The Development of Animals Homozygous for a Mutation Causing Periodic Albinism (Ap) 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. HOPERSKAYA1 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).

Pigmentation Stage of onset Stage of from egg to of pigmenta- appearance of Stage of complete neurula (oocyte- tion in melanophores depigmentation Genotype derived pigment) eyes in skin in skin +/+ Animal pole and 29-30 32 Depigmentation derived structures does not occur are pigmented ap/ap Eggs and embryos 39-40 43 Between 48 and are milk-white 63 according to colour all over if spawning derived from ap/ap mother 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 (Hum- phrey, 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 essen- tial 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

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 17-2 260 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 corre- spond to those of normal larvae (Bagnara, 1957). The character of xanthophore distribution is also unaffected in this mutation.

Breeding experiments In numerous crosses of ap/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 Fig. 8. (A) Larvae of the av strain. Stages 33-34; x 16. (B) Larvae of av ? x

17-3 262 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). Investiga- tions 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, vacuo- lized, and loose their cytoplasmic processes. Ultrastmctural analysis is indis- pensable 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 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 their expert technical assistance. I am indebted to Dr J. B. Gurdon for his invaluable help in the correction of the English.

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{Received 24 February 1975)