/ . Embryol exp. Morph. Vol. 60, pp. 173-188, 1980
Printed in Great Britain © Company of Biologists Limited 1980
173
The spatio-temporal framework of
melanogenic induction in pigmented retinal cells of
Xenopus laevis
ByOLGA A. HOPERSKAYA 1 AND OLGA N. GOLUBEVA1
From the Laboratory of Organogenesis, N. K. Koltzov Institute of
Developmental Biology of the Academy of Sciences of U.S.S.R.,
Moscow
SUMMARY
Using methods involving transplantation of neuroectoderm and eye rudiments between
ap/ap mutants and + / + embryos of Xenopus laevis, new aspects of melanogenic induction
have been ascertained. The possibility of separate induction of a melanocyte cell type and of
the product of terminal differentiation in it - melanosomes, has been demonstrated. Melanogenic induction starts at the late gastrula stage. It has an irreversible character which has been
shown using the technique of double transplantation. Melanogenic factor (MgF) is produced
only by the region of endomesoderm which is adjacent to eye rudiments in the course of
normal development. Competence for melanogenic induction lasts till stage NF 30. Melanogenic induction appears to be species-specific; at least after transplantation of eye vesicles
from av/av Xenopus laevis to Rana temporaria the melanoprotein synthesis cannot be switched
on. L-Tyrosine, under the conditions of the experiments, is not a factor in the induction of
melanogenesis.
INTRODUCTION
The mechanism of determination for melanoprotein production in cells, and
thus of melanocyte differentiation, has not been studied. Most investigations
have concerned cells already determined as melanocytes, but knowledge of
early determinative events is of primary importance for complete understanding
of cell differentiation (Whittaker, 1974).
Reactions by melanosomes within cells, synthesizing melanin itself, are
governed by light, peptide hormones and cAMP (Fujii, Nakazawa & Fujii,
1973; Whittaker, 1974; Chen et ah 1974) and pigment cells of different origins
show similar reactions (Burlakova, 1978). But the initial formation of melanosomes is primarily determined by inductive signals (Hoperskaya, 1978, 1980),
with several groups of genes apparently participating (Doezema, 1973). The
contribution of these genes can be studied by use of mutations affecting pigmentation (Vatti & Alekseevich, 1976), and this also allows us to discriminate
between genes operative in the inducing tissue and those operative in the
1
Author's address: Institute of General Genetics Ac. Sci. U.S.S.R., Gubkin Street 3,
0333 Moscow, VGSP-1 117809, U.S.S.R.
12
EMB 60
174
O. A. HOPERSKAYA AND O. N. GOLUBEVA
responding tissue. We have used embryos of the albino periodic mutant
(a p /a p ) of Xenopus laevis (Hoperkskaya, 1975), which is a mutation of a regulatory gene, deficient in a specific inducing factor for melanogenesis (MgF)
(Hoperskaya, 1980). Absence of this factor results in amelanotic development
in both mutant and wild-type pigmented epithelium. The wild-type product is
synthesized in endomesoderm and influences the character and numbers of
melanosomes in the induced pigmented epithelial cells (Hoperskaya, 1977, 1978,
1980). In affecting primarily an inducing signal, the a p mutant thus resembles c
(cardiac lethal) and o (ova deficient) in Ambystoma mexicanum (Briggs, 1973).
Adult flP/tfp animals are practically albino, possessing very small numbers of
melanosomes in dorsal regions only of the retinal pigmented epithelium. At
early developmental stages melanosomes and premelanosomes are lacking
(Bluemink & Hoperskaya, 1975) while at larval stages 39-40 (Nieuwkoop &
Faber, 1956) melanosomes begin to form in the pigmented epithelium and also
in the iris and the choroid coat (Hoperskaya, 1975). Some skin melanophores are
pigmented as well. All the machinery of melanin synthesis is clearly available
in the mutant and indeed tyrosinase, a key enzyme is more abundant in mutant
oocytes and larval cells than in normals (Wyllie & De Robertis, 1976; Tompkins,
Knight & Burke, 1977). The melanosomes initially formed are nevertheless
abnormal and disappear at later stages (Hoperskaya, 1980, and unpublished).
Determination of the eye region within the neural plate starts in the late
gastrula (Nieuwkoop, Oikawa & Boddingius, 1958) and we have evidence that
induction of melanoprotein synthesis within this region may start around the
same time. Following reciprocal transplantations of eye material at tail-bud
stage (Nieuwkoop & Faber (1956) stage 21) only the ventral eye regions can still
be affected (i.e. be induced or fail to be induced according to genotype of the
mesodermal environment), implying that in other parts of the eye melanogenic
induction has already occurred before eye-vesicle formation.
The purposes of the present investigation were: (1) to determine the temporal
characteristics of melanogenic induction; (2) to find out if the induction process
can be reversed in cells of pigmented epithelium; (3) to show the possibility of
separate induction for the melanocyte cell type itself and for the products of
terminal differentiation within it; (4) to find out whether melanogenic factor is
produced in parts of the endoderm and mesoderm other than those which
contact eye material in normal development; (5) to study the duration of competence for melanogenic induction and the species specificity of the signal; (6) to
find out whether L-tyrosine is an adequate inducer of the melanin-producing
machinery.
MATERIALS AND METHODS
Wild-type ( + / + ) and white mutant (av/av) embryos of Xenopus laevis were
obtained by gonadotrophin injection (Brown, 1970). Stages of development
were determined from the Normal Table (Nieuwkoop & Faber, 1956; =NF).
Melanogenic induction in pigmented retinal cells
175
Table 1. Determination of the start of melanoprotein induction and its irreversibility in Xenopus laevis
Number of embryos
Series
la
Type of experiment
Operated
Survived
Results
Transplantations at gastrula stage
ap/ap
+/+
>
st. 10
st. 10
25
15
30
18
Pigmented epithelium is
heavily pigmented, and in
most cells it is not distinguished from pigmented
epithelium of + / +
embryos
Pigmented epithelium
contains melanosomes only
of oocyte origin
25
19
Ib
st. 10
st. 10
Secondary transplantations
II
aP/ap
st. 10
> +/+ : :
st. 10
st. 21
st. 21
Synthesis of melanoprotein
induced at the gastrula
stage remains very high but
not uniform in all pigment
cells
In series IV we also used embryos of Rana temporaria, obtained by injection of
hypophyseal hormones, and staged according to Dabagyan & Sleptsova, 1975
( = DS). In series VI we also used embryos of Ambystoma mexicanum, staged
according to Harrison's Normal Table (Rugh, 1962; =H).
Operations were performed in Niu-Twitty saline, and the embryos transferred
after 2-3 h to sterile water + antibiotics. Tadpoles were later kept in dechlorinated tap water at room temperature. In series VI early embryonic cells of
Ambystoma were cultured in Holtfreter solution, while early embryonic cells of
Xenopus were cultured in Niu-T witty (Rugh, 1962).
L-Tyrosine was added to the culture medium in the following concentrations:
1/tg/ml, 10/6g/m], 100/tg/ml. We treated with L-tyrosine explants of early
gastrula ectoderm (NF 10) and eye vesicles (NF 21) of Xenopus laevis, and
explants of late blastula ectoderm (H 8), and anterior neural plate (H 16) of
Ambystoma mexicanum.
Material was fixed for histology in Bouin's fluid, at intervals from 1 week to
6 months after operation. Serial sections, cut at 5 /im, were stained with
azocarmine and counterstained according to Mallory.
RESULTS
I. Temporal characteristics and irreversibility of melanogenic induction
Pieces of neuroectoderm corresponding to future eye vesicles were reciprocally
exchanged between wild-type and mutant embryo at early gastrula stages
O. A. HOPERSKAYA AND O. N. GOLUBEVA
I 0-1 mm %|
Melanogenic induction in pigmented retinal cells
111
(NF 10) (Fig. 1, Table 1, series la). The pieces were slightly larger than the
implantation sites to promote good healing. Disturbance to the axial structures
led to some decrease in the viability of embryos after these operations. Grafted
regions subsequently formed eye vesicles, diencephalon and small pieces of
head skin, all easily distinguishable from the host contribution because of the
different content of egg melanosomes up to the stages of skin melanophore
pigmentation (NF 35-40). Embryos where some part of the eyes had been
formed from the host were excluded from the results.
Where transfer had been from white to wild-type embryos, the subsequent
development of melanophore migration was normal, confirming previous
observations (Hoperskaya & Golubeva, 1977; MacMillan, 1979). Melanoprotein synthesis was dramatically activated in the pigmented epithelium of the
eyes of mutant genotype in these embryos, being indistinguishable from wildtype intensity in most regions (Fig. 3). However in some regions, perhaps as a
consequence of imperfect contact between neuroectoderm and endomesoderm
the pigmentation intensity remains typical of the mutant (Fig. 3).
Where transfer had been from wild-type to white embryos, there was a failure
of melanogenic induction, the pigmented epithelium of the wild-type dorsal iris
containing even fewer newly synthesized melanosomes than are normally found
in this region in mutants (cf. Figs. 2,4 and 5), despite the normal morphogenesis
which brought av/ap lens epithelium into close contact with iris cells. Comparison of these specimens with normal ones allows quantitative assessment of the
contributions to melanogenesis made by melanosomes of egg origin and those
newly induced (Table 1, series Ib, Fig. 9).
FIGURES
1-6
Fig. 1. Experimental scheme of future eye region transplantation at early gastrula
stage from mutant embryo to wild type; secondary transplantation of eye vesicle
formed, at the expense of mutant material at tail-bud stage. Grafted eye, which had
been in contact with endomesoderm of + / + embryo, irreversibly preserves a high
level of melanoprotein synthesis.
Abbreviations for this and later figures: Che, choroid coat; endom, endomesoderm; EV, eye vesicle; 1, iris; Int, interzone; 1PE, induced pigmented epithelium;
L, lens; n-f eyes, newly formed eyes; PE, pigmented epithelium; R, retina; Unll,
uninduced iris; UnlPE, uninduced pigmented epithelium; VR, visual receptors.
Fig. 2. Mutant amelanotic pigmented. epithelium (NF 47).
Fig. 3. Pigmented epithelium of a tadpole eye (NF 47), which has been formed from
mutant eye material after its transplantation at early gastrula stage.
Fig. 4. Pigmented epithelium of a tadpole which developed from + / + eye material,
transplanted to a mutant endomesoderm at early gastrula stage. Only individual
melanosomes of oocyte origin are seen.
Fig. 5. Iris of tadpole eye totally free of melanosomes. It arose from + / + material
after its grafting to mutant endomesoderm at early gastrula stage.
Fig. 6. Mutant eye after the secondary transplantation at stage NF 22. In those
parts where induction occurred, it had an irreversible character.
178
O. A. HOPERSKAYA AND O. N. GOLUBEVA
Fig. 7. Absence of the pigmented epithelial cell type, appearing as a consequence of
eye material transplantation from mutant to + / + embryo at early gastrula stage.
Fig. 8. Aggregates of pigmented epithelial cells appeared from mutant eye material
after its transplantation to + / + embryo. Some cells are induced to melanoprotein
synthesis, others are not.
Fig. 9 Group of pigmented epithelial cells which appeared from + / + material
after its transplantation to mutant embryo. Cells contain pigment granules only of
oocyte origin.
Fig. 10. Inverted retina appearing in case of a disturbance of normal morphogenesis after presumptive eye material transplantation at the early gastrula stage.
Choroid coat does not form.
In certain of these reciprocally chimaeric animals, eye morphogenesis was not
normal, and cells of the pigmented epithelial layer were absent or were gathered
in large groups, causing abnormal contact relationships between the various eye
layers and the surroundings. All types of pigmented epithelial differentiation
were, therefore, obtained with successful or unsuccessful melanogenic induction
independent of the cells' genotype (Fig. 11). Where the retinal layers themselves
were inverted, the choroid coat was not formed at all (Fig. 10).
Melanogenic induction in pigmented retinal cells
179
This set of results demonstrates that subsequent to stage 10, selective induction of the pigmented epithelial zone of eye milage, with its distinctive cell type,
occurs, to be followed only later after fin»her inductive influences by the
activation of melanoprotein synthesis in this cell type. The influences of lens,
ectomesenchymal cells and circulation (Lopashov, 1963) and arrangement of
primary eye cells in a layer (Lopashov & Hoperskaya, 1967) formerly considered
decisive factors in the determination of the pigmsnted epithelial cell type,
now seem to be secondary influences governing the full expression of its
differentiation (Hoperskaya & Golubeva, 1979).
Mutant eye vesicles which had developed in a wild-type environment up to
stage NF 21, after ap/av to wild-type grafts of the sort described above, were
then back-transferred to mutant hosts to replace their own eye vesicles, removed
via a dorsal slit. Eyes in such embryos developed much more intense pigmentation than that of control a?la? eyes, showing that a substantial amount of
irreversible melanogenic induction occurs by early-eye-vesicle stages, before
embryonic melanoprotein synthesis actually starts (Fig. 6, Table 1, series II).
II. Duration of competence of melanocytes for melanogenic induction, and speciesspecificity of the signal
Eye rudiments from stages NF 30 and 32 were grafted to replace the eye
vesicles of stage-2l embryos, thus contacting their inducing endomesoderm.
Small regions only of the pigmented epithelium of av/ap rudiments, grafted to
wild-type hosts in this way, were activated to form melanosomes (Table 2,
series Ilia). Five hours later, at stage 32, induction of melanogenesis failed
completely (Table 2, series Illb) (Fig. 12), showing that pigmented cells are
by this time impermeable, or in some other way resistant, to the inducing signal.
We did not observe melanogenesis following transplantation of eye vesicles
from Xenopus to Rana temporaria (Fig. 13, Table 2, series IVa) although
transplantation to +•/+ Xenopus at the same stages causes dramatic induction
of melanin synthesis (Hoperskaya, 1977,1978). This apparent species-specificity
of the induction process may relate to (1) qualitative specificity of MgF;
(2) inability of this factor to interact across species with other factors affecting
competence, or (3) absence of an MgF synthesis in endomesodermal cells
adjacent to Rana temporaria eye vesicles at the stage concerned (DS 26). This
situation may be analogous to the quantitative variations in distribution of lens
inducing capacity in head endomesoderm and eye vesicles as between different
amphibian species (Tahara, 1962a, b; Lopashov, 1963).
Following implantation of heterospecific eyes, host ectomesenchyme cells
migrate only poorly from R. temporaria host onto the grafted eye vesicles, at
those morphological stages where this migration is still intensive in Xenopus
(Hoperskaya, 1978). The timing of processes crucial to melanogenic induction
may differ in the two species.
The reciprocal eye vesicle transplantation to the above, i.e. stage-26 (DS) R.
180
O. A. HOPERSKAYA AND O. N. GOLUBEVA
11
om
,
unIPE
ChC
*•'
Melanogenic induction in pigmented retinal cells
181
temporaria to a^/a? Xenopus stage 21, gives eyes whose pigmentation is hardly
distinguishable from that of control Rana eyes (Fig. 14, Table 2, series lVb). Since
a p /aP Xenopus endomesoderm cannot induce melanoprotein synthesis we can
conclude that melanogenic induction of future retinal pigmented epithelium in
Rana has already occurred by DS stage 26.
III. Heterotopic transplantation of eye vesicles and anterior neural plate pieces
to the belly region
Our aim, in the transplantation of eye vesicles and eye-forming regions of the
neural plate to ectopic sites, was to ascertain whether mesoderm or endoderm
other than that normally coming to be adjacent to the eye possesses melanogenic
inducing capacity. Data in Table 3 show that such tissues located ventral and
caudal to the normal region possess no such capacity, since mutant material
transplanted from eye vesicle or antero-lateral stage-16 neural plate under the
belly epidermis of stage-21 + / + embryos, remained ap/ap in pigmentation. In
the reciprocal transplantation, the fully melanized pigmented zones of the
ectopic eye structures developed (Figs. 16, 17) showed that melanogenic induction, as well as the induction of the pigmented epithelial cell type (see part I),
had occurred in the donor by stage 16 (Table 3, series Va).
In one series of operations, av/aP stage-21 vesicles were grafted into contact
with the anterior side of the left vesicle of a + / + host of the same stage. The
transfer of the induced state, i.e. melanization of the a p /a p epithelium contacting
the host eye and brain but devoid of contact with endomesoderm, was never
observed (Figs. 18, 19). We conclude that induced wild-type eye vesicle,
or brain, cannot itself furnish the inductive signal (Table 3, series Vc, d).
FIGURES
11-14
Fig. 11. Schematic representation of all possibilities for pigmented epithelium
differentiation independently of the cell genotype as a result of presumptive eye
material transplantation at early gastrula stage; absence of pigmented epithelium
cell type at all; pigmented epithelium in a form of aggregate, amelanotic; pigmented
epithelium in a form of monolayer, amelanotic; pigmented epithelium in a form of
aggregates, overloaded with melanosomes; pigmented epithelium in a form of
monolayer, overloaded with melanosomes.
Fig. 12. Pigmented epithelium of av/av embryo after eye rudiment transplantation
(NF 32) to endomesoderm of wild-type embryo (NF 21). Absence of melanogenesis.
Fig. 13. Pigmented epithelium of Xenopus laevis, transplanted at st. NF 21 to Rana
temporaria St. DS 26. Activation of melanogenesis does not proceed.
Fig. 14. Pigmented epithelium of Rana temporaria eye transplanted at st. DS 26 to
Xenopus laevis st. NF 21.
182
O. A. HOPERSKAYA AND O. N. GOLUBEVA
15
•A
0-1 mm
17
•1t
Int
<
18
Melanogenic
induction in pigmented
retinal cells
183
Table 2. Duration of melanocyte competence and species-specifity of melanogenic
induction
Number of embryos
Series
Type of experiment
Operated
Survived
15
15
10
10
10
10
Results
Transplantations between
embryos of various age
Ilia
st. 30
st. 21
111b
Induction of melanosome
formation in some regions
of pigmented epithelium
No induction
st. 21
st. 32
Transplantations between
two species
IVa
IVb
X.I. a?/a?
st. 21
R.t.
st. 26
>
>
R.t.
st. 26
X.I. a*/ap
st. 21
Induction of melanogenesis
does not take place
Pigment epithelium has
already been induced to
melanogenesis at the stage
of operation
1Y. Test of L-tyrosine as a possible specific melanogenic inductor
Landstrom & Lovtrup (1978) have suggested that L-tyrosine might be the
melanogenic inducer in the embryo. It is an initial substrate for melanin synthesis
(Riley, 1977), and may be an activator for melanogenesis in certain cells already
differentiated to produce melanin (Gilbert et al. 1973; van Woert, 1973).
In two series of experiments we have incubated the early gastrula ectoderm cells
of Ambystoma mexicanum and Xenopus laevis in L-tyrosine solutions. In large
groups of A. mexicanum cells we did not observe the appearance of pigment
granules, whereas in small groups of early embryonic cells, melanin granules
were found approximately to the seventh day of cultivation.
In cultures of various age cells of Xenopus laevis, treated with L-tyrosine even
FIGURES
15-19
Fig. 15. Contact of ventral surface of eye vesicles with endomesoderm in normal
development of Xenopus laevis St. NF 21.
Fig. 16. 'Tnterzone' formed as a result of the transplantation of the anterior piece of
+ / + neural plate to a^/aP embryo heterotopically.
Fig. 17. Eye formed after transplantation of the anterior piece of the + / + neural
plate to a mutant embryo (NF 21) heterotopically.
Fig. 18. Contact between mutant eye and normal eye. Early stage. Increase of a
pigmentation in a mutant eye does not occur.
Fig. 19. The same situation. Advanced stage.
184
O. A. HOPERSKAYA AND O. N. GOLUBEVA
Table 3. Heterotopic transplantations of eye material (to ascertain the localization
of melanogenic activity on Xenopus embryos)
Number of embryos
Series
Type of experiment
Operated
Survived
Va
Transplantations at neurula stage
+/+
>
ap/ap
st. 16
st. 16
12
11
Yb
av/a?
st. 16
15
14
15
12
12
11
Vc
Vd
>
+/+
st. 16
Transplantations of eye vesicles
+/+
> ap/av
st. 21
st. 21
ap/av
>
+/+
st. 21
st. 21
Results
Induced level of melanoprotein synthesis remains
very high, i.e. corresponds
to + / + type
Synthesis of melanoprotein
does not change, i.e.
corresponds to av/aP type
Synthesis of melanoprotein
corresponds to + / + type
Activation of melanoprotein
synthesis does not take
place
in the highest concentration, we did not observe any signs of melanization. This
suggests that the inducer of melanogenesis cannot be such an elementary
compound as L-tyrosine. Apparently without L-tyrosine (or L-phenylalanine) the
process of melanogenesis in cells cannot proceed. Therefore, the availability of
these animo acids is an obligatory but insufficient condition for melanogenesis.
Moreover, it is unlikely that the # p mutation, which is quite viable, could be for a
synthetic step in amino-acid metabolism, and also unlikely that so ubiquitous a
molecule as tyrosine should be topographically restricted, as our results show
that the melanogenic inducing signal is.
DISCUSSION
Our results have given us insight into the timing and positioning of melanogenic induction. This starts to a considerable extent at the middle-late gastrula
stage, evidently as a component of primary embryonic induction. During this
stage the pigmented epithelium cell type emerges and melanogenesis in it starts,
but these processes are not identical and do not automatically follow each other.
The induction of a selective cell type of retinal pigmented epithelium can be
separated from the induction of terminal differentiation in it. As a result of
such separation the amelanotic pigmented epithelium both in the form of a
layer and in the form of aggregates can arise, independently of the genotype of
reactive tissue.
These two different aspects of the induction of the retinal pigmented epithelial
Melanogenic induction in pigmented retinal cells
185
cells are mediated by different influences coming from the same sources. As far
as concerns the retinal pigmented epithelium there are two sources of melanogenic induction. The first source is prechordal plate underlying eye material in
the course of gastrulation and neurulation and the second source is an endomesodermal region situated behind the eye vesicles at tail-bud stage (Fig. 15).
Transplantations of eye material into alien surroundings shows that only these
zones possess melanogenic activity.
Experiments involving neural plate isolation show that up to the stage of
neurulation the process of melanogenesis in future melanocytes has already been
switched on earlier, at middle-late gastrula stage, but apparently not in all cells.
The process of melanogenic induction continues until tail-bud stages. It has
been shown (Hoperskaya, 1978) that mutant eye vesicles, transplanted at st. NF
21 to + / + endomesoderm, always contain retinal pigmented epithelium which
has been induced to melanogenesis, either in all cells or only in the most ventral
cells. The inherited amelanotic state of these cells was overcome by the endomesodermal influences of + / + embryos. In reciprocal eye transplantations at
this stage only the ventral parts of + / + pigmented epithelium were amelanotic,
whereas those parts which were subjected to melanogenic induction at earlier
stages were unable to reverse their melanized state. These results clearly show
that the ventral part of the retinal pigmented epithelium undergoes melanogenic
induction last.
Therefore, the temporal framework of melanogenic induction in normal
development stretches only between the stages of middle-late gastrula and tail
bud and depends on two sources of equivalent strength. The results of the present
investigation show that competence of eye material for melanogenic induction
lasts till st. NF 30-32.
Grafting of presumptive eye material of + / + embryos to an a^/a* environment causes failure of embryonic melanoprotein induction in pigmented
epithelial cells, and thus allows us to estimate quantitatively the contribution of
pigment granules of egg origin to the overall population of melanosomes.
Like many other induction processes, that for melanogenesis, and its expression as melanin synthesis, is irreversible once accomplished, demonstrating the
switch-like character of this cellular response in development.
The selective induction of the pigmented epithelial cell type may be better
understood if we consider the mutation of eyeless (e) in Ambystoma. The
eyeless mutation, which is expressed, among other peculiarities, in the complete
absence of eyes, affects the competence of neuroectodermal material (van
Deusen, 1973; Epp, 1978). If we suppose that a certain competence might
depend on the synthesis of specific proteins (Grunz, 1970), the absence of the
protein synthesis, coded for by gene e, located in neuroectoderm would make
the formation of eye material impossible. In our experiments with <2p mutants
the competence for eye material formation is available, but the absence of the
factors mediating melanogenic induction may result in the absence of the
186
O. A. HOPERSKAYA AND O. N. GOLUBEVA
product of terminal differentiation within cells of the normally pigmented
retinal epithelium. Generally speaking we can suppose that the formation of the
pigmented epithelial cell type might depend on the interaction of factors
determining competence, and localized in neuroectodermal cells, with factors
determining induction and entering these cells from their environment. Investigation of the process of melanocyte differentiation can serve as the starting
point for deeper insight into mechanisms of the correlation between induction
and competence in the emergence of individual cell types.
One of the most intriguing questions is that of the nature of the factors
mediating melanogenic induction. The present results show that L-tyrosine
cannot be the activator of melanogenesis, and apparently does not play such a
role in normal development.
The emergence of melanin-containing cells among the early embryonic cells of
Ambystoma treated with L-tyrosine or some other chemicals (Landstrom &
L0vtrup, 1978; Barth & Barth, 1974) tells us nothing of the normal induction of
melanogenesis. It is known that living embryonic cells contain many more
potential-inducing factors than those which are normally active (Born, Tiedemann & Tiedemann, 1972; Tiedemann, 1975). The release of inducing factors
which are normally inactive, together with the ease with which Ambystoma
embryonic cells in cultures can deteriorate may account for unspecific inductions
without inducers.
The approach developed in the present communication suggests how the true
determination of melanocytes arises, how an individual melanocyte cell type is
brought about by different inductive actions, and how it is possible to turn on or
off melanin-biosynthesizing machinery in cells at certain stages of development
by the action of a melanogenic factor, localized in endomesoderm.
We express here our gratitude to Professor G. V. Lopashov for his advice and critical
reading of the manuscript, and to Dr Jonathan Cooke for his editorial work.
REFERENCES
BARTH, L. G. & BARTH, L. J. (1974). Ionic regulation of embryonic induction and cell
differentiation in Ranapipiens. DevlBiol. 39,1-22.
BLUEMINK, J. G. & HOPERSKAYA, O. A. (1975). Ultrastructural evidence for the absence of
premelanosomes in eggs of the albino mutant (aP) of Xenopus laevis. Wilhelm Roux Archiv.
Entw Mech. Org. Ill, 75-79.
BORN, J., TIEDEMANN, H. & TIEDEMANN, H. (1972). The mechanism of embryonic induction:
Isolation of an inhibitor for vegetalising factor. Biochem. biophys Acta 270, 175-183.
BRIGGS, R. (1973). Developmental genetics of the axolotl. In Genetic Mechanisms of Development (ed. by F. H. Ruddle), pp. 169-199. New York: Academic Press.
BROWN, A. L. (1970). The African Clawed Toad. London: Butterworths.
BURLAKOVA, O. V. (1978). Influence of hormones, caused the pigment migrations in dermal
melanophores, on retinal epithelium. Bull. Moscow Univ., Ser. Biol. 3, 51-57.
CHEN, S., WAHN, H., TURNER, W. A., TAYLOR, D. & TCHEN, T. T. (1974). MSH, cyclic AMP
and melanocyte differentiation. Rec. Prog, in Hormone Res. 30, 319-345.
Melanogenic induction in pigmented retinal cells
187
N. V. & SLEPTSOVA, L. A. (1975). Common European frog: Rana temporariaL.
In Objects of Developmental Biology (ed. by T. A. Detlaff), pp. 442-462. Moscow: Nauka.
DEUSEN, E. VAN (1973). Experimental studies on a mutant gene (e) preventing the differentiation of eye and normal hypothalamus primordia in the axolotl. Devi Biol. 34, 135-158.
DOEZEMA, P. (1973). Protein from melanosomes of mouse and chick pigment cells. /. cell
Physiol. 82,65-74.
EPP, L. G. (1978). A review of the eyeless mutant in the Mexican axolotl. Amer. Zool. 18,
267-272.
FUJII, R., NAKAZAWA, T. & FUJIE, J. (1973). Effects of ultraviolet radiation on melanophore
system of fish. In Mechanisms of Pigmentation: Pigment Cell (ed. by V. I. McGovern &
P. Russel), pp. 195-201. Basel: Karger.
DABAGYAN,
GILBERT, E. F., MCGORD, R. G., SKIBBA, J. L., FALLON, J. F., GROFT, W. A. & JAUSCHKE,
W. F. (1973). Local recurrence and diffuse multiple melanoma following chronic oral
administration of L-Dopa. In Mechanisms of Pigmentation: Pigment Cell (ed. by J. M.
McGovern and P. Russel), pp. 300-307. Basel: Karger.
GRUNZ, H. (1970). Abhangigkeit der Kompetenz des Amphibien-Ektoderms von der
Proteinsynthese. Wilhelm Roux Arch. Entw Mech. Org. 165, 283-293.
HOPERSKAYA, O. A. (1975). The development of animals homozygous for a mutation causing
periodic albinism (ap) in Xenopus laevis. J. Embryol. exp. Morph. 34, 253-264.
HOPERSKAYA, O. A. (1977). Normalization of the melanin synthesis in the pigmented epithelium of «p embryos in Xenopus laevis. C. R. Ac. Sci. U.S.S.R. 236, 998-1000.
HOPERSKAYA, O. A. (1978). Melanin synthesis activation dependent on inductive influences.
Wilhelm Roux Arch. devlBiol. 184,15-28.
HOPERSKAYA, O. A. (1980). Induction - the main principle of melanogenesis in early development. Differentiation (In the Press.).
HOPERSKAYA, O. A. & GOLUBEVA, O. N. (1977). Experimental study of mutant gene aP
preventing normal differentiation of melanin-synthesizing cells. Genetics (U.S.S.R.) 13,
2129-2140.
HOPERSKAYA, O. A. & GOLUBEVA, O. N. (1979). Determination of temporal parameters and
irreversibility of the process of melanoprotein induction in retinal pigmented epithelium in
Amphibia. C. R. Ac. Sci. U.S.S.R. 249,726-729.
LANDSTROM, U. & LGVTRUP, S. (1978). Tyrosine and the synthesis of melanin in embryonic
cells from Ambystoma mexicanum. Wilhelm Roux Arch, devl Biol. 185, 201-207.
LOPASHOV, G. V. (1963). Developmental Mechanisms of Vertebrate Eye Rudiments. Oxford:
Pergamon Press.
LOPASHOV, G. V. & HOPERSKAYA, O. A. (1967). On the character of cell interaction during
differentiation of eye rudiments in amphibians. C. R. Ac. Sci. U.S.S.R. 175, 962-965.
MACMILLAN, G. J. (1979). An analysis of pigment cell development in the periodic albino
mutant of Xenopus. J. Embryol. exp. Morph. 52,165-170.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus laevis (Daudin). Amsterdam :
North-Hoil. Publ. Co.
NIEUWKOOP, P. D., OIKAWA, J. & BODDINGIUS, J. (1958). The anterior transverse neural fold
in amphibians. Arch. Neerl.Zool. 13,167-184.
RILEY, P. (1977). The mechanism of melanogenesis. Symp. Zool. Soc. Lond. 39, 77-96.
RUGH, R. (1962). Experimental Embryology. Minneapolis: Burgess.
TAHARA, J. (1962 a). Formation of the independent lens in Japanese amphibians. Embryologia,
7, 127-149.
TAHARA, J. (19626). Determination of the independent lens rudiment in Rhacophorus schlegelli arboreus Okada et Kawana. Embryologia 7,150-168.
TOMPKINS, R., KNIGHT, R. & BURKE, M. (1977). Tyrosinase activity and melanin synthesis in
the periodic albino mutant of Xenopus laevis. Amer. Zool. 17,920.
TIEDEMANN, H. (1975). Substances with morphogenetic activity in differentiation of vertebrates. In Biochemistry of Animal Development 3,258-287.
VATTI, K. V. & ALEKSEEVICH, J. A. (1976). Comparative genetics and ontogenetics of colours
in amphibians. In Physiological Genetics (ed. by M. E. Lobashov & S. G. Inge-Vechtomov),
pp. 326-350. Moscow: Medizina.
188
O. A. HOPERSKAYA AND O. N. GOLUBEVA
WHTTTAKER, J. R. (1974). Aspects of differentiation and determination in pigment cells. In
Concepts of Development (ed. by L. Lash & I. R. Whittaker). pp. 163-178. Stamford,
Sinauer Association.
WYLLIE, A. H. & DE ROBERTIS, E. M. (1976). High tyrosinase activity in albino Xenopus
laevis oocytes. /. Embryol. exp. Morph. 36,555-559.
WOERT, M. H. VAN (1973). Some properties of the outer melanosomal membrane. In Mechanisms of Pigmentation: Pigment Cell (ed. by V. J. Govern & P. Russel), pp. 125-133.
Basel: Karger.
{Received 20 November 1979, revised 12 May 1980)