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Volume 11, Issue 15, Pages 1202-1206 (August 2001) The medaka rs-3 locus required for scale development encodes ectodysplasin-A receptor Shu Kondo, Yoshikazu Kuwahara, Mariko Kondo, Kiyoshi Naruse, Hiroshi Mitani, Yuko Wakamatsu, Kenjiro Ozato, Shuichi Asakawa, Nobuyoshi Shimizu, Akihiro Shima Current Biology Volume 11, Issue 15, Pages 1202-1206 (August 2001) DOI: 10.1016/S0960-9822(01)00324-4

Figure 1 The phenotype of rs-3 mutant fish. (a,c) Wild-type. (b,d)rs-3 mutant. (a,b) Side views of 3-month-old fish. Mutant fish look more transparent, owing to the loss of melanophores associated with scales. (c,d) Transverse sections of 2-month-old fish stained with alizarin. The calcium contained in the scales is stained red in (c) wild-type fish Current Biology 2001 11, 1202-1206DOI: (10.1016/S0960-9822(01)00324-4)

Figure 2 An analysis of medaka EDAR. (a) The sequence alignment of the medaka and human EDAR proteins. The residues in blue and red letters are the signal peptide and the transmembrane domain, respectively. (b) A comparison of human Chr 2q and medaka linkage groups (LG) 2 and 21. The genes on LG2 and LG21 are written in blue and red letters, respectively. Medaka orthologs of 14 human genes on Chr 2q were cloned and placed on the reference map (middle column; [5]). HOXDA and 5 other genes that did not recombine with HOXD in the reference panel were put to linkage analysis with the rs-3 locus (right column) using 192 meioses of F2 progeny. (c) A schematic diagram depicting the genomic structure of medaka EDAR. The vertical line in exon 2 indicates the initiation codon. (d–i) The arrows indicate the positions of primers used for RT-PCR analysis. In the rs-3 mutant genome, a transposon is inserted about 100 bp downstream of the splice donor of the first intron. (d–h) RT-PCR analysis of the wild-type and mutant EDAR transcripts. M, size marker; 1, wild-type RT+; 2, wild-type RT−; 3, rs-3 RT+; and 4, rs-3 RT−. (i) The sequence analysis of the transposon insertion in the rs-3 locus. The 4-bp target site duplication is shown in red (underlined), and the 18-bp inverted repeats are shown in blue and are indicated by arrows Current Biology 2001 11, 1202-1206DOI: (10.1016/S0960-9822(01)00324-4)

Figure 3 The expression of EDAR. The expression of EDAR in a wild-type 30-day-old fish was visualized by whole-mount in situ hybridization. (a) The expression is seen in scale placodes and the posterior margins of growing scales. The expression is also observed in fin rays. (b,c) Close-up views corresponding to the white and black boxes in (a), respectively. (b) An example of a scale placode expressing EDAR is indicated by an arrowhead. Developing pectoral fins also express high levels of EDAR (arrow). (c) Epithelial cells of scale placodes (arrowheads) are clusters of cells that express EDAR Current Biology 2001 11, 1202-1206DOI: (10.1016/S0960-9822(01)00324-4)