If you dissect a planarian (Schmidtea mediterranea)—a flatworm about half an inch long—into 279 tiny fragments, each fragment can regrow into an entire animal.

New research from the lab of Alejandro Sánchez Alvarado, PhD, of the Stowers Institute for Medical Research, shows whole-body regeneration involves transcriptional changes in cells from all three germ layers (muscle, epidermis, and intestine) of the body, and that tissue close to and far from the site of injury contribute to the regenerative process.

The study is published in the Nature Cell Biology article titled, “Identification of rare, transient post-mitotic cell states that are induced by injury and required for whole-body regeneration in Schmidtea mediterranea.

Blair Benham-Pyle, PhD, a postdoctoral scientist in Alvarado’s lab and lead author of the paper says,It turns out that a number of genes that we characterized, for instance in the intestine, have also been implicated in immune evasion in the context of cancer, or in wound healing. A lot of the same mechanisms that stem cells use to avoid the immune system and to fuel proliferation and growth during regeneration may be the same mechanisms that are co-opted by tumors. By understanding what non-stem cell states and tissue types are helping to create that signaling environment, we might eventually find new targets for either stimulating healthy and normal wound healing in contexts where regenerative capacity is limited, or, limiting growth capacities of things that we don’t want to grow, like tumors.”

Capturing the cellular complexity of planarian regeneration: The authors captured 299,998 individual cells regenerating planarian tissue fragments and profiled their gene expression. The image shows an ‘atlas’ with each cell represented by a dot and colored by the time point from which it was collected. Distance in space represents the difference in gene expression between the cells, with cells closer together being more similar in gene expression and cells far apart, more different [Sánchez Alvarado Lab/ Nature Cell Biology]

Studying gene expression at a single-cell level across all the cell types of regenerating planaria over time, is the approach Alvarado’s team adopted.

“Regeneration was a little bit of a black box before—we knew some genes that were important, and we could look at how some genes were altered globally in response to amputation and during regeneration, but we didn’t know how individual cell types across the animal were changing their behavior or function. That’s what this experiment allowed us to characterize.”

Benham-Pyle adapted a new single-cell sequencing method (SplitSeq) for this largescale gene-expression study. Using this approach, the team captured 299,998 single-cell transcriptomes across eight different tissues in planaria that are capable versus incapable of regenerating into the entire animal.

“This allowed us to look at all of the different cell types across the entire animal to see which responded to amputation and what genes were marking these cells as they changed and responded to regeneration,” says Benham-Pyle.

The team characterized five cell types from all three germ layers that transiently altered gene expression after amputation. When genes enriched in these cell types were knocked down, says Benham-Pyle, “we found that all of them contribute to regeneration in different ways, being activated at different times and in different parts of the body.”

In an unexpected finding, the authors uncover muscle is important for patterning, and the epidermis is important for early stem cell proliferation bursts during regeneration. The researchers show muscle regulates patterning by controlling tissue polarity through changing the expression of the genes notum, follistatin, evi/wls, glypican-1 and junctophilin-1.

The researchers discover rare cellular states induced during whole-body regeneration, called transient regeneration-activating cell states (TRACS) that can exist in tissues near to and distant from a wound site during planarian whole-body regeneration. RNAi depletion of TRACS-enriched genes produced regeneration defects.

Transient Regeneration-Activated Cell States (TRACS) are distributed across germs layers and uniquely activated in space and time. The images show TRACS in the planarian muscle, epidermis, and intestine (yellow) important for patterning and stem cell proliferation during regeneration different [Sánchez Alvarado Lab/ Nature Cell Biology]

The team was also found that the intestine is important for both stem cell maintenance and regulating tissue remodeling after amputation. Although this can appear surprising, Benham-Pyle says, “It does make sense.” When the planarian is cut into pieces the intestine changes its gene expression to “scavenge material from dying cells within the animal, and to convert those materials into new healthy cells in a regenerated worm.”

“What this paper does is take a global look at what sorts of cells need to be in a signaling environment to stimulate stem cells to create new tissue and replace missing tissue,” says Benham-Pyle. “It was already known that the wound-induced epidermis and the wound-induced muscle played different roles in regeneration, but we wanted to understand the big picture.”

In future studies, the team intends to explore how the cells talk to each other and the tasks they perform to accomplish this astounding scale of regeneration.

Financial support for the work came from the Stowers Institute for Medical Research, the Howard Hughes Medical Institute, the National Institute of General Medical Sciences of the National Institutes of Health, and the Jane Coffin Childs Memorial Fund.