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Gross morphological analysis of limb regeneration in postmetamorphic adult Ambystoma.

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THE ANATOMICAL RECORD 2062955406 ( 1983)
Gross Morphological Analysis of Limb Regeneration in
Postmetamorphic Adult Ambystoma
HENRY E. YOUNG, CLAUDIA F. BAILEY, AND BERNELL K. DALLEY
Department ofAnatomy, Texas Tech University Health Sciences Center,
Lubbock, TX 79430 IH.E. Y., B.K.D.! and Department of Zoology, 2Jniuersit.y
ofArkansas, Fayettedle, A R 72701 (H.E.X, C.EB.I
ABSTRACT
Due to the great disparity between regeneration times for the
larval salamander (40 days), axolotl (30+ days), newt (44 days), and adult
salamander (155 to 370 days), a staging system was devised so correlative
comparisons could be made between regenerative model systems. The sequence
was based on two criteria: 1)the stages should be similar to previously reported
sequences for the newt, axolotl, and larval salamander, and 2) the stages must
be readily recognizable by examination of the external morphology in the
living state. Postmetamorphic adult land-phase A mbystoma were amputated
through the forearm, placed within survival conditions, and observed until
regeneration was completed. Of the initial 160 salamanders, 157 (98%) progressed through 11 clearly defined stages of regeneration. A side-by-side comparison of the staging sequence for land and aquatic phase urodeles is given
along with a summary of key external morphological characteristics for the
adult salamander. It was noted that as the length of time for regeneration
decreased, the relative ratio of the nerves innervating the limb (spinal nerves
4,5, and 6) increased for the four species of Ambystoma examined: A. annulatum, 324 to 370 days postamputation (dpa) with a 1 : l : l neural tissue ratio; A .
maculatum, 255 to 300 dpa with a 2 2 1 ratio; A . texanum, 215 to 250 dpa with
a 2:2:2 ratio; and A . tigranum, 155 t o 180 dpa with a 2:3:3 ratio.
Previously, only aquatic-phase urodeles,
i.e., the newt, axolotl (both young and mature), and larval salamander, have been used
as model systems for studying complete limb
regeneration (Wallace, 1981). The relative
rapidity of limb regeneration in these forms
was considered a valuable attribute in the
examination of the various aspects of limb
regeneration, including neurotrophic influence (Singer, 19781, regenerate epidermal influence (Singer and Salpeter, 19611, hormonal conditions (Schotte, 19611, environmental conditions (Schauble, 19721, and pattern development (Tank and Holder, 1981).
Young and co-workers (1983a) have shown
that adult postmetamorphic land-phase salamanders retain the intrinsic capacity to
completely regenerate a limb if maintained
under the proper laboratory environmental
conditions and observed for extended periods of time, 155 to 370 days postamputation. Since the increased size of these
organisms and the extended regenerative
time periods appeared ideal €or future stud-
‘ J 1983 ALAN
K LISS. INC
ies concerning possible tissue-tissue and/or
tissue-macromolecular interactions during
limb regeneration, we began a detailed examination of forelimb regenerative capabilities of the adult salamander, comparing and
contrasting the results with those previously published for aquatic urodeles. Unfortunately, as shown by the diversity of the
regeneration time periods for the adult salamander and the previously reported regeneration times for aquatic based regeneration
model systems (i.e., 44 days for the newt,
“30+” days for the axolotl, and 40 days for
the larval salamander), comparisons of specific tissueitissue interactions between systems were impossible based on days postamputation as the sole criterion. We therefore began a study to determine a staging
sequence, comparable to previously reported
sequences, for complete limb regeneration
in the postmetamorphic adult land-phase
Recclved July 7. 1982. accepted March 22. 1983
296
H.E. YOUNG, C.F. BAILEY, AND B.K. DALLEY
putation of the forearm on one side while the
opposite side underwent a sham operation
and was designated as a control (Young et
al., 1983a).
For the initial establishment of gross staging regenerating forelimbs of 20 anesthetized salamanders (five per species) were
examined daily for the first 66 days and then
every other day until complete limb regeneration occurred. Daily anesthetization did
not affect the morphology of the regenerate,
since the internal morphology did not differ
from that of the other respective salamanders which were only anesthetized twice
(Young, 1977a).
Throughout the study, the remaining 35
salamanders per species were examined periodically without anesthetic and their regeneration times recorded for each stage
through which they progressed At the termination of each experimental period, the
forearms of all salamanders within each
group were reamputated 1-2 ml proximal to
the original amputation site, fixed in either
Lillie’s fluid or Lillie’s-10% cetylpyridinium
chloride (CPC), washed in 50% ethanol with
or without 3% CPC (Young et al., 1983131, examined at the gross morphological level,
staged, their respective regeneration times
recorded, and photographed using either a
Wild or a n American Optical Stereo Dissecting Scope both modified for photomicrography).
The fixed salamanders (a minimum of five/
species) were dissected to obtain the following information: number, pathway, and area
innervated by each spinal nerve; number,
MATERIALS AND METHODS
course, and areas supplied to the extremities
Two hundred postmetamorphic adult Am- by the vasculature; osteological patterns of
bystoma (annulaturn, maculatum, texanum, all extremities; and muscle compartmentaland tigranurn) were obtained and housed as ization in all extremities. After dissection,
reported by Young et al. (1983a). The sala- the nerves within the brachial plexus remanders were allowed to acclimate to these gions of all salamanders (twolsalamander)
conditions for a period of 6 months. During were photographed, printed at a constant
this acclimation time period, any salaman- enlargement factor ( x 5), the widths meaders that appeared unhealthy were removed sured, and standardized to similar measurefrom their terraria and fixed in 10%formalin ments from A . annulaturn.
for gross dissection. At the end of the acclimation period, 160 salamanders of comparaRESULTS
ble size and weight were chosen as the
Under controlled environmental conditions
experimental population. The remaining salamanders were also fixed for gross dissec- 157 of 160 salamanders (98%) of the four spetion. The 160 salamanders in the experimen- cies of postmetamorphic adult Amb.ystoma
tal population, consisting of 40 postmeta- progressed through 11 clearly defined stages
morphic adults of each species, were treated of limb regeneration. The values in the paas follows: Four salamanders per species per rentheses represent the range of days postmonth, for 8 months, were subjected to am- amputation (dpa) for all four species (see
form. Two main criteria were used in devising this system: 1)the stages should be similar to published staging systems for the
newt (Iten and Bryant, 19731, the axolotl
(Tank et al., 1976), and the larval salamander (Stocum, 1979); and 2) the stages must
be readily recognizable by examination of
the external morphology in the living state.
Gross dissections of each of four species of
Ambystoma (annulatum, maculatum, texanum, and tigranurn) were made to determine osteology, innervation, musculature,
and vascularization patterns of the intact
limb and determine whether correlations
were possible between the tissues examined
and the differences in the regeneration rates
previously shown in these species. We determined that the postmetamorphic adult salamander, Ambystoma, progressed through
11clearly defined stages with structures visualized a t the gross morphological level paralleling structures present in the newt,
axolotl, and larval salamander. Gross dissections of the limb revealed that although the
osteology, musculature, and vascularization
were equivalent in all four species exaniined, the amount of nervous tissue in the
limbs differed. The A . tigranum contained
the largest complement of nervous tissue
within its brachial plexus, while the other
salamanders contained lesser amounts. In
decreasing order they were A . texanum, A .
maculatum, and A. annulatum. The amount
of nervous tissue within the brachial plexus
of each salamander appeared to correlate
with their respective regeneration rates.
297
LIMB REGENERATION ANALYSIS OF ADULT AMBYSTOMA
TABLE 1 Regeneration time periods for Ian&phace Ambystoma’
S-V (Avg.?
Stage
Wound healing
Early bud”
Middle bud
Late bud
Early palette
Middlc palette
Late palette
Early digit
Middle digit
Late digit
Coniulete reeenei.ate
A annulatun)
A nintulatum
A texannin
A t~~rrarzirni
85
94
102
128
0- 34
32- 66
60- 96
90-1 30
124-160
156-198
190-226
220-256
250-292
286-330
324-370
0- 30
25- 50
44- 68
63- 89
86-113
110-143
140-175
167-207
203-235
230-263
255-300
0- 24
20- 40
35- 62
56- 80
77-103
97-121
116-143
140-165
160-190
185-220
215-250
0- 20
18- 43
38- 52
48- 71
68- 91
87-109
103-127
124-140
138-152
143-160
155-180
i N u m h w s represent days postamputation.
‘S-V lrlvi.. I is t h e average snout-vent len@h, in rnillimetcrs, for each species of A n i h ~ ~ t o iexamined
ii~~
‘Atiih,v,s/r,iriol a n g c value for early bud s t a g e (see Table 31 is 18-66, i.e., the lowest value for A. Iiymtiuni is t a k e n with
t h e highest value for A anriulntuni. The A r i i a c ~ i l ~ z t ~and
i n i A. tmai/uni have interinediatr t i m e s between t h e other t w o ,
a n d tlic,wtoi,e fit within the r a n g e value given for each stage.
Table 1). The dpa for each individual species
was determined by pooling their respective
regeneration times per stage from three
sources: the examination with anesthesia of
five salamandersispecies, examination without anesthesia of 35 salamandersispecies
throughout the experimental periods, and
examination of 40 regenerate limbsispecies
at the end of the experimental periods. The
morphological characteristics for each stage
were determined from examination of both
the 20 anesthetized salamanders throughout
the process and the 40 regenerate limbshpecies at the termination of each experimental
period. The stages are as follows (see also
Tables 1 and 2).
Wound-HealingStage (0-34 dpa)
After 3 to 5 days the necrotic portions of
the radius and ulna were easily recognized
by their whitish appearance. The stump epidermis bordering the periphery of the amputation site became slightly swollen. It then
migrated centrally and completely covered
the wound within 12 to 22 days following the
amputation. Centrally, the epidermis was
thin and transparent; peripherally, it was
thicker and opaque. During this period, vascular stasis and subsurface hemorrhagic foci
were frequently visible beneath the transparent portion of the regenerate epidermis.
By 20 to 34 days postamputation, the entire
wound surface was covered by a layer of
thickened opaque regenerate epidermis, designated the “apical epidermal cap.” The dorsal epidermal surface was nonpigmented,
while the ventral surface was highly pig-
mented. Along the distal surface of the apical
epidermal cap a ridgelike swelling, designated the “epidermal ridge,” extending from
the preaxial (radial) to the postaxial (ulnar)
border. By the end of this stage, the “blastema1 outgrowth” had appeared (Fig. 1).
Early Bud Stage (18-66 dpa)
The earliest externally discernible blastema appeared as a slight swelling. The apex
of the apical epidermal cap covering the blastema was whitish, avascular, and nonpigmented. At the distal tip, a distinct epidermal
ridge covered the blastema. The epidermal
cells of the stump bordering the amputation
site were highly pigmented, and the area
was well vascularized. At the border between
the regenerate epidermis and stump epidermis was a demarcation line externally denoting the site of the amputation. By the end of
this stage, the blastema had grown slightly
t o form a symmetrical, rounded mass with
the epidermal ridge at its apex (Fig. 2).
Middle Bud Stage (35-96 dpai
A visible demarcation line existed only
along the dorsal surface of the stump-regenerate border. The apical epidermal cap with
associated epidermal ridge retained its whitish nonpigmented appearance. Tension lines
running longitudinally from the stump epidermis t o the epidermal ridge were visible
on the ventral surface of the regenerate. The
blastema with its continued outgrowth
became more symmetrically cone shaped
(Figs. 3,4).
TABLE 2. Key morphological events i n the regeneration stages of postmetamorphic adult Ambystoma
Stage
Description
____
Wound healing-Starts a t amputation; overgrowth of wound surface by regenerate epidermis forming
epidermal cap; ridgelike structure (designated “epidermal ridge”) appeared within epidermal cap,
extended from preaxial (radial) to post-axial (ulnar) borders.
Early bud-Starts with budding of blastemal outgrowth; “demarcation line,” separating stump tissues
from newly regenerating tissues, occurs along dorsal surface of limb; ends with rounded outgrowth
of blastema with epidermal ridge a t distal tip.
Middle bud-“Tension lines” appear along ventral surface of stump-regenerate complex; rounded
outgrowth of blastema transforms into symmetrical-shaped pyramidal cone.
Late bud-Pyramidal cone shape becomes flattened dorsoventrally; epidermal ridge maintaining presence
along dorsal distal tip of regenerate.
Early palette-Further elongation and flattening of bud produces paddle-shaped palette segregated into
two regions: proximal one third of palette bulged outward along dorso-ventral axis, distal two thirds
of palette retained a flattened contour.
Middle palette-Palette elongates; epidermal ridge area replaced with four small swellings along distal
tip.
Late palette-Swellings increase in size with “interdigital” grooves beginning to appear between
swellings; ventral regenerate epidermis beginning pigmentation patterns equivalent to respective
species.
Early digit-Swellings approximate future digits; “interdigital” wedges of flattened epidermis persist
between future digits; the most medial (radial side) and most lateral (ulnar side) future digits begin
to separate from two midline future digits.
Middle digit-All four future digits separated from each other by interdigital wedges of epidermis and
interdigital grooves.
Late digit-Outgrowth of midline pair of digits; most medial and most lateral digits completely separated
from midline digits.
Complete regenerate-All digits separated from each other; indistinguishable from controls; demarcation
line persists along dorsal surface of stump-regenerate complex.
Late Bud Stage (48-130 dpai
The stump epidermis was pigmented, while
the regenerate epidermis remained nonpigmented. A distinct epidermal ridge was visible along the dorsal distal tip of the bud. The
blastema outgrowth began as a symmetrical
pyramidal cone and, by the end of this stage,
became flattened dorsoventrally (Fig. 5 ) .
Early Palette Stage (68-160 dpa)
Both the demarcation line and epidermal
ridge continued to persist during this stage.
Further elongation and flattening of the bud
produced a paddle-shaped palette. The newly
formed palette possessed two distinct regions: the first, nearest the amputation site
and extending approximately one third the
length of the palette, bulged outward along
A hhrcr,iattons
APE Apical palette
DE
DL
ER
FD
IG
R1
epidermis
Dorsal
regenerative
epidermis
Demarcation line
Epidermal ridge
Forming digits
Interdigital grooves
Distal one third of
outgrowth, region 1
Rz
RGE
SE
SW
TL
VE
Proximal two
thirds of
outgrowth, region 2
regenerate
epidermis
Stump epidermis
Swellings
Tension lines
Ventral regenerate
epidermis
Fig. 1. Wound-healing stage, dorsal oblique view,
shows a slight swelling in the regenerate epidermis a t
the distal tip and the first evidence for the bud outgrowth, Arnhystorrio annulaturn. x 100.
Fig. 2. Early bud stage, ventral view, shows the
rounded outgrowth of the blastema with the epidermal
ridge a t its distal tip, A . nnnctlafu,rn. X 70.
Fig. 3. Middle bud stage, dorsal view. shows a demarcation line separating the stump tissues from a symmetrical cone-shaped hlastemal
outgrowth. A .
nnnulotum. x 94.
Fig. 4. Middle bud stage, ventral view of the same
hud as in Figure 3. Tension lines are visible from the
epidermal ridge to the stump tissues. A . annulaturn.
x 90.
Fig. 5 . Late bud stage, dorsal view of a flattened coneshaped outgowth. The demarcation line continues to
separate the blastemal outgrowth from the remaining
portion of the limb, A. annulaturn. x 60.
Fig. 6. Early palette stage, dorsal view shows a paddlelike palette-shaped outgrowth which can be segregated into two regions: region 1 comprises the area of
the future digits; region 2 comprises the area of the
future “hand,” A . onnulaturn. x 70.
Fig. 7. Middle palette stage, dorsal view of a n elongated paddle-shaped outgrowth. The apical palette epidermis covers the outgrowth and a demarcation line
separates stump tissues from regenerate outp-owth tissues. Along the distal ridge of the outgrowth, slight
swellings begin to replace t h e epidermal ridge, A . annulatum. x 70.
Fig. 8. Late palette stage, dorsal view of a n elongated
palette-shaped outgrowth. A demarcation line separates
proximal stump tissues fi-om the distal regenerate tissues. Distinct swellings are visible along the distal border and are indicative of forming digits, A . arznuiaturn.
x 90.
LIMB REGENERATION ANALYSIS OF ADULT AMBYSTOMA
299
300
H.E. YOUNG, C.F. BAILEY, AND B.K. DALLEY
a dorsoventral axis; the second, the remaining two thirds of the palette, had a flattened
contour except for a series of bulges which
appeared along the distal lateral edge (Fig.
6).
Middle Palette Stage (87-198 dpa)
The demarcation line was not as sharply
defined as in earlier stages. The regenerate
epidermis showed pigmentation, but less
than that observed in the stump epidermis.
The area of the epidermal ridge was replaced
with four small swellings separated by three
indentations, designated as early “interdigjtal grooves” (Fig. 7).
Late Palette Stage (103-226 dpa)
The regenerate had increased in length
from one and one half to two times that observed in the middle bud stage. The diameter
of the palette was less than the diameter of
the stump. Indentations, designated “interdigital grooves,” were interposed between the
swellings seen earlier. Excluding the nonpigmented epidermis covering the distal tips of
the swellings, the pigmentation of the palette was beginning to match the pigmentation patterns found on the respective stumps,
i.e., annular rings for the A. annulaturn, spots
for the A . maculatum, mottled appearance
for the A . texanum, and stripes for the A .
tigranum (Fig. 8).
Early Digit Stage (124-256 dpa)
Although the respective pigmentation patterns of the regenerate and stump were similar, the demarcation line observed i n earlier
stages remained visible. There was continued growth of the regenerate distal t o the
demarcation line. The number of swellings
visible at this distal tip coincided with the
original number of digits of the limb and the
regenerate assumed a digital appearance for
the first time. Wedges of flattened regenerate epidermis, designated the interdigital
wedges, persisted between each of the future
digits. Only the first (thumblike) digit was
separated from the remaining three or four
(fingerlike) digits (Fig. 9).
Middle Digit Stage (138-292 dpai
The digits were more recognizable in the
regenerate and were equally separated at
their distal orders. The interdigital wedges
of tissue were present at the base of the interdigital grooves and extended proximally
into the regenerate epidermis as dark triangular masses (Fig. 10).
Late Digit Stage (143-330 dpa)
The pigmentation pattern for each regenerate almost matched the respective pattern
of the stump. Large and small pigmented
areas were visible along the ventral surface
of the limb and the palmar surface of the
hand and digits. Digits 2 and 3 remained
fused as seen in the middle digit stage, while
digits 1 and 4 (5) were completely separated
from their neighbors. The inside pair of digits (digits 2 and 3) projected forward and were
longer than the two outside digits (digits 1
and 4) (Figs. 11, 12).
Complete Regenerate Stage (155-370 dpa)
The demarcation line was still faintly visible along the dorsal surface. The pigmentation pattern on the regenerate portion was
indistinguishable from the respective pig-
C
CI,
DL
FD
FL
A hhr.er.icztions
Chromatophores
IW’ Interdigital wrdpe
Crease lines
LD
L a t e r a i t w o digits
Demarcation line
MD Middle two digits
Forming digits
1 H Thumb-like digits
Fingerlike digits
Fig. 9. Early digit stage, dorsal view of a regenerate
structure that has t h e distinct appearance of a forming
“hand” containing digits. The forming digits a r e beginning to separate from each other, A . annulaturn. x 60.
Fig. 10. Middle digit stage, dorsal view of a definitive
“hand”-like regenerate structure consisting of separated
digital regions, A . a n u n l a f u m . x 70.
Fig. 11. Late digit stage, a dorsal view showing continued separation of the digits, A . ntzriulafrtm. x TO.
Fig. 12 Late digit stage. a ventral view of the same
regenerate as seen i n Fig. 11. Patches of chromatophores
are appearing along this surface. Transverse crease lines
overlie future joint areas, such a s t h e interphalangeal
joints, A . atinu/otum. x 50.
Fig. 13. Complete regenerate stage, a dorsal view
showing all digits separated from each other with t h e
regenerate forelimb indistinguishable fi-on1 either the
original or sham-operated control limbs. Within the regenerated limb the demarcation line remains and marks
the spot of the original amputation site, A . a r i r ~ u / a t c i n ~ .
x 60.
Fig. 14. Cutnplete regenerale stage, a ventral view
of Figure 13. At t h e beginning of this stage. patches of
chromatophores appear along t h e ventral surface, hut
by t h e end of this stage t h e Pigmentation pattern assumes t h a t oC i t s respective species: a n n u l a r rings for
A . nnnululum, spots i’or A . niaculc~tum,a rriottled appearance for A . texanum, a n d striptts Tor A . trgronutlz,
A . nirnulatutn. x 60.
LIMB REGENERATION ANALYSIS OF ADULT AMBYSTOMA
301
302
H.E. YOUNG, C.F. BAILEY, AND B.K. DALLEY
mentation patterns of the stumps. On each
digit along the ventral surface opposite the
interphalangeal joints were transverse
crease lines. In the early part of this stage
the regenerate portion (the distal forearm,
wrist, hand, and digits) was shorter and
broader than those of the control limbs. In
the latter part of this stage, however, the
regenerate portion assumed the same proportions as those of either the original or the
sham-operated controls (Figs. 13, 14).
Three anomalous regenerates (approximately 2% of the population) formed from
the 160 experimental animals and were previously reported by Young (1977b).
Comparison of the time periods for each
stage of regeneration for the four species of
Ambystoma are shown in Table 1. A summary of key external morphological characteristics of the adult salamander regenerating limb is given in Table 2. Staging comparisons between observed adult salamanders and aquatic phase urodeles (as reported
in the literature) are given in Table 3.
Gross dissections revealed that all four species of adult salamander examined exhibited
similar patterns of osteology, musculature,
vascularization, and innervation. The osteology of the normal forelimb consisted of a
humerus in the arm; a radius and ulna in
the forearm; two rows of cuboidal-shaped carpal bones in the wrist area, composed of three
bones in the proximal row and four bones in
the distal row; while the digits were com-
TABLE 3. Comnarison o f reperreration staging sequences'
Land phase
A mh,ystrma
Larval
A nr hynto ma
Present study
(Stocum, 1979)
Wound healing
0-34
Initial
dedifferentiation
0-6
Early bud
18-662
Early bud
5-7
Middle bud
35-96
Late bud
48-130
Early palette
68-160
Middle palette
87-198
Medium bud
6-9
Late palette
103-226
Early digit
124-254
Middle digit
128-292
Late digit
143-330
Complete regenerate
155-370
Late bud
7-9
Early
redifferentiation
7-11
Notch
8-11
Two-fingerbud
11-13
Three-fingerbud
14-16
Four-fingerbud
15-20
Newt
(Iten and Bryant,
1973)
Wound healing
0-?
(Tank et al.,
1976)
Wound healing
0-5
Early
dedifferentiation
'-?
Late
dedifferentiation
?-?
. .
Moderate early
bud lo?
Early bud
?-17
Medium bud
14-20
Late bud
18-24
Palette
22-28
Dedifferentiation
6-11
Early digits,
earliest
24-?
Early digits,
Late ?-25
Medium digits
30-40
Late digits
34-44
'Nunihers reprrsent days postamputation
'For explanation, see Table 1 .
Axolotl
Early bud
12-15
Medium bud
16-20
Late bud
21-24
Palette
24-28
Digital outgrowth
30 +
LIMB REGENERATION ANALYSIS OF ADULT AMBYSTOMA
posed of the distal portion of a row of rectangular-shaped metacarpals and two rows of
rectangular-shaped phalanges.
The musculature was compartmentalized
with anterior and posterior compartments in
the arm, forearm, and hand. There were two
major blood vessels enteringiexiting the limb,
and they remained close to the humerus
throughout the length of the arm. In the
most proximal part of the forearm the blood
vessels became divided into three groups and
followed the pathway of the nerves within
the remainder of the forearm and into the
hand.
The first spinal nerve innervates the area
along the back of the head. The second spinal
nerve innervates the area along the side of
the neck. The third spinal nerve innervates
the area around the shoulder. The forelimb
is innervated by spinal nerves 4, 5, and 6.
These nerves (4-6) combine to form a “brachial” plexus, in the axillary region deep t o
the cartilaginous scapula, prior to sending
three major branches to innervate the forelimb. The nerves enter the tissue of the forelimb along the anterior side of the limb and
diverge from each other along different pathways. One nerve curves around the humerus
and then travels down the posterior side of
the arm and forearm. A second nerve runs
along the anterior midline side of the arm
and forearm. The third nerve runs along the
postaxial border of both the arm and forearm. All three nerves converge on the hand,
one from the posterior side, the second from
the anterior side, and the third from the postaxial border. The four species of Ambystoma
appeared t o contain different amounts of
nervous tissue within their respective nerve
plexus. Relative amounts were based on a
ratio with the nerves of the A . annulaturn
brachial nerve plexus. The A. annulaturn appeared to have the least amount (1:l:l).The
other species in order of increasing amounts
of nervous tissue were A. rnaculatum
(2:2:1),A. texanum (2:2:2), and A . tigranum
(2:3:3)(see Figs. 15-18).
303
tal procedures, sampling, or comparative correlations. This disparity can be fully appreciated when comparisons are made within
the same genus, Ambystoma. The larval salamander, Ambystoma maculatum, requires
34 to 44 days to completely regenerate a
limb (Stocum, 19791, and the axolotl, Ambystoma mexicanurn, a neotenic salamander, requires 30+ days (Tank et al., 1976). In
contrast, postmetamorphic land-phase Ambystoma salamanders require much longer
regeneration periods, 155 to 370 days.
Therefore, within this study we have defined regeneration stages based on external
morphology to be used as a reference series
for comparisons between this and previously
reported regeneration model systems.
Although the number of stages described
within this sequence was increased over
those of the larval and neotenic salamanders,
the selected stages were designed t o closely
approximate those reported for the aquatic
forms. This divisibility of stages was especially evident during the latter phases of regeneration (middle palette through complete
regenerate, Table 3). Another difference
among the sequences concerned the woundhealing stage of adult salamander regeneration which encompassed both the woundhealing and dedifferentiation stages of the
newt and axolotl staging sequences. The extended time period of this stage was required
by the four species reported here for the regenerate epidermis to migrate over the
wound surface and form the apical epidermal
cap-epidermal ridge complex. Although histological examination of the wound-healing
stage showed the migration of the peripheral stump epidermis over the wound surface with a concomitant degeneration of the
internal tissues (dermis, fibrous connective
tissue, skeletal muscle, cartilage, tendon,
and bone) at the amputation surface and a n
accumulation of non-differentiated stump
tissues proximal to the amputation site
(Young et al., 1983b),no external morphological features allowed this distinction of the
separate events. Therefore, this period was
DISCUSSION
designated simply as the wound-healing
From these results it becomes apparent stage.
that comparisons between aquatic- and landSeveral notable morphological distinctions
phase limb regeneration model systems existed between the regenerates of the adult
based solely on the number of days postam- land-phase salamander and those of the
putation are unrealistic (see Table 3). In this aquatic urodeles. For example, the middle
heterogenous order of amphibia (Urodele), (medium) and late bud stage blastemas of the
staging according to time postamputation is adult salamander and the newt were symunreliable as the basis for critical experimen- metrical, whereas those of the large axolotl
304
H.E. YOUNG, C.F. BAILEY. AND B.K. DALLEY
LIMB REGENERATION ANALYSIS O F ADULT A M B Y S T O M A
were characterized by a pronounced “dorsal
to posterodorsal curvature” (Tank et al.,
1976). The apical epidermal cap was present
to some extent in all forms. The epidermal
ridge at the distal tip of the apical epidermal
cap was similar to the epidermal lobe in the
mature (Tank et al., 1976)and young (Farber,
1959) axolotls, but a homologue was missing
in both the newt (Iten and Bryant, 1973) and
larval salamander (Stocum, 1979).
The increased time seen for limb regeneration in the land-phase salamander (155 to
370 days postamputation, depending on the
species; Table l), versus the larval salamander (44 days), axolotl (30+ days), and the
newt (40 days) might be postulated to be due
in part to the increased size of the organism.
Pritchett and Dent (19721, studying regeneration in the adult newt, postulated a n inverse relationship between the size of the
organism and the rate of regeneration, and
inferred that as the size of the newt increased, the ability to regenerate decreased.
Tank et al. (1976), studying adult axolotl
limb regeneration, reported a similar situation. By the time the regenerates had
reached the digital outgrowth stage (30+
days), morphogenesis was virtually complete. However, the regenerate still required
a prolonged period of growth during which
time some internal changes occurred (i.e.,
ossification of the cartilaginous skeleton).
The extent of the growth period was inversely related to the size of the axolotl. This
inverse relationship between size and regeneration rate was consistent within each spe-
Fig. 15. Arubystorna cmnulatnm, left dorsolateral view
showing dissection of t h e region of the brachial nerve
plexus, with cartilaginous scapula removed. Spinal
nerves 4, 5, and 6 form a n intertwining plexus before
sending three branches into the forelimb and one to two
branches posteriorly down the lateral body wall. The
ratio of nervous tissue within t h e nerve plexus (based on
the relative size of t h e A . annulaturn nerves) is 1:l:l.
x 2.8.
Fig. 16. Amb,ystonm niaculatum, left dorsolateral view
showing dissection of the region of the nerve plexus. The
ratio of nervous tissue within t h e plexus (see Fig. 15)is
2:2:1. x 3.0.
Fig. 17. Arnb,ystorrzu texanum, left dorsolateral view
showing dissection of the region of the nerve plexus. The
ratio of nervous tissue within the plexus (see Fig. 15) is
2:2:2. x 3.0.
Fig. 18. Ambystoma tcgranuni, left dorsolateral view
showing dissection of the region of the nerve plexus. The
ratio of nervous tissue within the plexus (see Fig. 15)is
2 : 3 : 3 . x 3.0.
305
cies of land-phase salamander and was
reflected by the fact that the smaller salamanders within each species regenerated a t
a faster rate than their larger counterparts.
However, this inverse relationship was not
substantiated when comparisons were made
between size and regeneration rates for different species within the same genus, The
size range for the four species examined
here, ranking from smallest to largest, was
A. annulatum, A . maculatum, A. texanum,
and A . tigranum (see Table 1).The order of
regeneration rates for the four species, ranking from slowest to fastest, was identical.
Thus the smallest of the four species, A .
annulatum, regenerated the slowest, while
the largest of the four species, A. tigranum,
regenerated the fastest. Therefore, among
species of the same genus, size alone cannot
be used to predict the regeneration rate, or
even the ability of a n organism to regenerate.
Factors other than the overall size of an
organism, such as the amoant of neurotrophic stimulus, interaction between neurotrophic stimulus and wound epithelium, the
presence of extracellular macromolecules or
the competency of the tissue to respond to
these stimuli might dictate the regenerative
potential of a n organism. Gross dissection of
the original controls and the remaining 40
salamanders from the initial population revealed a greater amount of nervous tissue
within the brachial plexus of the A. tigranum than the A. annulaturn. Ambystoma
tigrunum was the largest in overall size
(snout-vent length, Table l),had the largest
apparent ratio of nervous tissue in the limb
(compare Figs. 15-18), and regenerated the
fastest. Thus, when the parameters of overall
size versus ratio of nervous tissue are compared against regeneration rates in the four
species of land phase salamanders, the ratio
of nervous tissue within the limb appeared
to be a better correlate for regeneration rate
than the overall size of the organism. Indeed,
Singer and co-workers (Singer and Egloff,
1949; Singer and Mutterperl, 1963) showed
that regeneration was possible in the amphibian limb if it contained three times the
amount of neural tissue needed to support
maintenance (without regeneration) of the
limb.
An additional factor related to the regeneration rate might be the quality and/
or quantity of matrical macromolecules
throughout tissue regions undergoing the regenerative processes. Toole and co-workers
306
H.E. YOUNG, C.F. BAILEY. AND B.K. DALLEY
(Toole and Gross, 1971; Smith et al., 1975)
have shown that isotopically labeled hyaluronate (an extracellular matrical macromolecule) and its endogenous enzyme, hyaluronidase, accumulate during limb regeneration
in the newt. Our own studies (Young and
Dalley, 1979, 1981; Young et al., 1983b,c,d)
have shown the patterned presence of glycoconjugates (glycoproteins and five families of
glycosaminoglycans) preceding tissue-tissue
interactions within both regenerate and
stump tissues during the three phases of limb
regeneration, i.e., initiation, growth, and redifferentiation. We are currently engaged in
studies to ascertain the possible interrelationships between the nervous tissue, wound
epithelium, extracellular macromolecular
substances, and limb regeneration in landphase salamanders of the genus Ambystoma.
ACKNOWLEDGMENTS
We would like to thank Dr. P.M. Johnston
for his technical expertise, Mr. Oxford for
allowing us to collect A . annulaturn on his
property, Mrs. V.E. Young for assistance in
field collection, Mr. A.G. Frisbie for photographic assistance, and Dr. H. Weitlauf for
review of the manuscript.
LITERATURE CITED
Fdrber. tJ. (1959) An experimental analysis of regional
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phthalamus c.irzdl,scens: Length, rate, stage. Wilhelm
Roux Axhiv., 173.77-89.
Pritchett, W.H., and J.N. Dent (1972) The role o f size in
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Schauble, M.K. 11972) Seasonal variation of the newt
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Smith, G.N., Jr., B.P. Toole, and J. Gross (1975, Hyaluronidase activity and glycosaminoglycan synthesis in
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Wallace, H. (1981 Vertebrate Limb Regeneration. John
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Young, H.E., R.K. Dalley, and R.R. Markwald (1983~1
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