Fig. 6. Kbtbd7 regulates Vangl2 in zebrafish convergent extension movement.

(A to C) Kbtbd7 degraded Vangl2 and caused convergent extension (CE) defects in zebrafish. (A) Three hundred picograms of zebrafish Kbtbd7 WT or M99A mutant mRNA was synthesized and microinjected into one-cell stage of embryos. Endogenous Vangl2 was examined. (B) Forty-eight hours after injection, zebrafish embryos were analyzed and classified into four groups (normal, mild, moderate, and severe) according to their CE phenotype. (C) Low level of Vangl2 (20 pg) partially rescued the CE defects caused by Kbtbd7 expression. The results are summarized from three independent experiments (t = 3). The total number of injected embryos (N =) for each group is labeled on the top. The error bars are the SD values. Scale bar, 1 mm. (D) The effects of Vangl2 MOs and HA-Vangl2 mRNA in zebrafish embryos were examined by Vangl2 and HA immunoblotting, respectively. (E) Knockdown of Vangl2 showed synergistic effects with Kbtbd7 expression in causing CE defects. Vangl2 MO (0.25, 0.5, or 2 ng) was injected alone or with Kbtbd7 mRNA (300 pg). (F) Two or 4 ng of Kbtbd7 MO was injected into one-cell stage of embryos. Kbtbd7 knockdown increased endogenous Vangl2 protein levels in a dose-dependent manner. (G) Typical morphological defects of Kbtbd7 MO–injected embryos, exhibiting ventral body curvature. Scale bar, 1 mm. (H) Vangl2 expression exhibited synergistic effects with Kbtbd7 MO in causing CE defects. Twenty or 60 pg of Vangl2 mRNA was injected alone or with 4 ng of Kbtbd7 MO. The results are summarized from three independent experiments (t = 3). The total number of injected embryos (N =) for each experimental setting is labeled on the top. The error bars are the SD values.