New knowledge about the regenerative powers of newts is overturning 250 years of conventional scientific wisdom and may one day lead to unlocking a similar capacity in humans.
In 1994 Goro Eguchi headed out the door of his research laboratory in Okazaki, Japan, on a hunt that had become familiar to him over the course of his long career. Eyes trained downward, Eguchi, 61 years old at the time, searched ponds and puddles for the Japanese fire-bellied newt. The creatures aren’t easy to spot. Although their underbellies are dotted in bright orange from chin to tail, their backs are brownish-black, helping them blend with muddy water.
Few developmental biologists in the world are as familiar with these newts, also known as Cynops pyrrhogaster, as Eguchi. He’s devoted his career to studying a biological phenomenon known as regeneration, the ability of some animals to regrow a lost body part. Other animals can regenerate — including salamanders, frogs and worms — but newts are the champions. Remove part of a limb or tail and another one grows. Take away the lens on the eye? No problem. In one month, a new lens grows back.
The fire-bellied newt’s ability to restore certain tissue has fascinated scientists for more than 250 years. In 1768, Lazzaro Spallanzani studied regeneration in newts and frogs, cutting off limbs and watching new ones return. Sometimes, though, the limbs that regrew in Spallanzani’s experiments were missing some bones or didn’t otherwise grow back properly. So for a long time, researchers studying regeneration were convinced that as animals aged, their ability to regenerate limbs, lenses and even hearts diminished over time.
No one had ever designed an experiment to test that conclusion. Eguchi and his colleague Panagiotis Tsonis had their suspicions about how aging would affect regeneration because they’d both worked with the newt for decades. Tsonis, director of UD’s Center for Tissue Regeneration and Engineering at Dayton, had done doctoral work in Eguchi’s lab and has devoted his career to figuring out how lens regeneration in newts works.
To test such an idea, of course, they needed to take the long view. That’s why, 17 years ago, Eguchi began collecting adult newts. He needed newts that were nearly full-sized to make certain they would be old enough at the start of the experiment. The fire-bellied newt grows slowly, reaching about 4 inches long — about 90 percent of its mature length — after 14 years.
Japanese fire-bellied newts were the perfect research subjects for this type of experiment. Unlike American newts, which don’t live very long and don’t tolerate captivity well, Japanese newts can live more than 30 years in captivity and thrive in laboratory life.
Eguchi’s lab took responsibility for the animal maintenance and planning of surgery, and Tsonis collaborated with researchers at the Sanford Children’s Health Research Center in La Jolla, Calif., to analyze the animals’ DNA, molecular profile and the structure of their lenses.
“American newts have such a short lifespan in captivity, so keeping them around in the lab for a continued experiment is tricky,” says Tsonis. “It’s the type of collaboration that could not happen otherwise.”
Starting such an experiment was a leap of faith. Seventeen years ago, the DNA techniques needed to analyze the data either hadn’t been developed or were too expensive to even consider. That’s why up until a decade or so ago, regeneration science had been mostly descriptive, says Alejandro Sánchez Alvarado, a regeneration specialist at the Stowers Institute for Medical Research in Kansas City, Mo. Scientists had been chopping off limbs or heads and tails of worms or removing lenses and then watching them grow back, but they couldn’t do much more.
Whether one is watching newts regrow lenses or watching worms regrow heads and tails, regeneration makes for great videos. But those videos don’t tell researchers what is going on at the molecular level, nor can it identify the genes responsible. Over the past 10 years, though, DNA sequencing — the technique that allows scientists to “read” the genetic code — has become less expensive, and other molecular techniques that allow scientists to add or remove genes or switch genes off have helped the regeneration field in general.
The progress at the molecular level has been slow because animals that regenerate well (newts and a species of worm known as planaria) have not been amenable to study with traditional genetics, either because their sexual reproductive cycles are too long or because traditional genetics and molecular resources were not available. So newts and their regenerating brethren began to fall behind other research animals, such as mice or even zebra fish.
Researchers like Tsonis spent painstaking years getting these genetic and molecular techniques to work in the newts, his lab supported by continuous funding from the National Institutes of Health since 1995. As other molecular techniques became available (such as ways to silence genes), the field of regeneration technology slowly became less descriptive and researchers started to piece together the networks of genes and molecules involved in rebuilding lost tissues.
As the 16-year experiment continued, Tsonis was able, through painstaking work, to use these techniques to analyze lens regeneration. Eventually, Tsonis could compare whether the same genes were switched on or off year after year as the newts grew older.
The technique to remove the lens (called a lentectomy) is simple. Just a tiny slit in the cornea followed by a light pinch with fine forceps, and the entire lens comes out in one piece. The cornea heals in 24 hours, and a lens has been differentiated within a month.
Over the first six years of the experiment, Eguchi’s team performed 12 lentectomies (two a year on the same eye of each newt). After carefully examining the lenses from those surgeries, the researchers determined that repetition was not a problem: The lens architecture (the size and shape of the tissue) and molecules in each lens were exactly the same. After that, Eguchi removed the lenses only once a year, and the team focused on the effects of aging. In 2011, after the experiment had been going for 16 years, Tsonis felt it was time to stop. “We had quite clear data,” he says.
In a study published this summer in Nature Communications, Eguchi and Tsonis concluded that newts’ ability to regenerate lenses was practically limitless: the 17th and 18th lenses (the last two lenses removed) were exactly the same as lenses removed when the experiment began 16 years earlier, they found. And even newts that were at least 30 years old — comparable to a 90-year-old human — showed no decline in their ability to regrow lenses every bit as good as those they started out with as young’uns. Each lens regrew with equal speed and vigor.
“This is a fundamental paper,” says Sánchez Alvarado. “It’s going to become a classic for two reasons, a practical reason and a scientific reason.” Such lengthy, basic science experiments are extremely unlikely to be funded in the United States, he says, because such research grants are given for five years and renewals for four years. Funding is also rare for a single experiment. “It’s very difficult to accomplish long-term experiments.”
But the experiment is notable not only for the researchers’ perseverance but also for its scientific significance, Sánchez Alvarado says. “Here is a real experiment with real data that essentially says, ‘Vertebrates can actually do this; they are aging chronologically, the animals are 30 years old, but biologically they’re young.’ To me that’s a remarkable paradigm shift because it provides incontrovertible evidence that chronological and biological age are not necessarily the same thing. It’s nice to go to your list of things we don’t know about regeneration and scratch that one off the list.”
Sánchez Alvarado says the list of what scientists don’t know about regeneration is still quite long. Now, with all of the information from genome sequencing on so many species, researchers know there’s a finite collection of genes, and those genes are coming together in some organized fashion to produce a finite collection of attributes that are shared throughout all animal species. For Sánchez Alvarado, the take-home message is that “we’re incredibly closely related to each other, so it should be feasible to understand why some animals can do certain things and others cannot; why some animals can regenerate so well now becomes part of the landscape for our interrogation.’’
Even though people don’t regenerate body parts like newts, the regenerative capacities we do possess begin to diminish with age. Hair recedes, wrinkles increase, muscle mass goes away. None of this happened in the newts’ lenses. None of the newts got cataracts. Since humans, mice and so many organisms share genes, regeneration scientists say that we may be able to figure out why some organisms regenerate limbs and heads and others don’t. Researchers suspect that we all have the capability, but in humans that capacity is genetically turned off for most tissues. People can regenerate liver and skin, and children can regenerate fingertips. Now that researchers know that aging newts can churn out fresh lenses, Tsonis says they may be able to figure out how to restore specific tissues lost to degeneration and aging.
Over the past 16 years, Tsonis has collaborated on not only the lens aging experiment. He’s also continued to make his own mark in lens regeneration. He finds the lens attractive because it provides a more clear-cut way for the research to proceed than limb regeneration because the process happens faster. Even more alluring was the way the lens regenerates. For limb regeneration, part of the limb is removed. In the lens, the entire organ is removed and then rebuilt from a different group of cells in the eye tissue. That phenomenon has allowed Tsonis a unique opportunity to study how one tissue stops in its tracks and then recreates an entirely different kind of tissue.
“That’s quite unique, even in the newt,” he says.
Studying regeneration in the lens offered another advantage over limb regeneration: the newt lens always regenerates from cells in the dorsal, or upper, part of the eye and never the ventral, or lower part, even though they’re the same type of cell.
Regeneration starts as a group of cells responsible for pigment in the iris begin a process that turns them into completely different cells and then back again. Scientific lingo for these twin processes are dedifferentiation (when cells slip back to a less specialized form) and transdifferentiation (when one cell type converts into another cell type). Tsonis wants to know everything about how these processes work to understand fundamental biological questions about how and why cells grow old and die, and why some turn cancerous.
David Stocum, a regeneration researcher at the Indiana University Center for Regenerative Biology and Medicine, compares the capacity of newts’ lenses to regenerate to a human’s ability to regrow the liver. Researchers can remove a fairly substantial fraction of the liver in lab experiments, and it will regenerate over and over — but he says the Tsonis team has regenerated the lens in the same animal many more times than anyone has repeatedly generated the liver.
As newts age, explains Stocum, their capacity to regenerate limbs declines. Either regeneration slows or the new limb grows with mistakes, such as an extra digit. In the long newt experiment the cells that built lens after lens made no mistakes, suggesting that the problems with limb regeneration might result from its more complex structure or external factors such as infection. “It tells us though that all of these old dogmas — and there have been lots of them — are not viable anymore. So the possibility exists that we will find out how to manipulate things at the site of an injury or disease to regenerate the tissue.”
Tsonis plans on going down some of those research avenues. He says finding answers in one area of regeneration will answer basic questions in other areas. For instance, Tsonis wants to see what’s going on with DNA repair and aging. He’s intrigued by cancer formation in the newts. While in Eguchi’s lab during his doctoral studies, Tsonis gave the newts all sorts of cancer-inducing chemicals, but the newts never got cancer. Now, he wants to return to those experiments so he can figure out why. “If that process is regulated, then I can trace it.”
He also wants to investigate the relationship between what newt cells do during regeneration and how stem cells work.
“There’s no doubt in my mind that nature invented common strategies and then modified them in different animals according to needs. I don’t think they’re completely different strategies.”
Investigating such strategies can spark ideas for research in mice and eventually people, says Tsonis. Although that’s a long way off, cellular pathways are similar and so are cell physiologies. He wants to discover whether newts and people have the same genes and cellular mechanisms.
One day, in the distant future, Tsonis hopes to use this research to find a way to treat eye disease, such as macular degeneration. “It’s not that easy, but that’s the ultimate goal of regeneration, to treat people.”
Jeanne Erdmann is a medical science writer in Wentzville, Mo. Her work has appeared in Scientific American, Nature and Science News.
Center for Tissue Regeneration and Engineering at Dayton http://trend.udayton.edu/
Coverage of the research in Discover http://bit.ly/p8T6K9
Read the paper in Nature Communications http://bit.ly/p86LiI