Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome
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Evaluation Summary:
This paper will be of great interest to scientists within the fields of developmental biology and evolution, as well as to researchers that generally use the sea urchin as a model system or those employing single-cell mRNA-sequencing technology. The work provides a comprehensive analysis of the cell state specification of a whole deuterostome organism and proof of principle of the use of single-cell sequencing to identity deep homologies of cell type.
(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. Reviewer #1, #2, and #3 agreed to share their names with the authors.)
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Abstract
Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single-cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4 , we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.
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Evaluation Summary:
This paper will be of great interest to scientists within the fields of developmental biology and evolution, as well as to researchers that generally use the sea urchin as a model system or those employing single-cell mRNA-sequencing technology. The work provides a comprehensive analysis of the cell state specification of a whole deuterostome organism and proof of principle of the use of single-cell sequencing to identity deep homologies of cell type.
(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. Reviewer #1, #2, and #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
The paper by Paganos and collaborators is an interesting one. It deals with the characterization of cell types (using single cell sequencing) in the sea urchin larvae. The paper combines different sets of data, from the transcriptome data to detailed analysis of gene expression domains. The advantage, and the strength of the paper, and the biological system, the California purple urchin, is that the cell transcriptome can be consistently related to specifically located cells in the larvae. The well characterized genome, transcriptomes, gene expression domains and gene regulatory networks, allows a clear mapping of cells and activities to specific areas of the embryo. This possibility is only amenable in a very few systems and, among them, the larvae of Strongylocentrotus purpuratus.
Though it is a bit …
Reviewer #1 (Public Review):
The paper by Paganos and collaborators is an interesting one. It deals with the characterization of cell types (using single cell sequencing) in the sea urchin larvae. The paper combines different sets of data, from the transcriptome data to detailed analysis of gene expression domains. The advantage, and the strength of the paper, and the biological system, the California purple urchin, is that the cell transcriptome can be consistently related to specifically located cells in the larvae. The well characterized genome, transcriptomes, gene expression domains and gene regulatory networks, allows a clear mapping of cells and activities to specific areas of the embryo. This possibility is only amenable in a very few systems and, among them, the larvae of Strongylocentrotus purpuratus.
Though it is a bit peripheral to the main intention of the paper it is particularly interesting that the authors show that given the extensive knowledge of territorial and cell type markers known in S. purpuratus, the clusters and their expression domains are strictly correlated. In fact, a very nice validation of the technology that others (working with other animal systems) can take as an example.
On the down side (a very minor "down") is the lack of information for other developmental time points. This would have allowed the authors to follow the lineage of many of the cell types characterized, with a more thorough description of developmental trajectories.
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Reviewer #2 (Public Review):
Development of the sea urchin larva offers a particularly useful model to unravel the gene regulatory interactions that drive the developmental process, and has served in the past to unravel basic principles of how gene regulatory networks control the specification of cell types and how these networks evolve. Paganos et. al. take full advantage of the model system to generate a novel and highly valuable data set with important evolutionary implications. The authors employ single-cell mRNA-sequencing to thoroughly characterize the cell types of the sea urchin larva at a critical developmental stage. By exploiting the rich previous knowledge in the model system, Paganos et. al. carefully validate the transcriptomics data, and convincingly establish a high level of reliability of the data set. In this manner, …
Reviewer #2 (Public Review):
Development of the sea urchin larva offers a particularly useful model to unravel the gene regulatory interactions that drive the developmental process, and has served in the past to unravel basic principles of how gene regulatory networks control the specification of cell types and how these networks evolve. Paganos et. al. take full advantage of the model system to generate a novel and highly valuable data set with important evolutionary implications. The authors employ single-cell mRNA-sequencing to thoroughly characterize the cell types of the sea urchin larva at a critical developmental stage. By exploiting the rich previous knowledge in the model system, Paganos et. al. carefully validate the transcriptomics data, and convincingly establish a high level of reliability of the data set. In this manner, the study provides a complete and very valuable map of cell types and transcriptional profiles, and is able to: i) add potentially important regulators to well established gene regulatory networks that control the developmental process in the sea urchin; and ii) unravel previously undescribed neuronal and immune cell types. At the technical level, the authors convincingly establish a higher sensitivity of the single-cell mRNA-sequencing data in comparison to in situ hybridization experiments.
One of the major strengths of the paper is the detailed characterization of a previously described population of neurons, which shares a large number of transcription factors with vertebrate pancreatic cells. The transcriptional profile of this enigmatic neuronal population is likely to reflect an interesting evolutionary event with broad implications. Although the hypothesis of a pancreatic transcriptional profile in this population of neurons in the sea urchin was already proposed in a previous paper by the same group, the current study provides important novelty at two levels: i) it adds strong support to the hypothesis; and ii) it unravels the expression in these neurons of a set of very relevant genes, allowing the authors to propose new evolutionary scenarios as well as to contribute to a more general and currently very active debate about how cell types evolve.
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Reviewer #3 (Public Review):
This manuscript is very well written and with beautiful high quality images to represent the data. The SC-seq and cluster analysis is done well with six samples and four biological replicates and reasonable parameter choice, cuttoffs, and controls. The extensive GRN and previously published expression data in the sea urchin allow the authors to convincingly provide multiple examples of "proof of principle" that their single cell data analysis is working well. Their ability to identify 12 neuronal subclasses further demonstrates the quality of the data. The authors provide extensive whole mount insitu images to verify their SC clustering. The analysis reveals the cell type complexity of this simple deuterostome larvae, and provides a robust dataset for those working with the sea urchin model system and also …
Reviewer #3 (Public Review):
This manuscript is very well written and with beautiful high quality images to represent the data. The SC-seq and cluster analysis is done well with six samples and four biological replicates and reasonable parameter choice, cuttoffs, and controls. The extensive GRN and previously published expression data in the sea urchin allow the authors to convincingly provide multiple examples of "proof of principle" that their single cell data analysis is working well. Their ability to identify 12 neuronal subclasses further demonstrates the quality of the data. The authors provide extensive whole mount insitu images to verify their SC clustering. The analysis reveals the cell type complexity of this simple deuterostome larvae, and provides a robust dataset for those working with the sea urchin model system and also for those studying cell type evolution. The most significant new finding is in the last section where they identify the putative GRN of the neuroendocrine cell in the sea urchin and to show the extraordinary similarity in gene expression profiles between this cell and vertebrate pancreatic cell. This suggests an ancient origin on neuroendocrine cell types and is a great example of using this approach to understand cell type evolution.
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