Xenbase Image ID: 172689

Fig. 4. Inducible depletion demonstrates that PFKFB4 has independent roles in NC specification and migration.We co-injected a UV-cleavable senseMO (PhMO) to block PFKFB4MO until the desired developmental stage and monitored twist1 expression at st.18. (A-H) Strategy of phMO validation: lacZ control injections (A), unmasked PFKFB4MO, ‘high’ dose (B), PhMO/MO without UV illumination (C; uninduced, u.i.); UV illumination at early NC specification st.12.5 (D-G). D-F show two examples of diminished phenotype and one example of abolished phenotype at st.14;Gshows an example of diminished phenotype at st.19. (H) Percentage of embryos with each phenotype, i.e. abolished, diminished, increased or normal twist1 expression. lacZ injections did not alter twist1 pattern. PFKFB4MO severely blocked twist1 expression. twist1 expression was decreased in PhMO/MO-injected embryos UV illuminated immediately after injection (a.i.) compared with uninduced PhMO/MO-injected embryos. Pfkfb4 depletion, activated at st.12.5, decreased twist1 expression strongly or moderately. This effect persisted until EMT and early NC emigration (G; st.20). (A-G): dorsal views. (I-W) To deplete pfkfb4 during EMT/early migration, st.18 neurulas were UV illuminated and analyzed at tadpole st.24. twist1 expression was normal in embryos injected with lacZ (I,J), or with PhMO/MO but not illuminated (M,N). Cells injected with PFKFB4MO alone died as expected (K,L). PhMO/MO-injected embryos UV illuminated at st.18 exhibited three phenotypes classes: ‘migration’ (O, P), ‘staining and migration’ (Q,R) and ‘severe staining’ defects (S,T). We compared the percentage of each phenotype (U), of migration distance (V) and of NC stream area (W; twist1-expressing area) with the contralateral side. Scale bars: 500 μm. All phenotype differences were statistically significant between illuminated PhMO/MO-injected embryos, and either lacZ-injected, or uninduced PhMO/MO-injected embryos (*P<0.05; **P<0.01; ***P<0.001; ns, not significant). Error bars represent s.e.m. Phenotype scores are shown in Table S9. Image published in: Figueiredo AL et al. (2017) Copyright © 2017. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Gene Synonyms Species Stage(s) Tissue twist1.L twist, twist1-a, twist1-b, X-twi, Xtwi, Xtwist X. laevis Sometime during NF stage 14 to NF stage 19 neural crest twist1.L twist, twist1-a, twist1-b, X-twi, Xtwi, Xtwist X. laevis Throughout NF stage 24 migratory neural crest cell cranial neural crest mandibular crest hyoid crest Image source: Published Larger Image Printer Friendly View

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