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First published online March 21, 2005
Journal of Experimental Biology 208, 1337-1345 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01532
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Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development

Masaki Takechi and Shoji Kawamura*

Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan



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Fig. 1. Whole-mount ISH of RH2 and M/LWS opsin probes to embryonic or larval eyes of zebrafish. (A–D) Lateral views of embryonic eyes (nasal side to the left and ventral to the bottom) hybridized with the RH2-1 CDR probe, where progress of the gene expression is staged as follows: stage 1, expression is detected in a patch of ventronasal retina; stage 2–3, another patch appears in nasal retina and the two fuse to fill the ventronasal retina; stage 4, expression extends from the ventronasal region to the central region and, less extensively, in dorsal and ventrotemporal directions; stage 5–6, expression is detected in more than three-fourths of the retina (Raymond et al., 1995Go; Takechi et al., 2003Go). Dotted line indicates rim of the eye. Arrowhead indicates location of the choroids fissure. (E) A lateral view of an embryonic eye (nasal side to the left and ventral to the bottom) hybridized with the RH2-4 CDR probe at 72 hpf. Hybridized area is indicated with a bracket. (F) A ventral view of a larval eye (nasal side to the left) hybridized with LWS-1 3'-UTR probe at 5.5 dpf. All scale bars, 50 µm.

 


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Fig. 2. ISH of RH2 opsin probes to transverse sections of zebrafish eyes. (A–C) Serial eye sections at 16 dpf hybridized with the RH2-1 3'-UTR (A), RH2-2 3'-UTR (B) and RH2-4 CDR (C) probes. Double arrows in (A) and (B) indicate the areas where the expression of RH2-1 and RH2-2 are disappearing, respectively. Arrows in (C) indicate the scarce hybridization signals detected for RH2-3/RH2-4. (D–F) Serial eye sections at 1 month pf hybridized with RH2-1 3'-UTR (D), RH2-2 3'-UTR (E) and RH2-4 CDR (F) probes. The single stars, double stars, and triple stars are position markers showing their corresponding locations between sections. (G–H) Serial sections at 2 years pf hybridized with the RH2-1 3'-UTR (G), RH2-2 3'-UTR (H) and RH2-4 3'-UTR (I) probes. Arrowheads in (H) and (I) point to the same location between the sections. An arrow in (I) indicates the hybridization signal of RH2-4 at ciliary marginal zone. In all panels, the dorsal side is to the top and ventral is to the bottom. Scale bars, 50 µm (A–F) or 200 µm (G–I).

 


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Fig. 3. Expression of RH2-3 and RH2-4 in an adult zebrafish retina. (A) A transverse section of an eye at 2 years pf hybridized with the RH2-3 3'-UTR probe. Double arrows indicate the areas of RH2-3 expression. (B,C) Neighboring transverse sections at higher magnification hybridized with the RH2-3 3'-UTR (B) and RH2-4 3'-UTR (C) probes. In all panels, the dorsal side is to the top and ventral is to the bottom. Scale bars, 200 µm (A) or 50 µm (B and C).

 


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Fig. 4. ISH of M/LWS opsin probes to transverse sections of zebrafish eyes. (A,B) Serial eye sections at 1 month pf hybridized with the LWS-1 3'-UTR (A) and LWS-2 3'-UTR (B) probes. Arrows in (A) indicate the sparse hybridization signals of LWS-1. Corresponding locations between the two sections are indicated with a star. (C,D) Serial sections at 2 years pf hybridized with the LWS-1 3'-UTR (C) and LWS-2 3'-UTR (D) probes. The solid and dotted double arrows in (C) indicate the areas where LWS-1 is expressed robustly and sparsely, respectively. The bracket in (D) indicates the area where LWS-2 expression disappeared on the dorsal side of the retina. In all panels, the dorsal side is to the top and ventral is to the bottom. Scale bars, 50 µm (A,B) or 200 µm (C,D).

 


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Fig. 5. Progression of gene expression of the five opsin types (RH1, M/LWS, SWS1, RH2 and SWS2) in embryonic zebrafish retina sampled at 40, 45, 50, 55, 60 and 72 hpf. Embryos sampled at each hpf were examined for staging opsin expression by whole-mount ISH with CDR probes of the five opsin genes (total number of embryos examined, N, are indicated in each panel). The number of embryos (vertical axis) showing certain expression stages (horizontal axis) was counted at each hpf for each gene. One eye was examined per embryo. Stage 0 indicates that no expression was detected in an observed retina. For RH1, subsequent stages are defined as follows (Raymond et al., 1995Go; Hamaoka et al., 2002Go): stage 1–2, expression is detected in a patch of ventronasal retina; stage 3, expression is detected across the choroid fissure from the primary ventronasal patch; stage 4, hybridization signal increases in the ventrotemporal patch and scatters outside the ventral region; stage 5–6, ventrotemporal and ventronasal patches are at equivalent density and fuse across the fissure to form a dumbbell shape, and density of the signal outside the ventral region further increases. For cone opsins (M/LWS, SWS1, RH2 and SWS2), refer to Fig. 1 legend for definitions of stages.

 


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Fig. 6. Schematic representation of the spatiotemporal expression patterns of M/LWS opsin subtypes in zebrafish retina (lateral views) reconstructed from ISH to serial sections. Expression areas of LWS-1 and LWS-2 are indicated with black and gray, respectively. Black dots indicate the sparse expression of LWS-1 in the central area of the retina. D, dorsal; V, ventral; N, nasal; T, temporal.

 


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Fig. 7. Schematic representation of the spatiotemporal expression patterns of RH2 opsin subtypes in zebrafish retina (lateral views) reconstructed from ISH to serial sections. Expression areas of RH2-1, RH2-2 and RH2-3 are indicated with different shades of gray (light, middle and dark, respectively). That of RH2-4 is indicated with black. Dots in RH2-2 expression at two weeks pf indicates sparse expression in the central retina. Up to 1 month pf, expression of RH2-3 and RH2-4 was not distinguishable. D, dorsal; V, ventral; N, nasal; T, temporal.

 

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© The Company of Biologists Ltd 2005