Bioelectricity and epimorphicregenerationScott Stewart,1 Agustin Rojas-Munoz,1 and Juan Carlos Izpisua Belmonte1,2*

SummaryAll cells have electric potentials across their membranes,but is there really compelling evidence to think that suchpotentials are used as instructional cues in developmen-tal biology? Numerous reports indicate that, in fact,steady, weak bioelectric fields are observed throughoutbiology and function during diverse biological proc-esses, including development. Bioelectric fields, gener-ated upon amputation, are also likely to play a key roleduring vertebrate regeneration by providing the instruc-tive cuesneeded to directmigrating cells to formawoundepithelium, a structure unique to regenerating animals.However, mechanistic insight is still sorely lacking inthe field. What are the genes required for bioelectric-dependent cell migration during regeneration? Thepower of genetics combined with the use of zebrafishoffers the best opportunity for unbiased identification ofthe molecular players in bioelectricity. BioEssays29:1133–1137, 2007. � 2007 Wiley Periodicals, Inc.

Introduction

Living organisms may be viewed as biological batteries since

their epithelial tissues function to maintain relatively high

concentrations of electrical charge, in the form of ions, inside

their bodies. This property endows epithelia with the remark-

able property of being able to generate an electric field,

or wound potential, when tissue damage leaks electrical

charge (freely diffusible ions) from inside the body to the

outside environment. Wound potentials are just one example

of themanydocumented bio-generated electric fields that play

roles in a surprisingly wide array of biological processes,

including cell–cell communication in the nervous system, cell

migration, differentiation, division, animal development and

organ regeneration.(1–6)

In contrast to action potentials of the nervous system, now

considered integral to the study of neurobiology, wound

potentials and other relatively weak, steady, long-lasting

bioelectric fields have been somewhat overlooked by scien-

tists and clinicians alike. This is in spite of the fact that they

were elegantly described decades ago. Thismost likely can be

attributed to a lack of insight into the mechanism of action of

bioelectricity at themolecular level. However, at the time these

experimentswere first performed, thewidearray sophisticated

molecular tools used today were not yet available to biologists

and geneticists.

A number of years ago, investigators observed that bio-

electric fields are generated by vertebrate tissues when they

undergo two intimately related, but seemingly opposite

biological processes, namely epimorphic regeneration and

non-regenerative wound healing.(1–6) However, the trail

leading to the identification of the molecular mechanisms

functioning downstream of bioelectricity during these two

events has gone cold andmany questions remain unanswered

even though research in regenerative biology has increased

exponentially in the last 25 years.Weare ultimately left with the

reality that little is still knownabout howcells and tissues derive

information from these weak electrical cues and turn it into a

biological outcome.

With the advent of modern molecular, cellular and genomic

methodologies, scientists and clinicians alike now find them-

selves in an ideal position to revisit these pressing questions in

a sorely neglected field. The most glaring of these is what

are the factors that are responsible for sensing, interpreting

and translating weak bioelectric signals into languages well

understood by biologists, namely gene expression and signal

transduction? Is there a difference between the magnitude,

direction or duration of wound potentials or the cellular

response to them by a regenerating animal when compared

to a non-regenerating one? Might variations in bioelectricity-

mediated signaling andphysiologybe factors that contribute to

understanding why humans fail to undergo the mysterious

process of epimorphic regeneration in response to organ loss

and injury?

Epimorphic regeneration

Certain organs and tissues in vertebrates cope with everyday

wear and tear by being continuously renewed. Such is the

case for the skin, blood and the lining of the gut. This type of

1Gene Expression Laboratory, The Salk Institute for Biological

Studies, La Jolla, CA 92037, USA.2Center for Regenerative Medicine of Barcelona 08003 Barcelona, Spain.

Funding agency: S. S. is a Scholar of the California Institute of

Regenerative Medicine. This work was supported by funds from

Fundacion Cellex, the National Institutes of Health, and the G. Harold

and Leila Y. Mathers Charitable Foundation to J. C. I. B..

*Correspondence to: Juan Carlos Izpisua Belmonte, Gene Expression

Laboratory, The Salk Institute for Biological Studies, La Jolla, CA

92037. E-mail: belmonte@salk.edu

DOI 10.1002/bies.20656

Published online in Wiley InterScience (www.interscience.wiley.com).

BioEssays 29:1133–1137, � 2007 Wiley Periodicals, Inc. BioEssays 29.11 1133

Problems and paradigms

self-renewal is mediated exclusively by stem cells. The cells

that will repopulate the organ undergoing renewal are the

progeny of resident or circulating progenitor (stem) cells, with

varying degrees plasticity. The cellular mechanisms that

underlie the process of tissue turnover greatly depend on the

specific tissue in question and may include cell migration,

proliferation and differentiation. Although the end-result of this

increased tissue turnover process is often referred to as tissue

‘‘regeneration’’, the term ‘‘regeneration’’, as it is used in this

article, is reserved for an entirely different biological process

altogether that is only observed in select vertebrate species,

such as salamanders and zebrafish.

In contrast to stem-cell-mediated tissue restorative proc-

esses described above, bona fide vertebrate regeneration,

technically referred to as epimorphic regeneration (Fig. 1A),

involves several unique biological events, namely dediffer-

entiation of post-mitotic cells, activation of multipotent

progenitor cells, cell proliferation, pattern formation and, in

some cases, transdifferentiation of specialized cells to rebuild

parts of the body plan after amputation or injury.(7–10) The

beauty and extraordinary complexity of regeneration has

fascinated and attracted the interest of biologists for centuries

and, indeed, it marked the beginning of the field of exper-

imental developmental biology.

The amazing capacity of urodele amphibians, such as the

newt and axolotl, to regenerate limbs, tail, jaws, heart or lens,

has made these animals the preferred subject for research on

vertebrate regeneration. This process is commonly divided

into four sequential steps: (1) formation of a wound epidermis,

in which the amputation site is covered by epithelial cells,

(2) disorganization and dedifferentiation of mesenchymal

tissue near the wound, (3) Formation of a mass of undiffer-

entiated cells, known as the blastema, primarily by dediffer-

entiation of cells in the surrounding tissue and (4) proliferation

of the dedifferentiated cells concomitantly with re-develop-

ment, in which the correct pattern is formed in the blastema

resulting in the regeneration of the amputated portions of the

organ (Fig. 1). Importantly, the first two steps outlined abovedo

not require cell proliferation and, therefore, depend on the

properties inherent to the existing cells. Working models of

regeneration using various experimental systems, suggest

that the re-development stages of regeneration are recapit-

ulations of early development. The same signaling molecules

and mechanisms, under the control of highly complex gene

regulatory mechanisms, appear to function in regeneration as

they do in early embryonic development.(7–10)

What then are the factors that initiate this complicated

biological program of organ regeneration? This is perhaps the

Figure 1. Epimorphic regeneration. A diagram of regeneration in newt limb. Formation of the wound epithelium is one of the first

cytologically discernable steps in regeneration and proceeds blastema formation and cell proliferation. Adapted from Essential

Developmental Biology, Slack J. W. Second Edition. Blackwell Publishing.

Problems and paradigms

1134 BioEssays 29.11

most-compelling question in the field of regeneration. If

observed from a temporal perspective, the first steps of

regeneration that redeploy developmental signaling proc-

esses can be thought of as truly ‘‘regeneration specific’’

phenomena whose molecular basis is completely unknown.

Insight into these processeswould yield clues as to the identity

of the driving force behind the regenerative response. There-

fore, elucidation of the mechanisms underlying the early

regenerative response is likely to be crucial in explaining the

huge disparities between regenerating and non-regenerating

organisms. Due to the extremely rapid and dramatic nature of

the early regenerative response, one cannot help sense that

something fundamentally different between these two types of

organisms exists, yet continues to be overlooked.

Wound potentials

Sometimebefore 1849, theGermanphysiologist Emil DuBois-

Reymond found that, when hemade a cut in one of his fingers,

he could cause a deflection of a galvanometer by putting

the injured finger and a contralateral unwounded finger into

the circuit.(11) This was the first documented experimental

evidence of endogenous wound electric fields, also known as

wound potentials. We now know that such electric fields are

generated when the epithelial layer is cut and the lesion short-

circuits the trans-epithelial potential (TEP). The TEP arises

because specializedepithelial cells have evolvedmechanisms

to selectively transport ions from the extracellular space to the

inside of the body.(1–6)

Let us consider the example of frog skin, which contains

epithelial sodium channels on its apical surface and Naþ/Kþ

ATPase on its basolateral surface (Fig. 2A). In this setting, Naþ

channels and Naþ/Kþ pumps act in concert to maintain a

charge differential on the inside of the animal with respect to

the outside environment.(1–6) This charge separation, com-

bined with the high electrical resistance of the plasma

membrane and the presence of tight junctions between

epithelial cells, results in the formation of a TEP with the

inside of the animal being electrically positive (the anode) with

respect to the outside environment of consisting of pond water

(the cathode). Upon damage to the tissue, ion (charge) flux to

the extracellular space ensues driven by an electrochemical

gradient (Fig. 2B). This is, of course, an electrical current with

ions, rather than electrons, being the carriers of charge and

this creates an electrical field with a magnitude on the order of

Figure 2. A:Epithelia can behave as biological batteries. Schematic representation of the skin epithelia of a frog. The combinedactivities

of Naþ channels andNaþ/Kþ pumps create a charge differential resulting in the inside of the skin having high electric potential (shaded red)

with respect to the outside surroundings of lower potential (shaded blue). This is known as a trans-epithelial potential (TEP) and the system

as a whole can be thought of as a biological battery. Note that the uni-directional transport of Naþ across the plasmamembrane precludes

completion of the circuit and thus current cannot flow. Rt and Rm refer to the inherent electrical resistance of the tissue and pond water,

respectively. B: Generation of an electric field by tissue injury. Upon damage to the epithelia, ion leakage results in an outward-directed

current and creates an electric field. Note that electric potential (shaded red) remains high at regions distal to the wound, whereas the

electric field (shaded green) is highest at regions proximal to the wound. A linear relationship between the distance from the wound and the

magnitude of both the potential and the electric field is indicated for the sake of simplicity only. Since positive charge (Naþ) flows towards the

site of injury, thewound is defined as the cathode. Adapted fromMcCaig CD, Rajnicek AM, SongB, ZhaoM2005Physiol Rev 85:943–978.

Problems and paradigms

BioEssays 29.11 1135

tens of millivolts. The phenomena of current flow and the

presence of an electric field are two sides of the same coin;

they are one and the same and you cannot have one without

the other. The two are, in fact, quantitatively related through

Ohm’s law. The phenomena of TEPs can be applied to all ion-

transporting epithelia, including mammalian skin.

Bioelectricity in regeneration:

directed cell migration

During the initial stages of epimorphic regeneration in

salamanders, a strong outward-directed electric current driven

through the stump is generated by the injury. Furthermore, the

current generated upon wounding is necessary for normal

regeneration to proceed.(3,12) Unlike wound healing in a non-

regenerating species, the intensity and direction of the electric

current generated during regeneration is maintained for

several days after the formation of the wound epithelium,(3,12)

further suggesting that the electric current may play a role in

later processes of epimorphic regeneration. Based on these

observations, one can envision that the generation of a steady,

weak electric field upon wounding may well be a regenerating

animal’s first response to such trauma. This can be attributed

to the fact that the extremely rapid leakage of ions from the

body to the outside is driven by electrochemical forces, as

opposed to the relatively slow interactions between biological

macromolecules. This crucial piece of information, we would

argue, makes it imperative to decipher the role of bioelectricity

during the early stages of regeneration.

One of the first cytologically distinguishable events during

regeneration is cell migration. Migration of epidermal cells in

amputated salamanders limbs and zebrafish caudal fins,

covers the wounded surface and forms the wound epithelium

that is essential for regeneration.(7–10). Strikingly, this initial

cell migration event in a regenerating animal, greatly contrasts

with the response of a non-regenerating animal, which secrete

an extracellular matrix to seal off the wound from the outside.

We propose that this crucial cell migration event, witnessed

only in regenerating animals or in early stages of vertebrate

development, is also mediated by bioelectrical cues.(13–16)

Indeed, recent experiments using in vitro models of non-

regenerating wound healing suggest that cell migration during

this process ismediated by bioelectric pathways.(13–16) These

studies indicate that wound-induced electric cues activate

signaling pathways similar to those reported for chemo-

taxis.(17) In both scenarios, the migratory response of cells

depends on the opposing activity of two enzymes, phospha-

tidylinositol-3-OH kinase-g (PI3Kg) and the lipid phophatase

PTEN (phosphatase and tensin homolog). Overall, these

observations indicate that, on one hand, PI3Kg activity is

essential for the ability of cells to respond to bioelectric fields

and, on the other hand, loss of PTEN activity enhanced the

migration of cells towardselectrical cues. For instance, it would

be interesting if epithelial cell migrate towards a wound in

PTEN mutant mice. Additionally, the PI3 kinase inhibitor

LY294002 can be administered to regenerating salamanders

and fish to determine if cell migration is normally involved in

wound epithelia.

In contrast, the observations of Donaldson and co-workers

have suggested thatmigration of axolotl epidermal cells invitro

can occur without bioelectric cues, since the culture system

used in these experiments is likely to short circuit any current

generated.(18–20) However, sensitive assays are required to

measure possible weakelectric fields in order to conclude that

they play no role in cellmigration. These observations can also

be interpreted that it is possible to coax cells to migrate

without bioelectric cues by in vitro culture. The multitudes of

researchers who have derived cell lines from tumor samples

ona tissue culture dish can readilyattest to theexistenceof cell

migration in an in vitro setting where bioelectric cues are likely

to be minimized. The question that needs to be addressed is

not what can occur in vitro but what does occur in vivo during

regeneration.

Unfortunately, at the present moment, researchers have

not uncovered any mechanisms as to how electrical signals

are received and converted into activation of cell signaling

pathways. It is therefore essential to isolate the factors

responsible for responding to bioelectrical signals as a first

step in understanding this area of biology. One of the great

advantages of the zebrafish as a model system is, of course,

the ability to perform sophisticated genetic screens.We would

argue that thorough unbiased genetic screens are likely to be

one of the most-efficient and productive means of uncovering

the genes required for the bioelectric response. As an

experimental system, genetic screens in zebrafish should

prove to be fruitful in identifying genes that function in

bioelectric pathways. A straight-forward approach along these

lines is to screen mutagenized fish for cell migration defects in

vivo upon caudal fin amputation using genetically encoded

fluorescent reporter genes. These animals could then be

crossed to fish engineered to express mutant alleles of PI3Kgor PTEN to perform epistasis experiments. This type of assay

has theadvantageof being simple andquite rapid since it takes

less than 24 hours for wound epithelial formation in the

zebrafish caudal fin.

A pressing question is how is PI3Kg activated by bioelectricsignals? PI3Kg has been shown to function mainly down-

stream of heterotrimeric G-proteins. Thus a logical guess

would be that a G-protein-coupled receptor and its regulators

may function in bioelectric signal transduction. This is an

attractive hypothesis due to the fact that this large family of

proteins has been extensively studied from biochemical and

physiological standpoints and a large number of pharmaco-

logical agents that specifically target G-protein-coupled

receptors are readily available, some in the form of FDA-

approved drugs. Libraries derived from these small molecules

could rapidly screen for effects on bioelectricity-mediated cell

Problems and paradigms

1136 BioEssays 29.11

migration in fish. Any ‘‘hits’’ from these types of screenswould

provide useful mechanistic information about bioelectric

signaling and regeneration, as well as immediately raise

the possibility of therapeutic intervention with antagonists/

agonists of these receptors. For instance, the mouse, a non-

regenerating animal, could be treated with pharmacological

agents identified in zebrafish screens and cell migration could

be tested after limb amputation. The use of ectoderm-specific

fluorescent reporter line would provide an efficient assay to

monitor cell migration in mice during wound healing.

The last decade has shed light on the basic cellular events

that initiate a regenerative response. Yet, research has still not

uncovered the mystery as to why only some animals can

regenerate whereas humans cannot. Regeneration appears

to proceed by a complex cascade of gene activity, but what

initiates this cascade? Is bioelectricity the key to regeneration?

It is too early to say, but it is clear that it is essential and should

be accounted for by researchers in regenerative biology.

Acknowledgments

We gratefully acknowledge Dr.Yasuhiko Kawakami insightful

discussion and helpful comments on the manuscript. We

apologize to any authors’ work whose was not cited or

overlooked due to space limitation. Interested readers should

refer to the excellent reviewarticles cited here, aswell as to the

primary literature cited therein for more insight.

References1. Altizer AM, Moriarty LJ, Bell SM, Schreiner CM, Scott WJ, et al. 2001.

Endogenous electric current is associated with normal development of

the vertebrate limb. Dev Dyn 221:391–401.

2. Borgens RB, Roederer E, Cohen MJ. 1981. Enhanced spinal cord

regeneration in lamprey by applied electric fields. Science 213:611–617.

3. Borgens RB, Vanable JW Jr, Jaffe LF. 1977. Bioelectricity and regene-

ration: large currents leave the stumps of regenerating newt limbs. Proc

Natl Acad Sci USA 74:4528–4532.

4. McCaig CD, Rajnicek AM, Song B, Zhao M, 2005. Controlling cell

behavior electrically: current views and future potential. Physiol Rev 85:

943–978.

5. Nuccitelli R. 2003. A role for endogenous electric fields in wound healing.

Curr Top Dev Biol 58:1–26.

6. Nuccitelli R, Robinson K, Jaffe L. 1986. On electrical currents in

development. Bioessays 5:292–294.

7. Alvarado AS, Tsonis PA. 2006. Bridging the regeneration gap: genetic

insights from diverse animal models. Nat Rev Genet 7:873–884.

8. Brockes JP, Kumar A. 2005. Appendage regeneration in adult verte-

brates and implications for regenerative medicine. Science 310:1919–

1923.

9. Poss KD, Keating MT, Nechiporuk A. 2003. Tales of regeneration in

zebrafish. Dev Dyn 226:202–210.

10. Tanaka EM. 2003. Regeneration: if they can do it, why can’t we? Cell

113:559–562.

11. DuBois-Reymond E. 1843. Vorlaufiger Abriss einer Untersuchung uber

den sogenannten Froschstrom und die electomotorischen Fische. Ann

Phy U Chem 58:1–30.

12. Borgens RB, Vanable JW Jr, Jaffe LF. 1977. Bioelectricity and

regeneration. I. Initiation of frog limb regeneration by minute currents.

J Exp Zool 200:403–416.

13. Zhao M, Song B, Pu J, Wada T, Reid B, et al. 2006. Electrical signals

control wound healing through phosphatidylinositol-3-OH kinase-gamma

and PTEN. Nature 442:457–460.

14. Zhao M, Dick A, Forrester JV, McCaig CD. 1999. Electric field-directed

cell motility involves up-regulated expression and asymmetric redistrib-

ution of the epidermal growth factor receptors and is enhanced by

fibronectin and laminin. Mol Biol Cell 10:1259–1276.

15. Zhao M, Bai H, Wang E, Forrester JV, McCaig CD. 2004. Electrical

stimulation directly induces pre-angiogenic responses in vascular

endothelial cells by signaling through VEGF receptors. J Cell Sci 117:

397–405.

16. Zhao M, Agius-Fernandez A, Forrester JV, McCaig CD. 1996. Orientation

and directed migration of cultured corneal epithelial cells in small electric

fields are Serum dependent. J Cell Sci 109:1405–1114.

17. Van Haastert PJ, Devreotes PN. 2004. Chemotaxis: signalling the way

forward. Nat Rev Mol Cell Biol 5:626–634.

18. Donaldson DJ, Mahan JT, Tsilibary EC, McCarthy JB, Dixit SN, et al.

1994. Migratory interaction of amphibian epidermal cells with compo-

nents of the basement membrane. J Cell Physiol 158:79–86.

19. Mahan JT, Donaldson DJ. 1992. Divalent cations and extracellular matrix

receptor function during newt epidermal cell migration. J Cell Sci 101:

173–181.

20. Donaldson DJ, Mahan JT, Smith GN Jr. 1988. Newt epidermal cell

migration over collagen and fibronectin involves different mechanisms.

J Cell Sci 90:325–333.

Problems and paradigms

BioEssays 29.11 1137