Sci. Aging Knowl. Environ., 1 September 2004
Vol. 2004, Issue 35, p. ns6
[DOI: 10.1126/sageke.2004.35.ns6]


Regenerating Regeneration

Salamanders, flatworms, and other creatures engage in dazzling feats of renewal. Now, researchers are beginning to uncover the molecular bases for these body-building tricks, hoping to decipher how humans might perform similar stunts

R. John Davenport

Abstract: Humans possess a limited capacity to restore missing or injured body parts. Stimulating this capability might circumvent some of the tissue deterioration that accompanies old age. Other organisms, such as salamanders and planaria, boast remarkable regenerative powers, sprouting limbs or producing entire new individuals. Once a scientific backwater, study of these creatures is maturing. As researchers uncover the secrets behind regeneration, they hope to conjure up similar forces in people.

A salamander has a few tricks up its sleeve. If it loses a leg or a tail to the jaws of a predator, it develops a new one. That appendage grows to the appropriate size and proportion of the original. Other creatures perform similarly: Starfish replace missing arms, bits of planaria produce whole new flatworms, and zebrafish repair clipped fins and injured hearts.

Humans don't possess the same prowess. Many researchers hope that prying into the mysteries of stem cells might reveal strategies to keep our own systems fresh. But other scientists are banking on the idea that studying regeneration artists might uncover the information necessary to breathe new life into old tissues and organs. Innovations in genetic tools are helping researchers gain insight into the molecular underpinnings of regeneration in these creatures. "You really have to know how it works in animals that know how to do it," says David Stocum, a developmental biologist at Indiana University-Purdue University in Indianapolis.

Regeneration has perplexed biologists for hundreds of years. Study of the phenomenon blossomed in the 18th century. In 1744, Swiss scientist Abraham Trembley described how the hydra--a small tubular freshwater animal that spends its life clinging to rock or wood--developed into two new organisms after being cut. This finding was a "spectacularly important piece of work," says Phillip Newmark, a regeneration researcher at the University of Illinois, Urbana-Champaign. "A lot of historians of science say that that was the birth of experimental biology." Later that century, Italian Lazzaro Spallanzani noted that salamanders regrew missing tails. At the end of the 1800s, Thomas Hunt Morgan devoted years of effort to studying regeneration in planaria. But realizing what a difficult problem it was, he left the flatworm behind and switched his attention to the fruit fly, a future darling of the genetics laboratory. Organisms such as hydra, planaria, and salamanders haven't shared the recent scientific limelight with flies, nematodes, and mice, hearty species that reproduce well under lab conditions. However, a relatively small but dedicated band of researchers has kept up the venerable tradition and continued to tackle regeneration in these animals.

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Updated classic. Researchers are bringing 21st century approaches to organisms such as the hydra, whose regenerative power was first described in the 1700s. [Credit: A. Trembley, Mémoires pour servir à l'histoire d'un genre de polypes d'eau douce à bras en forme de cornes (1744)]


Arrested Development Back to Top

Those efforts are beginning to reveal why some animals are superstars at rejuvenating ailing tissues whereas others, such as humans, are not. The key, researchers say, is the capacity to rekindle mechanisms responsible for an organism's embryonic development. A hydra, for instance, is "a kind of permanent embryo," says Brigitte Galliot, a molecular biologist at the University of Geneva in Switzerland. Even before an injury occurs, it has the cells it needs to step in and reconstruct the organism. Similarly, planaria maintain a cadre of stem cells--perhaps as many as 20% of its total number--ready to attend to a wound. "The planarian invests all its energy in maintaining adult tissues," says Newmark. "It's an animal that has taken stem cells to an extreme." Planarian stem cells collect at an injury site and form a blastema, a ball of unspecialized cells that can regenerate organs and tissues.

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Getting a head. In 7 days, a planarian grows a new noggin. Black dots in the upper three panels are photoreceptors. [Credit: A. S´┐Żnchez Alvarado/University of Utah]

Salamanders take a more complicated path. When these scuttlers require a new limb or organ, they deprogram specialized cells rather than recruit stem cells that already exist. "[Regeneration] comes down to how you convert an adult differentiated cell back into an embryonic underdifferentiated one," says Ken Muneoka, a cell biologist at Tulane University in New Orleans, Louisiana. As in planaria, these cells congregate at the site of a severed limb or injured organ and form a blastema, which regrows the missing part. But until recently, researchers hadn't pinpointed how these creatures maintain such pliability.

Back to the Future Back to Top

New approaches are aiding researchers in their hunt for the molecules that enable hydra, planaria, and salamander cells to harness powers usually reserved for infancy. Study of these creatures has suffered because they're less amenable to genetic manipulations--such as removing or adding genes--than are flies, worms, or even mice. For decades, scientists have been "relegated to old-style experiments," says cell biologist Mark Keating of Harvard University. "That means cutting off a limb and watching it grow." Those kinds of endeavors have been crucial, "but they don't tell you about mechanisms," he says. "To get at mechanisms, you need to have genetics."

Organisms that lend themselves to genetic manipulation, however, stink at regeneration. They invest their energy in reproduction--a boon for geneticists--at the expense of tissue maintenance. Except for sperm and eggs, cells in fruit flies and nematodes don't divide, and, without a source of new cells, these organisms can't replenish decaying tissue. "They're designed to lay eggs and as soon as they lay eggs, they're done," says Alejandro Sánchez Alvarado, a molecular biologist at the University of Utah, Salt Lake City. Mice can rejuvenate some types of tissue, such as skin and bone marrow, but are nowhere near as proficient as salamanders. And superregenerators don't pass muster in the genetics lab; for instance, salamanders have a relatively long generation time compared with those of flies or nematodes, which slows experiments that require breeding and analyzing offspring. As a result, researchers have faced a difficult decision: "Continue old style, give up on regeneration ... or roll up their sleeves and develop new technology in organisms that have robust regenerative capacity," says Keating.

Now, investigators are devising the tools they need to identify and manipulate the molecules that are responsible for extraordinary powers of rejuvenation. For instance, scientists are in the throes of sequencing the planaria genome. And Newmark, Sánchez Alvarado, and co-workers have successfully applied RNA interference, a now-standard method for turning genes off in numerous organisms, to planaria, they reported last year. They are using these tools to systematically blunt genes one by one and identify those whose absence quells regeneration. Although this hunt is in its early stages, the team has already found that several genes known to stimulate embryonic development also foster tissue renewal in the adult. Newmark is also looking for genes that either crank up or shut down their activity in regenerating planarian tissue; those results will presumably lead to the genes that control the process.

Similarly, Galliot and her colleagues are tracking down genes that goad hydra regrowth. They've focused on DNA sequences that influence gene activity, which they hope will point them toward the molecules that orchestrate renewal in these freshwater creatures. For instance, they've found that a gene-control molecule called cAMP response element-binding protein (CREB) grabs onto DNA more frequently in regenerating tissue than in nonregenerating tissue, suggesting that it might crank on genes more robustly during regrowth; furthermore, during tissue replenishment, more CREB molecules carry phosphate groups, modifications that prompt CREB to spark gene activity, the team reported on 24 February in the Proceedings of the National Academy of Sciences (PNAS). The study also revealed a potential trigger point for renewal of certain structures but not others: Blocking the enzyme that knits phosphates onto CREB halted the growth of hydra heads, but feet still formed. The team is now trying to pinpoint which types of cells kick CREB into action; that information will help them determine which tissue types organize the regeneration process.

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New kid on the regeneration block. The zebrafish, a laboratory favorite for studies of embryonic development, is now revealing feats of adult tissue renewal. [Credit: M. Keating/Harvard University]

Other scientists are bringing new animals into the regeneration fold. For instance, Keating scouted around for genetically pliable organisms that can regenerate and found that "the best one is zebrafish," he says. "Fins, spinal cord, heart, retina, pancreas--it regenerates all of these." His team has probed for mutations that cripple regrowth of clipped fins. Like the salamander, the zebrafish sparks specialized cells to deprogram and form a blastema. These cells turn on a gene called msx, Keating and his colleagues have found. This gene apparently keeps cells in a malleable state by dampening programs needed for specialization. It also helps repair broken hearts, Keating says, suggesting that similar machinery controls restoration in different organs. Like other champion regenerators, zebrafish readily retreat to an embryonic state--at least on the cellular level. When gauged against heart cells from mammals, for example, those from zebrafish seem more like fetal cells than adult ones, says Keating. Among other similarities, they are small and divide quickly, as baby heart muscle cells do. "There are gradations of differentiation," he says, and less specialized cells might more readily back up and assume stem-cell-like potency.

Other work suggests that similar pathways organize regeneration in other species. A salamander's mature muscle cells, which contain multiple nuclei, break apart into many cells with one nucleus each, and these cells divide and grow to replenish muscle. Cells that undergo this transition activate the Msx1 gene, reported regeneration researcher Jeremy Brockes of University College London and colleagues on 17 August in PLoS Biology, and blocking its activity prevents the dispersal. Mouse embryos, which can reconstitute severed digits, also spark Msx1, as well as another gene called Bmp4, Muneoka and colleagues found last year. And humans also harbor Msx1 and Bmp4, hinting that results from model organisms might inspire ways to stimulate regeneration in people.

Use It or Lose It Back to Top

But this observation raises the issue of why humans don't already exploit the potential of these genes. That question is difficult to answer, in part because scientists haven't teased apart the forces that encourage organisms to maintain the ability to regenerate.

"We don't understand how regeneration plays out in evolution," says Brockes. Regeneration likely arose very early, say some experts. Almost all major groups of multicellular animals contain at least one species with the capacity to regenerate, and it's unlikely that so many species developed the power independently. More likely, evolutionary pressure acted on animals to either maintain or lose that ancient skill.

Humans might have traded regeneration for other strengths. "One explanation is that the lack of nervous system regeneration is the price you pay for a highly sophisticated nervous system," says marine biologist Michael Thorndyke of Kristineberg Marine Research Station in Fiskebackskil, Sweden. Cells that specialize to a high degree must retrace many steps to return to a form that can rekindle regeneration, he says, so animals might have sacrificed renewal proficiency for ornately developed tissues. However, for every good regenerator, "there are closely related species that have lost the ability to regenerate," says Brockes, suggesting that complexity alone doesn't dispense with the talent. Others suggest that humans couldn't afford to carry the rapidly reproducing cells necessary to bolster tissues. People might have lost the ability to regenerate because maintaining such large numbers of dividing cells would increase the risk of cancer, posits Newmark. Although planaria are practically immortal, they maintain exquisite control of cell division, he says--a power that might have waned as our tissues took on specialized tasks.

Amphibian, Heal Thyself Back to Top

Alternatively, humans' injury response might impede renewal. "To have big wounds really is life threatening," says Muneoka. "To solve that problem, maybe we abandoned regeneration and went to immediate wound closure." Supporting that idea, "one thing that we know about regeneration is that animals that regenerate don't scar," says Muneoka. Scar formation, he adds, shows how rushed and disorganized the mammalian injury response is.

Other observations bolster the idea that mammals have regenerative potential, but that our rapid healing process blocks renewal. For instance, after a spinal cord injury, mammalian neurons start to grow, but glial cells--the helpers of the nervous system--prompt the formation of a scar at the injury and "regeneration stops cold," says Muneoka. If researchers cut the spinal cord of a mouse in such a way that hinders scarring, neurons reconnect. This observation suggests that mammalian cells harbor a latent capacity to grow, but their surroundings quell that potential. "It's all a matter of context," says Sánchez Alvarado.

Researchers have identified key wound-healing molecules that favor rejuvenation. For instance, thrombin--a protein that controls blood clotting--is a "pivotal signal for regeneration," says Brockes. Newts trigger thrombin activity in their eyes after an injury, and blocking thrombin's workings prevents lens regeneration, Brockes and colleagues reported last year in Current Biology. In addition, salamanders--which can't grow new lenses--spark thrombin activation in limbs but not in lenses, suggesting that thrombin helps determine whether a tissue can renew itself. The protein is apparently part of the signal that prompts specialized cells to morph into jacks-of-all-trades, says Brockes.

Regeneration researcher David Gardiner of the University of California, Irvine, proposes that fibroblasts--connective tissue cells--orchestrate renewal after an organism sustains a wound. "Fibroblasts are very plastic," he says, and they behave in ways that would aid regeneration; for instance, they are relatively unspecialized, compared with other cell types. Fibroblasts create the framework for a new limb or organ, he says, and then other cell types, such as muscle and nerve cells, take up residence in that structure and reactivate genes that control embryonic development. Fibroblasts are underappreciated, Gardiner adds: "They're always seen as kind of boring, but they're kind of a glue that holds us together."

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Sleight of hand. Prodding arms to grow where they don't belong might help scientists tease apart the salamander's secrets. [Credit: D. Gardiner/University of California, Irvine]

To further delineate the steps in regeneration, Gardiner and his team are using a new approach, which they describe in the April 2004 issue of Developmental Dynamics. In salamanders, small skin wounds typically heal over. But when scientists reroute a nerve to those areas, they can create burgeoning limbs where none should exist. A blastema forms at the wound site, but it grows into a full arm only if the researchers cover it with a patch of skin. The presence of the nerve appears to speed the splitting of cells, and the patch of skin seems to provide the impetus for cells to specialize and form a fully developed limb. By manipulating the molecules present during each stage of the process, Gardiner hopes to delineate which ones orchestrate each step and perhaps decipher where mammalian wound healing diverges.

Reversal of Fortune Back to Top

As scientists tease apart how regeneration unfolds in the salamander and other creatures, they aim to one day apply that knowledge to coax the same feats out of human tissues. Nevertheless, provoking regeneration in mammals might not be a simple task. "I'm not sure I believe that there's any single mechanism that ... would fix our ability to regenerate," says Brockes. And even if researchers can kick-start tissue renewal in people, they must also keep the process under control and stop it when appropriate; unharnessed growth might lead to cancer. "The potential is there to turn pathways on, but how do you turn them back off again?" says Thorndyke. "Stopping growth is pretty darn important, and it's one of the real dangers that isn't often highlighted in the embryonic stem cell debate."

Again, model creatures might provide clues. After losing a limb, an amphibian grows a new one that's proportioned for the size of the beast. It also reconstructs only the missing part. "If you cut it off at the wrist, you get a hand; if you cut it off at the shoulder, you get an arm," says Brockes. "They're able to derive local cues that are important for guiding regeneration." Researchers are beginning to make inroads into identifying potential signals. Fibroblasts might be crucial, says Gardiner, because recent studies suggest that fibroblasts are not all alike. Those from different anatomical positions display unique patterns of gene activity, according to a 2002 report in PNAS by genome researcher Patrick Brown and colleagues at Stanford University.

Models such as the salamander might advance practical efforts to spur regeneration in people. Reinvigorating insulin-producing {beta} cells in the pancreas and replenishing substantia nigra neurons in patients with Parkinson's disease using stem cells "are very real goals," says Muneoka. "But those cells need instructions," and regeneration studies will likely illuminate the tutoring those cells need, he says. Those insights might be especially important as researchers attempt to reconstruct entire organs rather than just one type of cell. Future work on the regenerative magicians of the animal world will perhaps reveal the rules that guide reformation of complex structures, findings that could eventually allow diseased human hearts or other organs to rebuild themselves. And in the short term, Muneoka adds, as regeneration studies lend a greater understanding of healing processes, they might help doctors treat wounds to minimize scarring. Says Brockes: "I would be astonished if in the future of regenerative medicine something like the [salamander] mechanism didn't have an important contribution to make." And that contribution might help people conjure up their own magic to help age-related tissue deterioration disappear.

September 1, 2004

R. John Davenport is an associate editor of SAGE KE in Santa Cruz, California. He'd rather no one applied regeneration technology to bad '80s music.

Suggested ReadingBack to Top

  • S. G. Lenhoff and H. M. Lenhoff, Hydra and the birth of experimental biology, 1744: Abraham Trembley's Memoires concerning the polyps (Boxwood Press, Pacific Grove, CA, 1986).
  • A. Trembley, Mémoires pour servir à l'histoire d'un genre de polypes d'eau douce à bras en forme de cornes [Memoirs concerning the natural history of a type of freshwater polyp with arms shaped like horns] (Jean and Herman Verbeek, Leiden, the Netherlands, 1744).
  • SmedDb
  • Zebrafish Information Network
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Citation: R. J. Davenport, Regenerating Regeneration. Sci. Aging Knowl. Environ. 2004 (35), ns6 (2004).

Stem cells and ageing: The potential of stem cells to overcome age-related deteriorations of the body in regenerative medicine.
A. D. Ho, W. Wagner, and U. Mahlknecht (2005)
EMBO Rep. 6, S35-S38
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