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Fig. 4. Depletion of XSufu induces expansion of the neural plate, stimulates anterior neural markers and suppresses eye development. (A) Targeting sequence of the XSufu morpholino oligonucleotide (XSufu-MO). The non-targeted XSufu* mRNA construct lacks the 5′ untranslated region. Blue letters indicate start codon. (B) In vitro transcription–translation assay. The XSufu-MO, but not an unspecific control-MO, inhibits XSufu protein synthesis. XSufu-MO does not reduce translation of XSufu* mRNA. (C,C′) At the tadpole stage, XSufu-MO-injected embryos have reduced head and tail structures. (D–D′) Dorsal view of early neurula embryos. Injection was performed animally into one blastomere at the 2-cell stage together with nlacZ mRNA as tracer. XSufu-MO, but not control-MO, causes expansion of Sox2 expression on the injected side (arrowhead); this effect is reverted by co-injection with XSufu* mRNA. (E,E′) XSufu-MO leads to a reduction of Cytokeratin expression. (F–H,F′–H′,G′,H'') Anterior view of neurula (F,F′,G–G′) and dorsal view of tailbud embryo (H–H′). XSufu-MO causes expansion of Otx2, reduced intensity of En2, and a posterior shift of En2 and Krox20 expression, while FoxG1 expression remains unaffected. Co-injection of XSufu-MO and XSufu* mRNA rescues normal En2 and Krox20 expression. (I,I′) Dorsal view of early neurula. XSufu-MO induces reduction of MyoD expression (arrowhead). (J,J′) Anterior view of tail bud embryo. XSufu-MO leads to a reduction of Rx2A expression. (K–M,K′–M′) A single injection of XSufu-MO causes reduction of eye structures (arrowheads), which is reverted by co-injection of XSufu* mRNA. The indicated phenotypes were observed in: C, 70/83; C′, 21/43; J, 22/24; D, 50/50; D′, 71/77; D′, 82/87; E, 24/27; E′, 39/40; F, 23/24; F′, 20/20; G, 39/39 (FoxG1), 55/55 (En2); G′, 42/42 (FoxG1), 54/54 (En2); G′, 31/31; H, 65/67; H′, 79/79; H′, 34/40; I, 24/26; I′, 27/28; J, 40/42; J′, 67/73; L,L′, 25/25; M,M′, 21/43. |
