Figure 2. A–N: Effects of overexpression of XlMitfα-M or dnXlMitfα on Dct, Sox10 and Slug expression. A-1, B-1 and C: At stage 31, Dct was normally expressed in melanoblasts on the dorsal side of the neural tube and in the RPE. A-2: On the XlMitfα-M-injected side, an increase in the number of Dct-positive melanophores (A-2, arrowheads) was observed ectopically covering the brown-colored melanized cells (A-2, arrows). B-2: On the dnXlMitfα-injected side, Dct-positive melanoblasts/melanophores were detected on between the dorsal side of the neural tube and the surface of the yolk sac in the trunk (B-2, arrowheads) contrasting with the noninjected side (B-1). An arrow indicates the eye expressing no signal (B-2). F,I: At stage 23, expression of Sox10 (F) and Slug (I) was detected in neural crest cells at the dorsal midline and in migrating cranial neural crest cells. D: In XlMitfα-M-overexpressed embryos, the number of Sox10-expressing premigratory neural crest cells was increased on the injected side (D, arrowheads). G-1, G-2: Similarly, Slug-expressing premigratory neural crest cells was increased by XlMitfα-M overexpression (G-2, arrowheads), although Slug expression was not detected in ectopic melanized cells (G-1, arrow) contrasted with Dct expression in these cells (A-2,E,H-1). H-2: In dnXlMitfα-injected embryos, the expression patterns of Sox10 (E, black arrowheads) and Slug (H-1 and H-2, black arrowheads) were disturbed and the signals shifted laterally contrasted with the noninjected side (H-1 and H-2, blue arrowheads). White arrows (E and H-1) and a black arrow (H-2) show the midline of each embryo. Green arrowheads (H-2) show the regions between the strong Slug-expressing regions (H-2, black arrowheads). J,K,L: BrdU immunostaining detecting proliferating cells. M, N: single-strand DNA (ssDNA) immunostaining detecting apoptotic cells. These stainings were performed in transverse sections prepared from the same embryos (G-2 and H-2) after in situ hybridization. J: The number of BrdU-positive cells (shown by red cells) on the injected side was increased (black arrowheads, contrasting with those in the noninjected side pointed by blue arrowheads) in the region where Slug expression was promoted. In the Slug-expressing region shifted laterally on the dnXlMitfα-injected side (H-2, black arrowheads), both the number of BrdU-positive cells (K, black arrowheads) and ssDNA-positive cells (M, black arrowheads) were larger than those in Slug-expressing area on the noninjected side (K and M, blue arrowheads). In the region where Slug signal became very weak on the injected side (H-2, green arrowheads), neither a decrease in the number of BrdU-positive cells (L, green arrowheads) nor an increase in the number of ssDNA-positive cells (N, green arrowheads) were observed, contrasted with Slug-expressing regions on the noninjected side (L and N, blue arrowheads). C,F,I: Noninjected embryos. A-1,A-2,B-1,B-2,C: Stage 32. D,E,F,H-1,I: Stage 23. G-1: Stage 21. G-2,H-2: Stage 25. NO, notochord; NT, neural tube.
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