Fig 1.
Phylogenetic tree for Opn5 genes.
The phylogenetic tree of Opn5 genes was constructed using the Neighbor-Joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in terms of the number of amino acid substitutions per site.
Fig 2.
Synteny of Opn5m2 gene in ray-finned fish species.
Phylogeny and syntenic orthologues in genomic regions containing the Opn5m2 gene of ray-finned fishes are shown. Phylogenetic relationship of ray-finned fish species was drawn based on Near, et al [17]. Pentagons represent the genes and the direction of the complementary strand. Blue, green, red, and orange pentagons correspond to orthologues of iffo1b, Opn5m2, gapdh, and nppcl, respectively.
Fig 3.
UV-visible absorption spectral properties of zebrafish Opn5m2.
A, Absorption spectra of zebrafish Opn5m2 purified after addition of 11-cis-retinal. Spectra were recorded in the dark (curve 1, black) and after UV light irradiation (curve 2, red), subsequent yellow light (>480 nm) irradiation (curve 3, green), and following repetition of UV (curve 4, cyan) and yellow light irradiations (curve 5, magenta), respectively. B, Difference spectra calculated based on the spectra in panel A. Curve 1 (red) is the difference spectrum calculated by subtracting curve 2 from curve 1 in panel A. Curves 2, 3, and 4 also show the spectral changes from curve 2 to 3, from curve 3 to 4, and from curve 4 to 5, in panel A, respectively. C, The calculated absorption spectra of zebrafish Opn5m2 in the dark and after UV light irradiation. The absorption spectrum of visible light-absorbing pigment (red curve) was calculated by fitting the visible spectral region of curve 3 in panel B according to the method described in a previous study [8]. The gray curve was acquired by subtracting the best-fitted curve from curve 3 in panel B, which is fitted by a Gaussian function (black curve). The spectra were normalized to the peak absorption of the visible-light absorbing pigment. The calculated absorption maxima were 360 and 462 nm for UV- and visible light-absorbing pigments, respectively. D, Absorption spectra of zebrafish Opn5m2 purified following addition of all-trans-retinal. Spectra were recorded in the dark (curve 1, black), after yellow light (>480 nm) irradiation (curve 2, red), and subsequent UV light irradiation (curve 3, green). Inset, Difference spectra calculated by subtracting curve 2 from curve 1 (curve 1, red), and curve 3 from curve 2 (curve 2, green) in panel D.
Fig 4.
In situ hybridization analysis of Opn5m and Opn5m2 mRNA in the retina of medaka fish, zebrafish, and spotted gar.
A-D, Detection of medaka fish Opn5m in the retina. Frontal consecutive sections were hybridized with Opn5m antisense (A) and sense (B) probes. Enlarged views of the boxed areas, ventral and dorsal sides, in (A) are shown in (C) and (D), respectively. E-H, Detection of zebrafish Opn5m in the retina. Frontal consecutive sections were hybridized with Opn5m antisense (E) and sense (F) probes. Enlarged views of the boxed areas, ventral and dorsal sides, in (E) are shown in (G) and (H), respectively. I-L, Detection of zebrafish Opn5m2 within the retina. Frontal consecutive sections were hybridized with Opn5m antisense (I) and sense (J) probes. Enlarged views of the boxed areas, ventral and dorsal sides, in (I) are shown in (K) and (L), respectively. M-P, Detection of spotted gar Opn5m within the retina. Ventral regions were hybridized with Opn5m antisense (M) and sense (N) probes. Dorsal regions were hybridized with Opn5m antisense (O) and sense (P) probes. Q-T, Detection of spotted gar Opn5m2. Ventral regions were hybridized with Opn5m antisense (Q) and sense (R) probes. Dorsal regions were hybridized with Opn5m antisense (S) and sense (T) probes. All the sections shown in this figure were counterstained with Nuclear Fast Red. Scale bar: A, B, E, F, I, J, 200 μm; C, D, G, H, K, L, M-T 50 μm
Fig 5.
Distribution of Opn5m mRNA within medaka fish brain.
A, Schematic drawing of medaka fish brain, dorsal view. Numbered lines indicate the positions of cross sections shown in B-J. B-J, Localization of medaka fish Opn5m in the brain. Expression signals were detected within frontal sections cut along lines 1 (B), 2 (E) and 3 (H). Sections were hybridized with Opn5m antisense (B, C, E, F, H, I) and sense (D, G, J) probes. Enlarged views of regions around preoptic area in panel B, pituitary in panel E, and habenula in panel H are shown in panels C, F, and I, respectively. Panels D, G, and J show the consecutive tissue sections to C, F, and I hybridized with Opn5m sense probe, respectively. All sections shown in this figure were counterstained with Nuclear Fast Red. Scale bar: B, E, H, 200 μm; F, G, I, J, 100 μm; C, D, 50 μm.
Fig 6.
Distribution of Opn5m mRNA within zebrafish brain.
A, Schematic drawing of zebrafish brain, dorsal view. Numbered lines indicate the positions of cross sections shown in B-M. B-M, Localization of zebrafish Opn5m within the brain. Expression signals were detected within frontal sections cut along lines 1 (B), and 2 (E). Sections were hybridized with Opn5m antisense (B, C, E, F, H, J, L) and sense (D, G, I, K, M) probes. Enlarged views of regions around entopeduncular nucleus in panel B, optic tectum, pretectal nucleus, paraventricular organ, and periventricular nucleus in panel E are shown in panels C, F, H, J, and K, respectively. Panels D, G, I, K, and M show consecutive tissue sections to C, F, and I hybridized with Opn5m sense probe, respectively. We could not detect any hybridization signals for Opn5m2 in the zebrafish brain. All sections shown in this figure were counterstained with Nuclear Fast Red. Scale bar: B, E, 200 μm; C, D, F-M, 50 μm.
Fig 7.
Distribution of Opn5m and Opn5m2 mRNA within spotted gar brain.
A, Schematic drawing of spotted gar brain, dorsal view. Numbered lines indicate the positions of cross sections shown in B-G. B-D, Localization of spotted gar Opn5m within the brain. Expression signals were detected within the frontal section cut along line 1. Sections were hybridized with Opn5m antisense (B, C) and sense (D) probes. Enlarged view of the region around the optic chiasm in B is shown in panel C. Panel D shows the consecutive tissue section to C hybridized with Opn5m sense probe. The broken line indicates the border between optic chiasm and brain parenchyma. E-G, Localization of spotted gar Opn5m2 in the brain. Expression signals were detected in the frontal section cut along line 2. Sections were hybridized with Opn5m2 antisense (E, F) and sense (G) probes. Enlarged view of the region around third ventricle in panel E is shown in F. Panel G shows the consecutive tissue section to F hybridized with Opn5m sense probe. All sections shown in this figure were counterstained with Nuclear Fast Red. Scale bar: B, E, 200 μm; C, D, F, G, 50 μm.