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Fig. 4. Hairy2 promotes NC markers and proliferation through Delta1 activation. (A) Q-PCR analysis of Sox10, Snail2 and SoxD expression in NC induced intact or dissociated animal caps derived from Hairy2-GR mRNA (250 pg/blastomere) injected embryos (−, Hairy2-GR mRNA injected minus DEX). Sox10 induction observed upon Hairy2-GR overexpression in intact stage 28 NC induced caps is not observed in dissociated NC induced explants (a). However, repression of Snail2 or induction of SoxD still occurs in dissociated stage 14 NC induced caps (b,c). (B) Whole-mount in situ hybridization of embryos co-injected with Hairy2-GR and Delta1Stu mRNA (1 ng) or Delta1 MO (7.5 ng). Co-injection of Hairy2-GR with Delta1Stu mRNA (1 ng) or Delta1 MO (7.5 ng) affects its ability to induce Sox10 in tailbud embryos, but not its capacity to repress Snail2 or activate SoxD in neurula embryos. Respective repression: (a) 63%, n = 23; (b) 43%, n = 21; (c) 64%, n = 24 and (d) 65%, n = 20. Respective induction: (e) 87%, n = 16 and (f) 60%, n = 28. Lateral views with control and injected sides (a, b, e, f) or dorsal views (c, d) with the injected side on the right are shown. LacZ was used as lineage tracer. (C) Percentage of embryos injected with Hairy2-GR mRNA, Hairy2-GR plus Delta1Stu mRNA (1 ng) or Delta1 MO (7.5 ng) showing increased pH3 staining. Note that co-injection of Delta1Stu mRNA or Delta1 MO blocks Hairy2 ability to promote cell proliferation (−, Hairy2-GR mRNA injected minus DEX). Number of embryos examined is indicated. (D) Percentage of embryos injected as indicated with Bax mRNA (50 pg), Hairy2-GR mRNA (500 pg), Delta1Stu (1 ng) or Delta1 MO (7.5 ng) developing normally or showing a low or high number of dying cells at gastrula stage as shown in Fig. 1C (−, Hairy2-GR mRNA injected minus DEX). Note that Hairy2-GR rescues Bax mRNA mediated embryonic lethality when Notch signaling is blocked. Number of embryos examined is indicated. |
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Fig. 5. Rescue of Hairy2 depletion by Delta1. (A) Whole-mount in situ hybridization of embryos injected with Hairy2 MOs (10 ng) alone or with Delta1 mRNA (1 ng) as indicated. Delta1 restores Snail2 (a, b) and Sox10 (c, d) expression in Hairy2 MOs injected embryos. Respective inhibition: (a) 76%, n = 17; (b) 30%, n = 24; (c) 50%, n = 16; (d) 15%, n = 28. Dorsal views of neurula embryos (a, b) with the injected side on the right or lateral views of tailbud embryos (c, d) with control and injected sides revealed by LacZ staining are shown. (B) Percentage of embryos injected with Hairy2 MOs (10 ng) alone or together with Delta1 mRNA (500 pg) showing reduced pH3 staining. Delta1 injection partially rescues the inhibition of proliferation observed in Hairy2 depleted embryos (−, rhodamine dextran injected alone). Number of embryos examined is indicated. (C) Whole-mount in situ hybridization of tailbud embryos injected with Hairy2 MOs (10 ng) with Su(H)Ank-GR mRNA (500 pg). Su(H)Ank-GR efficiently restores Sox10 expression in Hairy2 MOs injected embryos. Rescue of Sox10 is not observed in − DEX control embryos. Respective inhibition: a, 77% n = 18; b, 42% n = 21. Lateral views with control and injected sides identified by LacZ staining are shown. |
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Fig. 6. Id3 is required for NC development downstream of Delta1. (A) Whole-mount in situ analysis of embryos injected with Delta1 mRNA (1 ng), DeltaStu mRNA (1 ng) or Delta1 MO (7.5 ng) as indicated. Note that Delta1 overexpression increases Id3 (a, 50% induced, n = 16). Conversely, Id3 is reduced in Delta1Stu mRNA or Delta1 MO injected embryos (b, 55%, n = 24; c, 67%, n = 18). (B) Activation of Notch signaling by injection of Delta1 mRNA (1 ng) rescues Id3 and Snail2 inhibition by Hairy2-GR in neurula embryos (a, 60% reduced, n = 23; b, 93% rescued, n = 15; c, 78% inhibited n = 18; d, 67% rescued, n = 30). Q-PCR analysis of Id3 (e) and Snail2 (f) expression in NC induced explants derived from embryos injected with Hairy2-GR mRNA (250 pg/blastomere) with or without Delta1 mRNA (500 pg/blastomere) as indicated (−, uninjected NC induced caps). Note that co-injection of Delta1 mRNA rescues Id3 and Snail2 repression by Hairy2-GR. (C) Id3 MO (15 ng) blocks Delta1 (1 ng mRNA, a–d; 500 pg mRNA, e) induction of Snail2, Sox10 and cell proliferation in embryos. Percentage of embryos with the indicated phenotype: a, 55% increased, n = 20; b, 70% inhibited, n = 26; c, 68%, increased n = 19; d, 62% reduced, n = 33. Percentage of embryos injected with Delta1 mRNA (500 pg) with or without Id3 MO (15 ng) showing increased pH3 staining (e). Note that co-injection of Id3 MO reduced Delta1 ability to stimulate cell proliferation (−, rhodamine dextran injected alone). Number of embryos examined is indicated. (D) Id3-GR mRNA (1 ng) injection partially rescues Snail2 in Delta1 (7.5 ng) depleted neurula embryos and Sox10 at tailbud stage but not in − DEX control embryos. Respective inhibition and rescue: a, 87% inhibited, n = 30; b, 70% unaffected, n = 37; c, 62% inhibited, n = 16; d, 67% unaffected, n = 21. Percentage of embryos injected with Delta1 MO (7.5 ng) with Id3-GR mRNA (1 ng) showing decreased pH3 staining (e). Note that Id3 partially rescues the inhibition of cell proliferation observed in Delta1 MO injected embryos. Number of embryos examined is indicated. In all panels, neurula embryos are viewed from the dorsal side, with the injected side revealed by LacZ staining oriented to the right. Tailbud embryos are viewed from the lateral side. Control and injected sides are shown. |
