/. Embryol. exp. Morph. 77, 273-295 (1983)
273
Printed in Great Britain © The Company of Biologists Limited 1983
The effect of vitamin A on the regenerating axolotl
limb
By M. MADEN 1
From the National Institute for Medical Research, London
SUMMARY
These experiments describe further investigations into the effects of vitamin A on
regenerating limbs. The effects of different retinoids, the time of administration, concentration of vitamin A and histological, autoradiographic and histochemical studies are reported.
The most obvious result of vitamin A treatment is to cause proximal elements to regenerate
from distal amputation levels, that is to cause serial reduplication of pattern in the proximodistal axis. Retinoic acid was the most potent of the analogues tested and longer times of administration or higher concentrations cause a greater amount of serial reduplication. Various
tissue changes have been found which include the inhibition of cell division, loss of cartilage
metachromasia, changes in the mucous-secreting properties of the epidermis and an increased
packing in the blastemal cells. The significance of these cellular effects in relation to the
pattern-formation changes is discussed.
INTRODUCTION
Recent experiments on the effects of vitamin A on developing and regenerating limbs have presented us with the possibility of advancing our understanding
of the molecular basis of pattern formation. Administration of vitamin A seems
to change the positional information of the cells of the limb. In the regenerating
axolotl limb (Maden, 1982, 1983a), instead of replacing just those elements
removed by amputation, vitamin A causes extra elements to be produced in the
form of serial repetitions. As the concentration of vitamin A and/or the time of
administration is increased, the degree of serial reduplication increases such that
a complete limb including part of the girdle can be regenerated from a distal
amputation plane.
In the developing chick limb, instead of affecting the proximodistal axis,
vitamin A causes mirror imaging in the anteroposterior axis (Tickle, Alberts,
Wolpert & Lee, 1982; Summerbell & Harvey, 1983). This is a different type of
pattern abnormality to a serially repeated structure, but still must involve a
respecification of positional information in the cells of the developing limb. A
third type of result is produced by the regenerating hindlimbs of Rana tadpoles.
Here, serial reduplications in the proximodistal axis are produced by increasing
1
Author's address: Division of Developmental Biology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
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M. MADEN
concentration and/or times or vitamin A administration, as in axolotls, but in
addition mirror-imaged limbs in the anteroposterior axis appear too (Maden,
1983ft).
In order to decide upon the most profitable biochemical approach to understanding these vitamin A effects, further work must be performed on these
systems to characterize their responses more fully. The experiments described
below were conducted on the regenerating limbs of axolotls with this in mind.
They explore in greater detail concentration and time effects with different
retinoids and analyse the cellular effects of vitamin A with histological,
autoradiographic and histochemical studies.
MATERIALS AND METHODS
All experiments were performed on young axolotl larvae, Ambystoma
mexicanum of sizes varying between 50—100 mm. Although different-sized
animals were used between experimental series, within any one series uniformly
sized animals were specially selected ( ± 5 mm snout to tail length).
The details of individual experimental designs are given in the Results as each
series is described. General techniques only will be considered here. Solutions
of all-trans retinol palmitate (type VII, Water dispersible, Sigma) were made at
varying concentrations by tipping the solid into tap water which then formed a
white, cloudy solution. Solutions of all-trans retinoic acid (Type XX, Sigma) did
not involve an intermediary solvent, instead the solution was prepared by
sonicating the crystals in tap water. This forms a clear, yellow solution. Solutions
of retinol (vitamin A alcohol) (Type X, all-trans) were prepared by dissolving the
required amount firstly in ethanol and then in dimethyl sulphoxide in order to
obtain an aqueous solution. Solutions of retinol acetate (Type 1, all-trans,
Sigma) were prepared by dissolving in water as in the case of retinol palmitate.
For the histological study animals had their forelimbs amputated through the
radius and ulna. Half served as controls and half were placed in a 300mg/l
solution of retinol palmitate for 15 days. Every other day from 0-30 days the
forelimbs of one animal (two samples) from each series were fixed in neutral
formalin, embedded in wax, sectioned at lO^um and stained with haematoxylin
and eosin.
For the autoradiographic study 16 animals had their forelimbs amputated
through the humerus, half placed in 300mg/l retinol palmitate for the duration
of the experiment and half in water. One animal from each series was used as a
sample (two limbs) every other day for 0-14 days. At the same time on each
sample day the animals to be sacrificed were injected with 10/il [methyl3
H]thymidine, 103/iCi//ig (Amersham). A labelling time of 4h was permitted
before the limbs were fixed in 3:1 alcohol: acetic acid. Samples were embedded
in wax, sectioned at 10 /im and stained with Feulgen. Slides were then prepared
for autoradiography by dipping in Ilford K2 emulsion. They were exposed in the
Vitamin A and regeneration
275
dark for 5 weeks, developed in Kodak D19 for 8min, fixed for 8min and then
counterstained with light green and mounted. To determine labelling and mitotic
indices per thousand cells, ten sections from each sample were used and 300*500
cells counted on each section. Therefore each data point on the graph is from
6-10000 cells (two samples at each point).
For the histochemical studies 28 animals were used. Half of them were
controls, half had their forelimbs amputated through the radius and ulna and
were placed in 30mg/l (0-1 mM) retinoic acid. Every other day for 14 days one
experimental and one control animal had both regenerates fixed in neutral formalin for Alcian blue staining. At the same time the limbs of another experimental and control animal were fixed in Susa for van Geisen, Mallory trichrome
and toluidine blue staining. Limbs were sectioned at 10 /im. Alcian blue staining
was performed both at pH 1 and pH2-5.
RESULTS
Scoring system
For the presentation of subsequent data a scoring system was devised, called
the degree of repetition. This is based upon the progressive effects of vitamin A
upon the proximodistal axis (Maden, 1982) and is a measure of the degree to
which the regeneration level had been shifted proximally. The score varies from
0 to 5. A normal regenerate (Fig. 1A) scores 0, a regenerate with extra carpals
(Fig. IB) scores 1, with a part extra radius and ulna (Fig. 1C scores 2, a complete
extra radius and ulna (Fig. ID) scores 3, a part extra humerus (Fig. IE) scores
4 and a complete limb (Fig. IF) scores 5. Where hindlimbs are also used the
scoring system is the same. Thus the higher the average degree of repetition for
any sample, the more proximal the level from which regeneration commenced
in those limbs.
Series 1. The effect of different retinoids
This data has already been presented in tabular form (Maden, 1982) but for
comparative purposes it is reproduced here in the form of a histogram recording
the average degree of repetition (Fig. 2). Each column is the average of between
15-20 samples regenerating in a 0-12mM solution for 15 days after amputation
through the radius and ulna. It is clear that retinoic acid is the most potent,
closely followed by retinol, then there is a big drop in potency to retinol palmitate
and retinol acetate. Retinoic acid has about x8 the potency of retinol palmitate.
Series 2. The effect of concentration
Two comparable concentrations of retinol palmitate have been tested for their
effect, 0-24 mM (120mg/l) and 0-6 mM (300mg/l) each administered for 10 or
15 days after amputation through the radius and ulna. These data are also a
276
M. MADEN
reformulation of that presented before (Maden, 1982) and reveals that increasing the concentration increases the degree of serial repetition (Fig. 3).
2^3
u
B
D
111
Vitamin A and regeneration
4-
o
2 3-
&
Q
2
'
Ra
Rp
RA
Fig. 2. Average degree of repetition (reduplication) of limbs after amputation,
through the radius and ulna and immersion in a 0-12 mM solution of four different
retinoids for 15 days. Ra = retinol acetate, Rp = retinol palmitate, R = retinol,
RA = retinoic acid. Each column represents the average of 15-20 cases. See text and
Fig. 1 for details of degree of repetition and scoring.
Series 3. Increasing time of administration
This effect has been investigated both with retinol palmitate and retinoic acid.
All animals had their limbs amputated either through the radius and ulna or tibia
and fibula. For the retinol palmitate experiment they were immersed in a 0-6 mM
solution for 0-20 days at 2-day intervals. Each sample consisted of two or three
animals (four to six limbs). For the retinoic acid experiment the same amputation
level but a lower concentration of 0-05 mM was used since this analogue is more
potent (Fig. 2). In this case samples of two animals were removed from the
solution every day.
Fig. 4 records the results. In retinol palmitate (Fig. 4A) there was a progressive
increase in the degree of serial repetition with time of administration, reaching
Fig. 1. Increasing serial reduplication in the proximodistal axis after amputation
through the radius and ulna and treatment with retinol palmitate. Broken lines mark
the amputation plane, h = humerus, r = radius, u = ulna, c = carpals, 1234 = digits.
A, Control which replaced the distal ends of the radius and ulna, eight carpals and
four digits. Mag. x 18. B, Regenerate with three extra carpal-like elements (arrows).
Mag. xl5. C, Regenerate with a radius and ulna which are too long. Mag. xl5. D,
Regenerate with an extra radius and ulna in tandem. Mag. xl3. E, Regenerate with
the distal end of the humerus produced at the amputation plane. Mag. xl2. F, A
complete limb has regenerated from the amputation plane. Mag. xlO.
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M. MADEN
a maximum at 12 days. Both forelimbs and hindlimbs behaved in the same
fashion. The occasional dips in the data lines (at days 8 and 16) are caused
partially by variation in the degree of serial reduplication, but mostly by the
death of one of the two samples. The same applies to Fig. 4B. Thus animals need
to be in this solution for approximately two weeks before complete limbs can be
induced, to regenerate from the distal amputation plane. If the time of administration is too long, however, (> 18 days) then a permanent inhibition of
regeneration occurs (Fig. 5), hence the graph falls off rapidly.
In retinoic acid, by contrast, the effect was much more immediate (Fig. 4B).
Maximal degrees of serial repetition could be caused by only 4 days immersion
in this solution. Again, both forelimbs and hindlimbs behaved similarly. Increasing times still produced maximal effects until day 10 when there was a sudden and
permanent inhibition of regeneration as described for retinol palmitate (Fig. 5).
Thus retinoic acid has a much more immediate effect on pattern formation, a
phenomenon which is considered further in the Discussion.
Series 4. Delayed administration
This experiment was undertaken to further define the period when vitamin A
exerts its effect on pattern formation. Increasing periods of time from amputation through the radius and ulna (0-20 days) were allowed to elapse before
placing animals in vitamin A.
4-
•S
3 •
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<L>
Q
1-
120
300
10 days
120
300
15 days
Fig. 3. Average degree of repetition after amputation through the radius and ulna
and immersion in two concentrations of retinol palmitate for 10 or 15 days.
120= 120mg/l, 300 = 300mg/l retinol palmitate. Each column is the average of
8-10 cases.
Vitamin A and regeneration
5-1
279
Retinol palmitate
432-
1-
1-
20
Days of exposure
Fig. 4. Average degree of repetition of limbs amputated through the radius and ulna
or tibia and fibula and placed in 0-6mM-retinol palmitate (A) or 0-05mM-retinoic
acid (B) for increasing lengths of time after amputation. Solid line,
circles = forelimbs; broken line, crosses = hindlimbs.
Retinol palmitate and retinoic acid effects were again compared. A 0-6mMretinol palmitate solution was used with delays of 0-20 days in 2-day intervals
before immersing the animals (two in each sample) for 15 days. A 0-05 mMretinoic acid solution was used with delays of 0-12 days in daily intervals before
immersing the animals (two in each sample) for 6 days. These administration
times (15 days and 6 days) were chosen from previous data (Fig. 4) for maximal
effectiveness.
In retinol palmitate (Fig. 6A) maximum effects were still produced if limbs
were allowed to regenerate for up to 8 days before administration of vitamin A.
After that the effectiveness declined because regeneration had progressed too
far, and only relatively minor disturbances such as digit loss occurred (Fig. 7).
In retinoic acid a similar picture emerged (Fig. 6B). Good reduplications arose
280
M. MADEN
I
I
_
Fig. 5. Limb treated with 300mg/l retinol palmitate for 20 days after amputation
through the radius and ulna (broken line). Regeneration has been inhibited although
some growth of the ends of the radius and ulna has occurred. Mag. x20.
after delays of up to 8 days, but later than that minor disturbances were seen (Fig.
7). This similarity between the two retinoids is in contrast to the data of Series
3 and reveals that vitamin A must be present during the first week of regeneration
for maximal pattern reduplications to occur.
What morphological period of regeneration does this correspond to? A parallel study of normal regeneration stages revealed that by day 8 limbs had reached
the late-cone stage (Tank, Carlson & Connelly, 1976). By day 10 they had
reached the palette stage when differentiation of cartilage had begun internally
and the outline of two digital primordia could be seen externally. Therefore
vitamin A needs to be administered during the early phases of regeneration,
during dedifferentiation, establishment of the blastema and blastemal cell
division for the effects on pattern formation to be manifest.
Series 5. Histology of vitamin A effects
What effect is vitamin A having upon the cells of the regenerating limb? One
obvious difference between control and limbs treated with vitamin A was that
there was a concentration-dependent inhibition of regeneration for the duration
of the treatment. This is shown in Fig. 8 with camera-lucida drawings of the
Vitamin A and regeneration
281
external appearance of regenerating limbs. Controls (top row) have virtually
completed regeneration by day 18, whereas experimental limbs in 0-6 mM-retinol
palmitate for 15 days have not regenerated anything by day 15 (bottom row).
Once they have been removed from Vitamin A, regeneration commenced and
progressed normally (except for the change in proximodistal level).
Histologically, there were no differences between control and vitamin A limbs
during the early phases of regeneration. Wound healing occurred normally within
24 h after amputation and an apical cap of normal proportions and consistency
developed (Fig. 9A and E). Dedifferentiation of the internal tissues commenced
(Fig. 9A, B, E, F) and there was no obviously excessive dedifferentiation of
cartilage in the presence of vitamin A as might be expected from reports on other
systems (see Discussion). An early blastema was thus established.
Retinol palmitate
A
Days before exposure
Fig. 6. Average degree of reduplication of limbs amputated through the radius and
ulna and allowed to regenerate for increasing periods of time before being placed in
0-6 mM-retinol palmitate (A) or 0-05 mM-retinoic acid (B). Note the similarity in this
experiment compared to the differences between the two retinoids in Fig. 4.
282
M. MADEN
B
Fig. 7. Limbs treated with (A) 0-05 mM-retinoic acid 12 days after amputation for 6
days or (B) 0-6 mM-retinol palmitate 10 days after amputation for 15 days. Amputation was through the radius and ulna (broken lines). If the time between amputation
and administration of vitamin A is too long, then only digital abnormalities appear
such as missing or fused phalanges (A) or missing digits (B). Mag. A, xl5;B, X12.
As blastemal cells in control limbs now commenced rapid cell division and the
blastema elongated the first signs of vitamin A effects appeared (Fig. 9C).
Blastemas on experimental limbs did not grow, instead the dedifferentiated cells
remained stable at the tip of the limb, often clumping together in a tight ball (Fig.
9G). As control limbs rapidly progressed through blastemal development and
commenced redifferentiation, experimental limbs remained at this stable, early
blastema stage with the clump of cells becoming more prominent (Fig. 9H). The
apical cap remained stable too.
After removal from vitamin A, however, development of the blastema recommenced but the direction of outgrowth was often eccentric relative to the longitudinal axis of the limb and the position of the apical cap (Fig. 10A). Growth
continued and redifferentiation occurred as normal although the cartilage elements which redifferentiated were not the radius and ulna as in control limbs, but
Vitamin A and regeneration
283
a single, central element, the humerus (Fig. IOC). As mentioned above and is
apparent from the whole mount in Fig. IF, the longitudinal axis of the new
humerus was at an angle to that of the radius and ulna.
Series 6. Autoradiography
The reason for the inhibition of development in vitamin A was apparent from
counts of labelling indices (Fig. 11A) and mitotic indices (Fig. 11B).
The labelling indices of normal and experimental limbs did not greatly differ
for the first week after amputation. Both reached a reasonably high labelling
level of about 40 %. This limited amount of cell cycling is responsible for the
generation of the early blastema in both control and experimental limbs. But
during the second week when control limbs began to increase further their
cycling rate, experimental limbs decreased dramatically in labelling indices,
falling back to preamputation levels by the end of the second week.
The same principle was apparent with mitotic indices (Fig. 11B). There was
some increase in mitosis over basal levels in experimental limbs, but during the
second week after amputation when controls began rapid cell division, limbs
treated with vitamin A declined to preamputation levels of mitosis.
Epidermal labelling and mitotic indices were also determined and the same
day 10
day 15
day 18
day 12
day 22
day 18
day 24
day 28
day 32
Fig. 8. Camera-lucida drawings of control (top row) and experimental (bottom row)
regenerates at various times after amputation through the radius and ulna. Experimental limbs were placed in 0-6 mM-retinol palmitate for 15 days, then removed into
pure water, whereupon regeneration commenced. Compare with histological sections in Figs 9 and 10.
284
M. MADEN
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Vitamin A and regeneration
285
Fig. 10. Haematoxylin- and eosin-stained sections of vitamin-A-treated limbs. A, 18
days after amputation. Having been removed from vitamin A on day 15, dedifferen*
tiated cells now begin to divide and a recognizable blastema develops. It is, however,
eccentrically placed and not under the apical cap as would be expected. B, 20 days
after amputation. The blastema continues to develop. C, 22 days after amputation,
Cartilage redifferentiation commences with the formation of a humerus (h), not a
radius and ulna as in controls. Mag. x65.
patterns were observed (data not shown), namely during the second week when
control levels greatly increased vitamin A levels began to decrease. As far as the
apical cap was concerned no labelled cells at all were seen either in control or
experimental caps. This aspect of regeneration is therefore unaffected by vitamin
A (Hay & Fischman, 1961; Maden, 1975).
Series 7. Histochemistry
Aldan blue staining: In normal control limbs Alcian blue at pH2-5 stained the
thin mucous layer on the external surface of the epidermis, the dermal matrix,
Fig. 9. Haematoxylin- and eosin-stained sections of control (A-D) and vitamin-Atreated (E-H) limbs after amputation through the radius and ulna. A, Control limb
14 days after amputation showing an apical cap and dedifferentiation of the radius
and ulna. Mag. x70. B, Control limb 6 days after amputation showing dedifferentiation of muscle. Mag. xl50. C, Control limb 10 days after amputation showing a
palette-stage blastema. Mag. x62. D, Control limb 12 days after amputation showing early cartilage condensations of the wrist and digit elements. Mag. x55. E,
Vitamin A limb 4 days after amputation showing dedifferentiation of cartilage and
an apical cap. Mag. x75. F, Vitamin A limb 6 days after amputation showing dedifferentiation of muscle. Mag. X160. G, Vitamin A limb 12 days after amputation
showing the inhibition of blastemal development and the clumping of blastemal cells
into an eccentric ball (b). Note the prominent apical cap remains. Mag. x70. H,
Vitamin A limb 15 days after amputation showing the clump of blastemal cells
becoming more prominent and the apical cap still present. By now control limbs had
essentially completed regeneration. Mag. x70.
EMB77
286
M. MADEN
connective tissue matrix, periochondrium and cartilage matrix (Fig. 12A, D). At
pH 1 the staining of the dermis and connective tissue was virtually non-existent,
but the external mucous layer, periochondrium and cartilage still stained
heavily. Over the 14-day period of regeneration, this picture did not change. The
epidermis over the regenerate remained unstained, except for the mucous layer
and as the basement membrane redifferentiated beneath, it stained too. The
extracellular matrix of the blastema also took up stain, but not the blastemal cells
themselves.
After vitamin A treatment, no significant changes in the staining of the extracellular matrix could be detected. There was no development of the blastema
due to the inhibition by vitamin A and it remained at the early bud stage of
regeneration. But an obvious increase or decrease in the Alcian-blue-stainable
material surrounding the cells was not observed.
However, two effects of vitamin A on the other tissues of the limb were noted.
Firstly, there was a marked change in the state of the epidermis. In control limbs
only the external mucous layer of the epidermis stained (Fig. 12A), but after 8
days of vitamin A treatment Alcian-blue-stainable material appeared in patches
in the intercellular spaces of the outer layer of epidermal cells (Fig. 12B). The
presence of this material gradually spread deeper into the epidermis such that by
day 12 it was present within cells adjacent to the basal layer (Fig. 12C). The
stimulation by retinoids of mucopolysaccharide synthesis in epidermal cells is a
60-i
40-
20
•tf
2
4
6
8
Days after amputation
10
12
14
Vitamin A and regeneration
287
30 - I
Days after amputation
Fig. 11. Graphs of percent-labelling indices (A) and mitotic indices per thousand
cells (B) of control (solid lines, crosses) and vitamin-A-treated (broken lines, circles)
limbs.
well-known phenomenon (see Discussion).
The second effect of vitamin A was on the cartilage of the limb where a loss
of Aclian blue staining was detected. The control cartilage stained deeply with
Alcian blue (Fig. 12D) whereas after 6 days of vitamin A cartilage staining
became patchy (Fig. 12E). By day 12 (Fig. 12F) the cartilage stained only very
lightly. This loss of metachromasia by cartilage was subsequently confirmed with
toluidine blue staining and, again, is a well-known effect of retinoids (see
Discussion).
Connective tissue staining: van Giesen's and Masson's trichrome stains were
employed to investigate possible changes in the extracellular matrix induced by
vitamin A. No significant alterations were detected with either of these stains
despite the fact that limbs treated with vitamin A did not develop over the 14day period of investigation. They simply remained in a juvenile stage of
regeneration.
288
M. MADEN
Fig. 12. Alcian-blue staining at pH2-5. A, Control limb showing the epidermis (e),
dermis (d) and muscle (m) underneath. Only the outer mucous layer of the
epidermis (arrow) and thefibresof the dermis take up stain. Mag. X125. B, Section
through the epidermis after 8 days of vitamin A treatment. Alcian-blue-staining
material is beginning to appear between the outer layers of cells (arrows). Mag.
xl90. C, The epidermis after 12 days of vitamin A treatment showing the
appearance of Aldan blue material within the cells throughout the epidermis
(arrows). Mag. xl90. D, Control limb showing heavy staining of the cartilage.
Mag. xl50. E, Cartilage after 6 days of vitamin A treatment showing patchy
staining. Mag. x 150. F, Cartilage after 12 days of vitamin A treatment showing
the virtual absence of staining. Mag. xl50.
Vitamin A and regeneration
289
DISCUSSION
It has been demonstrated (Maden, 1982) that the effect of vitamin A on
regenerating axolotl limbs is to cause serial reduplications specifically in the
proximodistal axis. Instead of replacing those elements removed by amputation,
regeneration commences from a more proximal level after vitamin A administration such that a complete limb can be regenerated from the lower arm. The
experiments described here were performed with the intention of exploring this
phenomenon in greater detail. The points that emerged will be discussed both
for their relevance to the analysis of pattern formation and their relation to the
effects of vitamin A on other, completely different systems.
The effect of different retinoids: Four retinoids were tested and the sequence of
efficacy was found to be retinoic acid > retinol > > retinol palmitate > retinol
acetate. That is, at the particular concentration tested, retinol acid caused the
level at which regeneration commenced to be more proximal than the other
analogues. This sequence of potency is typical of a wide variety of other
biochemical studies on the effects of retinoids. For example Lotan, Neumann &
Lotan (1980) have studied the growth inhibitory properties of 27 different
retinoids on cultured melanoma cells and obtained the sequence retinoic
acid > retinol > > retinol acetate > retinol palmitate. In summarizing twelve
studies (their table 2) it is clear that great similarities in the relative efficacies of
retinoids exist despite highly diverse activities. These activities involve inhibition
of normal and neoplastic growth, reversal of keratinization, breakdown of cartilage matrix, increase in RNA synthesis, enhancement of cell adhesion and
stimulation of differentiation. In all cases retinoic acid is the most effective,
usually followed by retinol, with the acetate and palmitate least effective.
Two points can therefore be made in relation to the results on limb regeneration reported here. Firstly, the similarity in relative activities suggests that a
common biochemical mechanism underlies the diverse effects of retinoids on
growth, differentiation, cell adhesion and we can now add pattern formation to
that list. Secondly, since the in vivo efficacy of the retinoids used here is the same
as that found in the in vitro studies discussed above, the cause of the variation
in potency is not due to systemic effects such as differences in uptake from the
water or differences in pharmacodynamics within the body. Therefore the
relative potencies most likely reflect specific effects on the cells of the limb
blastema, for example the ability of the retinoids to bind to cellular retinoic-acidbinding protein (Lotan et al. 1980).
Concentration and time effects: It was demonstrated that increasing the concentration of retinol palmitate caused increased degrees of serial reduplication,
that is, caused the level at which regeneration commenced to become more
proximal. The same was true when the time of administration was increased.
290
M. MADEN
These effects can be described in terms of a monotonic positional information
gradient (Wolpert, 1969) along the proximodistal axis of the limb. If the concentration of a morphogen determines the level-specific properties of limb cells
and vitamin A stimulates the synthesis of this substance, then the more vitamin
A there is available or the longer it is present for, the more proximal the cells at
the level of the cut will become because their morphogen concentration will have
been increased (Maden, 1983c).
The experiments in which an increasing period of time was allowed to elapse
before placing regenerates in vitamin A combined with those just discussed
revealed that it is the early phases of regeneration, during the first 8 days, which
are susceptible to vitamin-A-induced change. These include dedifferentiation
and the establishment of blastema, precisely those phases when we would
expect pattern-forming mechanisms to be operating. If regeneration has
progressed too far past these stages then serial reduplications can no longer
be induced.
It is interesting to compare the time effects of retinol palmitate and retinoic
acid. Retinoic acid, although at a x l 2 weaker concentration produced much
more immediate effects. It only needed to be present for 4 days before a nearmaximum reduplication was produced compared with 12 days for retinol palmitate. The reason for this difference between the two retinoids could be due to
their different pharmacodynamics within the body. Retinol palmitate is stored
in the liver (Mahadevan & Ganguly, 1961) and would therefore not be available
to limb cells in high concentrations in the blood until the liver stores (or plasma
retinol-binding protein levels - Smith & Goodman, 1976) are full. On the other
hand, retinoic acid is not deposited in the tissues of the body (Zachman, Dunagin
& Olson, 1966; Fidge, Shiraton, Ganguly & Goodman, 1968) and would
therefore be available more immediately to the cells of the regenerating limb.
However, I have argued above that since the in vitro relative potencies of those
retinoids are the same as their in vivo properties, considerations such as liver
storage are irrelevant. Perhaps then, the more immediate effect of retinoic acid
in the time series experiments is simply due to a concentration phenomenon and
even smaller doses would produce a more gradual increase in serial reduplication
as found for retinol palmitate.
Effects on cell division: External observations of limbs regenerating in concentrations of vitamin A which induce maximal serial reduplications, revealed
that no development took place during the period of vitamin A administration.
Only when the animals were removed from retinoid solutions did regeneration
commence. This paradoxical nature of vitamin A in inhibiting regeneration and
yet causing extra pattern to form was also observed if animals were left in vitamin
A for too long. After 18 days in retinol palmitate or 10 days in retinoic acid
regeneration was permanently inhibited. The reason for this was revealed in an
autoradiographic study. In vitamin A, DNA synthesis and mitosis are severely
Vitamin A and regeneration
291
inhibited, although a significant stimulation above normal levels in the first few
days after amputation is not prevented.
This inhibition of cell division is a well-recognized effect of retinoids on a wide
variety of cell types (Lotan, 1980). Although most research on growth inhibition
has been performed on cultured cells rather than in vivo systems, the antitumour
properties of retinoids (Mayer, Bollag, Hanni & Ruegg, 1978) may well result
from a direct inhibition of tumour cell growth (Schroder & Black, 1980). Again,
the similarity of effects between the results related here and those on other
system may be significant.
Effects on cartilage: Despite the fact that cell division is severely reduced in the
presence of vitamin A, other early events of regeneration were unaffected. Both
wound healing and the dedifferentiation of muscle, cartilage and other internal
tissues occurred as normal. From other studies on the effects of retinoids on
cartilage it would be reasonable to expect that excessive dedifferentiation of
cartilage might occur. It has been shown that, in culture, retinoids cause the
breakdown of cartilage (Fell & Mellanby, 1952; Fell & Dingle, 1963) resulting
in the loss of metachromatic staining and the release of proteoglycans into the
medium (Dingle, Fell & Goodman, 1972; Goodman, Smith, Hembry & Dingle,
1974). As in the case of the inhibition of cell division, although these studies were
performed in vivo, a similar loss of metachromatic staining was demonstrated
here in a histochemical study with Alcian blue and toluidine blue. Nevertheless
an abnormal amount of cartilage dedifferentiation was not detected. Again,
retinoid effects on axolotl limbs conform with well-established data on other
systems.
Extracellular matrix and cell surface effects: Once dedifferentiated cells were
liberated at the tip of the amputated limb the inhibition of cell division discussed
above was manifest. However, instead of simply remaining stationary as the
dedifferentiated cells in denervated (Butler & Schotte, 1941) or irradiated (Butler, 1933; Maden, 1975) limbs, which similarly experience an inhibition of cell
division, after vitamin A treatment the cells clumped together into a tight ball.
This effect is presumably mediated either via changes in the extracellular matrix
of the early blastema or via changes in the surface properties of dedifferentiated
cells. Histochemical studies with Alcian blue, toluidine blue, van Giesen or
trichrome stains, did not produce any positive results regarding extracellular
matrix changes. This suggests that alterations in cell surface properties rather
than in the extracellular matrix are more likely the cause of this abnormal cell
behaviour.
Once again both of these retinoid effects have previously been described with
in vitro studies. In a study particularly relevant to the effects described here,
Lewis, Pratt, Pennypacker & Hassell (1978) found that retinoic acid inhibited
chondroflenesis in cultures of mouse limb-bud cells. Furthermore, relative to
292
M. MADEN
control cultures retinoid-treated cells were tightly packed together with little
surrounding extracellular space. There was a greater area of close contact between the plasma membranes of treated cells and more specialized junctions had
developed. This is precisely what was found here, although ultrastructural
studies were not performed to confirm the latter aspects. Changes in the extracellular matrix were not, apparently, the cause of the tight packing, rather
they suggested that specific changes in high-molecular-weight cell-surface
glycoproteins were responsible, perhaps mediated by retinoid stimulation of
glycosylation (Pennypacker, Lewis & Hassell, 1978). If the same applies to the
axolotl limb then the failure to find any gross changes in the extracellular matrix
is to be expected.
Epidermal effects: As discussed above, histochemical studies failed to reveal any
changes in the extracellular matrix of the early blastema caused by vitamin A.
However, one effect that was noted in Alcian-blue-stained sections was a significant alteration in the state of the epidermis. In the epidermis of the normal
limb, Alcian blue material is present only as a thin layer on the outer surface. But
after vitamin A treatment this material appears within the epidermis, right down
to the basal layer of cells. It is presumably a mucopolysaccharide whose synthesis
by epidermal cells is switched on by vitamin A.
Once again this is a well-known effect of vitamin A: the stimulation of
mucopolysaccharide production has been observed in histochemical and
electron microscopic studies using other animal systems (Bellows & Hardy,
1977; Yuspa & Hains, 1974; Fell & Mellanby, 1953; Sweeney & Hardy, 1976)
and has been shown to occur after topical application of retinoic acid in humans
(Elias & Williams, 1981; Orfanos et al. 1981). This effect is presumably a mild
form of mucous metaplasia which vitamin A induces in epithelia of many types
(Fell & Mellanby, 1953; Elias & Williams, 1981). It is interesting to note that this
phenomenon is associated with the widening of intercellular spaces and decreasing area of intercellular contact, exactly the opposite of the effect of vitamin A
on mesodermal cells (see earlier). Thus despite being administered to the same
animal system and at the same external concentration, vitamin A effects are
tissue specific.
CONCLUSION
Despite the wide variety of methods and cell types used to establish the effect
of retinoids on living tissues the majority of those effects were found to occur
here within the tissues of the regenerating axolotl limb. These were a typical
series of relative potencies of four retinoids, the inhibition of cell division, loss
of metachromatic staining in cartilage, increase in mesodermal cell packing and
the stimulation of mucous metaplasia in the epidermis. From the point of view
of furthering our understanding of pattern formation, which, if any, of these
Vitamin A and regeneration
293
cellular effects causes the serial reduplication of pattern elements in the
proximodistal axis and is thus responsible for controlling pattern formation,
remains to be determined. Some clues, nevertheless, are already to hand.
The production of serial reduplications is unlikely to be due to the inhibition
of cell division and the cell-cycle delay thus incurred because other methods of
reversibly inhibiting or slowing down cell division in regenerating limbs, such as
denervation (Singer, 1978) do not cause pattern abnormalities (Maden, 1983a).
If the breakdown of cartilage was responsible for pattern control, for example
by the local concentration of a proteoglycan-breakdown product which would
increase in the presence of vitamin A and therefore produce more pattern, then,
in the absence of cartilage, organized pattern formation should not occur. However, when cartilage is removed from the limb, regeneration takes place normally (Bischler, 1926; Thornton, 1938), thus denying any role for cartilage.
There is no specific evidence to rule out epidermal effects in the control of
pattern in limbs treated with vitamin A. Experiments are currently under way in
an attempt to answer this question by grafting epidermis or mesoderm between
treated and normal limbs. But it is generally accepted that epidermis plays no
role in pattern formation during regeneration (Carlson, 1975; Tank, 1977),
rather it is the mesoderm which is responsible.
We are therefore left (apart from other hitherto undiscovered effects) with
mesodermal cell surface and membrane glycoprotein synthesis effects as possible
candidates for the control of pattern. It is interesting that there has been a recent
trend towards the focusing of our attention on the cell surface and the part it plays
in pattern formation. Coincidentally, there has even been a theory proposed in
which pattern is determined by glycoprotein molecules on the cell surface (Slack,
1980). The validity of these speculations await to be tested, but at least we now
have a system in which such questions can be approached.
REFERENCES
C. G. & HARDY, M. H. (1977). Histochemical evidence of mucosubstances in the
metaplastic epidermis and hair follicles produced in vitro in the presence of excess Vitamin
A. Anat. Rec. 187, 257-272.
BISCHLER, V. (1926). L'Influence du squelette dans la r6g6n6ration et les potentialities des
divers territories du membre chez Triton cristatus. Rev. Suisse Zool. 33, 431-560.
BUTLER, E. G. (1933). The effects of X-radiation on the regeneration of the fore limb of
Amblystoma larvae. /. exp. Zool. 65, 271-316.
BUTLER, E. G. & SCHOTT£, O. E. (1941). Histological alterations in denervated nonregenerating limbs of Urodele larvae. /. exp. Zool. 88, 307-329.
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.
DINGLE, J. T., FELL, H. B. & GOODMAN, D. S. (1972). The effect of retinol and of retinolbinding protein on embryonic skeletal tissue in organ culture. J. Cell Sci. 11, 393-402.
ELIAS, P. M. & WILLIAMS, M. L. (1981). Retinoids, cancer and the skin. Arch. Dermatol. 117,
160-180.
FELL, H. B. & DINGLE, J. T. (1963). Studies on the mode of action of excess Vitamin A. 6.
Lysosomal protease and the degradation of cartilage matrix. Biochem. J. 87, 403-405.
BELLOWS,
294
M. MADEN
H. B. & MELLANBY, E. (1952). The effect of hypervitaminosis A on embryonic limbbones cultivated in vitro. J. Physiol. 116, 320-349.
FELL, H. B. & MELLANBY, E. (1953). Metaplasia produced in cultures of chick ectoderm by
high Vitamin A. /. Physiol. 119, 470-488.
FIDGE, N. H., SHIRATON, T., GANGULY, J. & GOODMAN, D. S. (1968). Pathways of absorption
of retinal and retinoic acid in the rat. J. Lipid Res. 9, 103-109.
GOODMAN, D. S., SMITH, J. E., HEMBRY, R. M. & DINGLE, J. T. (1974). Comparison of the
effects of Vitamin A and its analogs upon rabbit ear cartilage in organ culture and upon
growth of the Vitamin A-deficient rat. /. Lipid Res. 15, 406-414.
HAY, E. D. & FISCHMAN, D. A. (1961). Origin of the blastema in regenerating limbs of the
newt, Triturus viridescens. An autoradiographic study using tritiated thymidine to follow
cell proliferation and migration. Devi Biol. 3, 26-59.
LEWIS, C. A., PRATT, R. M., PENNYPACKER, J. P. & HASSELL, J. R. (1978). Inhibition of limb
chondrogenesis in vitro by Vitamin A: alterations in cell surface characteristics. Devi Biol.
64, 31-47.
LOTAN, R. (1980). Effects of Vitamin A and its analogues (retinoids) on normal and neoplastic
cells. Biochim. Biophys. Acta 605, 33-91.
LOTAN, R., NEUMANN, G. & LOTAN, D. (1980). Relationships among retinoid structure,
inhibition of growth and cellular retinoic acid-binding protein in cultured S91 melanoma
cells. Cancer Res. 4, 1097-1102.
MADEN, M. (1975). Transformation during amphibian development and regeneration. Ph.D.
thesis, University of Birmingham.
MADEN, M. (1982). Vitamin A and pattern formation in the regenerating limb. Nature 295,
672-675.
MADEN, M. (1983#). Vitamin A and the control of pattern in regenerating limbs. In "Limb
Development and Regeneration'. Part A, pp. 445-454. New York: Alan R. Liss Inc.
MADEN, M. (1983ft). The effects of Vitamin A on the developing limbs of Rana temporaria.
Devi Biol. (in press).
MADEN, M. (1983C). Retinoids as probes for investigating the molecular basis of positional
information. In "Primers in Developmental Biology". Vol. 1, Pattern Formation, (eds G.
M. Malacinski & S. V. Bryant). New York: Macmillan. (in press).
MAHADEVAN, S. & GANGULY, J. (1961). Further studies on the absorption of Vitamin A.
Biochem. J. 81, 53-58.
MAYER, H., BOLLAG, W., HANIN, R. & RUEGG, R. (1978). Retinoids, a new class of compounds with prophylactic and therapeutic activities in oncology and dermatology. Experientia 34, 1105-1119.
FELL,
ORFANOS, C. E., BRAUN-FALCO, O., FARBER, E. M., GRUPPER, C., POLANO, M. K. & SCHUPPLI,
R. (1981). "Retinoids: Advances in Basic Research and Therapy'. Berlin, Heidelberg:
Springer-Verlag.
PENNYPACKER, J. P., LEWIS, C. A. & HASSELL, J. R. (1978). Altered proteoglycan metabolism
in mouse limb mesenchyme cell cultures treated with Vitamin A. Arch. Biochem. Biophys.
186, 351-358.
SCHRODER, E. W. & BLACK, P. H. (1980). Retinoids: Tumor preventers or tumor enhancers?
/. natn. Cane. Inst. 65, 671-674.
SINGER, M. (1978). On the nature of the neurotrophic phenomenon in urodele limb regeneration. Am. Zool. 18, 829-841.
SLACK, J. M. W. (1980). A serial threshold theory of regeneration. /. theor. Biol. 82,105-140.
SMITH, F. R. & GOODMAN, D. S. (1976). Vitamin A transport in human Vitamin A toxicity.
New Eng. J. Med. 194, 805-808.
SUMMERBELL, D. & HARVEY, F. H. (1983). Vitamin A and the control of pattern in developing
limbs. In "Limb Development and Regeneration', Part A, pp. 109-118. New York: Alan R.
Liss Inc.
SWEENEY, P. R. & HARDY, M. H. (1976). Ciliated and secretory epidermis produced from
embryonic mammalian skin in organ culture by Vitamin A. Anat. Rec. 185, 93-100.
TANK, P. W. (1977). The timing of morphogenetic events in the regenerating forelimb of the
axolotl, Amblystoma mexicanum. Devi Biol. 57, 15-32.
Vitamin A and regeneration
295
P. W., CARLSON, B. M. & CONNELLY, J. G. (1976). A staging system for forelimb
regeneration in the axolotl, Ambystoma mexicanum. J. Morph. 150, 117-128.
THORNTON, C. S. (1938). The histogenesis of the regenerating fore limb of larval Amblystoma
after exarticulation of the humerus. /. Morph. 62, 219-242.
TICKLE, C , ALBERTS, B., WOLPERT, L. & LEE, J. (1982). Local application of retinoic acid to
the limb bond mimics the action of the polarizing region. Nature 296, 564-566.
WOLPERT, L. (1969). Positional information and the spatial pattern of cellular differentiation.
/. theor. Biol. 25, 1-47.
YUSPA, S. H. & HARRIS, C. C. (1974). Altered differentiation of mouse epidermal cells treated
with retinyl acetate in vivo. Expl Cell Res. 86, 95-105.
ZACHMAN, R. D., DUNAGIN, P. E. & OLSON, J. A. (1966). Formation and enterohepatic
circulation of metabolites of retinol and retinoic acid in bile duct-cannulated rats. /. Lipid
Res. 7, 3-9.
TANK,
{Accepted 2 June 1983)