585
Development 105, 585-593 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Organization of connective tissue patterns by dermal fibroblasts in the
regenerating axolotl limb
NIGEL HOLDER
Anatomy & Human Biology Subject Group, Division of Biomedical Sciences, King's College, Strand, London WC2R 2LS, UK
Summary
A set of tendons, aponeurotic sheets and retinaculae,
which transduce muscle action from proximal limb
levels to flexion and extension of the digits, is found in
limbs of many vertebrates. This set of structures, here
termed the digit tendon complex, is described for the
axolotl foreUmb. We show that the complex forms
autonomously in muscleless axolotl limb regenerates
produced from a cuff of unirradiated dermis surrounding an irradiated limb stump, and persists for up to a
year after amputation. The pattern of other connective
tissue structures, including the skeleton, is also normal.
Fibroblast condensations that may represent sets of
these cells normally associated with muscles in the
extensor and flexor compartments of the carpal region
also form in muscleless limbs. The results are discussed
in terms of the importance of the dermis in pattern
regulation, seiforganization of connective tissues in general and autonomous development of the digit tendon
complex in particular.
Introduction
vallier & Kieny, 1982), and that the pattern of wing
muscles is determined by the somatopleure from which
connective tissues differentiate (Chevallier et al. 1977).
The tendons that form in muscleless chick wings are
those associated with flexion and extension of the digits
by muscles originating at more proximal segments,
from the humerus or radius and ulna (Kieny & Chevallier, 1979; Fig. 1). Although clearly described by these
authors, the fact that tendons for muscle attachment
within the fore and upper limb levels failed to form was
not discussed. In this paper, the distal limb tendons,
here referred to as the digit tendon complex, is considered as a separate set of structures the development
of which may be autonomously controlled. In all
vertebrate limbs, such a group of tendons is present,
often in combination with specialized connective tissue
structures such as expanded fascial sheets, retinaculae
and aponeuroses (Fig. 1). Tendons within these complexes may produce very complicated patterns with
branching, overlapping, fusion and, as is the case with
tendons in the human hand, situations where one
tendon pierces another before insertion.
The digit tendon complex provides an excellent
system for study of the pattern-organizing characteristics of connective tissue cells. In the present study, we
examine the ability of connective tissues to undergo
morphogenesis in regenerating axolotl limbs which are
deprived of muscle, looking in detail at the formation of
the digit tendon complex. Muscleless regenerates were
created by amputating limbs in which all tissues except
When the limb of a urodele amphibian is amputated, a
new limb regenerates which comprises exactly those
parts that were removed. A number of recent studies
indicate that the positional information necessary for
pattern regulation to occur during regeneration lies
within the connective tissue of the limb. Such studies
have focused primarily on the connective tissue dermis,
the mesodermal component of the skin. Surgical manipulations in which the limb skin is rotated (Carlson,
1974, 1975), made symmetrical with respect to the
transverse limb axes (Tank, 1979; Maden & Mustafa,
1982; Slack, 1980, 1983), or used as positionally mismatched implants (Tank, 1981; Rollman-Dinsmore &
Bryant, 1984) all demonstrate that the dermis dramatically affects the pattern-regulation process. Taken
together with the results of these experimental procedures perhaps the strongest evidence for a central
role for the dermal connective tissue cells in pattern
regulation is the observation that this tissue contributes
relatively more cells to the blastema than would be
expected from relative cell numbers in the stump
(Muneoka et al. 1986; see also Tank & Holder, 1979).
Consistent with this notion of the importance of
connective tissues in pattern formation are the observations that tendons can form autonomously in chick
wings devoid of muscle (Kieny & Chevallier, 1979), that
the connective tissue in such limbs is able to organize
grafted myogenic cells into recognizable muscles (Che-
Key words: limb regeneration, connective tissue,
fibroblasts, pattern formation.
586
N. Holder
th.m
hth.
Fig. 2. The experimental procedure involved removing a
cuff of upperarm skin (epidermis and dermis), X-irradiating
the arm, replacing the unirradiated skin and amputating the
limb at the distal end of the graft one week after the
operation.
edct
eux, 1983) but little attention has been paid to pattern
regulation in such limbs. We demonstrate that patterning of the connective tissue components of muscleless
regenerates is remarkably normal in a number of
respects, including the overall pattern of the skeleton
and the structure of the digital tendon complex which,
unlike the case in the chick wing where tendon primordia degenerate before hatching, remains well formed
for a long period. These results add considerable
support to the hypothesis (Bryant, 1978; Bryant et al.
1981) that fibroblasts (and principally dermal fibroblasts) play a pivotal role in limb pattern regulation and
that they are able to selforganize into complex tissue
patterns.
Materials and methods
General
All experiments were performed on larval axolotls (Ambystoma mexicanum) which were spawned at King's College.
Animals were maintained in individual plastic containers in
standing tap water through the duration of the experiment
and were fed twice weekly on chopped heart. During surgery
animals were anaesthetized in MS222 (Sigma).
Fig. 1. The digit tendon complex varies in extent in
different vertebrate groups. (A) In humans, the complex
comprises 22 tendons from 14 muscles which are evident in
this schematic cross section through the wrist (after Snell,
1986). The tendons are grouped together in synovial sheaths
and surrounded by flexor and extensor retinaculae. These
retinaculae and the tendons are black in this drawing. In
the chick, fewer tendons are evident than in the human in
the extensor (B) and flexor (C) compartments, and they
appear separate, not associated with any fascial sheets. The
only tendons to form in developing muscleless chick wings
(Kieny & Chevallier, 1979) are those of flexor digitorum
superficialis (fdst) and profundus (fdpt) and extensor
digitorum communis (edct) and extensor metacarpales
ulnares (emut). These tendons and others in the wrist area
are highlighted in black, hth.m, hypothenar muscles; th.m,
thenar muscles; ham, hamate; cap, capitate; traz, trapezoid;
trap, trapezium. 2, 3, 4 represent digit numbers.
the skin had been X-irradiated. This method has been
used previously to examine questions of metaplasia
(see, for example, Dunis & Namenworth, 1977; Lheur-
Experimental
Operations were carried out on the upper arm region from
which a complete skin cuff (epidermis and dermis) was
removed from approximately the mid two thirds. The cuff was
laid aside while the remaining limb tissues received 2500 rads
of soft X-rays. Any adhering muscle fibres visible in the
dissecting microscope were cleaned from the skin which was
then returned to the upper arm and sutured into place in a
normal orientation (Fig. 2). Following a period of between 8
and 16 days for skin healing, the limb was amputated through
the distal edge of the skin cuff and the protruding humerus
trimmed flush with surrounding soft tissues. Control operations involved the same procedure except the limb was not
X-irradiated. In addition, a number of normal limbs were
fixed for wax histology (see below) to help assess the normal
pattern of tendons and fascial sheets.
After a period of between 3 and 12 months the limbs were
fixed for light and electron microscopic analysis.
(1) Analysis of skeletal and tendon patterns
In order to reconstruct the overall skeletal and tendon pattern
in the regenerated limbs they were fixed in Bouin's fluid,
decalcified in EDTA, dehydrated, stained with Victoria blue
and cleared in methyl salicylate. At this point, the overall
skeletal pattern was drawn with a camera lucida and the gross
Limb regeneration from dermal
ftbroblasts
587
Table 1
No. of digits
Experimental
Control
Total
4
3 Spike
30
7
25
7
2
-
3*
-
Muscleless
Partly Normal
muscled muscle
12(40%) 8(2(5%) 7(23%)
7
*Not included in muscle pattern analysis.
appearance of muscle was assessed using cross polaroid filters
set to pick up birefringence in myotubes. The limbs were then
returned to alcohol and processed for wax histology. Serial
transverse 10 /an sections were cut on a rotary microtome and
stained either with haematoxylin and eosin or Sirius red, a
stain that reveals collagen. Camera-lucida drawings of every
tenth section were then made of selected specimens and the
tendon and fascia! pattern reconstructed.
(2) Electron microscopy
A number of digits were removed from operated and control
limbs and fixed in 2-5% glutaraldehyde. The tissue was
postfixed in osmium tetroxide, dehydrated and embedded in
Araldite. Sections for light microscopy were cut at I/an and
stained with toluidine blue, and the block was then trimmed
and ultrathin sections taken.
Results
General
A total of 30 experimental limbs were produced, 25 of
which regenerated 4-digit limbs, two were 3-digit limbs
and three were short spikes (Table 1). Seven operated
controls all regenerated 4-digit limbs. The detailed
structure of the experimental limbs and the normal
anatomy derived from the operated controls and five
normal limbs are presented below.
(1) Overall appearance of regenerates and skeletal
patterns
The great majority of regenerates (25 of 30-83 %) had
grossly normal appearance with 4 digits. Of the 30
experimental limbs, 11 were stained with Victoria blue
to reveal the skeleton to ascertain whether it could form
normally in the absence of muscle. Of the 11 cleared
specimens, four proved to be muscleless after subsequent sectioning, five had partial muscle patterns,
one had a normal musculature, and one was a spike.
The ten cases with digits all had essentially normal
skeletons (Fig. 3); minor variations in carpal number
and position were seen, and two cases had a fused
elbow joint (Fig. 3D).
(2) Muscle patterns
Muscle patterns were examined in each of the 27
experimental regenerates with digits and three classes
of muscle distribution were evident. Twelve limbs had
no muscle distal to the amputation plane; seven had
normal muscle patterns and eight had muscle present
only in part of the regenerates. The structure of
muscleless limbs will be described in detail in the next
section, where a comparison is made with the normal
Fig. 3. Skeletal patterns of four regenerates, three of which
(B,C,D) were shown to be muscleless upon subsequent
histological analysis. A is a normal regenerate from a
control operation showing the normal axolotl limb skeleton.
Magnification is x44 in all cases.
limb; however, the structure of the partly muscled limbs
is worthy of comment. In six of these eight cases,
muscle was present in a restricted position within the
forearm in either the extensor (dorsal) or flexor (ventral) compartment (Fig. 6A). When present it was
continuous with stump musculature and formed identifiable forearm muscles when sufficient myofibres were
present. The muscle in all of these examples petered out
at more distal levels and in only one regenerate did it
reach the wrist. In all six cases, therefore, no muscle
was present in the proximal carpals and hand. The
remaining two regenerates showed a different pattern,
with small amounts of muscle present only in the flexor
(ventral) compartment of the metacarpals. Thus, in
588
N. Holder
B
D
Fig. 4. Landmarks in the normal limb illustrating the digit tendon complex in transverse sections stained with Sirius red.
(A) In the distal forearm, the palmar aponeurosis (arrowhead) begins at the ventral surface of the palmaris superficialis (ps)
ventral to the radius (r) and ulna (u). Other tendons appear clearly at this level (arrows). (B) High-power view of the
palmar aponeurosis at the proximal carpal level showing the thick collagenous band (arrows). (C) At the midmetacarpal
level, the tendons of ebd (having merged with the edc middle tendon) are present dorsally (arrow) and of ps ventrally
(arrow). (D) At the metacarpophalangeal joint, the ps tendon lies within the joint capsule (jc) and superficial to the expanded
tendinous insertion of fdbs and fdbm (arrowed, see also Fig. 5). Abbreviations (see also in text): muscles: edc, extensor
digitorum communis; ps, palmaris superficialis; edb, extensor digitorum brevis; fdbs, flexor digitorum brevis superficialis;
fdbm, flexor digitorum brevis medii; t indicates a tendon for the appropriate muscle; d, dorsal; v, ventral. Bar, lOjan.
these examples, cells of the myogenic lineage may have
been carried distally during regeneration and differentiated in the absence of more proximal muscle.
(3) Patterns of tendons, aponeurotic sheets and fascia
In this section, we concentrate on muscleless limbs and
describe the normal connective tissue pattern assessed
from normal and control limbs only where necessary to
clarify these. Throughout the muscleless regenerates
the mesodermal limb regions where muscle would
normally exist contained blood vessels, peripheral
nerves and accumulations of fibroblasts arranged in
condensations of various sizes and densities. The nerve
pattern will be discussed in detail elsewhere. No tendons were evident at upper or forearm levels; however,
fibroblast condensations were present in the forearm
although they did not have any obvious pattern from
limb to limb excepting the presence of a thin expansion
in the flexor (ventral) compartment in the distal third,
which corresponds to the beginning of the palmar
aponeurosis, a specialization of the tendon of the large
digital flexor muscle, palmaris superficialis (ps - Fig. 4;
muscles named after Francis, 1934 and Grim & Carlson,
1974). This structure is the first part of a complex
arrangement of connective tissues comprising the tendons, aponeurosis and fascia that transduce muscle
action from the forearm, carpus and metacarpals into
flexion or extension of the digits; the digit tendon
complex (Fig. 5A). To be able to describe the appearance of this connective tissue complex in muscleless
limbs, it is necessary first to clarify its normal structure.
In the axolotl, extension of the digits is brought about
Limb regeneration from dermal fibroblasts 589
muscleless limbs (Fig. 6). In the flexor compartment, it
comprised the palmar aponeurosis, the tendons of ps
extending to the distal phalanx and the tendons of fdbs
and fdbm at the metacarpophalangeal joint. A capsule
was also present at each of the digital joints. In the
extensor compartment, the long tendons of edc and the
shorter tendons of edb were clearly present. The middle
edc tendon (Fig. 5A) of each digit began in the midcarpus and appeared continuous with that of edb, forming
a long tendon running to the distal phalanx, exactly as it
does in the normal limb. In the case of both the palmar
aponeurosis and the tendons of edc, they begin at the
appropriate proximodistal level, forming in 'mid-air'
with no obvious structure or connective tissue cells
located more proximally.
Fig. 5. The digit tendon complex in the axolotl forelimb.
(A) Lateral view showing the principal muscles. (B) The
metacarpophalangeal joint of either digit 2 or 3, showing
the tendons inserting onto theflexorside, t', central tendon
of fdbs; fdbm tendon is arrowed. Other abbreviations as for
Fig. 4.
by two principal muscles; the large extensor digitorum
communis (edc), originating from the humerus, and the
smaller distal extensor brevis digitorum (ebd - associated with abductor digit for digit I) which arise from the
carpals (Fig. 4A,C,D). The edc myofibres end at the
midcarpal level and form a set of three tendons, one for
each of digits 2, 3 and 4; two lateral tendons insert into
the proximal region of the metacarpals and a large
medial tendon attaches to the dorsal side of the ebd
fascia. The ebd tendons form at the distal metacarpal
level where they merge fully with the middle edc
tendons.
As mentioned above, the flexor component of the
digit tendon complex begins with the formation of the
palmar aponeurosis at the beginning of the carpals
(Fig. 4A). Palmaris superficialis fibres have run out by
the midcarpus and several muscles originate and insert
into the aponeurosis within the carpal area. Two
muscles, flexor digitorum brevis superficialis (fdbs) and
flexor digitorum brevis medii (fdbm), originate from
the aponeurosis and insert into the proximal phalanx at
the metacarpophalangeal joint (Fig. 5B). With the
passage of the ps tendon, which inserts into the distal
phalanx, the metacarpophalangeal joint has a particularly complex set of tendon insertions on the flexor side
(Figs 4D, 5B). In addition to the muscles mentioned, a
number of other muscles are found in the carpal and
digit regions that insert into either the palmar aponeurosis or the metacarpals (see Grim & Carlson, 1974).
These muscles are not described here because they have
very short tendons or fleshy insertions and, for the sake
of simplicity, are not considered part of the digit tendon
complex.
The digit tendon complex in muscleless limbs
The digit tendon complex was present in each of the 12
Structure of the tendons in muscleless limbs
In order to ensure that the tendons seen in muscleless
limbs at the light microscopic level were of normal
structure, thin sections from digits from muscleless
limbs and control limbs were examined in the electron
microscope. Normal tendons of the ps and edb muscles
comprise stellate fibroblasts in a sea of oriented collagen fibrils, typical of tendons in other vertebrates
(Fig. 7A and see for example Chaplin & Greenlee,
1975; Birk & Trelstad, 1986). The same tendons in
muscleless limbs have an identical ultrastructure and
appeared as well formed in all respects as their normal
counterparts (Fig. 7B).
Additional connective tissue condensations
In all of the muscleless limbs, two additional condensations of fibroblasts were present within the carpal
region which were not identifiable as part of the digit
tendon complex (Fig. 8). They are worthy of note
because of their consistent appearance and the possibility that they represent fibroblasts normally constituting the peri-, endo- and epimysia of muscles associated
with the complex. The first appears in the flexor
compartment of the carpus (Fig. 8B-D). In the normal
limb, the flexor compartment contains more muscles
than the extensor compartment (Grim & Carlson, 1974)
and, in muscleless limbs, the ventral side of the carpals
contains more connective tissue cells than the dorsal
side. Within this ventral region, a single clear oblique
band of fibroblasts was seen emerging from the surface
of the ulnare and intermedium carpal elements and
joining the dorsal side of the palmar aponeurosis. No
single muscle normally lies in this location; however,
three muscles, palmaris profundus I, II and III (pp
I-III) (Grim & Carlson, 1974; Fig. 8A), originate from
the posterior carpals and distal ulna and run obliquely,
pp III and II inserting into the dorsal palmar aponeurosis and pp I inserting into the first metacarpal. The
oblique connective tissue band, therefore, lies in the
overall position of these three muscles and may represent the fibroblasts normally associated with them.
The second fibroblast condensation was located dorsal
to the anterior carpals, the radiale and prepollicis, in
the normal location of abductor minimi I (Grim &
Carlson, 1974; Fig. 8A,E).
590
N. Holder
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B
Fig. 6. The digit tendon complex in muscleless limbs shown through transverse sections stained with haematoxylin and
eosin. (A) A partially muscled limb with dorsal muscle masses (arrows) in the extensor compartment which ran out prior to
the wrist. (B-F) A sequence of sections of a completely muscleless regenerate at progressively more distal levels. (B) No
muscle is evident at the forearm level. However, peripheral nerve fascicles (arrowed in the higher power view of the same
section shown in C) are clearly evident in dorsal and ventral positions. (D) In the midcarpus, the palmar aponeurosis is
present (arrows). (E) The midmetacarpal level showing the principal tendons, pst ventrally and edbt dorsally (arrows).
(F) The metacarpophalangeal joint showing the tendons of the complex on extensor and flexor sides of the proximal end of
the third phalanx of digit 3. The central tendons of fdbs and fdbm are arrowed. Abbreviations as for Figs 4 and 5. Bar,
10/on.
Limb regeneration from dermal fibroblasts 591
7A
Fig. 7. Ultrastructure of ps tendons from normal (A) and muscleless limbs (B). Both show stellate fibroblasts with long
processes running into a 'sea' of orientated collagen fibrils. Bar, 10 [xm.
Discussion
The differentiation of the digit tendon complex in the
muscleless axolotl limb regenerates confirms and
expands the original observations of Kieny & Chevallier (1979), who described tendon formation in developing muscleless chick wings. In the chick, the tendons are
transient primordia, surviving a couple of days, whereas
in the axolotl the digit tendon complex survives for at
least a year. In either case, however, the formation of
tendons in the absence of muscle indicates the piecemeal manner in which limb patterns form; parts of the
pattern develop independently and then connect
together. The organization of connective tissues into
recognizable patterns in muscleless limb regenerates
adds to the growing body of evidence implicating the
fibroblast as a cell type of major importance in the
patterning process. Evidence is strong that in the chick
wing muscle patterns are determined by somatopleurally derived connective tissue (Chevallier et al. 1977)
and, although such a role for fibroblasts has not been
demonstrated directly in the axolotl limb, these cells
have been implicated in pattern regulation in a number
of ways including positional mismatch and cell contribution experiments (see Introduction).
It is intriguing that only the tendons and aponeurosis
of the digit tendon complex formed and not tendons
within the forearm. This result is exactly that described
by Kieny & Chevallier (1979) in the chick, although
these authors did not place any significance in their
observation that the only tendons to form were those of
the digit tendon complex (refer to Fig. 1B,C where the
tendons formed in the Kieny and Chevallier experiments are indicated). The similar results in the comparable experiments in chick and axolotl indicate that not
all tendons may be formed in the same way; the long
and complex tendons, aponeurotic sheets and retinaculae typical of digit tendon complexes may be considered
as a separate and identifiable part of the limb pattern in
terms of the information necessary to build them during
development and the cellular mechanisms involved in
morphogenesis.
A unique feature of the results is the appearance of
connective tissue bands which may represent the fibroblasts normally associated with particular muscles
(Fig. 8). Like the tendons, these do not form at forearm
levels and, in the case of the ventral oblique band,
approximates the position of a group of three muscles,
those of the palmaris profundus set; however, they were
present in each limb examined, suggesting that they are
examples of sets of fibroblasts fated to package myoblasts into recognizable muscles during normal regeneration. Their presence is, therefore, consistent with a
pattern-organizational role for these cells. Such bands
were not seen in muscleless chick wings (cf. Kieny &
Chevallier, 1979).
The present experiments provide strong evidence for
a controlling role of fibroblasts in pattern regulation
because these cells, as the major component of the
dermis, give rise to the whole limb regenerate. In those
limbs that are completely muscleless, the skeleton
forms essentially normally as well as the digit tendon
complex. This result extends the initial findings of
Dunis & Namenworth (1977) and Lheureux (1983) who
first created muscleless limbs using non-irradiated dermis as a sole source of cells for regeneration although
these authors were concerned with metaplasia rather
than the pattern per se and did not discuss the overall
organization of the regenerates. It is interesting to note
that the success rate for producing muscleless limbs
(40 % - Table 1) is approximately that achieved in these
other studies. It is unclear why partial or completely
muscled limbs should regenerate. In previous studies
(for example Maden, 1979; Holder et al. 1979), 2500
rads have been shown to prevent limb regeneration; but
the conditions may be different if a population of nonirradiated cells is present at the stump. It is interesting
to note that in partially muscled regenerates, in the
majority of cases, muscle was found proximally in the
regenerate and was continuous with the stump musculature in the appropriate flexor or extensor compartment. This arrangement suggests that a subpopulation
N. Holder
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Fig. 8. Formation of additional connective tissue condensations in the flexor and extensor compartments of the carpus.
(A) Camera-lucida drawing of a transverse section of the midcarpal region showing the normal muscle pattern at this level.
(B) Photomicrograph of a muscleless limb at a level corresponding to that shown in A. The oblique connective tissue band
(arrows) emerges from the ventral side of the ulnare (ul) and intermedium (i) and inserts into the palmar aponeurosis (pa).
(C) Photograph of a section from a different muscleless limb to that shown in B cut at a slightly more distal level,
emphasizing the consistent appearance, origin and insertion of the oblique band. (D) High power of the oblique band shown
in C to illustrate the clear condensation of fibroblasts separating capillaries and nerve bundles (arrows) which lie in the less
condensed mesenchyme. (E) The fibroblast condensation (outlined) in the extensor compartment which lies in the normal
location of abductor digiti I (ad I in A) seen in a section through the distal carpals. Abbreviations not already indicated: pp
I, II, III, palmaris profundus muscles; p, prepollicis; c, centrale; a, anterior; p, posterior; d, dorsal; v, ventral. Bar,
of cells from the muscle lineage escaped the immediate
effects of X-irradiation (which damages DNA - Maden
& Wallace, 1976) but could only undergo a limited
number of cell divisions. In two cases, myotubes were
seen in distal limb regions in the absence of muscle at
more proximal levels of the regenerate. The origin of
these rare cells is unclear but their existence raises the
possibility that small numbers of myoblasts exist in
distal locations in other, apparently muscleless, regenerates. The detection of a low number of myoblasts at a
density too low to promote fusion into myotubes is
beyond the resolution of the histological methods used
in this paper and would require a myoblast-specific cell
marker. We cannot completely rule out the possibility,
therefore, that the presence of unfused myoblasts may
influence the organization of connective tissue patterns
in some way.
Finally, the organization of tendons and fascial sheets
in the absence of external mechanical influences normally provided by muscle contraction highlights the
Limb regeneration from dermal fibroblasts 593
selforganizational capacities of connective tissue cells.
The normal appearance of organized collagen fibrils
and the normal stellate appearance of tendon fibroblasts in the ps tendon of muscleless limbs (Fig. 7) is
reminiscent of the behaviour of fibroblasts in culture,
where fibroblast traction models collagen matrices into
organized arrays (Harris et al. 1981; Stopak & Harris,
1982). The results support the notion that the organization of collagen matrices within the tendons and fascial
sheets of the digit tendon complex is a property of the
fibroblasts that generate that matrix. This view is
entirely consistent with the conclusions of Stopak et al.
(1985) who used labelled injected collagen to assess the
principal factors affecting its morphogenesis in chick
limbs.
It is a pleasure to thank Meena Jesani and Charleston
Weeks for technical assistance, Jon Clarke for critically
reading the manuscript and Malcolm Maden for arranging
and assisting me with the X-ray machine. This work was
supported financially by an SERC project grant to Nigel
Holder.
ontogenesis and regeneration in the axolotl (Ambystoma
mexicanum). 1. Anatomical description of muscles of the
forearm and hand. Z. Anat. EntwGesch. 145, 137-145.
HARRIS, A. K., STOPAK, D. & WILD, P. (1981). Fibroblast traction
as a mechanism for collagen morphogenesis. Nature, Lond. 290,
249-251.
HOLDER, N., BRYANT, S. V. & TANK, P. W. (1979). Interactions
between irradiated and unirradiated tissues during
supernumerary limb formation in the newt. J. exp. Zool. 208,
303-310.
KIENY, M. & CHEVALLIER, A. (1979). Autonomy of tendon
development in the embryonic chick wing. J. Embryol. exp.
Morph. 49, 153-165.
LHEUREUX, E. (1983). The origin of tissues in the X-irradiated
regenerating limb of the newt, Pleurodeles waltlii. In Limb
Development and Regeneration. Progress in Clinical and
Biological Research, Vol. 110A (ed. J. Fallon & A. I. Caplan),
pp. 455-465. New York: A. Liss.
MADEN, M. (1979). The role of irradiated tissue during pattern
formation in the regenerating limb. J. Embryol. exp. Morph. 50,
235-242.
MADEN, M. & MUSTAFA, K. (1982). Axial organisation of the
regenerating limb: asymmetrical behaviour following skin
transplantation. J. Embryol. exp. Morph. 70, 197-213.
MADEN, M. & WALLACE, H. (1976). How X-rays inhibit amphibian
limb regeneration. J. exp. Zool. 197, 105-114.
MUNEOKA, K., Fox, W. F. & BRYANT, S. V. (1986). Cellular
References
BIRK, D. E. & TRELSTAD, R. L. (1986). Extracellular compartments
in tendon morphogenesis: Collagen fibril, bundle and
macroaggregate formation. J. Cell Biol. 103, 231-240.
BRYANT, S. V. (1978). Pattern regulation and cell commitment in
amphibian limbs. In Clonal Basis of Development. Symp. Soc.
Devi Biol. 36, 63-82.
BRYANT, S. V., FRENCH, V. & BRYANT, P. J. (1981). Distal
regeneration and symmetry. Science 212, 993-1002.
CARLSON, B. M. (1974). Morphogenetic Interactions between
rotated skin cuffs and underlying stump tissues in regenerating
axolotl forelimbs. Devi Biol. 39, 263-285.
CARLSON, B. M. (1975). The effects of rotation and positional
change of stump tissues upon morphogenesis of the regenerating
axolotl limb. Devi Biol. 47, 269-291.
CHAPLIN, D. M. & GREENLEE, T. K. (1975). The development of
human digital tendons. J. Anal. 120, 253-274.
CHEVALLIER, A. & KIENY, M. (1982). On the role of the connective
tissue in the patterning of the chick limb musculature. Wilhelm
Roux' Arch, devl Biol. 191, 277-280.
CHEVALLIER, A., KIENY, M. & MAUGER, A. (1977). Limb-somite
relationship: origin of the limb musculature. J. Embryol. exp.
Morph. 41, 245-253.
DUNIS, O. A. & NAMENWORTH, M. (1977). The role of grafted skin
in the regeneration of X-irradiated axolotl limbs. Devi Biol. 56,
97-109.
FRANCIS, E. T. B. (1934). The Anatomy of the Salamander. Oxford:
Clarendon Press.
contribution from dermis and cartilage to the regenerating limb
blastema in axolotls. Devi Biol. 116, 256-260.
ROLLMAN-DINSMORE, C. & BRYANT, S. V. (1984). The distribution
of marked dermal cells from small localised implants in limb
regenerates. Devi Biol. 106, 275-281.
SLACK, J. M. W. (1980). Morphogenetic properties of the skin in
axolotl limb regeneration. J. Embryol. exp. Morph. 58, 265-288.
SLACK, J. M. W. (1983). Positional information in the forelimb of
the axolotl: properties of the posterior skin. J. Embryol. exp.
Morph. 73, 233-247.
SNELL, R. S. (1986). Clinical Anatomy for Medical Students. Third
Edition Little Brown. Boston.
STOPAK, D. & HARRIS, A. K. (1982). Connective tissue
morphogenesis by fibroblast traction. 1. Tissue culture
observations. Devi Biol. 90, 383-398.
STOPAK, D., WESSELLS, N. K. & HARRIS, A. K. (1985).
Morphogenetic rearrangement of injected collagen in developing
chicken limb buds. Proc. natn. Acad. Sci. U.S.A. 82, 2804-2808.
TANK, P. W. (1979). Positional information in the forelimb of the
axolotl; Experiments with double-half tissues. Devi Biol. 73,
11-24.
TANK, P. W. (1981). The ability of localised implants of whole or
minced dermis to disrupt pattern formation in the regenerating
forelimb of the axolotl, Ambystoma mexicanum. Am. J. Anat.
162, 315-326.
TANK, P. W. & HOLDER, N. (1979). The distribution of cells in the
upper forelimb of the axolotl, Ambystoma mexicanum. J. exp.
Zool. 209, 435-442.
GRIM, M. & CARLSON, B. M. (1974). A comparison of
morphogenesis of muscles of the forearm and hand during
(Accepted 16 December 1988)