Single-cell RNA sequencing of the holothurian regenerating intestine reveals the pluripotency of the coelomic epithelium

Reviewer #1 (Public Review):

Summary:
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 potential identity. Based merely 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 for gaining a better understanding of the holoturian intestine regeneration. The conclusions of this paper are supported only by bioinformatic analyses, since the in vivo validation through HCR does not sufficiently support them.

Strengths:
- The Authors are providing a single cell dataset obtained from sea cucumber regenerating their intestine. This represents a 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 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.
- 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.
- 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 decided to validate only a few clusters and in many cases there are no positive controls (such as specific localization, specific function, changes between control and regenerating animals, co-stain) that could actually validate the cluster identity and the specificity of the selected marker. There is no validation of the trajectory analysis and there is no validation of the proliferating cluster with H3P or BrdU stainings.
- It is not clear what is already known about holothurian intestine regeneration and what are the new findings in this manuscript. The Authors reference several papers throughout the whole result sectioning mentioning how the steps of regeneration, the proliferating cells, some of the markers and some of the cell composition of mesenteries and anlages was already known.

Reviewer #2 (Public Review):

Summary:
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.

Weaknesses:
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.

Reviewer #3 (Public Review):

Summary:
The authors have done a good job of 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

Weaknesses:
• 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
• Better microscopy is needed for many figures to be convincing
• Some minor additions to the figures will help readers understand the data more clearly

Author response:

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. We are partly responsible for this confusion, since we did not state the number of animals used for the HSC-FISH 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 will be changed to color-blind friendly colors.

Diagrams, arrows and new photos will be included as to guide readers to the signal

or labeling in cells. In the original manuscript 6 out of 7 cluster validations included a photo of a normal, non-eviscerated control. We will make certain that this is highlighted in the resubmission and that ALL figures with HCR-FISH labeling will include data from control animals.

- 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 Reviewer has correctly identified the limitations of using cell percentages in scRNA-seq analyses. However, these percentages do offer a general overview of the sequenced cell populations and highlight potential differences between samples. In addition, these percentages, as addressed by the Reviewer, not only emphasize the shortcommings of the dissociation methods but at the same time provide some explanation for the absence of particular cell populations, as we describe in the manuscript. In our future resubmission, we will acknowledge these limitations and inform readers of any potential biases introduced by relying on these numbers.

- The Authors decided to validate only a few clusters and in many cases there are no positive controls (such as specific localization, specific function, changes between control and regenerating animals, co-stain) that could actually validate the cluster identity and the specificity of the selected marker. There is no validation of the trajectory analysis and there is no validation of the proliferating cluster with H3P or BrdU stainings.

We validated the seven clusters that were important to reach our conclusions. Six of these had controls of normal (uneviscerated) intestine. Nonetheless we will increase the number of cluster validations and include the dividing cell cluster using BrdU.

- It is not clear what is already known about holothurian intestine regeneration and what are the new findings in this manuscript. The Authors reference several papers throughout the whole result sectioning mentioning how the steps of regeneration, the proliferating cells, some of the markers and some of the cell composition of mesenteries and anlages was already known.

The manuscript presents several novel findings on holothurian intestine regeneration, including:

- The integration of multiple cellular processes, reported for the first time within a single species, along with the identification of the specific mRNAs expressed by each involved cell population.

- A comparative analysis of the sea cucumber anlage structure, highlighting its similarities to previously described blastemal structures.

- The identification of the potential dedifferentiated cell populations that form the foundation of the anlage, serving as the epicenter for proliferating and differentiating cells.

We will ensure that these and other significant findings are prominently emphasized in the resubmitted manuscript.

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 will make an effort to provide a better understanding of the cellular/tissue localization of the labeled cells. Similarly, we will revise 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 will emphasize 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 will make an effort to provide a better

understanding of the cellular/tissue localization of the labeled cells. Similarly, we will revise the conclusions so that the statements made are well justified.