Download (a ) in Xenopus laevis

/ . Embryol. exp. Morph. Vol. 34, 1, pp. 253-264, 1975
253
Printed in Great Britain
The development of animals homozygous
for a mutation causing periodic albinism (ap) in
Xenopus laevis
ByOLGA A. HOPERSKAYA 1
From the institute of Developmental Biology, Academy of Sciences
of U.S.S.R., Moscow
SUMMARY
This paper describes the development of a mutant strain associated with periodic albinism
(ap) in the clawed toad Xenopus laevis. The most outstanding feature of this mutation is the
instability of the albino state. In the course of the development there is a succession of three
periods of pigment expression: (1) complete absence of melanin pigment, (2) appearance of
melanin in the pigmented epithelium of the eyes and in small quantities in skin melanophores,
(3) disappearance of most pigment granules. Repeated spawnings show that the mutant
syndrome is inherited as a recessive trait. Possible ways of analysing pigment cell differentiation with the use of the mutation described are discussed.
INTRODUCTION
The value of the pigment-synthesizing system in vertebrates as a model for the
study of gene expression is well known. The pattern of pigmentation as well as
the quantitative aspects of its manifestation are properties which allow the
analysis of gene function and the study of external influences on these functions
during development. Coloration in amphibia results from the interrelations of
different pigments synthesized in melanophores, xanthophores, erythrophores
and iridophores (Bagnara, 1966). Colour mutations in amphibia are relatively
rare and they are of great importance for investigations in developmental
biology (Markert & Ursprung, 1971).
Albinism presents one of the more valuable pigment mutations. It is characterized by the lack of melanin synthesis by pigmented cells of two types: (1)
pigmented epithelium cells of the eye; (2) melanophores originating from the
neural crest from where they migrate all over the body. True albinos do not
contain pigmented granules (melanosomes) either in the skin or in pigmented
epithelium. Their eyes appear pink because of the colour of blood in small
vessels in the iris stroma and choroid coat. In amphibia, albino mutations have
been described up to this time in frogs (Rana temporaria and R. pipiens) and in
1
Author's address: Instituteof Developmental Biology Ac. Sci., U.S.S.R., Vavilov Street 26,
Moscow 117334, U.S.S.R.
254
O. A. HOPERSKAYA
Fig. 1. Adult toads of Xenopus laevis carrying the mutation of periodic albinism, av.
the axolotl (Smallcombe, 1949; Browder, 1972; Smith-Gill, Richards & Nace,
1972; Humphrey, 1967). In Xenopus, albino mutations have not yet been reported
(Gallien, 1968; Gurdon & Woodland, 1974; Droin, 1974).
An advantageous model for study of melanin synthesis is provided by the
mutation periodic albinism (ap) which appeared spontaneously in 1972 in the
Xenopus laevis colony of the Institute of Developmental Biology in Moscow.
The purpose of the present communication is to describe the normal development of animals carrying this mutation. Special attention will be paid to the
development of melanocytes of the pigmented epithelium of eyes as well as to
dendritic melanophores developing in skin and in the choroid coat of the eye.
The mutation periodic albinism (ap) is characterized by (1) the complete absence
of melanin in oocytes and embryos, (2) the appearance of melanin at larval
stages and (3) the almost complete disappearance of melanin in metamorphosed
animals.
OBSERVATIONS
Coloration of adult animals
'Albino' adults (Fig. 1) are characterized by a cream colour dorsally and a
silvery-white colour ventrally. Immediately after metamorphosis, animals have
pink eyes and pinkish skin; the skin becomes somewhat cream in colour as
Periodic albinism in Xenopus
255
1 //m
Fig. 2. em, Expelled melanosomes; chc, choroid coat; m, melanosomes; ope, outgrowths of pigmented epithelium; pe, pigmented epithelium; phr, photoreceptors;
r, retina; v, vacuoles.
(A-D) Details of pigmented epithelium structure in adult Xenopus laevis of av strain.
(A) Thickened pigmented epithelium without pigmented granules.
(B) Part of pigmented epithelium with melanin granules and outgrowths in the
vicinity of optic nerve.
(C) Vacuolization of pigmented epithelium cells.
(D) Clumps of the expelled pigmented granules.
(E) Structure of pigmented epithelium in normal adult Xenopus laevis.
17
EMB 34
256
O. A. HOPERSKAYA
Table 1. Comparative table of data on pigmentation
Numbers refer to stage numbers of Nieuwkoop & Faber (1956).
Genotype
+/+
ap/ap
Pigmentation
from egg to
neurula (oocytederived pigment)
Animal pole and
derived structures
are pigmented
Eggs and embryos
are milk-white
colour all over if
derived from ap/ap
mother
Stage of onset
Stage of
of pigmenta- appearance of Stage of complete
tion in
melanophores depigmentation
eyes
in skin
in skin
29-30
32
Depigmentation
does not occur
39-40
43
Between 48 and
63 according to
spawning
they grow. The cream colour is determined apparently by the presence of
xanthophores. In adults, black melanin is found only in horny claws on three
toes. In addition, males may have a small weakly pigmented strip on the inner
surface of forelimbs; i.e. on the nuptial pads. Eyes of adult animals are
pink, though around the limbus and in the vicinity of optic nerve pigmented
epithelium contains a small quantity of melanin granules. In respect to this
attribute, animals carrying the ap mutation differ from true albinos (Humphrey, 1967) which would have melanin in their horny claws but not in their
pigmented epithelium. In aP animals, those cells of the pigmented epithelium
which contain melanin are concentrated in the vicinity of the optic nerve. The
number of pigmented granules per cell varies from 3-5 to 30-40 (Fig. 2).
Melanophores of the choroid coat contain only a few pigmented granules.
Neither internal organs nor coelomic linings contain melanin. A remarkable
individual variability in the intensity of pigmentation is observed. Thus av
'albinos' cannot be classified as true albinos.
Growth rate and changes in the pattern of pigmentation during development
After the standard injection of gonadotropic hormone, adult animals lay
milk-white eggs. Their appearance differs sharply from that of normal eggs in
which the animal hemisphere is dark and the vegetal hemisphere white (Fig. 3).
In the wild-type embryo melanin derived from the oocyte remains during
embryogenesis, particularly in ectoderm. avjav and wild-type embryos develop
at the same rate during early development, but at later stages, aplav embryos
develop more slowly. Though the presence of the pigment melanin is not essential for normal development (Balinsky, 1970), its complete absence is correlated
with slower growth and development. This was clearly observed in the course of
Periodic albinism in Xenopus
257
Fig. 3. (A) Milk-white eggs of a mutant strain av of Xenopus laevis; x 16.
(B) Pigmented eggs of wild-type Xenopus laevis; x 16.
development of two synchronously obtained spawnings of av\av and wild-type
embryos which were allowed to develop in the dark at a constant temperature of
+ 20 °C. A noticeable difference in the rate of growth was found at about stage 45
(Nieuwkoop & Faber, 1956) when embryos of both spawnings had achieved the
same morphological stage, but wild-type embryos had reached a greater length
(12 mm, while a'^a" embryos were only 8 mm). At later stages, avjav embryos
also develop slower than wild-type ones. Embryos which are the progeny of the
cross a"la" $ x + / + J are pigmented like wild-type embryos but develop at the
rate typical for white embryos.
The first indication of pigmentation appears in avjav embryos of all spawnings
at stage 39-40. First of all, pigmented granules appear in the eye rudiments. Up
till stage 42 the dorsal part of a'}jav eyes is pigmented much more heavily than
the ventral part (Fig. 4). The pigmented epithelium of dP\av embryos does not
become uniformly pigmented while in + / + embryos the pigmented epithelium
is uniformly pigmented from as early as stage 38. At stage 43 the eyes ofaplap
embryos are becoming heavily pigmented (Fig. 5). At this same stage melanophores appear but they are only just visible and are not at first dendritic. In
the course of further development the form of skin melanophores becomes more
complicated. At stage 45-46 the skin melanophores acquire a complex stellate
morphology. However, even at the climax of skin pigmentation av\av mutants
never reach the same degree of pigmentation as wild-type tadpoles. In the course
of further development up to approximately stage 56, the pigmentation of avjav
embryos increases, though the rate and the intensity of their melanin synthesis
display the individual variability. Stage 49 is characterized by the start of pigmentation in the choroid coat in av\av tadpoles, whereas in wild-type tadpoles the
choroid coat is already fully loaded with pigment at this stage. At stage 55,
the iris o>{av\av mutants is becoming bleached; it looses pigment earlier than the
258
O. A. HOPERSKAYA
A
01 mm
01 mm
Fig. 4. Successive stages of differentiation of pigmented epithelium in the a? mutant
strain in comparison with those of wild-type tadpoles.
(A) Pigmented epithelium of mutant ap larvae before the onset of the appearance of
pigmented granules. Stages 37-38.
(B) Pigmented epithelium of wild-type larvae. Stages 37-38.
(C) Pigmented epithelium of tadpole of av strain. Dorsal part of eye is more pigmented. Stage 42.
(D) Pigmented epithelium of a wild-type tadpole of the same stage.
Periodic albinism in Xenopus
259
Fig. 5. White larvae at the stage of completely black eyes. Stage 43; x 10.
Fig. 6. (A) Bleaching of eyes in larvae of the mutant strain av. Stage 45; x 12.
(B) The same stage of wild-type larvae, x 12.
Fig. 7. Illustration of variability in the process of depigmentation in different
batches of tadpoles of the mutant strain av at stage 57-58. (A) Tadpole with completely depigmented eyes; x 2. (B) Tadpole with black eyes; x 2.
pigmented epithelium, which at this stage remains uniformly pigmented. At
about this stage, the involution of melanophores begins.
The first visible pigmented granules appear at the same stage in all av\av
spawnings but depigmentation takes place earlier than usual in some spawnings.
Consequently a shortening of the period of melanogenesis is observed. In the
course of depigmentation a number of brilliant colourless structures similar to
dendritic melanophores without melanin appear on the body of tadpoles. The
more pigmented were mutant embryos the more clearly such structures are
expressed. Melanophores change shape during regression in the reverse order
to that in which they arise. During regression, the form becomes progressively
simpler; at first they have a dendritic form, then they become round like small
spots; at last they disappear altogether. In some avjav spawnings almost complete eye depigmentation occurs at stage 63 whereas in others light-grey eyes are
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O. A. HOPERSKAYA
found as early as stage 53. Up to these stages, pigment in the skin is almost
absent.
In ap/av larvae, the gradual disappearance of melanin granules in the pigmented epithelium and in melanophores appears to proceed in different ways. As
far as could be concluded from visual observations and from light microscopy,
the gradual disappearance of melanosomes in melanophores occurs in situ. In
the course of depigmentation of the pigmented epithelium, the formation of
different-sized clumps of pigmented granules in the choroid coat takes place.
This suggests the loss of melanosomes without the mediation of melanophages,
a process which is, however, less frequent than one in which melanophages are
involved (Eguchi, 1963; Dumont & Yamada, 1972; Keefe, 1973). This suggests
that melanosome elimination in ap/ap larvae is analogous to the process of egg
pigment expulsion from the retina and cornea in embryonic development of
Rana pipiens (Hollyfield, 1973) or in the process of lens regeneration in vitro
from the iris in newts (Yamada, Reese & McDevitt, 1973).
Here I shall not describe all peculiarities of the structure of the pigmented
epithelium of adult ap/ap Xenopus but only aspects that are pertinent. Cells of
the pigmented epithelium are almost devoid of melanin granules. However, some
pigment exists mainly in cells which are situated ventrally to the optic nerve.
Pigment-containing cells of the pigmented epithelium form clearly visible outgrowths. Those cells which lack melanin granules stand in marked contrast to the
normal pigmented epithelium as regards their thickness. They form a cylindrical
epithelium which is twice as large as that of wild-type animals. Probably the
formation of such cells is a consequence of thickening and fusion of their
outgrowths (Fig. 2). Such a thickened pigmented epithelium contains a lot of
vacuoles which are not arranged in any definite order (Fig. 2). The thickening
and vacuolization of the pigmented epithelium is a peculiarity of ap/ap animals.
These properties are not typical either of true albino forms (Dowling & Gibbons,
1962) or of species in which the pigmented epithelium becomes free of pigmented granules in the course of normal differentiation (Baburina &
Vybornykh, 1968).
The formation and character of iridophore distribution in ap/ap larvae correspond to those of normal larvae (Bagnara, 1957). The character of xanthophore
distribution is also unaffected in this mutation.
Breeding experiments
p
In numerous crosses of a /ap females x ap/ap males, uniformly white embryos
were always obtained, which differed only in intensity of pigmentation and in the
stage of depigmentation. The main features characterizing this mutation were
retained.
In crosses of apjap females with + / + males, the resulting eggs and embryos
do not contain any pigment at early stages of development, but at advanced
stages the character of their pigmentation gradually becomes like that of + / +
Periodic albinism in Xenopus
261
9C
v
Fig. 8. (A) Larvae of the a strain. Stages 33-34; x 16. (B) Larvae of av ? x <y of
wild-type. Stages 33-34; x 16. (C) Larvae of wild-type. Stages 33-34; x 16.
Fig. 9. (A) Larvae of the mutant strain av. First pigmented granules in eyes. Stage 39;
x 16. (B) Larvae of a p $ x £ of wild type. Stage 39; x 16. (C) Larvae of wild-type.
Stage 39; x 16.
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O. A. HOPERSKAYA
individuals. The first pigment granules appear in their eyes at the same time as
normal embryos, but the pigmentation is less intense (Figs. 8, 9). This suggests
that mutant syndrome is inherited as typical recessive trait.
DISCUSSION
The mutation described is peculiar because it differs both from a true albino
where melanin synthesis is inhibited completely (Reed, 1938; Humphrey, 1967;
Browder, 1972) and from the local suppression of melanin-synthesizing genes
known in white Ambystoma and White Leghorn chickens (Dalton, 1953;
Rawles, 1948). The most outstanding feature of the a1J mutation is the succession
of periods of albinism and pigmentation, manifestated at different stages of development. There is little doubt that the gene system responsible for melanin
synthesis persists in cells of avjav animals; evidently some factors must inhibit
the expression of these genes during two periods of development. An important
question is whether the inhibition of synthesis of new melanin granules results
necessarily in the degradation of those already formed, or whether the degradation of these requires some additional influences.
One important distinction that can be drawn between stages where melanin
synthesis proceeds and those where it is absent concerns hormone activity.
Oocytes developing in the body of a female are exposed to the action of hormones;
at early stages of development there is no hormonal activity but hormones again
begin to act at stages of advanced development close to metamorphosis when
the endocrine glands of larvae become active themselves. It is possible that the
genes responsible for melanin synthesis are controlled by regulator genes whose
activity may be hormone-dependent. The av mutation may, of course, affect
a regulatory gene (Britten & Davidson, 1969), and not genes directly responsible
for melanin synthesis.
Other possible reasons for the phenotypic manifestation of the av mutant
include (a) a deficiency or inhibition of tyrosinase activity (Whittaker, 1968) or
(b) an inability to form the first generation of premelanosomes and melanosomes
which are known (Eppig & Dumont, 1974) to form during oogenesis. Mutations
disturbing these processes can occur independently (Moyer, 1966). Investigations that could help to find the real explanation among those enumerated
include (1) the injection of different hormones at stages before pigmentation,
and reciprocal eye and skin exchange between normal and mutant strains, (2)
the study of tyrosinase activity at different stages of development during pigmentation and depigmentation, and (3) electronmicroscopic studies of premelanosomes. All these approaches are now in progress.
The last comment concerns the role of melanosomes and premelanosomes
in cell morphology. It appears from the study of the a'J mutation that they may
determine the morphology of pigmented epithelium and melanophores. In the
course of depigmentation melanophores get smaller and seem to disappear if
Periodic albinism in Xenopus
263
not converted into mesenchymal dermal cells. In the process of melanosome
elimination from pigmented epithelium, sharp differences are observed between
pigmented and depigmented cells. Depigmented cells become thicker, vacuolized, and loose their cytoplasmic processes. Ultrastmctural analysis is indispensable for the study of fine structure of pigmented epithelium, but already we
can ask if the developmental capacities of the normal and depigmented eye
epithelium are identical. It is known that the pigmented epithelium of tadpoles
and adult Xenopus, in contrast to that of newts, cannot transform into retina
(Sologub, 1975). The elimination of melanin granules is a prerequisite for the
transformation of iris cells into lens (Yamada et al. 1973) and of the pigmented
epithelium into retina (Lopashov & Sologub, 1972; Keefe, 1973). It cannot be
excluded a priori that processes leading to depigmentation can change the metaplastic properties of pigmented epithelium of Xenopus laevis carrying the a1'
mutation.
I wish to express my gratitude to Mrs T. I. Shumilina and Mrs N. K. Pastuhova for their
expert technical assistance. I am indebted to Dr J. B. Gurdon for his invaluable help in the
correction of the English.
REFERENCES
BABURINA, E. A. & VYBORNYKH, G. S. (1968). Development of eyes in Trygon pastinaca. In
Morpho-Ecological Investigations of Fish Development, pp. 21-39. Moscow: Nauka.
BAGNARA, J. T. (1957). Hypophysectomy and the tail darkening reaction in Xenopus. Proc.
Soc. exp. Biol. Med. 94, 572-575.
BAGNARA, J. T. (1966). Cytology and cytophysiology of non-melanophore pigment cells.
Int. Rev. Cytol. 20, 173-205.
BALINSKY, B. T. (1970). An Introduction to Embryology. Philadelphia: Saunders.
BRITTEN, R. J. & DAVIDSON, E. H. (1969). Gene regulation in higher cells: a theory. Science,
N. Y. 165, 349-357.
BROWDER, J. W. (1972). Genetic and embryological studies of albinism in Ranapipiens. J. exp.
Zool. 180, 149-155.
DALTON, H.C. (1953). Relations between developing melanophores and embryonic tissues
in the Mexican axolotl. In Pigment Cell Growth (ed. M. Gordon), pp. 17-27. New York:
Academic Press.
DOWLING, J. E. & GIBBONS, I. R. (1962). The fine structure of the pigment epithelium in the
albino rat. / . Cell Biol. 14, 459-474.
DROIN, A. (1974). Trois mutations recessives letales, 'dwarf-F (dw-I), 'dwarf-W (dw-II) et
'precocious oedema' (p.oe) affectant les tetards de Xenopus laevis. Annls Embryol. Morph.
7, 141-150.
DUMONT, J. M. & YAMADA, T. (1972). Dedifferentiation of iris epithelial cells. Devi Biol. 29,
385-401.
EGUCHI, G. (1963). Electronmicroscopic studies on lens regeneration. 1. Mechanism of
depigmentation of the iris. Embryologia 8, 45-62.
EPPIG, J. J. & DUMONT, J. N. (1974). Oogenesis in Xenopus laevis (Daudin). II. The induction
and subcellular localization of tyrosinase activity in developing oocytes. Devi Biol. 36, 330342.
GALLIEN, L. (1968). Mutations spontanees obtenues chez les Amphibiens Ambystoma
mexicanum Shaw, Pleurodeles waltlii Michah., Xenopus laevis Daudin. Archs Anat. Histol.
Embryol. 51, 249-253.
GURDON, J. B. & WOODLAND, H. R. (1974). Xenopus. In Handbook of Genetics, vol. 3 (ed.
R. C. King). New York: Plenum Press. (In the Press.)
264
O. A. HOPERSKAYA
J. G. (1973). Elimination of egg pigment from developing ocular tissues in the
frog Ranapipiens. Devi Biol. 30, 115-128.
HUMPHREY, R. R. (1967). Albino axolotls from an albino tiger salamander through hybridization./. Hered. 58, 95-101.
KEEFE, J. R. (1973). An analysis of Urodelian retinal regeneration. III. Degradation of
extruded melanin granules in Notophtalmus viridescens. J. exp. Zool. 184, 233-237.
LOPASHOV, G. V. & SOLOGUB, A. A. (1972). Artificial metaplasia of pigmented epithelium
into retina in tadpoles and adult frogs. J. Embryol. exp. Morph. 28, 521-546.
MARKERT, C. L. & URSPRUNG, H. (1971). Developmental Genetics. Englewood Cliffs: PrenticeHall.
MOYER, F. (1966). Genetic variations in the fine structure and ontogeny of mouse melanin
granules. Am. Zool. 6, 43-66.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal table of Xenopus laevis (Daudin). Amsterdam:
North-Holland Publ. Co.
RAWLES, M. E. (1948). Origin of melanophores and their role in development of color pattern
in vertebrates. Physiol. Zool. 28, 383-408.
REED, S. C. (1938). Determination of hair pigment. III. Proof that expression of the black and
tan gene is dependent upon tissue organization. /. exp. Zool. 79, 337-346.
SMALLCOMBE, W. A. (1949). Albinism in Rana temporaries. J. Genet. 49, 286-290.
SMITH-GILL, S. J., RICHARDS, C. M. & NACE, G. W. (1972). Genetic and metabolic bases of
two 'albino' phenotypes in the leopard frog, Ranapipiens. J. exp. Zool. 180, 157-168.
SOLOGUB, A. A. (1975). Mechanisms of repression and derepression and of spatial organization of the regenerate upon artificial transformation of pigmented epithelium into retina in
Xenopus laevis. Wilhelm Roux Archiv. EntwMech. Org. (Submitted.)
WHITTAKER, J. R. (1968). The nature and probable cause of modulations in pigment cell
culture. In The Stability of Differentiated State, pp. 25-35. Berlin: Springer.
YAMADA, T., REESE, D. H. & MCDEVITT, D. S. (1973). Transformation of iris into lens in
vitro and its dependency on neural retina. Differentiation 1, 65-82.
HOLLYFIELD,
{Received 24 February 1975)