/. Embryol. exp. Morp/i. Vol. 26, 3, pp. 571-585, 1971
571
Printed in Great Britain
The behaviour of the egg pigment in wild-type and
'rusty' tadpoles of Xenopus laevis
By V. UEHLINGER,* M L BEAUCHEMIN 1 AND A. DROIN 1
From the Station de Zoologie experimentale, Universite de Geneve
SUMMARY
The behaviour of the egg pigment was studied by histological analysis of wild-type and
'rusty' embryos and tadpoles of Xenopus laevis as well as by experimental procedures.
The histological analysis of the wild-type animals showed that the various tissues, notably
the skin, neural tube, alimentary system and cement gland go through progressive stages of
egg pigment migration and concentration at the apical ends of the cells. In the 'rusty'
mutants the migration and concentration of pigment occur to a slight extent only, the
majority of the pigment granules remaining dispersed.
The experiments (tail cultures, squashes of cement gland mucus and of meconium) showed
that in wild-type animals the pigment, after migration and concentration, is eliminated from
the cells by expulsion. In 'rusty' animals, this expulsion does not take place.
Parabiotic tadpoles of a 'rusty' wild-type combination possess a coloration corresponding
to their genotype. Ectodermal grafts performed at the neurula stage between 'rusty' and
wild-type embryos develop according to their origin.
The amount of egg pigment found in wild-type and 'rusty' tadpoles, and the exceptional
case of the cement gland are discussed.
It is concluded that the behaviour of the egg pigment is an active cell-specific process, and
that the pigment is eliminated by expulsion. The non-elimination of the egg pigment in the
'rusty' mutant, accounting for its characteristic colour, appears to be due to a failure of the
expulsion mechanism.
INTRODUCTION
The pigmentation of the amphibian egg is due to the presence of pigment
granules which are produced during the later stages of oogenesis. This pigment
which we shall call egg pigment is found mainly in the cortex of the egg, the
vegetative surface being less pigmented than the animal one. The role of the egg
pigment in embryogenesis is still unknown but, like any pigmentation, it
certainly has a protective function. Many authors have also considered the
possibility of a relation with yolk digestion but as yet no definite role of the
pigment in the breakdown of the yolk platelets has been established (Barth &
Barth, 1954; Flickinger, 1956; Nass, 1962; Karasaki, 1963; Denis, 1964;
Lanzavecchia, 1966).
* The co-authors deeply regret the death of Dr Verena Uehlinger, while this work was
being carried out.
1
Authors' address: Station de Zoologie experimental, Universite de Geneve, 154 route de
Malagnou, 1224 Chene-Bougeries, Geneva, Switzerland.
572
V. UEHLINGER, M. L. BEAUCHEMIN AND A. DROIN
The behaviour of the egg pigment in Xenopus laevis has not been studied
extensively. Nieuwkoop & Faber (1956) mentioned that the pigment disappears
from the tissues in the developing embryo. Pigment granules have been observed
in the cerebrospinal fluid by Adam (1954), Komnick (1961), Millot & Lynn
(1966) and Kordylewski (1969).
We have been led to study the behaviour of the egg pigment in Xenopus after
the discovery of a mutation, 'rusty', which affects this behaviour but does not
otherwise interfere with the normal development of the tadpoles or frogs. The
mutant exhibits a reddish brown colour which is due to the persistence of egg
pigment in various tissues at times when it has already disappeared from the
tissues of the wild-type tadpoles. This persistence of egg pigment is particularly
obvious in the tail fin (Fig. 1). The origin and heredity of this mutation have
already been described (Uehlinger & Droin, 1969). The comparison of wildtype and 'rusty' tadpoles facilitated the interpretation of the results.
MATERIALS AND METHODS
Wild-type embryos of Xenopus were obtained from our laboratory stock
and the 'rusty' embryos from two different homozygous ry\ry strains. Oviposition and fertilization were induced by injection of gonadotropic hormones.
The embryos were reared at room temperature until they reached the required
stages. They were then fixed in Zenker's solution, cut transversely at 7 /mi
and stained with Mayer's haemalun, The staging follows the normal table of
Nieuwkoop & Faber (1956).
To test the behaviour of the egg pigment the following methods were devised
using normal and 'rusty' tadpoles.
Tails of stage 39 embryos were cut off and cultured in a slide provided with a
perforation which was closed in the lower side with a coverslip sealed on with
paraffin wax. The tails were cultured at room temperature in normal Niu &
Twitty solution (Flickinger, 1949). Except for the medium, no sterility precautions were taken. Twenty-four to forty-eight hours later the tails were removed
and the coverslip was detached from the culture slide and placed upside-down
on a normal slide to facilitate microscopic inspection.
The secretion of the cement gland was collected from 20 tadpoles of stages
37-40. These were kept at room temperature in a small dish filled with aquarium
water to a height of 1 cm. At these stages the tadpoles adhere to the surface
by strands of mucus secreted by the cement gland. If one places a coverslip on
the water surface, 24 h later it is covered with mucus strands. The coverslip is
then removed and placed on a normal slide.
The meconium (the intestinal waste-product of the embryo) of stage 46-47
tadpoles was collected from Petri dishes containing clear water in which the
tadpoles were kept without food for a period of 24 h. The fragments of
meconium were put on a slide and squashed with a coverslip.
The egg pigment o/Xenopus
573
To test the specificity of the egg pigment parabiosis and grafting experiments were performed on neurulae (stages 20-22) in normal Niu & Twitty
solution.
OBSERVATIONS
The following description is concerned with the characteristic features of the
egg pigment in several tissues. As melanophores develop normally in 'rusty'
animals, they will not be mentioned.
The various tissues chosen for analysis are derived from all three germ layers,
each showing particular aspects of egg pigment behaviour: skin, neural tube,
cement gland, alimentary canal, notochord and muscles. The observations
concerning the amount of pigment granules are based on rough approximations
established by comparing the sections.
The general condition of the egg pigment from the egg until stage 24 of
normal and ' rusty' embryos
The pigment is found throughout the whole egg as small granules of variable
size, which are easily recognizable in stained and unstained tissues as small
round black dots. A large proportion of the pigment is concentrated in the
cortical layer, the remainder being dispersed throughout the cytoplasm. During
cleavage the cortical pigment becomes located mainly in the prospective
ectoderm. After gastrulation, a layer of pigment-bearing ectodermal cells
covers the whole embryo. Inside these cells, as well as inside the cells of the
mesoderm and endoderm, the pigment granules are dispersed between the
numerous yolk platelets, sometimes accumulating around the nucleus. The
quantity of egg pigment remains approximately constant during these early
stages in all the tissues, except for the cement gland, which from stage 15
onwards exhibits a progressive accumulation of black pigment.
The condition of the egg pigment in various tissues from stage 24 onwards
Skin.
Normal individuals. At stage 24 the thick outer epithelial cell-layer bears a
large number of granules dispersed throughout the cells. Some of them are
found to be accumulating near the external cell-wall. In the thinner sensorial
cell-layer, the granules are less numerous. At stage 29-30 in the epithelial layer,
the pigment condensation against the external cell-walls becomes obvious. At
stage 35-36, this condensation continues. In some cells of the sensorial layer
one can now also see a fine layer of pigment granules against the apical cellwall. By stage 39 all the pigment is accumulated against the cell-walls while
some cells in the crest of the tail-fin appear devoid of pigment. At stage 41 when
the cells have become more flattened and stretched, one begins to notice a
distinct decrease in the quantity of pigment granules, many cells being already
574
V. UEHLINGER, M. L. BEAUCHEMIN AND A. DROIN
1
2
The egg pigment of Xenopus
575
devoid of them. Some cells show a protruding surface containing a cloud of
pigment granules while others seem to extrude the granules into the exterior.
The disappearance of pigment progresses both rostrally and dorso-ventrally,
the cells of the tail and back already having lost their egg pigment while the
majority of the ventral cells are still retaining it. By stage 45 no more pigment
granules can be found (Fig. 2).
'Rusty' individuals. Until stage 35-36 no significant difference in egg pigment
behaviour can be observed between normal and 'rusty' animals except that the
concentration of pigment against the external cell-wall is less marked. From
stage 39 to stage 45 all cells still contain many dispersed pigment granules
showing very little concentration at the external cell-walls (Fig. 3). At stage 48
and even after stage 50 a few isolated granules can be found in the epithelium.
Neural tube
Normal individuals. After the closure of the neural folds (stage 21) irregular
groups of pigment granules are found throughout the whole neural tube. By
stage 24 they have started to aggregate against the wall of the lumen. At stage
29-30 some granules are still dispersed between the yolk platelets but most of
them form an irregular black border around the lumen. Some epithelial cells
have ruptured walls with pigment pouring out into the neural lumen. A few
granules are observed in the cerebrospinal fluid, sometimes forming clusters in
the IVth ventricle; this becomes much more obvious by stage 32 when pigment
granules can be found throughout the whole canal. At stage 35-36 the egg
pigment is almost completely gone from the lumen of the canal and from the
neural tissue, except in the tail tip where the elimination of the pigment is
slightly delayed. At stage 39 a few pigment granules can still be found but by
stage 48 they have completely disappeared (Fig. 4).
'Rusty' individuals. Prior to stage 29-30 no differences can be observed as
compared with normals. At these stages and until stage 32 the pigment is less
strongly condensed against the inner wall of the neural tube and no granules can
be observed in the lumen. After stage 32 the amount of pigment granules becomes
Fig. 1. Tadpoles of Xenopus laevis at stage 44. The transparent tail fin of the wildtype animal (right) is scarcely visible on the light background, whereas the pigmented
tail fin of the 'rusty' tadpole (left) is distinct. (Reprinted from the 20th annual
report of the Societe Suisse de Genet ique 1969.) x 7 5 .
Fig. 2. The skin of + / + stage 45, lacking pigment granules, x 1160.
Fig. 3. The skin of ry/ry stage 45, showing many pigment granules in the peridermal
layer, x 1160.
Fig. 4. + / + neural tube at stage 41, showing a few isolated pigment granules
against the wall of the lumen. On the roof of the neural tube, two heavily pigmented
melanophores. x464.
Fig. 5. Neural tube ry/ry stage 41, showing patches of aggregated pigment granules.
On the roof of the neural tube some heavily pigmented melanophores. x 464.
576
V. UEHLINGER, M. L. BEAUCHEMIN AND A. DROIN
* *
10
The egg pigment of Xenopus
577
slowly diluted as the cells multiply. No pigment granules are found in the lumen
(Fig. 5).
Cement gland
Normal individuals. The nearly fully differentiated gland consists of very
elongated cells bearing an enormous quantity of pigment granules which are
more concentrated than in any other tissue. Only the area surrounding the
nucleus, in the basal part of the cell, is free of pigment. By stage 29-30 the
gland is fully differentiated and mucus secretion has begun. The cells contain
even more pigment than before except at their apical ends where it has disappeared. By stage 35-36 the amount of pigment has diminished. In the basal
parts of the cells some vacuoles have appeared becoming more numerous as the
cement gland begins to show signs of degeneration at stage 41 (Fig. 6). Isolated
pigment granules can occasionally be found in the vacuoles. At stage 52 the
gland has completely disappeared.
'Rusty' individuals. There is no obvious difference as compared with normals
until stage 39. From this stage until stage 45 there is always a concentration of
pigment in the basal parts of the cells (Fig. 7). During stages 45 to 50 the gland
degenerates in an unorganized manner, due to the presence of large pigment
clusters.
The alimentary system
Normal individuals. At stage 29-30 the epithelium surrounding the pharyngeal
cavity is studded with pigment granules, many of which are concentrated against
the wall of the cavity. During stages 35 to 39 some pigment granules can be seen
inside the cavity accumulating against its edges. From stage 39 to stage 41
crusts of pigment peel off, forming large clusters of pigment granules inside the
cavity (Fig. 8). By stage 45 no more pigment granules are present in the
pharynx.
With the beginning of the intestinal coiling, pigment granules are found
dispersed throughout the whole endoderm mass. As the intestine continues to
differentiate (stages 45-47) the pigment moves toward the apical walls of the
cells, thus becoming more and more condensed around the lumen. By stage 46-47,
Fig. 6. Cement gland of + / + stage 41 showing a few remaining granules, x 256.
Fig. 7. Cement gland of ry/ry stage 41 showing a dense concentration of pigment
granules, x 320.
Fig. 8. Pharynx of + / + stage 39 showing a large cluster of pigment inside the
cavity. x464.
Fig. 9. Pharynx of ry/ry stage 39 showing pigment granules dispersed in tissue,
x 464.
Fig. 10. Pigment granules found on the coverslip after culturing a stage 39 +/ +
tail for 24 h. x 528.
578
V. UEHLINGER, M. L. BEAUCHEMIN AND A. DROIN
the most differentiated parts of the intestine have lost most of their pigment
which has passed into the meconium except for the rectum whose epithelium
loses its pigment at a much earlier stage (stage 41). In the other parts of the
intestine the layer of pigment becomes progressively thinner from stage 48
onwards and finally disappears at stage 52.
1
Rusty' individuals. Until stage 41 the pigment appears to be more dispersed
in the pharyngeal and intestinal epithelium than in normal animals but some
condensation against the walls of the cavities can be observed (Fig. 9). No
pigment granules are ever observed in the pharyngeal cavity but some rare
granules can be found in the meconium. Isolated pigment granules can be
observed even at stage 55 in the epithelium throughout the whole alimentary
canal.
Notochord and muscles
Normal and 'rusty' individuals. Pigment granules are dispersed throughout
these tissues showing no obvious condensation.
As vacuolization proceeds in the notochord (stage 29-30) the pigment
becomes attached to the cytoplasmic strands and the corners of the cell junctions.
From stage 39 and stage 45 onwards (notochord and muscles respectively)
the pigment gradually diminishes, but some isolated granules persist until stage
52.
EXPERIMENTAL RESULTS
(1) Pigment elimination
To test the hypothesis that the pigment is eliminated by some sort of excretion,
the following experiments were performed.
(a) Skin. The elimination of pigment from the skin was tested by amputating
tails of stage 39 embryos and culturing them. Twenty-four hours later the
normal tails have lost their pigmentation and become transparent. The coverslip
at the bottom of the culture slide is covered with isolated or clustered pigment
granules, their amount being proportional to the number of tails cultured
(Fig. 10).
The culturing of 'rusty' tails shows that the epithelial cells of the 'rusty' skin
Fig. 11. Pigment granules attached to mucus strands secreted by the cement gland
of a + / + stage 40 animal. x464.
Fig. 12. Meconium of + / + stage 46-47 containing a large amount of pigment
granules, x 183.
Fig. 13. Junction of the skin in a ryjry to + / + parabiotic combination at stage 45.
The skin of the 'rusty' partner contains pigment granules, x 656.
Fig. 14. The brain of a + / + parabiont without any pigment granules. On the roof of
the brain, two melanophores. x 464.
Fig. 15. The brain of the ry/ry partner showing many clusters of pigment granules.
On the roof of the brain, a large melanophore. x 464.
Fig. 16. Junction of a + / + graft with the skin of a ryjry host at stage 42. x 1160.
The egg pigment o/Xenopus
14
37
E M B 26
580
V. UEHLINGER, M. L. BEAUCHEMIN AND A. DROIN
do not excrete any pigment. After 24-48 h of culturing (stage 39) the skin
becomes transparent but it still contains pigment and exhibits the reddish brown
colour typical of 'rusty' larvae. The coverslip at the bottom of the culture slide
is devoid of pigment granules. Occasionally whole epithelial cells break away
as a result of damage inflicted at the time of amputation, liberating a few
pigment granules on the coverslip.
(b) Cement gland. The mucus strands secreted by the cement glands of normal
tadpoles which were collected on the coverslip are full of black pigment granules
as well as a rich microflora (Fig. 11).
The same test failed to show pigment granules in the mucus strands of 'rusty'
tadpoles.
(c) Intestine. The elimination of pigment from the intestine was tested by
taking samples of meconium excreted by normal tadpoles of stage 46-47.
Besides abundant yolk debris, the samples contain a large quantity of black
pigment granules (Fig. 12).
Squash preparations of meconium of 'rusty' tadpoles were found to contain
some black pigment granules. Their number, however, is very small as compared
with the amount present in the meconium of normal tadpoles.
(2) Specificity of pigment behaviour
In order to determine whether the factor controlling the pigment behaviour is
body-specific, tissue-specific or cell-specific, parabiosis and grafting experiments
were carried out.
(a) Parabiosis. Parabiosis was performed between + / + and ryjry embryos
at stages 20-22. The parabionts were fixed at stages 41-45. Some + / + to + / +
and ryjry to ryjry pairs served as controls.
All parabionts developed according to their own genotype. In the + / + to
ryjry combination, the skin of the + / + individual is devoid of pigment while
the skin of its ryjry partner is full of pigment granules. Morphologically and
histologically the junction is easily recognizable (Fig. 13). The brain and the
neural tube are without pigment in the + / + partner (Fig. 14), whereas many
granules occupy the ryjry brain (Fig. 15). The same holds for the pharynx and
the rectum. However, pigment granules are present throughout the intestinal
tract which is partly common to the two partners. Since the pigment does not
normally disappear from the intestine of the + / + before stage 47, the different
parts cannot be allocated to the + / + or ryjry parabionts. The muscles and
notochord of the two partners still bear pigment granules which is in agreement
with our observations on individual + / + and ryjry tadpoles.
(b) Grafts. Pieces of ventral ectoderm were grafted from rusty donors on to
+ / + host embryos and conversely at stage 20-21.
The .+ / + host larvae develop normally until stage 40. When the yolk
granules in the skin begin to disappear, the skin becomes colourless all over the
body except in the ventral graft region, which retains a typical 'rusty' coloration.
The egg pigment o/Xenopus
581
The outlines of the 'rusty' grafts are sharply delimited. On the other hand, the
'rusty' host larvae develop their typical colour except in the region of the + / +
grafts which becomes colourless.
The histological sections show that in those cases in which smooth healing
has taken place, the boundary between the host and the graft periderm can be
easily recognized. On one side of the boundary the cells are loaded with pigment
granules while the cells of the other side are completely devoid of pigment.
Figure 16 shows the junction of a + / + graft with the skin of a 'rusty' host.
When healing is less perfect some intermingling of host and graft cells takes
place, the ryjry cells always being characteristically full of pigment and the
+ / + cells free of pigment.
DISCUSSION
The pigment granules which are abundant in the embryonic skin of most
amphibian species have their origin in the oocyte. This pigment called embryonic
pigment by Nieuwkoop & Faber (1956) is synthesized in the growing oocyte.
According to Balinsky & Devis (1963) the synthesis begins when the yolk
platelet precursors become recognizable in the subcortical cytoplasm of
oocytes having a diameter of Ca. 300 jura and ceases by the time they attain a
diameter of 500 /un. The ultrastructure of the pigment granules has been described by several authors (Dollander, 1954, 1956; Wischnitzer, 1957, 1965,
1966; Wartenberg & Schmidt, 1961; Wartenberg, 1962; Balinsky & Devis,
1963; Karasaki, 1963; Eppig, 1970). However, the mechanism underlying the
formation of the egg pigment still awaits complete elucidation; this also holds
for the question whether the egg pigment granule is identical with the melanin
granule of the melanocyte (Wilde, 1961; McCurdy, 1969; Eppig, 1970).
The amount of egg pigment synthesized in the oocyte is determined by the
genotype of the oocyte itself. It has often been observed that the intensity of
pigmentation of the eggs laid by any one female is constant but that it varies
from one female to another. Blackler & Fischberg (personal communication) grafted primordial germ cells from one Xenopus laevis embryo to another.
Host embryos which developed into females spawned donor and host eggs which
differed in pigmentation. This experiment in which the Oxford nucleolar mutant
(Elsdale, Fischberg & Smith, 1958) was used as marker, clearly showed that the
oocyte genotype and not the maternal ovary determines the amount of egg
pigmentation. It has further been observed that a phenotypically normal female
heterozygous for the 'rusty' mutant will lay eggs of uniform pigmentation.
When fertilized by a homozygous ry\ry male, 50 % of the eggs will give rise to
phenotypically normal embryos and 50% to 'rusty' mutants (Uehlinger &
Droin, 1969). The normal and 'rusty' embryos of this spawning initially must
contain the same amount of egg pigment, since it was found by Balinsky &
Devis (1963) that pigment formation is restiicted to a certain period during
oogenesis. Moreover, our observations on various developmental stages of
37-2
582
V. UEHLINGER, M. L. BEAUCHEMIN AND A. DROIN
normal animals indicate that there is probably no renewed synthesis of pigment
granules in the embryo. Thus it seems more logical to name this pigment which
is present in the embryo 'egg pigment' rather than 'embryonic pigment'.
The only exception in this respect seems to be the cement gland which, during
its differentiation, accumulates a larger amount of pigment. Nieuwkoop &
Faber (1956) mention a 'concentration' of embryonic pigment in the differentiating cement gland. However it seems unlikely that the pigment granules move
from one cell into another. The experiments of Holtfreter (1943) who turned
paits of the embryo insideout showed that the pigment granules move towards
the outer surface of the cell without leaving it. Moreover, in poorly differentiated
cells of the oral 'sucker' of Hyla regilla embryos treated with actinomycin D,
Eakin (1964) observed a reduction in the amount of pigment. This suggests that
most of the pigment granules present in the cement gland are synthesized during
the differentiation of its cells so that in this case the name 'embryonic pigment'
is acceptable.
Several authors have observed a decrease in the number of pigment granules
during anuran development (Elias, 1937; Sung, 1962; Karasaki, 1963; Millot &
Lynn, 1966). In Xenopus laevis, Nieuwkoop & Faber (1956) pointed out that
the embryonic pigment in the skin disappears rather fast during a definite
period of development (stages 41-44). Adam (1954) observed cells filled with
pigment granules floating in the cerebrospinal fluid. Kordylewski (1969) made
the same observation but, unlike Adam, he believes that they contain only
pigment granules originating from the neural tissue and that there is no additional
pigment resulting from synthesis in these cells. Komnick (1961) also described
the excretion of the egg pigment into the cerebrospinal canal by the breaking
open of the apical walls of the neuro-epithelial cells and the pouring out of
the egg pigment together with some yolk platelets. He states that this is the
means by which the embryonic brain can clear itself from excess pigment. Our
observations are in accordance with his findings but it seems that it is not only
an excess but the totality of the egg pigment which is expelled from the neural
tissue.
The similarity between pigment movements in neural tissue and in the majority
of all other tissues has led us to assume that egg pigment is eliminated by
expulsion, as was also suggested by Kordylewski (1969). Our three experiments
devised to test this (tail cultures, squashes of cement gland mucus and meconium)
confirm the expulsion hypothesis. Additional evidence for pigment expulsion
has been provided by H. Kobel (personal communication) in Xenopus mulleri. In
this species, the hatching takes place at a later stage than in X. laevis, i.e. after
the embryo has normally lost its egg pigment. The intact egg membranes of
X. mulleri with the tadpoles inside were observed to have a definite brownish
colour; after puncturing the membranes to free the tadpoles, a cloud of brownish
material burst into the surrounding medium. Inspection under the microscope
revealed that this brown substance consisted of egg pigment granules.
The egg pigment of Xenopus
583
In conclusion we can define three different components in the behaviour of
the egg pigment: migration, concentration at the apical border of the cell and
expulsion into the exterior or into a lumen. These three components are
characteristic and time specific for nearly all the tissues.
In tissues which are not in contact with the exterior, either directly or by a
lumen, such as the notochord or the muscles, pigment granules were observed
at much later stages than in the other tissues. In these cases, the granules
remain more or less dispersed, are neither concentrated nor expulsed, and their
density decreases with time in the growing tissues.
An indirect confirmation of the occurrence of the &gg pigment movements
in wild-type tadpoles is provided by the abnormal behaviour of the pigment in
the 'rusty' mutants. In embryos homozygous for 'rusty', migration and
concentration of the pigment do occur but to a much lesser extent. Elimination
never takes place (except to a very small extent in the intestine), as was evident
from our observations and confirmed by our experiments. Thus the 'rusty'
colour is the result of the non-elimination of pigment. However, in later stages,
the pigment concentration gradually decreases; as the tadpoles grow, the
granules are distributed passively among the dividing cells and the 'rusty'
colour subsequently disappears.
With regard to the yolk platelets, we have observed that the disappearance
of the yolk platelets in normal animals occurs shortly before that of the pigment
granules. In 'rusty' animals no difference in the behaviour of yolk platelets has
been observed.
The parabiosis and grafting experiments show that the action of the ' rusty'
factor is not only tissue-specific but also cell-specific. No influence of 'nonrusty' host tissues on the 'rusty' effect can be observed. The mechanism controlling the elimination of the egg pigment, the nature of which is still unknown,
is thus located within the cells. The sudden onset and speed of the elimination
indicates an active process. Since the pigment granules are of maternal origin,
the expulsion controlling factor cannot be connected with the pigment granules
themselves but must be related to the genotype of the cells. The lack of expulsion
observed in the 'rusty' mutants could result from a mechanism of inhibition.
Further investigations will be necessary to elucidate this problem.
RESUME
L'6tude histologique de tetards de Xenopus laevis, sauvages et 'rusty', et une serie d'experiences ont permis d'analyser le comportement du pigment de l'oeuf.
L'analyse histologique des tetards sauvages revele que, dans differents tissus tels que la
peau, le tube nerveux, la papille et le tube digestif, le pigment presente un mouvement de
migration puis de concentration au bord apical des cellules. Chez les mutants 'rusty', ces
mouvements sont moins prononces, la majorite des granules de pigment reste dispersee.
Les cultures de queue, les 'squashes' du mucus de la papille et de meconium montrent que,
chez les tetards sauvages, apres migration et concentration, il y a elimination du pigment par
expulsion. Cette expulsion n'a pas lieu chez les tetards 'rusty'.
584
V. U E H L I N G E R , M. L. BEAUCHEMIN AND A. DROIN
Dans les cas de parabioses et de greffes effectuees au stade neurula entre tetards sauvagcs
et 'rusty', les parabiontes et les greffes se developpent selon leur genotype respectif.
C'est un processus actif, specifique de la cellule qui controle le comportement du pigment de
1'oeuf. Ce dernier est elimine par expulsion. La non-elimination du pigment chez les tetards
'rusty', leur donnant leur couleur caracteristique, semble resulter d'un defaut du mecanisme
d'expulsion.
We are indebted to Professor M. Fischberg, in whose Department the material was put at
our disposal, for his advice and criticism, to Dr J. Faber (Utrecht) for valuable discussions
and to the Library of the Hubrecht Laboratory, International Embryological Institute, for
bibliographical assistance. We wish to thank Misses M. van Schaik, C. Voll and H. Jutte for
technical help, and Miss M. Maye for the photographic work.
This work was supported by the Fonds national suisse de la Recherche scientifique
(requetes no. 4411 and 3.60.68).
REFERENCES
ADAM, H. (1954). Freie kugelformige Pigmentzellen in den Gehirnventrikeln von Krallenfroschlarven Xenopus laevis (Daudin). Z. mikrosk.- anat. Forsch. 60, 6-32.
BALINSKY, B. I. & DEVIS, R. J. (1963),. Origin and differentiation of cytoplasmic structures in
the oocytes of Xenopus laevis. Acta Embryol. Morph. exp. 6, 55-108.
BARTH, L. G. & BARTH, L. J. (1954). The Energetics of Development. New York: Columbia
University Press.
DENIS, H. (1964). Phosphoproteine-phosphatase et resorption du vitellus chez les amphibiens:
une etude cytochimique, electrophoretique et immunologique. J. Embryol. exp. Morph. 12,
197-217.
DOLLANDER, A. (1954). La structure du cortex de l'oeuf de Triton observee sur coupes fines
et ultrafines au microscope ordinaire et au microscope electronique. C. r. Seanc. Soc. Biol.,
Paris US, 152-154.
DOLLANDER, A. (1956). Ultrastructure de la region corticale de l'ovocyte et de l'oeuf feconde
symetrise chez le Triton. C. r. Seanc. Soc. Biol., Paris 150, 998-1001.
EAKIN, R. M. (1964). Actinomycin-D inhibition of cell differentiation in the amphibian
sucker. Z. Zellforsch. mikrosk. Anat. 63, 81-96.
ELIAS, H. (1937). Zur vergleichenden Histologie und Entwicklungsgeschichte der Haut der
Anuren. Z. mikrosk.- anat. Forsch. 41, 359.
ELSDALE, T., FISCHBERG, M. & SMITH, S. (1958). A mutation that reduces nucleolar number
in Xenopus laevis. Expl Cell Res. 14, 642-643.
EPPIG, J. J. Jr. (1970). Melanogenesis in Amphibians. III. The buoyant density of oocyte and
larval Xenopus laevis melanosomes and the isolation of oocyte melanosomes from the eyes
of PTU-treated larvae. /. exp. Zool. 175, 487-492.
FLICKINGER, R. A. (1949). A study of the metabolism of amphibian neural crest cells during
their migration and pigmentation in vitro. /. exp. Zool. 112, 465-484.
FLICKINGER, R. A. (1956). The relation of phosphoprotein phosphatase activity to yolk
platelet utilization in the amphibian embryo. /. exp. Zool. 131, 307-332.
HOLTFRETER, J. (1943). Properties and functions of the surface coat in Amphibian embryos.
/. exp. Zool. 93, 251-323.
KARASAKI, S. (1963). Studies on amphibian yolk. 5. Electron microscopic observations on the
utilization of yolk platelets during embryogenesis. /. Ultrastruct. Res. 9, 225-247.
KOMNICK, H. (1961). Uebsr Herkunft, Bedeutung und Schicksal der Melanocyten im
Cerebralliquor von Krallenfroschlarven (Xenopus laevis). Wilhelm Roux Arch. EntwMech.
Org. 153, 14-31.
KORDYLEWSKI, L. (1969). The fate of egg pigment in tadpoles of Xenopus laevis (Daudin)
(Amphibia, Anura). Bull. Acad. pol. Sci. Cl. If. (Ser. Sci. biol.) 17, 347-348.
LANZAVECCHIA, G. (1966). Osservazioni sulla formazione et sulla demolizione del vitello
negli Anfibi. Atti Accad. naz. Lincei Memorie. Ser. vm, 8, sez. in<7, 1-47.
The egg pigment of Xenopus
585
H. M. (1969). Enzyme localization during melanogenesis. J. Cell Biol. 43,
220-228.
MILLOT, N. & LYNN, W. G. (1966). Ubiquity of melanin and the effect of phenylthiourea.
Nature, Loud. 209, 99-101.
NASS, S. (1962). Localization and properties of phosphoprotein phosphatase in the frog egg
and embryo. Biol. Bull. mar. biol. Lab., Woods Hole 111, 232-251.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus laevis (Daudin). Amsterdam:
North Holland Publishing Company.
SUNG, H. S. (1962). Electron microscopic studies on structural changes of developing cells of
the anuran embryos. Embryologia 7, 185-200.
UEHLINGER, V. & DROIN, A. (1969). Origine et heredite de la mutation 'rusty' (ry) du
Batracien anoure Xenopus laevis D. Archiv der Julius Klaus-Stift. 43/44, 48-54.
WARTENBERG, H. (1962). Elektronenmikroskopische und histochemische Studien iiber
Oogenese der Amphibieneizelle. Z. Zellforsch. mikrosk. Anat. 58, 427-486.
WARTENBERG, H. & SCHMIDT, W. (1961). Elektronenmikroskopische Untersuchungen der
strukturellen Veranderungen im Rindenbereich des Amphibieneies im Ovar und nach der
Befruchtung. Z. Zellforsch. mikrosk. Anat. 54, 118-146.
WILDE, C. E. (1961). The differentiation of vertebrate pigment cells. In Advances in Morphogenesis, vol. l (ed. M. Abercrombie and J. Brachet), pp. 267-300. New York and London:
Academic Press.
WISCHNITZER, S. (1957). The ultrastructure of yolk platelets of amphibian oocytes. /. biophys.
biochem. Cytol. 3, 1040-1042.
WISCHNITZER, S. (1965). The cytoplasmic inclusions of the salamander oocyte. I. Pigment
granules. Acta Embryol. Morph. exp. 8, 141-149.
WISCHNITZER, S. (1966). The ultrastructure of the cytoplasm of the developing amphibian egg.
In Advances in Morphogenesis, vol. 5 (ed. M. Abercrombie and J. Brachet), pp. 131-179.
New York and London: Academic Press.
MCCURDY,
(Manuscript received 15 April 197T)