Figure 3. The instructive role of Wnt5a in the establishment of Pk3 polarity.

(A) Experimental scheme. RNAs encoding GFP-Pk3 (150 pg) and Xenopus HA-Vangl2 (60 pg) were injected animally into a ventral blastomere of 32-cell embryos, followed by injection of TurboFP635 (TFP) RNA (150 pg, lineage tracer) with or without Wnt5a RNA (500 pg) into a blastomere either to the right (L–R) or anterior (A–P) of the Pk3-injected blastomere. The injected embryos were fixed at indicated stages, ectodermal explants were dissected, and the orientation of Pk3 crescents was evaluated by GFP fluorescence. (B–D) Cell orientation in L-R-positioned clones. (B) Low magnification view of a stage 11.5 explant. Orientation of individual cells was quantified relative to the dashed line approximating TFP clone border (boxed area). The antero-posterior and left-right axes are indicated. (C) Control embryo, (D) Wnt5a-expressing embryo. (E–H) Cell orientation in A–P-positioned clones at indicated stages. (E) Low magnification view. (F) Control embryo, (G, H) Wnt5a RNA-injected embryos. Arrows indicate cell orientation relative to the TFP clone (D, G, H). Scale bar, 50 µm. (I, K) Rose diagrams show Pk3 patch orientation in L–R (I) or A–P (K) experimental groups. Cell orientation was defined by an angle between the line joining the two ends of each Pk3 patch and the line approximating TFP clone border. (M) Orientation of Pk3 crescents in the A–P group at stage 15. See Figure 1F–H legend for quantification details. n, number of scored cells. p values were obtained by comparing the Wnt group to the control group using Chi-squared test. (J, L, N) Polar plots derived from (I, K, M), respectively, depict the mean Pk3 orientation in individual embryos. Arrow length is 1 minus the circular variance around the mean. Data were collected from two independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.16463.006

Figure 3—figure supplement 1. Effect of Wnt5a on microtubule orientation.

Clip170-GFP RNA (150 pg) was injected into the left animal-ventral blastomere of eight-cell embryos, followed by injection of TFP RNA with or without Wnt5a RNA (500 pg) into the right animal-ventral blastomere. The movement of Clip170-GFP foci in 3–10 ectodermal cells located within 5 cell diameters to the TFP clone border was traced at stage 11 to 11.5. (A) Rose diagrams show the directions of Clip170-GFP foci movement. The proximal (P)-distal (D) axis is perpendicular to the TFP clone border. Data are collected from three independent experiments. Numbers of analyzed traces and cells (in parentheses) are indicated. p value was obtained using Chi-squared test. (B) Polar plots display mean axial vectors of Clip170 traces in individual embryos (each embryo represented by a blue arrow). Arrow length is 1 minus the circular variance around the mean. Black arrows are mean axial vectors for each experimental group.

DOI: http://dx.doi.org/10.7554/eLife.16463.007

Figure 3—figure supplement 2. ECD8 does not direct GFP-Pk3 polarization.

Eight-cell embryos were injected at the left ventral-animal blastomere with RNAs encoding GFP-Pk3 and HA-Vangl2, followed by injection of RNAs for TFP and either Wnt5a (500 pg) or ECD8 (500 pg) into the right ventral-animal blastomere. GFP and TFP fluorescence in the epidermal ectoderm of stage 11.5 embryos is shown. Images are representative of two independent experiments. Scale bar, 20 µm.

DOI: http://dx.doi.org/10.7554/eLife.16463.008