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doi: 10.1242/10.1242/dev.00519

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Formation of the adult pigment pattern in zebrafish requires leopard and obelix dependent cell interactions

Max-Planck-Institut für Entwicklungsbiologie, Abt. III/Genetik, Spemannstrasse 35, 72076 Tübingen, Germany

View larger version (93K): [in a new window]   Fig. 1. Adult pigment pattern in mutants lacking one pigment cell type. (A,D) The wild-type pattern consists of four or five alternating stripes of blue melanophores and yellow xanthophores. (B,E) fms mutants lack all xanthophores and melanophores occur in clusters as well as scattered in between. (C,F) nac mutants lack melanophores and xanthophores form a coherent longitudinal domain (arrowhead in C) as well as clusters more ventrally (arrows in C). Large areas are devoid of xanthophores (black outline in F). Ontogeny of the pigment pattern in wild type (G,H), fms (I) and nac (J) during larva-to-adult transition. Images were taken from individual fish in corresponding regions of the body with the anterior edge of the image coinciding with the anterior edge of the dorsal fin. The first number given indicates the size in millimetres and the second the age in days. (G) In wild type (9 mm/23 d), melanophores initially appear scattered in the flank, with some persisting in the horizontal myoseptum from larval stages (arrowhead). Subsequently (11 mm/27 d), melanophores start to aggregate and to converge towards the horizontal myoseptum. Concomitantly, xanthophores appear (11 mm/27 d, arrowhead). (H) Xanthophores are (13.5 mm/25 d) seen in a stripe around the horizontal myoseptum and later (15 mm/27 d) start appearing in more ventral regions (arrowhead), initiating a second xanthophore stripe. (I) In fms (10 mm/24 d), melanophores appear differentiating throughout the flank, similar to wild type. At later stages (14.5 mm/30 d) melanophores start aggregating but fail to be cleared from ventral areas and around the horizontal myoseptum. (J) In nac mutants (10 mm/24 d) xanthophores appear in the same position as in wild type (compare with Fig. 2B). Later (13 mm/28 d), the xanthophores have only slightly increased in number.

View larger version (120K): [in a new window]   Fig. 2. Reintroduction of the missing cell type restores stripes in fms and nac. (A,B) Transplantation of ß-Actin GFP expressing golden mutant cells into fms hosts. (A) Normally pigmented, donor-derived fms mutant melanophores form a stripe pattern if they are in proximity of xanthophores. Melanophores further away from the xanthophore clone form the typical melanophore clusters seen in fms mutants. (B) UV-illumination shows GFP+, wild-type xanthophores forming stripes with fms mutant melanophores. The xanthophores form a coherent domain; however, several xanthophores have invaded the melanophore stripe (arrows). Some of the GFP signal within the melanophore stripe is due to transplanted cells underlying the melanophore stripe. (C,D) Transplantation of bpeGFP cells into nac hosts. (C) Melanophores form stripes of relatively normal position and size. (D) Fluorescence image of the same sample. Host-derived xanthophores (arrows) display pteridine autofluorescence around their nuclei. GFP+ xanthophores (arrowheads) show fluorescence in the entire cell. In the proximity of melanophores, both donor- and host-derived xanthophores organise into stripes, whereas outside the melanophore clone, xanthophores form the nac mutant pattern.

View larger version (90K): [in a new window]   Fig. 3. Mutants affecting the adult pigment pattern in zebrafish. (A,E) Homozygous obe mutants display fewer and wider stripes of loosely clustered melanophores, which contain intermingled xanthophores. Partially, the melanophore stripes show a higher density of cells (arrowhead in A). (B,L) obe heterozygotes display fewer and wider stripes and melanophore stripes are interrupted. (C,H) In homozygous mutants for a strong leo allele, melanophores form spots that are surrounded by xanthophores. (D,K) In an obe;leo double mutant, melanophores are singled out between xanthophores, apart from minor melanophore stripe remainders (arrowhead in D). (M) In mutants for a weak leo allele (tw28), melanophore stripes are undulating and interrupted. (F) fms;obe double mutants lack the melanophore clusters seen in fms single mutants (Fig. 1C,D). (G) obe;nac double mutants still contain xanthophore-free areas (black outline) similar to nac single mutants (Fig. 1E,F). (I) In a leo;fms double mutant, the melanophore clustering is lost. (J) In leo;nac mutants xanthophore-free areas are absent.

View larger version (85K): [in a new window]   Fig. 4. Ontogeny of the pigment pattern in obe (A,B) and leo (C,D) during larva to adult transition. (A) In obe, the initial distribution of melanophores at the onset of stripe formation (9 mm/22 d) is similar to the wild-type situation. Later (11 mm/24 d), melanophores fail to aggregate and remain evenly scattered. After the onset of xanthophore differentiation (11.5 mm/26 d), melanophores fail to be cleared from within the xanthophore stripe (13.5 mm/29 d, arrowhead). (B) The early (13.5 mm/29 d) distribution of xanthophores in obe is similar to wild type. Later (15 mm/31 d), differentiating xanthophores appear in between the scattered melanophores, giving rise to a mingled pattern. (C) leo mutants show a similar early (10 mm/23 d) melanophore pattern as in wild type. Later (11.5 mm/26 d), melanophores fail to cluster and converge towards the horizontal myoseptum and remain within the xanthophore domain (13.5 mm/29 d, arrowhead). (D) The early positioning of xanthophores in leo is similar to wild type, but later (14 mm/30 d) xanthophores start to differentiate within the melanophore domains, with some xanthophores encircling several melanophores (arrowhead).

View larger version (94K): [in a new window]   Fig. 5. Comparison of melanophore movements in a region ventral to the horizontal myoseptum in 1 day intervals. In the lower panels, cells that changed their position are marked with red bars, indicating the direction and extent of movement. (A) In wild type, 12 out of 26 melanophores changed position, some for over one cell-diameter. (B) In obe only 4/18 melanophores changed their position, indicating that obe mutant melanophores still are able to translocate. (C) In addition, leo mutant melanophores are able to move, as 9/24 melanophores changed their position.

View larger version (120K): [in a new window]   Fig. 6. Cell-type-specific requirements for obe and leo. (C,F,I,L) Diagrammatic representation of the outcomes of the mosaic analyses. Melanophores are represented in blue, xanthophores in yellow. Wild-type cells are shaded darker than mutant ones. (A-C) Transplantation of golden bpeGFP cells into nac;obe double mutant hosts generates a wild-type pattern. (A) A clone of wild-type melanophores organising the surrounding xanthophores into stripes of wild-type appearance. (B) A variety of cell types displaying GFP expression in the vicinity of the clone, but the majority of xanthophores is GFP negative and thus mutant for obe (arrows). (D-F) Upon transplantation of obe; bpeGFP cells into nac mutants, an obe like pattern is formed. (D) A melanophore clone with loosely clustered melanophores and intermingled xanthophores. (E) Only a few xanthophores are GFP positive (arrowhead), whereas the majority only displays autofluorescence (arrow) and is thus wild type for obe. (G-I) Transplantation of bpeGFP cells that are wild type for leo into nac;leo hosts generates a leo like pattern. (G) A clone of wild-type melanophores displaying key features of the leo pattern such as undulating interrupted stripes and spots. (H) Many different GFP positive cells are discernible around the clone, but most of the xanthophores are GFP negative (arrows) and thus mutant for leo. (J-L) Transplantation of leo; bpeGFP cells into nac single mutant hosts results in a leo like pattern. (J) A clone of leo mutant melanophores forming spots and undulating stripes. (K) Both GFP positive leo mutant xanthophores (arrowheads) as well as GFP-negative, wild type xanthophores (arrows) participate in formation of leo like pattern elements.

View larger version (82K): [in a new window]   Fig. 7. A model of cell behaviours underlying formation of the adult pigment pattern in zebrafish. Melanophores are depicted in blue, xanthophores in yellow. Double headed arrows indicate homotypic interactions and single headed arrows heterotypic ones. The horizontal myoseptum is represented by broken lines. (A) At the onset of pattern formation, melanophores are scattered. (B) Melanophores start aggregating by obe- and leo-dependent homotypic interactions and also exert a positive effect on xanthophore differentiation (green arrow). (C) Xanthophores that also display leo-dependent homotypic interactions positively attract melanophores over a distance (green arrows), but repel them in short range (red bars). (D) The boundary between melanophores and xanthophores is shaped by leo-dependent heterotypic interactions.

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