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Fig. 1. Ednrb2 and melanoblast marker gene expression patterns in Xenopus embryos by WISH. A,B: Mitf expression is detected (A), but there is no Ednrb2 expression in a stage-19 embryo. C: Ednrb2 expression (arrowheads) is first detected in the NT at stage 21. D–F: Weak Dct (D) and strong Ednrb2 (E,F) expression is detected at stage 25. G–I: Dct (G) and Ednrb2 (H,I) expression is detected in the NT and only Ednrb2 is expressed in the head region at stage 28 (red arrowheads). J–R: The expression pattern of Ednrb2 (K,O,R) resembles that of Dct (J,M,P) except in the RPE (Dct only). Blue arrowheads indicate expression in the epiphysis. Note that after stage 35, melanophores are pigmented, but because the embryos are bleached in these embryos, the melanins are not visible. Insets are magnified images in the squares (scale bar ¼ 100 mm) and show dendritic pigmented melanophores expressing both Dct (P) and Ednrb2 (R). F, I, L, O, and R are lateral views of E, H, K, N, and Q, respectively. Heads are on the right side of each picture. Scale bar ¼ 1 mm.
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Fig. 2. Expression patterns of Ednrb2 and Dct A–D: Serial sections of an embryo at stage 26.
Dct-expressing cells also express Ednrb2 in the rostral trunk region (A, B) and in the caudal
trunk region (C, D). A melanoblast initiating migration is observed in A, B (arrowheads). E,F:
Both Dct (E) and Ednrb2 (F) are expressed in the same cells in serial sections of the trunk region
at stage 32. G,H: RPE cells express Dct (G) but not Ednrb2 (H) in serial sections of the eye at
stage 32. Note that the brown color of H in the RPE is not the ISH signal but is due to the melanin.
Scale bar ¼ 100 mm.
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Fig. 3. Ectopic induction of Ednrb2 expression due to Mitf over-expression. A,B: Embryos in which Mitf-GR was injected, stained by Ednrb2 WISH (violet). Mitf-GR activity was induced with DEX treatment at stage 25 (A), or not induced (B). DEX-treated embryo showing ectopic Ednrb2 expression in cranial NCCs (A). This expression is not observed in B. The blue stain indicates lacZ staining as a tracer. C,D: Serial sections in the eye region of a Mitf-GR injected embryo.
DEX treatment was performed at stage 22. White arrowheads illustrate the ectopic expression of Ednrb2 (A) and Dct (B). Scale bar ¼ 100 mm.
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Fig. 4. Expression patterns of ET3. A,C: ET3 is expressed in the rostral trunk region, where melanophores later migrate to the yolk at stages 25 (A) and 31 (B). B,D: ET3 is expressed in thecaudal trunk region where melanophores later localize in the NT. E,F: Dct expression represents
migrating melanoblasts. E and F are serial sections of C and D, respectively. In the rostral region, melanoblasts seek yolk migrating through the ventral pathway (E). In the caudal region, melanoblasts localize in the NT or migrate to the fin (F). Scale bar ¼ 100 mm.
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Fig. 5. Phenotypes of Ednrb2-over-expressed embryos. A,B: An Ednrb2 over-expressed embryo on the injected side (A) and on the non-injected side (B). C,D: Magnification of A in the trunk region (C) and in the tail region (D). The injected side has fewer melanophores in the yolk
(C), while no difference is observed in the tail region (D). Each inset shows Venus fluorescence as a tracer. E–I: A series of sections (8 mm each at 40-mm intervals, anterior to posterior) of an Ednrb2 over-expressed embryo stained with Dct ISH. The injected side is on the right side of each image. Red arrowheads illustrate melanoblasts migrating through the ventral pathway in the non-injected side. No melanoblasts are observed in the ventral pathway on the Ednrb2 -
injected side. Scale bar ¼ 100 mm.
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Fig. 6. Cell proliferation and apoptosis in a representative Ednrb2 over-expressed embryo at stage 30. A–E: Serial sections around the otic vesicle (A, B) and in the trunk region (C–E). Red, green, and blue signals represent cell proliferation (M-phase) detecting phospho-Histone H3,
H2B-Venus as a tracer co-injected with Ednrb2 and DNA by DAPI staining, respectively (A, C). Violet signals represent apoptosis with the TUNEL assay (B, D). Ednrb2 signal represents melanoblasts
(E). Red arrows indicate a TUNEL-positive melanoblast in Ednrb2 over-expressed cells (C–E). Scale bar ¼ 100 mm.
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Fig. 7. Inhibition of ET3 activity by ET3-MO. A,B: The injected side of an ET3 MO-injected embryo showing the yolk (A) and the tail (B) regions. Blue signals indicate the X-gal stain as a tracer. No melanophore is observed in either region. Insets in A and C show each side of the
whole image of the embryo, respectively. C,D: The non-injected side view of the ET3 MOinjected embryo. The yolk (C) and the tail (D) regions are shown. These images are mirror-reversed for easy comparison. E: Percentage of each phenotype in the injected embryos. The
numbers in each column indicate the number of the observed embryos. Columns indicate ET3MO-injected embryos (left), ET3MO and ET3 mRNA-injected embryos (middle), and controlMO-injected embryos (right). Blue, no melanophore; red, reduced number of melanophores;
yellow, normal melanophore distribution are observed in the injected sides.
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Fig. 8. ET3 promotes melanoblast invasion in vitro. A–C: An NT placed in the top well of a trans-filter migration chamber after 1 day; 1 nM (C) or
2.5 nM (A, B) ET3 was added to the bottom well. A and C show melanophores on both side. These cells are observable because of transparency of the membrane. B represents only melanoblasts that had invaded to the bottom side of the filter in A. This was observed only when 2.5 nM ET3 had been added (B). D: Ratio between the numbers of invaded melanophores/total melanophores in samples that showed invaded melanophores. NTs were cultured with (+) or without (-) ET3 (2.5 nM). Top, top well; bottom, bottom well. Note that this graph does not include samples that did not show melanophore invasion. Error bars represent standard errors. Scale bar ¼ 100 mm.
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ednrb2 (endothelin receptor B subtype 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, dorsal view, anterior right, dorsal up.
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ednrb2 (endothelin receptor B subtype 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior right, dorsal up.
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ednrb2 (endothelin receptor B subtype 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
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ednrb2 (endothelin receptor B subtype 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 39, lateral view, anterior right, dorsal up.
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edn3 (endothelin-3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 31, coronal section, dorsal up.
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