Planar polarized distribution of Crumbs2a is retained at the level of cone inner segments in tbx2b mutants despite loss of pentameric organization of red, green, and blue cones.
Panels A–C and E–G are confocal images of retinal flat-mounts immunostained for Crb2a (white) in a wild-type, transgenic (trβ2:tdTomato) fish (A–C) in which red cones express the tdTomato reporter (red), and a tbx2b mutant (E–G), which expressed the sws1:EGFP reporter, although no UV cones are present in the region imaged. Each panel is a projection of 3 to 12 optical sections selected from a complete, vertical z-stack of the Crb2a immunolabeling: panels A, E are at the level of the OLM; panels B, F are 2–4 μm apical to the OLM, at the level of the red, green, and blue cone inner segments; panels C, G are 6–7 µm apical to the OLM, at the level of the red and green inner segments. A) Selected UV and blue cones are identified with magenta and blue dots, respectively, based on their relative sizes and positions in the mosaic array at the OLM. B) At the level of inner segments, Crb2a is preferentially localized to interfaces between red/green double cones and between a blue cone and the two flanking red cones (arrows). The inner segments of UV cones (magenta dots) are short and do not extend this far apically. C) Only the longest cones (red/green double cones) extend to this level, and Crb2a remains polarized. E) Retinal flat-mount from a tbx2b mutant at the OLM. This mutant did not carry the sws2:mCherry transgene, so the blue cones cannot be distinguished from the red and green cones. F, G) Short, curved segments of Crb2a, which resemble red/green double cone interfaces in the wild-type extend to the most apical level (G), but other Crb2a segments (yellow arrows) are at the intermediate level (F) but not higher (G). These may represent blue cones. Panels D and H show tangential cryosections through the level of nuclei of red, green, and blue cones; nuclei are stained with Hoechst (grey); red and green cones are immunolabeled with the membrane-associated, specific antibody, zpr1 (red). Both the wild-type (D) and tbx2b mutant (H) fish carried the UV and blue cone transgenes, sws1:EGFP (magenta) and sws2:mCherry (blue), although this retinal region lacked UV cones in the mutant. A single pentameric unit is encircled by an oval (D) and white arrows point to the interface between red and green double cone pairs. Scale bars: 10 μm (A, B, C, E, F, G) and (D, H).