After experiencing an injury or amputation, an organism’s cells must determine how much tissue needs to be regenerated and where, but how they know where to grow remains a mystery. “It's a classic question in limb regeneration of salamanders or fin regeneration of fish, or you can apply it to almost any type of regeneration. How do you know how much to grow back and what shape it should be and when to stop?” said Ken Poss, a regeneration biologist at the University of Wisconsin-Madison. Although it is well documented that many organisms regenerate through positional memory, the process by which cells remember their developmental spatial identities and use that information to restore tissue correctly, scientists are still piecing together the mechanisms.1
In work published in iScience, a research team led by molecular biologist Alejandro Sánchez Alvarado at the Stowers Institute for Medical Research turned to single-cell RNA sequencing to identify the targets involved in positional memory.2 They investigated how amputation position informs regenerating tailfins in African killifish (Nothobranchius furzeri) and identified a cell state in the basal epidermis that is transiently amplified based on amputation distance from the body. They also found extracellular matrix (ECM) components that govern regenerative capacity, and evidence that growth rate can be uncoupled from amputation position.
The researchers began by investigating the connection between fin amputation position and appendage regeneration speed. “If you cut just a tiny, little sliver at the very, very end of the tailfin, it'll regenerate just fine. If you cut it closer to the trunk of the fish, closer to the body of the fish, it'll take a little longer,” said Sánchez Alvarado. “We wanted to see if we could shed some cellular and molecular light onto this developmental process.”
Sánchez Alvarado and his team found minor transcriptional changes when they compared the cells remaining after distal versus proximal amputation, but the biggest difference was in cell state abundance. They identified a new basal epidermal cell state unique to regenerating tissue, which appeared in greater abundance after amputation closer to the body. This subpopulation that the researchers described as a transient regeneration-activated cell state (TRACS) showed gene enrichment for ECM modifiers and embryonic morphogenesis pathways. Additionally, when researchers deleted a TRACS marker and ECM modifier called sequestosome 1 (sqstm1) using CRISPR-Cas9, they observed enhanced regenerative capacity exclusively in distal injuries, suggesting that regeneration rates can be modified independent from amputation position if the correct pathways are targeted.
“The field does not have a lot of targets for what might be controlling position during regeneration,” said Poss, who investigates similar processes in zebrafish models, and who was not involved in the study. “This study took a lot of work, a lot of infrastructure, and I think it's interesting. I think it's a great resource for the field, not only what they showed, but it's a lot of data, and we can use that.”
In addition to identifying a novel TRACS involved in positional memory, Sánchez Alvarado’s research team observed that amputating the tailfin also triggered a regeneration-related transcriptional response in uncut dorsal fin epithelial cells, which leads to more questions for the future. “What was interesting was that within a very short period of time, that response dissipated to the point where it was undetectable in the dorsal fin, but it was sustained in in the tail, suggesting that there really are mechanisms for sensing injury and for sensing the extent of the injury,” said Sánchez Alvarado. “I think the genes are probably there in the expression profile that we define. But now we need to start establishing cause and effect relationships, hierarchies of interactions, and so forth, and that's going to be part of future work in the lab.”
- Otsuki L, Tanaka EM. Positional memory in vertebrate regeneration: A century’s insights from the salamander limb. Cold Spring Harb Perspect Biol. 2021;14(6):a040899.
- Ortega Granillo A, et al. Positional information modulates transient regeneration-activated cell states during vertebrate appendage regeneration. iScience. 2024;27(9):110737.
