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Fig. 3. Hairy2 overexpression represses NC marker at early stages but expands a subset of them in later embryos. (A) Snail2 expression is initially repressed by Hairy2-GR overexpression (500 pg mRNA) and increased in later embryos (a, stage 13, 65% decreased, n = 17; b, stage 15, 58% decreased, n = 24; c, stage 21, 27% reduced n = 22; d, stage 27, 53% increased n = 19). Dorsal view (a, b) or high magnification of the head region are shown (c, d). β-galactosidase was used as a lineage tracer. (e) Q-PCR analysis of Snail2 expression in animal caps injected with noggin+ Wnt8 (control) or injected with noggin+ Wnt8 and Hairy2-GR (250 pg/blastomere) and harvested at different stages. Snail2 is repressed by Hairy2 during neurula stages but is progressively upregulated at later stages. (B) Injection of 500 pg of Hairy2-GR mRNA increases Sox10 (a) but decreases Sox9 (b) and Trp2 (c) (78% induced for Sox10, n = 29; 60% inhibited n = 20 for Sox9 and 62%, n = 21 for Trp2) at tailbud stages. Lateral views with control and injected sides are shown. β-galactosidase was used as a lineage tracer. (C) Q-PCR analysis of animal caps derived from embryos injected with noggin+ Wnt8 mRNA and cultured until the equivalent of stage 28. Hairy2-GR (250 pg/blastomere) represses Sox9 but increases Snail2 and Sox10 while Hairy2 depletion (5 ng/blastomere) reduces all markers. (D) Temporal ability of Hairy2 activity to activate late NC development. Note that Hairy2-GR ability to increase Sox10 is only observed when DEX is added before st14. (E) Eight-cell-stage embryos injected radially at the animal pole with Hairy2-GR (250 pg/blastomere) (b, d) display reduced pigmentation. Cranial cartilages were also reduced compared to uninjected controls (a, c). Respective inhibitions: (b) 58%, n = 24; (d) 54%, n = 21. (F) Extracts from stage 32 (a) or 45 (b) embryos injected radially with Hairy2-GR were analysed using anti-GFAP antibody. GAPDH was used as loading control. Note that GFAP is first reduced and later increased by Hairy2 overexpression. |
