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Differentiation (1984) 28 :87-93 DifferentiationQ
Springer-Verlag 1984
Model and hypothesesAre limb development and limb regeneration
both initiatedby an integumentary wounding?A hypothesisRichard B.
BorgensDepartment of Anatomy, School of Veterinary Medicine, Purdue
University, West Lafayette, IN 47907, USA
Abstract. It is proposed that, whereas an actual wound toa
salamander limb may initiate limb regeneration, a localand
developmentally programmed integumentary woundmay initiate limb
development. The electrophysiologicalchanges induced by these
lesions of the skin may be a com-mon denominator linking limb
regeneration and limb devel-opment. Such early electrical events
are considered to initi-ate or guide the early accumulation of
cells, and to helpto produce the local environment in which a limb
will arise.This scheme provides a self-limiting
positive-feedbackmechanism for the production of a localized area
whereother developmental mechanisms act in concert with endog-enous
electrical fields (or in their complete absence), therebyleading to
limb differentiation. This hypothesis may notbe restricted to limb
formation; it may be of more generalsignificance, i.e. in the
process of organogenesis n embryos.One might reasonably suggest
that, by such a mechanism,any developing placode (for example,
auditory or olfactoryplacodes) might form and localize.
IntroductionWhat is a wound? A wound is usually regarded as a
breakin an organisms integument produced by an acute trauma.(This
concept can also be applied to a single cell, whenan external force
disrupts the membrane.) In animals,wounds can also be thought of as
signals that elicit a multi-tude of biological phenomena designed
to prevent furtherinjury and restore functional integrity to the
tissues. Themobilization of cells designed to phagocytose cellular
de-bris, and cellular migrations (sometimes also cell
division)which close lesions are initiated by sublethal wounds
inmany, if not most, metazoa. In more complex animals,wounds can be
associated with higher orders of biologicalresponses, such as
inflammation reactions and immunologi-cal responses. The wound
itself is a signal of damage, andin still-viable organisms, the
biological mobilization thatresults often leads to repair.In many
systems, the cellular responses to woundingmay be nearly
instantaneous; for example, cell migrationin Xenopus skin begins
within secondr after the injury [26].There have been many
suggestions as to what wound-in-d u d mechanisms initiate these
phenomena (for example,wound hormones, chalones, or other
biochemical events[24]). I would like to focus on one instantaneous
physical
event that is often overlooked, but which accompanies
allwounding- namely, a dramatic change in the electrophysio-logical
character of undamaged skin near the wound. Anydisruption of an
integument produces a steep electrical fieldadjacent to the lesion,
and this is associated with a steadyflow of ionic (electric)
current [2, 41. This is because integu-ments (like cellular
membranes) support large (about48-80 mV) potential differences
across themselves. Whena physical opening (an electrical leak) is
produced in theintegument, current will flow. This current will be
persis-tent, because it is driven by the same cellular batteries
lo-cated in the epidermis which initially support the separationof
charge across the skin. After a new membrane or woundepithelium has
covered the open leak, the current flow willpersist for varying
lengths of time, because such initial cov-erings are usually
ionically leaky. Current flow andwounding are inextricably linked.
I know of no wound toa cell or an organism which is not associated
with immedi-ate electrical responses- he two are inseparable.
Moreover,there is evidence to suggest that the electrica l
consequencesof wounding may be critical for wound healing or
regenera-tive responses [4]. This idea has come from studies of
am-phibian limb regeneration (4, 91. The earliest signalwhich
induces an area of local change in ontogeny leadingto limb
formation may also be a wounding, i.e. a pro-grammed developmental
wounding in which the integrityof limb-forming flank integument is
disrupted. It is thisresponse to integumentary wounding that may
initiate boththe generation and regeneration of limbs in
ampibians.Skin batteries and the regeneration of limbsIt is
well-known to students of amphibian limb regenerationthat a
wounding of the integument initiates this developmental sequence of
events. In experiments in whichaccesso-ry limbs are produced in
intact salamanders and newts bythe deviation of nerve tissue to a
location beneath the flankskin, the success of such procedures is
greatly enhancedby (or is perhaps even dependent on) wounding [3,
341.Skin flaps can be experimentally produced which cover theface
of a limb stump and inhibit limb regeneration. How-ever, if a small
area is incompletely covered with skin ofa full thickness (by
accident or design), a simple epitheliumwill form, and a limb will
arise from this area [22]. Thus,limbs regenerate from an area of
integumentary disruptionwhere the wound is left open to the
environment or is onlycovered by a simple epithelium.
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88
I t is well-known that amphibian limb regeneration isdependent
both on innervation and a wound epithelium[28, 31, 32, 351. In
recent years, it has also been suggestedthat an ionic current
traversing the core tissues of the stumpis necessary (but not
sufficient) for the regeneration of thelimb [4]. This stump current
is produced by amputation,and the voltage source driving this
current is the internallypositive transcutaneous potential known to
exist across am-phibian skin (as well as the skin of most animals,
includingman [2, 4, 5, 171). When a break in the continuity of
theskin is produced, current (defined as moving in the directionof
positive charge) flows through the low-resistance path-way of the
wound produced by amputation (Fig. 1a). Theevidence fo r the
necessity of this current flow for limb re-generation is as
follows:1. The current traversing the core tissues of the stumpin
adult frogs (nonregenerators) is strikingly reduced whencompared to
the density of the current (hence electric fields)within the core
tissues of the stump in salamanders andnewts. (This is due to a
shunting of current through subder-ma1 lymph spaces that is found
in anura but not urodeles(71).
Fig. 1. In sahm anders, the skin isthe electrom otive force that
drivcselectric current (charg e) out of theregenerating stum p end
(a) or outof presumptive limb regions anddeveloping buds in larvae
(b). Anelectric field associated with thiscurrent flow is produced
withinthe core tissues of theregenerating limb an d the lo caleof
the developing limb. It is thisnaturally produced vo ltagegradient
that is thought to be acritical factor in limbdevelopment. (The
circuit iscompleted via the conductivepondwater or the surfacc
moistureof semiterrestrial adults)2. Enhancing the fields within
adult-frog limb stumps(by implantation of batteries and electrodes)
can initiatea measure of limb regeneration [6] r improve the
externalform of limb regeneration in hypomorphically
regeneratingspecies [8].3. Topical applications of amiloride,
benzamil, ormethyl ester of lysine, chronic immersion in a
Na+-depletedmedium, and the imposition of a counter current
within
limb stumps all serve to inhibit or retard limb regeneration,or
cause it to be abnormal [4, 10, 331. What all of thesedifferent
techniques have in common is that they reducethe currents
traversing the salamander limb stump.4. Although there are
unreconciled differences betweenthe regeneration and development of
the amphibian limb,it is probable that these processes share
certain mechanismsof control. Both are characterized by the
amassing of cellsto form a limb rudiment (by cell division and
migration),and both share certain anatomical similarities (such as
theapical cap of the limb blastema and the apical ectodermalridge
of many developing limb buds). One may wonderif developing limbs
and regenerating limbs share a similarphenemonology of endogenous
current flow. It is significant
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89
Fig. 2a , b. Electronmicrographs of the basal domain of larval
axolotl epidermis.a Ventrolateral, non-limb-forming flank.b
Limb-formingflank approximatly 800 pm caudal to th e area depicted
in a (same animal). In a, note th e numerous hemidesmosome (h)
attachmentsto the basal laminae (arrow), the vast arrays of
tonofilaments (I) within the basal cell, and a well-ordered
sublamellar matrix (sm).In b, although a basal laminae (arrow) is
still evident, note the absence of hemidesmosornes and
tonofilaments, and the large intracellularspace between the two
opposed basal cells. The sublamellar matrix (sm) is
characteristically disorganized beneath the epidermis ofthe
limb-forming flank. N, ucleus of basal cell. x 18,OOOthat limb
development in the larvae of both a salamander(the axolotl [ll])
and a frog (Xenopus [27]) is predictedby a local exodus of current
from the exact area from whicha limb will arise.Ionic batteries and
limb developmentThe outcurrents that predict limb formation are
intriguingfor several reasons. First, they are remarkably similar
intheir phenomenologies: both bud currents predict theexact
location where the limb will arise (Fig. 1b). In thedevelopment of
frogs, the hindlimbs are the first to appearand form rapidly.
Robinson [27] has detected peaks in theoutcurrent over limb-forming
regions in stage45 (prebud)Xenopus embryos. In the slowly
developing hindlimb of theaxolotl [l 11, peaks in current density
accurately predict thelocalization of bud formation about 6 days
prior to its ap-pearance. In both species, the current densities
declinesteadily during the growth of the bud and localize aboutit.
In studies of axolotls with large limb buds, the currentsreversed
their direction in half of the animals. It is alsointriguing that
the main source of the current may be differ-ent in the two
species. In Xenopus, the gill epithelium appar-ently drives the
extracellular current that leaves the flank(inward currents were
measured at the gill; large outwardcurrents were measured at the
bud). In the axolotl, diffuseincurrents were usually measured over
the skin of non-limb-forming regions. This is the usual direction
of current overamphibian integument, thus suggesting a skin
batterysimilar to that which drives the stump currents in
adults
Lastly, the exodus of current from this localized areaof
apparently intact skin is curious. It is understandable
how a lesion could produce a low-resistance current leak.In the
intact larvae, it is less clear what changes in theintegument may
allow or produce such localized outcur-rents.The anatomy and
physiology of limb-bud currentsThe integuments of animals usually
possess a high transcu-taneous resistance (relative to other organs
and tissues [2,161). Most of this resistance to charge movement (as
inmost epithelia) is due to tight junctions between the apicalcells
of the epidermis. Although there are numerous junc-tional complexes
within the cell strata of epithelia (or epi-dermis), it is the
tight-junction complex which is relativelyimpermeable to the flow
of ions, thus allowing the separa-tion of fluid compartments and
the presence of a potentialdifference across this cellular
syncytium. Moreover, the for-mation, degradation, and electrical
characteristics of tightjunctions are known to differ in various
regions of the sameepithelium (proximal and distal convoluted
tubule of themouse kidney), to exhibit a process of maturation in
anarea of epithelial renewal (in the crypt region of
intestinalepithelia), and to appear and disappear during early
em-bryogenesis (blastulation) and organ regeneration
(liver)(reviewed by Staehelin [29]).It is possible that prior to
the anatomical changes asso-ciated with limb formation, a decrease
in junctional resis-tance in the local area where limbs will arise
allows currentto leak out through the epidermis. The fact that
there isa local efflux of current across this special area is
good(if not direct) evidence for a decrease in
tight-junctionalresistance. Subsequent changes in the anatomy of
the inte-gument associated with early limb-bud development
could
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90
Fig.3a-e. Hypothetical scheme for early limb-bud formation. a In
a region of presumptive limb formation, cells of the
embryonicepidermis are closely opposed and reside on a well-defined
basement membrane which overlays a well-ordered sublamellar
matrix.The apical cells are attached t o each o ther by tight
junctions. T his forms a barrier to extracellular charge movement,
while an inwardlypositive transcutaneous potential (of tens of
millivolts) is supported by cellular ionic pumps. b A programmed
reduction in tight-junctionresistance allows a short-circuit
current to flow through this electrically leaky area of flank. c
This short-circuit current would lowcrand, perhaps, reverse the
normal potential supported across this now leaky area of epidermis,
thus resulting in further cellular disorganiza-tion. d Th e
subepidermal voltage gradient associated with this increasing
current flow would be negative immediately below the expanseof
leaky epidermis, thereby providing a rough guide for migratory
mesenchyme cells and the subsequent projections of axons. e Asthe
subepidermal cellular plaque grows in mass and cellular packing, it
would become an increasing resistance in the path of theleakage
current. The total voltage imposed across this developing ar ea
would then be shared by the plaque (placode) and the
overlyingepidermis. This might result in a reduction in the net
effects of this imposed voltage on the overlying epidermis and lead
to its returnto a more normal structural and physiological state.
This postulated positive-feedback system, in which a change in the
electricalcharacter of the embryonic epidermis leads to an anato
mically distinct ar ea of flank (the presump tive limb), may
eventually be self-limiting
also explain the persistent nature of the localized
currentleak.We (Borgens RB, Callahan L, Rouleau M F ; unpub-lished
results) have recently completed a light- and electron-microscope
investigation of changes in the anatomy of limb-forming skin which
compared this flank skin with non-limb-forming flank of the same
salamander. The very localizedregion of skin overlying the limb
mesenchyme placodes (orvery early limb buds) showed a remarkable
degree of disor-ganization and, sometimes, deterioration. There was
an in-crease in the extracellular space, a disappearance of
thehemidesmosome complexes between the basal cells and bas-al
laminae, a gross disorganization of the sublamellar ma-trix, an
apparent decrease in the overall junctional complex-ity of the
epidermal cells, and a curious deterioration andsloughing of apical
epidermal cells (Fig. 2). These changes
were usually restricted to th c small (diameter, about100-200
pm) patch of skin overlying the early accumulationof mesenchyme and
very early bud protuberances. Someof these structural changes have
been observed by others,but in larger or differentiating limb buds;
for example,Kelly and co-workers have noted changes in the
characterof cell attachments and the disorganization of the
sublamel-lar matrix in Xenopus [20]. A necrosis of individual
cellsnear the apical ridge in human limb buds has also beennoted
[21]. Such zones of cell necrosis are a well-knownfeature of
developing limbs [18].The necrosis and sloughing of apical cells
explains howa local area of integument can stay electrically leaky.
Inaxolotls, patches of apical epithelium are lost, and withthis
group of cells, the tight junctions coupling them arealso lost.
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91The early initiation of limb buds: A hypothesisThe already
mentioned facts and observations suggest thefollowing hypothesis:1.
Prior to bud mesenchyme accumulation, developmen-tally programmed
alteratons are initiated in a local areaof presumptive limb-field
integument. These changes canbe summarized as a decrease in the
resistance of tight junc-tions coupling cells of the epidermis
(Fig. 3b) followed bythe local deterioration, disorganization,
loosening, andsloughing of epidermal cells. The net result of these
changesis the production of an electrical leak.
2. Current driven by the internally positive transcutan-eous
potential in adjacent skin or by more distant ion-pumping epithelia
(such as gill epithelia) will leak out ofthis local area. This
current leak may additionally causegreater disorganization of this
localized area of integument,thereby producing a positive-feedback
loop leading to stillgreater cellular disorganization and greater
densities of cur-rent leakage (Fig. 3c).3. The direction of current
flow is outwardly directedtowards this leak; thus, the voltage
gradient beneath larvalskin will be negative in the vicinity of the
outwardly directedleak compard to that of more distant subcutaneous
areas(Fig. 3d). Since most migratory and developmentally
activecells, such as fibroblasts and neural-crest cells, are
knownto move toward the negative pole of an applied electricalfield
in culture [13, 23, 301, one might reasonably suggestthat this
subcutaneous extracellular voltage gradient mayprovide a rough
guide for limb-forming mesenchyme cells.The field may ultimately
serve to direct and consolidatethe mesenchyme in this local area.
Additionally, nerves areknown to project into the growing bud. I t
is well establishedthat nervous tissue in vivo or in vitro can be
affected byapplied electric fields[4]. It is worth pointing out
that devel-oping single axons (in culture) strikingly deviate their
axisof growth towards the negative pole of an applied
field[19,25].Thus, endogeneous extracellular fields may providea
rough vector which influences axons to enter the regionof the
developing limb bud.4. As the number of cells in the bud increases
greatly,the impedence to current flow should rise (relative to
earlierstages, when few cells occupy this area). The total
voltagedrop associated with the endogenous current leakingthrough
this local area would then be shared between theepidermis and the
mesenchyme plaque forming beneath it(a new resistance in the path
of the leakage current). There-fore, the magnitude of the potential
imposed across thislocal epidermis by the leakage current may
steadily decreaseas the placode becomes an increasing resistance
due to itsincreasing size and cellular packing. This may lead to
areduction in the net effects of fields and leakage currenton the
overlying skin (Fig. 3e). This overlying integumentmay then begin
to return to a more normal structural andphysiological state, thus
reducing the leakage current stillfurther. Such a return to
normalcy is suggested by the reap-pearance of incurrents over large
buds in the axolotl [ll].Since normal skin usually takes up cations
from pondwater,one usually observes currents entering it [5 , 11,
121. Thus,the positive-feedback loop which may aid in the
productionof a local condition of cell accumulation (the early
bud)may eventually be self-limiting.Overall, it is both convenient
and instructive to viewthe deterioration of a small localized patch
of larval flank
integument as a wound- a physiological wound (perhapssimilar to
the preprogrammed cell death that occurs inthe intradigit tissues
during the morphogenesis of extremi-ties in some vertebrates [18]).
In this case, whether thewound to the amphibian integument is
produced by a sharpobject or the local destruction and
disorganization of cellsis not as important as its consequences. An
injury currentflowing out the lesion is the direct result in both
cases.As limb buds and limb blastema all share similar problems(and
probably similar solutions) when producing a limb,it is fair to
suggest that the available data points to a rolefor limb-bud
currents in limb development. How can thisbe tested?Testing the
modelCan one interfere with or eliminate bud currents? Suchan
experimental maneuver should retard or inhibit limbdevelopment, if
the endogenous fields are critical to theprocess. We have not been
able to reduce larval bud cur-rents by eliminating various ions
from the external culturemedia or by the use of specific blocking
agents (such asAmiloride for Na+ , Verapamil for Ca ++ ,etc. [ll]).
Thisis because larval skins (perhaps neotenous urodeles as well)are
very nonspecific to the ions pumped across the integu-ment [11,
121.Another approach would be to attempt to induce limbbuds in
amphibia that do not possess them in nature (caeci-lians or sirens)
or snake embryos by artificially imposinga field across
limb-forming flank regions which is compara-ble in magnitude and
polarity to the fields associatd withdeveloping limbs. Could an
appropriately placed weak elec-tric field induce supernumerary
limbs in tetrapod urodeles?Measurements of extracellular current
could also be madeabout the flank regions of avian limb mutants
availablefor study. Would current leave flank areas in limbless
ani-mals? Would there be extra foci of current in
polydactylidmutants? It is interesting that there are known regions
oflimb potency in adult salamander flanks; at distances farfrom the
fore or hind limb, this potency diminishes. Couldthe areas of
potency correspond to the expanse of flankwhere outcurrents can be
measured in larvae?One might also be able to determine whether the
localchanges in flank integument are intrinsic to this
particulararea. This could be accomplished by simple grafting
proce-dures commonly used by embryologists. A patch of headskin
could be exchanged for flank skin at times well beforelimb
formation. Such procedures along with the measure-ment of
extracellular current using a vibrating electrodeand anatomical
techniques could determine:
1. Whether the local structural and physiological chan-ges in
the skin are intrinsic. If so, one would expect tosee current
leaking from the patch of flank skin removedto its new location on
the head. Likewise, such currentleaks should be absent from the
head skin grafted in placeover limb-forming regions.2. By varying
the times of the grafts prior to limb forma-tion, one might be able
to observe at what time these chan-ges are determined or
developmentally programmed.3. One might also be able to determine
whether inappro-priate skin (perhaps not leaking outcurrents)
prevents limbformation. Textbook accounts [l] of such classical
experi-ments suggest that limbs develop even when
presumptivelimb-bud ectoderm is replaced with skin from another
re-
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92gion of the body. A close inspection of R.G. Harrisonsoriginal
paper of 1918 [I41 eveals that this experimentalmanipulation was
never performed. In all trials, Harrisonreplaced the ectoderm
overlying well-developed limb budsin Ambystomapunctatum, but not
the ectoderm of a prelimbregion. Tantalizing evidence that foreign
skin might haveinhibited the formation of limbs if the grafts had
been madeearlier can be found in Harrisons further studies on
thedevelopment of gills in Ambystoma larvae [15]; lank ecto-derm,
when grafted in place of presumptive brachial ecto-derm, prevented
the formation of gills. This result has re-cently been repeated
using axolotls and the larvae of Tari-clta torosa (L.E. DeLanney,
personal communication).
Concluding remarks and reservationsI have suggested that a
steady electrical field existing be-neath the larval flank may
electrophorese or galvano-tactically guide cells into a local area
of accumulation. Inthis regard, the polar ity is correct, i.e.,
negative in regionsof cell accumulation. However, the fields used
to experi-mentally guide various neuronal and nonneuronal cells
inculture are in the order of 10mV/mm to several hundredmillivolts
per millimeter. The fields beneath larval am-phibian skin may be
much weaker than even 2mV/mm[l 1, 271. Thus, at a first glance, th
e endogenous fields seemtoo weak to be biologically active.
Overall, we have littleinsight into the responsiveness of cells to
electric fields invivo. An electrically induced hypertrophy of
nerves in frogstumps resulted when 1 0G 20 0n A of total current
waspulled through the limb-stump tissues. Since one electrodewas
metallic [ 6 ] , it is certain that polarization occurrredbetween
this electrode and the wick electrode stimulatingthe stump. The
total current may have radically declinedby 10- to 100-fold of this
figure within hours. This suggestscellular responses in vivo to
extremely minute electricalfields.
Altogether, most of the experiments discussed here poin tto the
necessity of an integument specific to local develop-mental events.
This is not a novel idea, since the develop-ment of a variety of
organs and extremities depends ona close relationship between
specific embryonic integumentsand mesenchyme. The exact nature of
this relationship isstill an active subject of investigation. A
novel proposalis that the role of the integument in these events m
ay beintimately involved with its electrical character; i.e., th
egeneration of extracellular ionic currents and electricalfields in
regions of growth and development (see also thediscussionin [11,
19,23, 27, 301). The mechanisms for thegeneration of such
endogenous fields m ay be a prepro-grammed uncoupling of the
embryonic integument (produc-ing an electrical leak)at various
times and at various loca-tions in embryos. The net result is a
localized current flowthrough various local areas (and a t various
times of devel-opment) which may produce a local area that is
distinctfrom adjacent areas, thereby setting the stage for
subse-quent epimorphic processes.
Acknowledgements.I wish to thank Marie Rouleau for the
electronmicroscopy, and David Williams for the excellent artwork. I
alsothank Gene McGinnis and Joseph Vanable for a careful readingof
this manuscript. This work was supported by an N.I.H.
grant(NS-19598-01).
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Received April 1984 / Accepted in revised form July 1984
