Fig 2: Binding of PTK7 with FGFR1, EGFR, and ROR2 in HEK293 cells. The binding of PTK7 to FGFR1, EGFR, or ROR2 in HEK293 cells co-expressing PTK7-FLAG and FGFR1-HA, EGFR-HA, or ROR2-HA was analyzed. Cell lysates were pulled down with FLAG. The numbers below the HA or FLAG panels indicate the densitometric values of HA or FLAG bands relative to the corresponding GAPDH bands.

Fig 3: Effect of PTK7 knockdown on the phosphorylation of FGFR1 and EGFR in MDA-MB-231 cells. (A) The expression levels of FGFR1, EGFR, and ROR2 polypeptides in MDA-MB-231 cells were analyzed by Western blotting. FGFR1, EGFR, and ROR2 polypeptides expressed in HEK293 cells were used as positive controls. GAPDH levels are displayed for normalization. The numbers below the FGFR1, EGFR, or ROR2 panel indicate the densitometric values of FGFR1, EGFR, or ROR2 bands relative to the corresponding GAPDH bands. (B) Serum-starved MDA-MB-231 cells with or without PTK7 knockdown were stimulated with 10 ng/mL aFGF or 10 ng/mL EGF for 5 min. The phosphorylation of FGFR1 and EGFR was analyzed in the growth-factor-stimulated MDA-MB-231 cells by Western blotting with pFGFR1 and pEGFR. GAPDH levels are displayed for normalization. Each bar represents the mean ± standard deviation (SD) from three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. growth-factor-stimulated cells transfected with control vector. ++ p < 0.01 and +++ p < 0.001 vs. cells without growth factor stimulation. # p < 0.05 vs. vector-transfected cells. n.s.: not significant.

Fig 4: Principles of convergence and extension assay.a. An example maximum projection image to illustrate the proximity of the lineage tracer labeled clones (red) to Tg(Gsc-GFP-CAAX) expressing cells in the embryo shown in Fig. 5a–i. The yellow arrow shows the proximity. Scale bar is 50 µm b. Circularity assay. Some example cells illustrate how the width of a cell in parallel to the body axis (white lines) or perpendicular to the body axis (yellow line) was measured in a WT embryo or an embryo with a Ror2 expressing clone at 24 h. The ratio between the yellow line and the white line was calculated. A perfect circular cell has a circularity of 1.0, while below or above 1.0 indicates a noncircular, elongated shape. Scale bar is 20 µm. c. Cell circularity was measured within each embryo (n = 173, 292, 194, 200, 149, 176, 305, 458, 607 n = numbers per cell, 3 biological repeats). A perfect circular cell has a circularity of 1.0, while below or above 1.0 indicates a noncircular, elongated shape. d. Wild-type zebrafish embryos injected with mRNA for the indicated constructs plus Mini-Emerald (lineage tracer), subsequent in situ hybridization against pax6a. White dashed lines indicate the course of the midline, and the white bars indicate the width of the hindbrain domain. Scale bar 200 µm. e. The quantitative analysis of phenotype severity in zebrafish embryos. The phenotype severity is classified into none (e.g. ctrl), mild (e.g., +Ror2), and severe (e.g., +Wnt5b/Ror2). Ctrl, +Ror2 and +Wnt5b/Ror2 are representative examples of the different severity classifications, respectively. Numbers in bars represent the percentage of total embryos in each group. f. Hindbrain width was measured in embryos. n = 13, 23, 19, 18, 23, 11, n = numbers per embryo, 3 biological repeats. Significance is calculated by a one-way ANOVA test plus a Tukey multiple comparisons test. Box and whisker plots (c,f) show median, upper and lower quartile ranges with whiskers for minimum and maximum values.

Fig 5: Principles of FLIM-FRET analysis of Wnt5b/Ror2 complexes.a. FRET efficiency, FRETeff, is defined as the fraction of photons that is transferred from a donor to an acceptor. τFRET, a lifetime of the donor in the presence of the acceptor; τdonor, lifetime if the donor is without the acceptor. b. Bi-exponential fitting of the fluorescence decay demonstrates that the lifetime of a donor is decreased in the presence of a FRET acceptor. c. Close packaging in the Wnt5b-GFP/mCherry-Ror2 complex allows FRET to the acceptor and, thus, reduces donor FLIM. d. A C-terminally fused Ror2-mCherry can be used as a negative control like the distance between donor and acceptor is too large, and the plasma membrane acts like as insulator. e. Quantification of FRET efficiency (n = 0, 17, 24, 0, 36, 22, 30, 11, n = regions of interests in three biological repeats). Significance is calculated by a one-way ANOVA test plus Dunnett multiple comparisons test. p-values shown on the graph are compared to the corresponding negative controls, indicated on the graph. Quantification of fluorescent GFP lifetime (n = 8, 47, 25, 21, 36, 23, 33, 11, n = regions of interest in three biological repeats). Significance is calculated by a one-way ANOVA test plus Tukey multiple comparisons test. p-values shown on the graph are compared to the corresponding negative controls indicated on the graph. f. Schematic representation of the confocal detection volume positioned on the plasma membrane in a living zebrafish embryo to measure ligand-receptor interactions. g. Fluctuations in the detected fluorescence intensities with time. h. Auto- and cross-correlation analysis of fluorescence fluctuations over time. The Grey curve represents the cross-correlation between Wnt5b-GFP and Ror2-mCherry. i. The dissociation constant KD is defined as the fraction of the concentration of the unbound ligands and unbound receptors to the concentration of the ligand-receptor complexes. j. Autocorrelation curves of ATTO 488 and ATTO 565 in solution to determine the FCCS volume. k. Determination of the FCS volume obtained from n = 10 independent samples of labeled protein stock solutions. l. For FCCS analysis, the auto-correlation curves for Wnt5b-GFP and Ror2-mCherry are shown in red and green, respectively. The cross-correlation curve is shown in blue. The corresponding average dissociation constant (KD) is indicated on the graph. m-n. Representative fluorescence images for FCCS measurements and example measuring spots (yellow circle) in the zebrafish embryo at 6hpf, injected with indicated mRNA. C1 indicates the Wnt5b-GFP/Ror2 positive clone, and C2 indicates a clone expressing Ror2-mCherry. Scale bar 10 µm. o. Relative binding affinities measured by FCCS for indicated fluorescent protein combinations from Fig. 2h. Significance is calculated by one-way ANOVA together with Dunnett multiple comparisons test. Box and whisker plots (e,k,o) show median, upper and lower quartile range with whiskers for minimum and maximum values. Source Data