J. Embryol. exp. Morph. 95,117-130 (1986)
117
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The pigmentary system of developing axolotls
IV. An analysis of the axanthic phenotype
S. K. F R O S T , L. G. EPP* AND S. J. ROBINSON
Center for Biomedical Research and Department of Physiology and Cell Biology,
Haworth Hall Addition, University of Kansas, Lawrence, KS 66045, USA
SUMMARY
The axanthic mutant in the Mexican axolotl (Ambystoma mexicanum) was analysed with
respect to the differentiation of pigment cells. Transmission electron micrographs revealed the
presence of melanophores and cells that are described as unpigmented xanthophores in axanthic
skin. Iridophores apparently failed to differentiate in axanthic axolotls (a pattern similar to that
observed in melanoid axolotls). Chromatographic analyses of skin extracts confirmed that there
are no pteridines (xanthophore pigments) in axanthic skin, suggesting that the axanthic gene
may affect pteridine biosynthesis at some point early in the biosynthetic pathway. Why
iridophores fail to differentiate in these animals is not known, but this, too, may be related to
an inability to synthesize pigments properly. Xanthophore and iridophore pigments both
presumably derive from purine precursors.
Finally, all axanthic animals were found to be infected by a virus. Electron microscopic results
demonstrated the presence of numerous macrophages in the dermis of the skin, occupying
positions typical of pigment cells. The virus was localized primarily in macrophages, but was also
observed in pigment cells. The virus is, as yet, uncharacterized but is thought to contribute to the
low survivability of axanthic adults.
INTRODUCTION
The axanthic mutation in axolotls was first described by Lyerla & Dalton in
1971. Axanthic is a simple Mendelian recessive trait. Axolotls homozygous for this
mutation appear normal except for lack of visible xanthophores and iridophores
(Lyerla & Dalton, 1971). Axanthic animals also lack the yellow pteridine pigments
that are characteristic of normal xanthophores.
Subsequent to the initial description of the axanthic phenotype, Dalton &
Hoerter (1974) examined purine synthesis in wild-type, melanoid and axanthic
axolotl skin in an effort to determine why iridophores fail to develop in either
melanoid or axanthic axolotls. They concluded only that the chromatographic
patterns of purines observed in these three types of animals were different;
however, they were unable to identify positively any of the purines. Thus, little
* Present address: Department of Biology, Mount Union College, Alliance, OH 44601, USA.
Key words: axolotl, Ambystoma mexicanum, pigmentary system, axanthic mutant, mutant,
iridophore, melanophore.
118
S. K. FROST, L . G. E P P AND S. J. ROBINSON
additional information was gained which might have contributed to further
defining either the melanoid or axanthic defects. Moreover, it seems unlikely that
more will be learned about purine pigment synthesis and iridophore differen­
tiation until the process is better understood in wild-type animals.
Dunson (1974) searched for structural evidence of either xanthophores or
iridophores in axanthic skin using transmission electron microscopy. Iridophores
were not observed anywhere in axanthic skin. Cells that were present in 'positions
occupied by xanthoblasts in wild type larvae...' (Dunson, 1974) were identified
as potential xanthoblasts in axanthic skin. These cells generally lacked distinc­
tive organelles, especially organelles resembling pterinosomes (characteristic of
xanthophores). Based on the apparent presence and characteristics of presumed
xanthoblasts, Dunson speculated that the axanthic mutation might be responsible
for either (1) an early block in xanthophore differentiation (prior to pteridine
biosynthesis or pterinosome formation), (2) a block in pterinosome formation
leading to failure of pteridines to accumulate or (3) a block in pteridine bio­
synthesis leading to a failure of pterinosomes to form.
The goal of this study was to define more carefully and completely the chemical
and structural features of skin in developing axanthic axolotls, and to determine
which, if any, of Dunson's (1974, see above) speculative explanations for the
axanthic defect might be valid. We also report two additional significant findings
regarding the axanthic phenotype. (1) Defective xanthophores are clearly present
in axanthic skin. They are fragile cells, identifiable on the basis of location and
morphological criteria, such as the presence of pterinosome-like organelles in the
cell processes. (2) Some cells in the dermis (and epidermis) of axanthic axolotls are
heavily contaminated with a virus. The virus particles are most often packaged in
lysosome-like vesicles within cells that appear to be macrophages. Occasionally, in
older axanthic animals, virus is also observed to be packaged within vesicles
(resembling pigment granules) in melanophores. Finally, we suggest that the
axanthic defect is most likely to be a defect associated with pteridine/purine
pigment biosynthesis.
MATERIALS AND METHODS
Animals
Axolotls homozygous for the axanthic gene (ax) were obtained from the Indiana University
Axolotl Colony, Bloomington, Indiana. Feeding, maintenance and the categorization of
axolotls into three arbitrary age classes (larva, juvenile, adult) are described in Frost et al.
(1984a).
Electron microscopy
Axolotl skin was prepared for transmission electron microscopy (TEM) as described
previously (Frost, Epp & Robinson, 1984tf). Thefixativeused was 2-5% glutaraldehyde in
0-1 M-cacodylate buffer, pH7-4, with postfixation in 2% osmium tetroxide in the same buffer,
and en bloc staining in 2% aqueous uranyl acetate.
Axanthic phenotype of axolotl
119
Pigment extraction
Axanthic axolotls, by definition are pteridine deficient (Lyerla & Dalton, 1971). To confirm
this, axolotl skin was extracted in 70% ethanol and analysed by thin-layer chromatography
(TLC) as described in detail by Frost & Bagnara (1978) and Frost et al. (1984a). TLC plates were
coated with a mixture of cellulose: silica gel G (1:1). The solvent used for pigment separations in
one dimension was n-propanol: 7 % ammonia (2:1, v/v). Pigments were identified by comparing
u.v.-fluorescent properties (colour) and chromatographic mobility with similar data from
commercially purchased standards (see Frost et al. 1984a for a list of the pteridine pigments that
have been identified in wild-type axolotls and for which we searched in axanthics).
RESULTS
Structural analysis of pigment cells
The only well-differentiated pigment cell types observed in axanthic skin are
melanophores (Fig. 1). In young larval axanthic axolotls, melanophores are
localized beneath the basement membrane of the epidermis, surrounded by a
loose collagen matrix (Fig. 1A,B). In older axanthics, the collagen layer is much
thicker and melanophores are often observed embedded within the collagen as
shown in Fig. IC. Morphologically, melanophores from axanthic skin are similar
to those described from wild-type skin (Frost et al. 1984a). 'Mature' melano­
phores (like the one shown in Fig. IC) contain primarily pigment granules
(melanosomes), occasional mitochondria, few premelanosomes and little else in
the way of cell organelles (Fig. IC). In young (larval and young juvenile) axanthics
melanophores are generally 'less differentiated' and contain a variety of cyto­
plasmic organelles including numerous melanosomes, premelanosomes, large and
small vesicles, extensive Golgi, some endoplasmic reticulum (ER), mitochondria,
occasional microtubules and intermediate filaments (see Figs 1A,B, 2A,B). In
addition to organelles that are characteristic of axolotl melanophores in general,
axanthic melanophores also have been observed to have virus-containing organ­
elles (Figs IC, 2), some of which are nearly indistinguishable from pigment
granules (melanosomes) (see the virus-containing particle (arrow) in Fig. IC).
Although clearly infected by a virus, the melanophores appear to be otherwise
healthy (Fig. 2).
Evidence of xanthophores in axanthic skin is rare, but when such cells are
encountered their morphology is distinctive (Fig. 3). The characteristics of the
cells shown in Fig. 3 and those'of the cell processes that are parts of xanthophores
(Fig. 4) include: numerous prepterinosome-like (large, empty) vesicles (the
prepigment organelles of xanthophores), a cytoplasm typical of pigment cells and
distinctly different from fibroblasts or blood cells (the only other cell types
commonly encountered in axolotl dermis; see also Frost et al. 1984a,b, 1986),
numerous small, electron-dense multivesicular bodies (MVBs), many small empty
vesicles, mitochondria, smooth and rough ER (much of which is swollen),
microtubules, intermediate filaments and occasional Golgi complexes that appear
to be underdeveloped compared to those observed in melanophores (compare
Golgi in Fig. 3B,C with that in Fig. 2A, for example). Axanthic melanophores also
120
S. K. F R O S T , L . G. E P P AND S. J. R O B I N S O N
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Fig. 1. Melanophores from axanthic axolotl skin. Nuclei (N), melanosomes (m),
collagen (c). Note the basement membrane (b) of the epidermis at the top of Fig. 1B,C.
C contains a membrane-bound hexagonal array of virus particles (arrow) that
resembles (at this magnification) a melanosome. Bar, l^m.
Axanthic phenotype of axolotl
121
do not contain electron-dense MVBs, they exhibit little evidence of ER (no
evidence of swollen ER) and have numerous melanosomes. (Compare Figs 1, 2
with Figs 3, 4.) Thus far, xanthophore cell bodies have only been observed to
occur beneath the collagen layer of the dermis, and not within the collagen layer as
is the normal case for melanophores in older axanthic animals and all pigment cells
in older wild-type axolotls (Frost etal. 1984A). The positional location of axanthic
Fig. 2. (A) Higher magnification of a melanophore from axanthic axolotl skin con­
taining a packaged array of virus particles (arrow). Although virus is present in this
cell, the normal 'cellular machinery' appears to be intact. Golgi (G), mitochondria (*),
nucleus (N) and melanosomes (ra). Also present are microtubules, intermediate
filaments and smooth and rough ER. Bar, 1 jum. (B) A melanophore embedded in the
collagen matrix. Bar, ljum.
122
S. K. F R O S T , L. G. E P P AND S. J.
ROBINSON
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Axanthic phenotype of axolotl
123
xanthophores is typical of pigment cells in larval skin, wherein pigment cells are
initially located beneath the dermis and subsequently migrate to just below the
basement membrane of the epidermis (Frost etal. 1984a). It is possible, based on
this observation, that axanthic xanthophores may be defective in their ability to
migrate to their proper location.
In addition to melanophores and occasional xanthophores, a specific blood cell
type is frequently encountered in the dermis of axanthic axolotl skin. Based on the
morphology and contents of this cell, we suggest that it is a macrophage (Fig. 5). In
axanthic skin macrophages are most often observed adjacent to or near melano­
phores (a position often occupied by xanthophores). Such cells contain numerous
empty vesicles (presumed lysosomes) and a variety of vesicles containing electrondense materials (Fig. 5A). Close inspection of the electron-dense vesicles reveals
that they are filled with virus particles close packed in hexagonal array (Fig. 5B,C).
Individual particles measure approximately 40 nm in diameter and are
morphologically identical to the virus particles observed to occur occasionally in
melanophores (Figs 1C, 2). Aside from the presence of numerous electron-dense
virus-containing particles, the macrophage shown in Fig. 5A is similar in general
features to the xanthophore shown in Fig. 3A. The macrophage, however, does
not possess extensive cell processes as is typical of pigment cells.
Pigment analyses
Pigment extraction and analyses confirmed the earlier findings of Lyerla &
Dalton (1971). There are no pteridine pigments detectable in axanthic skin at any
stage during development. Riboflavin (a yellow compound) is, however, present
in skin as shown on the thin-layer chromatogram in Fig. 6. Also, there appears to
be more riboflavin in skin from older axanthic axolotls compared to that from
younger animals.
DISCUSSION
From 1983 to 1985 we examined skin from 17 different axanthic axolotls. These
animals represent a variety of ages and were from a number of different spawnings
carried out during this two-year period. Every axanthic animal examined was
found to be virus infected, some more severely so than others.
During the course of the past two years we also tried repeatedly to rear axanthic
larvae to sexual maturity. From more than 50 axanthic larvae only one survived to
attain adult secondary sexual characteristics (a female), and this animal was never
bred successfully. The animal eventually died in apparently healthy condition and
Fig. 3. Xanthophore and xanthophore processes from axanthic axolotl skin. Devoid of
pteridines, these cells exhibit most of the characteristics of healthy pigment cells with
the notable exceptions of swollen ER (arrows), relatively underdeveloped Golgi (G),
and numerous electron-dense multivesicular bodies (*). Empty-appearing 'prepterinosomes' are common in the cytoplasmic processes of axanthic xanthophores
(Fig. 3C). Bar, lfim.
124
S. K. F R O S T , L. G. E P P AND S. J.
ROBINSON
Axanthic phenotype of axolotl
125
was subsequently found also to be virus infected. The Indiana University Axolotl
Colony has also had similar difficulties in rearing axanthic animals to sexual
maturity (F. Briggs, personal communication), and only recently have two homo­
zygous axanthic adults been successfully bred by colony personnel.
It seems likely that the virus found in axanthics is responsible for the re­
duced survivability of axanthic animals. The virus does not, however, appear
to be immediately lethal to these animals, because otherwise healthy-appearing
axanthics have been found to be virus-infected. During our survey of the
development of pigmentation in wild-type, melanoid, albino and axanthic axolotls
(Frost et al. 1984A,b, 1986, and the present study), we encountered only one
nonaxanthic, nonexperimental (see below) animal that was virus-infected (Frost &
Robinson, unpublished data). This animal, phenotypically wild-type, was possibly
ax/+, having arisen from a cross involving two ax/+ parents. Considering the
large number of animals examined during these studies (>100 of all phenotypes
over a 4-year period), it seems likely that the defect caused by the axanthic gene is
severe enough that viral expression and this mutant phenotype are coincidental.
More recently, we have undertaken experiments designed to alter the pigment
phenotype of wild-type axolotls by administering either allopurinol (Frost &
Bagnara, 1979) or guanosine to young larval axolotls (see below). In a significant
number of cases these drug-treated animals eventually developed virus infections
(especially those animals receiving guanosine) (Frost & Robinson, unpublished
data). Thus, it seems that all axolotls may carry the genetic information for virus
expression, but only ax/ax animals and those animals that have been 'stressed' by
drug treatment actually express the virus. Furthermore, it is intriguing that
guanosine (a precursor to both purine and pteridine pigment biosynthesis) and
allopurinol (an inhibitor of purine/pteridine synthesis; Spector, 1977) appear to
allow the virus to be expressed.
The significance of the virus (which remains unidentified) to the axanthic
phenotype is obscure at the present time. That it may, in fact, be linked, perhaps
indirectly, to the expression of the axanthic gene is plausible in view of the fact that
all axanthic animals express the virus. It is anticipated that future studies will be
designed (1) to isolate and identify the virus and (2) to determine what factors
control the expression of the virus.
With regard to axanthic xanthophores, the cytoplasmic contents of these cells
differ distinctly from other pigment cells, including other xanthophores (see
descriptions and figures of wild-type xanthophores: Frost et al. 1984a; melanoid
xanthophores: Frost et al. 19846; albino xanthophores: Frost et al. 1986),
iridophores (Frost et al. 1984a, 1986) and melanophores (e.g. the present study)
that we have observed. Based on these observations, it appears that pterinosomes
do form in axanthic xanthophores, although no pigment is synthesized in such
cells. Furthermore, axanthics appear to have all of the standard 'cellular
Fig. 4. Micrographs of presumed xanthophore processes from axanthic axolotl skin.
Note the large empty vesicles that are presumed to be prepterinosomes (p), the
multivesicular bodies (*), and swollen smooth and rough ER (arrows). Bar, 1 fim.
126
S. K. F R O S T , L. G. E P P AND S. J.
ROBINSON
Axanthic phenotype of axolotl
111
machinery' found in other pigment cells although some organelles, notably the
Golgi, ER and electron-dense MVBs, contribute to the 'abnormal' appearance of
these cells.
In view of these observations, we assert that none of Dunson's (1974) hypoth­
eses regarding the underlying mechanism of the axanthic defect (see Introduction)
are likely because these speculations were based on the presumed absence of
pterinosome-like organelles in axanthic skin. This is clearly not the case, and we
would thus offer the suggestion that the axanthic defect is one that blocks pigment
biosynthesis (pteridines in particular) at a point very early in the biosynthetic
pathway, but does not affect organelle formation. To our knowledge this is the
first case in which pigment biosynthesis and organelle formation have been clearly
separated from each other as independent cellular events in cells other than
melanophores. [Albinism is the classic example wherein 'unpigmented' pigment
cells occur (see Frost et al. 1986).]
Iridophores have not been observed in axanthic skin and may fail entirely to
differentiate. Iridophores are also absent in melanoid axolotl skin (Frost et al.
19846), and in neither case is there evidence to suggest why these cell types are
missing. It may be that iridophores do differentiate in both mutant axolotl types
but have not been identified. 'Prereflecting platelets' may not resemble mature
reflecting platelets enough to facilitate identification of unpigmented iridophores
(for an example of what a 'prereflecting platelet' might look like see Frost et al.
1986). Nevertheless, in both melanoid and axanthic axolotls, pteridine metabolism
is abnormal (Frost et al. 1984a,b) and it is further known that both xanthophore
and iridophore pigments derive from purine precursors (Frost & Malacinski,
1980). Consequently, we suggest that the synthesis of the purine precursor(s) may
be the key to understanding these defects. The next obvious step towards gaining a
better understanding of these defects is thus to examine iridophores more closely
and attempt to characterize purine pigments precisely in terms of those purines
that function as pigments and how they are synthesized.
CONCLUDING STATEMENTS
It is well known that all pigment cells share a common embryonic origin (the
neural crest) and it has been further hypothesized that the various types of pigment
organelles also share a common origin, i.e. at the very least they all arise from a
combination of Golgi and ER components (Bagnara et al. 1979). If this is true,
Fig. 5. (A) A macrophage containing virus-laden cellular components in the dermis of
axanthic axolotl skin. Some of the invaded structures resemble melanosomes; others
resemble secretory granules from leukocytes. Note lysosomes (L) throughout the
cytoplasm. Bar, 1 /mi. (B) A closer view of a typical hexagonal array of virus particles
within a former secretory granule. Some of the crystalline structure of the granule is
still intact in the upper left corner of the micrograph (arrow). Bar, 1 fun. (C) Another
close-up of the virus particles packed into an organelle. Bar, 0-5 jum.
128
S. K. F R O S T , L . G. E P P AND S. J. R O B I N S O N
Fig. 6. (A) Photograph of a one-dimensional thin-layer chromatographic separation of
ethanol-extracted pigments from larval (la) and adult (ad) axanthic axolotl skin.
The minor fluorescent bands observed beneath the main riboflavin band are trails
of riboflavin (all fluoresce yellow at 360nm). O, origin; arrowheads, riboflavin. (B)
Photograph of a one-dimensional thin-layer chromatographic separation of ethanol-
extracted pigments from larval (la) and adult (ad) wild-type axolotl skin. Each of the
pteridines present in wild-type skin appears as a distinctfluorescentband under u.v.
light (see Frost et al. 1984a for details).
then the different cell types might be suspected to arise as a result of differential
synthesis of specific pigments. If the axanthic defect resulted simply from an
inability to synthesize pteridines, then one would expect to observe more- or lessnormal-appearing pigment cells with organelles devoid of pigment. This, in fact,
seems to be the case; however, this simple explanation raises a great number of
questions.
For example, given the ubiquitous nature of pteridines and their, role as essential
enzymatic cofactors (Kaufman, 1967), could an animal survive if it were totally
unable to synthesize these compounds? Are axanthic axolotls, in fact, totally
unable to synthesize pteridines? Regarding the xanthophores, what is the signifi­
cance of swollen ER, sparse Golgi and numerous electron-dense MVBs? Are
these characteristics typical of a pigment cell unable to synthesize pigment? Also,
Axanthic phenotype of axolotl
129
why do unpigmented xanthophores occur only rarely in axanthic skin, and why do
they fail to migrate up to their normal position just beneath the basement
membrane of the epidermis? Finally, given the known plasticity of pigment cell
differentiation (Ide, 1978, 1979), why do xanthophores (or their precursors) not
simply differentiate into melanophores as we suspect may happen in melanoid
animals (Frost et al. 1984e)? [This possibility is unlikely because melanophores
have not been shown to be present in abnormally large numbers in axanthic skin
(Lyerla & Dalton, 1971).]
At the present time very little is known about the actual mechanism of differ­
entiation of pigment cell types. Why some chromatoblasts become melanophores
while others become xanthophores is largely speculative. It is hoped that further
investigations into the nature of the axanthic defect coupled with studies that are
currently in progress in this laboratory whereby pigment phenotypes are being
altered by specific drugs will eventually elucidate the mechanisms responsible for
altering pathways of cell differentiation.
The authors wish to thank Fran Briggs (IU Axolotl Colony; recently deceased) for providing
the animals used in all of these studies. We dedicate this series of four papers to her memory.
This work was supported by NSF PCM80-22599, NIH AM34478 and the KU Biomedical
Research Fund. The Center for Biomedical Research (KU) provided equipment and facilities
and Mrs Lorraine Hammer provided invaluable assistance with the electron microscopy.
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LYERLA,T.
(Accepted 19 February 1986)