Ascidians, marine invertebrates more commonly known as tunicates or sea squirts, come in two flavors: solitary or colonial. Colonial species are known for their extensive ability to rebuild damaged tissue and even generate entirely new individuals through budding, while solitary species have long been thought to be much more limited in what they can regrow. A study published April 15 in Frontiers in Cell and Developmental Biology has documented for the first time the ability of a solitary tunicate to generate as many as three new individuals in response to amputation.
Tal Gordon, a marine biologist at Tel Aviv University in Israel who completed this work as part of her dissertation (she’s now a postdoc at the same institution), has been studying the tunicate Polycarpa mytiligera since her undergraduate research, and it was during that time when she first noticed something unique about her study species. One day, while trying to pry a specimen off a rock, the tunicate eviscerated itself, a defense tactic that involves ejecting most of its internal organs. The gruesome, but not lethal, move serves up a tasty snack to satisfy would-be predators. Previously, this behavior had only been noted in sea cucumbers, distant evolutionary relatives that are able to regenerate their lost body parts. When Gordon brought the tunicate into the lab, it too seemed to heal within a few weeks.
“For colonial [species], we expect that this is how they live. . . . We expect them to have these high regenerative capabilities,” says Ayelet Voskoboynik, a molecular biologist at Stanford University’s Hopkins Marine Station who has previously collaborated with Gordon but was not involved in the current work. Colonial species can reproduce both sexually and asexually by budding, which requires them to grow new organs. In solitary species that only reproduce sexually, “we thought that it’s more limited, so this is beautiful work establishing essentially a new model system from the perspective of regeneration,” Voskoboynik tells The Scientist.
That assumption is rooted in observations of the most commonly studied tunicate, the solitary Ciona intestinalis. When cut in half along the midline, the bottom, posterior portion of Ciona that contains the gut can repair its missing siphons and central nervous system, but the top, anterior part cannot do the same and ultimately dies. After watching P. mytiligera spill its guts and survive, Gordon tells The Scientist, she “was curious in finding out the limits of its regenerational ability.”
For the latest study, Gordon divided tunicates up in different ways. She cut individuals into two pieces either vertically or horizontally, and cut others horizontally into three pieces. She tracked their regeneration at 7, 30, and 40 days post-amputation, analyzing the segments under a microscope.
In all cases, each fragment was able to regenerate into a whole, functioning animal within a month—growing entirely new hearts, guts, siphons, and nerves—although in some cases the regrown organs were smaller than those in nonamputated controls. This ability of Polycarpa to regenerate two or even three body segments shows, for the first time, “that there is something special here,” Gordon says, particularly that the upper body is able to generate the lower, which has never been shown in a solitary species.
“This is a novel result, and it opens new areas . . . for people to look at a number of different ascidians going forward,” says William Jeffery, a developmental biologist at the University of Maryland who was not involved in the current study. Jeffery has spent many years using Ciona to study regeneration, and after so long, he adds, “it was surprising to see that a solitary [species] would regenerate in the way it did.”
Gordon next wanted to understand what was happening at a cellular level. She used one dye to stain the nucleus of each cell, and another dye called 5-ethynyl-2′-deoxyuridine, or EdU, to mark only those cells that are proliferating. Prior to being amputated, the number of proliferating cells in circulation were evenly distributed throughout the tunicate, but in the first five days after being cut into pieces, they quickly migrated to the margins of the incisions and began to divide.
While Gordon wasn’t able to study the cells in more detail, their behavior, according to Voskoboynik, “really suggests that we are talking about stem cells.” Voskoboynik is a stem cell specialist, and her prior work with the colonial species Botryllus schlosseri has shown that tunicates often use these adult stem cells to regenerate damaged tissues, as has been shown by other researchers to occur in Ciona. More work will be needed to confirm this for Polycarpa, but “I will be very surprised if solitary tunicates are behaving differently,” Voskoboynik says.
As a final component of her study, Gordon used RNA-seq to create a phylogenetic tree. She wanted to see which species were most closely related to Polycarpa. Surprisingly, it turned out that the tunicate’s nearest relative was a colonial species, Polyandrocarpus zorritensis. Her working hypothesis, Gordon tells The Scientist, is that Polycarpa may actually be in a transitional state between a solitary and a colonial species. “This is only a hypothesis, but if we look at the tree, it’s reasonable to assume.”
All three scientists who spoke with The Scientist stressed that the next step will be a comparative analysis of Polycarpa and other tunicates to study the evolution of regeneration in tunicates more broadly—when regeneration first emerged, how often the ability has been lost or regained, and which species are transitioning, as Polycarpa might be. And because Polycarpa has bucked the usual trend, they say, it could prompt researchers to study solitary species more scrupulously.
T. Gordon et al., “And then there were three…: Extreme regeneration ability of the solitary chordate Polycarpa mytiligera,” Front Cell Dev Biol, doi:10.3389/fcell.2021.652466, 2021.