Dynamics and Compositions of the PAAs and OAAs
(A) Diagrams illustrate the principle of GFP-assisted sagittal serial microscopy for visualizing proteins at the opposing apical surfaces: the opposing apical surfaces at the midline region (SM), which contain the OAAs, must be flanked by two spatial references—PAA regions of the left and right halves of the tissue (SL and SR). Green represents transiently expressed GFP in some cells. The bottom panels are images collected with this technique: N-Cad and β-catenin were detected at the PAA regions as circumferential belts (SL and SR, arrows) as well as in the OAA regions as punctate foci (SM; arrowheads). By contrast, ZO-1 and actin were detected in the PAA regions (SL and SR, arrows) but not in the OAA regions (SM, arrowheads).
(B) Diagrams illustrate the strategy to visualize transiently expressed N-Cadwt-GFP or N-Cadm117-GFP at 14-ss. Note that N-Cadwt-GFP enriched on the opposing apical surfaces (illustrated by the drawing on the right).
(C) The PAAs (arrows) and the opposing apical surfaces (arrowheads indicating the OAAs) survived the Triton X-100 extraction of the anti-GFP immunogold electron microscopic procedure, whereas the lateral membranes were dissolved (basal of asterisks).
(D) The magnification of the boxed region in C revealed the labeling of N-Cad-GFP with 5-nm gold particles at the PAAs (arrows, 72% of the PAAs were labeled with gold particles) and OAAs (arrowhead). Insets are magnifications of boxed regions.
(E) Crb2a juxtaposed with ZO-1 sites at the apical ends (26-ss).
(F) The table summarizes the developmental and compositional changes of the OAAs and PAAs. Crb*, Crb-based adhesion; NaKATPase*, Na+/K+-ATPase-based adhesion; +, presence; -, absence. Also see Figure S2.