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Humans possess a limited capacity to restore miss-ing or injured body parts. Stimulating this capabilitymight circumvent some of the tissue deteriorationthat accompanies old age. Other organisms, such assalamanders and planaria, boast remarkable re-generative powers, sprouting limbs or producing en-tire new individuals. Once a scientific backwater,study of these creatures is maturing. As researchersuncover the secrets behind regeneration, they hopeto conjure up similar forces in people.

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

Humans don’t possess the same prowess. Many researchershope that prying into the mysteries of stem cells might revealstrategies to keep our own systems fresh. But other scientistsare banking on the idea that studying regeneration artists mightuncover the information necessary to breathe new life into oldtissues and organs. Innovations in genetic tools are helping re-searchers gain insight into the molecular underpinnings of re-generation in these creatures. “You really have to know how itworks in animals that know how to do it,” says David Stocum, adevelopmental biologist at Indiana University–Purdue Univer-sity in Indianapolis.

Regeneration has perplexed biologists for hundreds of years.Study of the phenomenon blossomed in the 18th century. In1744, Swiss scientist Abraham Trembley described how the hy-dra—a small tubular freshwater animal that spends its life cling-ing to rock or wood—developed into two new organisms afterbeing cut. This finding was a “spectacularly important piece ofwork,” says Phillip Newmark, a regeneration researcher at theUniversity of Illinois, Urbana-Champaign. “A lot of historians ofscience say that that was the birth of experimental biology.” Lat-er that century, Italian Lazzaro Spallanzani noted that salaman-ders regrew missing tails. At the end of the 1800s, Thomas HuntMorgan devoted years of effort to studying regeneration in pla-naria. But realizing what a difficult problem it was, he left theflatworm behind and switched his attention to the fruit fly, a fu-ture darling of the genetics laboratory. Organisms such as hydra,planaria, and salamanders haven’t shared the recent scientificlimelight with flies, nematodes, and mice, hearty species that re-produce well under lab conditions. However, a relatively smallbut dedicated band of researchers has kept up the venerable tra-dition and continued to tackle regeneration in these animals.

Arrested DevelopmentThose efforts are beginning to reveal why some animals aresuperstars at rejuvenating ailing tissues whereas others, such ashumans, are not. The key, researchers say, is the capacity torekindle mechanisms responsible for an organism’s embryonicdevelopment. A hydra, for instance, is “a kind of permanentembryo,” says Brigitte Galliot, a molecular biologist at the Uni-versity 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 asmany as 20% of its total number—ready to attend to a wound.

Regenerating RegenerationSalamanders, 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(Published 1 September 2004)

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Updated classic. Researchers are bringing 21st century ap-proaches to organisms such as the hydra, whose regenerativepower was first described in the 1700s.

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“The planarian invests all its energy in maintaining adult tis-sues,” says Newmark. “It’s an animal that has taken stem cellsto an extreme.” Planarian stem cells collect at an injury site andform a blastema, a ball of unspecialized cells that can regener-ate organs and tissues.

Salamanders take a more complicated path.When these scuttlers require a new limb or or-gan, they deprogram specialized cells rather thanrecruit stem cells that already exist. “[Regenera-tion] comes down to how you convert an adultdifferentiated cell back into an embryonic under-differentiated one,” says Ken Muneoka, a cell biologist at Tulane University in New Orleans,Louisiana. As in planaria, these cells congregateat the site of a severed limb or injured organ andform a blastema, which regrows the missingpart. But until recently, researchers hadn’t pin-pointed how these creatures maintain such pliability.

Back to the FutureNew approaches are aiding researchers in theirhunt for the molecules that enable hydra, pla-naria, and salamander cells to harness powersusually reserved for infancy. Study of thesecreatures has suffered because they’re lessamenable to genetic manipulations—such as re-moving or adding genes—than are flies, worms,or even mice. For decades, scientists have been“relegated to old-style experiments,” says cellbiologist Mark Keating of Harvard University.“That means cutting off a limb and watching itgrow.” Those kinds of endeavors have been cru-cial, “but they don’t tell you about mecha-nisms,” he says. “To get at mechanisms, youneed to have genetics.”

Organisms that lend themselves to geneticmanipulation, however, stink at regeneration.They invest their energy in reproduction—aboon for geneticists—at the expense of tissuemaintenance. Except for sperm and eggs, cells infruit flies and nematodes don’t divide, and, with-out a source of new cells, these organisms can’treplenish decaying tissue. “They’re designed tolay eggs and as soon as they lay eggs, they’redone,” says Alejandro Sánchez Alvarado, amolecular biologist at the University of Utah,Salt Lake City. Mice can rejuvenate some typesof tissue, such as skin and bone marrow, but arenowhere near as proficient as salamanders. Andsuperregenerators don’t pass muster in the genetics lab; for in-stance, salamanders have a relatively long generation time com-pared with those of flies or nematodes, which slows experi-ments 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 developnew technology in organisms that have robust regenerative ca-pacity,” says Keating.

Now, investigators are devising the tools they need to identi-fy and manipulate the molecules that are responsible for extraor-dinary powers of rejuvenation. For instance, scientists are in the

throes of sequencing the planaria genome. And Newmark,Sánchez Alvarado, and co-workers have successfully appliedRNA interference, a now-standard method for turning genes offin numerous organisms, to planaria, they reported last year.

They are using these tools to systematicallyblunt genes one by one and identify thosewhose absence quells regeneration. Al-though this hunt is in its early stages, theteam has already found that several genesknown to stimulate embryonic developmentalso foster tissue renewal in the adult. New-mark is also looking for genes that eithercrank up or shut down their activity in re-generating planarian tissue; those resultswill presumably lead to the genes that con-trol the process.

Similarly, Galliot and her colleagues aretracking down genes that goad hydra re-growth. They’ve focused on DNA se-quences that influence gene activity, whichthey hope will point them toward themolecules that orchestrate renewal in thesefreshwater creatures. For instance, they’vefound that a gene-control molecule calledcAMP response element–binding protein(CREB) grabs onto DNA more frequentlyin regenerating tissue than in nonregenerat-ing tissue, suggesting that it might crank ongenes more robustly during regrowth; further-more, during tissue replenishment, moreCREB molecules carry phosphate groups,modifications that prompt CREB to sparkgene activity, the team reported on 24February in the Proceedings of the NationalAcademy of Sciences (PNAS). The study al-so revealed a potential trigger point for re-newal of certain structures but not others:Blocking the enzyme that knits phosphatesonto CREB halted the growth of hydraheads, but feet still formed. The team isnow trying to pinpoint which types of cellskick CREB into action; that informationwill help them determine which tissuetypes organize the regeneration process.

Other scientists are bringing new ani-mals into the regeneration fold. For in-stance, Keating scouted around for geneti-cally pliable organisms that can regenerateand found that “the best one is zebrafish,”he says. “Fins, spinal cord, heart, retina,

pancreas—it regenerates all of these.” His team has probed formutations that cripple regrowth of clipped fins. Like the sala-mander, the zebrafish sparks specialized cells to deprogram andform a blastema. These cells turn on a gene called msx, Keatingand his colleagues have found. This gene apparently keeps cellsin a malleable state by dampening programs needed for special-ization. It also helps repair broken hearts, Keating says, sug-gesting that similar machinery controls restoration in differentorgans. Like other champion regenerators, zebrafish readily re-treat to an embryonic state—at least on the cellular level. Whengauged against heart cells from mammals, for example, those

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Getting a head. In 7 days, a pla-nar ian grows a new noggin.Black dots in the upper threepanels are photo receptors.

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from zebrafish seem more like fetal cells than adult ones, saysKeating. Among other similarities, they are small and dividequickly, as baby heart muscle cells do. “There are gradationsof differentiation,” he says, and less specialized cells mightmore readily back up and assume stem cell–like potency.

Other work suggests that similar pathways organize re-generation in other species. A salamander’s mature musclecells, which contain multiple nuclei, break apart into manycells with one nucleus each, and these cells divide and grow toreplenish muscle. Cells that undergo this transition activatethe Msx1 gene, reported regeneration researcher JeremyBrockes of University College London and colleagues on 17August in PLOS Biology, and blocking its activity preventsthe dispersal. Mouse embryos, which can reconstitute severeddigits, also spark Msx1, as well as another gene called Bmp4,Muneoka and colleagues found last year. And humans alsoharbor Msx1 and Bmp4, hinting that results from model organ-isms might inspire ways to stimulate regeneration in people.

Use It or Lose ItBut this observation raises the issue of why humans don’t al-ready exploit the potential of these genes. That question is dif-ficult to answer, in part because scientists haven’t teased apartthe forces that encourage organisms to maintain the ability toregenerate.

“We don’t understand how regeneration plays out in evo-lution,” says Brockes. Regeneration likely arose very early,say some experts. Almost all major groups of multicellularanimals contain at least one species with the capacity to re-generate, and it’s unlikely that somany species developed thepower independently. More like-ly, evolutionary pressure actedon animals to either maintain orlose that ancient skill.

Humans might have tradedregeneration for other strengths.“One explanation is that the lackof nervous system regenerationis the price you pay for a highlysophisticated nervous system,”says marine biologist MichaelThorndyke of Kristineberg Ma-

rine Research Station in Fiskebäckskil, Swe-den. Cells that specialize to a high degreemust retrace many steps to return to a formthat can rekindle regeneration, he says, so an-imals might have sacrif iced renewal profi-ciency for ornately developed tissues. Howev-er, for every good regenerator, “there areclosely related species that have lost the abili-ty to regenerate,” says Brockes, suggestingthat complexity alone doesn’t dispense withthe talent. Others suggest that humans could-n’t afford to carry the rapidly reproducingcells necessary to bolster tissues. Peoplemight have lost the ability to regenerate be-cause maintaining such large numbers of di-viding cells would increase the risk of cancer,posits Newmark. Although planaria are prac-tically immortal, they maintain exquisite con-

trol of cell division, he says—a power that might have wanedas our tissues took on specialized tasks.

Amphibian, Heal ThyselfAlternatively, humans’ injury response might impede renew-al. “To have big wounds really is life threatening,” says Mu-neoka. “To solve that problem, maybe we abandoned regener-ation and went to immediate wound closure.” Supporting thatidea, “one thing that we know about regeneration is that ani-mals that regenerate don’t scar,” says Muneoka. Scar forma-tion, he adds, shows how rushed and disorganized the mam-malian injury response is.

Other observations bolster idea that mammals have re-generative potential, but that our rapid healing process blocksrenewal. For instance, after a spinal cord injury, mammalianneurons start to grow, but glial cells—the helpers of the ner-vous system—prompt the formation of a scar at the injury and“regeneration stops cold,” says Muneoka. If researchers cutthe spinal cord of a mouse in such a way that hinders scarring,neurons reconnect. This observation suggests that mammaliancells harbor a latent capacity to grow, but their surroundingsquell that potential. “It’s all a matter of context,” says SánchezAlvarado.

Researchers have identified key wound-healing moleculesthat favor rejuvenation. For instance, thrombin—a protein thatcontrols blood clotting—is a “pivotal signal for regeneration,”says Brockes. Newts trigger thrombin activity in their eyes afteran injury, and blocking thrombin’s workings prevents lensregeneration, Brockes and colleagues reported last year in Cur-

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New kid on the regeneration block. The zebrafish, a laboratory favoritefor studies of embryonic development, is now revealing feats of adult tissuerenewal.

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Sleight of hand. Prodding arms to grow where they don’t belong might help scientists teaseapart the salamander’s secrets.

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rent Biology. In addition, salamanders—which can’t grow newlenses—spark thrombin activation in limbs but not in lenses, sug-gesting that thrombin helps determine whether a tissue can renewitself. The protein is apparently part of the signal that promptsspecialized cells to morph into jacks-of-all-trades, says Brockes.

Regeneration researcher David Gardiner of the University ofCalifornia, Irvine, proposes that fibroblasts—connective tissuecells—orchestrate renewal after an organism sustains a wound.“Fibroblasts are very plastic,” he says, and they behave in waysthat would aid regeneration; for instance, they are relatively un-specialized, compared with other cell types. Fibroblasts createthe framework for a new limb or organ, he says, and then othercell types, such as muscle and nerve cells, take up residence inthat structure and reactivate genes that control embryonic devel-opment. Fibroblasts are underappreciated, Gardiner adds:“They’re always seen as kind of boring, but they’re kind of aglue that holds us together.”

To further delineate the steps in regeneration, Gardiner andhis team are using a new approach, which they describe in theApril 2004 issue of Developmental Dynamics. In salamanders,small skin wounds typically heal over. But when scientistsreroute a nerve to those areas, they can create burgeoning limbswhere none should exist. A blastema forms at the wound site,but it grows into a full arm only if the researchers cover it with apatch of skin. The presence of the nerve appears to speed thesplitting of cells, and the patch of skin seems to provide the im-petus for cells to specialize and form a fully developed limb. Bymanipulating the molecules present during each stage of the pro-cess, Gardiner hopes to delineate which ones orchestrate eachstep and perhaps decipher where mammalian wound healing diverges.

Reversal of FortuneAs scientists tease apart how regeneration unfolds in the sala-mander and other creatures, they aim to one day apply thatknowledge to coax the same feats out of human tissues. Never-theless, provoking regeneration in mammals might not be asimple task. “I’m not sure I believe that there’s any single mech-anism 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 whenappropriate; unharnessed growth might lead to cancer. “The po-tential is there to turn pathways on, but how do you turn themback off again?” says Thorndyke. “Stopping growth is prettydarn important, and it’s one of the real dangers that isn’t oftenhighlighted in the embryonic stem cell debate.”

Again, model creatures might provide clues. After losing alimb, an amphibian grows a new one that’s proportioned for thesize of the beast. It also reconstructs only the missing part. “Ifyou cut it off at the wrist, you get a hand; if you cut it off at theshoulder, you get an arm,” says Brockes. “They’re able to derivelocal cues that are important for guiding regeneration.” Re-searchers are beginning to make inroads into identifying poten-tial signals. Fibroblasts might be crucial, says Gardiner, becauserecent studies suggest that fibroblasts are not all alike. Thosefrom different anatomical positions display unique patterns ofgene activity, according to a 2002 report in PNAS by genomeresearcher Patrick Brown and colleagues at Stanford University.

Models such as the salamander might advance practical ef-forts to spur regeneration in people. Reinvigorating insulin-producing β cells in the pancreas and replenishing substantia

nigra neurons in patients with Parkinson’s disease using stemcells “are very real goals,” says Muneoka. “But those cellsneed instructions,” and regeneration studies will likely illumi-nate the tutoring those cells need, he says. Those insightsmight be especially important as researchers attempt to re-construct entire organs rather than just one type of cell. Fu-ture work on the regenerative magicians of the animal worldwill perhaps reveal the rules that guide reformation of com-plex structures, findings that could eventually allow diseasedhuman hearts or other organs to rebuild themselves. And inthe short term, Muneoka adds, as regeneration studies lend agreater understanding of healing processes, they might helpdoctors treat wounds to minimize scarring. Says Brockes: “Iwould be astonished if in the future of regenerative medicinesomething like the [salamander] mechanism didn’t have animportant contribution to make.” And that contribution mighthelp people conjure up their own magic to help age-relatedtissue deterioration disappear.

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

Further ReadingS. G. Lenhoff and H. M. Lenhoff, Hydra and the birth of experimental biology,

1744: Abraham Trembley’s Memoires concerning the polyps (BoxwoodPress, Pacific Grove, CA, 1986).

A. Trembley, Mémoires pour servir à l’histoire d’un genre de polypes d’eaudouce à bras en forme de cornes [Memoirs concerning the natural historyof a type of freshwater polyp with arms shaped like horns] (Jean and Her-man Verbeek, Leiden, the Netherlands, 1744).

SmedDb planaria.neuro.utah.eduZebrafish Information Network zfin.org

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2. H. Y. Chang et al., Diversity, topographic differentiation, and positionalmemory in human fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 99,12877–12882 (2002).

3. T. Endo, S. V. Bryant, D. M. Gardiner, A stepwise model system for limb re-generation. Dev. Biol. 270, 135–145 (2004).

4. M. Han, X.Yang, J. E. Farrington, K. Muneoka, Digit regeneration is regulat-ed by Msx1 and BMP4 in fetal mice. Development 130, 5123–5132 (2003).

5. Y. Imokawa, J. P. Brockes, Selective activation of thrombin is a critical de-terminant for vertebrate lens regeneration. Curr. Biol. 13, 877–881 (2003).

6. Y. Imokawa, A. Simon, J. P. Brockes, A critical role for thrombin in verte-brate lens regeneration. Phil. Trans. R. Soc. Lond. B 359, 765–776 (2004).

7. K. Kaloulis, S. Chera, M. Hassel, D. Gauchat, B. Galliot, Reactivation of de-velopmental programs: The cAMP-response element-binding protein path-way is involved in hydra head regeneration. Proc. Natl. Acad. Sci. U.S.A.,101, 2363–2368 (2004).

8. A. Kumar, C. P. Velloso, Y. Imokawa, J. P. Brockes, The regenerative plasti-city of isolated urodele myofibers and its dependence on Msx1. PLoS Biol.2 (8), e218 (2004).

9. P. A. Newmark, P. W. Reddien, F. Cebria, A. Sánchez Alvarado, Ingestion ofbacterially expressed double-stranded RNA inhibits gene expression inplanarians. Proc. Natl. Acad. Sci. U.S.A. 100, 11861–11865 (2003).

10. P. A. Newmark, A. Sánchez Alvarado, Not your father’s planarian: A classicmodel enters the era of functional genomics. Nat. Rev. Genet. 3, 210–219(2002).

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