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).

Ectopic gene expression in the pineal gland driven by the zebrafish rh chimeric promoter carrying the PIPE sequence. (A) Schematic representation of the constructs, Rh(–1084), Rh(–1084)/PIPE, and PIPE-Rh(–1084). (B) Comparison of PIPE and nearby sequence in Ex(–1055) with those in the corresponding region of Rh(–1084) or Rh(–1084)/PIPE. Bold characters in Rh(–1084)/PIPE represent five nucleotides (four substitutions and a single insertion) modified from Rh(–1084) to create an ectopic PIPE sequence. (C–F) Dorsal views (anterior up) of Rh(–1084) (C and D) and Rh(–1084)/PIPE (E and F) transgenic larvae at 7 dpf. Nomarski (C and E) and fluorescent (D and F) images were taken at the same focal plane without moving the larvae. EGFP fluorescence signals in the pineal gland of Rh(–1084)/PIPE transgenic larva are marked by an arrowhead (F). (F Inset) High-magnification image of the EGFP-positive pineal structure. The larvae in C–F were not treated with 1-phenyl-2-thiourea, so that strong EGFP fluorescence signals in the pigmented eyes (D and F) are only visible through the pupil. [Bars = 100 μm (C–F), 20 μm (F Inset).]

5′-Deletion analysis of the exorh promoter. (A) Schematic representation of the constructs, Ex(–1055), Ex(–301), and Ex(–147). (B–G) Dorsal views (anterior up) of Nomarski (B, D, and F) and EGFP fluorescent (C, E, and G) images of Ex(–1055) (B and C), Ex(–301) (D and E), and Ex(–147) (F and G) transgenic larvae at 7 dpf. The larvae in C, E, and G were photographed under the same exposure conditions. The larvae were treated with 0.003% 1-phenyl-2-thiourea. (Bars = 100 μm.)