Fz7 interacts with C-cadherin and PAPC at the cell membrane in vivo, shown by BiFC. (A) Primary structure of the different split YFP constructs used in the BiFC assay. The putative interacting partners were either fused to YFP fragment consisting of the first N-terminal 155 amino acids (YN) or the last C-terminal 83 amino acids (YC) of YFP. All constructs contained a myc tag that served as a control for successful expression. β, β-catenin binding site; CRD, cysteine-rich domain; cyto, cytoplasmic region; EC, extracellular cadherin repeat; p120, p120 binding site; SS, signal peptide sequence; TM, transmembrane domain. (B) Expression of different combinations of the split YFP constructs in DMZ explants to monitor complex formation in vivo during convergent extension. DMZ explants were either coinjected with GAP43-mCherry (GAP43) to label cell membranes or fixed and fluorescently immunostained for split YFP constructs using myc antibody (9E10) to control their successful expression. Nuclei were stained with DAPI. Protein–protein interaction was displayed by the YFP fluorescence (BiFC). Full-length xFz7 interacted with xC-cadherin, whereas xFz7-TM1 interacted with xPAPC at the cell membranes. No direct interaction between xC-cadherin and xPAPC was observed. (C–G) In vivo xFz7–xC-cadherin complex formation in the presence or absence of xPAPC or xWnt-11. Cell membranes were labeled with GAP43-mCherry, and protein–protein interaction was displayed by YFP fluorescence (BiFC). Depletion of xPAPC promoted the interaction between xFz7 and xC-cadherin, whereas in Wnt11 morphants, xFz7–xC-cadherin complex formation was abolished. Injection amount was as follows: 1 ng xPAPC-YN/YC RNA, 1 ng xC-cadherin-YC/YN RNA, 500 pg xFz7-YN RNA, 500 pg xFz7-TM1-YC, 20 pg xWnt-11 RNA, 1 pmol xWnt-11 MO, 1 ng xPAPC RNA, 1.6 pmol xPAPC MO, and 200 pg GAP43-mCherry RNA. Bars, 20 µm.