New hypotheses of cell type diversity and novelty from orthology-driven comparative single cell and nuclei transcriptomics in echinoderms
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eLife assessment
This study is an excellent example of how one can use high-throughput technologies to address animal evolution from a cell-type perspective. The authors present a beautiful analysis of single-nucleus RNA-seq data from three embryonic stages in a sea star; this is the first single-cell data from this large group of echinoderms and the first single-nucleus transcriptomic study in any echinoderm. The authors demonstrate the presence of unique as well as previously unnoticed homologous cell types between a sea star and a sea urchin, discovery very intriguing to echinoderm developmental biologists, which will also be of great interest to scientists in the broad fields of evolution and development.
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Abstract
Cell types are the building blocks of metazoan biodiversity and offer a powerful perspective for inferring evolutionary phenomena. With the development of single-cell transcriptomic techniques, new definitions of cell types are emerging. This allows a conceptual reassessment of traditional definitions of novel cell types and their evolution. Research in echinoderms, particularly sea star and sea urchin embryos has contributed significantly to understanding the evolution of novel cell types, through the examination of skeletogenic mesenchyme and pigment cells, which are found in sea urchin larvae, but not sea star larvae. This paper outlines the development of a gene expression atlas for the bat sea star, Patiria miniata , using single nuclear RNA sequencing (snRNA-seq) of embryonic stages. The atlas revealed 23 cell clusters covering all expected cell types from the endoderm, mesoderm, and ectoderm germ layers. In particular, four distinct neural clusters, an immune-like cluster, and distinct right and left coelom clusters were revealed as distinct cell states. A comparison with Strongylocentrotus purpuratus embryo single-cell transcriptomes was performed using 1:1 orthologs to anchor and then compare gene expression patterns. The equivalent of S. purpuratus piwil3+ Cells were not detected in P. miniata , while the Left Coelom of P. miniata has no equivalent cell cluster in S. purpuratus . These differences may reflect changes in developmental timing between these species. While considered novel morphologically, the Pigment Cells of S. purpuratus map to clusters containing Immune-like Mesenchyme and Neural cells of P. miniata , while the Skeletogenic Mesenchyme of S. purpuratus are revealed as orthologous to the Right Coelom cluster of P. miniata . These results suggest a new interpretation of the evolution of these well-studied cell types and a reflection on the definition of novel cell types.
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eLife assessment
This study is an excellent example of how one can use high-throughput technologies to address animal evolution from a cell-type perspective. The authors present a beautiful analysis of single-nucleus RNA-seq data from three embryonic stages in a sea star; this is the first single-cell data from this large group of echinoderms and the first single-nucleus transcriptomic study in any echinoderm. The authors demonstrate the presence of unique as well as previously unnoticed homologous cell types between a sea star and a sea urchin, discovery very intriguing to echinoderm developmental biologists, which will also be of great interest to scientists in the broad fields of evolution and development.
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Reviewer #1 (Public Review):
The study by Meyer and collaborators is tackling the question of cell type evolution between sea urchins and sea stars. To address this question, they generated single nuclei RNA sequencing libraries originating from early developmental time points of the sea star Patiria miniata. The resulting cell type atlas recapitulated the cell types previously known to exist as indicated by traditional methods in the past and revealed hidden cell type complexity. The authors provide evidence for the existence of previously not described sea star neuronal types and provide a thorough characterization of their molecular signature. Once validating the sea star cell type atlas through means of WMISH they computationally compared the sea star cell types to the sea urchin ones by taking advantage of already available …
Reviewer #1 (Public Review):
The study by Meyer and collaborators is tackling the question of cell type evolution between sea urchins and sea stars. To address this question, they generated single nuclei RNA sequencing libraries originating from early developmental time points of the sea star Patiria miniata. The resulting cell type atlas recapitulated the cell types previously known to exist as indicated by traditional methods in the past and revealed hidden cell type complexity. The authors provide evidence for the existence of previously not described sea star neuronal types and provide a thorough characterization of their molecular signature. Once validating the sea star cell type atlas through means of WMISH they computationally compared the sea star cell types to the sea urchin ones by taking advantage of already available single-cell RNA sequencing data, carried out at equivalent stages of Strongylocentrotus purpuratus development. Using 1-1 orthologs they integrated the sea star and sea urchin datasets and provided evidence for the presence of novel cell types that are not shared between the two animals (at least novel for the specific developmental window analyzed) such as the left coelomic pouch in sea urchin. Moreover, their analysis suggests that sea urchin skeletal cells, a population known to not exist in sea stars, correlate transcriptionally to other mesodermal cell types of the sea star, while sea urchin pigment cells appear to be very similar to sea star immune cells and neurons. Overall, the data of this study demonstrate how single-cell RNA sequencing can be used as a tool to study cell type evolution and provide complete molecular evidence of cell type diversification between the two echinoderm species. Lastly, their P. miniata cell type atlas will be of great importance for the evo-devo field and contribute to a better understanding of the development and evolution of novelties.
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Reviewer #2 (Public Review):
A comparison of sea stars and sea urchins has been shown in the past to be a very fertile ground to understand the evolution of cell types. Among other reasons, this is due to the rich amount of information on the gene regulatory networks that control the establishment of cell types in the sea urchin embryo, the experimental amenability of both the sea urchin and sea star embryos, and the fact that embryos of these two animal groups show homologous cell types as well as morphological innovations. The study by Meyer et. al. takes full advantage of these features and takes the comparison of the sea urchin and the sea star to a new technological level by implementing single-cell technologies in the sea star embryo for the first time. The authors employ a single-nuclei RNA-sequencing protocol to profile the …
Reviewer #2 (Public Review):
A comparison of sea stars and sea urchins has been shown in the past to be a very fertile ground to understand the evolution of cell types. Among other reasons, this is due to the rich amount of information on the gene regulatory networks that control the establishment of cell types in the sea urchin embryo, the experimental amenability of both the sea urchin and sea star embryos, and the fact that embryos of these two animal groups show homologous cell types as well as morphological innovations. The study by Meyer et. al. takes full advantage of these features and takes the comparison of the sea urchin and the sea star to a new technological level by implementing single-cell technologies in the sea star embryo for the first time. The authors employ a single-nuclei RNA-sequencing protocol to profile the transcriptomes of all cell types in the sea star embryo at three stages of development and very convincingly show that the generated dataset is able to capture known cell types as well as previously undescribed cell types. In this context, the study significantly advances the molecular characterization of the previously known cell types and draws convincing conclusions about the biological significance of the newly discovered cell types. By using the newly generated sea star dataset, and a previously published sea urchin single-cell RNA-sequencing dataset at equivalent developmental stages, Meyer et. al. compare cell types between the two animals. Three important claims arise from this comparison: 1. The unanticipated discovery of a cell cluster in each species that has no counterpart in the clusters of the other species. 2. That the primary mesenchyme cells (PMCs) of the sea urchin, thought to be a novel cell type in the sea urchin, share significant transcriptomic profiles with the cells of the right coelom of the sea star; 3. That pigment cells of the sea urchin also thought to be a novelty in the sea urchin, shares transcriptomic signatures with immune and neural cells of the sea star.
The strength of the study by Meyer et. al. is the robustness of the newly generated sea star single-nuclei RNA-sequencing dataset, as well as the rigorous validation and biologically meaningful interpretation of the data. As a result, the conclusions of Meyer et. al. concerning the description of sea star cell types are convincing, robust, and biologically important. A potential weakness of the study is the method used for integrating this data with that of the sea urchin. The integration method employed is based on generating a list of genes with 1:1 orthology between the two species and then computing a common cell type atlas by using only the genes with 1:1 orthology. Given the relatively large evolutionary distance between sea urchins and sea stars, and the growing evidence suggesting that paralogs may be more functionally similar than orthologs across species, the method employed for integrating the two datasets might limit the depth and robustness of the comparison.
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Reviewer #3 (Public Review):
Overall, the data quality and analyses are solid. The authors have extracted a lot of detailed information about gene expression in specific cell types of the sea star embryo, and this descriptive narrative forms much of the Results section. However, the most interesting analyses will be the between-species comparisons. The authors identify several striking differences in the apparent presence or absence of specific cell types between seastar and sea urchins. Some confirm well-known differences, such as the absence of pigmented and skeletogenic mesenchyme cells in seastar embryos based on morphological comparisons. Other findings are novel, such as transcriptionally distinct left and right coelomic pouches as early as late gastrula and the apparent absence of germ cells in seastar embryos. These findings are …
Reviewer #3 (Public Review):
Overall, the data quality and analyses are solid. The authors have extracted a lot of detailed information about gene expression in specific cell types of the sea star embryo, and this descriptive narrative forms much of the Results section. However, the most interesting analyses will be the between-species comparisons. The authors identify several striking differences in the apparent presence or absence of specific cell types between seastar and sea urchins. Some confirm well-known differences, such as the absence of pigmented and skeletogenic mesenchyme cells in seastar embryos based on morphological comparisons. Other findings are novel, such as transcriptionally distinct left and right coelomic pouches as early as late gastrula and the apparent absence of germ cells in seastar embryos. These findings are based on solid evidence, highly informative regarding molecular details, and will no doubt inspire many future studies, both into developmental mechanisms per se and into the evolution of development. While the descriptive part of this study is solid and highly informative, the evolutionary interpretations are more problematic. The Abstract and Introduction emphasize the promise of sc/snRNAseq to shed light on the evolution of cell types and novelty, but the data themselves tell a less clear-cut story. Indeed, for me, the biggest takeaway from reading this manuscript is that it is quite difficult to identify when a novel cell type has evolved based solely on analysis of embryonic stages. The last stage examined is late gastrula, which means that some cell types may appear to be missing simply because they have not yet begun to differentiate transcriptionally. An example would be germ cells since adults make gametes. Another limitation is that just two species are compared. This means that for any given difference in cell type composition, it is not possible to distinguish whether this represents a novel cell type in one species or the loss (or delay in differentiation) of a cell type in the other species. The authors are generally careful to identify these limitations when presenting results, but it does lead me to wonder why they did not choose to examine later stages of development when more cells are clearly differentiated.
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