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. 2016 May 3;590(9):1291–1303. doi: 10.1002/1873-3468.12180

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© 2016 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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WDR26 is involved in the canonical Wnt pathway. (A) Phenotypes of xWDR26‐MO injected embryos. Control‐MO (40 ng) or xWDR26‐MO (40 ng) was coinjected with or without 5‐mis‐xWDR26 plasmids (20 pg) into two dorsal animal blastomeres of eight‐cell embryos. Phenotypes are categorized into normal, mild (small eyes), or severe (no eye). The injection of 5‐mis‐xWDR26 plasmids had no effect on head formation. Coinjection of 5‐mis‐xWDR26 plasmids with xWDR26‐MO rescued the phenotypes of the xWDR26‐morphants. The ratio of each phenotype in the injected embryos was indicated in the graph. Lane 1: n = 21, normal (100%). Lane 2: n = 45, normal (8.9%), mild (35.5%), severe (55.6%). Lane 3: n = 16, normal (81.3%), mild (18.7%). Lane 4: n = 14, normal (50.0%), mild (21.4%), severe (28.4%). (B–E) Quantitative RT‐PCR analysis. The value obtained for each marker gene or Wnt target gene was normalized to the level of xODC or hGAPDH. The value of control (lane 1) was set to 100, and other values were computed (other lanes). Error bars represent standard deviation of the mean in three experiments. Statistical significances of values among indicated lanes were determined by Mann–Whitney U test. (B) Quantitative RT‐PCR analysis of anterior neural marker genes, xNCAM, xOTX‐2, xPAX‐6, xSIX‐3, xRX‐1 in the head region of each molpholino‐ or plasmid‐injected embryo at stage 28. The injection was performed with similar methods indicated in (A). The value of neural marker gene/xODC of Control‐MO injected embryos (lane 1) was set to 100 and other values were computed. P < 0.01: all neural marker genes (between lane 1 and lane 2). P < 0.01: neural marker genes except for xRX‐1, P > 0.1: xRX‐1 (between lane 1 and lane 3). P < 0.01: all neural marker genes (between lane 2 and lane 4). (C) Quantitative RT‐PCR analysis of early dorsal Wnt target genes (Xtwn, Siamois, Xnr3) at stage 10. Control‐MO (40 ng), xWDR26‐MO (40 ng), and Xwnt‐8 (0.5 pg) mRNA were ventrally coinjected with xWDR26 mRNA (500 pg). The value of Wnt target gene/xODC of dorsal sectors (lane 1) was set to 100, and other values were computed. The value of uninjected ventral sectors was used as negative control. P < 0.005: all Wnt target genes (between lane 2 and lane 3). P < 0.005: Xnr3 and Siamois, P > 0.1: Xtwn (between lane 3 and lane 4). P < 0.05: all Wnt target genes (between lane 3 and lane 5). P < 0.005: Siamois and Xtwn, P > 0.05: Xnr3 (between lane 5 and lane 6). (D) Quantitative RT‐PCR analysis of early dorsal Wnt target genes (Xtwn, Siamois, Xnr3). Control‐MO (40 ng), xWDR26‐MO (40 ng) and Xwnt‐8 (0.5 pg) mRNA were ventrally coinjected with hWDR26 mRNA (500 pg). The value of Wnt target gene/xODC of ventral sectors (lane1) of Control‐MO injected embryos was set to 100, and other values were computed. P < 0.005: all Wnt target genes (between lane 1 and lane 2). P < 0.005: all Wnt target genes (between lane 1 and lane 3). P < 0.005: all Wnt target genes (between lane 2 and lane 4). (E) Quantitative RT‐PCR analysis of hAxin2 in HEK 293T cells. The siRNA‐transfected cultured cells were stimulated with Wnt3A conditioned medium from L‐Wnt‐3A cells for 6 h. The conditioned medium from L cells was used for unstimulated control. The value of hAxin2/hGAPDH of unstimulated control (lane1) was set to 100, and other values were computed. P > 0.1 (between lane 1 and lane 2). P < 0.005 (between lane 1 and lane 3). P < 0.005 (between lane 3 and lane 4).