Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome
- Stazione Zoologica Anton Dohrn, Department of Biology and Evolution of Marine Organisms, Italy
- European Molecular Biology Laboratory, Developmental Biology Unit, Germany
Figures
Figure 1 with 1 supplement
Cell type family atlas of the three dpf S. purpuratus larva.
(A) Single-cell RNA sequencing pipeline from gamete fertilization to computational analysis. (B) UMAP showing three dpf larval cells colored by their assignment to the initial set of 21 distinct …
Figure 1—figure supplement 1
Overlap of the different replicates and characterization of the undefined cluster.
A) UMAPs split by original identity showing the overlap of the different biological and technical replicates. (B) Overlay UMAP showing the overlap of the different biological and technical …
Figure 2 with 4 supplements
Validation of scRNA-seq predictions and novel expression domains.
(A) FISH of S. purpuratus three dpf larvae with antisense probes for Sp-FbsL_2 (A1), Sp-Chrna9_4 (A2), Sp-Frizz5/8 (A3), Sp-FoxABL (A4), Sp-Bra (A5), SPU_006199 (A6), Sp-FcolI/II/III (A7), Sp-Hypp_12…
Figure 2—figure supplement 1
Expression pattern of genes used to annotate the clusters.
FISH of S. purpuratus larvae with specific probes detecting the mRNAs for Sp-Alpi (A), Sp-IrxA (B), SPU_002797 (C), SPU_021898 (D), Sp-ManrC1A (E), Sp-Nkx6.1 (F), Sp-FoxP (G), Sp-Emx (H), Sp-Pks1 (I)…
Figure 2—figure supplement 2
ScRNA-seq predicted expression patterns of genes used to annotate the clusters.
A) Dotplot showing the average expression of genes used as markers to identify the cell type families. Cell type families are grouped based on their developmental origins: ectodermally derived, in …
Figure 2—figure supplement 3
ScRNA-seq is able to detect expression patterns, previously undetectable by in situ hybridization.
Dotplot of genes known for their involvement in germline determination and maintenance showing overall enrichment of their average expression in the coelomic pouches cell type family.
Figure 2—figure supplement 4
Proliferation status and dynamics of the larval cell type families.
A) Dotplot showing the average expression of genes encoding cdk1, pcna, DNA polymerases, DNA ligases, condensins, and centromere proteins. The cell type families are grouped based on their …
Figure 3 with 1 supplement
Regulatory states of the three dpf S. purpuratus larva.
(A) Comparison of the transcription factor content per germ layer. Venn diagram showing the shared and unique transcription factors per germ layer. Ectodermally derived cell type families are shown …
Figure 3—figure supplement 1
Cell type family trees of the three dpf pluteus larva.
Cell type family trees reconstructed using all expressed genes (A) and only transcription factors (B).
Figure 4 with 2 supplements
Localization of major transcription factor family members.
Dotplot showing the average scaled expression of members of the Homeobox, Forkhead and Ets transcription factor families. The developmental origins of each cell type family are shown in blue for …
Figure 4—figure supplement 1
Distribution of the Zinc finger TF family members across the cell type families.
Dotplot showing the mRNA average expression of the zinc finger transcription family members. The cell type families are grouped based on their developmental origins: ectodermally derived, in blue, …
Figure 4—figure supplement 2
Differentially expressed TFs across cell type families.
Dotplot showing the average scaled expression of differentially expressed TFs with p-value less than 0.5 among the 21 clusters. The cluster order is according to the transcription factor-based tree …
Figure 5 with 1 supplement
Validation of preexisting GRNs and putative novel function of specific gene regulatory modules.
(A) Dotplot showing the mRNA localization of genes involved in the homing of small micromeres to the coelomic pouch and novel apical plate domain. (B) Dotplot of aboral ectoderm regulatory module …
Figure 5—figure supplement 1
Reconstruction of the molecular signature of the gut at a single-cell resolution.
Dotplot showing the regionalized average expression of marker genes labeling specific endodermal domains. The scRNA-seq prediction recapitulates the previously described compartmentalization of gene …
Figure 6 with 3 supplements
Neuronal complexity of the three dpf S. purpuratus larva.
(A) (From left to right and top to bottom) UMAP highlighting the neurons cluster (green), immunohistochemical detection for the paneuronal sea urchin marker 1E11 (green), UMAP showing the 12 …
Figure 6—figure supplement 1
Subclustering of the skeletal cell type family.
Dotplot showing the mRNA average expression of 22 skeletal gene markers and their distribution among five skeletal subtypes.
Figure 6—figure supplement 2
Subclustering of the immune cells cluster.
(A) UMAP showing eight distinct immune cells populations as revealed by our subclustering analysis. (B) Dotplot showing gene markers expressed in the different immune cell populations. Pigment cells …
Figure 6—figure supplement 3
Co-localization of Sp-Fgf9/16/20 and Sp-FgfR1 in the cardiac sphincter region.
FISH using antisense probes for Sp-Fgf9/16/20 (magenta) and Sp-FgfR1 (green). Nuclei are labeled with DAPI (in blue). All images are stacks of merged confocal Z sections. Cs, cardiac sphincter; Es, …
Figure 7 with 1 supplement
ScRNA-seq reveals a Pdx-1-dependent neuroendocrine cell type.
(A) Molecular characterization of a Sp-Pdx1/Sp-Brn1/2/4 double positive neuronal population. (A1) Double FISH of S. purpuratus 3 dpf larvae with specific antisense probes for Sp-Pdx1 and Sp-An. (A2) …
Figure 7—figure supplement 1
Pancreatic genes expression significance testing in the ‘post oral and lateral neurons’ subcluster.
Barplots showing the number of genes expressed in the actual data and the randomized data.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70416/elife-70416-transrepform1-v2.docx
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Supplementary file 1
Differentially expressed genes per cell type family and putative target genes in the PDX1 positive neurons.
- https://cdn.elifesciences.org/articles/70416/elife-70416-supp1-v2.xlsx
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Supplementary file 2
Primers used to generate specific antisense RNA probes.
- https://cdn.elifesciences.org/articles/70416/elife-70416-supp2-v2.xlsx
