Development of the cone mosaic pattern. Laser scanning confocal images (3D projections) from whole eyes of zebrafish embryos, processed for in situ hybridization with red and green opsin RNA probes to visualize individual cone photoreceptors. After processing, whole eyes are isolated from the embryonic heads and visualized using a Zeiss 510 laser scanning confocal microscope, by collecting a set of optical sections through the entire thickness of the eye (~100 µm). Computer reconstructions of three-dimensional and projection images were then generated from various angles of view. (A) The earliest photoreceptors appear at ~2 days postfertilization (dpf) in the outer nuclear layer of the ventral-nasal retina adjacent to the optic stalk, and the first cone opsin is red (TSA method, FITC green fluorophore). The lens is visible in the center, and the inset shows the individual red cones at higher magnification, in orthogonal views. Note the regularly spaced rows of red cones in the rotated (planar) view. (B) Double in situ hybridization with red opsin (Fast Red) and green opsin (TSA-FITC) probes, showing (at high magnification) the ventral patch of initial cones, adjacent to the future optic disc. (C) By ~3 dpf, the front of the wave of differentiating photoreceptors has nearly reached the dorsal-temporal edge of the retina at the upper left corner, with red cones at the leading edge, trailed by green cones.
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