Wnt-11 regulates PAPC cell membrane localization through Fz7 during convergent extension. (A) Scheme showing time-lapse xPAPC localization analyses in DMZ explants. The two dorsal blastomeres of 16-cell–stage embryos were injected with 500 pg xPAPC-mCherry RNA alone or in combination with 20 pg xWnt-11 RNA, 1 pmol xWnt-11 MO, 500 pg xFz7 RNA, 1.6 pmol xFz7 MO, and/or 500 pg dnDyn1 RNA. 50 pg GAP43-GFP RNA was injected in all samples as a cell membrane tracer. At stage 10.25, DMZ explants were cut, and subcellular localization of xPAPC-mCherry was analyzed. (B–G) Representative images of time-lapse videos are shown. xWnt-11 stabilized xPAPC at the cell membrane, and this activity required xFz7. Bars, 60 µm. (H) Counting of DMZ explants showing xPAPC subcellular localization according the observed phenotypes showed in Fig. S1 A. n, number of DMZ explants. # exp., number of independent experiments. (I) Western blot (WB) analysis of total xPAPC-myc (gray arrowhead) protein amount performed with cell lysates of stage-11 embryos. xWnt-11 did not influence total xPAPC-myc protein amount, whereas xFz7 depletion reduced the overall protein level of xPAPC-myc, even in the presence of xWnt-11. GAP43-GFP (black arrowhead) served as an injection control, α-tubulin (black star) as a loading control, and PonceauS as a transfer control. Injection amount was as follows: 1 ng xPAPC-myc RNA, 40 pg xWnt-11 RNA, 1 pmol xWnt-11 MO, 500 pg xFz7 RNA, 1.6 pmol xFz7 MO, and 100 pg GAP43-GFP RNA. (J) Relative xPAPC-myc signal intensity in dependency of xWnt-11 and xFz7. For more details, see Materials and methods. Error bars show SEM. Student’s t test was performed (*, P < 0.05; **, P < 0.005).