|
Figure 2. A-N: Effects of overexpression of XlMitf alpha -M or dnXlMitf alpha 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 XlMitf alpha -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 dnXlMitf alpha -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 XlMitf alpha -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 XlMitf alpha -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 dnXlMitf alpha -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 dnXlMitf alpha -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.
|
|
Figure 3. Effects of Slug overexpression on melanophore development. A,B: In Slug-injected embryos, the number of melanophores was increased (B) compared with noninjected embryos (A). C,D: Overexpression of Slug induced an increase in the number of XlMitf alpha -positive cells on the injected side (C,D, arrowheads). D: A transverse section of the trunk region of C, plane D. E: The number of Sox10-positive cells was also increased on the injected side (E, arrowheads). Yellow arrows in C and E show the midline. NT, neural tube.
|
|
Figure 6. Effects of XlMitfM and dnXlMitf alpha mRNA microinjection on expression of eye-related genes. A-E: XlMitf alpha -M-injected embryos. A: The facial region expressing Pax6 on the injected side (black arrow) was smaller than that on the noninjected side (blue arrow). Black dashes show the midline, and the eye region where Pax6 is normally expressed is shown by white dashes. B,C: Similarly, Rx1 (B) and Six6 (C) were repressed on the injected side (black arrow). D: Expression of Otx5b was repressed both in the optic vesicle (black arrow) and the pineal body (white dashes) on the injected side. Blue arrow shows normal Otx5b expression in the optic vesicle on the noninjected side, and black dashes show the midline. E: In contrast, Pax2 expression was activated in the optic vesicle (black arrow), although those in the otic vesicle (green dashes) and midbrain-hindbrain boundary (pink dashes) were repressed on the injected side compared with those on the noninjected side (blue, green, and pink arrows, respectively). F-J: dnXlMitf alpha -injected embryos; overexpression of dnXlMitf alpha induced the reduction of Pax6 (F), Rx1 (G), Six6 (H), Otx5b (I), and Pax2 (J) in the optic vesicle on the injected side (black arrows). Pax2 expression (J) in the otic vesicle (green dashes) and the midbrain-hindbrain boundary (pink dashes) and Otx5b expression in the pineal body (I, white dashes) were also repressed on the injected side. A-C,E-H,J: Stage 23. D,I: Stage 25. CG, cement gland; MH, midbrain-hindbrain boundary; OT, otic vesicle; PB, pineal body.
|
