Paganos P et al. (2021), Single-cell RNA sequencing of the Strongylocent...

ECB-ART-50672

Elife 2021 Nov 25;10. doi: 10.7554/eLife.70416.

Show Gene links Show Anatomy links

Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome.

Paganos P , Voronov D , Musser JM , Arendt D , Arnone MI .

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.

PubMed ID: 34821556
PMC ID: PMC8683087
Article link: Elife

Antibodies: LOC763123 Ab1 acetyl-LOC590586 Ab14 chat Ab1 th Ab2

Article Images: [+] show captions

	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 replicates. (C) Distribution of cells per cell type family. Color-code is the same as indicated on the UMAP in Figure 1B. (C) Table showing the percentage of WHL gene models expressed per cell type family compared to the total number of genes we identified (15,578). (E) Violin plot showing the number of genes (nFeature_RNA) and number of molecules (nCount_RNA) across the cell type families. (F) Dotplot of the total marker genes (69) expressed in the undefined cluster, that shows high similarity with ectodermally derived cell type families.
	Figure 1. 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 cell clusters. (C) UMAP with cells colored by germ layer of origin: endoderm (yellow), mesoderm (red), and ectoderm (blue). (D) Dotplot of gene markers specific to cell clusters. (E) Illustration depicting location of cell type families on different larval domains. Color-code is the same as in (B).
	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), SPU_016308 (J), Sp-Six1/2 (K), Sp-MacpfA2 (L), SPU_010640 (M), Sp-FoxJ2 (N), Sp-Chordin (O), and Sp-Tob (P). Nuclei are labeled with DAPI (in blue). All images are stacks of merged confocal Z sections.
	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 blue, mesodermally derived, in red, and endodermally derived, in yellow. (B) FISH using the antisense probe of Sp-Hbn (B1) paired with immunofluorescent detection of Msp130 (B2). Merged channels of both signals show localization of Sp-Hbn transcripts in Msp130 positive PMCs (B3). Nuclei are labeled with DAPI (in blue). All images are stacks of merged confocal Z sections. PMCs: Primary mesenchyme cells.
	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 developmental origins: ectodermally derived, in blue, mesodermally derived, in red and endodermally derived cell types, in yellow. (B) Proliferating cells as indicated by EdU labeling. (B1) Larva labeled with EdU and the skeletogenic marker Msp130. (B2) Selected confocal sections of larva in oral view labeled with EdU. (B3) Selected confocal sections showing the internal part of the same larva as in B2 in oral view labeled with EdU. (B4) Larva labeled with EdU and the endodermal marker 5c7. Abo, Aboral ectoderm; Ap, Apical plate; Cb, Ciliary band; Es, Esophagus; Oe, Oral ectoderm; St, Stomach.
	Figure 2. 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_1249 (A8), Sp-Hypp_2386 (A9), Sp-Mlckb (A10), Sp-MsxI (A11), SPU_008104 (A12), Sp-Ahrl (A13), Sp-Serp2; Sp-Serp3 (A14), Sp-Hnf4 (A15), Sp-Cyp2L42 (A16), Sp-Rfxc1l (A17), Sp-Pdx1 (A17) and Sp-Cdx (A18). Color-code indicates germ layer embryonic origin: endoderm (yellow), mesoderm (red), ectoderm (blue). Immunofluorescent detection of acetylated tubulin in ciliary band (green). (B) Dotplot of Sp-Sip1, Sp-SoxC, Sp-Hbn, and Sp-Fgf9/16/20 expression showing previously described and novel expression domains. (C) FISH of S. purpuratus three dpf larvae with antisense probes for Sp-SoxC (C1–C2), Sp-Hbn (C4–C5), and Sp-Fgf9/16/20 (C7–C8). Illustrations depicting all the expression domains of Sp-SoxC (C3), Sp-Hbn (C6), and Sp-Fgf9/16/20 (C9). Illustrations depicting all the expression domains of Sp-SoxC (C3), Sp-Hbn (C6), and Sp-Fgf9/16/20 (C9). Nuclei are labeled with DAPI (in blue). All images are stacks of merged confocal Z sections. (D) FISH of S. purpuratus three dpf larvae with antisense probes for Sp-SoxC and Sp-Fgf9/16/20 (D1–D4), for Sp-Hbn combined with immunohistochemical detection for the skeletal cells marker Msp130 (D5–D9) and Sp-Fgf9/16/20 with immunohistochemical detection for the midgut and hindgut protein Endo1 (D10–D13). A, Anus; Cs, Cardiac sphincter; I, Intestine; M, Mouth; PMCs, Primary mesenchyme cells; St, Stomach.
	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 3. 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 in blue, mesodermally derived in red, and endodermally derived in yellow. (B) Comparison of the transcription factor content across mesodermal lineages and cell type families. Venn diagram showing the shared and unique transcription factors per comparison. (C) Transcription factor content comparison of pyloric sphincter (endodermally derived) and skeletal cells (mesodermally derived), used as a negative control of our comparison. (D) Comparison of the transcription factor content per endodermal lineage and endodermally derived cell type families. Venn diagram showing the shared and unique transcription factors per comparison. (E) TF signature comparison of ectodermally derived cell type families. Venn diagram showing the shared and unique transcription factors per comparison. Cartoons indicated the relative position of each cell type family/lineage. Mesodermal cell type families/lineages are shown in shades of red, endodermal ones in shades of yellow and endodermal ones in shades of blue.
	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, mesodermally derived, in red and endodermally derived, in yellow.
	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 as depicted in Figure 3—figure supplement 1B.
	Figure 4. 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 ectodermally derived, red for mesodermally derived and yellow for endodermally derived ones.
	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 expression along the digestive tract of the 3 dpf sea urchin larva.
	Figure 5. 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 genes showing novel apical plate expression. (C) Pre-gastrula gene regulatory network enriched in skeletal cells of the sea urchin pluteus larva. Asterisks indicate larval genes involved in biomineralization, putative members of this GRN.
	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 gene markers are highlighted in purple (box), while non pigmented globular cells are evident in yellow (box).
	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, esophagus; M, mouth.
	Figure 6. 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 distinct neuronal subclusters. (B) Schematic representation of the three dpf pluteus larva showing the localization of neuronal subclusters (colors as in A). (C) Dotplot of signaling molecules, transcription factors, and neurotransmitters involved in sea urchin neuronal function and neurogenesis (colors as in A). (D) FISH of S. purpuratus three dpf larvae with antisense probes for the neuronal genes Sp-Delta (D1), Sp-SoxC (D2), Sp-Brn1/2/4 (D3), Sp-Ddc (D4), Sp-Ngn (D5), Sp-Prox1 (D6), Sp-Nacha6 (D7), Sp-Isl (D8), Sp-An (D8 and D14), Sp-Chrna9_4 (D9), Sp-Hbn (D10), Sp-SoxB2 (D11), Sp-Otx (D12), Sp-Tph (A13), Sp-Salmfap (D14), Sp-NeuroD1(D15), Sp-Six3 (D16), Sp-Trh (D17), Sp-Kp (D17), and Sp-Rhox3 (D18). FISH shown in figures D1-3 are paired with immunohistochemical detection of the neuropeptide Sp-An. Nuclei are labeled with DAPI (in blue). All images are stacks of merged confocal Z sections. A, anus; M, mouth.
	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.
	Figure 7. 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) Double immunohistochemical detection of the Sp-An and Synaptotagmin (1E11) proteins. (A3) Close up caption of the Sp-An PON neurons shown in A2. (A4) Double FISH of S. purpuratus three dpf larvae with specific antisense probes for Sp-FbsL_2 and Sp-An. Double immunohistochemical detection of the Sp-An and acetylated tubulin proteins. (A6) Close-up caption of the Sp-An PON neurons shown in A5. (A7) FISH of S. purpuratus three dpf larvae with a specific antisense probe for Sp-Chrna9_4 paired with immunodetection of Sp-An. (A8) FISH of S. purpuratus three dpf larvae with a specific antisense probe for Sp-Prox1 paired with immunohistochemical detection of Sp-An. (A9) Close-up caption of the Sp-An PON neurons shown in A8. (A10) Double immunohistochemical staining for the neuropeptide Sp-An and the skeletal cells marker Msp130. (A11) FISH of S. purpuratus three dpf larvae with a specific antisense probe for Sp-MacpfA2 paired with immunohistochemical detection of Sp-An. (A12) Double immunohistochemical staining for the neuropeptide Sp-An and the enzyme Sp-TH. (A13) Double FISH of S. purpuratus three dpf larvae with specific antisense probes for Sp-Nk1 and Sp-An. (A14) FISH of S. purpuratus three dpf larvae with a specific antisense probe for Sp-Otp paired with immunohistochemical detection of Sp-An. Double immunohistochemical staining for the neuropeptide Sp-An and the enzyme Sp-Chat. Nuclei are labeled with DAPI (in blue). All images are stacks of merged confocal Z sections. LGN, lateral ganglion neurons; M, Mouth; PON, Post-oral neurons. (B) Dotplot of genes important in endocrine pancreas differentiation and function in vertebrates. (C) Bar plot of selected Sp-Pdx1 target genes in the Sp-Pdx1/Sp-Brn1/2/4 positive population as revealed by differential RNA sequencing analysis of Sp-Pdx1 knockdown larvae. (D) Schematic representation of the Sp-Pdx1/Sp-Brn1/2/4 neurons genetic wiring as revealed by the combination of scRNA-seq and differential RNA-seq analysis after Sp-Pdx1 knockdown, highlighting an important role of Sp-Pdx1 in the differentiation of this cell type.

References [+] :

Adomako-Ankomah, Growth factors and early mesoderm morphogenesis: insights from the sea urchin embryo. 2014, Pubmed, Echinobase