An atlas of spider development at single-cell resolution provides new insights into arthropod embryogenesis
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Abstract
Spiders are a diverse order of chelicerates that diverged from other arthropods over 500 million years ago. Research on spider embryogenesis, particular studies using the common house spider Parasteatoda tepidariorum , has made important contributions to understanding the evolution of animal development, including axis formation, segmentation, and patterning. However, we lack knowledge about the cells that build spider embryos, their gene expression profiles and fate. Single-cell transcriptomic analyses have been revolutionary in describing these complex landscapes of cellular genetics in a range of animals. Therefore, we carried out single-cell RNA sequencing of P. tepidariorum embryos at stages 7, 8 and 9, which encompass the establishment and patterning of the body plan, and initial differentiation of many tissues and organs. We identified 20 cell clusters, from 18.5k cells, which were marked by many developmental toolkit genes, as well as a plethora of genes not previously investigated. There were differences in the cell cycle transcriptional signatures, suggestive of different proliferation dynamics, which related to distinctions between endodermal and some mesodermal clusters, compared with ectodermal clusters. We found many Hox genes were markers of cell clusters, and Hox gene ohnologs often were present in different clusters. This provided additional evidence of sub- and/or neo-functionalisation of these important developmental genes after the whole genome duplication in the arachnopulmonate ancestor (spiders, scorpions, and allies). We also examined the spatial expression of marker genes for each cluster to generate a comprehensive cell atlas of these embryonic stages. This revealed new insights into the cellular basis and genetic regulation of head patterning, hematopoiesis, limb development, gut development, and posterior segmentation. This atlas will serve as a platform for future analysis of spider cell specification and fate, and studying the evolution of these processes among animals at cellular resolution.
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In this study, the authors examine spider development using ACME dissociation and SPLiT-seq at three developmental stages associated with segmentation and regionalization. The authors cluster cells in their data to identify groupings of cell identity across timepoints. The authors examine the expression of AP genes such as Hox genes and DV genes as well as newly-identified markers from their scRNA-seq data using in situ hybridization and fluorescent in situ hybridization.
In generating and analyzing their data, the authors uncover expression of genes in the precheliceral region and in the posterior SAZ, which gives rise to the opisthosomal segments.
This study and the data it generates provide an exciting window into spider development and should greatly accelerate future investigations.
One thing that could be added to the manuscript …
In this study, the authors examine spider development using ACME dissociation and SPLiT-seq at three developmental stages associated with segmentation and regionalization. The authors cluster cells in their data to identify groupings of cell identity across timepoints. The authors examine the expression of AP genes such as Hox genes and DV genes as well as newly-identified markers from their scRNA-seq data using in situ hybridization and fluorescent in situ hybridization.
In generating and analyzing their data, the authors uncover expression of genes in the precheliceral region and in the posterior SAZ, which gives rise to the opisthosomal segments.
This study and the data it generates provide an exciting window into spider development and should greatly accelerate future investigations.
One thing that could be added to the manuscript to provide a greater understanding of its impact would be a more thorough engagement with and discussion of the current arthropod comparative developmental literature.
For example, it would be interesting to consider how the data presented for the SAZ corresponds to the sequential addition of segments during development of the flour beetle Tribolium castaneum, a system for which thorough investigations of this process have been conducted.
It would also be interesting to hear consider how the authors might decode the logic of Hox gene co-expression in the spider appendages based on their RNA-Seq expression data, or how the data from precheliceral patterning might provide some additional insights into the arthropod head problem.
Overall, this study provides a wealth of data for future developmental biology work and will be a valuable resource for other researchers.
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(ref)
missing reference
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g30822
What kind of protein is produced by g30822? It would be interesting to determine if this is a transcription factor, secreted factor, etc. and what orthologs it has in other arthropods.
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Expression of marker genes, colour bars represent theclusters they are associated with relative to figures B and C
Given the large number of embryo images in several of these figures, it might be helpful to provide larger versions of relevant groups of images as supplementary figures. As is, the images are quite small and features of the embryos can be difficult to distinguish. There also appears to be some debris (perhaps fibers?) in some of these images; it would be helpful to indicate those where present.
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Fig 2
It might just be a consequence of file compression or something by bioRXiv, but it's very hard to interpret some of the expression data presented due to their low resolution in this format.
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hunchback (hb)
Did the authors look for enrichment of other genes homologous to insect gap genes? It would be really interesting to see whether there is a differential AP distribution of those ohnologs and how their expression relates to Hox expression.
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A) Cell cycle scoring shows four clusters, 5, 12, 19 and 20, with more than 25% G1 phasecells (red)
It might be helpful to repeat the cluster number label beneath the bar chart, as well as below the bubble chart, for ease of reading.
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scoring
It appears that the data used for performing the analysis are included in the methods. It might be helpful to mark that this information is included by adding "(described in Methods, Cell Cycle Scoring)" or some other parenthetical to this sentence.
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Seurat rPCA, CCA and Harmony produced similar results, with only Harmonyappearing to not integrate stage 9.1 as strongly as rPCA/CCA (Sup Fig 1).
I'm curious how different one would expect the stage 9.1 cells to be from the other stages. Might it be the case that all cell types undergo some global changes in gene expression that result in the cells appearing more separated, and that Harmony better captures that separation?
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