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Fig. 2 Pineal-specific EGFP expression in Ex(–1055) transgenic zebrafish. (A) Schematic representation of Ex(–1055) construct in a linearized form used for microinjection. (B) Dorsolateral views (bright field image) of a WT larva (upper) and Ex(–1055) transgenic larva (lower) at 7 dpf. (C) Fluorescent image of B. The transgene-dependent fluorescence signal was observed specifically in the pineal gland of Ex(–1055) transgenic larva (arrowhead), and autofluorescence signals observed in transgenic and WT fish are marked by arrows. (D) High-magnification confocal image (dorsal view) of EGFP-positive pineal cells of 7-dpf-larva, with anterior to the left. The outer segment-like extrusion is indicated by each arrowhead. (E) Dorsal view of Ex(–1055) transgenic adult fish illuminated with both tungsten lamp and blue light. EGFP fluorescence signals were observed only in the pineal gland throughout its life. (F–I) Frontal views (dorsal up) of living transgenic embryos observed at 28 hpf (F and G) or 43 hpf (H and I) by using Nomarski optics (F and H) or fluorescence microscopy (G and I). Arrowheads in G and I indicate EGFP-positive cells in the pineal gland, and arrows in I point to EGFP-positive cells in the retina. The embryos in G and I were photographed under the same exposure conditions. (J) 4′,6-diamidino-2-phenylindole staining of a 10-μm-thick cross-section of the head of Ex(–1055) transgenic larva at 7 dpf. (K) EGFP fluorescent image of J. [Bars = 1 mm (B and C), 10 μm (D), 2 mm (E), and 100 μm (F–K).] For a clear demonstration, pigmentation of the embryos and larvae was reduced by treatment with 0.003% 1-phenyl-2-thiourea (Nacalai Tesque, Kyoto).