AMER. ZOOL., 18:883-896 (1978).
Organization of the Morphogenetic Field in Regenerating Amphibian Limbs
DAVID L. STOCUM
Department of Genetics and Development,
University of Illinois,
Urbana, Illinois 61801
SYNOPSIS. The amphibian limb is an example of a secondary embryonic field that can be
reactivated during larval or adult life so that amputated parts are regenerated. Two major
questions are: (1) what is the origin of the morphogenetic field of the regeneration
blastema, and (2) what is the nature of this field and how does it specify the spatial pattern
of blastemal redifferentiation? Evidence is analyzed here which leads to the following
propositions: (1) the field is represented in latent form by properties of the mature limb
cells, and these properties are activated and inherited by the blastemal cells after
amputation and dedifferentiation. At the same time, the inherited field is sensitive to the
mature stump tissues and its spatial organization can be altered by a stump pattern alien to
the one from which it was derived. (2) The properties of the mesodermal limb tissues
represent positional values that are arranged in gradients along the'proximal-distal,
anterior-posterior and dorsal-ventral axes. These properties allow dedifferentiated
mesodermal cells to change their positional value to any value between their original one in
the limb and the value of any neighboring cell after creation of a discontinuity. The
direction of change is always from proximal to distal in the PD axis; it is uncertain as to
whether change can take place only centripetally or both centripetally and centrifugally
along the AP and DV axes. (3) Epidermal cells have the same positional value everywhere
in the limb and act as the distal and circumferential boundaries up to which the
mesodermal cells may change their positional values. The proximal boundary is represented by the level-specific properties of the mesodermal cells at the maximum extent of
distal to proximal dedifferentiation. Normal regeneration can then be visualized as
occurring in the following way. When deletions are made in the limb pattern, cells with
widely different positional values are confronted. During regeneration, blastema cells
increase in number and continually interact with their neighbors to adjust their positional
values within the boundaries until discontinuities are eliminated. The multiple limbs
resulting from rearrangement of stump tissue patterns can also be accounted for by using
these propositions. It is suggested that positional information is encoded on the cell surface
and/or in the extracellular matrix.
INTRODUCTION
The major mystery for developmental
biologists is how to account for pattern
formation, the process whereby embryonic
cells differentiate in the precise spatial orders that we recognize as tissues and organs. We have long recognized that the
fertilized egg develops according to an
epigenetic plan that enables cells to know
their positions within the whole and to
react to this positional information (Wolpert, 1969, 1971) by differentiating in the
Most of the original work described here was sup-
5^5.NM^Kjrrp^SlDi£i
copy of his manuscript describing the averaging
model in advance of its publication.
appropriate spatial patterns. This organization was conceptualized by Weiss (1939)
as the morphogenetic field, an abstraction
borrowed from physics to denote certain
similarities in the behavior of electromagnetic fields and organ districts after
perturbation. Two kinds of morphogeneticfieldmay be distinguished: (1) primary
fields, which are represented by the
cytoplasmic or cortical heterogeneity of the
egg (for example, ectoderm, mesoderm,
and endoderm); and (2) secondary fields,
which are subdivided out of the primary
fields during and after gastrulation. Fields
have two major properties by which they
? " be analyiedP(.)Pthey can' regulate Z
iorm
normally proportioned wholes atter
some of their parts have been deleted o r
883
884
DAVID L. STOCUM
added in excess, and (2) they can interact
with neighboring fields and become altered in the process. These properties are
very evident in the early embryo but
eventually disappear as axial polarities and
cell phenotypes become progressively determined.
The urodele amphibian limb is an
example of a secondary embryonic field
that can be reactivated by amputation
during larval or adult life so that the missing parts are regenerated. Limb regeneration occurs by the formation of a blastema
composed of embryonic-like mesenchyme
cells which form a continuum with the
remaining differentiated tissues of the
stump. These cells arise by the dedifferentiation of mature limb tissues for a considerable distance proximal to the amputation
plane. The dedifferentiated cells multiply
mitotically and then redifferentiate in the
spatial patterns of exactly those structures
that were removed. Blastema cells thus
normally redifferentiate in patterns more
distal to that of the level from which they
were derived, an observation known as the
"rule of distal transformation" (Rose,
1970).
Two questions of major importance immediately arise. (1) What is the origin of
the morphogenetic field of the regenerate?
(2) What is the nature of this field and how
does it specify new structural patterns?
The state of our knowledge with respect to
these questions is the subject of this paper.
ORIGIN OF THE REGENERATION FIELD
The morphogenetic field of the regenerate could arise in three possible ways.
First, positional information representing
the field might be directly inherited by the
blastema from the mature limb tissues via
dedifferentiation, and the field would thus
have an autonomous existence throughout
the regenerative process. Alternatively, all
positional information might be completely erased from dedifferentiating limb
cells and then reimposed upon them by
extracellular signals emanating from the
remaining differentiated stump tissues
after a blastema had formed. Third, the
field could be inherited from the mature
limb tissues, but also be susceptible to alteration by the stump tissue pattern.
It was originally believed that the
blastema was nullipotent (devoid of selforganizing capacity) and that its redifferentiation required the action of inducer
substances diffusing from the limb stump.
The blastema was also believed to be
pleuripotent, able to develop into several
different kinds of non-limb structures if in
contact with the appropriate inducers. The
notion of nullipotency arose from experiments in which young blastemas either
failed to develop at all or developed only
partially if grafted to a non-regenerating
(neutral) site in the absence of stump tissues, but did develop if a segment of stump
were included in the graft or if the
blastema were transplanted at a stage
where it had already begun to redifferentiate (Milojevic, 1924; Weiss, 1930; David,
1932; Mettetal, 1952; Faber, 1960; Skowron and Walknowska, 1960). The claim for
pleuripotency was derived from experiments in which young limb or tail blastemas grafted to foreign regions capable of
regeneration (limb blastema to tail stump
or vice versa, limb or tail blastema to lentectomized eye) were observed to regenerate according to the character of the host
site (Weiss, 1927; Schotte and Hummel,
1939). It was later shown, however, that all
these results were probably due to conditions of transplantation which favored resorption of the grafted blastemas, with
regeneration subsequently taking place
from the host site (Polezhaev, 1936; Stone,
1966; Stocum, 1968). It is now generally
accepted that a regeneration blastema can
form only the type of organ from which it
is derived. Piquing one's interest in this
regard, however, is the little-known report
of Emerson (1940), who claimed that
fragments of medulla transplanted into
young tail blastemas induced otic vesicles
from the mesenchyme and that similar
transplants of otic vesicles induced cartilaginous capsules. Thus, the possibility of
blastemal pleuripotency does not appear to
be strictly ruled out.
Regardless of the question of pleuripotency, the blastema is certainly not nullipotent. When undifferentiated larval
LIMB REGENERATION FIELD
salamander limb blastemas are grafted to
the dorsal fin in a way that minimizes
conditions leading to resorption, they develop quite normally in the absence of
stump tissues (Stocum, 1968; Michael and
Faber, 1971; Stocum and Dearlove, 1972).
Similar results have been obtained with
Xenopiis tadpole limb blastemas implanted
into the brain (Jordan, 1960). These experiments show that the blastema is a selforganizing entity from the time it becomes
visible at the tip of the limb. Two other
experiments suggest that this self-organizational ability is present during pre-blastemal dedifferentiation as well. Regenerates which have begun redifferentiation
can be halved transversely and the proximal halves transplanted to a foreign location in either normal or inverted orientation (Faber, 1960; Michael and Faber,
1961; Stocum, 1968), or several whole
regenerates can be combined in the orbit
of the eye (DeBoth, 1970). In either case,
the regenerates dedifferentiate completely, yet subsequently form relatively
normal limb parts. Collectively, these results lead to the conclusion that the morphogenetic field is represented in latent
form by intrinsic properties of mature limb
cells which are activated by dedifferentiation and directly inherited by the resulting
blastema cells. Positional information
sufficient to restore a complete pattern is
not erased during dedifferentiation and
extracellular signals from the stump tissues
are not necessary for specification of positional information in the regenerate.
The autonomy of the regeneration field
does not, however, mean that it does not or
cannot interact with the remaining differentiated stump tissues. There is evidence that the inherent axial polarity of
the blastema can be influenced by the axial
polarity of the stump. Only the anteriorposterior (AP) and dorsal-ventral (DV)
axes of the blastema appear to be labile,
whereas the proximal-distal (PD) axis appears to be stable. Stability of the PD axis
has been inferred from the results of experiments in which blastemas were transplanted either distally or proximally to
their level of origin within the limb (Iten
and Bryant, 1975; Stocum, 19756; Stocum
885
and Melton, 1977). When shifted distally,
the blastemas clearly developed according
to their level of origin, forming a second
set of limb structures in series with those of
the stump. When shifted proximally, normal limbs were produced by a process
of intercalary regeneration between graft
and stump levels. Preliminary autoradiographic studies utilizing 3 H-thymidine
labeled grafts suggested that in these
limbs the graft also developed according to
its level of origin, with the intercalated
parts being derived from the host stump
(Stocum, 19756). The stump level was thus
unable to alter the PD pattern of the
grafted regenerates in either experiment.
However, when the AP axis alone or
both the AP and DV axes of an undifferentiated blastema are reversed with respect to the same axes of the stump, the
axiation of the resulting regenerate often
conforms to that of the stump (Iten and
Bryant, 1975). Furthermore, Tank (1977)
has shown that if the stump skin of an
axolotl upper arm is rotated 180° around
the PD axis after a medium bud or palette
blastema has formed, the regenerate develops with multiple digits, just as does a
limb amputated immediately after skin
rotation. The palette blastema has initiated
redifferentiation and this result indicates
that the stump influence is strong enough
to disturb the inherited pattern of blastemal organization even at late stages of
development. It seems unlikely that the
effect of the rotated skin in Tank's experiment was mediated by a contribution
of additional (and axially disharmonious)
cells to the blastema by the skin. This is
further indicated by the finding that a
normalforelimb blastema grafted to asymmetrical hindlimb stump composed of two
anterior halves sometimes forms a symmetrical double anterior regenerate composed entirely of forelimb structures
(Stocum, unpublished observations).
To summarize, the pattern ot the regenerate would appear to be generated by an
autonomous morphogenetic field inherited by the blastema from the stump tissues
but which is nevertheless susceptible to the
influence of the pattern of the remaining
differentiated stump tissues. The nature of
886
DAVID L. STOCUM
erate (Stocum, 1975/;). This suggests that
blastema cells are unable to form patterns
proximal to their level of origin, and that
changes in their positional values must
always proceed unidirectionally, i.e., proxNATURE OF THE REGENERATION FIELD
imal to distal. However, Bohn (1976) has
Numerous observations suggest that the conclusively demonstrated that blastema
morphogenetic field of the regenerating cells of cockroach legs routinely form inlimb is represented by intrinsic cellular tercalary regenerates with patterns that are
properties which delimit a three-dimen- proximal to their level of origin, and Desional set of positional boundary values Both (1970) has claimed that combining
corresponding to the physical borders several axolotl wrist blastemas in the orbit
of the dedifferentiated portion of the limb, of the eye sometimes leads to their forming
while at the same time allowing change in lower arm structures, in addition to wrist
positional value within those limits. The and hand parts. I have repeated DeBoth's
boundary properties prevent blastema experiment (Stocum, unpublished), but
cells from having their positional informa- have been unable to confirm his results. At
tion respecified in patterns other than the present time, there is insufficient evthose of the missing limb structures. This idence to conclude that amphibian limb
idea will now be elaborated upon, first with regeneration blastema cells can undergo
respect to the PD and then the AP and DV proximal transformation. The evidence is
more consistent with the notion that these
axes of the limb.
blastema cells have intrinsic properties
which restrict them to the formation of
The PD axis
distal patterns.
It is obvious that positional information
Indirect evidence from the literature
for several structural levels along this axis suggests that the epidermis may have a
must be respecified using blastema cells distal boundary function. It should be emthat are derived from only one level of the phasized that this function is distinct from
limb. I suggest (see Stocum, 1975ft) that the the function of the epidermis as a nonmost distal positional value of the blas- specific promoter of blastemal outgrowth
temal mesenchyme is specified by unique (Stocum, 1977). Both epidermis and
properties of the apical epidermis, and nerves are required to keep blastema cells
that level-specific properties of the meso- cycling mitotically (Mescher, 1976; Tassava
dermal cells constrain dedifferentiated and Mescher, 1976). Withdrawal of either
cells derived from them to the formation nerves or epidermis during dedifferentiaof only those patterns distal to their level of tion prevents formation of a blastema, yet
origin. The latter properties form a gra- mitotically inactive dedifferentiated cells
dient of positional values along the PD axis remain in the limb stump for weeks (Tasof the limb, while the properties of the sava and Loyd, 1977). If nerves are withepidermal cells are constant everywhere in drawn after a blastema has formed, the
the limb. Dedifferentiated mesodermal blastema fails to undergo further growth
cells may change their positional value to in volume, but is often able to form a
any value between their original one in the complete PD array of missing limb parts in
limb and the distal boundary represented miniature (Singer and Craven, 1948; Powby the epidermis.
ell, 1969). If the edpidermis is removed
The evidence that level-specific proper- from a blastema and the latter is grafted to
ties constrain blastema cells to the forma- a tunnel in the dorsal fin, growth in voltion of patterns distal to their level of ume also ceases or is vastly reduced. Howorigin comes from the experiments, dis- ever, a distally incomplete regenerate is
cussed earlier, in which a proximally formed in this case (Stocum and Dearlove,
transplanted blastema did not contribute 1972). The fact that withdrawal of either
jo the formation of the intercalary regen- nerves or epidermis after blastema formathis influence is unknown, but it is likely to
involve cell surface interactions between
stump and blastema (see below).
LIMB REGENERATION FIELD
tion halts further growth, but only the
withdrawal of epidermis interferes with
completion of the PD pattern suggests that
the epidermis has a boundary function in
pattern formation. This function must be
constant at every level of the limb, since PD
reversal of the limb skin prior to amputation has no effect on the quality of the
regenerated parts (Carlson, 1974; Lheureux, 1975«).
How are positional values of blastema
cells respecified along the PD axis during
regeneration? Several models have been
proposed and applied to regenerating
and/or embryonic limbs: positional information is specified by (1) a sequential chain
of mesenchymal inductive interactions
between specified and unspecified parts of
the pattern (Rubin and Saunders, 1972),
(2) the sequential and autonomous counting of cell divisions in a "progress zone" at
the tip of the limb bud or blastema, each
division laying down a segment of the limb
(Summerbell, et ai, 1973; Summerbell
and Lewis, 1975), (3) the linear amount of
mesenchyme present in the blastema
(Faber, 1976), or (4) gradients of diffusable
morphogens, each concentration on the
gradient representing a positional value
(Stocum, 1975a). The first model is that of
stump induction, which has already been
shown to be unnecessary for blastemal
development. The second model has been
faulted on mechanistic grounds by Maden
(1976), who found that the number of cell
cycles during regeneration from a given
limb level was about two less than the
number of segments regenerated. This
model is, in addition, incompatible with
certain regulatory phenomena (Stocum,
1975*r/). The third model is based on DeBoth's (1970) finding that combining blastemas leads to the formation of a larger
pattern, a result which I have been unable
to repeat. This model also assumes that
blastema development is not size-independent, which has been shown not to
be the case (Maden, 1976). The last model
is compatible with much of the data concerning regeneration of the PD pattern,
but in the regenerating limbs of large
animals the distance over which diffusion
gradients would have to form might make
887
such a mechanism impractical. Finally,
none of these models account for the fact
that the extent of dedifferentiation is
greater at proximal than at distal levels
(Stocum, unpublished observations).
During recent years, evidence has been
accumulating that many of the interactions
between embryonic cells which were previously thought to occur by molecular diffusion actually require cell contact or are
mediated by extracellular matrix material
(Saxen, 1975; Toivonen et ah, 1976). The
cell surface and its extracellular matrix
have long been known to play an essential
role in cell recognition, cell-specific movement and intercellular adhesion, species
and tissue-specific cell sorting and the regulation of cell division during the ontogenetic development of form, all phenomena which can also be expected to play a
part in limb regeneration (Holtfreter,
1948; Townes and Holtfreter, 1955; Steinberg, 1970; Moscona, 1975; Trinkaus,
1976). Cell surfaces and extracellular matrices contain a variety of complex carbohydrates (glycoproteins, mucins and glycolipids). Roseman (1970) has suggested
interactions between surface located enzyme (specifically, glycosyltransferases)acceptor-substrate complexes as a molecular mechanism responsible for intercellular
adhesion and Roth (1973) has reviewed evidence which suggests the operation of
such a mechanism in contact inhibition,
cell adhesion and recognition and embryonic induction. It is thus logical to postulate that positional information may also be
encoded on the surface and/or in the extracellular matrix of the cells of the mature
amphibian limb, as has been proposed for
the patterning of retino-tectal connections
(Marchase et al., 1975). In the mesodermal
cells, this information would be in the form
of a PD gradient of stable cell surface
states, while the epidermal cells would represent only the most distal state. The inherited properties which delimit the
boundaries of the blastemal morphogenetic field would thus be cell surface states.
The idea that the morphogenetic gradients found in appendages (gradients of
developmental capacity) are manifestations of gradients in cell surface properties
888
DAVID L. STOCUM
was indeed put forward by Bohn in 1970
on the basis of his work on intercalary
regeneration in insect legs. In support of
this idea, Nardi and Kafatos (1976«, b)
have recently provided the first evidence
for a PD gradient of cell adhesivity in the
wing of the tobacco horn worm, Manduca.
They interchanged proximal and distal
blocks of pupal wing cells and observed
that the further away from their origin, the
smaller and more rounded up the grafts
subsequently became. The graft cells also
exhibited distinct changes in polarity,
forming rosettes with respect to the surrounding tissue. These results were better
explained by the differential adhesion
model of Steinberg (1970) than by diffusion gradient models, and Nardi and
Kafatos state the idea that proximal and
distal boundary values of cellular adhesiveness could generate a gradient of adhesiveness via mitosis "accompanied by an
orderly partitioning of surface properties"
during the development of the wing.
Recently, Maden (1976, 1977) has developed a model for the respecificadon of
positional information along the PD axis of
the regenerating limb that is based on cell
surface interactions within proximal and
distal boundaries. He proposes that the
mesodermal tissues of the mature limb
represent a stable PD gradient of cell surface states in which adjacent cells (or blocks
of cells) differ by a value of one. The
epidermis overlying these tissues has a
value of zero everywhere in the limb and
this value represents the distal boundary of
the regenerate. Upon wound healing, the
widely disparate positional values represented by the epidermis and mesodermal
cells are confronted. Recognition of this
positional disparity leads to dedifferentiation of the mesodermal cells and they
display their previously inactive surface
configurations. Maden postulates that
when a blastema cell senses a surface disparity greater than one positional value
between itself and its neighbors, it "averages" its surface state to assume the positional value half-way between those of the
adjacent cells. The initial averaging interaction takes place between epidermal
and blastema! cells shortly after the onset
of dedifferentiation and continues in a
synchronous fashion as dedifferentiation
progresses proximally and the blastema
cells divide, until no positional discontinuities remain (Fig. 1). The changes in
positional value that occur during averaging are always from proximal to distal, in
conformity with the rule of distal transformation. Averaging is predicted to occur in
an essentially morphallactic fashion during
the first few days prior to formation of a
visible blastema and mainly epimorphically
thereafter, since the mitotic index is at first
very low, rising sharply as blastema cells
accumulate at the tip of the limb stump
(Maden, 1977; Stocum, unpub. observ.).
The pattern in which intercalation occurs in this model accounts for two major
features of regeneration, the cessation of
dedifferentiation and switch to redifferentiation, and the proximal to distal sequence
of redifferentiation. The formation of the
initial blastema cell population by dedifferentiation, coupled with morphallactic
averaging, results in a more rapid intercalation of positional discontinuities proximally than distally (Fig. 1). Dedifferentiation will thus cease and redifferentiation be
initiated proximally after fewer rounds of
averaging, thereby determining the proximal extent of the regenerate. Elimination of positional discontinuities then
spreads distally, giving rise to the observed
redifferentiation sequence. Since a limb
amputated distally has fewer positional
values to replace than one amputated proximally, it is clear that the averaging model
predicts the observation that the proximal
extent of dedifferentiation will not be so
great in the former as it is in the latter.
The model also accounts for the failure
of the blastema to form terminal parts
after epidermal removal (Stocum and
Dearlove, 1972) and for intercalary regeneration after shift-level grafting (Iten and
Bryant, 1975; Stocum, 19756). In the
former case, the distal boundary of the
regenerate is deleted, and the positional
value represented by the surface state of
the distal-most mesenchyme cell at the
time of deletion would now become the
distal boundary. In the latter case, averaging of cell surface states would occur
889
LIMB REGENERATION FIELD
] 9 M o l [Confrontation of disparate positional values
Preblastemic dedifferentiation and Morphallactic averaging
Blastema stages and Epimorphic averaging
Divide
Redifferentiation
FIG. 1. Respecification of positional values along the
PD axis by an averaging method (after Maden, 1977).
The square cell to the extreme right represents the
epidermis which has positional value O (distal boundary) everywhere on the limb. The triangular cell to
the extreme left represents the mature stump tissues. The round cells between these two represent
blastema cells. The x's indicate those cells which are
changing positional value, and the short arrows the
new (always more distal) positional values of these
cells. When a cell divides, one daughter maintains its
etc.
positional value, while the other is respecihed to a
more distal value. Redifferentiation sets in when positional discontinuities are eliminated and this will
first occur in the most proximal part of the blastema
(heavy bar). Note that several cells immediately distal
to cell 20 in the bottom row differ from one another
by only slightly more than one positional value. It is
possible that once these close approximations to continuity are achieved, the next cell division results in
only slight, non-averaging adjustments on the part of
the daughter cells.
among the blastema cells derived from the
stump tissues, with the most proximal positional value of the grafted blastema as the
"distal" boundary for the intercalary regenerate and the positional value at the
most proximal extent of stump dedifferentiation as the proximal boundary.
can take place in the AP and DV axes
under experimental conditions, providing
further examples of the cellular boundary
properties and interactions which constitute the regeneration field. A complete
complement of AP and DV positional information need not be present in a limb
stump for it to form a completely normal
The AP and DVaxes
regenerate pattern, as long as the tissues that
are present represent all four quadrants of its
In contrast to the PD axis, a complete set
of AP and DV positional information is
inherited by the blastema after simple amputation, since nothing is lost along these
.axes. However, intercelary regeneration
circumference. Blastemas derived from
upper arms from which the bone or muscle has been completely removed regenerate a normal skeleton and musculature
(Bischler and Guyenot, 1925; Weiss, 1925;
890
DAVID L. STOCUM
Carlson, 1972). X-irradiated upper arms
supplied with unirradiated pure cartilage
or skin form regenerates of normal asymmetry containing cartilage, general connective tissue and muscle (Wallace et al.,
1974; Maden and Wallace, 1975;
Lheureux, 1975ft; Dunis and Namenwirth,
1977). Removal of either the internal tissues or skin from the posterior half of the
lower arm does not prevent regeneration
of a complete limb as long as a complete
complement of one or the other remains.
However, if the posterior halves of both
skin and internal tissue are removed, only
1/2 Regenerate
FIG. 2. Intercalation along the AP axis of a regenerating upper arm after removal of various tissues. A = anterior; P = posterior; D = dorsal; V =
ventral; Ep = epidermis; Dr = dermis; Ms = muscle;
Bn = bone. Positional information in the crosssectional plane can be represented as a grid of discrete values, of which only those along the AP and DV
axes are shown. The arrows indicate the possible
directions of positional value change after dedifferentiation. (a) After removal of a bone (dashed
line). Interaction takes place initially between 3 and 7
and intercalation is centripetal from both anterior
and posterior cells; (b) after removal of muscle alone
from the anterior half of the limb. Interaction initially
takes place between 0 and 4. Two cases are possible:
(1) intercalation takes place only centripetally (from
the dermis) in which case AP compaitment bound-
aries are not crossed, or (2) intercalation also takes
place centrifugally from the posterior muscle, in
which case there are no AP compartment boundaries
to be respected; (c) after removal of skin and muscle
from the anterior half of the limb. Only a posterior
half limb regenerates in this case. It is not known
whether this is because mesodermal and epidermal
cells do not interact around the circumference of the
limb (lack of boundary properties of the epidermis),
or because a compartment boundary cannot be crossed by the dedifferentiated mesodermal cells. For this
reason, the epidermal cells have not been assigned a
positional value in these diagrams. In (a) and (b), the
positional values bounding the deletions are drawn as
separated for convenience; in reality the cells with
those values would be brought into intimate contaci
b\ collapse in the region of deletion.
LIMB REGENERATION FIELD
the anterior half of the limb regenerates
(Weiss, 1926; Goss, 1957a, b), and upper
arms or thighs consisting of two anterior or
posterior halves either fail to regenerate or
regenerate double half limbs (Bryant,
1976; Stocum, 1978). These observations
suggest that inherited surface properties,
representing positional values of blastema
cells derived from both skin and internal
tissues are arranged in gradients along the
AP and DV axes of the limb, just as they
are along the PD axis, and that these
properties allow the blastema cells to interact and change their positional values to
resolve discontinuities in the pattern (Fig.
2). These changes could also involve an
averaging mechanism of the type postulated by Maden (1977).
In the deletion experiments described
above, it is obvious that the boundaries of
the intercalated portion of the pattern are
determined by the positional values of the
mesodermal cells which physically bound
the discontinuity. Restoration of the pattern in these experiments, therefore, is the
same as intercalary regeneration in the PD
axis when a blastema is grafted to a more
proximal stump level. This brings up an
interesting and important question. Might
the epidermis have a boundary function in
pattern formation along the AP and DV
axes? That such a role of the epidermis is
possible is not immediately apparent, for
several reasons. First, the epidermis does
not contribute to the mesenchymal cell
population of the blastema (Hay and Fischman, 1961), and a complete set of AP
and DV positional information is inherited
from the mesodermal tissues by the
blastema during normal regeneration.
Second, even when deletions of all tissues
except the skin are made within the crosssection of the limb, the positional information inherited by the blastema is representative of the whole circumference of the
limb, allowing intercalary regeneration to
occur between the most peripheral mesodermal (dermal) positional values. This is in
contrast to the situation along the PD axis,
where the most distal mesodermal positional value of the limb is not inherited by
the blastema cells, and where a boundary
function of the epidermis is thus more
891
logical. Third, rotation of the epidermis
180° round the PD axis does not cause the
multiple limb formation that occurs when
dermis is similarly rotated (Carlson, 1975).
The latter observation suggests that, in
contrast to the asymmetric and morphogenetically active qualities of the dermis,
the epidermis has a singular and morphogenetically neutral quality around
the circumference of the limb. However,
this singular quality may well have a boundary function similar to that postulated for
the PD axis, representing periphery, rather
than anterior, etc. Then, if all mesodermal
tissues along a portion of the AP or DV
axis were deleted, bringing the remainder
in contact with the epidermis, boundary
interactions analogous to those postulated
to occur along the PD axis could be involved in regenerating the missing part of
the pattern along these axes after amputation.
The fact that a half lower limb does not
regenerate a whole (Goss, 1957) (Fig. 2)
would appear to be against this notion, but
a further consideration complicates the
matter. It is possible that blastema cells
might belong to anterior, posterior, dorsal
or ventral compartments (see Lawrence,
1975), the boundary lines of which cannot
be crossed when positional discontinuities
are to be filled in. That such may be the
case is suggested by the fact that irradiated
limbs covered with unirradiated skin taken
from only one quadrant of the limb produce quantitatively subnormal regenerates
that lack asymmetry (Lheureux, 19756).
Two kinds of experiments might provide
information on whether the epidermis
could have a circumferential boundary
function and whether blastema cells might
belong to compartments. First, the skin
might be stripped from the upper arm and
a portion of the muscle uniformly removed from around the circumference, so
that the limb is heavily deficient but has all
four quadrants represented in the remaining muscle. After epidermal healing,
the limb would be amputated to see if a
normal regenerate formed. If so, an epidermal boun '.ary function would be indicated. Second, marking experiments
designed to reveal the extent of the con-
892
DAVID 1.. STOCUM
tribution to the blastema of different limb
sectors after deletions of half or more than
half of the internal tissues have been made
could tell whether or not compartment
lines are crossed.
Although the blastema field can compensate for the deletion of stump tissues, it
apparently cannot compensate for the addition of extra tissues to the stump in
disharmonious locations, or for rearrangement of the normal complement of
tissues along the AP and DV axes. If an
extra ulna .is- add sd ^anterior to thea'adius
or if the normal ulna is simply displaced to
that position, an extra ulna (but not more
distal structures) develops in the regenerate after amputation through the altered
region (Goss, 1956). Positional displacement of skin or muscles by 180° around the FIG. 3. Intercalation after reversing the AP axis of
skin with respect to the internal tissues of an
PD axis results in the production of multi- the
upper arm. The operation confronts normally oppople regenerates in Notophthalmus viridescens site positional values of the dermis and underlying
(Settles, 1967, 1970), Pleurodeles waltlii muscle on both the anterior and posterior sides of the
(Lheureux, 1972) and Ambystoma mexica- limb. Intercalation is envisaged as taking place cenand centrifugally at both these loci, thereby
num (Carlson, 1974, 1975). Carlson (1975) tripitally
creating two accessory morphogenetic fields (arrows)
has shown that the dermis is responsible in addition to the main field. A maximum of three
for the effect of the rotated skin.
limbs can thus be produced by this operation.
The results of these experiments again
suggest that AP and DV positional information is asymmetrically borne in both 1956). The blastema field would interpret
skin and internal tissues. When the AP or this manipulation as both a deletion and
DV axial relationships of the skin and insertion of a part of the pattern; it would
internal tissues are made disharmonious intercalate to fill in the deletion but igby 180°, the limb stump regenerates as if nore the displaced ulna for its lack of
under the control of more than one field. boundary activity; the latter would simply
This can be explained by assuming that regenerate that part of the pattern which it
skin rotation or muscle exchange results in contained.
the confrontation of normally opposite
French et al., (1976) have recently depositional loci, each of which can act as a veloped a formal general model (the polar
boundary. This creates several secondary coordinate model) for regeneration based
fields, as illustrated in Figure 3, within on the number, location, orientation and
which cellular interactions can specify handedness of supernumerary limbs arisextra sets of AP and DV positional values. ing after blastema rotations in amphibian
Although bones represent a portion of the limbs (Bryant and Iten, 1976) and rotation
circumferential pattern, their cells appar- of limb segments in cockroaches (French,
ently do not have boundary functions, 1976). The model can also account for
since when they are displaced or added to duplication and regeneration of imaginal
a stump, there is either no effect on regen- discs in Drosophila (Bryant and Hsei, 1977).
eration (Carlson, 1975) or only that por- They propose that each cell of the retion of the pattern which they represent is generating limb has information with rereduplicated in the regenerate (Goss, spect to its angular position around the
1956). This would explain why simply dis- circumference of the limb and its position
placing an ulna results in the regeneration along a radius of the circumference. The
of two ulnae, but not multiple limbs (Goss, angular component specifies the axial
LIMB REGENERATION FIELD
"
patterns in the cross-sectional plane of the
limb and the radial component specifies
the PD axis. The model postulates interactions between cells of widely disparate
positional values that are confronted after
blastema rotations, and specifies two formal rules (complete circle rule and shortest
intercalation rule) which govern these interactions, but does not specify any particular mechanism for the generation of
positional information. It will not be described further, since it is discussed
elsewhere in this symposium. At this point,
it is sufficient to say that all of the evidence
so far obtained indicates that the cellular
interactions which generate normal limbs
after deletions of stump tissues and multiple limbs after rearrangements of stump
tissues are of the same kind that produce
multiple limbs after blastema rotations.
Whether the rules governing these interactions are exactly as the polar coordinate- model proposes, especially from the
limbs of one species to another, remains to
be seen.
CONCLUDING REMARKS
Our present knowledge of limb regeneration leads to the following working
hypothesis about the organization of the
morphogenetic field of the limb regeneration blastema. The blastema field is in the
form of a three-dimensional set of cell
surface-associated properties which delimit pattern boundaries while at the same
time allowing change to positional values
enclosed by these boundaries whenever a
discontinuity arises. These properties reside in both the mesodermal and epidermal tissues of the limb, forming gradients
of positional values along the AP, DV, and
PD axes in the mesodermal cells, but remaining constant in the epidermal cells
everywhere on the limb. The surface states
of the epidermal and mesenchymal cells of
the blastema are inherited from the mature limb cells and are confronted during
wound healing and dedifferentiation. The
mesenchymal cells recognize positional!}'
disparate surface states between themselves and their neighbors and respond to
the discontinuity by continually changing
893
their surface states until a complete gradient of states is restored up to the boundary values. This is a self-sustaining process
which is initiated whenever dedifferentiated cells with widely disparate surface
states confront one another in the presence of all the systemic factors (nerve, hormones) required for regeneration.
A great deal of work remains to be done
at all levels of biological organization to
explore the ramifications of this hypothesis. First, we need to further investigate the phenomenon of field inheritance and stump-blastema interaction. For
example, are the multiple fields created by
the rearrangement of a limb stump both
self-organizing and labile in the same way
that a normal single field appears to be? Or
are they completely dependent on a
stump-blastema interaction, so that a blastema derived from a rearranged stump
might actually be capable of forming a
unified field during dedifferentiation? Answers to these questions should provide us
with more detailed knowledge of how the
morphogenetic field of the blastema arises.
Second, better evidence is required to
substantiate how the blastema field is
bounded. There is presently no direct evidence that the epidermis functions as a
boundary, and experiments which will differentiate between boundary and nonspecific outgrowth functions of the
epidermis must be devised in order to
confirm or deny this idea. Most of the
available evidence from amphibian limbs is
consistent with the concept that levelspecific mesodermal cell properties determine the proximal boundary of the
regenerate, but other evidence from insect
appendages suggests that proximal transformation of blastema cells occurs, leaving
open other possible mechanisms for the
determination of proximal boundaries in
regenerates of the latter. Further experimentation is required to determine
whether amphibian limb regeneration
blastema cells and their insect counterparts
do or do not have similar capacities for
proximal transformation.
Third, the hypothesis that the molecular
mechanism for the specification of positional information by the blastema field
894
DAVID L. STOCUM
resides on or is expressed at the cell surface and/or extracellular matrix is completely untested. Nevertheless, it is a highly
plausible one which bears investigating by
the techniques now available for probing
the structure and function of the cell surface and extracellular matrix. For example, dedifferentiating and converted dorsal
iris cells of regenerating amphibian lenses
have already been shown to have different
electrophoretic mobilities (Zalik and Scott,
1972), altered surface structural characteristics (Dumont and Yamada, 1972), and
vastly increased glycosyltransferase activity
(Idoyaga-Vargas et at., 1976), compared to
iris cells not involved in regeneration. It
would seem profitable to look for gradients
of ultrastructural and biochemical differences which correspond to the morphogenetic gradients along the PD axis of
the limb and regeneration blastema using
scanning and transmission electron microscopy and agents such as lectins (Sharon, 1975), which would bind to cell surfaces
and block their interaction without violating the integrity of the cells.
We are only beginning to get a glimpse
of the mechanisms of field action. It is
unrealistic to expect any quick solutions to
the problems posed by the relationship
between the whole and its parts; nevertheless, these problems arc the most fascinating ones in developmental biology
and will continue to be a challenge for a
long time to come
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