Intestinal regeneration in H. glaberrima.

(A) Schematic view of normal and regenerating tissues as viewed in cross sections of normal and regenerating intestines. After evisceration (1hr) the tip of the mesentery is torn but eventually is covered by coelomic epithelium (24hrs). A thickening of the mesentery tip forms the intestinal anlage which grows in size for the next two weeks, initially (3-5 dpe) by cellular dediFerentiation and later by cellular proliferation (7-9 dpe). Eventually, the luminal epithelium is formed from migrating cells of the esophagus and cloacal ends of the digestive tract. (B) DAPI-stained section of 9-dpe anlage and mesentery showing the coelomic epithelium (CE) and connective tissue (CT) layers. (C-D) Sections with fluorescently-labeled phalloidin show the muscle (Mu) labeled in the (C) 9-dpe anlage and in the (D) normal intestine. The normal intestine is made of three distinct layers: mesothelium (Me) (that includes the CE and Mu), CT, and luminal epithelium (not shown). Me and CT are continuous throughout the mesentery and body wall. CE - Coelomic epithelium, CT - Connective tissue, Me - Mesothelium, Mu - Muscle, dpe - days post evisceration. Bar= 10µm

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

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

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.

HCR-FISH for (A, C) FBCD1 or (B, D) BAR3 in holothurian (A, B) normal or (C, D) regenerating tissues. Cells (arrowheads) expressing FBCD1 mRNA in the connective tissue (CT) layer of (A) normal intestine and (C) anlage. Cells (arrowheads) expressing BAR3 mRNA in the connective tissue of (B) normal (non-eviscerated) animal and (D) the coelomic epithelium (CE) of the anlage. Insets provide the approximate localization in the connective tissue of the normal intestine (top) and the anlage (bottom). Cyan - DAPI, Magenta - HCR-FISH. CE - Coelomic epithelium, CT - Connective tissue, Mu - Muscle. Bar= 10 µm.

Expression profile of mesenchymal cell types.

Cells (arrowheads) expressing HMCNT1 mRNA in the connective tissue (CT) layer of (A) normal intestine, (B) regenerating mesentery, and (C) intestinal anlage of H. glaberrima. In all tissues, the cells are found within the connective tissue (CT) layer. Notice in C that no expression is found in the cells of the coelomic epithelium (CE). Insets provide the approximate localization of the cells in the adjacent photos. Cyan - DAPI, Magenta - HCR-FISH. CE - Coelomic epithelium, CT - Connective tissue, Mu - Muscle. Bar= 10µm

Expression profile of proliferating cells.

Cells (arrowheads) expressing Ki67 mRNA in the coelomic epithelium (CE) layer of regenerating (A) mesentery and (B) anlage of H. glaberrima. Most of the labeled cells are within the CE tissue layer. (C) BrdU-labeled cells (arrowheads) are also mainly found in the CE layer. Insets provide the approximate localization of the cells in the adjacent photos. Cyan - DAPI, Magenta - HCR-FISH. CE -Coelomic epithelium, CT- Connective tissue. Bar = 10µm

Myosin mRNA expression in regenerating and mature muscle cells.

(A-C) Cells expressing MYH7 mRNA (arrowheads) in the (A) normal intestine, (B) regenerating mesentery, and (C) basal area of the anlage coelomic epithelium (CE). Arrowheads point to expression in cells of regenerating tissues. Insets provide the approximate localization of the cells in the adjacent photo. (D-G) Double labeling of (D, F) MYH7, and (E, G) muscle-specific antibody (HgM2), in (D, E) normal intestine, and (F, G) regenerating mesentery, showing the co-expression of both markers in the same cells. Note the two cells (arrowheads in F-G) that express both the MYH7 marker and the muscle-specific antibody (HgM2), representing cells initiating their diFerentiation toward enteric muscle cells. Cyan - DAPI, Magenta - HCR-FISH, Yellow - muscle antibody (HgM2). CE - Coelomic epithelium, CT - Connective tissue. Mu - Muscle. Bars = 10μm.

STARD-10 expression in differentiating neuroepithelial cells of the coelomic epithelium.

(A) Cells (arrowhead) expressing STA10 mRNA in the coelomic epithelium (CE) of the regenerating mesentery. (B) Double labeling with STA10 HCR-FISH and STARD-10 antibody (RN1) shows the presence of a cell (arrowhead) in the CE of the regenerating mesentery. (C-E) Fibers (arrow) in the anlage, also express both the (C) STA10 mRNA and (D) the protein, as shown in (E) the corresponding overlay. Insets provide the approximate localization of the cells or fibers in the adjacent photos. Cyan - DAPI, Magenta - HCR-FISH, Yellow-STARD-10 antibody (RN1). CE - Coelomic epithelium, CT - Connective tissue. Bar- B = 10μm; A, C-E = 5 μm.

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

(A) HCR-FISH for UNC5C mRNA labeled most if not all cells in the coelomic epithelia (CE) of the normal intestine. Strong labeling is also observed in various regions of (B, C) the CE of the regenerating mesentery, while a few cells (arrowhead) are found in (D) the CE of the anlage (arrowhead). Insets provide the approximate localization of the cells in the adjacent photos. Cyan - DAPI, Magenta - HCR- FISH. CE - Coelomic epithelium, CT - Connective tissue. Bar = 10µm.

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

TRFM mRNA (left column) is expressed by (A) cells (arrowheads) of the coelomic epithelium (CE) in the normal intestine. In regenerating tissues, there is a high expression in (C) CE cells (arrowheads) close to the body wall. This expression decreases toward the anlage, with some expression in (E) CE cells of the mid part of the mesentery and very little if any in (G) the CE of the anlage. In contrast, NET1 (right column) is expressed in fewer CE cells (arrowheads) of the (B) normal intestine, and (D, F) mesentery, but its expression is extremely high in most of (H) the CE cells of the anlage. Insets provide the approximate localization of the cells in the adjacent photos. Cyan - DAPI, Magenta - HCR-FISH. CE - Coelomic epithelium, CT - Connective tissue. Ba r= 10µm.

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 of the intestinal anlage.

Various cell populations can be identified in the connective tissue (CT) and coelomic epithelium (CE) in the anlage of the sea cucumber H. glaberrima at 9 dpe. These populations correspond to those identified by the scRNA-seq that are described in the text. Most of the cell populations are found within the CE, some of them showing particular localizations; differentiating muscle cells are found in the basal part of the CE while differentiating coelomic epithelia are found in the apical region. The two mesenchymal populations are shown together. The colors of cells and cluster numbers (C#s) correlate with those used on the UMAP shown in Fig 2. Inset provides the approximate localization of the diagram cells.

Overview of the intestinal regeneration process in the sea cucumber H. glaberrima.

The non-eviscerated (normal) digestive tract is shown as a continuous tube beginning at the mouth, followed by an esophagus which is continuous with the intestine ending in the cloaca. This digestive tract is attached to a mesentery (light blue) which attaches the digestive tract to the body wall. Following evisceration, the tip of the mesentery begins to heal by 24 hrs. After 3 days post evisceration (dpe) the anlage (salmon) begins to form at the free tip of the mesentery and continues growing in the following days. Once the anlage is formed, the lumen develops at around 14-dpe. By 21-dpe the full formed gut lumen (green) can be traced from the esophagus to the cloaca.

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 SphAA12 and toluidine blue). E. Immunocytochemistry using the SphAA12 antibody F. Overlay of E (UV light) with classical histochemical stain toluidene blue (visible light) identifies a diFerent coelomocyte population (see dark cell on lower left, labeled with arrowhead). Cyan-DAPI, Magenta-antibody or phalloidin marker. Bar= 20um.

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. Cyan-DAPI, Magenta-antibody or phalloidin marker. Bar= 20um.

Quality control assessment of H. glaberrima scRNA-seq data.

(A) Scatter plot of the correlation of unique genes detected per cell (nFeature_RNA) versus the total number of transcripts (nCount_RNA). Each point in the plot represents a single cell. (B) Bar plot displaying the total number of cells per sample. (C) Density plot of log-transformed genes per UMI (log10GenesPerUMI) showing the sequencing complexity across sample. (D) Density plot of the total number of UMIs (nUMI) per cell showing the distribution of UMI across the four samples. (E) Density plot of the total number of detected genes (nGenes) per cell. Vertical lines in plot C-E represents the minimum threshold cut-off of expected values for each component. (F) Principal component analysis (PCA) plot of the scRNA-seq data after data filtering and integration. M1 and M2 represent mesentery replicates in red and blue, respectively. R1 and R2 represent rudiment/anlage replicates in green and purple, respectively.

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.

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 of other clusters. Gene identifiers starting with “g” correspond to uncharacterized gene models of H. glaberrima

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.

Enrichment of GO terms across cellular populations of the regenerative intestine of H. glaberrima.

Top Go enriched terms of biological processes for (A) mesenchymal populations C2 and C7, (B) anlage precursor populations C0, C1, C3, and C4, (C) specialized cell populations C5, C8, C9, and (D) immune populations C6, C10, C11, C12. Dot size corresponds to the enrichment score while the color reflects the adjusted p-value.

HCR-FISH for Poly-A mRNA as a positive control.

Labeling of Poly A-mRNA in the (A) normal intestine, (B) regenerating mesentery, and (C) anlage, provides evidence for distinct labeling of cells in the coelomic epithelia (CE) and connective tissue (CT). Note that some cells express higher intensities of the marker, while others express scarce or no labeling. A particular example is observed in (C) the cells undergoing epithelial-mesenchymal transition in the anlage where little mRNA expression is observed. Insets provide the approximate localization of the cells in the adjacent photos. Cyan- DAPI, Magenta- HCR-FISH. CE- Coelomic epithelium, CT-Connective tissue. Bar= 10µm.

HCR-FISH negative control.

Treatment of tissue sections with only the fluorescent hairpins used for HCR- FISH shows little or no labeling in (A) normal intestine, (B) regenerating mesentery, and (C) anlage. Non-specific labeling is observed in some coelomocytes (arrowheads) found mainly in the connective tissue (CT) and some associated with the coelomic epithelium (CE). Insets provide the approximate localization of the cells in the adjacent photos. Cyan- DAPI, Magenta- HCR-FISH. CE- Coelomic epithelium, CT- Connective tissue. Bar= 10µm.

Double labeling for muscle markers in intestinal muscle cells.

Muscle cells in the normal intestine are labeled using (A) fluorescent phalloidin, and (B) muscle-specific antibody (HgM2). (C) The overlay shows that while there is co-expression of both markers in the muscle cells, the markers recognize diFerent structures; phalloidin is known to bind to polymerized actin, while the epitope recognized by the muscle antibody (HgM2), which remains unknown, appears to be associated with the membrane component. Cyan- DAPI, Magenta- fluorescent phalloidin, Yellow- muscle-specific antibody (HgM2). CE- Coelomic epithelium, CT- Connective tissue. Mu- Muscle. Bar= 20μm.

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.

General statistics of scRNA-seq data after mapping with Cell Ranger.