Vertebrate ancient-long opsin: a green-sensitive photoreceptive molecule present in zebrafish deep brain and retinal horizontal cells

Abstract

Nonretinal/nonpineal photosensitivity has been found in the brain of vertebrates, but the molecular basis for such a "deep brain" photoreception system remains unclear. We conducted an extensive search for brain opsin cDNAs of the zebrafish (Danio rerio), a useful animal model for genetic studies, and we have isolated a partial cDNA clone encoding an ortholog of vertebrate ancient (VA) opsin, the function of which is unknown. Subsequent characterization revealed the occurrence of two kinds of mRNAs encoding putative splicing variants, VA and VA-Long (VAL) opsin, the latter of which is a novel variant of the former. Both opsins shared a common core sequence in the membrane-spanning domains, but VAL-opsin had a C-terminal tail much longer than that of VA-opsin. Functional reconstitution experiments on the recombinant proteins showed that VAL-opsin with bound 11-cis-retinal is a green-sensitive pigment (lambdamax approximately 500 nm), whereas VA-opsin exhibited no photosensitivity even in the presence of 11-cis-retinal. Immunoreactivity specific to this functionally active VAL-opsin was localized at a limited number of cells surrounding the diencephalic ventricle of central thalamus, and these cells were distributed over approximately 200 micrometer along the rostrocaudal axis. Taken together with the previous study on the locus of the teleost brain photosensitivity (von Frisch K, 1911), it is strongly suggested that the VAL-positive cells in the zebrafish brain represent the deep brain photoreceptors. The VAL-specific immunoreactivity was also detected in a subset of non-GABAergic horizontal cells in the zebrafish retina. The existence of VAL-opsin, a new member of the rhodopsin superfamily, in these tissues may indicate its multiple roles in visual and nonvisual photosensory physiology.

Fig. 1.

The deduced amino acid sequences of zebrafish VA- and VAL-opsin. A, A secondary structural model for VA- and VAL-opsin. VA- and VAL-opsin have a common core sequence (M1-Q303) and diverged C-terminal tails, which are shown on the gray background. Putative transmembrane segments (helices I–VII) are indicated with roman numerals. The lysine residue at 287 (K287) and the glutamic acid at 106 (E106) are the putative retinal binding site and the counter ion, respectively (white characters on the black background).B, Comparison of C-terminal amino acid sequences of zebrafish VAL-opsin (zVAL; V293-M377) with those of other opsins: catfish parapinopsin (cfPP; GenBank accession number AF028014), chicken pinopsin (cP;U15762), chicken red (cR; X57490), chicken violet (cV; M92039), chicken blue (cB; M92037), chicken green (cG; M92038), chicken rhodopsin (cRh; D00702), salmon VA-opsin (sVA;AF001499), and zebrafish VA-opsin (zVA). The multiple alignment was conducted with the Clustal W 1.8 program (Thompson et al., 1994). The residues identical to that of zVAL at each position are shown with white characters on black background. The C termini of the sequences are shown withasterisks. C, A schematic drawing of VN and VC regions in VA/VAL-opsin. VN region (M1-S31) and VC region (Q303-M377 in VAL-opsin) were used for raising antibodies (see Materials and Methods).

Fig. 2.

Light-induced changes in absorption spectra of reconstituted VA- and VAL-opsin. The recombinant proteins of zebrafish VA- and VAL-opsin produced in the 293S cells were incubated with an excess amount of 11-cis-retinal for reconstitution. Then the sample was irradiated with an orange light (>520 nm) in the presence of 50 mm hydroxylamine (see Materials and Methods). Shown are the difference absorbance spectra before and after complete bleaching of VA-opsin (curve 1) and VAL-opsin (curve 2). Equivalent sample from mock-transfected cells showed no spectral change on irradiation (data not shown).

Fig. 3.

Immunohistochemical localization of VAL-opsin in the zebrafish brain. A, A Nomarski image of the zebrafish brain cross-section including the central posterior thalamic nucleus (CP), which is located near the diencephalic ventricle (V).B, Immunofluorescence labeling of the CP region with VC antibody. The VC immunoreactivity was monitored as fluorescence signals. The positive signals in the cell bodies and axon-like fibers are indicated by arrows and arrowheads, respectively. C, A control section. The section of the CP region was immunolabeled as in B without the primary antibody. Background fluorescence signals with week intensities should be ascribed to the endogenous fluorescence. D, A magnified image of B. Ce, Cerebellum;MO, medulla oblongata; OB, olfactory bulb; P, pineal gland; Tel, telencephalon; TeO, tectum opticum. Scale bar, 50 μm.

Fig. 4.

RT-PCR analysis of VA- and VAL-opsin gene expression in the zebrafish brain and eye. The amplification reaction was performed after incubation with (+RTase) or without (−RTase) reverse transcriptase (see Materials and Methods).

Fig. 5.

Immunohistochemical localization of VA/VAL-opsin in the zebrafish retina. A, A Nomarski image of the zebrafish retinal section. B, Immunofluorescence labeling with the VC antibody. The VC immunoreactivity was detected as fluorescence signals in subpopulation of horizontal cells (arrows) in the inner nuclear layer (INL). C, Immunofluorescence labeling with the VN antibody. The VN antibody labeled not only the horizontal cells (arrows) but also the subpopulation of amacrine cells (arrowheads) in the inner nuclear layer (INL). D, Immunofluorescence labeling with AS-Rh, an antibody against bovine rhodopsin. E, A control section. The retinal section was immunolabeled as inD with replacement of the primary antibody by mouse preimmune serum. Background fluorescence signals in the photoreceptor layer (PRL) with weak intensities were attributed to the endogenous fluorescence, because similar signals were detected even without treatment of the secondary antibody. ONL, Outer nuclear layer; OPL, outer plexiform layer;IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 50 μm.

Fig. 6.

Double-immunofluorescence labeling of the zebrafish retinal section with the VC and GAD antibodies. The VC antibody, specific to VAL-opsin, labeled a subset of horizontal cells (A, red), and the GAD antibody labeled horizontal and amacrine cells (B, green).C is a merged image of A andB, demonstrating the mutually exclusive distribution of the VC-positive cells (arrows) and the GAD-positive horizontal cells (arrowheads). D is a control section that was treated with both mouse and rabbit preimmune sera instead of the primary antibodies. In each section, cell nuclei were stained by TO-PRO-3 (blue), and images were viewed with a confocal laser scanning microscopy (Leica, TCS-NT). Scale bar, 50 μm.

Fig. 7.

A regular geometric pattern of VAL-opsin-expressing horizontal cells in the zebrafish retina. A section tangential to the retinal surface through the distal margin of the inner nuclear layer was immunolabeled with the VC antibody, specific to VAL-opsin, as in Figure 5B. The regular arrangement of the VC-positive horizontal cells is represented in theinset, where their cell bodies are depicted byopen circles. Scale bar, 20 μm.