In holothurians, the regenerative process following evisceration involves the development of a ‘rudiment’ or ‘anlage’ at the injured end of the mesentery. This regenerating anlage plays a pivotal role in the formation of a new intestine. Despite its significance, our understanding of the molecular characteristics inherent to the constituent cells of this structure has remained limited. To address this gap, we employed state-of-the-art scRNA-seq and hybridization chain reaction fluorescent in situ hybridization analyses to discern the distinct cellular populations associated with the regeneration anlage. Through this approach, we successfully identified 13 distinct cell clusters. Among these, two clusters exhibit characteristics consistent with putative mesenchymal cells, while another four show features akin to coelomocyte cell populations. The remaining seven cell clusters collectively form a large group encompassing the coelomic epithelium of the regenerating anlage and mesentery. Within this large group of clusters, we recognized previously documented cell populations such as muscle precursors, neuroepithelial cells, and actively proliferating cells. Strikingly, our analysis provides data for identifying at least four other cellular populations that we define as the precursor cells of the growing anlage. Consequently, our findings strengthen the hypothesis that the coelomic epithelium of the anlage is a pluripotent tissue that gives rise to diverse cell types of the regenerating intestinal organ. Moreover, our results provide the initial view into the transcriptomic analysis of cell populations responsible for the amazing regenerative capabilities of echinoderms.
sea cucumberregenerationblastemasingle cellholothurianintestineResearch organismOtherhttp://dx.doi.org/10.13039/100000002National Institutes of Health5R25GM061151-22Miranda-NegrónYamilhttp://dx.doi.org/10.13039/100000057National Institute of General Medical SciencesP20 GM103475Medina-FelicianoJoshua GValentín-TiradoGriselleLuna-MartínezKiaraBeltran-RiveraAlejandraMiranda-NegrónYamilGarcia-ArrarasJosé Ehttp://dx.doi.org/10.13039/100000002National Institutes of Health2R15GM124595Medina-FelicianoJoshua GValentín-TiradoGriselleGarcia-ArrarasJosé EThe funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Author impact statementThe intestinal coelomic epithelium provides dedifferentiated precursor cell populations that drive the adult holothurian intestinal regeneration.publishing-routeprcIntroduction
Animals exhibit a remarkable diversity in their responses to injury, ranging from basic wound healing to the complete regeneration of lost structures. At one end of the spectrum are species that heal wounds without regenerating the missing part, while at the other end are those capable of recreating structures identical to the original. Despite these differences, all animals possess some level of regenerative ability. This capacity for regeneration can vary not only between different species but also within the same species, depending on the tissue or organ involved.
For decades, scientists have tried to understand these vast differences in regeneration capacities across the animal kingdom. For this they have focused on those species that show amazing regenerative abilities (Tanaka and Reddien, 2011), including coelenterates (hydra) (Vogg et al., 2019), flatworms (planaria) (Reddien, 2018), fish (zebrafish) (Gemberling et al., 2013), and amphibians (Brockes and Kumar, 2002; Roy and Gatien, 2008; Beck et al., 2009; Joven et al., 2019), among others. These studies have uncovered various mechanisms that regeneration-competent species exhibit to regenerate tissues, entire organs, body parts, and, in some cases, complete bodies. Key findings include the discovery of essential processes, such as the formation of a blastema – a mass of proliferating cells that plays a crucial role in regenerating the lost structure (Min and Whited, 2023; Poleo et al., 2001; Santos-Ruiz et al., 2002; Seifert and Muneoka, 2018; Stocum, 2004; Zenjari et al., 1996).
Among deuterostomes, echinoderms are considered prime exponents of regenerative capability (Candia-Carnevali et al., 2024). Within this group, holothurians, commonly known as sea cucumbers, exhibit an extraordinary form of regeneration. They can regenerate their internal organs following evisceration, a process in which they expel their viscera in response to stress or predation (Byrne, 2023). This extraordinary ability makes them a valuable model for studying regeneration in complex organisms. The regeneration of the digestive system in holothurians, in particular, has garnered significant interest (Dolmatov, 2021; Mashanov et al., 2014; Medina-Feliciano and García-Arrarás, 2021; Quispe-Parra et al., 2021a; Su et al., 2022). Upon evisceration, the holothurian intestine, which constitutes nearly their entire digestive tract, begins to regenerate from the mesentery, a supportive tissue layer where the original intestine was attached (García-Arrarás, 1998). Figure 1 andFigure 1—figure supplement 1 provide a schematic view of the regeneration process and identify key structures for those not familiar with the holothurian model. A thickening at the injured end of the mesentery, known as an ‘anlage’ or ‘rudiment’, initiates this process (García-Arrarás, 1998). As will be discussed later, this structure is analogous to a blastema but differs in that the cell proliferation mainly occurs in the surrounding epithelium, rather than in the mesenchymal cells, as observed in classical blastemas (Carlson, 2007; García-Arrarás, 1998).
Intestinal regeneration in <italic>H. glaberrima</italic>.
(A) Schematic view of normal and regenerating tissues as viewed in cross sections of normal and regenerating intestines. After evisceration (1 hr), the tip of the mesentery is torn but eventually is covered by CE (24 hr). A thickening of the mesentery tip forms the intestinal anlage, which grows in size for the next 2 weeks, initially (3–5 dpe) by cellular dedifferentiation 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 CE and 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 = 10 µm.
Antibody/markers used for immune- and cytochemical labeling of dissociated cell suspension.
Overview of the intestinal regeneration process in the sea cucumber <italic>H. glaberrima</italic>.
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 hr. 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 toluidine blue were used to identify various cell populations among the cells dissociated from the mesentery and 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 toluidine blue (visible light) identifies a different coelomocyte population (see dark cell on lower left, labeled with arrowhead). Cyan, DAPI; magenta, antibody or phalloidin marker. Bar = 20 um.
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 = 20 um.
Histologically, the holothurian intestinal anlage forms from dedifferentiated cells within the mesentery, which revert to a more stem-cell-like state before proliferating and migrating to form a new intestinal structure (Candelaria et al., 2006; Mashanov et al., 2005). This dedifferentiation process is crucial for regeneration, involving a spatial and temporal gradient starting at the injury site and extending along the mesentery border (García-Arrarás et al., 2011). The new coelomic epithelial layer that forms around the anlage is distinct from the original mesothelium and shows significant morphological and molecular changes compared to the mesenteric tissue (Candelaria et al., 2006; García-Arrarás, 1998; Mashanov et al., 2005). Further examination of holothurian regeneration reveals that most cellular division occurs within the anlage’s coelomic epithelium (García-Arrarás, 1998; García-Arrarás et al., 2011). These proliferating cells are hypothesized to differentiate into various cell types, including myocytes and neurons, as well as mesenchymal cells, through an epithelial to mesenchymal transition (EMT) (García-Arrarás, 1998; García-Arrarás et al., 2011). The gene expression profiles during the formation and growth of the anlage suggest extensive reprogramming, leading to a more plastic cell phenotype (Ortiz-Pineda et al., 2009; Quispe-Parra et al., 2021b; Rojas-Cartagena et al., 2007).
The process of intestinal regeneration in holothurians raises fundamental questions of regenerative phenomena, particularly concerning the identity, origin, and fate of progenitor cells, involved in the process (Alvarado and Tsonis, 2006; Candia-Carnevali et al., 2024). In this context, the role of the anlage, and specifically the mesothelium (also named celothelium) in echinoderms, deserved particular attention (Candia-Carnevali et al., 2024; Smiley, 1994). This tissue, mainly composed of coelomic epithelia and myocytes, exhibits significant morphological and gene expression changes that are associated with the dedifferentiation process. These dedifferentiated cells form the coelomic epithelium of the anlage and appear to be the principal source of cells for the new intestine. Despite these findings, little is known about the cell composition and dynamics of the anlage nor of its coelomic epithelium.
Single-cell RNA sequencing (scRNA-seq) offers tremendous promise to dissect the cellular contributions of the holothurian intestinal anlage and identify the specific cells involved in generating a new intestine. It is a powerful tool for dissecting cellular composition and dynamics that has been used in related species to explore regenerating or developing tissues, in mammals (Ayyaz et al., 2019; Beumer and Clevers, 2021; Capdevila et al., 2021; Parigi et al., 2022), axolotl and planaria (Gerber et al., 2018; King et al., 2024; Leigh et al., 2018; Rodgers et al., 2020), and echinoderm embryos (Cocurullo et al., 2023; Paganos et al., 2022a; Paganos et al., 2021; Satoh et al., 2022; Tominaga et al., 2023). These applications highlight the groundbreaking role of scRNA-seq in advancing our knowledge of the cellular mechanisms in both regenerative and developmental contexts.
In this study, we employ scRNA-seq to analyze the regenerating intestinal anlage of the sea cucumber Holothuria glaberrima, aiming to delineate its constituent cellular populations. We corroborate our findings using hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) to verify the presence and location of specific cell types (Choi et al., 2018). The characterization of the cellular populations and their gene expressions serves to answer various questions such as: What are the cellular precursors? What is the role of the coelomic epithelium? What are the similarities and differences of the anlage and a classical blastema? This research not only advances our understanding of the unique regenerative capabilities of holothurians but also contributes to the broader field of regenerative biology, highlighting the diverse strategies employed by different organisms to restore lost tissues.
Results
Previous work from our laboratory has shown that the rudiment or anlage that forms at the tip of the mesentery plays a pivotal role in the formation of the new intestine (García-Arrarás et al., 2019). This transient mass of cells is thought to give rise to most intestinal cell types, the sole exception being the luminal cells. Therefore, to maximize the characterization of the cells in the regenerative anlage, we chose to perform scRNA-seq in the tissues of 9-day post evisceration (dpe) regenerating animals (García-Arrarás, 1998; García-Arrarás et al., 2011). At this stage, a well-formed anlage consists of epithelial cells surrounding a large area of connective tissue populated with mesenchymal cells (Figure 1A). More importantly, different cell populations undergoing proliferation or differentiation can be found at this stage. Since some of the cellular processes during regeneration occur in a spatio-temporal gradient along the length of the mesentery, we separately surveyed the anlage and the mesentery tissues accordingly. Thus, for each animal, the anlage was separated from the mesentery, and both tissues were processed independently, for a total of four scRNA-seq runs.
Cell heterogeneity: Immuno- and cytochemical analyses
The strength of the scRNA-seq data depends mainly on the dissociation and isolation of the cell populations from the dissected tissue. Since our focus was on the cells of the regenerating anlage, we devised a dissociation protocol that favored the isolation of the cells within this structure. To determine, at least partially, the cell types in our original dissociation, we performed immuno- and cytochemical analyses on the enzyme-dissociated cell suspension that was used for the scRNA-seq. The labeling obtained for each marker is shown in Figure 1—figure supplement 2.
We found that, in the dissociated anlage, 5% of the cells were labeled with a spherulocyte (immune cell) marker, 2–7% with phalloidin (a muscle marker), 40% with a mesenchymal marker (KL14-antibody), and a large number of cells (32–60%) with a mesothelial marker (MESO-antibody). Similar populations were found in the mesentery, although in this tissue, 5–10% of cells expressed the neuronal markers, heptapeptide GFSKLYFamide (Blanco et al., 1995), and RN1 (Díaz-Balzac et al., 2007). This analysis suggested that most of the cells originate from the regenerate coelomic epithelium.
Different populations were also observed with three different tubulin antibodies (Figure 1—figure supplement 3). In the 9-dpe anlage, anti-acetylated alpha tubulin labeled about 7% of the cells, anti-beta tubulin labeled about 70% of the cells, while an anti-alpha tubulin labeled about 80% of the cells. Except for the acetylated alpha tubulin, which labeled about 1% of the cells dissociated from the mesentery versus 7% of those from the anlage, anti-alpha and beta labeling percentages were similar in both mesentery and anlage dissociated cells.
Finally, a cell population labeled with fluorescently labeled phalloidin accounted for about 7% of the cells in both mesentery and anlage. These cells, however, did not correspond to the elongated muscle cells of the mesentery. Instead, they were rounded cells with labeling found in the cytoplasm surrounding one side of the nuclei.
Two cell populations from the mesentery were absent or greatly underrepresented in the scRNA-seq. Firstly, the muscle cells of the mesentery, due to their non-dissociation by the protocol used, were not found in the dissociated cell suspension. Their elongated morphology would have further complicated their passage via cell separation system for sequencing. Secondly, the majority of neurons from the neuronal network associated with the mesentery (Nieves-Ríos et al., 2020) could not be dissociated. We did observe structures that resembled a tangled mass of cells immunoreactive to some of our neuronal markers. This suggests that the mesentery nervous component, being unable to be isolated as single cells, was not sequenced.
In summary, the immuno- and cytochemical results show that the dissociated cell populations sequenced correspond to cellular phenotypes that have been previously described within the regenerating anlage. The abundance of these cells in the sequenced samples corresponds to the ease of their dissociation by trypsin. Thus, dedifferentiated cells of the mesentery and anlage epithelium (which are loosely connected to each other) and those of the connective tissue are probably over-represented compared to differentiated cell types.
Cell populations defined by scRNA-seq
Analysis of scRNA-seq data resulted in a total of 3844 cells, with 2392 originating from the two anlage samples and 1452 from the two mesentery samples (Figure 2—figure supplement 1). Upon dataset integration and graph-based clustering, we identified 13 clusters, each thought to represent singular cell types or cell states in the regenerating intestine (Figure 2A, Figure 2—figure supplement 2). The percentage of cells that form each cluster differs from cluster to cluster, ranging from 21% (cluster 0) to 1% (cluster 12) of the total cells. Nonetheless, each cluster consists of cells from both the mesentery and anlage samples (Figure 2B and C). The number of clusters did not change dramatically under various parameters (resolution, dimensionality, and number of variable features), constantly around 12–15 clusters. Moreover, except for C3 and C4, the clusters were supported by the clustering significance analysis performed with scSHC (Grabski et al., 2023), a model-based hypothesis testing method for scRNA-seq that evaluates the probability of each individual cluster being unique (Figure 2—figure supplement 2). The uniqueness of C3 and C4 is suggested by additional analyses as will be addressed below.
Overview of single-cell RNA sequencing of regenerating intestinal tissue of <italic>H. glaberrima</italic>.
(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.
General statistics of scRNA-seq data after mapping with Cell Ranger.
Quality control assessment of <italic>H. glaberrima</italic> 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 Figure 1B.
UMAP visualization of clusters highlighting the expression of their top genes.
UMAPs A-M represent the 13 cell populations described in the text, while N and O describe the epithelial and mesenchymal supraclusters, respectively. 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.
Each identified cluster exhibited a distinctive gene expression profile relative to cells in other clusters (Figure 2—figure supplement 3). This shows the relative expression of the top gene for each cluster based on two factors: (1) difference in percentage of representation and (2) log2 fold-change (log2FC) against all other clusters. Interestingly, each cluster shows dramatic differential expression values (>2 log2FC) and differences in representation percentages over 50%, except for C0 through C3, with differences in representation around 30%.
Prior to characterizing each of the 13 cell clusters, we sought to understand what, in a broad view, appeared to be a segregation of ~90% of the cells into two distinct supra-clusters. One of them encompassing seven clusters (C0, C1, C3, C4, C5, C8, C9) that corresponded to 69.6% of all cells and the other encompassing two clusters (C2 and C7) that corresponded to 19.1% of cells. The remaining 11.3% of cells were distributed in four distinct isolated clusters (C6, C10, C11, and C12). As stated earlier, all clusters have representation from mesentery and anlage tissues (Figure 2B and C), thus excluding the possibility that the two supra-clusters represented mesentery versus anlage cells.
Mesenchymal versus epithelial clusters
These two supra-clusters are of interest as they appear to represent the two main cell types found in the regenerating intestine: coelomic epithelial cells and mesenchymal cells (Figure 2A). Comparison between these two supra-clusters showed distinct expression profiles that allowed us to characterize their cell types (Figure 3, Figure 2—figure supplement 3). For instance, the supra-cluster composed of C2 and C7, showed ERG (transcriptional regulator ETS-related gene) as the top expressed gene, an oncogene that is associated with mesenchymal cells in other echinoderms (Meyer et al., 2023; Tominaga et al., 2023). While this gene has a strong expression in C2 and C7, it also appears to be expressed in C6 and C10. However, it is important to highlight that ERG as a transcription factor has been shown to have roles in additional processes including inflammation and apoptosis.
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 of marker genes previously documented in the sea cucumber.
(A–B) UMAP highlighting the cells expressing (A) Proteoglycan-4 and (B) Wnt9.
Enrichment of GO terms across cellular populations of the regenerative intestine of <italic>H. glaberrima</italic>.
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.
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.
The top marker gene in the other supra-cluster is AHNK, known as neuroblast differentiation-associated protein AHNAK. Reports have shown AHNK to have a role in calcium regulation, cellular migration, and carcinogenic transformation of colon epithelial cells (Dumitru et al., 2013). Furthermore, this gene is overexpressed in regenerating rat muscle compared to normal muscle (Huang et al., 2007). However, the localized expression of WNT9 in the C1, C3, C5, and C8 of this supra-cluster more clearly favors its classification as a marker for coelomic epithelial cell types (Figure 3B, Figure 3—figure supplement 1). A previous study from our laboratory using in situ hybridization details the expression of WNT9 during intestinal regeneration in H. glaberrima, where it was shown to be localized to the coelomic epithelium of the anlage and adjacent mesentery (Mashanov et al., 2012). In addition, correlating with what was shown by the in situ hybridization results, the population of cells that differentially expresses WNT9 makes 15% of those in the mesentery. However, it is close to 30% of the cells in the anlage. Additional analyses, discussed below, further strengthen the coelomic epithelium identity of cells in this supra-cluster.
In summary, results show 13 individual cell clusters with distinct expression profiles in the regenerative intestinal tissue, with most of them corresponding to either mesenchymal or coelomic epithelia cells. The rest of the populations show top expressed genes that are immune-related, suggesting that these must be coelomocyte populations such as those that have been previously associated with both normal and regenerating intestinal tissues and that were also detected by immune and histochemistry in the cell samples used for the scRNA-seq (Figure 1—figure supplement 2; García-Arrarás et al., 2006; Ramírez-Gómez et al., 2010).
Cluster identities
Rather than considering single genes, the uniqueness of each cluster can be assessed in terms of their transcriptomic profile (Figure 3A) and by the expression of transcription factors and intercellular signaling molecules (Figure 3B). As shown in Figure 3A and B, each cluster has a unique transcriptomic are clusters C0, C1, C3, and C4 of the epithelial layer populations, which share many top genes, albeit at different expression levels. Similarly, C8 and C9 have low expression of genes expressed by C0, C1, C3, and C4 but have expression of other genes that are not expressed by any other cluster. This is also true for the mesenchymal clusters, where these two clusters have overlap of some genes, that are not expressed by other clusters. Therefore, these results show the interaction of these clusters and their transcriptomic relationship depending on where they are localized within the regenerating intestinal tissue.
Identifying the top genes expressed by each cluster also provides essential, notwithstanding limited, information for their complete identification. To further characterize each cluster, we have used other analyses or performed additional experiments, including (1) multiple gene expression patterns, (2) enriched ontology, (3) HCR-FISH, and (4) pseudo-trajectory. Initially, we describe in depth the potential identity of these clusters based on their expression pattern and enriched ontology, and we will end with their potential interactions.
An in-depth analysis of the various clusters and their possible relation to cell populationsCoelomocyte populations
Coelomocytes are specialized cells found in the coelomic fluid and within organs of echinoderms. These cells have been associated with immunological roles including pathogen recognition, encapsulation, phagocytosis, debris removal, cytokine production, and secretion, among others (Barela Hudgell et al., 2022; Courtney Smith et al., 2018; García-Arrarás et al., 2006; Smith et al., 1995), In holothurians, these cells have been shown to be present at injury sites and in the mesentery and regeneration anlage (García-Arrarás et al., 2006). Coelomocytes can be subdivided into different populations by using morphological, physiological, and molecular characteristics (Ramírez-Gómez et al., 2010).
Many of the coelomocyte characteristics correlate with the top differentially expressed genes of cell populations within our data. These immune-like clusters (C6, C10, C11, and C12) have high expression of genes related to the immune system that are not shared with any other cluster. For example, C6 embodies a distinct cell population that represents a substantial number of cells (6% of all cells, 8% of the mesentery, and 4% of the cells in the anlage). These cells are the only population expressing FBCD1 (fibrinogen C domain-containing protein 1). Cells in C6 also express other genes such as various tyrosine protein phosphate receptors, integrin alpha-8 (ITA8), platelet glycoprotein V (GPV), leucine-rich repeats, and immunoglobulin-like domains protein 2 (LRIG2) (Figure 3). The immune identity of C6 is also supported by the resulting gene set enrichment of gene ontology (gseGO) terms related to immune responses, such as ubiquitin-dependent ERAD pathway, innate immune response activating cell surface receptor signaling pathway, respond to endoplasmic reticulum stress, phagocytosis, and many more related to defense mechanisms (Figure 3—figure supplement 2). Similarly, C10 and C12 transcriptomic profiles suggest they correspond to immune-like populations (Figure 3). Specifically, C10 shows various uncharacterized genes along with HCK (tyrosine-protein kinase HCK), DMBT1 (deleted in malignant brain tumors 1), ALS (insulin-like growth factor-binding protein complex acid labile subunit), GPV, and IRF8 (interferon regulatory factor 8), FER (tyrosine-protein kinase Fer). The top GO-enriched terms of this cluster support its involvement in immune process, some of these being lipase activity regulation, Fc receptor-mediated stimulatory signaling pathway, cellular pigmentation, B cell activation involved in immune response, and Fc receptor signaling pathway (Figure 3—figure supplement 2). The transcripts expressed by C12 show a more complex profile, where most of the top genes are uncharacterized. Nonetheless, among the annotated genes are BAR3 (Balbiani ring protein 3), LYS1 (lysozyme 1), TRPM3 (transient receptor cation channel subfamily M member 3), PGCA (aggrecan core protein), and MRC1 (macrophage mannose receptor 1). The top genes of C11 include a great number of immune genes such as MUC5A (mucin-5AC), FCGBP (IgGFc-binding protein), SSPO (SCO-spondin), FCN1A (ficolin-1-A), MUC5B (mucin-5B), and TIE1 (tyrosine-protein kinase receptor Tie-1). However, the top genes of this cluster also include several genes involved in neuronal activity, which is evident in the top enriched GO terms of this cluster that include regulation of postsynaptic membrane potential, excitatory postsynaptic potential, chemical synaptic transmission, endoplasmic reticulum to Golgi vesicle-mediated transport, adult behavior, and regulation of neurotransmitter levels (Figure 3—figure supplement 2).
To partially confirm the prediction that cells from C6, C10, C11, and C12 corresponded to coelomocyte populations, we used HCR-FISH to identify the cell types expressing the top gene in two of the clusters. We focused on the expression of FBCD1 and BAR3, the genes that are the most represented by cells of C6 and C12, respectively. For each probe, in situ hybridization identified a distinct cell type in both regenerating and non-regenerating tissues (Figure 4, Figure 4—figure supplements 1 and2). The FBC1-expressing cells showed round or oval morphologies with a central round nucleus. In some cases, short extensions could be observed. The cells were heterogeneously distributed in all tissues, including the nervous system and the body wall, and could be found associated with either epithelial tissues or with the extracellular matrix (ECM) in the normal intestine, the intestinal anlage and in the mesentery of normal or regenerating animals (Figure 4A and C). The BAR3-expressing cells were also distinct, isolated cells found in different tissues of the normal and regenerating animals (Figure 4B and D). Their numbers were not as high as those of the FBC1-labeled cells and their labeling was more punctuated within the cytoplasm. In the regenerating tissues, they were mostly associated with the mesentery. The widespread distribution of both cell types hinted at a cell function consistent with patrolling the body to detect and respond to potential threats such as injury or bacterial invasion.
Expression profile of coelomocyte cell types.
Hybridization chain reaction fluorescent in situ hybridization (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 CT layer of (A) normal intestine and (C) anlage. Cells (arrowheads) expressing BAR3 mRNA in the CT of (B) normal (non-eviscerated) animal and (D) the CE (CE) of the anlage. Insets provide the approximate localization in the CT 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.
Hybridization chain reaction fluorescent in situ hybridization (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 CE and 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 = 10 µm.
Hybridization chain reaction fluorescent in situ hybridization (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 CT and some associated with the 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 = 10 µm.
Mesenchymal cell populations
Mesenchymal cells of the intestinal anlage are yet to be well studied. They are known to be less proliferative than those in the overlying epithelium (García-Arrarás, 1998; García-Arrarás et al., 2011) and involved in ECM remodeling (Quiñones et al., 2002). Some of the mesenchymal cells are thought to migrate from the connective tissue of the mesentery to the connective tissue in the anlage (Cabrera-Serrano and García-Arrarás, 2004), while others have been shown to originate via EMT from the overlying epithelium (García-Arrarás et al., 2011). Some of these cells will form a mesenchymal cellular layer associated with the luminal epithelial cells as the lumen forms (García-Arrarás et al., 2011). As explained earlier, we propose that C2 and C7 form a separate supra-cluster, clearly identified by ERG expression. These two clusters also share many marker genes, including multiple ECM genes (Figure 3). For instance, highest expressed gene of C2 is TIMP3 (metalloproteinase inhibitor 3), followed by NPNT (nephronectin), which has been reported to be an integrin ligand during kidney development (Sun et al., 2018). Additionally, this cluster has overexpression of ECM2 (extracellular matrix protein 2), KLKB1 (plasma kallikrein), MMP14 (matrix metalloproteinase 14), MMP24 (matrix metalloproteinase 24), HMCN1 (hemicentin-1), and ITA8 (integrin alpha-8), all of which are explicitly related to the ECM component (Bökel and Brown, 2002; Dolmatov and Nizhnichenko, 2023; Dong et al., 2006; Stamenkovic, 2003; Volkert et al., 2014). Many of these genes are also highly represented in C7. However, here we also find ITIH2 (inter-alpha-trypsin inhibitor heavy chain H2), DUOX1 (dual oxidase 1), SVEP1 (sushi, von Willebrand factor type A, EGF, and pentraxin domain-containing protein 1), SEPP1 (selenoprotein P), and KLH20 (Kelch-like protein 20), suggesting that cells have gained some specialization and are more advanced in their differentiation compared to those of C2. Along with this, when analyzing the gseGO, for C2 we obtain GO terms of numerous ECM processes, such as cell adhesion mediated by integrin, integrin-mediated signaling pathway, and regulation of cell-substrate junction assembly (Figure 3—figure supplement 2). Similarly, C7 has an enrichment of regulation of cell-matrix adhesion, regulation of cell-substrate junction organization, and substrate adhesion-dependent cell spreading. neuroblast proliferation, among others (Figure 3—figure supplement 2).
To confirm our prediction that cells in C2 and C7 were those present within the mesenchyme of the connective tissue, we chose to localize the expression of HMCN1 mRNA. The mRNA for this protein, known to code for an ECM protein (Dolmatov and Nizhnichenko, 2023; Lindsay-Mosher et al., 2020; Welcker et al., 2021), is present in both cell clusters. HCR-FISH showed that cells expressing the HMCN1 mRNA were present in the normal intestine, the regenerating mesentery and the anlage (Figure 5). In the normal intestine and regenerating mesentery, labeled cells were present in the connective tissue (Figure 5A and B). In both tissues, cells were somewhat distanced from each other and had an irregular morphology with strong punctate labeling throughout the cytoplasm. A weaker labeling was observed in cells of the anlage, which were more densely packed and adjacent to the coelomic epithelium (Figure 5C), suggesting that they correspond to cells undergoing EMT on their way to differentiate into ECM-producing mesenchymal cells. A very similar pattern of expression was observed with HCR-FISH for ERG (not shown).
Expression profile of mesenchymal cell types.
Cells (arrowheads) expressing HMCNT1 mRNA in the 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 CT layer. Notice in (C) that no expression is found in the cells of the 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 = 10 µm.
Coelomic epithelia/mesothelial cell populations
The mesothelial layer of the intestine and mesentery is formed by cells (coelomic epithelium or peritoneocytes together with myocytes and neurons) in contact with the coelomic fluid. The coelomic epithelium of the regenerating tissues differs in morphology and gene expression (Mashanov et al., 2017; Mashanov et al., 2005; Mashanov et al., 2015; Mashanov et al., 2012; Mashanov et al., 2010) to the mesothelium that normally surrounds the organ. This coelomic epithelium, which is present mainly in the anlage and in areas of the adjacent mesentery, is made of dedifferentiated cells and is responsible for most of the cell division that takes place in the regenerating intestine (García-Arrarás et al., 2011). The analysis of the scRNA-seq data, in view of our knowledge of the ongoing events in the 9-dpe regenerating organ, strongly suggests that the seven clusters within the major supra-cluster represent the cells in the coelomic epithelium of the mesentery and the anlage. Here is our analysis.
C8 represents the proliferating cells
These cells mainly found within the anlage coelomic epithelia express proliferation markers such as PLK1 (serine/threonine-protein kinase PLK1), SMC2 (structural maintenance of chromosomes protein 2), PRI2 (PRIM2 – DNA primase large subunit), PCNA (proliferating cell nuclear antigen), and CDK1 (cyclin-dependent kinase 1) (Locard-Paulet et al., 2022). In addition, this cluster has high expression of TOP2A (DNA topoisomerase 2-beta), a gene that has also been seen to be overexpressed in proliferating basal cells of the human gastrointestinal epithelia (Busslinger et al., 2021). Other marker genes related to cell mitotic activity found here include CENPE (centromere-associated protein E), SMC4 (structural maintenance of chromosomes protein 4), KI67 (proliferation marker protein Ki-67), and CCNB3 (G2/mitotic-specific cyclin-B3) (Figure 3). Furthermore, this cluster expresses specific transcription factors such as E2F3, MCM10 (protein MCM10 homolog), PAF15 (PCNA-associated factor), and BRCA1 (breast cancer type 1 susceptibility protein) that are also associated with control of cell division (Humbert et al., 2000; Lõoke et al., 2017; Xie et al., 2014). The gseGO terms also confirm its proliferative identity with GO terms related to chromosome separation, condensation, mitotic cytokinesis, and regulation of cell cycle checkpoint (Figure 3—figure supplement 2). Moreover, the dividing cell population is higher in the anlage samples (4%) than in the mesentery samples (2%), which is in accordance with what we have observed in regenerating animals, that while cell division does take place in the mesentery, more cells are proliferating in the epithelial layer of the intestinal anlage (Bello et al., 2020; García-Arrarás, 1998; García-Arrarás et al., 2011).
To verify the epithelial nature of this cell cluster, we performed HCR-FISH for the KI67 mRNA. Multiple cells of the coelomic epithelium of the anlage were found to express the gene, as determined by a punctate labeling found throughout the cell body (Figure 6A and B). Some cells in the mesentery coelomic epithelium were also labeled but their number decreased in areas closer to the body wall. Similarly, few cells were labeled in the mesothelium of the normal intestine.
Expression profile of proliferating cells.
Cells (arrowheads) expressing Ki67 mRNA in the 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 = 10 µm.
Technical aspects of the protocol do not allow for double labeling of BrdU and HCR-FISH to be performed. (The HCl step that is needed in BrdU labeling to allow the antibody access to the BrdU-labeled DNA seems to interfere with the HCR-FISH labeling.) Nonetheless, we could show that, as previously documented (García-Arrarás, 1998; García-Arrarás et al., 2011), BrdU-labeled are found in the coelomic epithelium of the anlage (Figure 6C). The number of BrdU cells is much smaller than that of KI67 HCR-FISH-labeled cells. This is expected since BrdU is only incorporated into the DNA in cells undergoing the S-phase while KI67 is expressed during the whole duration of the mitotic cycle (Kee et al., 2002).
C5 represents muscle precursors
Enteric muscle precursors are known to originate from the coelomic epithelium during the second week of regeneration (Murray and García-Arrarás, 2004). This population can be recognized in our data by the expression of muscle-specific markers present in C5, such as TITIN, MYL1 (myosin light chain 1/3), MYH7 (myosin 7), ACTG (actin, cytoplasmic 2), and CNN3 (calponin-3) (Figure 3). Considering that this cell population is potentially a population of the coelomic epithelium actively undergoing differentiation toward muscle phenotype, they also share gene expression with other epithelial cell clusters (C0, C1, C3, C4), albeit at a lower fold-change. Similarly, this cluster shows a specific expression of transcription factors associated to muscle cells, namely FXL16 (F-box/LRR-repeat protein 16) and SCRT2 (transcriptional repressor scratch 2). Results of GO terms of this cluster also demonstrate enriched terms related to muscle tissue growth, such as muscle tissue morphogenesis, muscle development, myofibril assembly, and sarcomere organization (Figure 3—figure supplement 2). The focus on development and morphogenesis is to be expected, considering that these are still undergoing differentiation toward a muscle phenotype. HCR-FISH of MYH7 corroborates the muscle phenotype of the cells in this cluster (Figure 7). In control intestinal tissues, the labeling is specific to the muscle cell layer (Figure 7, Figure 7—figure supplement 1, also see Figure 1). In regenerating tissues, the labeling is observed in some cells in the basal part of the coelomic epithelium (Figure 6), the same regions as where myoblasts or muscle cells were previously identified (Murray and García-Arrarás, 2004; Figure 7). An additional experiment was performed to double label the tissue with MYH7 HCR-FISH and an antibody that labels the muscle fibers. The overlay of these markers showed the expression of the myosin mRNA in the normal intestine muscle cells (Figure 7D and E) and in cells of the regenerating tissues that have begun to form the actin-myosin contractile apparatus (Figure 7F and G).
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 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 differentiation toward enteric muscle cells. Cyan, DAPI; magenta, HCR-FISH; yellow, muscle antibody (HgM2); CE, coelomic epithelium; CT, connective tissue; Mu, muscle. Bars = 10 μm.
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 different 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 = 20 μm.
C9 represents the neuroepithelial cells
Cells in C9 represent another population of specialized cells that can be associated to cells previously described in the intestinal anlage. This small number (3%) of cells most likely corresponds to neuroepithelial cells that will eventually give rise to neurons. These cells express neuroepithelial or neuronal genes such as neurotrypsin (NETR), potassium gated-voltage channels, PRD10 (PRDM10 – PR domain zinc finger protein 10), ELAV2 and STA10 (STARD10 – START domain-containing protein 10) (Figure 3). The latter is a protein that we have characterized as being expressed by enteric neurons and nerve bundles (Rosado-Olivieri et al., 2017). The holothurian STA10 (STARD10) is recognized by our monoclonal antibody RN1. This antibody has been used to detect enteric neurons as they begin to differentiate in the coelomic epithelium during the second week of regeneration (Tossas et al., 2014). Among its associated GO terms are positive regulation of ion transmembrane transporter activity, cyclic nucleotide metabolic process, regulation of muscle contraction, regulation of membrane potential, and positive regulation of hormone secretion (Figure 3—figure supplement 2). Moreover, there is also a great representation of processes involved in development, differentiation, and growth of nerve cells, all of which together would be expected of a neuroepithelial layer. To verify the neuroepithelial nature of the cells, we performed HCR-FISH for the STA10 mRNA in the tissue and show that some cells in the regenerating mesentery and anlage coelomic epithelium express the gene (Figure 8). Moreover, double labeling with the HCR-FISH against STA10 and the RN1 antibody shows cells and fibers that express both the mRNA and the protein product (Figure 8) are forming fiber extensions typical of differentiating neurons (Figure 8C–E).
STARD-10 expression in differentiating neuroepithelial cells of the coelomic epithelium.
(A) Cells (arrowhead) expressing STA10 mRNA in the CE of the regenerating mesentery. (B) Double labeling with STA10 hybridization chain reaction fluorescent in situ hybridization (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) = 10 μm; (A, C–E) = 5 μm.
The characterization of these three clusters (C5, C8, C9), which represent cells undergoing differentiation or proliferation, leaves a group of four clusters (C0, C1, C3, and C4) that show some overlap in expressed genes and at the same time share some gene expression with some of the previously described clusters. Nonetheless, as seen in Figure 3A and B, the cells in these four clusters still have high expression and representation of specific transcripts.
C4 represents the intestinal coelomic epithelial cells
The gene expression profile of cells in C4 sets them slightly apart from the other three clusters (C0, C1, C3). It identifies cells that are more advanced in their development toward a particular phenotype. C4 shows high expression of genes such as KCNQ5 (potassium voltage-gated channel subfamily KQT member 5), SC6A9 (sodium and chloride-dependent glycine transporter 1), EFNB2 (Ephrin-B2), and UNC5C (Netrin receptor UNC5C) (Figure 3). Other than these, C4 also shows high expression of genes that are related to cell-cell interactions and ECM molecules such as LAMA2 (laminin subunit alpha-2), MEGF6 (multiple epidermal growth factor-like domains protein 6), FMN1 (formin-1), and NPHN (nephrin). This cluster, distinct from others, shows enrichment of GO terms related to more advanced stages of development, such as morphogenesis of epithelium, sensory perception, regulation of calcium ion transmembrane transport, among others (Figure 3—figure supplement 2). Noteworthy, cells from this cluster correspond mostly to cell from the mesentery samples (67%) rather than from the anlage (37%). Additionally, from all the mesentery cells, about 11% are part of this cluster, while only 4% of the anlage cells are represented here. HCR-FISH of UNC5C (Netrin Receptor), a gene differentially expressed in the cells of this cluster, provided a surprising result. The expression of this mRNA was observed as an intense labeling in cells of the normal intestine mesothelium (Figure 9A), In the regenerating tissue, the coelomic epithelium of the mesentery is labeled (Figure 9B and C) and some labeling is observed in the coelomic epithelium of the anlage (Figure 9D). This pattern of labeling clearly shows the cells to be part of the coelomic epithelium and strongly suggests that they correspond to cells that are in a differentiation pathway to become part of the coelomic epithelia (possibly the peritoneocytes) of the regenerated organ. This conclusion is strengthened by the pseudotime analyses presented in the following section.
Expression profile of coelomic cell populations in the regenerating intestine anlage and mesentery.
(A) Hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) for UNC5C mRNA labeled most if not all cells in the 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 = 10 µm.
C0, C1, and C3 represent differentiation stages of coelomic epithelial cells
The three remaining clusters to be analyzed are C0, C1, and C3. These three clusters share many of their top representative genes, which are associated with developmental, regenerative, or oncogenic processes (Bradford et al., 2009; Dunn et al., 2006; Lu et al., 2015; Nishimoto and Nishida, 2007; Oike et al., 2004; Zhao et al., 2008). These include, for C0: TRFM (melanotransferrin), TIMP4 (metalloproteinase inhibitor 4), and DMBT1; for C1: FGF13 (fibroblast growth factor 13), TGFB3 (transforming growth factor beta 3), HS90A (heat shock protein HSP 90-alpha), and ANGL1 (angiopoietin-related protein 1) (Figure 3); and for C3: SEM5B (semaphoring-5B), LRIG3 (leucine-rich repeats and immunoglobulin-like domains protein 3), TUTLB (protein turtle homolog B), and NET1 (netrin-1). Moreover, C0 and C1 share an over-representation of GO-enriched terms that highlights biological processes related to ribosomal activity such as cytoplasmic translation, and ribosome assembly, biogenesis, and assembly (Figure 3—figure supplement 2). In addition to these, C3 also showed enrichment of processes involved in the development of tubular lumen-containing structures such as mesonephric and ureteric ducts, differentiation and regulation of cell growth, and negative regulation of axogenesis (Figure 3—figure supplement 2).
C1 and C3 are also closely related in their localization, being overwhelmingly associated with the anlage. These two clusters are mostly composed of cells from the anlage tissue, where we expect to see the precursor cells that will give rise to specialized cells of the organ (Figure 1). Precisely, C1 and C3 cells together correspond to 37% of all anlage cells compared to 14.6% of all mesentery cells. In contrast, cells from C0 correspond to 27% and 18% of the mesentery and anlage cells, respectively.
To obtain insight into these cell clusters, we performed HCR-FISH for two different mRNAs; NET1 (Netrin), a chemotropic protein highly represented in C1 and C3, and TRFM (melanotransferrin) the top represented gene in C0. Both in situ hybridization experiments labeled cells in the coelomic epithelium of the regenerating intestine, supporting our contention that the large supra-cluster represents the coelomic epithelium layer (Figure 10). However, their spatial pattern of expression was unpredictably different. While NET1 was highly expressed in most of the coelomic epithelial cells of the anlage, little expression was found in the regenerating mesentery or in the coelomic epithelium of the normal intestine (Figure 10). TRFM, in contrast, was highly expressed in the coelomic epithelium of the normal intestine and poorly expressed in the intestinal anlage (Figure 10). In the regenerating mesentery, a gradient in expression of the TRFM is observed, where high levels of expression were found in the coelomic epithelium close to the body wall and diminished as one approached the anlage. Thus, the HCR-FISH results show that C0, C1, and C3 correspond to cells of the coelomic epithelium, but strongly suggest that C0 differs from C1 and C3 both in their gene expression profile and in the localization where they are found, both in the regenerating and in the normal intestine.
Hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) for TRFM and NET1 shows the differential gene expression of the anlage versus mesentery CE.
TRFM mRNA (left column) is expressed by (A) cells (arrowheads) of the 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. Bar = 10 µm.
Finally, it is essential to highlight the many signaling, or growth factors expressed by the coelomic epithelial clusters, in particular C0, C1, and C3 (Figure 3). These include Wnt, Hox, semaphorin, FGFs, TGF-beta, netrin, insulin-like growth factor (IGF), growth/differentiation factors (GDF), and angiopoietin-related proteins (ANGL) (e.g., WNT9, SEM5B, FGF13, NKx3.2, TGFB3, HOX9, IGF1, GDF8, ANGL1, and FOXF1) (Figure 3). This is important in view that the epithelium of the vertebrate blastema is characterized by its chemical modulation of the underlying mesenchyme, as will be discussed later. Likewise, other genes that serve as markers of specific cell types or cellular stages were also identified, including PIWL1 (piwi-like protein 1) in C1, YAP1 in C3 and C4, HES1 (transcription factor HES-1) in C1 and C3, and PRRX1 (paired mesoderm homeobox protein) in C0 and C4 (Figure 3, Figure 3—figure supplement 3).
In summary, of the 13 clusters identified, our data strongly suggest that 4 of them (C9, C10, C11, and C12) correspond to coelomocytes or immune cells, 2 of them (C2 and C7) correspond to cells with a mesenchymal phenotype, and the remaining 7 to cells of the coelomic epithelia. Of these seven, C5 corresponds to differentiating muscle, C9 to differentiating neuroepithelium, C4 to differentiating coelomic epithelia, and C8 to proliferating cells. C1 and C3 represent most of the cells found in the coelomic epithelium of the anlage. In contrast, C0 represents a coelomic epithelial phenotype more closely associated with the mesentery than the intestine.
Trajectory analysis: What populations are driving cell specification?
Among the many mysteries of the holothurian intestinal regeneration process is the identification of the precursor cells. In simple terms, what are the cells from which all nascent cells derive from? We performed a trajectory analysis of the data to provide some insights into this issue. This type of analysis is usually performed with samples at different stages or time points. However, we considered it feasible to conduct this analysis because in the 9-dpe regenerating anlage/mesentery we find cells at various stages of differentiation. These cells could provide crucial information on how the cell populations are associated with each other. To address this, we initially employed RNA velocity analysis. This method describes the temporal dynamics of gene expression based on the relative abundances of spliced and un-spliced mRNA across cell populations.
Our initial velocity analysis on all the clusters and samples (Figure 11A and B) provided three main results. First, the direction of arrows in our UMAP shows them flowing toward C5, C9, and C4. These arrows do not point toward any other cluster; thus, they are terminal arrows. Second, while arrows from C8 are not terminal, they are directed toward C1. This suggests that cells in C1 provide cells for the growth of the anlage via proliferation. Thus, these results support C5, C9, and C4 as terminal cell clusters that we have described as muscle, neuroepithelial, and the nascent coelomic epithelium cells, respectively. Third, velocity embedding shows shorter arrows that point from clusters 0 and 1 toward terminal populations previously described. Therefore, cells of C0 and C1 are not undergoing significant transcriptional changes. The RNA velocity results of the rest of the clusters are less interesting as they do not show directions toward any other clusters, mainly because of their individuality within the UMAP. However, it is interesting that arrows of the mesenchymal cell populations show distinct directions and lengths. Based on the results, it seems that portions of both mesenchymal clusters (C2 and C7) have gone or are undergoing more extensive differentiation changes.
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 reclustering 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 C9. (F) UMAP of reclustered anlage cells corresponding to C0, C1, C3, and C4. (G) RNA velocity results from the cluster results from panel (F). (H) UMAP of (F) plot overlayed with pseudotime results of Slingshot. Color represent pseudotime values from 0 (blue) to 60 (yellow). (I) Jitter plot showing the Slingshot pseudotime of cells from each cell cluster.
Pseudotime analysis of all clusters within the 9-dpe intestinal regeneration dataset.
Each color reflects the corresponding cluster from C0 to C12. The x-axis represents slingshot pseudotime and y-axis the corresponding lineage.
We then reclustered C0, C1, and C3 cells, along with the differentiating cell populations (C4, C5, and C9) to better understand their relationship (Figure 11C). The velocity assessment of these newly clustered populations resulted in a similar pattern (Figure 11D). Here, we can see that the differentiating cells (C4, C5, and C9) have long arrows, suggesting that these cells are going through an advanced stage of transcriptional change compared to others (Anderson et al., 2020; La Manno et al., 2018). It is clear from these results that C1 future states are cells of C3 and, to some extent, C4. Moreover, it seems that C0 has a closer relationship to cells of C5 and C9 and that some cells of these clusters could potentially differentiate into cells of C4 (the coelomic epithelium cells). In this case, to complement and confirm our RNA velocity interpretation, we also performed a pseudotime analysis using Slingshot, which relies on the expression data of each cluster. This analysis showed C1 in an earlier pseudotime than C3, C4, and C0 in the resulting two lineages (Figure 11D). The resulting lineages differed by the terminal clusters, one containing C5 (muscle) and the other C9 (neuroepithelial). Thus, it supports what we have already visualized on the RNA velocity embeddings.
The results described so far show that C0, C1, and C4 are cells in distinct differentiation states, but we wanted to have a clearer view of the cell clusters that potentially have an essential role in the regeneration process. For this, we made another subset of cells that corresponded to C0, C1, C3, and C4, but uniquely from cells of the anlage (Figure 11F). The rationale was that cells from the mesentery are certainly at a different state from those of the anlage and thus could interfere with the pseudotime of cells from the anlage. The RNA velocity analysis using this subset strengthened our previous inferences. First, C1 seems to be the least dedifferentiated cell cluster, whose future state will be cells of C3 and part of the population of C4. Second, that C0 seems to be in a specialized state of differentiation that has a relationship to C4 (Figure 11G). This would explain the relationship of this cluster to that of the differentiating cells of C5 and C9. Interestingly, portions of C1, C0, and C4 appear to be in an advance process of differentiation based on their longer arrows compared to C3 and another portion of C4 close to C3 (Figure 11G). The Slingshot analysis of the anlage cells from C0, C1, C3, and C4 revealed that the pseudotime starts at a point of convergence that contains a portion of cells from C1 and C0. Yet, it further supports the cells from C1 as the least differentiated (Figure 11H). C1 is then followed by cells of C3, C4, and, lastly, C0, which for the most part seems to be at a more advanced differentiation state with a closer relationship to differentiating cells (Figure 11I).
Discussion
In this study, we employed scRNA seq and HCR-FISH techniques to examine the cellular phenotypes in regenerating intestinal tissues of the sea cucumber H. glaberrima. These techniques have been seldom used to characterize echinoderm cells, and the few studies available are mainly limited to embryonic stages of sea urchins (Strongylocentrotus purpuratus, Lytechinus variegatus) and the sea stars (Patiria miniata) (Cocurullo et al., 2023; Foster et al., 2022; Meyer et al., 2023; Paganos et al., 2022a; Paganos et al., 2022b; Paganos et al., 2021; Tominaga et al., 2023). Nonetheless, these studies provide an excellent description of the cell population and dynamics arising from major germ lines during echinoderm development. We now apply the same techniques to explore cell phenotypes involved in intestinal regeneration in holothurians. This is, to our knowledge, the first time scRNA-seq and HCR-FISH have been used in adult echinoderms to analyze the cellular and molecular basis of their amazing regenerative properties. This research integrates the extensive cellular and molecular information on intestinal regeneration in holothurians collected over the past two decades, offering a comprehensive view of the cellular phenotypes and molecular changes involved.
Cell types and differentiation stages in the 9-dpe regenerating intestine
Our study has focused on the description of cells present in the 9-dpe regenerating intestine of the sea cucumber, where cells are known to have gone through dedifferentiation and are, at this timepoint, in the process of differentiating into specialized cells (García-Arrarás et al., 2011). Based on our analysis, we have identified 13 distinct populations that form part of the regenerating intestinal mesentery and anlage (Figure 2A). One intriguing finding was the lack of significant differences in the cell clusters between the anlage and the mesentery. However, this can be explained by two different facts. First, we have previously shown that many of the cellular processes that take place in the anlage, including cell proliferation, apoptosis, dedifferentiation, and ECM remodeling, occur in a gradient that begins at the tip of the mesentery where the anlage forms and extends significantly into the mesentery (García-Arrarás et al., 2019; García-Arrarás et al., 2006; García-Arrarás et al., 2011; Mashanov et al., 2012; Reyes-Rivera et al., 2024). Similarly, migrating cells move along the connective tissue of the mesentery to the anlage (Cabrera-Serrano and García-Arrarás, 2004). Thus, there is no clear partition of the two regions that would account for distinct cell populations associated with the regenerative stage. Second, the two cell populations that would have been found in the mesentery but not in the regenerating anlage, mature muscle and neurons, were not dissociated by our experimental protocol as to allow for their sequencing. Current experiments are being done using single-nuclei RNA sequencing to overcome this hurdle.
Among the 13 clusters we have described, the major divisions are clusters corresponding to the coelomocyte, coelomic epithelium, and mesenchyme cell types (Figure 3). Thus, we can now use the information that our laboratory and others have gathered to pinpoint the cellular mediators and their activity during regeneration. The coelomic epithelia, for example, is highly influential in the regeneration process as it is the site where major cellular events occur, particularly cell division, dedifferentiation, and differentiation (García-Arrarás et al., 2011). Moreover, the mesenchyme ECM is known to undergo remodeling during the regeneration process, which is also critical for the proper growth of the new tissue (Quiñones et al., 2002). However, while much of the events have been described using microscopic and histological tools, we need a more comprehensive understanding of the transcriptomic characteristics of specific cell populations involved in the regeneration process.
The sea cucumber contains various mesenchymal and coelomocyte populations
Among the phenotypes identified are clusters of mesenchymal (C2 and C7) and coelomocyte cell populations (C6, C10, C11, and C12). The mesenchymal populations demonstrated a unique expression of ERG and ETS-1. ERG, in particular, has been related to embryonic development, differentiation, angiogenesis, and apoptosis (Dhordain et al., 1995; Iwamoto et al., 2001; Vlaeminck-Guillem et al., 2000; Figure 2). More importantly, the expression of ERG has been associated with mesenchymal identity in other echinoderms (Meyer et al., 2023; Tominaga et al., 2023). In these studies, ERG has been reported as the marker gene of mesenchymal cells of sea urchin and sea star larva by scRNA-seq analyses, and the localization of ERG on embryonic precursor mesenchymal cells of the sea urchin was further confirmed by in situ analyses (Meyer et al., 2023). In addition, this latter group also reported these mesenchymal cells to express a GATA transcription factor (GATA3) and ETS1, which in our dataset are also being expressed only by populations within this supra-cluster (GATA2 and ETS1;Figure 3B). The expression of PRG4 (proteoglycan-4) reinforces the mesenchymal identity of this supra-cluster in the regenerating intestine (Figure 2—figure supplement 2), which correlates with previous studies that have identified a proteoglycan-like molecule in the mesenchyme of 7-dpe regenerating intestine (Vázquez-Vélez et al., 2016).
ETS-1 has long been associated with developmental processes of mesenchymal formation in sea urchin (Koga et al., 2010; Rizzo et al., 2006). In other studies, ERG, a member of the ETS gene families, has been found to be necessary for controlling mesenchymal identity and differentiation (Cox et al., 2014; Mochmann et al., 2014). Further analyses of their individual marker genes suggest that these cells might be involved in EMT. For instance, C2 expresses TIMP3, a metalloproteinase inhibitor that aids in the ECM remodeling (Dewing et al., 2020), and NPNT, a gene reported to be related to development and cancer processes (Magnussen et al., 2021). These genes are also crucial for cells undergoing EMT as the cells need to detach from the other cells and the basal lamina that forms the epithelium. Comparatively, the expression profile of C7 with genes such as PA21B (phospholipase A2), TMPS9 (transmembrane protease serine 9), SEPP1, DMBT1, ITIH3, and SVPE1, and its GO results suggest this mesenchymal population is undergoing different processes. Based on these contrasting expression profiles, we propose that C2 corresponds to cells that have recently undergone EMT from the coelomic epithelium and eventually differentiate into a more specialized phenotype (C7). Our pseudotime results further support this developmental transition as cells in C7 appears to be in a more advanced stage compared to those of C2 (Figure 11—figure supplement 1). Therefore, these two populations correspond to the first transcriptomic description of mesenchymal phenotypes reported in sea cucumber regenerating intestine.
Our dataset contains four distinct populations that we have characterized as coelomocytes. The coelomocyte populations reported in different holothuroid species ranged between 4 and 6 distinct types (Hetzel, 1963; Ramírez-Gómez et al., 2010; Xing et al., 2008). Studies from our laboratory previously revealed four different coelomocyte populations in H. glaberrima, distinguished by their morphology, histochemistry, and phagocytic activity. These were lymphocytes, phagocytes, spherulocytes, and a population named ‘giant cells’ (Ramírez-Gómez et al., 2010). The distinctive gene expression profile of each of the coelomocytes that we have identified can provide insights into the differences in their role as immune/circulating cells within the sea cucumber. For example, the C6 marker gene fibrinogen-like protein, which is part of a protein family known as FREP, makes this population of great interest. Mainly because these molecules have been vastly studied across invertebrates, and multiple immune roles have been proposed, including phagocyte recognition and encapsulation (Hanington and Zhang, 2011). A distinct example is that of C10, where the expression of HCK, FER, and ITF8 markers suggests this might be a macrophage-like activity (Chen et al., 2023; Dolgachev et al., 2018; Shuttleworth, 2018). Specifically, HCK, a member of Src family kinases (SFK), has been closely related to macrophage activation and polarization (Bhattacharjee et al., 2011; Poh et al., 2015). Interestingly, recent studies in A. japonicus found that an Src homolog mediates the phagocytosis of Vibrio splendidus, which further supports C10 immune identity (Wan et al., 2022). Thus, to our knowledge, this would be the first report of the expression profile of distinct coelomocyte populations in an adult echinoderm species, setting up the stage for integrating these populations with the previously described ones.
The coelomic epithelia of the intestinal anlage is composed of a heterogeneous population of cells
Our research findings align with previous microscopic descriptions of cells in the normal and regenerating mesothelium and coelomic epithelia. In our data, we can easily identify the cell population that forms the cluster exhibiting a proliferative phenotype (C8). Proliferative cells in the regenerating intestine are primarily localized within the coelomic epithelium of the anlage (García-Arrarás, 1998; García-Arrarás et al., 2011; Reyes-Rivera et al., 2024), and the gene expression profile documented here unequivocally identifies these as proliferative. These cell proliferation genes are specific to C8, with minimum representation in other populations. Interestingly, as mentioned before C8 expresses at lower levels many of the genes of other coelomic epithelium populations. Nevertheless, even if we mask the top 38 proliferation genes (not shown), this cluster is maintained as an independent cluster, suggesting that its identity is conferred by a complex transcriptomic profile rather than only a few proliferation-related genes. Therefore, the identity and potential role of C8 could be further described by two distinct alternatives: (1) cells of C8 could be an intermediate state between the anlage precursor cells (discussed below) and the specialized cell populations or (2) cells of C8 are the source of the anlage precursor populations from which all other populations arise. The pseudotime data is certainly complex and challenging to interpret with our current dataset, yet the RNA velocity analysis shown in Figure 11B would suggests that cells of C8 transition into the anlage precursor populations, rather than being an intermediate state. This is also supported by the Slingshot pseudotime analysis that incorporates C8 (Figure 11—figure supplement 1). Nevertheless, additional experiments are needed to confirm this hypothesis.
A second population of cells that can be well correlated to previously described cells is that with a muscle cell phenotype (C5). The evidence suggests that this cell population represents those cells from the coelomic epithelium that are differentiating into myocytes. This evidence includes (1) the top-expressed genes by the cells in this cluster are all muscle-associated genes; (2) the top-enriched terms are all related to muscle morphogenesis; (3) the cluster is mostly composed of cells that come from the anlage where muscle formation is known to be taking place at this stage (Murray and García-Arrarás, 2004); (4) differentiated muscle cells were not dissociated by the enzymatic procedure strongly suggesting that the muscle cells that we have identified in our data are those that are in a differentiation process, rather than fully differentiated cells closer to the body wall; and (5) the cells in this cluster were identified by HCR-FISH of MYH7. These cells are localized toward the basal region of the coelomic epithelium, the region where the differentiating myocytes are known to be present (Murray and García-Arrarás, 2004). Moreover, among the top expressed genes of this cluster is Troponin I (TNNI1), which has also been reported to be highly expressed in muscle precursor cells of the sea star embryo and immature cardiomyocytes of the chicken (Mantri et al., 2021; Tominaga et al., 2023). The high differentiation activity of these cells is also supported by our pseudotime analysis, where distinct cells within the cluster are in individual differentiation states (Figure 11D and E). Further analysis of these populations could allow us to understand the transcriptional changes these cells undergo to become fully specialized muscle cells.
An additional population in our dataset is the neuroepithelial population (C9). This population has STARD10, among its top expressed genes, known to be a phospholipid transfer protein present in a neural cell population. This population has been localized in the coelomic epithelium of the intestine, among other sites (García-Arrarás et al., 2019; Rosado-Olivieri et al., 2017). Furthermore, cells of this cluster have high expression of other genes reported to be expressed by neuronal cells of regenerative and developmental tissues, such as beta-tubulin in developing human gut and sea urchin larva, and synapsin in planaria regenerating tissue (Cocurullo et al., 2023; Elmentaite et al., 2020; King et al., 2024). Interestingly, an earlier study showing that beta-tubulin-positive cells arise from dedifferentiated cells of the regenerating intestinal tissue of the sea cucumber (García-Arrarás et al., 2019) also suggests that similar to the muscle population, these cells are neuroepithelial cells that are differentiating rather than fully specialized. Further support for this theory lies in the main contribution to C9 coming from anlage cells and their terminal differentiation state observed in the pseudotime analysis (Figure 11D).
The remaining four clusters of the coelomic epithelium supra-cluster are more challenging to characterize. Nonetheless, we will explore some hypotheses regarding their cellular phenotypes. The identity of C4 was put forward based on its close transcriptional and pseudotime correlation with populations of the coelomic epithelia (Figures 3 and 11). Additionally, in our in situ hybridization, its marker gene UNC5C as well as another gene SC6A5 (not shown here) are mainly expressed by the coelomic epithelial cells of the normal intestine, strongly suggesting that the cells expressing this gene in the anlage are those that will become the coelomic epithelial cells of the regenerated organ (Figure 9).
C0 remains an intriguing cell population. On one hand, it appears to be closely related, by its gene expression, to other coelomic epithelial populations (Figure 3). Thus, it may represent an unknown cell population or cell stage. The in situ expression suggests that this cluster represents the cellular population of the mesentery or peritoneal coelomic epithelium. In this case, we might be evidencing differences in the coelomic epithelium of the mesentery (exemplified by C0) from those of the coelomic epithelium of the intestine (exemplified by C4). Future experiments will be needed to address this controversy.
Lastly, C1 and C3 share many common genes and represent distinct populations but are still closely associated, regardless of the clustering parameters or the statistical assessment performed. Although we considered combining them into a single cluster, we ultimately decided against it as they likely represent different stages of cell development or plasticity in the regenerating intestine. One-to-one comparisons revealed that C1 expressed various ribosomal gene markers, while C3 expressed specific genes such as EPHA4 (ephrin type-A receptor 4) and LRIG3, indicating distinct transcriptomic states. Additionally, all trajectory analyses revealed that C1 cells appear to give rise to C3 cells.
Further characterization of C1 and C3, within the anlage coelomic epithelium, suggests that these cells probably serve as cellular precursors to differentiating cells. The evidence from pseudotime analyses and gene expression of C1 and C3 strongly supports this conclusion. These cells appear to exhibit pluripotency with the potential to form muscle, neurons, coelomic epithelia, and mesenchymal cells in the regenerating intestine. Their gene expression profiles include markers from gene families associated with embryonic development such as Hox, zinc fingers, basic helix-loop-helix, and others, some of which are linked to stemness and pluripotency. For example, HSP90A, a molecular chaperone essential for stem cell pluripotency, and markers like HES1 and TGFb, essential for maintaining stem cell proliferation, are present in these cell populations (Aztekin, 2021; Mishra et al., 2005). Moreover, the expression of PIWL1 (piwi-like protein 1) and YAP1 further supports the classification of these clusters as precursor cells of the intestinal anlage (Figure 3—figure supplement 3). In hydra, piwi-like molecules are exclusively localized in stem/progenitor cells (Juliano et al., 2014), and in mice, YAP1 is expressed only in multipotent cells during intestinal epithelium regeneration, vital for their emergence (Ayyaz et al., 2019). Ultimately, trajectory analysis also supports C1 as a precursor cell population, as the dividing cells (C8) appear to give rise to C1, which then progresses toward C3. This analysis aligns with evidence of a proliferation center in the coelomic epithelia that provides precursor cells essential for the growth of the anlage.
In summary, C1 and C3 cells are probably dedifferentiated cells from the mesentery mesothelium, retaining markers common to all epithelial cells. Similar to what is known in other highly regenerative organisms, these dedifferentiated cells can proliferate and then redifferentiate into the cells of the new organ. Future experiments will determine whether they retain the memory of their previous phenotype (muscle or coelomic epithelium) or are completely pluripotent.
The coelomic epithelium of the intestinal anlage is pluripotent
Our study reveals that the coelomic epithelium, as a tissue layer, is pluripotent. It fulfills the definition of pluripotency; a group of cells that has the ability to differentiate into many, but not all, cell types. Microscopy studies across different echinoderm species have consistently suggested that the coelomic epithelium can differentiate into various cell types. Among these are the formation of muscle cells and neurons in sea cucumbers (Dolmatov et al., 1996), mesenchymal cells in brittle star (Piovani et al., 2021), and even immune cells (coelomocytes) in sea star (Byrne et al., 2020; Sharlaimova et al., 2021). In some species, the coelomic epithelium has even been proposed to transdifferentiate into intestinal luminal cells (Mashanov et al., 2005). In our analysis, we can identify the cells in the coelomic epithelium that are differentiating toward muscle (C5), neurons (C9), coelomic epithelium (C4), and mesenchymal cells (C2 and C7) corroborating what has been previously described in H. glaberrima studies. Nonetheless, it remains unclear whether individual cells are pluripotent, thus a more definitive conclusion will be reached once lineage-tracing experiments can be done.
The coelomic epithelium of the regeneration anlage could also be the source of other cell types. This is exemplified in the brittle star Marthasterias glacialis, where it has been suggested that the coelomic epithelium is not only involved in arm regeneration but also serves as a source of immune cells (Guatelli et al., 2022). This report indicated that ‘residential stem cells’ in the regenerating arm of the brittle star originate from the coelomic epithelia (Candia-Carnevali et al., 2009). Similarly, in the sea cucumber Holothuria forskali, the injured mesothelial layer has been identified as the source of undifferentiated cells that differentiate into the cells of the growing organ and into phagocytic cells, now recognized as coelomocytes (VandenSpiegel et al., 2000).
Of particular interest, our study reveals that the mesentery coelomic epithelia population (C0) exhibits localized expression of SAA1, an immune response-related gene. This finding, in line with previous reports of SAA1 localization in the coelomic epithelium of the regenerating intestinal tissue (Santiago et al., 2000; Figure 3—figure supplement 3), raises the intriguing possibility that the coelomic epithelium of the sea cucumber regenerating intestine could also be the source of coelomocytes, as suggested by Guatelli et al., 2022. This hypothesis is further supported by the observation that in the sea star Asteria rubens, coelomocytes arise from its coelomic epithelium (Vanden Bossche and Jangoux, 1976). However, our current results, while suggestive, do not yet provide conclusive evidence to support this in the sea cucumber, at least not in the coelomic epithelia of the 9-dpe regenerating intestine.
Currently, there is no definitive evidence identifying the precursor cells that give rise to these cell populations in echinoderms. However, given our gene expression data and the interactions observed among the cell populations, we postulate that cells from C1 stand as the precursor cell population from which most of the cells in the anlage coelomic epithelium arise. This is based on their preferential localization within the anlage coelomic epithelium, their lack of any clear differentiated cell marker, and their association with the cell dividing cluster. Granted, much more experimental evidence will be needed to arrive at this conclusion. Nonetheless, it provides a focus on the most likely, and probably most intriguing, cell population. It also provides the path to explore multiple questions that arise from our results. Can C1 be subdivided into other cell populations? Are the cells in this cluster pluripotent? Do these cells originate from the same cells via dedifferentiation? These and many other questions will indeed be tested in future experiments.
The intestinal anlage as a regeneration blastema
The intestinal anlage is a particular regenerative structure that has long been described as a blastema-like structure due to its remarkable morphological resemblance to the ‘classical blastema’ (García-Arrarás, 1998). As stated previously, a blastema is usually described as a transient structure composed of a mass of proliferating undifferentiated cells. This structure is found at the site of injury and will give rise to the regenerated organ (Seifert and Muneoka, 2018). The blastema cells can originate from different lineages across species. For example, in some amphibians the blastemal cells originate from dedifferentiated muscle, cartilage, fibroblast, and other tissues, while in others they include cells that originate from muscle satellite cells, a type of stem cell (Globus et al., 1980; Sandoval-Guzmán et al., 2014). In Planaria, they originate from undifferentiated stem cells known as neoblasts (Baguñà, 2012; Wagner et al., 2011). The blastema is overlayed by a wound epidermis that is formed by a re-epithelization process following injury, which develops into a specialized wound epidermis that in amphibians is known as the apical epithelial cap (AEC) (Aztekin, 2021). Since its first description, more than 100 years ago, the blastema has been regarded as the best indicator of regeneration, and its presence is associated with tissues or organs that are highly regenerative. In fact, it has been proposed that the presence or absence of a blastema defines the regenerative success or failure of the regeneration process. However, as more regenerative species continue to be studied, the classical definition of a blastema has been reconsidered, moving toward its functional role rather than its histological or structural characteristics (Seifert and Muneoka, 2018).
The blastema of salamanders and newts have long served as models to describe a blastema cellular and molecular properties (Globus et al., 1980; Scimone et al., 2022; Tajer et al., 2023). The sea cucumber intestinal anlage stands as a different structure in terms of the tissue compartmentalization of certain activities. Yet, when examined closely, much of the processes and signaling molecules described in the amphibian blastema also take place in the holothurian anlage, although their spatial occurrence might differ. For instance, we and others have previously described the dedifferentiation process by which muscle cells dedifferentiate and proliferate (García-Arrarás et al., 2011; Dolmatov, 2021). Thus, similar to some amphibian blastema cells, the cells of the holothurian anlage coelomic epithelium are proliferative undifferentiated cells that originated via a dedifferentiation process. Moreover, some of the genes expressed by the amphibian blastema cells or the overlying AEC, such as Wnt, Tgf-beta, and Fgf, are also known to be expressed during sea cucumber regeneration (Auger et al., 2023; Zeng et al., 2023). In amphibians, some of these factors are thought to be released by cells of the AEC, serving as a way of modulating the blastema cell activity. For example, FGF1 is expressed in the cells of the AEC while FGF receptors were localized to blastema cells (Zenjari et al., 1996). Moreover, both FGF and Wnt signaling have been shown to be vital for the formation of the blastema (Makanae et al., 2014).
This expression of Fgf and Wnt and their possible functions correlate with what has been found in sea cucumbers. Not only are the same growth factors expressed in C1 and C3, but a recent study highlighted the role of FGF4 as a modulator of cell proliferation during the intestinal regeneration of the sea cucumber A. japonicus (Zeng et al., 2023). This group demonstrated that inhibiting FGF4 and its receptor, FGFR2, negatively affected cell proliferation of the mesothelial layer during intestinal regeneration. Similar results have been shown for Wnt, primarily a study by our laboratory demonstrating that Wnt pathway inhibition, either by pharmacological drugs or by RNAi, caused a reduction in cell proliferation (Alicea-Delgado and García-Arrarás, 2021; Bello et al., 2020). Thus, in the sea cucumber coelomic epithelium of the anlage, cells are also involved in intercellular communications that modulate cellular dynamics through factors similar to those found in the AEC-blastema cell modulation.
The holothurian anlage and the amphibian blastema exhibit some differences. In the latter, there is a clear separation of the AEC and the underlying blastema cells. In amphibians, the AEC has been shown to actively participate in the formation and maintenance of the blastemal cells, particularly in their proliferation and differentiation. In contrast, in holothurians, cells with both blastema and AEC characteristics can be found within the coelomic epithelium. In fact, both PRRX1 (paired mesoderm homeobox protein), a gene expressed by amphibian blastema cell precursors (Gerber et al., 2018; Lin et al., 2021), and HES1 (transcription factor HES-1), a marker gene of the amphibian AEC, are expressed by the holothurian coelomic epithelium (Aztekin, 2021; Figure 3, Figure 3—figure supplement 3).
Our data has allowed us to construct a model of the cell populations we identified in the 9-dpe intestinal anlage (Figure 12). This model presents the coelomic epithelium of the anlage as a heterogeneous layer of cells comprising of six cell populations corresponding to distinct differentiation states. Among these are cell populations that seem to be in the process of differentiation toward muscle (C5), neuroepithelium (C9), and intestine coelomic epithelial cells (C4). We also propose the presence of undifferentiated (C0, C1, C3) and proliferating cell populations (C8) in the coelomic epithelia, which give rise to the cells in this layer. Underneath the coelomic epithelia are the mesenchymal cells (C2, C7), some of which may have originated from the coelomic epithelial layer via EMT. Moreover, the sea cucumber intestinal anlage behaves as a blastema since it uses similar mechanisms to fulfill the same role in the regeneration of the new organ. In this case, the cells of the coelomic epithelium would be those considered akin to the blastemal cells. Our results, thus, provide an alternative view of cell precursors and regenerating structures with similarities and differences to those of better-studied regenerating animal models.
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. (A) A diagram showing the intestinal anlage and a portion encompassing coelomic epithelia and connective tissue that would be represented by the cell populations shown in (B). 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 Figure 2.
MethodsAnimals and sample collection
Two adult sea cucumbers were collected from the northeastern rocky region of Puerto Rico and were kept in aerated sea water for acclimatization prior to experiments. Evisceration of the sea cucumber was stimulated by intracoelomic injection of 0.35 M KCl as previously described (Reyes-Rivera et al., 2024). They were maintained in sea water aquaria for 9 days to undergo regeneration until dissection. Before the dissection, animals were anesthetized by immersion in ice-cold water for 45 minutes. Dissection was done through an initial dorsal incision which allowed exposition of the internal organs, upon which the growing anlage (or rudiment) and mesentery were dissected and separated from each other. Each tissue was kept in CMFSS on ice and treated separately during the tissue dissociation process. Thus, tissues were dissected for four scRNA-seqs; two anlage samples and two mesentery samples.
Tissue dissociation
Mesentery and anlage tissues were digested for 15 minutes, in rocking shaker at room temperature with 1 mL of 0.05% Trypsin/0.02% EDTA solution prepared in Ca++ and Mg++ free sea water (CMFSS) (Bello et al., 2015). Digestions were quenched by adding 500 µL of 0.2% bovine serum albumen (BSA) in CMFSS and then centrifuged for 2 minutes at 1500 rpm. The supernatants were discarded, the pellets resuspended with 500 µL of 0.04% BSA in CMFSS, and the cells were gently separated by pipetting using glass pipettes with fire-blunted tips. The cell suspensions were filtered using a nylon cell strainer with 70 mm mesh, and aliquots were taken for cell counting. Cell viability was assessed through manual cell counting with hemocytometer to confirm that a viability higher than 90% was maintained. Sample suspensions were adjusted to 1000 cells/mL using CMFSS as required for sequencing procedures.
Immuno- and cytochemistry
50 µL of dissociated cell sample (from the same samples that were used for scRNAseq) were placed on polysine-treated slides and fixed with 50 µL of 4% paraformaldehyde and left to dry overnight. Slides were washed in phosphate buffered saline (PBS) prior to use for immune and or cytochemistry. The methodology for the immunofluorescence techniques/preparation of slides was followed as published except for the time of PBS washes, which were of 10 minutes instead of 15 minutes (Díaz-Balzac et al., 2007; Reyes-Rivera et al., 2024). The primary and secondary antibodies used are in Figure 1—source data 1. Samples to be probed with fluorescent phalloidin were treated directly with Phalloidin-TRITC (1:1000; Sigma P1951) for 1 hr as described previously (Reyes-Rivera et al., 2024). DAPI was incorporated into the mounting medium as described previously (Reyes-Rivera et al., 2024). Cells were observed in a Nikon DS-Qi2 fluorescent microscope.
Single-cell RNA sequencing and data analysis
Single-cell libraries were prepared using the 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1 and the Chromium 10X instrument, following the protocol from manufacturer (CG000204). Sequencing was carried out with Illumina NextSeq 2000 at the Sequencing and Genotyping Facility of the University of Puerto Rico Molecular Science Building.
The raw sequencing reads from all four samples (two mesentery and two anlage) were processed individually using Cell Ranger (v7.1.0) (Zheng et al., 2017) using the reference genome and gene models of H. glaberrima available at http://blastkit.hpcf.upr.edu/hglaberrima-v1. Gene models were annotated against human reference proteins from UniProt and the entire UniProt reference protein database. Gene IDs throughout the study correspond to the annotations with the human reference protein sequences from UnitProt.
Samples quality was assessed through the number of UMI, genes, and overall complexity of gene expression. The number of UMI and genes was found to be over 500 and 300, respectively (Figure 2—figure supplement 1). Furthermore, the distribution of genes per cells was found to be unimodal. In general aspects, samples from each animal were similar, with the total number of cells from the anlage tissue higher than that of the mesentery. General sequencing statistics after mapping reads with Cell Ranger can be found in Figure 2—source data 1.
Initial quality filtering was conducted in R (v4.3.2) using SoupX (v1.6.2) with default parameters to remove ambient RNA (Young and Behjati, 2020). Subsequent data processing was carried out using Seurat (v4.3.0.1) (Hao et al., 2024; Hao et al., 2021; Satija et al., 2015; Stuart et al., 2019). After data normalization and identifying variable features (n=2000), 22,970 anchors were identified using 20 dimensions. The datasets were integrated using the canonical correlation analysis approach of Seurat, followed by data scaling for principal component analysis (npcs = 50) and Uniform Manifold Approximation and Projection (UMAP) dimensional reduction technique (dims = 1:20).
Neighbors were then identified with 20 dimensions, leading to cluster identification at a resolution of 0.5. Various resolution parameters (0.8, 1.2, and 1.4) and dimensions (up to 35 in increments of 5) were tested before the final resolution value. The statistical analysis tool scSHC was employed to assess the probability of each cluster being unique (Grabski et al., 2023). Marker genes of each cluster or supra-clusters were identified using the FindMarkers() function in Seurat and mapped to the appropriate UniProt IDs. Genes highlighted throughout the study were further validated using the NCBI non-redundant reference database and EchinoBase (Telmer et al., 2024) via BLAST.
Differential expression data across clusters was used to perform gene set enrichment analysis of Gene Ontology (gseGO) biological processes terms using clusterProfiler (v.10.0) (Yu et al., 2012) with a p-value cutoff of 0.05. For this analysis, BLASTp was used to map H. glaberrima gene models to human reference protein sequences from NCBI (GCF_000001405.40), facilitating the assignment of ENTREZ ID to the correct human GO terms in the AnnotationDbi (v1.64.1) human database (v2.1) (Pagès et al., 2024).
RNA velocity loom files were generated with velocyto (v0.17), which relied on genome-masked regions obtained from RepeatModeler (v2.0.5) and RepeatMasker (v4.1.5) (Smit et al., 2015; Smit and Hubley, 2015), along with the sea cucumber gene models and CellRanger dataset. These files were further analyzed with velocyto.R (v0.6) and SeuratWrappers (v0.2.0), using the ReadVelocity and RunVelocity functions. Pseudotime analysis for the distinct data subsets was conducted using Slingshot (v2.10.0) (Street et al., 2018). The code of the data analysis has been made available at GitHub (copy archived at Medina, 2024).
HCR-FISH
Regenerating intestines from animals eviscerated 8–9 days previously and intestines from non-eviscerated (controls) animals were collected and fixed in 4% (v/v) paraformaldehyde with phosphate-buffered saline (0.01 M PBS; 0.138 M NaCl; 0.0027 M KCl; pH7.4) overnight at 4℃. Tissues were then washed three times by PBS and treated overnight with 40% saccharose prior to cutting in the cryostat. Sections (20 mm) were prepared in a cryostat (Leica CM1850), as previously published for immunohistochemistry (Reyes-Rivera et al., 2024). Gene spatial expression was determined by designing 12 or 24 set split probes for each gene marker. To decrease probe unspecific binding and increase its signal to background ratio, probes were designed as described by H. Choi and colleagues (Molecular Instruments) (Choi et al., 2018). To ensure probe gene target specificity, all nucleotide regions selected for probe design underwent extensive validation using the newly developed H. glaberrima genome and transcriptome alignment tool (https://blastkit.hpcf.upr.edu/hglaberrima-v1/; Medina-Feliciano et al., 2021). Probes were used at a final concentration of approximately 20–25 nM.
Immediately after slide preparation, HCR-FISH v3 was carried out using a modified version of Molecular Instrument’s fresh fixed frozen tissues protocol. Probe hybridization was conducted overnight at 37℃, while DNA hairpin amplification (B1-546nm or B2-546nm) was done at 25℃ overnight with 3 pmol of h1 and h2, respectively. Our protocol modification involved replacing ethanol with methanol during sample permeabilization. Also, the use of proteinase K was omitted, and Tween 20 at 0.1% was added to all PBS wash buffers. Once slides were prepared, image acquisition was attained using a Nikon DS-Qi2 fluorescent microscope. Positive control probes (PolyA) were used as signal calibrators to define background from positive signal during image analysis (Figure 4—figure supplement 2). Negative controls with fluorescent hairpins only can be found in Figure 7—figure supplement 1. Each probe was studied in at least three different animals, none of which were the specimens used for the scRNA-seq.
Additional informationCompeting interests
No competing interests declared
Author contributions
Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
Conceptualization, Resources, Data curation, Supervision, Visualization, Methodology, Project administration, Writing – review and editing
Investigation, Methodology
Investigation
Methodology
Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing
Ethics
This study involves the use of sea cucumbers (Holothuroidea), which are marine invertebrates. As such, they do not require approval by the Institutional Animal Care and Use Committees (IACUC), which generally govern vertebrate animal research. Nonetheless, all procedures involving sea cucumbers were conducted in accordance with standard scientific practices for invertebrate species, ensuring ethical treatment and minimal harm to the animals involved.
Additional filesData availability
Data generated during this project has been made publicly available at Figshare (Medina-Feliciano et al., 2024) including raw and processed sequencing data. The code of the data analysis has been made available at GitHub (copy archived at Medina, 2024).
The following dataset was generated:
Medina-FelicianoJGGarcía-ArrarásJE2025Single-cell RNA sequencing of the holothurian regenerating intestinefigshare10.6084/m9.figshare.c.7289770.v1
Acknowledgements
We thank the funding support from the National Institute of Health (NIH) under the grant number 2R15GM124595. Also, funding support was provided by the NIH Research Initiative for Scientific Enhancement (RISE) program to YMN under grant number 5R25GM061151-22. We acknowledge the High-Performance Computing Facility of the University of Puerto Rico and the Sequencing and Genotyping Facility sponsored by the University of Puerto Rico and the Institutional Development Award (IDeA) INBRE grant P20 GM103475 from the National Institute for General Medical Sciences (NIGMS), a component of the NIH and the Bioinformatics Research Core of INBRE. We acknowledge Echinobase for providing access to valuable genomic and biological data that greatly supported our research. Their resources and tools were instrumental in achieving the results presented here. We would also like to acknowledge our colleague Joseph F Ryan for his constructive feedback and computational resources.
ReferencesAlicea-DelgadoMGarcía-ArrarásJE2021Wnt/β-catenin signaling pathway regulates cell proliferation but not muscle dedifferentiation nor apoptosis during sea cucumber intestinal regenerationDevelopmental Biology48010511310.1016/j.ydbio.2021.08.01134481794AlvaradoASTsonisPA2006Bridging the regeneration gap: genetic insights from diverse animal modelsNature Reviews Genetics787388410.1038/nrg1923AndersonDSkutPHughesAMFerrariETicknerJXuJMullinBHTangDMalingeSKeesURKotechaRSLassmannTCheungLC2020The bone marrow microenvironment of pre-B acute lymphoblastic leukemia at single-cell resolutionScientific Reports101917310.1038/s41598-020-76157-433154494AugerNAMedina-FelicianoJGQuispe-ParraDJColón-MarreroSOrtiz-ZuazagaHGarcía-ArrarásJE2023Characterization and expression of holothurian wnt signaling genes during adult intestinal organogenesisGenes1430910.3390/genes1402030936833237AyyazAKumarSSangiorgiBGhoshalBGosioJOuladanSFinkMBarutcuSTrckaDShenJChanKWranaJLGregorieffA2019Single-cell transcriptomes of the regenerating intestine reveal a revival stem cellNature56912112510.1038/s41586-019-1154-y31019301AztekinC2021Tissues and cell types of appendage regeneration: A detailed look at the wound epidermis and its specialized formsFrontiers in Physiology1277104010.3389/fphys.2021.77104034887777BaguñàJ2012The planarian neoblast: the rambling history of its origin and some current black boxesThe International Journal of Developmental Biology56193710.1387/ijdb.113463jb22252540Barela HudgellMAGrayferLSmithLC2022Coelomocyte populations in the sea urchin, Strongylocentrotus purpuratus, undergo dynamic changes in response to immune challengeFrontiers in Immunology1394085210.3389/fimmu.2022.94085236119116BeckCWIzpisúa BelmonteJCChristenB2009Beyond early development: Xenopus as an emerging model for the study of regenerative mechanismsDevelopmental Dynamics2381226124810.1002/dvdy.2189019280606BelloSAAbreu-IrizarryRJGarcía-ArrarásJE2015Primary cell cultures of regenerating holothurian tissuesMethods in Molecular Biology118928329710.1007/978-1-4939-1164-6_1925245701BelloSATorres-GutiérrezVRodríguez-FloresEJToledo-RománEJRodríguezNDíaz-DíazLMVázquez-FigueroaLDCuestaJMGrillo-AlvaradoVAmadorAReyes-RiveraJGarcía-ArrarásJE2020Insights into intestinal regeneration signaling mechanismsDevelopmental Biology458123110.1016/j.ydbio.2019.10.00531605680BeumerJCleversH2021Cell fate specification and differentiation in the adult mammalian intestineNature Reviews. Molecular Cell Biology22395310.1038/s41580-020-0278-032958874BhattacharjeeAPalSFeldmanGMCathcartMK2011Hck is a key regulator of gene expression in alternatively activated human monocytesThe Journal of Biological Chemistry286367093672310.1074/jbc.M111.29149221878628BlancoREDíaz-MirandaLGarcía-ArrarásJE1995Localization of the heptapeptide GFSKLYFamide in the sea cucumber Holothuria glaberrima (Echinodermata): a light and electron microscopic studyThe Journal of Comparative Neurology35262664010.1002/cne.9035204107722004BökelCBrownNH2002Integrins in development: moving on, responding to, and sticking to the extracellular matrixDevelopmental Cell331132110.1016/s1534-5807(02)00265-412361595BradfordDKColeSJCooperHM2009Netrin-1: diversity in developmentThe International Journal of Biochemistry & Cell Biology4148749310.1016/j.biocel.2008.03.01418455953BrockesJPKumarA2002Plasticity and reprogramming of differentiated cells in amphibian regenerationNature Reviews. Molecular Cell Biology356657410.1038/nrm88112154368BusslingerGAWeustenBLABogteABegthelHBrosensLAACleversH2021Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolutionCell Reports3410881910.1016/j.celrep.2021.10881933691112ByrneMKoopDStrbenacDCisternasPBaloghRYangJYHDavidsonPLWrayG2020Transcriptomic analysis of sea star development through metamorphosis to the highly derived pentameral body plan with a focus on neural transcription factorsDNA Research27dsaa00710.1093/dnares/dsaa00732339242ByrneM2023Autotomy, evisceration, and regeneration in dendrochirotid holothuroidsByrneMThe World of Sea Cucumbers: Challenges, Advances, and InnovationsAcademic Press81983910.1016/B978-0-323-95377-1.00034-5Cabrera-SerranoAGarcía-ArrarásJE2004RGD-containing peptides inhibit intestinal regeneration in the sea cucumber Holothuria glaberrimaDevelopmental Dynamics23117117810.1002/dvdy.2011215305297CandelariaAGMurrayGFileSKGarcía-ArrarásJE2006Contribution of mesenterial muscle dedifferentiation to intestine regeneration in the sea cucumber Holothuria glaberrimaCell and Tissue Research325556510.1007/s00441-006-0170-z16541286Candia-CarnevaliMDThorndykeMCMatrangaV2009Regenerating Echinoderms: A Promise to Understand Stem Cells potentialStem Cells in Marine OrganismsSpringer Netherlands10.1007/978-90-481-2767-2_7Candia-CarnevaliMDSugniMBonasoroF2024Frontiers in Invertebrate Physiology: A Collection of ReviewsApple Academic Press10.1201/9781003405184-5CapdevilaCTrifasMMillerJAndersonTSimsPAYanKS2021Cellular origins and lineage relationships of the intestinal epitheliumAmerican Journal of Physiology Gastrointestinal and Liver Physiology321G413G42510.1152/ajpgi.00188.202134431400CarlsonBM2007Principles of Regenerative BiologyElsevier10.1016/B978-0-12-369439-3.X5000-4ChenMMenonMCWangWFuJYiZSunZLiuJLiZMouLBanuKLeeSWDaiYAnandakrishnanNAzelogluEULeeKZhangWDasBHeJCWeiC2023HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagyNature Communications14429710.1038/s41467-023-40086-337463911ChoiHMTSchwarzkopfMFornaceMEAcharyaAArtavanisGStegmaierJCunhaAPierceNA2018Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robustDevelopment145dev16575310.1242/dev.16575329945988CocurulloMPaganosPAnnunziataRVoronovDArnoneMI2023Single-cell transcriptomic analysis reveals the molecular profile of go-opsin photoreceptor cells in sea urchin larvaeCells12213410.3390/cells1217213437681865Courtney SmithLArizzaVBarela HudgellMABaroneGBodnarAGBuckleyKMCunsoloVDheillyNMFranchiNFugmannSDFurukawaRGarcia-ArrarasJHensonJHHibinoTIronsZHLiCLunCMMajeskeAJOrenMPagliaraPPinsinoARaftosDARastJPSamasaBSchillaciDSchrankelCSStabiliLStensvägKSuttonE2018Echinodermata: The Complex Immune System in echinodermsAdvances in Comparative ImmunologySpringer International Publishing10.1007/978-3-319-76768-0_13CoxMKAppelboomBLBanGISerraR2014Erg cooperates with TGF-β to control mesenchymal differentiationExperimental Cell Research32841041810.1016/j.yexcr.2014.08.01525139621DewingJMCarareROLoteryAJRatnayakaJA2020The diverse roles of TIMP-3: Insights into degenerative diseases of the senescent retina and brainCells93910.3390/cells9010039DhordainPDewitteFDesbiensXStehelinDDuterque-CoquillaudM1995Mesodermal expression of the chicken erg gene associated with precartilaginous condensation and cartilage differentiationMechanisms of Development50172810.1016/0925-4773(94)00322-e7605748Díaz-BalzacCASantacana-LaffitteGSan Miguel-RuízJETossasKValentín-TiradoGRives-SánchezMMeslehATorresIIGarcía-ArrarásJE2007Identification of nerve plexi in connective tissues of the sea cucumber Holothuria glaberrima by using a novel nerve-specific antibodyThe Biological Bulletin213284210.2307/2506661617679718DolgachevVPanickerSBalijepalliSMcCandlessLKYinYSwamySSureshMVDelanoMJHemmilaMRRaghavendranKMachado-ArandaD2018Electroporation-mediated delivery of FER gene enhances innate immune response and improves survival in a murine model of pneumoniaGene Therapy2535937510.1038/s41434-018-0022-y29907877DolmatovIYEliseikinaMGGinanovaTTLamashNEKorchaginVPBulgakovAA1996Muscle regeneration in the holothurian Stichopus japonicusRoux’s Archives of Developmental Biology20548649310.1007/BF00377230DolmatovIY2021Molecular aspects of regeneration mechanisms in holothuriansGenes1225010.3390/genes1202025033578707DolmatovIYNizhnichenkoVA2023Extracellular matrix of echinodermsMarine Drugs2141710.3390/md2107041737504948DongCMurielJMRamirezSHutterHHedgecockEMBreydoLBaskakovIVVogelBE2006Hemicentin assembly in the extracellular matrix is mediated by distinct structural modulesThe Journal of Biological Chemistry281236062361010.1074/jbc.M51358920016798744DumitruCABankfalviAGuXZeidlerRBrandauSLangS2013AHNAK and inflammatory markers predict poor survival in laryngeal carcinomaPLOS ONE8e5642010.1371/journal.pone.005642023409183DunnLLSekyereEOSuryo RahmantoYRichardsonDR2006The function of melanotransferrin: A role in melanoma cell proliferation and tumorigenesisCarcinogenesis272157216910.1093/carcin/bgl04516704991ElmentaiteRRossADBRobertsKJamesKROrtmannDGomesTNayakKTuckLPritchardSBayraktarOAHeuschkelRVallierLTeichmannSAZilbauerM2020Single-cell sequencing of developing human gut reveals transcriptional links to childhood crohn’s diseaseDevelopmental Cell5577178310.1016/j.devcel.2020.11.01033290721FosterSOulhenNFresquesTZakiHWesselG2022Single-cell RNA-sequencing analysis of early sea star developmentDevelopment149dev20098210.1242/dev.20098236399063García-ArrarásJE1998Cellular mechanisms of intestine regeneration in the sea cucumber, Holothuria glaberrima selenka (Holothuroidea:Echinodermata)Journal of Experimental Zoology28128830410.1002/(SICI)1097-010X(19980701)281:43.0.CO;2-KGarcía-ArrarásJESchenkCRodrígues-RamírezRTorresIIValentínGCandelariaAG2006Spherulocytes in the echinoderm Holothuria glaberrima and their involvement in intestinal regenerationDevelopmental Dynamics2353259326710.1002/dvdy.2098317061269García-ArrarásJEValentín-TiradoGFloresJERosaRJRivera-CruzASan Miguel-RuizJETossasK2011Cell dedifferentiation and epithelial to mesenchymal transitions during intestinal regeneration in H. glaberrimaBMC Developmental Biology116110.1186/1471-213X-11-6122004330García-ArrarásJEBelloSAMalavezS2019The mesentery as the epicenter for intestinal regenerationSeminars in Cell & Developmental Biology92455410.1016/j.semcdb.2018.09.00130193995GemberlingMBaileyTJHydeDRPossKD2013The zebrafish as a model for complex tissue regenerationTrends in Genetics2961162010.1016/j.tig.2013.07.00323927865GerberTMurawalaPKnappDMasselinkWSchuezMHermannSGac-SantelMNowoshilowSKageyamaJKhattakSCurrieJDCampJGTanakaEMTreutleinB2018Single-cell analysis uncovers convergence of cell identities during axolotl limb regenerationScience362eaaq068110.1126/science.aaq068130262634GlobusMVethamany-GlobusSLeeYCI1980Effect of apical epidermal cap on mitotic cycle and cartilage differentiation in regeneration blastemata in the newt, Notophthalmus viridescensDevelopmental Biology7535837210.1016/0012-1606(80)90169-47372003GrabskiINStreetKIrizarryRA2023Significance analysis for clustering with single-cell RNA-sequencing dataNature Methods201196120210.1038/s41592-023-01933-937429993GuatelliSFerrarioCBonasoroFAnjoSIManadasBCandia CarnevaliMDVarela CoelhoASugniM2022More than a simple epithelial layer: multifunctional role of echinoderm coelomic epitheliumCell and Tissue Research39020722710.1007/s00441-022-03678-x36083358HaningtonPCZhangSM2011The primary role of fibrinogen-related proteins in invertebrates is defense, not coagulationJournal of Innate Immunity3172710.1159/00032188221063081HaoYHaoSAndersen-NissenEMauckWMZhengSButlerALeeMJWilkAJDarbyCZagerMHoffmanPStoeckiusMPapalexiEMimitouEPJainJSrivastavaAStuartTFlemingLMYeungBRogersAJMcElrathJMBlishCAGottardoRSmibertPSatijaR2021Integrated analysis of multimodal single-cell dataCell1843573358710.1016/j.cell.2021.04.04834062119HaoYStuartTKowalskiMHChoudharySHoffmanPHartmanASrivastavaAMollaGMadadSFernandez-GrandaCSatijaR2024Dictionary learning for integrative, multimodal and scalable single-cell analysisNature Biotechnology4229330410.1038/s41587-023-01767-y37231261HetzelHR1963Studies on holothurian coelomocytes. I. A survey of coelomocyte typesThe Biological Bulletin12528930110.2307/1539404HuangYLavalSHvan RemoortereABaudierJBenaudCAndersonLVBStraubVDeelderAFrantsRRden DunnenJTBushbyKvan der MaarelSM2007AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regenerationFASEB Journal2173274210.1096/fj.06-6628com17185750HumbertPOVeronaRTrimarchiJMRogersCDandapaniSLeesJA2000E2f3 is critical for normal cellular proliferationGenes & Development1469070310733529IwamotoMHiguchiYEnomoto-IwamotoMKurisuKKoyamaEYehHRosenbloomJPacificiM2001The role of ERG (ets related gene) in cartilage developmentOsteoarthritis and Cartilage9S41S4710.1053/joca.2001.0443JovenAElewaASimonA2019Model systems for regeneration: salamandersDevelopment146dev16770010.1242/dev.16770031332037JulianoCEReichALiuNGötzfriedJZhongMUmanSReenanRAWesselGMSteeleRELinH2014PIWI proteins and PIWI-interacting RNAs function in Hydra somatic stem cellsPNAS11133734210.1073/pnas.132096511124367095KeeNSivalingamSBoonstraRWojtowiczJM2002The utility of Ki-67 and Brad as proliferative markers of adult neurogenesisJ Neurosci Meth11597105KingHOOwusu-BoaiteyKEFincherCTReddienPW2024A transcription factor atlas of stem cell fate in planariansCell Reports4311384310.1016/j.celrep.2024.11384338401119KogaHMatsubaraMFujitaniHMiyamotoNKomatsuMKiyomotoMAkasakaKWadaH2010Functional evolution of Ets in echinoderms with focus on the evolution of echinoderm larval skeletonsDevelopment Genes and Evolution22010711510.1007/s00427-010-0333-520680330La MannoGSoldatovRZeiselABraunEHochgernerHPetukhovVLidschreiberKKastritiMELönnerbergPFurlanAFanJBormLELiuZvan BruggenDGuoJHeXBarkerRSundströmECastelo-BrancoGCramerPAdameykoILinnarssonSKharchenkoPV2018RNA velocity of single cellsNature56049449810.1038/s41586-018-0414-630089906LeighNDDunlapGSJohnsonKMarianoROshiroRWongAYBryantDMMillerBMRatnerAChenAYeWWHaasBJWhitedJL2018Transcriptomic landscape of the blastema niche in regenerating adult axolotl limbs at single-cell resolutionNature Communications9515310.1038/s41467-018-07604-030514844LinTYGerberTTaniguchi-SugiuraYMurawalaPHermannSGrosserLShibataETreutleinBTanakaEM2021Fibroblast dedifferentiation as a determinant of successful regenerationDevelopmental Cell561541155110.1016/j.devcel.2021.04.01634004152Lindsay-MosherNChanAPearsonBJ2020Planarian EGF repeat-containing genes megf6 and hemicentin are required to restrict the stem cell compartmentPLOS Genetics16e100861310.1371/journal.pgen.100861332078629Locard-PauletMPalascaOJensenLJ2022Identifying the genes impacted by cell proliferation in proteomics and transcriptomics studiesPLOS Computational Biology18e101060410.1371/journal.pcbi.101060436201535LõokeMMaloneyMFBellSP2017Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicaseGenes & Development3129130510.1101/gad.291336.11628270517LuHShiXWuGZhuJSongCZhangQYangG2015FGF13 regulates proliferation and differentiation of skeletal muscle by down-regulating Spry1Cell Proliferation4855056010.1111/cpr.1220026230950MagnussenSNToraskarJHadler-OlsenESteigedalTSSvinengG2021Nephronectin as a matrix effector in cancerCancers1395910.3390/cancers1305095933668838MakanaeAMitogawaKSatohA2014Co-operative Bmp- and Fgf-signaling inputs convert skin wound healing to limb formation in urodele amphibiansDevelopmental Biology396576610.1016/j.ydbio.2014.09.02125286122MantriMScuderiGJAbedini-NassabRWangMFZMcKellarDShiHGrodnerBButcherJTDe VlaminckI2021Spatiotemporal single-cell RNA sequencing of developing chicken hearts identifies interplay between cellular differentiation and morphogenesisNature Communications12177110.1038/s41467-021-21892-z33741943MashanovVSDolmatovIYHeinzellerTMashanovVSDolmatovIYHeinzellerT2005Transdifferentiation in holothurian gut regenerationThe Biological Bulletin20918419310.2307/359310816382166MashanovVSZuevaORRojas-CatagenaCGarcia-ArrarasJE2010Visceral regeneration in a sea cucumber involves extensive expression of survivin and mortalin homologs in the mesotheliumBMC Developmental Biology1011710.1186/1471-213X-10-11721114850MashanovVSZuevaORGarcia-ArrarasJE2012Expression of Wnt9, TCTP, and Bmp1/Tll in sea cucumber visceral regenerationGene Expression Patterns12243510.1016/j.gep.2011.10.00322079950MashanovVSZuevaOGarcía-ArrarásJE2014Postembryonic organogenesis of the digestive tube: why does it occur in worms and sea cucumbers but fail in humans?Current Topics in Developmental Biology10818521610.1016/B978-0-12-391498-9.00006-124512710MashanovVSZuevaORGarcía-ArrarásJE2015Expression of pluripotency factors in echinoderm regenerationCell and Tissue Research35952153610.1007/s00441-014-2040-425468557MashanovVZuevaOMashanovaDGarcía-ArrarásJE2017Expression of stem cell factors in the adult sea cucumber digestive tubeCell and Tissue Research37042744010.1007/s00441-017-2692-y28986650MedinaJG2024ScRNAseq_Hglaberrimaswh:1:rev:a180414331fd36f6a23769377e427d64298f7d81Software Heritagehttps://archive.softwareheritage.org/swh:1:dir:efa43e64ba1fb5908b8e3fc0d9df70e3a356d245;origin=https://github.com/devneurolab/scRNAseq_Hglaberrima;visit=swh:1:snp:beebe8cc47f801e74a15940a868bbf107e035338;anchor=swh:1:rev:a180414331fd36f6a23769377e427d64298f7d81Medina-FelicianoJGGarcía-ArrarásJE2021Regeneration in echinoderms: Molecular advancementsFrontiers in Cell and Developmental Biology976864110.3389/fcell.2021.76864134977019Medina-FelicianoJGPirroSGarcía-ArrarásJEMashanovVRyanJF2021Draft genome of the sea cucumber Holothuria glaberrima, a model for the study of regenerationFrontiers in Marine Science860341010.3389/fmars.2021.60341038741925Medina-FelicianoJGValentín-TiradoGLuna-MartinezKMiranda-NegronYGarcia-ArrarasJE2024Single-Cell RNA sequencing of the holothurian regenerating intestineFigshare1728977010.6084/m9.figshare.c.7289770MeyerAKuCHatlebergWLTelmerCAHinmanV2023New hypotheses of cell type diversity and novelty from orthology-driven comparative single cell and nuclei transcriptomics in echinodermseLife12e8009010.7554/eLife.8009037470227MinSWhitedJL2023Limb blastema formation: How much do we know at a genetic and epigenetic level?The Journal of Biological Chemistry29910285810.1016/j.jbc.2022.10285836596359MishraLDerynckRMishraB2005Transforming growth factor-b signaling in stem cells and cancer roles of TGF-b Family members in neural stem cellsScience310111838910.1126/science.1118389MochmannLHNeumannMvon der HeideEKNowakVKühlAAOrtiz-TanchezJBockJHofmannWKBaldusCD2014ERG induces a mesenchymal-like state associated with chemoresistance in leukemia cellsOncotarget535136210.18632/oncotarget.144924504051MurrayGGarcía-ArrarásJE2004Myogenesis during holothurian intestinal regenerationCell and Tissue Research31851552410.1007/s00441-004-0978-315480798Nieves-RíosCAlvarez-FalcónSMalavezSRodriguez-OteroJGarcía-ArrarásJE2020The nervous system component of the mesentery of the sea cucumber Holothuria glaberrima in normal and regenerating animalsCell and Tissue Research380677710.1007/s00441-019-03142-331865468NishimotoSNishidaE2007Fibroblast growth factor 13 is essential for neural differentiation in Xenopus early embryonic developmentThe Journal of Biological Chemistry282242552426110.1074/jbc.M70427720017584734OikeYYasunagaKSudaT2004Angiopoietin-related/angiopoietin-like proteins regulate angiogenesisInternational Journal of Hematology80212810.1532/ijh97.0403415293564Ortiz-PinedaPARamírez-GómezFPérez-OrtizJGonzález-DíazSSantiago-De JesúsFHernández-PasosJDel Valle-AvilaCRojas-CartagenaCSuárez-CastilloECTossasKMéndez-MercedATRoig-LópezJLOrtiz-ZuazagaHGarcía-ArrarásJE2009Gene expression profiling of intestinal regeneration in the sea cucumberBMC Genomics1026210.1186/1471-2164-10-26219505337PaganosPVoronovDMusserJMArendtDArnoneMI2021Single-cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostomeeLife10e7041610.7554/eLife.7041634821556PaganosPRonchiPCarlJMizzonGMartinezPBenvenutoGArnoneMI2022aIntegrating single cell transcriptomics and volume electron microscopy confirms the presence of pancreatic acinar-like cells in sea urchinsFrontiers in Cell and Developmental Biology1099166410.3389/fcell.2022.99166436060803PaganosPUllrich-LüterECaccavaleFZakrzewskiAVoronovDFournon-BerodiaICocurulloMLüterCArnoneMI2022bA new model organism to investigate extraocular photoreception: opsin and retinal gene expression in the sea urchin Paracentrotus lividusCells11263610.3390/cells1117263636078045PagèsHCarlsonMFalconSLiN2024Manipulation of sqlite-based annotations in bioconductor3.20AnnotationDbihttps://bioconductor.org/packages/release/bioc/html/AnnotationDbi.htmlParigiSMLarssonLDasSRamirez FloresROFredeATripathiKPDiazOESelinKMoralesRALuoXMonasterioGEngblomCGaglianiNSaez-RodriguezJLundebergJVillablancaEJ2022The spatial transcriptomic landscape of the healing mouse intestine following damageNature Communications1382810.1038/s41467-022-28497-035149721PiovaniLCzarkwianiAFerrarioCSugniMOliveriP2021Ultrastructural and molecular analysis of the origin and differentiation of cells mediating brittle star skeletal regenerationBMC Biology19910.1186/s12915-020-00937-733461552PohARO’DonoghueRJJErnstM2015Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cellsOncotarget6157521577110.18632/oncotarget.419926087188PoleoGBrownCWLaforestLAkimenkoMA2001Cell proliferation and movement during early fin regeneration in zebrafishDevelopmental Dynamics22138039010.1002/dvdy.115211500975QuiñonesJLRosaRRuizDLGarcía-ArrarásJE2002Extracellular matrix remodeling and metalloproteinase involvement during intestine regeneration in the sea cucumber Holothuria glaberrimaDevelopmental Biology25018119710.1006/dbio.2002.077812297105Quispe-ParraDValentínGGarcía-ArrarásJE2021aA roadmap for intestinal regenerationThe International Journal of Developmental Biology6542743710.1387/ijdb.200227dq32930367Quispe-ParraDJMedina-FelicianoJGCruz-GonzálezSOrtiz-ZuazagaHGarcía-ArrarásJE2021bTranscriptomic analysis of early stages of intestinal regeneration in Holothuria glaberrimaScientific Reports1134610.1038/s41598-020-79436-233431961Ramírez-GómezFAponte-RiveraFMéndez-CastanerLGarcía-ArrarásJE2010Changes in holothurian coelomocyte populations following immune stimulation with different molecular patternsFish & Shellfish Immunology2917518510.1016/j.fsi.2010.03.01320412860ReddienPW2018The cellular and molecular basis for planarian regenerationCell17532734510.1016/j.cell.2018.09.02130290140Reyes-RiveraJGrillo-AlvaradoVSoriano-LópezAEGarcía-ArrarásJE2024Evidence of interactions among apoptosis, cell proliferation, and dedifferentiation in the rudiment during whole-organ intestinal regeneration in the sea cucumberDevelopmental Biology5059910910.1016/j.ydbio.2023.11.00137925124RizzoFFernandez-SerraMSquarzoniPArchimandritisAArnoneMI2006Identification and developmental expression of the ets gene family in the sea urchin (Strongylocentrotus purpuratus)Developmental Biology300354810.1016/j.ydbio.2006.08.01216997294RodgersAKSmithJJVossSR2020Identification of immune and non-immune cells in regenerating axolotl limbs by single-cell sequencingExperimental Cell Research39411214910.1016/j.yexcr.2020.11214932562784Rojas-CartagenaCOrtíz-PinedaPRamírez-GómezFSuárez-CastilloECMatos-CruzVRodríguezCOrtíz-ZuazagaHGarcía-ArrarásJE2007Distinct profiles of expressed sequence tags during intestinal regeneration in the sea cucumber Holothuria glaberrimaPhysiological Genomics3120321510.1152/physiolgenomics.00228.2006.-RepairRosado-OlivieriEARamos-OrtizGAHernández-PasosJDíaz-BalzacCAVázquez-RosaEValentín-TiradoGVegaIEGarcía-ArrarásJE2017A START-domain-containing protein is A novel marker of nervous system components of the sea cucumber Holothuria glaberrimaComparative Biochemistry and Physiology Part B, Biochemistry & Molecular Biology214576510.1016/j.cbpb.2017.08.00428864221RoySGatienS2008Regeneration in axolotls: a model to aim for!Experimental Gerontology4396897310.1016/j.exger.2008.09.00318814845Sandoval-GuzmánTWangHKhattakSSchuezMRoenschKNacuETazakiAJovenATanakaEMSimonA2014Fundamental differences in dedifferentiation and stem cell recruitment during skeletal muscle regeneration in two salamander speciesCell Stem Cell1417418710.1016/j.stem.2013.11.00724268695SantiagoPRoig-LópezJLSantiagoCGarcía-ArrarásJE2000Serum amyloid A protein in an echinoderm: its primary structure and expression during intestinal regeneration in the sea cucumber Holothuria glaberrimaThe Journal of Experimental Zoology28833534410.1002/1097-010X(20001215)288:4<335::AID-JEZ6>3.0.CO;2-111144282Santos-RuizLSantamaríaJARuiz-SánchezJBecerraJ2002Cell proliferation during blastema formation in the regenerating teleost finDevelopmental Dynamics22326227210.1002/dvdy.1005511836790SatijaRFarrellJAGennertDSchierAFRegevA2015Spatial reconstruction of single-cell gene expression dataNature Biotechnology3349550210.1038/nbt.319225867923SatohNHisataKFosterSMoritaSNishitsujiKOulhenNTominagaHWesselGM2022A single-cell RNA-seq analysis of Brachyury-expressing cell clusters suggests A morphogenesis-associated signal center of oral ectoderm in sea urchin embryosDevelopmental Biology48312814210.1016/j.ydbio.2022.01.00535038441ScimoneMLCloutierJKMaybrunCLReddienPW2022The planarian wound epidermis gene equinox is required for blastema formation in regenerationNature Communications13272610.1038/s41467-022-30412-635585061SeifertAWMuneokaK2018The blastema and epimorphic regeneration in mammalsDevelopmental Biology43319019910.1016/j.ydbio.2017.08.00729291973SharlaimovaNShabelnikovSBobkovDMartynovaMBystrovaOPetukhovaO2021Coelomocyte replenishment in adult Asterias rubens: the possible waysCell and Tissue Research3831043106010.1007/s00441-020-03337-z/PublishedShuttleworthEE2018The role of integrin αvβ8 on human monocytes and macrophages in intestinal immune homeostasisStudent thesisSmileyS1994HolothuroideaHarrisonFWChiaFSMicroscopic Anatomy of InvertebratesNew YorkWiley-Liss401471SmitAHubleyR2015RepeatModeler open1.0Scienceopenhttps://www.scienceopen.com/document?vid=c1050e3d-8dc1-48ba-81c8-b372af25be50SmitAHubleyRGreenP2015RepeatMasker open4.0RepeatMaskerhttps://www.repeatmasker.org/SmithLCBrittenRJDavidsonEH1995Lipopolysaccharide activates the sea urchin immune systemDevelopmental and Comparative Immunology1921722410.1016/0145-305x(95)00009-i8595820StamenkovicI2003Extracellular matrix remodelling: the role of matrix metalloproteinasesThe Journal of Pathology20044846410.1002/path.140012845612StocumDL2004Amphibian regeneration and stem cellsCurrent Topics in Microbiology and Immunology28017010.1007/978-3-642-18846-6_114594207StreetKRissoDFletcherRBDasDNgaiJYosefNPurdomEDudoitS2018Slingshot: cell lineage and pseudotime inference for single-cell transcriptomicsBMC Genomics1947710.1186/s12864-018-4772-029914354StuartTButlerAHoffmanPHafemeisterCPapalexiEMauckWMHaoYStoeckiusMSmibertPSatijaR2019Comprehensive integration of single-cell dataCell1771888190210.1016/j.cell.2019.05.03131178118SuFYangHSunL2022A review of histocytological events and molecular mechanisms involved in intestine regeneration in holothuriansBiology11109510.3390/biology1108109535892951SunYKuekVQiuHTicknerJChenLWangHHeWXuJ2018The emerging role of NPNT in tissue injury repair and bone homeostasisJournal of Cellular Physiology2331887189410.1002/jcp.2601328513838TajerBSavageAMWhitedJL2023The salamander blastema within the broader context of metazoan regenerationFrontiers in Cell and Developmental Biology11120615710.3389/fcell.2023.120615737635872TanakaEMReddienPW2011The cellular basis for animal regenerationDevelopmental Cell2117218510.1016/j.devcel.2011.06.01621763617TelmerCAKarimiKChessMMAgalakovSArshinoffBILotayVWangDZChuSPellsTJVizePDHinmanVFEttensohnCA2024Echinobase: a resource to support the echinoderm research communityGenetics227iyae00210.1093/genetics/iyae00238262680TominagaHNishitsujiKSatohN2023A single-cell RNA-seq analysis of early larval cell-types of the starfish, Patiria pectinifera: Insights into evolution of the chordate body planDevelopmental Biology496526210.1016/j.ydbio.2023.01.00936717049TossasKQi-HuangSCuyarEGarcía-ArrarásJE2014Temporal and spatial analysis of enteric nervous system regeneration in the sea cucumber Holothuria glaberrimaRegeneration1102610.1002/reg2.1527499861Vanden BosscheJPJangouxM1976Epithelial origin of starfish coelomocytesNature26122722810.1038/261227a0VandenSpiegelDJangouxMFlammangP2000Maintaining the line of defense: regeneration of Cuvierian tubules in the sea cucumber Holothuria forskali (Echinodermata, Holothuroidea)The Biological Bulletin198344910.2307/154280210707812Vázquez-VélezGERodríguez-MolinaJFQuiñones-FríasMCPagánMGarcía-ArrarásJE2016A proteoglycan-like molecule offers insights into ground substance changes during holothurian intestinal regenerationThe Journal of Histochemistry and Cytochemistry6438139310.1369/002215541664578127126824Vlaeminck-GuillemVCarrereSDewitteFStehelinDDesbiensXDuterque-CoquillaudM2000The Ets family member Erg gene is expressed in mesodermal tissues and neural crests at fundamental steps during mouse embryogenesisMechanisms of Development9133133510.1016/s0925-4773(99)00272-510704859VoggMCGalliotBTsiairisCD2019Model systems for regeneration: HydraDevelopment14617721210.1242/dev.177212VolkertGJahnADinkelCFahlbuschFZürnCHilgersKFRascherWHartnerAMarekI2014Contribution of the α8 integrin chain to the expression of extracellular matrix componentsCell Communication & Adhesion21899810.3109/15419061.2013.87601224460181WagnerDEWangIEReddienPW2011Clonogenic neoblasts are pluripotent adult stem cells that underlie planarian regenerationScience33281181610.1126/science.120398321566185WanJZhaoXLiuJChenKLiC2022Src kinase mediates coelomocytes phagocytosis via interacting with focal adhesion kinase in Vibrio splendidus challenged Apostichopus japonicusFish & Shellfish Immunology12441142010.1016/j.fsi.2022.04.02435462003WelckerDSteinCFeitosaNMArmisteadJZhangJLLütkeSKleinriddersABrüningJCEmingSASengleGNiehoffABlochWHammerschmidtM2021Hemicentin-1 is an essential extracellular matrix component of the dermal-epidermal and myotendinous junctionsScientific Reports111792610.1038/s41598-021-96824-434504132XieCYaoMDongQ2014Proliferating cell unclear antigen-associated factor (PAF15): A novel oncogeneThe International Journal of Biochemistry & Cell Biology5012713110.1016/j.biocel.2014.02.02424631643XingKYangHSChenMY2008Morphological and ultrastructural characterization of the coelomocytes in Apostichopus japonicusAquatic Biology2859210.3354/ab00038YoungMDBehjatiS2020SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing dataGigaScience9giaa15110.1093/gigascience/giaa15133367645YuGWangLGHanYHeQY2012clusterProfiler: an R package for comparing biological themes among gene clustersOMICS1628428710.1089/omi.2011.011822455463ZengCGuoMXiangYSongMXiaoKLiC2023Mesentery AjFGF4-AjFGFR2-ERK pathway modulates intestinal regeneration via targeting cell cycle in echinodermsCell Proliferation56e1335110.1111/cpr.1335136263902ZenjariCBoilly-MarerYDesbiensXOudghirMHondermarckHBoillyB1996Experimental evidence for FGF-1 control of blastema cell proliferation during limb regeneration of the amphibian Pleurodeles waltlThe International Journal of Developmental Biology409659718946244ZhaoHTanegashimaKRoHDawidIB2008Lrig3 regulates neural crest formation in Xenopus by modulating Fgf and Wnt signaling pathwaysDevelopment1351283129310.1242/dev.01507318287203ZhengGXYTerryJMBelgraderPRyvkinPBentZWWilsonRZiraldoSBWheelerTDMcDermottGPZhuJGregoryMTShugaJMontesclarosLUnderwoodJGMasquelierDANishimuraSYSchnall-LevinMWyattPWHindsonCMBharadwajRWongANessKDBeppuLWDeegHJMcFarlandCLoebKRValenteWJEricsonNGStevensEARadichJPMikkelsenTSHindsonBJBielasJH2017Massively parallel digital transcriptional profiling of single cellsNature Communications81404910.1038/ncomms140492809160110.7554/eLife.100796.3.sa0eLife AssessmentBenayounBérénice AReviewing EditorUniversity of Southern CaliforniaUnited StatesConvincingValuable
This article describes a resource detailing the econstitution of Holothuria glaberrima gut following self-evisceration in response to a potassium chloride injection, using scRNAseq and fluorescent RNA localization in situ. It provides some new findings about organ regeneration, as well as the origins of pluripotent cells, and places these findings in the context of regeneration across species. The article’s schematic model and HCR images are a valuable foundation for future work. The authors provide convincing RNA localization images to validate their data and provide spatial context. These validation experiments are of good quality but remain challenging to connect to the complex spatial organization of complex tissues. This resource will be of interest to the field of regeneration, particularly in invertebrates, but also in comparative studies in other species, including evolutionary studies.
Joshua G. Medina-Feliciano et al. investigated the single-cell transcriptomic profile of holoturian regenerating intestine following evisceration, a process used to expel their viscera in response to predation. Using single-cell RNA-Sequencing and standard analysis such as "Find cluster markers", "Enrichment analysis of Gene Ontology" and "RNA velocity", they identify 13 cell clusters and their potential cell identity. Based on bioinformatic analysis they identified potentially proliferating clusters and potential trajectories of cell differentiation. This manuscript represents a useful dataset that can provide candidate cell types and cell markers for more in-depth functional analysis of the holoturian intestine regeneration.
The conclusions of this paper are supported only by bioinformatic analyses since the in vivo validation through HCR is not sufficient to support them.
Strengths:
- The Authors are providing a single-cell dataset obtained from sea cucumbers regenerating their intestines. This represents the first fundamental step to an unbiased approach to better understand this regeneration process and the cellular dynamics taking part in it.
- The Authors run all the standard analyses providing the reader with a well digested set of information about cell clusters, potential cell types, potential functions and potential cell differentiation trajectories.
Weaknesses:
- The Authors frequently report the percentage of cells with a specific feature (either labelled or expressing a certain gene or belonging to a certain cluster). This number can be misleading since that is calculated after cell dissociation and additional procedures (such as staining or sequencing and dataset cleanup) that can heavily bias the ratio between cell types. Similarly, the Authors cannot compare cell percentage between anlage and mesentery samples since that can be affected by technical aspects related to cell dissociation, tissue composition and sequencing depth.
- The Authors did not validate all the clusters.
- There is no validation of the trajectory analysis and there is no validation of the proliferating cluster with H3P or EdU co-labeling.
This research offers a comprehensive analysis of the regenerative process in sea cucumbers and builds upon decades of previous research. The approach involves a detailed examination using single-cell sequencing, making it a crucial reference paper while shedding new light on regeneration in this organism.
Strengths:
Detailed analysis of single-cell sequencing data and high-quality RNA localization images provide significant new insights into regeneration in sea cucumbers and, more broadly, in animals. Identifying a proliferating cluster of cells is very interesting and may open avenues to identify the cell lineage history and deeper molecular properties of the cells that regenerate the intestine.
Weaknesses:
The spatial context of the RNA localization images is challenging to interpret in this spatially complex tissue organization. Although the authors have taken care to perform RNA localization staining, it is still challenging to relate these data to their schematic model. This is only a minor weakness that will almost certainly be clarified by future work from the authors as they follow up on findings.
The authors have done a good job at creating a "resource" paper for the study of gut regeneration in sea cucumbers. They present a single-cell RNAseq atlas for the reconstitution of Holothuria glaberrima gut following self-evisceration in response to a potassium chloride injection. The authors provide data characterizing cellular populations and precursors of the regenerating anlage at 9 days post evisceration. As a "Tools and Resources" contribution to eLife, this work, with some revisions, could be appropriate. It will be impactful in the fields of regeneration, particularly in invertebrates, but also in comparative studies in other species, including evolutionary studies. Some of these comparative studies could extend to vertebrates and could therefore impact regenerative medicine in the future.
Strengths:
• Novel and useful information for a model organism and question for which this type of data has not yet been reported
• Single-cell gene expression data will be valuable for developing testable hypotheses in the future
• Marker genes for cell types provided to the field
• Interesting predictions about possible lineage relationships between cells during sea cucumber gut regeneration
• Authors have done a good job in the revision of making sure not to overstate the lineage claims in absence of definitive lineage-tracing experiments
• Authors have improved the figures and the overall readability of the figures and text
Specific questions:
- Is there any way to systematically compare these cells to evolutionarily-diverged cells in distant relatives to sea cucumbers? Or even on a case-by-case basis? For example, is there evidence for any of these transitory cell types to have correlate(s) in vertebrate gut regeneration?
• Authors acknowledged this would be interesting and important, but they say in the response document this is outside the scope of the current manuscript and more data would be needed to do this well.
- Line 808: The authors may make a more accurate conclusion by saying that the characteristics are similar to blastemas or behaves like a blastema rather than it is blastema. There is ambiguity about the meaning of this term in the field, but most researchers seem to currently have in mind that the "blastema" definitions includes a discrete spatial organization of cells, and here these cells are much more spread out. This could be a good opportunity for the authors to engage in this dialogue, perhaps parsing out the nuances of what a "blastema" is, what the term has traditionally referred to, and how we might consider updating this term or at least re-framing the terminology to be inclusive of functions that "blastemas" have traditionally had in the literature and how they may be dispersed over geographical space in an organism more so than the more rigid, geographically-restricted definition many researchers have in mind. However, if the authors choose to elaborate on these issues, those elaborations do belong in the discussion, and the more provisional terminology we mention here could be used throughout the paper until that element of the revised discussion is presented. We would welcome the authors to do this as a way to point the field in this direction as this is also how we view the matter. For example, some of the genes whose expression has been observed to be enriched following removal of brain tissue in axolotls (such as kazald2, Lust et al.), are also upregulated in traditional blastemas, for instance, in the limb, but we appreciate that the expression domain may not be as localized as in a limb blastema. Additionally, since there is now evidence that some aspects of progenitor cell activation even in limb regeneration extend far beyond the local site of amputation injury (Johnson et al., Payzin-Dogru et al.), there is an opportunity to connect the dots and make the claim that there could be more dispersion of "blastema function" than previously appreciated in the field. Diving a bit more into these nuances may also enable a better conceptual framework of how blastema function may evolve across vast evolutionary time and between different injury contexts in super-regenerative organisms.
• Authors addressed this comment and agree it is interesting, but given how much territory they had to cover and space limitations, they will save this type of discussion and comparative theoretical work for the future.
Overall, the manuscript is much improved.
10.7554/eLife.100796.3.sa4Author responseMedina-FelicianoJoshua GAuthorUniversity of Puerto Rico at Río PiedrasSan JuanPuerto RicoValentín-TiradoGriselleAuthorUniversity of Puerto Rico at Río PiedrasSan JuanPuerto RicoLuna-MartínezKiaraAuthorUniversity of Puerto Rico at Río PiedrasSan JuanPuerto RicoBeltran-RiveraAlejandraAuthorUniversity of Puerto Rico at Río PiedrasSan JuanPuerto RicoMiranda-NegrónYamilAuthorUniversity of Puerto Rico at Río PiedrasSan JuanPuerto RicoGarcia-ArrarasJosé EAuthorUniversity of Puerto Rico at Río PiedrasSan JuanPuerto Rico
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1:
The entire study is based on only 2 adult animals, that were used for both the single cell dataset and the HCR. Additionally, the animals were caught from the ocean preventing information about their age or their life history. This makes the n extremely small and reduces the confidence of the conclusions.
This statement is incorrect. While the scRNAseq was indeed performed in two animals (n=2), the HCR-FISH was performed in 3-5 animals (depending on the probe used). These were different animals from those used for the scRNAseq. The number of animals used has now been included in the manuscript.
All the fluorescent pictures present in this manuscript present red nuclei and green signals being not color-blind friendly. Additionally, many of the images lack sufficient quality to determine if the signal is real. Additional images of a control animal (not eviscerated) and of a negative control would help data interpretation. Finally, in many occasions a zoomed out image would help the reader to provide context and have a better understanding of where the signal is localized.
Fluorescent photos have been changed to color-blind friendly colors. Diagrams, arrows and new photos have been included as to guide readers to the signal or labeling in cells. Controls for HCR-FISH and labeling in normal intestines have been included.
Reviewer #2:
The spatial context of the RNA localization images is not well represented, making it difficult to understand how the schematic model was generated from the data. In addition, multiple strong statements in the conclusion should be better justified and connected to the data provided.
As explained above we have made an effort to provide a better understanding of the cellular/tissue localization of the labeled cells. Similarly, we have revised the conclusions so that the statements made are well justified.
Reviewer #3:
Possible theoretical advances regarding lineage trajectories of cells during sea cucumber gut regeneration, but the claims that can be made with this data alone are still predictive.
We are conscious that the results from these lineage trajectories are still predictive and have emphasized this in the text. Nonetheless, they are important part of our analyses that provide the theoretical basis for future experiments.
Better microscopy is needed for many figures to be convincing. Some minor additions to the figures will help readers understand the data more clearly.
As explained above we have made an effort to provide a better understanding of the cellular/tissue localization of the labeled cells.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
- Page 4, line 70-81: if the reader is not familiar with holothurian anatomy and regeneration process, this section can be complicated to fully understand. An illustration, together with clear definitions of mesothelium, coelomic epithelium, celothelium and luminal cells would help the reader.
A figure (now Figure 1) detailing the holothurian anatomy of normal and regenerating animals has been added. A figure detailing the intestinal regeneration process has also been included (S1).
- Page 5 line 92-104: this paragraph could be shortened. It would be more important to explain what the main question is the Authors would like to answer and why single cell would be the best technique to answer it, than listing previous studies that used scRNA-Seq.
The paragraph has been shortened and the focus has been shifted to the question of cellular components of regenerative tissues in holothurians.
- Page 6, line 125-127 and line 129-132: this belongs to the method section.
This information is now provided in the Materials and Methods section.
- Page 11, line 210-217: this belongs to the discussion.
This section has now been included in the Discussion.
- How many mesenteries are present in one animal?
This has now been included as part of Figure S1.
- In the methods there are no information about the quality of the dataset and the sequencing and the difference between the 2 samples coming from the 2 animals. How many cells from each sample and which is the coverage? The Authors provided this info only between mesentery and anlage but not between animals.
We have added additional information about the sequencing statistics in S4 Fig and S15 Table. Description has also been added in the methods in lines 922-926 under Single Cell RNA Sequencing and Data Analysis section.
- The result section "An in-depth analysis of the various cluster..." is particularly long and very repetitive. I would encourage to Authors to remove a lot of the details (list of genes and GO terms) that can be found in the figures and stressed only the most important elements that they will need to support their conclusions. Having full and abbreviated gene names and the long list of references makes the text difficult to read and it is challenging to identify the main point that the Authors are trying to highlight.
This section has been abbreviated.
- Figure 1: I would suggest adding a graph of holothurian anatomy before and after the evisceration to provide more context of the process we are looking at and remove 1C.
Information on the holothurian anatomy has been included in a new Fig 1 and in supplementary figure S1
- Figure 2: I would suggest removing this figure that is redundant with Figure 3 and several genes are not cluster specific. Figure 3 is doing a better job in showing similar concepts.
Figure 2 was removed and placed in the Supplement section.
- In figure 3 how were the 3 cell types defined? Was this done manually or through a bioinformatic analysis?
The cell definition was done following the analysis of the highly expressed transcripts and comparisons to what has been shown in the scientific literature.
- Figure 2O shows that one of the supra-cluster is made of C2, C7, C6 and C10. This contradicts the text page 9, line 195.
The transcript chosen for this figure gives the wrong idea that these 4 clusters are similar. We have now addressed this in the manuscript.
- Figure 4A and 4C: if these are representing a subset of Figure 3, they should be removed in one or the other. The same comment is valid also for Figures 5, 6 and 7. In general the manuscript is very redundant both in terms of Figures and text.
These are indeed subsets of Fig 3 that were added with the purpose of clarifying the findings, however, in view of the reviewer’s comment we have deleted the redundant information from all figures.
- Figure 9: since the panels are not in order, it is difficult to follow the flow of the figure. - All UMAP should have the number of the cluster on the UMAP itself instead of counting only on the color code in order to be color-blind friendly.
The figure has been modified and clusters are now identified in the UMAP by their number.
- Figure S1F seems acquired in very different conditions compared to the other images in the same figure.
Fig S1F (now S2 Fig) is an overlay of fluorescent immune-histochemistry (UV light detected) with “classical” toluidine blue labeling (visible light detected). This has now been explained in the figure legend.
- Table S7 is lacking some product numbers.
The toluidine blue product number has now been added to the table. The antibodies that lack product number correspond to antibodies generated in our lab and described in the references provided.
- The discussion is pretty long and partially redundant with the result section. I would encourage the Authors to shorten the text and shorten paragraphs that have repeating information. - It might be out of the scope of the Authors but the readers would benefit from having a manuscript that focuses more on the novel aspects discovered with the single-cell RNA-Seq and then have a review that will bring together all the literature published on this topic and integrating the single-cell data with everything that is known so far.
We have tried to shorten the discussion by eliminating redundant text.
Reviewer #2 (Recommendations For The Authors):
- An intriguing finding is the lack of significant difference in the cell clusters between the anlage and mesentery during regeneration. This discovery raises important questions about the regenerative process. The authors should provide a more detailed explanation of the implications of this finding. For example, does it suggest that both organs contribute equally to the regenerated tissues?
The lack of significant differences in the cell clusters between the anlage and the mesentery is somewhat surprising but can be explained by two different facts. First, we have previously shown that many of the cellular processes that take place in the anlage, including cell proliferation, apoptosis, dedifferentiation and ECM remodeling occur in a gradient that begins at the tip of the mesentery where the anlage forms and extends to various degrees into the mesentery. Similarly, migrating cells move along the connective tissue of the mesentery to the anlage. Thus, there is no clear partition of the two regions that would account for distinct cell populations associated with the regenerative stage. Second, the two cell populations that would have been found in the mesentery but not in the regenerating anlage, mature muscle and neurons, were not dissociated by our experimental protocol as to allow for their sequencing. Our current experiments are being done using single nuclei RNA sequencing to overcome this hurdle. This has now been included in the discussion.
- Proliferating cells are obviously important to the study of regeneration as it is assumed these form the regenerating tissue. The authors describe cluster 8 as the proliferative cells. Is there evidence of proliferation in other cell types or are these truly the only dividing cells? Is c8 of multiple cell types but the clustering algorithm picks up on the markers of cell division i.e. what happens if you mask cell division markers - does this cluster collapse into other cluster types? This is important as if there is only one truly proliferating cell type then this may be the origin of the regenerative tissues and is important for this study to know this.
As the reviewer highlights, we also believe this to be an important aspect to discuss. We have addressed this in the manuscript discussion with the following: “Our data suggest that there appears to be a specific population of only proliferative cells (C8) characterized by a large number of cell proliferation genes, which can be visualized by the top genes shown in Fig 3. These cell proliferation genes are specific to C8, with minimum representation in other populations. Interestingly, as mentioned before C8 expresses at lower levels many of the genes of other coelomic epithelium populations. Nevertheless, even if we mask the top 38 proliferation genes (not shown), this cluster is maintained as an independent cluster, suggesting that its identity is conferred by a complex transcriptomic profile rather than only a few proliferation-related genes. Therefore, the identity and potential role of C8 could be further described by two distinct alternatives: (1) cells of C8 could be an intermediate state between the anlage precursor cells (discussed below) and the specialized cell populations or (2) cells of C8 are the source of the anlage precursor populations from which all other populations arise. The pseudotime data is certainly complex and challenging to interpret with our current dataset, yet the RNA velocity analysis showed in Fig 11B would suggests that cells of C8 transition into the anlage precursor populations, rather than being an intermediate state. This is also supported by the Slingshot pseudotime analysis that incorporates C8 (S13 Fig).
Nevertheless, additional experiments are needed to confirm this hypothesis.”
- The schematic model presented in Fig 10 is essential for clarifying the paper's findings and will provide a crucial baseline model for future research. However, the comparison of the data shown in the HCR figures with the schematic is challenging due to the lack of spatial context in the HCR figures. The authors should find a way to provide better context in the figures, such as providing two-color in situ images to compare spatial relationships of cell types and/or including lower resolution and side-by-side fluorescent and bright field images if possible.
The figure has been modified to explain the spatial arrangement of the tissues.
The authors make several strong statements in the discussion that weren't well connected to the findings in the data. Specifically:
“Regardless of which cell population is responsible for giving rise to the cells of the regenerating intestine, our study reveals that the coelomic epithelium, as a tissue layer, is pluripotent.”
This has now been expanded to better explain the statement.
738 “…we postulate that cells from C1 stand as the precursor cell population from which the rest of the cells in the coelomic epithelium arise”.
This has now been expanded to better explain the statement.
748 “differentiation: muscle, neuroepithelium, and coelomic epithelium cells. We also propose the presence of undifferentiated and proliferating cell populations in the coelomic epithelia, which give rise to the cells in this layer…”
This has now been expanded to better explain the statement.
777 “amphibians, the cells of the holothurian anlage coelomic epithelium are proliferative undifferentiated cells and originated via a dedifferentiation process…”
This has now been expanded to better explain the statement.
Reviewer #3 (Recommendations For The Authors):
Specific questions:
- Is there any way to systematically compare these cells to evolutionarily-diverged cells in distant relatives to sea cucumbers? Or even on a case-by-case basis? For example, is there evidence for any of these transitory cell types to have correlate(s) in vertebrate gut regeneration?
This is a most interesting question but one that is perhaps a bit premature to answer due to multiple reasons. First, most of the studies in vertebrates focus on the regeneration of the luminal epithelium, a layer that we are not studying in our system since it appears later in the regeneration process. Second, there is still too little data from adult echinoderms to fully comprehend which cells are cell orthologues to vertebrates. Third, we are only analyzing one regenerative stage. It is our hope that this is just the start of a full description of what cell types/stages are found and how they function in regeneration and that this will lead us to identify the cellular orthologues among animal species.
Major revisions:
- If lineage tracing is within the scope of this paper, it would provide more definitive evidence to the conclusions made about the precursor populations of the regenerating anlage.
Response: This is certainly one of the next steps, however at present, it is not possible due to technical limitations.
Minor revisions:
- Line 47: "for decades" even longer! Could the authors also cite some other amphibians, such as other salamanders (newts) and larval frogs?
References have been added.
- Line 85: "specially"-could authors potentially change to "specifically"
Corrected
- Line 122: Authors should add the full words of what these abbreviations stand for in the caption for Figure 1 or in Figure 1A itself.
Corrected
- Lines 153: What conclusions are the authors trying to make from one type of tubulin presence compared to the others? It's unclear from the text.
The authors are not trying to reach any particular conclusion. They are just stating what was found using several markers, and the possibility that what might be viewed first hand as a single cell population might be more heterogenous. Although the tubulin-type information might not be relevant for the conclusions in the present manuscript, it might be important for future work on the cell types involved in the regeneration process.
- Line 226: Could the authors clarify if "WNT9" is "WNT9a". Figure 3 lists WNT9a but authors refer to WNT9 in the text.
The gene names in Fig 3 are based on the human identifiers. H. glaberrima only has one sequence of Wnt9 (Auger et al. 2023) and this sequence shares the highest similarity to human Wnt9a, thus the name in the list. We have now identified the gene as Wnt9 to avoid confusion.
- Lines 236-237: Can authors rule out that some immune cells might infiltrate the mesenchymal population?
No, this cannot be ruled out. In fact, we believe that most of the immune cells found in our scRNA-seq are indeed cells that have infiltrated the anlage and are part of the mesenchyma. This has been reported by us previously (see Garcia-Arraras et al. 2006). We have now included this in the text.
- Line 452-453: The over-representation of ribosomal genes not shown. Would it be possible to show this information in the supplementary figures?
The sentence has been modified, the data is being prepared as part of a separate publication that focuses on the ribosomal genes.
- Line 480: Could authors clarify if it's WNT9a or just WNT9?
It is indeed Wnt9. See previous response above.
- Line 500: In future experiments, it would be interesting to compare to populations at different timepoints in order see how the populations are changing or if certain precursors are activated at different times.
We fully agree with the reviewer. These are ongoing experiments or are part of new grant proposals.
- Line 567-568: Choosing 9-dpe allowed for 13 clusters, but do authors expect a different number of clusters at different timepoints as things become more terminally differentiated?
Definitely, we believe that clusters related to the different regenerative stages of cells can be found by looking at earlier or later regeneration stages of the organ. A clear example is that if the experiment is done at 14-dpe, when the lumen is forming, cells related to luminal epithelium populations will appear. It is also possible that different immune cells will be associated with the different regeneration stages.
- Line 653: References Figure 10D (not in this manuscript). Are authors referring to only 1D or 9D or an old draft figure number?
As the reviewer correctly points out, this was a mistake where the reference is to a previous draft. It has now been corrected.
- Line 701: "our study reveals that the coelomic epithelium, as a tissue layer, is pluripotent." Phrasing may be better as referring to the cell population making up the tissue layer as pluripotent/multipotent or that the cells it contains would likely be pluripotent or multipotent. Additionally, lineage tracing may be needed to definitively demonstrate this.
This has been modified.
- Line 808: The authors may make a more accurate conclusion by saying that the characteristics are similar to blastemas or behave like a blastema rather than it is blastema. There is ambiguity about the meaning of this term in the field, but most researchers seem to currently have in mind that the "blastema" definition includes a discrete spatial organization of cells, and here these cells are much more spread out. This could be a good opportunity for the authors to engage in this dialogue, perhaps parsing out the nuances of what a "blastema" is, what the term has traditionally referred to, and how we might consider updating this term or at least re-framing the terminology to be inclusive of functions that "blastemas" have traditionally had in the literature and how they may be dispersed over geographical space in an organism more so than the more rigid, geographically-restricted definition many researchers have in mind. However, if the authors choose to elaborate on these issues, those elaborations do belong in the discussion, and the more provisional terminology we mention here could be used throughout the paper until that element of the revised discussion is presented. We would welcome the authors to do this as a way to point the field in this direction as this is also how we view the matter. For example, some of the genes whose expression has been observed to be enriched following removal of brain tissue in axolotls (such as kazald2, Lust et al.), are also upregulated in traditional blastemas, for instance, in the limb, but we appreciate that the expression domain may not be as localized as in a limb blastema. Additionally, since there is now evidence that some aspects of progenitor cell activation even in limb regeneration extend far beyond the local site of amputation injury (Johnson et al., Payzin-Dogru et al.), there is an opportunity to connect the dots and make the claim that there could be more dispersion of "blastema function" than previously appreciated in the field. Diving a bit more into these nuances may also enable better conceptual framework of how blastema function may evolve across vast evolutionary time and between different injury contexts in super-regenerative organisms.
We have followed the reviewer’s suggestion and stated that the holothurian anlage behaves as a blastema. Though we would love to elaborate on the blastema topic, as suggested by the reviewer, we believe that it would extend the discussion too much and that the topic might be better served in a different publication.
- In the discussion, it would be important not to leave the reader with the impression that all amphibian blastema cells originate via dedifferentiation. This is not the case. For example, in axolotls (Sandoval-Guzman et al.) and in larval/juvenile newts, muscle progenitors within the blastema structure have been shown to originate from muscle satellite cells, a kind of stem cell, in stump tissues (while adult newts use dedifferentiation of myofibers to generate muscle progenitors in the blastema). Most cell lineages simply have not been evaluated in the level of detail that would be required to definitively conclude one way or the other, and the door is open for a more substantial contribution from stem cell populations than previously appreciated especially because new tools exist to detect and study them. Providing the reader with a more nuanced view of this situation will not negatively impact the findings in this paper, but it will show that there is biological complexity still waiting to be discovered and that we don't have all the answers at this point.
This has now been corrected.
Figures: Overall, the figures need minor work.
- Figure 1A: Can the authors draw a smaller, full-body cartoon and feature the current high-mag cartoon as an inset to that? Can they label the axes and make it clear how the geometry works here?
Fig 1 has been re-done and now is split into Fig 1 and Fig 2.
- Figure 1B: Can the authors label the UMAP with cluster identities on the map itself? This will make it easier to identify each cluster (especially to make sure cluster 11 is easier to find).
This has been corrected.
- Figure 2: Could the authors put boxes/clearly distinguish panel labels around each cluster (AO), so that there are clear boundaries?
Fig 2 has been moved to Supplement, following another reviewer recommendation.
- "Gene identifiers starting with "g" correspond to uncharacterized gene models of H. glaberrima." - The sentence is from another figure caption but this figure would benefit from having this sentence in the figure caption as well.
This has been added to other figures as suggested.
- Figure 3A: Can the authors potentially bold, highlight, or underline genes you discuss in text, so it's easier for the reader to reference?
This has been added as suggested.
- Figure 3C: Can the authors please label the cell types directly on the UMAP here as well?
The changes were made following the reviewer’s recommendation.
- Figure 4D-E: There's not much context here to determine if this HCR-FISH validation can tell us anything about these cells besides some of them appear to be there. Do authors expect the coelomocyte morphology to look different in regenerating/injured tissue versus normal animals? Can the authors provide some double in situs, as well as some lower-magnification views showing where the higher-magnification insets are located? Is there any spatial pattern to where these cells are found? Counter stains would be helpful.
- Figure 6C: If clusters C5, C8, C9 are part of the coelomic epithelium, then authors could show a smaller diagram above with blue and grey to show types and then show clusters separately to help get their point across better.
- Figure 6G: This image appears to have high background- would it be possible for authors to repeat phalloidin stain or reimage with a lower exposure/gain. Additionally, imaging with Zstacks would help to obtain maximum intensity projections. It would greatly aid the reader if each image was labeled with HCR probes/antibodies that have been applied to the sample.
- Figure 7E: The cells appear to be out of focus and have high background. Additionally, they are lacking the speckled appearance expected to be seen with HCR-FISH. Would it be possible for authors to collect another image utilizing z-stacks?
HCR-FISH figures identifying the gene expression characteristic of cell clusters have been modified following the reviewer’s concerns. The changes include:
(1) Additional clusters have been verified with probes to gene identifiers. These include clusters 8, 9 and 12.
(2) Redundant information has been removed.
(3) Colors have been changed to make figures friendlier to color-impaired readers.
(4) Spatial context has been added or identified.
(5) In some cases, improved photos have been added
(6) Better labels have been included
(7) When necessary individual photos used for the overlay have been included.
This change was made to Fig 2A. UMAP in Fig 9A is the same and used just as reference of the subset.
- Figure 10: It could be useful if authors put a small map of the sea cucumber like in other images so that readers know where in the anlage this zoomed in model represents.
Added as suggested by the reviewer.
- Supplementary figure 1F: Could authors add an arrow to the dark cell that's being pointed out?
Changed made as suggested by the reviewer.
- Supplementary figure 1: Could authors label clearly what color is labeled with what marker?