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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
sox10
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xenopus
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NF stage 28
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otic vesicle
,
neural crest
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dct
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tropicalis
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NF stage 28
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brain
,
hindbrain
,
midbrain-hindbrain boundary
,
spinal cord
,
neural crest
,
[+]
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sox9
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xenopus
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NF stage 28
|
otic vesicle
,
neural crest
,
branchial arch
,
dorsal
,
branchial arch 1
,
[+]
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trpc2
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tropicalis
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NF stage 40
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neural crest
,
eye
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trpc2
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tropicalis
|
NF stage 28
|
otic vesicle
,
brain
,
hindbrain
,
spinal cord
,
neural crest
,
[+]
|
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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. |
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Fig. 6. Id3 interacts and blocks Hairy2 activity in NC. (A) Overexpressed Flag-tagged Hairy2 but not Hairy1, Hes2 or Hes6 interacts with HA-tagged Id3 in immunoprecipitation assays. (B–C) Mapping of the domains of Hairy2 and Id3 required for their interaction. Note that only the Hairy2 deletion mutants containing the Orange domain are able to interact with Id3 (B) and that the C-terminal part of the HLH domain of Id3 is required for Hairy2 interaction (C). (D) Co-injection of Id3-GR (1 ng) rescues Snail2 inhibition by Hairy2-GR (70% rescued, n = 26)(a, b). Q-PCR analysis of Snail2 in animal caps induced to form NC injected radially with Hairy2-GR (250 pg), Id3-GR (500 pg) or both (250 pg and 500 pg respectively. A control western blot showing that in each condition similar levels of overexpressed proteins Myc-Hairy2-GR and Flag-Id3-GR have been produced is shown (c). Co-injection of Id3-GR (1 ng) blocks the ability of Hairy2-GR to induce SoxD (d, 89% ectopic n = 18; e, 74% rescued n = 19). Dorso-anterior views with injected side to the right (a, b) or lateral views (d–g) of neurula embryos are shown. β-galactosidase was used as a lineage tracer. |
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Display additional annotations [+]
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Fig. 7. Id3 is required for Hairy2 to promote NC formation. (A) Hairy2-GR (500 pg) injection induces Sox10 (a; 55% increased, n = 24) in tailbuds and upregulates SoxD (e, 88%, n = 16), Pax3 (h, 65%, n = 32), Msx1 (k, 59%, n = 27) and Zic1 (n, 63%, n = 32) in neurula embryos. Hairy2-GR and Id3 MO co-injection(15 ng) decreases Sox10 (60% inhibition, n = 27) in embryos but still induces SoxD (g, 70% increased n = 20), Pax3 (j, 62%, n = 29), Msx1 (m, 49, n = 35) and Zic1(p, 67%, n = 33). Control minus DEX shows that the Id3 MO efficiently inhibits Sox10 (50% inhibited, n = 15;) and has no effect on SoxD (f, 76% normal n = 17) Pax3 (i, 70%, n = 23), Msx1 (l, 85%, n = 21) and Zic1(o, 65%, n = 23). Hairy2-GR was also unable to induce Sox10 with Id3 MO in NC induced animal explants (d). (B) Id3-GR (1 ng) partially rescues Snail2 and Sox10 in Hairy2 (10 ng) depleted embryos (a, b, d, e) and in NC induced animal caps explants analysed by Q-PCR (c, f). Respective inhibition and rescue: (a) 68% inhibited, n = 26; (b) 89% rescued, n = 27; (d) 56% inhibited, n = 18; (e) 75% rescued; n = 30. Dorso-anterior views of embryos with injected side to the right (a, b) or lateral views (d, e) of embryos are shown. β-galactosidase was used as a lineage tracer. |