/ . Embryol. exp. Morph. Vol. 34, 1, pp. 253-264, 1975 2 5 3

Printed in Great Britain

The development of animals homozygousfor a mutation causing periodic albinism (ap) in

Xenopus laevis

ByOLGA A. HOPERSKAYA1

From the institute of Developmental Biology, Academy of Sciencesof 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 theinstability of the albino state. In the course of the development there is a succession of threeperiods of pigment expression: (1) complete absence of melanin pigment, (2) appearance ofmelanin 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 mutantsyndrome is inherited as a recessive trait. Possible ways of analysing pigment cell differentia-tion with the use of the mutation described are discussed.

INTRODUCTION

The value of the pigment-synthesizing system in vertebrates as a model for thestudy of gene expression is well known. The pattern of pigmentation as well asthe quantitative aspects of its manifestation are properties which allow theanalysis of gene function and the study of external influences on these functionsduring development. Coloration in amphibia results from the interrelations ofdifferent pigments synthesized in melanophores, xanthophores, erythrophoresand iridophores (Bagnara, 1966). Colour mutations in amphibia are relativelyrare and they are of great importance for investigations in developmentalbiology (Markert & Ursprung, 1971).

Albinism presents one of the more valuable pigment mutations. It is charac-terized by the lack of melanin synthesis by pigmented cells of two types: (1)pigmented epithelium cells of the eye; (2) melanophores originating from theneural crest from where they migrate all over the body. True albinos do notcontain pigmented granules (melanosomes) either in the skin or in pigmentedepithelium. Their eyes appear pink because of the colour of blood in smallvessels in the iris stroma and choroid coat. In amphibia, albino mutations havebeen 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 themutation periodic albinism (ap) which appeared spontaneously in 1972 in theXenopus laevis colony of the Institute of Developmental Biology in Moscow.The purpose of the present communication is to describe the normal develop-ment of animals carrying this mutation. Special attention will be paid to thedevelopment of melanocytes of the pigmented epithelium of eyes as well as todendritic melanophores developing in skin and in the choroid coat of the eye.The mutation periodic albinism (ap) is characterized by (1) the complete absenceof melanin in oocytes and embryos, (2) the appearance of melanin at larvalstages and (3) the almost complete disappearance of melanin in metamorphosedanimals.

OBSERVATIONS

Coloration of adult animals

'Albino' adults (Fig. 1) are characterized by a cream colour dorsally and asilvery-white colour ventrally. Immediately after metamorphosis, animals havepink 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, out-growths 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 thevicinity 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

Pigmentationfrom egg to

neurula (oocyte-derived pigment)

Stage of onsetof pigmenta-

tion ineyes

Stage ofappearance ofmelanophores

in skin

Stage of completedepigmentation

in skin

+/+ Animal pole and 29-30derived structuresare pigmented

ap/ap Eggs and embryos 39-40are milk-whitecolour all over ifderived from ap/ap

mother

32

43

Depigmentationdoes not occur

Between 48 and63 according tospawning

they grow. The cream colour is determined apparently by the presence ofxanthophores. In adults, black melanin is found only in horny claws on threetoes. In addition, males may have a small weakly pigmented strip on the innersurface of forelimbs; i.e. on the nuptial pads. Eyes of adult animals arepink, though around the limbus and in the vicinity of optic nerve pigmentedepithelium contains a small quantity of melanin granules. In respect to thisattribute, animals carrying the ap mutation differ from true albinos (Hum-phrey, 1967) which would have melanin in their horny claws but not in theirpigmented epithelium. In aP animals, those cells of the pigmented epitheliumwhich contain melanin are concentrated in the vicinity of the optic nerve. Thenumber 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 remarkableindividual 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 laymilk-white eggs. Their appearance differs sharply from that of normal eggs inwhich the animal hemisphere is dark and the vegetal hemisphere white (Fig. 3).In the wild-type embryo melanin derived from the oocyte remains duringembryogenesis, particularly in ectoderm. avjav and wild-type embryos developat the same rate during early development, but at later stages, aplav embryosdevelop more slowly. Though the presence of the pigment melanin is not essen-tial for normal development (Balinsky, 1970), its complete absence is correlatedwith 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-typeembryos 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 thesame 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 embryosalso develop slower than wild-type ones. Embryos which are the progeny of thecross a"la" $ x + / + J are pigmented like wild-type embryos but develop at therate typical for white embryos.

The first indication of pigmentation appears in avjav embryos of all spawningsat stage 39-40. First of all, pigmented granules appear in the eye rudiments. Uptill stage 42 the dorsal part of a'}jav eyes is pigmented much more heavily thanthe ventral part (Fig. 4). The pigmented epithelium of dP\av embryos does notbecome uniformly pigmented while in + / + embryos the pigmented epitheliumis 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 melano-phores appear but they are only just visible and are not at first dendritic. Inthe course of further development the form of skin melanophores becomes morecomplicated. At stage 45-46 the skin melanophores acquire a complex stellatemorphology. However, even at the climax of skin pigmentation av\av mutantsnever reach the same degree of pigmentation as wild-type tadpoles. In the courseof further development up to approximately stage 56, the pigmentation of avjav

embryos increases, though the rate and the intensity of their melanin synthesisdisplay the individual variability. Stage 49 is characterized by the start of pigmen-tation in the choroid coat in av\av tadpoles, whereas in wild-type tadpoles thechoroid 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? mutantstrain in comparison with those of wild-type tadpoles.(A) Pigmented epithelium of mutant ap larvae before the onset of the appearance ofpigmented 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 pig-mented. 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 differentbatches of tadpoles of the mutant strain av at stage 57-58. (A) Tadpole with com-pletely depigmented eyes; x 2. (B) Tadpole with black eyes; x 2.

pigmented epithelium, which at this stage remains uniformly pigmented. Atabout 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 thecourse of depigmentation a number of brilliant colourless structures similar todendritic melanophores without melanin appear on the body of tadpoles. Themore pigmented were mutant embryos the more clearly such structures areexpressed. Melanophores change shape during regression in the reverse orderto that in which they arise. During regression, the form becomes progressivelysimpler; at first they have a dendritic form, then they become round like smallspots; at last they disappear altogether. In some avjav spawnings almost com-plete eye depigmentation occurs at stage 63 whereas in others light-grey eyes are

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260 O. A. HOPERSKAYA

found as early as stage 53. Up to these stages, pigment in the skin is almostabsent.

In ap/av larvae, the gradual disappearance of melanin granules in the pig-mented epithelium and in melanophores appears to proceed in different ways. Asfar as could be concluded from visual observations and from light microscopy,the gradual disappearance of melanosomes in melanophores occurs in situ. Inthe course of depigmentation of the pigmented epithelium, the formation ofdifferent-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 areinvolved (Eguchi, 1963; Dumont & Yamada, 1972; Keefe, 1973). This suggeststhat melanosome elimination in ap/ap larvae is analogous to the process of eggpigment expulsion from the retina and cornea in embryonic development ofRana pipiens (Hollyfield, 1973) or in the process of lens regeneration in vitrofrom the iris in newts (Yamada, Reese & McDevitt, 1973).

Here I shall not describe all peculiarities of the structure of the pigmentedepithelium of adult ap/ap Xenopus but only aspects that are pertinent. Cells ofthe pigmented epithelium are almost devoid of melanin granules. However, somepigment exists mainly in cells which are situated ventrally to the optic nerve.Pigment-containing cells of the pigmented epithelium form clearly visible out-growths. Those cells which lack melanin granules stand in marked contrast to thenormal pigmented epithelium as regards their thickness. They form a cylindricalepithelium which is twice as large as that of wild-type animals. Probably theformation of such cells is a consequence of thickening and fusion of theiroutgrowths (Fig. 2). Such a thickened pigmented epithelium contains a lot ofvacuoles which are not arranged in any definite order (Fig. 2). The thickeningand 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 pig-mented granules in the course of normal differentiation (Baburina &Vybornykh, 1968).

The formation and character of iridophore distribution in ap/ap larvae corre-spond to those of normal larvae (Bagnara, 1957). The character of xanthophoredistribution is also unaffected in this mutation.

Breeding experiments

In numerous crosses of ap/ap females x ap/ap males, uniformly white embryoswere always obtained, which differed only in intensity of pigmentation and in thestage of depigmentation. The main features characterizing this mutation wereretained.

In crosses of apjap females with + / + males, the resulting eggs and embryosdo not contain any pigment at early stages of development, but at advancedstages the character of their pigmentation gradually becomes like that of + / +

Periodic albinism in Xenopus 261

9C

Fig. 8. (A) Larvae of the av strain. Stages 33-34; x 16. (B) Larvae of av ? x <y ofwild-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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262 O. A. HOPERSKAYA

individuals. The first pigment granules appear in their eyes at the same time asnormal embryos, but the pigmentation is less intense (Figs. 8, 9). This suggeststhat mutant syndrome is inherited as typical recessive trait.

DISCUSSION

The mutation described is peculiar because it differs both from a true albinowhere melanin synthesis is inhibited completely (Reed, 1938; Humphrey, 1967;Browder, 1972) and from the local suppression of melanin-synthesizing genesknown in white Ambystoma and White Leghorn chickens (Dalton, 1953;Rawles, 1948). The most outstanding feature of the a1J mutation is the successionof periods of albinism and pigmentation, manifestated at different stages of de-velopment. There is little doubt that the gene system responsible for melaninsynthesis persists in cells of avjav animals; evidently some factors must inhibitthe expression of these genes during two periods of development. An importantquestion is whether the inhibition of synthesis of new melanin granules resultsnecessarily in the degradation of those already formed, or whether the degrada-tion of these requires some additional influences.

One important distinction that can be drawn between stages where melaninsynthesis 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 againbegin to act at stages of advanced development close to metamorphosis whenthe endocrine glands of larvae become active themselves. It is possible that thegenes responsible for melanin synthesis are controlled by regulator genes whoseactivity may be hormone-dependent. The av mutation may, of course, affecta regulatory gene (Britten & Davidson, 1969), and not genes directly responsiblefor melanin synthesis.

Other possible reasons for the phenotypic manifestation of the av mutantinclude (a) a deficiency or inhibition of tyrosinase activity (Whittaker, 1968) or(b) an inability to form the first generation of premelanosomes and melanosomeswhich are known (Eppig & Dumont, 1974) to form during oogenesis. Mutationsdisturbing these processes can occur independently (Moyer, 1966). Investiga-tions that could help to find the real explanation among those enumeratedinclude (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 pigmen-tation and depigmentation, and (3) electronmicroscopic studies of premelano-somes. All these approaches are now in progress.

The last comment concerns the role of melanosomes and premelanosomesin cell morphology. It appears from the study of the a'J mutation that they maydetermine the morphology of pigmented epithelium and melanophores. In thecourse of depigmentation melanophores get smaller and seem to disappear if

Periodic albinism in Xenopus 263not converted into mesenchymal dermal cells. In the process of melanosomeelimination from pigmented epithelium, sharp differences are observed betweenpigmented and depigmented cells. Depigmented cells become thicker, vacuo-lized, and loose their cytoplasmic processes. Ultrastmctural analysis is indis-pensable for the study of fine structure of pigmented epithelium, but already wecan ask if the developmental capacities of the normal and depigmented eyeepithelium are identical. It is known that the pigmented epithelium of tadpolesand adult Xenopus, in contrast to that of newts, cannot transform into retina(Sologub, 1975). The elimination of melanin granules is a prerequisite for thetransformation of iris cells into lens (Yamada et al. 1973) and of the pigmentedepithelium into retina (Lopashov & Sologub, 1972; Keefe, 1973). It cannot beexcluded a priori that processes leading to depigmentation can change the meta-plastic 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 theirexpert technical assistance. I am indebted to Dr J. B. Gurdon for his invaluable help in thecorrection of the English.

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{Received 24 February 1975)