/. Embryol. exp. Morph. Vol. 71, pp. 109-120, 1982
109
Printed in Great Britain © Company of Biologists Limited 1982
The effect of tail skin on the
morphology and morphogenesis of limb
regenerates in the red-backed salamander,
Plethodon cinereus
By CHARLES E. DINSMORE 1
From the Department of Anatomy, Rush Medical College, Chicago and
The Mount Desert Island Biological Laboratory,
Salsbury Cove, Maine
SUMMARY
Tail skin cuffs have been grafted to the upper forelimb of red-backed salamanders in
either normal or 180°-rotated dorsoventral orientation. Subsequent amputation through the
graft region resulted in arrested regeneration, distally deficient or typical four-digit regenerates.
Distribution was not substantially influenced by graft orientation nor were there any supernumerary limbs induced by the axially dislocated tail skin on the limb stumps. Furthermore,
regenerates bore no indication of tail-like structures other than large granular skin glands
proximally. These data indicate that, unlike limb skin, tail skin is not morphogenetically
active in the epimorphic process of limb regeneration. In addition, the species used in this
study is a fin-less, round-tailed salamander. It is therefore suggested that the previously
reported morphogenetic effects of tail skin on limb regeneration may be related to the
presence of tail fins on the species studied.
INTRODUCTION
The location of pattern information within various developmental fields and
the mechanisms by which morphogenesis is accomplished are only beginning
to be discovered. One of the means by which this area is being explored is
through the identification of obligatory morphogenetic tissue interactions at
various stages of developmental processes. By selectively dissociating the constituents or altering the spatial relationships of specific tissue components in a
developing system, whether early embryo or epimorphic field, one can determine which manipulations affect morphogenesis, thereby identifying both the
timing of morphogenetic interactions and the tissues involved in primary or
secondary patterning.
The regenerating urodele limb is a system which has proven amenable to such
experimental dissection. Building on the accumulated observations of many
earlier investigations, Carlson (1974, 1975) designed and performed a critical
1
Author's address: Department of Anatomy, Rush Medical College, 600 S. Paulina Street,
Chicago, Illinois 60612, U.S.A.
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C. E. DINSMORE
series of experiments from which the data and lucid analysis have provided
insight into many previously described but poorly understood regenerative
phenomena. By surgically isolating and positionally dislocating, in turn, the
individual components of the limb (i.e. epidermis, dermis, skeletal muscle or
bone), he demonstrated that only the dermis and skeletal muscle are morphogenetically active. When a 'positional disharmony' (Lheureux, 1972, 1975) is
created between either of these tissues and the rest of the limb stump, regenerates
are often abnormal and frequently multiple.
Recent efforts to confirm the morphogenetic activity of skin (Dinsmore,
1981 a, b) and skeletal muscle (Dinsmore, 1979, 1981 b) in another epimorphic
field, the urodele tail, have failed. Neither positional dislocation nor unilateral
ablation of these caudal tissues disrupts the regenerative process, in marked
contrast to the effects such procedures have on the limb field. There are several
possible explanations for this apparent difference in the modes by which these
epimorphic fields are governed. Morphogenetic information may not be intrinsic
to these tissues but rather dependent upon their field of origin; present in the
limb territory, absent in the tail. Another explanation previously considered
(Dinsmore, 1981 a) is that there are hierarchies among the tissues which comprise a particular field. Within the tail, the regenerating spinal cord (sine qua
non of this field) may present a dominant primary morphogenetic signal source
with other constituent tissues subordinate to its regulatory influence. This need
not assume a lack of positional information in other tissues. Rather, it may
simply be a field specific masking of the tissues' potentials. The expression of
a tissue's hidden potential might be affected by grafting it to a heterotypic field
in which its counterpart is normally active in regulating regenerate morphogenesis. It is also possible that a tissue such as skin bears information relative
to field morphology (e.g. tailness), as has been described by Glade (1957, 1963,
1978), yet lacks axial stability relying on underlying tissues for appropriate
differentiation at that level.
The present experiments address some of these possibilities by re-examining
the effect that grafted tail skin may have on urodele limb regeneration. Earlier
studies of this nature (Glade, 1957, 1963, 1978) employed urodeles with distinct
dorsal and ventral tail fins. This raised questions about the morphogenetic
influence of fin mesenchyme and how it might alter pattern regulation outside
of the tail field. In order to avoid such potential problems, the round-tailed
salamander, Plethodon cinereus, has been used exclusively in this study assuring
that grafts consist of whole tail skin uncontaminated with fin mesenchyme.
This terrestrial salamander regenerates its limbs in a typical urodele manner
and may produce supernumerary structures in response to limb skin rotation
and subsequent amputation (Fig. 1). It is thus comparable to those urodeles
used in the earlier studies by Lheureux (1972, 1975) and Carlson (1974, 1975)
relative to morphogenetic regulation during limb regeneration as determined by
that technique.
Effect of tail skin on limb regeneration
111
0-2 mm
Fig. 1. A supernumerary regenerate from an upper forelimb whose skin was
exchanged with that of the contralateral limb. Only the anteroposterior axis of the
graft was reversed.
Fig. 2. Frontal section through a 61 -day normal control regenerate. Four metacarpals
are clearly visible while the carpals and phalanges are out of the plane of section due to
normal wrist extension and digit flexion. Amputation level is indicated by the arrows.
Two specific questions addressed by the ensuing experiments are: (1) will
grafts of tail skin to the limb influence the morphology of limb regenerates by
introducing a degree of'tailness' into the internal organization of the regenerate,
and (2) does tail skin bear accessible positional information with an axial bias
such that a positionally dislocated graft of tail skin will alter limb regenerate
morphogenesis ?
MATERIALS AND METHODS
The experiments described below were performed on adult Eastern redbacked salamanders, Plethodon cinereus, ranging from about 8 to 10 cm in
total body length. Specimens collected in the wooded areas around the Mount
Desert Island Biological Laboratory, Salsbury Cove, Maine, were maintained
as in earlier studies (Dinsmore, 1979, 1981a).
Two series of experimental manipulations were employed. The basic procedure
consisted of anaesthetizing animals by immersion in 1-0% MS 222 (Eastman)
and removing them to the stage of a dissecting microscope. A circumferential
cuff of tail skin was then carefully dissected free by making a longitudinal midventral incision approximately 1-5 cm distal to the vent, elevating the skin
gently while cutting intervening myoseptal attachments and circumferential ly
incising the skin at the proximal and distal extents (5-6 mm) of the longitudinal
incision. The freed cuff was placed in a pool of Holtfreter's solution where
adhering myofibres and fat deposits were removed. The flattened piece of skin
was then trimmed to a square which would subsequently be fitted to a graft bed
on the limb. The limb was prepared for the graft of tail skin by completely
removing the skin from the upper forelimb from elbow to shoulder. Except for
a strong attachment by an anterior intercompartmental connective tissue septum,
the limb skin is only tenuously attached to the underlying musculature and is
easily removed. The graft tissue was then introduced onto the graft bed and a
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C. E. DINSMORE
final trim assured close fitting of the tail skin onto the limb. At this point,
grafts for specimens in series I (16 animals) were positioned so that their dorsal
surfaces corresponded with the dorsal aspect of the limb (normal orientation)
and were secured by two or three interrupted 7-0 sutures (Ethicon). Designation
of axes in this study is consistent with that applied to the whole body. The
craniocaudal orientation of the tail skin grafts relative to the proximodistal axis
of the limb was not recorded.
The animals in series II (24 animals) were prepared in the same manner as
those in series I except for the orientation of the graft. The squares of tail skin
were positioned with their dorsal surfaces covering the ventral side of the upper
forelimbs and, as above, secured in that orientation with two or three sutures.
The skin grafts were thus rotated 180° about the long axis cf the limbs producing
maximal dislocation of this tissue relative to the dorsoventral axis of the limb.
Subsequent treatment was the same for both groups of animals. The tails
were removed at the proximal edge of the denuded areas by pinching through
an autotomy plane. This reduced the risk of infection and death which was a
problem in early trials. The animals were then placed in petri dishes containing
Holtfreter's solution for recovery, after which they were returned to their
containers for a 10-day post-operative period to allow graft stabilization and
healing. At the end of this period, animals were again anaesthetized and under
low power magnification the limbs bearing tail skin were amputated through
the middle of the graft (mid-humerus) with the projecting humerus trimmed
even with the retracted soft tissues. Distal segments were usually fixed in Bouin's
solution for subsequent histological analysis of graft morphology and stability
at the time of amputation. The contralateral limbs were amputated at the same
time to serve as normal controls on each specimen.
Animals were again anaesthetized at intervals of from 50 to 98 days postamputation at which time both the experimental and contralateral control limbs
were dissected free at the shoulder. The limbs were examined closely under the
dissecting microscope for superficial morphology, noting also the thickness and
pigmentation of the stump and regenerate on the experimental limb relative to
that of the control. Due to the larger skin glands and heavier pigmentation of
the grafted tail skin, the experimental limbs were thicker and darker, at least
at the stump level, preliminarily verifying graft retention. The limbs were then
fixed in Bouin's solution, decalcified and prepared for routine paraffin sectioning.
Blocks were sectioned serially at 8 /*m and stained with haematoxylin and eosin
or by Mallory's trichrome method.
RESULTS
The control limbs which were simply amputated at the same time as the contralateral experimental limbs produced normal four-digit regenerates (Fig. 2)
with one exception. The single abnormal regenerate (1 out of 40) consisted of
Effect of tail skin on limb regeneration
113
Table 1. Summary of results comparing regenerative success versus
tail skin graft orientation
Graft orientation
Normal
Rotated 180°
Pooled date
Contralateral
limbs (no graft)
Number of
limbs
16
24
40
40
No epimorphic
regeneration
Deficient
regenerate
4-digit
regenerate
6(37-5%)
5 (20-8%)
11 (27-5%)
0(0%)
6 (37-5%)
11 (45-8%)
17(42-5%)
1(2-5%)
4 (25%)
8(33-3%)
12(30%)
30(97-5%)
a normal elbow joint with the proximal segments of radius and ulna articulating
appropriately. There was no development beyond mid-zeugopodium. However,
a low percentage of defective regenerates following simple amputation is not
unexpected (Dearlove & Dresden, 1976).
Regenerates from limbs bearing grafts of tail skin in either normal or 180°rotated orientation have been grouped into three general categories: no epimorphic regeneration, deficient regenerate, and four-digit regenerate (Table 1).
Stumps which were grossly truncated and upon histological examination showed
no sign of forming an elbow joint have been arbitrarily classified as showing no
epimorphic regeneration. The covering of the humeral stump in specimens from
this category ranged from a simple fibrous cap (Fig. 3) to an elongate cartilaginous rod (Fig. 4). These stumps were enclosed in a jacket of skin endowed
with thick and obviously tail-like glands (compare with Fig. 5).
Limb regenerates which were deficient bore at least an elbow joint but fewer
than four digits on the distal segment. The range of skeletal expression included
simple spikes (Fig. 6) to nearly complete, three-digit regenerates. Figure 7 shows
one plane in a serially sectioned, three-digit regenerate demonstrating the level
of amputation, the obvious retention of the tail skin graft and its influence on
regenerate skin gland morphology. Serial sectioning provided a more complete
means of evaluating the internal organization of regenerates. Distal fusion of
the radius and ulna as well as fusion of the carpal and metacarpal elements was
also observed in this group. Again, skin proximal to the level of amputation
was obviously of tail type. That covering the regenerate was more variable,
however, usually bearing progressively smaller, more limb-like glands in a
proximodistal gradient (compare with Figs. 2 and 3).
Although they were a minority, 30% of the amputated stumps bearing cuffs
of tail skin produced four-digit regenerates (Table 1). Examination of serial
sections showed that regenerates in this category bore elbow joints with synovial
cavities, a radius and ulna articulating appropriately, carpals, metacarpals, and
four phalanges. The absolute numbers of skeletal elements in the autopodia were
not counted and may be reduced in some cases. This also occurs in normal
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C. E. DINSMORE
Ei
LO
Effect of tail skin on limb regeneration
115
regenerates and was therefore not important to the fundamental observations of
this study. Skin gland morphology was again the criterion for ascertaining graft
retention at the level of the stump. Except at the proximodistal line along which
the skin cuff was sutured closed around the limb, typical tail skin was present
on the stump and proximally on most of the regenerates in this category (Fig. 8).
These data demonstrate that grafts of tail skin to the limb with subsequent
regeneration induced in the limb-tail chimaera do not, in this species, alter the
gross morphology of the limbs which regenerate. Although distal reduction was
the most common outcome in this procedure (70% producing either no regeneration or distally incomplete outgrowths), tail structures other than large
granular glands were not observed in any of the regenerates.
Table 1 shows a basic similarity of results irrespective of the graft orientation
indicating that, unlike rotated limb skin, tail skin is morphogenetically inert
relative to axial positional information. Regenerates developed grossly normal
axiation as judged by the elbow flexure and, when present, orientation of the
digits. No supernumerary limbs were induced by apposition of dorsal skin with
the ventral limb surface. A small digitiform outgrowth was observed, however,
on the palm of one four-digit regenerate. From the curious location of this supernumerary projection, it is unclear how its development may have been stimulated.
In summary, the data indicate that while morphogenesis of the limb regenerate may be arrested or stunted by a cuff of tail skin at the level of amputation, tailness has not been introduced into the system beyond the level of the
integument. Furthermore, a rotated cuff of tail skin which conceptually presents
positionally dislocated axial information to the limb stump, and therefore
creates potential morphogenetic disharmony, produces results which are not
substantially different from the normally oriented skin series. Under the constraints of the procedures used in this study, one may conclude that tail skin
is not a morphogenetically active tissue though it can support normal limb
regeneration in this species.
DISCUSSION
Regeneration of urodele limbs and tails requires not only a source of cells
with which to reconstruct the lost appendage but also a pattern by which the
appropriate appendage will be constructed and in proper alignment. There are,
Fig. 3. Limb stump bearing tail skin graft in inverted orientation 62 days postamputation. Regeneration has been totally arrested and an obviously tail-type
integument completely envelops the stump.
Fig. 4. Another example of arrested regeneration in a limb bearing an inverted
cuff of tail skin. Although the humeral stump produced an elongate cartilaginous
cap, no elbow was formed, hence its being categorized as showing no epimorphic
regeneration. Specimen was fixed 63 days postamputation.
Fig. 5. Cross section of a normal tail regenerate, the skin morphology of which may
be compared with that of both control and experimental limbs in this study. Dorsal
side at top; spinal cord in centre above vertebral centrum.
116
C. E. DINSMORE
8
Effect of tail skin on limb regeneration
117
however, apparent differences between limbs and tails in the ways in which
they regulate their respective patterns. While skeletal muscle and skin are the
predominant sites of morphogenetic signalling in the limb, the spinal cord
dominates tail regeneration with tail skin and muscle being relatively inert
(discussed in Dinsmore, 1979, 1981 b). This conclusion is based on several types
of experiments which were characterized and discussed early on by Goss (1961).
Three types are drawn on in this discussion as bearing directly upon the problem
of identifying morphogenetic activity in a tissue: deletion of a specific tissue,
rearrangement of that tissue relative to adjacent tissues and substitution with a
comparable tissue from another locus.
The importance of limb skin in pattern regulation during urodele limb regeneration has been demonstrated in several laboratories. A complete circumference of limb skin supports normal regeneration from a longitudinally split
half stump, while inclusion of the skin when the limb is split will most often
result in a half regenerate (Goss, 1957; Dinsmore, 1982). This example of
deletion indicates that a complete circumference of skin is sufficient for normal
pattern regulation in the absence of other stump constituents. Nevertheless, the
pattern itself is a mosaic in that a half stump produces an appropriately halved
regenerate, at least in the lower forelimb.
Positional dislocation of the limb skin by various degrees of cuff rotation
also produces distinct morphogenetic effects on limb regenerates. Supernumerary
limbs may develop on stumps bearing skin cuffs rotated 90° about the dorsoventral axis of the limb thereby presenting a complete circumference of dorsal
positional information (Settles, 1967, 1978). Several studies have used rotation
of limb skin about the longitudinal axis of limbs to demonstrate further the
morphogenetic activity of this tissue (Lheureux, 1972,1975; Carlson, 1974,1975).
Whether one axis (e.g. dorsoventral) or two axes (dorsoventral and anteroposterior) of the limb skin cuff are reversed relative to the internal limb tissues,
multiple or supernumerary regenerates are often produced (Lheureux, 1972,
1976), although this may vary depending upon the species employed (Carlson,
1974).
Fig. 6. An experimental limb regenerate from a stump with normally oriented tail
skin. Sacrificed 78 days postamputation, this regenerate differentiated an elbow
joint whose distal skeletal element was a single cartilaginous spike. The bonecartilage transition in the humerus marks the approximate level of amputation.
Fig. 7. Another example of distally deficient regenerate, this specimen had three
digits, the proximal segments of the metacarpals evident in this section. The tailtype skin is seen not only at the stump level but also over the proximal regenerate.
Compare with Fig. 1. The arrow again indicates the level of amputation.
Fig. 8. This frontal section through a four-digit regenerate bearing inverted tail
skin shows the bases of four metacarpals and obvious tail skin on the proximal part
of the regenerate. The arrow marks the approximate level of amputation.
Bar = 01 mm.
118
C. E. DINSMORE
Focusing on the potential of the skin.and recalling Goss's (1961) classification
of experiments, the protocol of the present study incorporates both' substitution'
of a tissue in the limb field by its counterpart from the tail and 'rearrangement'
of that tissue in the host field by axial rotation. By grafting tail skin to the limb
in the normal dorsoventral orientation, the question of whether or not general,
field-specific information is borne by this tissue was answered in the negative.
Although the skin in the area of the stump-regenerate interface has large
granular glands characteristic of the axial skin of this species, the morphology
of the limbs which regenerated (including the distal skin of the regenerate) was
appropriate for that appendage. This appears to conflict with earlier reports of
chimaeric regenerates from limbs bearing grafts of tail skin (e.g. Glade, 1957,
1963, 1978). A comparison of details actually provides insight into diversity of
morphogenetic control mechanisms among the tails of different urodele species.
The species in the present work, Plethodon cinereus, lacks a tail fin and its
associated fin mesenchyme. Both species employed by Glade, Notophthalamus
viridescens and Ambystoma mexicanum, have distinct caudal fins along the
dorsal and ventral margins of their tails. Without necessarily being 'contaminated' with fin mesenchyme, dermis from finned tails may carry a bias toward
organizing adjacent undifferentiated connective tissue or blastemal mesenchyme
into a fin-like structure. The physically reorganized materials could then have a
'second morphogenetic effect' (Glade, 1978) by mechanically disrupting the
host epimorphic field. Another way of interpreting the disrupted morphology
is to consider that competitive recruitment of cells into either a fin or a normal
limb regenerate by the two field influences causes the formation of unresolved
regions where anomalous structures result. Whatever the eventual explanation
for the latter phenomena, they do not arise when the experimental animal lacks
a caudal fin, at least not with the present experimental method.
Rearrangement or axial rotation of the substituted tissue is the other component of the foregoing protocol. The procedure asked what, if any, axial
information was present or accessible in tail skin when grafted to the limb field.
Again, the species employed showed no morphogenetic effect even though the
skin graft produced apposition of dorsal position (skin) with ventral (internal
limb tissues). Such maximal positional dislocation of limb skin results in a
majority of regenerates being multiple (Lheureux, 1972; Carlson, 1974, 1975).
In these cases, however, the anteroposterior as well as dorsoventral axis is
rotated 180°. Since tail skin lacks an anteroposterior axis, a more appropriate
comparison of the tail-skin-to-limb data may be found in the single-axisrotation experiments where cuffs of limb skin were grafted to the contralateral
limb with only one axis maximally dislocated. When only the dorsoventral axis
of the limb skin graft was dislocated 180°, Lheureux (1972) found that 46%
of the subsequent limb regenerates bore supernumerary structures. Maden &
Goodwin (1980) have also shown that when the tips of axolotl limb buds are
exchanged with the contralateral bud and rotated 180° such that only the dorso-
Effect of tail skin on limb regeneration
119
ventral axis is inverted, 40 % of the limbs develop supernumeraries. However,
Carlson (1974) repeated the skin graft experiment on ten axolotls and found
no supernumerary outgrowths. This may again indicate species differences or
differences between larval and adult forms in the way morphogenetic information
is maintained, transmitted, received or any combination of these and other
factors involved in regulating pattern formation. Supernumerary limb regenerates have been produced on Plethodon cinereus in this laboratory by reversing
either the dorsoventral or anteroposterior asis of the limb skin or both simultaneously (Fig. 1). However, the frequency and complexity of supernumeraries
in this species for any of the axial skin dislocations is not as great as those found
by Lheureux (1972, 1975) using Pleurodeles wait Hi.
One final analogy for the tail-skin-to-limb procedure takes into account the
larger circumference of the tail relative to the limb. Care was taken to assure
that the dorsal aspect of the tail skin was always included in the graft, as it
usually bore the red pigment which was a convenient marker for graft orientation. Trimming the tail skin to fit the graft bed reduced the ventral skin contribution but invariably produced a cuff representing, at the least, dorsal and
lateral caudal position.
Analogous with this reduced representation of normal axial position are other
experiments performed by Lheureux (1975). He removed cuffs of whole skin
or longitudinal strips of skin from the upper arm and repositioned them with
the proximodistal axis of the graft wrapped around the circumference of the
limb. By selecting either the level of amputation through the repositioned cuff
or the origin of the graft strip, he produced limb stumps whose skin represented
a single axial quality. When the stump bore skin of dorsal origin around its
perimeter, 28 % of the resulting regenerates had supernumerary structures but
only 34% approximated normal regeneration with three or four digits. Amputation through a cuff of posterior or postaxial skin had an even greater disruptive
influence producing 49% supernumerary regenerates. It is thus unlikely that
the reduced ventral representation in the tail skin grafts in the present experiment had any significant effect on the overall results. Morphogenetic influence
is thus considered to be a product not of a particular tissue in an epimorphic
field, but rather the result of its development in a particular field. Urodele tail
skin thus appears to be morphogenetically inert, at least in this species.
This work was supported in part by BRSG Grant S07RR 05477 from the NIH and a
Grant to the MDIBL from NSF.
I wish to thank Drs R. J. Goss, R. W. Glade and D. L. Stocum for their helpful comments
on and criticisms of the initial manuscript. Their thoughtful suggestions were greatly
appreciated.
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C. E. DINSMORE
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(Received 30 November 1981, revised 24 May 1982)