Overview of single cell RNA sequencing of regenerating intestinal tissue of H. glaberrima.

(A) Diagram of 9-day regenerating intestine depicting mesentery and anlage components (B) UMAP plot of population identities determined through unsupervised clustering of 9-day regenerating mesentery and anlage tissues. (C) Representation of cells per tissue of both samples. (C) UMAP projections of cluster cells separated by tissue of origin. (D) Percentage of cells per cluster based on their tissue of origin.

UMAP visualization of clusters highlighting the expression of their top genes.

Each gene corresponds to the top gene of each independent cluster based on the percentage of representation to other clusters.

Cluster characterization by gene expression.

(A) Top-expressed genes or (B) genes corresponding to transcription factors and intercellular signaling molecules are identified in the 13 cell clusters. Clusters are classified by their corresponding cell type, where blue corresponds to cells of the coelomic epithelium, red to those in the mesenchyme and green to coelomocytes. Color intensity shows the expression level of each gene in log2fold-change (log2FC) values. Dot size corresponds to percentage of representation of the gene in the respective cluster compared to all others. Gene identifiers starting with “g” correspond to uncharacterized gene models of H. glaberrima. (C) UMAP plot of clusters colored by cell type.

Expression profile of coelomocyte cell types.

(A) Dot plot showing the top expressed genes of C6, C10, C11, and C12. Color intensity reflects the expression levels in log2FC values. Dot size correspond to the percentage of representation of each gene in the respective cluster compared to all others. (B) Top enriched GO biological processes terms of C6, C10, C11, and C12. Dot size corresponds to the enrichment score while the color reflects the adjusted p-value. (C) UMAP highlighting the coelomocyte populations. (D-E) HCR-FISH for FBCD1 in holothurian intestinal tissues. Cells expressing FBCD1 mRNA in the connective tissue layer of (D) regenerating mesentery and (E) normal intestine of H. glaberrima are shown. Red = DAPI stain, green = HCR-FISH. Bar= 10 μm.

Expression profile of mesenchymal cell types.

(A) Dot plot showing the top expressed genes of C2 and C7. Color intensity reflects expression levels in log2FC values. Dot size correspond to the percentage of representation of each gene in the respective cluster compared to all others. (B) Top GO enriched terms of biological processes of C2 and C7. Dot size corresponds to the enrichment score while the color reflects the adjusted p-value. (C) UMAP highlighting the clusters that correspond to the mesenchymal population (D-F) Cells expressing HMCNT1 mRNA in the connective tissue layer of (D) regenerating mesentery, (E) intestinal anlage and (F) normal intestine of H. glaberrima. Red = DAPI stain, green = HCR-FISH. Bar= 10μm

Expression profile of cell clusters undergoing differentiation or proliferation in the coelomic epithelium.

(A) Dot plot showing the expression profile of C5, C8, and C9. Color intensity reflects expression levels in log2FC values. Dot size represents percentage of representation of each gene. (B) Top GO enriched terms of biological processes of each cluster. (C) UMAP highlighting C5, C8, and C9, which along with clusters in color blue correspond to the coelomic epithelium. (D-F) Cells expressing MYH7 mRNA in the (D) regenerating mesentery, (E) basal area of the anlage coelomic epithelium and (F) muscle layer of the normal intestine. (G) Phalloidin labeled differentiating muscle cells in basal area of the coelomic epithelium. (H) Cell labeled with RN1 antibody (neuronal marker) in coelomic epithelia of the anlage. Red = DAPI stain, green D-F= HCR-FISH. G= Fluorescent Phalloidin, H= RN1 antibody. Bar-D-F= 25μm, G-H=10μm.

Expression profile of coelomic cell populations in the regenerating intestine anlage and mesentery.

(A) Dot plot showing the top expressed genes of C0, C1, C3 and C4. Color intensity reflects expression levels in log2FC values. Dot size correspond to the percentage of representation of each gene in the respective cluster compared to all others. (B) Top GO enriched terms of biological processes of each cluster. Dot size corresponds to the enrichment score while the color reflects the adjusted p-value. (C) UMAP highlighting the clusters of the coelomic epithelial cells of the regenerating intestine. (D-E) HCR-FISH for SC6A5 mRNA was performed as a marker for cells of C4. Labeling is observed in the (D) regenerating mesentery, (E) the coelomic epithelium of the anlage and (F) coelomic epithelium of the normal intestine. Red = DAPI stain, green = HCR-FISH. Bar= 25μm.

HCR-FISH for TRFM and NET1 shows the differential gene expression of the anlage versus mesentery coelomic epithelia.

TRFM is mainly expressed in the mesentery coelomic epithelial cells that are distant from the regenerating anlage while NET1 is mainly expressed in the coelomic epithelial cells of the anlage TRFM mRNA expression (A-E) shows a gradient from (A) very strong expression in the coelomic epithelium at the attachment of the mesentery to the body wall, (B) high expression in the coelomic epithelia of the mesentery close to the body wall, (C) weaker expression in the mesentery close to the anlage to (D) weak or lack of expression in the coelomic epithelium of the anlage. (E) TRFM mRNA is expressed by the coelomic epithelium of the normal intestine. regenerating intestine. In contrast, few cells express (A) NET1 mRNA in the regenerating mesentery, while high expression is found in (B) the coelomic epithelium of the anlage (C) Little expression of NET1 is present in the coelomic epithelium of the normal intestine. Red = DAPI stain, green = HCR-FISH. Bar= 25μm.

Trajectory analysis of cell populations from the regenerating intestinal tissue.

(A) UMAP plot of all identified clusters. (B) RNA velocity embedded in UMAP of all main clusters. (C) UMAP of re-clustering of cells from C0, C1, C3, C4, C5, and C9. (D) RNA velocity analysis results from the subset from panel (C). (E) Jitter plot of Slingshot pseudotime of cells from C0, C1, C3, C4, C5 and C9. Pseudotime resulted in two lineages one containing C5 and the other C 9. (F) UMAP of re-clustered anlage cells corresponding to C0, C1, C3, and C4(G) RNA velocity results from the cluster results from panel F. (H) UMAP of panel F plot overlayed with pseudotime results of Slingshot. Color represent pseudotime values from 0 (blue) to 60 (yellow). (I) Jitter plot showing the Slingshot pseudotiume of cells from each cell cluster.

Model of cellular organization within the anlage of the regenerating intestine of the sea cucumber H. glaberrima at 9 dpe.

Schematic highlights the heterogeneity of the coelomic epithelia of the intestinal anlage and identified mesenchymal populations. The colors of cells and C# correlate with those used on the UMAP of Fig 1B.

Labeling of dissociated cell phenotypes with cell markers.

Immunocytochemistry, fluorescently labeled phalloidin and Toluidene blue were used to identify various cell populations among the cells dissociated from the mesentery an anlage sample. These include A. a mesenchymal marker (KL4), B. a neuronal marker (RN1), C. a mesothelial marker (Meso1), D. a muscle marker (Phalloidin) and E & F two coelomocyte markers (The antibody SphAA12and toluidine blue). E. Immunocytochemistry using the SphAA12 antibody F. Overlay of toluidene blue labeling (see dark cell on lower left) identifies a different coelomocyte population.

Immunocytochemical labeling of dissociated cell phenotypes using three different antibodies against tubulin.

A. anti-acetylated tubulin labels around 7% of the cells. B. Anti-beta-tubulin labels around 70% of the cells. C. Anti-alpha tubulin labels around 80% of the cells.

UMAP of clusters after statistical assessment with scSHC.

Results reflect that each of the identified clusters are unique, except for C3 and C4, which it suggests they correspond to a single cluster. All clusters, except for C3/4 (black), are colored as in Fig 1B.

Expression of marker genes previously documented in the sea cucumber.

(A-C) UMAP highlighting the cells expressing (A) Wnt9, (B) SAA, and (C) Proteoglycan-4.

Expression of marker genes associated with cell types or state.

(A-D) Violin plot highlighting the level of expression of (A) PIWL1, (B) YAP1, (C) HES1, and (D) SAA1 across clusters.

Pseudotime analysis of all clusters within the 9dpe intestinal regeneration dataset.

Each color reflects the corresponding cluster form C0-C12. The x-axis represents slingshot pseudotime and y-axis the corresponding lineage.

Antibody/Markers used for immune- and cytochemical labeling of dissociated cell suspension.