markers validation is superb. The finding that some NCCs initiate differentiation prior to migration seems to provide additional evidence toward a longstanding debate in the neural crest field.
The major weaknesses are that, while a subset of new markers are presented and extensively validated, there is not a broader presentation of some of the novel gene expression results contained in their data that might be more broadly useful to the field.
Additionally, the study leaves me wondering: if cell labeling experiments in zebrafish have suggested that NCCs are lineage restricted prior to migration, why are NCCs expressing cell-type specific markers only observed prior to migration for pigment cells and not for other neural crest derivative cell types?
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