Planarians employ diverse and dynamic stem cell microenvironments to support whole-body regeneration

written by: Rebecca Kern

Centre for Organismal Studies, Heidelberg University, Heidelberg. Germany.

‘Master’s of Molecular Biosciences’ program with major ‘Developmental and Stem Cell Biology’

Background

Some planarians – a group of free-living flatworms – show the astonishing capability of regenerating their entire body even from small fragments. They have a substantial population of adult pluripotent stem cells widely distributed throughout their body which can give rise to all organismal cell types. But is it the abundance of stem cells that is integral to regeneration? Apparently not, as it has been observed that certain small fragments do not regenerate successfully despite containing numerous stem cells. The answer may lie in the cells that are in close vicinity to stem cells, i.e., their microenvironment or niche. A common feature of many extensively studied adult stem cell niches is that they regulate the behaviour of residing stem cells, for instance, by causing tissue-specific and lineage-restricted proliferation following injury to replace the damaged tissue. Stem cell niches in planarians have not been well defined and it stands to reason that identifying the regulators of stem cell proliferation will aid in uncovering the cause of their regenerative capacities.

Key findings 

In this preprint, the authors employed a relatively novel technique, Slide-seqV2, to generate spatial transcriptomic data. It permits the mapping of RNA expression patterns directly from intact tissue sections. This allows the faithful recapitulation of in vivo expression of tissue-specific transcripts in intact and in regenerating animals at 6 and 48 hours post amputation (hpa), as mRNA transcripts from tissue sections are captured onto barcoded beads which contain spatial information. Hence, this was an apt method to study the stem cell microenvironment in an unbiased way.

Cells and tissues likely to contribute to regenerative stem cell niches were identified based on the assumption that cell types in proximity to stem cells co-deposit their mRNA onto the same beads. It is important to differentiate between single cells and beads, as the resolution of Slide-seqV2 is not at single cell level, so a single bead can contain the signature of several cells. The authors focused on beads that captured piwi-1, a well-established marker for planarian stem cells. Analysis of piwi-1⁺ beads showed heterogeneity in stem cell microenvironments, as mRNA from many other cell types had been co-captured extensively. The top two co-captured cell types were secretory and intestinal cells, which became the subject of further investigation.

To verify the spatial and temporal distribution of the stem cell microenvironments, the authors calculated the proportion of beads from 6 and 48 hpa and the average distance of beads from the wound, for every piwi-1⁺ bead. Some piwi-1⁺ bead clusters were evenly composed of beads from both time points, but several were dominated by beads from one time point. Most piwi-1⁺ bead clusters appeared at different distances from the wound at 6 or 48 hpa. This led the authors to conclude that the planarian stem cell microenvironments were characteristically “diverse and highly dynamic during regeneration”. The authors additionally used in situ hybridization and confocal imaging to confirm the spatial relationship between stem cells and the two cell types identified in their microenvironment, in intact and in regenerating animals.

Stem cells were observed to be closely associated with secretory cells in intact and regenerating animals at both timepoints, 6 and 48 hpa. To distinguish this particular population of secretory cells based on their close association with stem cells, they were named hecatonoblasts. Proliferating stem cells were found to be depleted immediately adjacent to the intestinal cells, while enriched 10-40 microns away from them. This enrichment was noticeably absent in regenerating animals at 48 hpa.

To functionally test the possible regulatory ability of the identified cell types, the authors focused on bead clusters that co-captured piwi-1, and selected 23 enriched genes which would be either intestinal or hecatonoblast markers. They then performed in situ hybridizations to visualize expression patterns of all genes and compared them to an existing single-cell RNA sequencing atlas. Subsequently, the authors employed RNAi to perform knockdown experiments to functionally test if the genes had any effect on stem cell proliferation during regeneration. The knockdown of two intestinal and four secretory genes led to various regeneration or survival defects, indicating that these genes are essential for regeneration. The authors concluded that intestinal cells and hecatonoblasts indeed express genes that functionally regulate stem cell proliferation during regeneration.

What I liked about this preprint

One major strength of this manuscript lies in the introduction of a novel technique and using several different methods to successfully verify results. Slide-seqV2 as a tool to analyze spatial transcriptomics at a near-cellular resolution has only been developed in recent years, and as of now, there have not been any other publications applying it to study regeneration in planarians.

Moreover, inferences made in the article did not rely solely on Slide-seqV2 data. For further validation and functional tests, well-established methods such as in situ hybridizations and RNAi were employed. All methods were adequately outlined in the “materials and methods” section. The Slide-seqV2 data and code used for analysis have been made accessible as well, which grants the option to validate and reproduce the results presented in the article.

The relevance of elucidating molecular mechanisms underlying regeneration for potential therapeutic targets cannot be understated. Even though the regenerative capabilities of planarians have been known for around 200 years, the research field underwent a temporary cutback during the 20^th century, then once again picked back up in the early 2000s. A considerable achievement of this article is that it managed to characterise stem cell micro-environments and challenge the paradigm of a spatially restricted niche. It also lays the foundation for the identification of other stem cell micro-environments and re-evaluates the approach of only looking into cell types in the immediate vicinity of stem cells. Furthermore, the article also highlights that it is not just the number of stem cells, but their diverse micro-environments that are of great importance during regeneration. Taking all aforementioned points into consideration, the work done in this article meaningfully fills a knowledge gap in addressing the role of stem cell micro-environments during regeneration primarily in planarians, and possibly in other organisms.

Questions to the authors

1. What was the reason for choosing 6 hpa and 48 hpa as timepoints to study regenerating animals? Could one have included more timepoints? Would you argue that one can infer the dynamics of the stem cell proliferation process following injury, just from looking at two timepoints?
2. Why was the 6 hpa time point not included in the RNAi experiment to verify the effect of selected genes on stem cell regulation during regeneration?
3. How could one study possible signalling pathways within the stem cell microenvironment? In the discussion, you made some assumptions on contact-dependent or -independent signalling that were inferred from the spatial relationships between stem cells and either intestinal cells or hecatonoblasts. Are there bioinformatic tools or resources available for planarians to study cell-cell communication/signalling which could be implemented?
4. After identifying genes where the knockdown visibly affects stem cell proliferation and the regenerative process, what are the next steps? Since the expression of some genes was shown to not be cell type specific (tubulin beta and cytoplasmic dynein), should other cell types be taken into consideration to more comprehensively characterise the stem cell microenvironment?

Tags: planarians, regeneration, slide-seqv2, stem cell niche

doi: https://doi.org/10.1242/prelights.34743

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