Unusual development of light-reflecting pigment cells in intact and regenerating tail in the periodic albino mutant of Xenopus laevis

Abstract

Unusual light-reflecting pigment cells, "white pigment cells", specifically appear in the periodic albino mutant (a(p) /a(p)) of Xenopus laevis and localize in the same place where melanophores normally differentiate in the wild-type. The mechanism responsible for the development of unusual pigment cells is unclear. In this study, white pigment cells in the periodic albino were compared with melanophores in the wild-type, using a cell culture system and a tail-regenerating system. Observations of both intact and cultured cells demonstrate that white pigment cells are unique in (1) showing characteristics of melanophore precursors at various stages of development, (2) accumulating reflecting platelets characteristic of iridophores, and (3) exhibiting pigment dispersion in response to α-melanocyte stimulating hormone (α-MSH) in the same way that melanophores do. When a tadpole tail is amputated, a functionally competent new tail is regenerated. White pigment cells appear in the mutant regenerating tail, whereas melanophores differentiate in the wild-type regenerating tail. White pigment cells in the mutant regenerating tail are essentially similar to melanophores in the wild-type regenerating tail with respect to their localization, number, and response to α-MSH. In addition to white pigment cells, iridophores which are never present in the intact tadpole tail appear specifically in the somites near the amputation level in the mutant regenerating tail. Iridophores are distinct from white pigment cells in size, shape, blue light-induced fluorescence, and response to α-MSH. These findings strongly suggest that white pigment cells in the mutant arise from melanophore precursors and accumulate reflecting platelets characteristic of iridophores.

Fig. 1

Pigment cells present in the wild-type tadpole tail (a−c) and the mutant tadpole tail (d−f) at stage 48. a The wild-type tail placed in BSS before α-MSH administration (transmitted light). b The wild-type tail after α-MSH administration (1 μg/ml) (transmitted light). c Ultrastructure of melanophores of the wild-type. d The mutant tail placed in BSS before α-MSH administration (incident light). e The mutant tail after α-MSH administration (1 μg/ml) (incident light). f Ultrastructure of white pigment cells in the mutant. While wild-type melanophores were filled with many mature melanosomes (c), white pigment cells in the mutant contained both irregular reflecting platelets (f, asterisk) and premelanosomes with internal lamellar structures (f, arrows). Note that white pigment cells (d,e) in the mutant responded to α-MSH and dispersed pigment organelles in the same manner as wild-type melanophores (a b)

Fig. 2

Differentiation of pigment cells from wild-type neural crest cells in culture using serum-free medium. a Differentiating melanophores and neural crest cells migrating out from the neural tube explant (nt) after 3 days in culture. b,c Differentiated melanophores and iridophores after 20 days in culture observed under transmitted light (b), or incident light (c). d,e Ultrastructure of differentiated melanophores (d) and iridophores (e) in culture. Wild-type melanophores, which differentiated first in culture, were dendritic and aggregated melanosomes (a,b). Note that melanophores also appeared on the neural tube explant (a). Wild-type iridophores, which differentiated later in culture, looked brown under transmitted light (b, arrowheads) and reflected light under incident illumination (c, arrowheads). Wild-type melanophores contained many melanosomes (d), while wild-type iridophores were filled with many rectangular reflecting platelets (e)

Fig. 3

Dopa and ammoniacal silver nitrate staining in mutant neural crest cell culture. a−c Differentiating melanophores in serum-supplemented medium on day 3 before dopa staining (a), and after dopa (b) and subsequent ammoniacal silver nitrate staining (c). d−f Melanophore precursors in serum-free medium on day 3 before dopa staining (d), and after dopa (e) and subsequent ammoniacal silver nitrate staining (f). Mutant melanophores differentiated in serum-supplemented medium and contained visible melanosomes (a). These melanophores showed strong dopa staining as well as ammoniacal silver nitrate staining (arrowheads). On the other hand, mutant neural crest cells were unmelanized in serum-free medium (d). These unmelanized cells were identified as melanophore precursors at various stages of development, because some cells were stained with both dopa and ammoniacal silver nitrate (arrows), whereas the other cells were stained with ammoniacal silver nitrate, but not with dopa (asterisks). The round bodies within the cells are yolk platelets

Fig. 4

Differentiation of pigment cells from mutant neural crest cells in culture using serum-free medium. a Neural crest cells migrating out from the neural tube explant (nt) after 3 days in culture. b,c Differentiated iridophores after 20 days in culture observed under transmitted light (b), or incident light (c). d−g Ultrastructure of melanophore precursors at various stages of development (d−f) and iridophores (g) in culture. In contrast to wild-type neural crest cells, melanophores did not differentiate from mutant neural crest cells in culture using serum-free medium (a,b). In the present culture condition, melanophore precursors at various stages of development were detected in the mutant (d−f). Mutant melanophore precursors at early to middle stages of development (d) contained stage I premelanosomes (m1) and stage II premelanosomes (m2). In contrast, mutant melanophore precursors at the late stage of development (f) contained stage II premelanosomes and a small number of partially melanized stage III melanosomes (m3). The number of premelanosomes varied among melanophore precursors (d−f). Mutant iridophores differentiated in culture (b,c, arrowheads) in the same manner as wild-type iridophores. However, reflecting platelets of mutant iridophores were irregular in size and shape (g, asterisk)

Fig. 5

Physiological and ultrastructural characteristics of white pigment cells which were cultured from mutant tadpole tails at stage 52. a,b Cultured white pigment cells before α-MSH administration observed under transmitted light (a), or incident light (b). c,d The same fields as (a) and (b), respectively, after α-MSH administration (1 μg/ml) observed under transmitted light (c), or incident light (d). e,f Ultrastructural variation of white pigment cells in culture. White pigment cells which had a few dendrites dispersed pigment organelles in response to α-MSH (a−d). Some white pigment cells (e) contained irregular reflecting platelets (asterisk), in addition to stage II premelanosomes (m2), whose organelles were characteristic of melanophore precursors at early to middle stages of development. The other white pigment cells (f) contained irregular reflecting platelets (asterisk), in addition to stage II premelanosomes and a small number of partially melanized stage III melanosomes (m3), whose organelles were characteristic of melanophore precursors at the late stage of development. Arrows indicate premelanosomes in which reflecting platelet formation seems to be occurring

Fig. 6

Expression of pigment cells in the 6-day regenerating tail in the wild-type and the mutant (amputated at stage 50). a,b The wild-type regenerating tail observed under transmitted light (a) or incident light (b). c,d The mutant regenerating tail observed under transmitted light (c) or incident light (d). e Ultrastructure of differentiated iridophores in the mutant regenerating tail. Dashed lines indicate the amputation level. Melanophores appeared in the wild-type regenerating tail (a,b), and their distribution was similar to that in the intact tadpole tail. In contrast, white pigment cells (arrows) appeared in the mutant regenerating tail (c,d), and their distribution was similar to that in the intact tadpole tail. A small number of iridophores (arrowheads), which were never present in the intact tadpole tail, appeared in the somites of the mutant regenerating tail in addition to white pigment cells. Differentiated iridophores in the mutant regenerating tail were filled with reflecting platelets, which were irregular in size and shape (asterisk)

Fig. 7

The number of newly differentiated pigment cells in the regenerating tail of the wild-type and the mutant. After amputation of the posterior half of the tadpole tail (stage 48/49), melanophores or white pigment cells were counted in the regenerating tail of either the wild-type (n = 15) or the mutant (n = 16), respectively, on days 4, 5, and 6 post-amputation. The number of white pigment cells in the mutant regenerating tail was not statistically different from that of melanophores in the wild-type regenerating tail on days 5 and 6 post amputation (t test, P > 0.05)

Fig. 8

Dopa staining in the 5-day regenerating tail in the mutant (amputated at stage 49). a,b The mutant regenerating tail before dopa staining observed under transmitted light (a), or incident light (b). c The mutant regenerating tail after dopa staining observed under transmitted light. Dashed lines indicate the amputation level. White pigment cells were appearing in the mutant regenerating tail on day 5 post-amputation (b). Dopa staining was observed in white pigment cells (arrowheads) as well as melanophore precursors which were not visible under transmitted light or incident light before dopa reaction (arrows)

Fig. 9

Differentiation of iridophores and white pigment cells in the 19-day mutant regenerating tail (amputated at stage 50). a,b The mutant regenerating tail observed under transmitted light (a) or incident light (b). c−e Enlarged view of the mutant regenerating tail of another tadpole observed under transmitted light (c), incident light (d), or blue light (e). Dashed lines indicate the amputation level. White pigment cells (arrows) which were small and aggregated pigment organelles, looked white under incident light and emitted green fluorescence under blue light. In contrast, iridophores (arrowheads) which were large and dispersed pigment organelles, reflected light under incident light, but did not emit green fluorescence under blue light