Fig. 5: Anterior accumulation of the Vangl2/Pk3 complex in the neuroectoderm is inhibited by N-terminal tyrosine phosphorylation of Vangl2.

A Mapping major phospho-tyrosine (pY) sites in Vangl2. Embryos were injected with 40 pg of RNA encoding FGFR1 and the indicated HA-Vangl2 constructs. Vangl2 phosphorylation was analyzed in anti-HA pull-downs from stage 13 embryo lysates. Immunoblotting (IB) was performed using anti-pY, anti-HA or anti-FLAG antibodies, as indicated. One representative set of duplicate samples is shown. The graph below shows average pY/HA intensity ratios for the duplicates. B Alignment of N-terminal amino acid sequences of Vangl2 from several chordate species and Drosophila Van Gogh. The N-terminal tyrosine cluster (in red) is conserved in vertebrates but not in Drosophila. C–E Planar polarization of Vangl2 tyrosine phosphosite mutants in the neuroectoderm. C Experimental scheme. Adapted from Xenopus illustrations © Natalya Zahn (2022), via Xenbase (www.xenbase.org, RRID: SCR_003280). Two dorsal blastomeres of 16-cell embryos were coinjected with myr-BFP RNA (80 pg) and RNAs encoding GFP-Pk3 (150 pg), HA-tagged Vangl2 (in D), Y7, 10, 12 > F (Y > F, in E) or Y7, 10, 12 > E (Y > E, in F) (20 pg each). Embryos were fixed at stages 14–15, and GFP and BFP fluorescence were imaged. Anterior enrichment of GFP-Pk3 is indicated by arrows (D–E’), while a non-polarized cell is marked by an asterisk (F–F’). The anteroposterior (AP) axis is indicated. Scale bar, 30 µm. (G) Quantification of GFP fluorescence for the GFP-Pk3-Vangl2 complexes in mosaically expressing cells is shown as a graph. Mean fluorescence is plotted along the cell circumference as a function of circular angle from 0 to 360 degrees relative to the AP axis, with fluorescence intensity shown in green (HA-Vangl2), pink (HA-Vangl2Y > F), and brown (HA-Vangl2Y > E) lines. The number of scored cells is indicated. The data are representative of four independent experiments.