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The Journal of Neuroscience, April 15, 2000, 20(8):2845-2851
Vertebrate Ancient-Long Opsin: A
Green-Sensitive Photoreceptive Molecule Present in Zebrafish Deep Brain
and Retinal Horizontal Cells
Daisuke
Kojima,
Hiroaki
Mano, and
Yoshitaka
Fukada
Department of Biophysics and Biochemistry, Graduate School of
Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo
113-0033, and CREST, Japan Science and Technology Corporation,
Tokyo, Japan
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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 ( max ~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
~200 µm 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.
Key words:
VAL-opsin; VA-opsin; deep brain photoreceptor; horizontal
cell; alternative splicing; zebrafish
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INTRODUCTION |
Various nonmammalian vertebrates
have photoreceptor cells not only in the retina (rod and cone) but also
in the extraretinal tissues such as pineal complex, deep brain, and
skin (Yoshikawa and Oishi, 1998 ). These nonvisual photoreceptor cells
seem to play important roles in diverse physiological responses
including photoentrainment of circadian rhythms, detection of seasonal
changes in the photoperiod, and regulation of body color. Several lines of evidence suggested that opsin-type molecules contribute to the
nonvisual photoreception of vertebrates (Deguchi, 1981; Lythgoe et al.,
1984; Foster et al., 1985), and in fact, opsin-type molecules have been
identified in the pineal complex (Okano et al., 1994; Max et al., 1995;
Blackshaw and Snyder, 1997; Mano et al., 1999), dermal melanophores
(Provencio et al., 1998), and the deep brain region of the pigeon (Wada
et al., 1998), frogs (Provencio et al., 1998; Yoshikawa et al., 1998),
and mouse (Blackshaw and Snyder, 1999). Despite evident expression of
opsin-type molecules in the deep brain region, their roles in
light-regulated physiological responses have not been precisely
determined because of (1) no clear evidence for physiological responses
attributable to the deep brain photoreceptors (in amphibians and
mammals) or (2) infeasibility of genetic approaches (in birds) or
both. With respect to these points, the zebrafish (Danio
rerio) is a useful animal model for future genetic studies on the
deep brain photoreceptive functions, which have been well characterized
especially in teleosts (von Frisch, 1911; Scharrer, 1928; Oksche and
Hartwig, 1975; van Veen et al., 1976; Hartwig and van Veen, 1979;
Kavaliers, 1980; Tabata et al., 1988, 1989).
The pioneering study on the vertebrate deep brain photoreception (von
Frisch, 1911) demonstrated that a light-induced change in skin color of
a teleost (European minnow) is not abolished by the removal of the eyes
and pineal complex, and the light sensitivity was ascribed to the
"deep brain photoreceptor" located at the ependyma of the
diencephalic ventricle. Despite this clear evidence, the molecular
identity and detailed location of the teleost deep brain photoreceptor
remain unknown. In the present study, we find a gene expression of
VA-Long (VAL) opsin, a novel variant of vertebrate ancient
(VA) opsin in the zebrafish brain. VA-opsin was originally identified
in the salmon as an ocular opsin (max: 451 nm), with its function
unknown, and the gene expression has been detected in a subset of
horizontal and amacrine cells in the retina (Soni and Foster, 1997;
Soni et al., 1998). Here we report that VAL protein is localized to a
limited number of diencephalic cells and retinal horizontal cells, the
location of the former being consistent with that of the deep brain
photoreceptor described by von Frisch (1911). Together with the light
sensitivity reconstituted with 11-cis-retinal in
vitro, it is strongly suggested that VAL-opsin contributes to the
deep brain photosensitivity as well as retinal physiology.
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MATERIALS AND METHODS |
PCR-based cDNA cloning. The RNA fraction extracted
from the brains of light-adapted zebrafish (Danio rerio) was
used for oligo(dT)-primed synthesis of the cDNA, and it was applied to
PCR with the degenerate primers Op-Fw and Op-Rv (Yoshikawa et al.,
1998), which were designed to amplify DNA fragments encoding various
vertebrate rhodopsin family members. This PCR amplified two kinds of
partial cDNA fragments: one encoded a pineal opsin termed exo-rhodopsin
(Mano et al., 1999), and the other (Z9-20) encoded the zebrafish
ortholog of VA-opsin (Soni and Foster, 1997). In the subsequent 3'
rapid amplification of cDNA end (RACE) of Z9-20, two kinds of cDNA
fragments (V14E9 and V14E3) were obtained. The sequences of V14E9 and
V14E3 were identical to each other in 119 nucleotides of their 5'-ends,
but they showed a divergence in the rest of the 3'-ends. In contrast, 5'-RACE, by using internal primers of Z9-20 with the zebrafish brain
cDNA template, amplified a single DNA fragment (V16E23). On the basis
of the sequences of Z9-20, V14E9, V14E3, and V16E23, we assumed the
presence of two kinds of VA-opsin mRNAs (VA and VAL) in the zebrafish
brain. Finally, the two variants of VA-opsin mRNAs were identified by
amplification of the entire coding sequences of VA and VAL, using the
zebrafish brain cDNA as a template. The primers used were
(1) the internal primers of V16E23
[5'-TCAGGGAGA(C/A)ATC(T/C)GAACTG-3'] and V14E9
(5'-AACTGAAATCAAATGCTCACTC-3') for amplification of VA sequence or
(2) the internal primers of V16E23
[5'-TCAGGGAGA(C/A)ATC(T/C)GAACTG-3'] and V14E3
(5'-CACTGAGATGAAGACTCTGC-3') for amplification of VAL sequence. In
this final series of PCR, four independent clones from each of the
three independent amplifications of either VA or VAL were sequenced on
both DNA strands. The nucleotide sequence data have been
deposited to the DNA Data Bank of Japan/European Molecular Biology
Laboratory/GenBank nucleotide sequence databases with the accession
numbers AB035276 (VAL-opsin) and AB035277 (VA-opsin).
Preparation of recombinant proteins and spectroscopic
measurements. The zebrafish VA- and VAL-opsin were produced in
human embryonic kidney cells 293S as described previously (Kojima et al., 1996), with the aid of a eukaryotic expression vector pUSR (Kayada et al., 1995) derived from pUC-SR (Shimamoto et al., 1993).
Briefly, five 10-cm-diameter dishes of 293S cells were co-transfected
with the opsin expression vector and pRSV-TAg and cultured for 48 hr at
37°C. Then, the cells expressing VA or VAL were collected in 400 µl
of buffer Pm (50 mM HEPES-NaOH, 140 mM NaCl, 3 mM MgCl2, 1 mM
dithiothreitol, 1 µg/ml aprotinin, 1 µg/ml leupeptin, pH 6.6, at
4°C). Subsequent procedures were performed under a dim red light
(wavelengths >660 nm) or in the dark. The collected cells were mixed
with 4 nmol of 11-cis-retinal and incubated at 4°C for 5 hr, and proteins were extracted by the addition of 500 µl of 2%
dodecyl- -D-maltoside dissolved in buffer Pm. Then the extract was
mixed with neutralized hydroxylamine (at a final concentration of 50 mM) and incubated at 0°C for 2 hr for
conversion of free 11-cis-retinal into retinal-oxime. A
difference absorption spectrum before and after bleaching of the sample
with an orange light (>520 nm) at 0°C for 60 sec was recorded with a
spectrophotometer (model MPS-2000; Shimadzu, Kyoto, Japan).
RT-PCR. For detection of VA and VAL mRNAs in the
zebrafish tissues, RT-PCR was performed by using TaqGold polymerase (PE
Biosystems, Foster City, CA) and oligo(dT)-primed cDNA template
(synthesized as described in the previous section). The primers used
were (1) 5'-TATGTGTTCATGAACAAACAG-3' and 5'-ACAAGTCAGTTTTAATGATGC-3' to amplify a 236 base pair fragment derived from VA mRNA or (2)
5'-TATGTGTTCATGAACAAACAG-3' and 5'-GACACACTTTGTTCTCAGG-3' to amplify a
238 base pair fragment from VAL mRNA. The cycling protocol used was
94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec for 35 cycles.
Preparation of antibodies. For preparation of
region-specific antibodies, three kinds of fusion proteins were
designed: (1) MBP-VC, which is composed of maltose-binding protein
(MBP) and the C-terminal region of VAL (termed VC region; Q303-M377)
(see Fig. 1A,C), (2) GST-VC,
composed of glutathione S-transferase (GST) and VC, and (3)
GST-VN, composed of GST and the N-terminal region common to VA and VAL
(termed VN region; M1-S31) (see Fig. 1A,C). MBP-VC was expressed in
Escherichia coli BL21 strain with the aid of a prokaryotic
expression vector pMAL-c2 (New England Biolabs, Beverly, MA) and
purified by affinity chromatography using amylose resin (New England
Biolabs). GST-VC, GST-VN, and GST were expressed in BL21 with the aid
of a prokaryotic expression vector pGEX-5X-1 (Amersham Pharmacia
Biotech, Tokyo, Japan) and were purified by affinity
chromatography using glutathione-Sepharose 4B (Amersham Pharmacia
Biotech). Five female mice (BALB/c strain, 5 weeks old) were immunized
with purified MBP-VC or GST-VN. For affinity purification of antisera,
GST-VC, GST-VN, and GST were separately immobilized to
glutathione-Sepharose 4B according to the method reported by Bar-Peled
and Raikhel (1996). Then, the antibodies specific to the VC region (VC
antibody) were purified by using the GST-VC-immobilized Sepharose to
eliminate anti-MBP activity. As for the antisera to GST-VN, anti-GST
fractions were removed by passing through a GST-immobilized Sepharose
column, and the antibodies specific to the VN region (VN antibody) were purified by using GST-VN-immobilized Sepharose.
Preparation of tissue sections. Tissue sections were
prepared as described (Barthel and Raymond, 1990) with some
modifications. Adult zebrafish were maintained in light (14 hr)/dark
(10 hr) cycles, and decapitated 6 hr after the onset of the light
phase. Then, the heads including the eyes and brain were
immersion-fixed in 4% paraformaldehyde, 5% sucrose in phosphate
buffer (PB; 0.1 M Na-phosphate buffer, pH 7.4) for 2 hr at
room temperature (23°C). After they were rinsed with 5% sucrose in
PB, the tissues were cryoprotected with increasing concentrations of
sucrose (10, 15, and 20%) in PB, embedded in a solution of 2:1 mixture
of 20% sucrose (in PB) and the OCT mounting medium (Sakura, Tokyo,
Japan), frozen by using liquid nitrogen, and stored at  80°C until
use. Finally, 10-µm-thick sections were cut out from the embedded
tissues, mounted on gelatin-coated glass slides, and air-dried.
Immunofluorescence analyses of tissue sections. The sections
on glass slides were pretreated with a blocking solution [1.5% horse
or goat normal serum, 0.3% Triton X-100 in PBS (10 mM
Na-phosphate buffer, 140 mM NaCl, 1 mM
MgCl2, pH 7.4)], and then incubated with a
primary antibody diluted in the blocking solution at 4°C for 3 d. After they were rinsed with PBS, the sections were treated with a
secondary antibody for 20 hr at 4°C and again washed with PBS. Then,
the sections were coverslipped with a solution of 1:1 mixture of 50%
glycerol (in PBS) and Vectashield Mounting Medium (Vector Laboratories,
Burlingame, CA). The primary antibodies used were VC antibody (diluted
to 0.6 µg/ml), VN antibody (diluted to 0.4 µg/ml), AS-Rh [(Kawata
et al., 1992) a mouse antiserum raised against purified bovine
rhodopsin, diluted 1:10,000], and GAD antibody (AB108, Chemicon,
Temecula, CA; a rabbit antiserum raised against feline glutamic acid
decarboxylase, diluted 1:1000). For a control staining, the primary
antibody was eliminated or replaced by a mouse IgG fraction (diluted to
0.6 µg/ml) purified from preimmune sera by Protein G-Sepharose column
chromatography, or replaced by rabbit preimmune serum (diluted 1:1000).
The secondary antibodies used were the horse anti-mouse IgG antibody
conjugated either with FITC (diluted to 7.5 µg/ml) or with Texas Red
(diluted to 15 µg/ml), and the goat anti-rabbit IgG antibody
conjugated with FITC (diluted to 7.5 µg/ml), all of which were
purchased from Vector Laboratories. TO-PRO-3 (Molecular Probes, Eugene, OR), diluted to 0.2 µM, was used for staining of cell nuclei.
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RESULTS |
Two isoforms of zebrafish VA-opsin ortholog
To identify an opsin-type photosensitive molecule expressed in the
zebrafish brain, we conducted an extensive PCR-based screening of cDNAs
prepared from the whole-brain RNA fraction (see Materials and Methods).
Then we isolated a cDNA clone (Z9-20) encoding a part of a putative
opsin, of which the deduced amino acid sequence was highly similar
(75% identical) to that of salmon VA-opsin (Soni and Foster, 1997). To
obtain a full-length cDNA, 5'- and 3'-RACE were used, resulting in
isolation of two distinct cDNA variants. The deduced amino acid
sequences (and the corresponding nucleotide sequences) of these
variants were identical to each other within the first 303 residues,
and they showed a divergence in the C-terminal sequence (Fig.
1A). These two forms
were probably derived from alternative RNA splicing from a single gene
(see Discussion), and the common core sequence (M1-Q303) showed higher similarity (73.4% identity) to salmon VA-opsin (Soni and Foster, 1997)
than to any other known opsin sequences (<50% identity). Interestingly, one of the isoforms had an extremely short cytoplasmic tail, just like salmon VA-opsin, and hence this isoform is most likely
to represent a counterpart of salmon VA-opsin. The cytoplasmic tail of
the other isoform was 67 amino acids longer than that of VA-opsin, and
thus we named it VAL-opsin (VA-Long-opsin). Such a longer
cytoplasmic tail is a common feature among all the members of rhodopsin
family except for VA-opsin. Furthermore, the proximal region in the
cytoplasmic tail of VAL-opsin had several characteristic residues (Fig.
1B, open triangles), which are well
conserved among the members of vertebrate rhodopsin family except for
salmon and zebrafish VA-opsin. These suggest a functional difference
between VA- and VAL-opsin.
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Figure 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 with
asterisks. 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).
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Functional reconstitution of VAL-opsin with
11-cis-retinal
A possible functional difference between the two isoforms was
examined by preparing recombinant proteins produced in the human embryonic kidney cells 293S. After being reconstituted with
11-cis-retinal, which is a common chromophore of the
rhodopsin family, VA- and VAL-opsin were subjected to photobleaching
experiments in the presence of hydroxylamine (Fig.
2). On light irradiation, VAL-opsin with
11-cis-retinal showed a significant change in the absorption spectrum, and the difference absorption spectrum before and after complete bleaching had its maximum at ~500 nm (Fig. 2, curve
2). This represents photobleaching of a green-sensitive
pigment (VAL-opsin with bound 11-cis-retinal) into opsin
plus all-trans-retinal-oxime. On the other hand, no
photobleaching signal was detected in the VA-opsin sample (curve
1). These data indicated that only VAL-opsin can bind
11-cis-retinal and form a functional (light-sensitive) pigment under the conditions.
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Figure 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).
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VAL-opsin expression in the diencephalon
To investigate localization of functionally active VAL-opsin in
the zebrafish brain, we prepared an antibody recognizing the C-terminal
tail of VAL-opsin (Fig. 1C, VC region). Judging
from the structure, this VC antibody is specific to VAL-opsin because the VC region shows no significant homology in sequence to any part of
VA-opsin and any known opsins in the zebrafish. The specificity was
confirmed by an immunoblot experiment, in which the VC antibody immunoreacted to the recombinant VAL-opsin (~50 kDa band) expressed in the 293S cells but not to the recombinant VA-opsin (data not shown).
An extensive search for VC immunoreactivity in the cross sections of
whole brain of the zebrafish revealed the existence of a limited number
of VC-positive cells in the diencephalic region, or more exactly in the
central posterior thalamic nucleus (Fig. 3B, CP), which was
proximal to the diencephalic ventricle of central thalamus. Noticeably,
their cell bodies and axon-like fibers were both immunoreactive (Fig.
3D), a pattern reminiscent of the opsin immunoreactivity in
the cerebrospinal fluid-contacting neurons in the pigeon deep brain
(Wada et al., 1998). Our analysis of the serial sections demonstrated
that the VC-positive neurons in CP are distributed over ~200 µm
along the rostrocaudal axis. These neurons could represent the deep
brain photoreceptor cells in the zebrafish (see Discussion).
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Figure 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.
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VAL-opsin expression in the retina
The RT-PCR-based examination of tissue-specific gene expression of
VA- and VAL-opsin indicated that both were expressed not only in the
brain but also in the eye (Fig. 4,
+RTase). We detected no amplified product when the reverse
transcriptase treatment was eliminated (Fig. 4, RTase),
confirming no detectable contamination of genomic DNA. Then, we
investigated localization of VA and VAL proteins in the zebrafish eye
(Fig. 5). The VC antibody labeled a
subset of horizontal cells in the distal margin of the inner nuclear
layer of the retina, but not the rod/cone photoreceptor layer (Fig.
5B). The VAL-opsin localization in the retina was confirmed
by using another antibody, VN, which was raised against the N-terminal
region common to VA- and VAL-opsin (Fig. 1C). The VN
antibody consistently labeled a subset of horizontal cells. This
antibody also immunoreacted to a small fraction of amacrine cells in
the inner nuclear layer (Fig. 5C). These amacrine cells were
not detected by the VC antibody, suggesting the specific localization
of VA-opsin.
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Figure 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).
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Figure 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 in
D 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.
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The horizontal cells in teleosts have been physiologically and
histologically classified into several classes (Dowling, 1987). One of
the good histochemical markers for the classification is glutamic acid
decarboxylase (GAD), which is responsible for synthesis of GABA (Lam et
al., 1979). By using an antibody to GAD, we examined whether the
VAL-opsin-expressing cells are GABAergic. The GAD antibody labeled a
subset of horizontal cells in addition to a subset of amacrine cells
(Fig. 6B), just as
reported previously (Connaughton et al., 1999). As shown in Figure
6C, however, the GAD- and the VC-immunoreactive horizontal
cells were mutually exclusive, indicating the presence of VAL-opsin in
non-GABAergic horizontal cells. Judging from their location and
non-GABAergic feature, the VAL-opsin-expressing cells seem to represent
the rod-type horizontal cells, which are generally found in the teleost retina (Dowling, 1987). In these analyses, we noticed that the VAL-opsin-expressing horizontal cells were regularly
distributed in the layer (Fig. 7) and
that their relative signal intensity showed a dorsoventral gradient,
i.e., the signal intensities in the horizontal cells are stronger in
the dorsal part of zebrafish retina (data not shown). The cellular
distribution of VAL-opsin in the zebrafish retina is not random but
seems to be spatially regulated for an unknown function (see
Discussion).
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Figure 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 and
B, 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.
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Figure 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 the
inset, where their cell bodies are depicted by
open circles. Scale bar, 20 µm.
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DISCUSSION |
The present study demonstrated that VAL-opsin, a newly identified
variant of VA-opsin, can bind 11-cis-retinal in
vitro and form a green-sensitive pigment (Fig. 2) and that
VAL-opsin is localized to a limited number of cells in CP of the
zebrafish deep brain (Fig. 3). Because 11-cis-retinal, a
common chromophore of vertebrate opsins, has been detected in the deep
brain regions such as telencephalon (Foster et al., 1993) and
diencephalon (Masuda et al., 1994), our reconstitution experiments
in vitro strongly suggest the photosensitivity of
VAL-opsin-expressing cells in the zebrafish CP. To our knowledge,
little is known about the neuronal function of CP in teleosts, and a
role for transmitting auditory information is only suggested (Echteler,
1984, 1985; Striedter, 1991). On the other hand, von Frisch (1911)
demonstrated that a fish deep brain photoreceptor responsible for the
regulation of skin color is present in the ependyma of the diencephalic
ventricle. This location nicely fits the localization of VAL-positive
cells in the ependyma or subependyma of the diencephalic ventricle of the zebrafish brain. Like the minnow (von Frisch, 1911), the zebrafish skin becomes pale in the dark and darkened on exposure to light. Therefore, one of the physiological roles of VAL-opsin in the zebrafish
deep brain could be the skin color regulation that is dependent on
environmental light conditions.
Our finding of the zebrafish VA/VAL-opsin provides the first example
for alternative RNA splicing generating opsin isoforms with distinct
amino acid sequences. This contrasts with the alternative splicing of
mouse rhodopsin mRNAs in the 3'-untranslated sequence, leaving the
protein sequence unaltered (Al-Ubaidi et al., 1990). The amino acid
sequences of VA- and VAL-opsin are different from each other only in
the C-terminal cytoplasmic tail (Fig. 1A), and this
boundary between the common and the isoform-specific region (Fig.
1B) corresponds exactly to the splice site conserved among vertebrate opsin genes (Nathans and Hogness, 1983; Nathans et
al., 1986; Max et al., 1995). Our preliminary sequence data of the
zebrafish VA/VAL-opsin gene (data not shown) indicate that the
zebrafish VA-opsin mRNA is produced by an intron retention at this
splice site, whereas the VAL-opsin mRNA is spliced at this conserved
splice site. Because the cytoplasmic tail is different between
zebrafish VA- and VAL-opsin, we anticipated a functional difference
between the two isoforms. In the case of other G-protein-coupled receptors such as the prostaglandin EP3 receptor
(Namba et al., 1993) and the endothelin ETB
receptor (Elshourbagy et al., 1996), similar alternative splicing
produces multiple receptor isoforms with distinct cytoplasmic tails,
each of which is coupled with a distinct set of G-protein subtypes. In
contrast, the present results demonstrated all-or-none
difference in ligand (11-cis-retinal) binding ability
between the isoforms (Fig. 2), although it remains unclear why
zebrafish VA-opsin formed no photopigment with
11-cis-retinal. A plausible explanation is that the
recombinant VA-opsin was misfolded and thus failed to bind the
chromophore, because the proximal region of the C-terminal tail (Fig.
1B, closed circle), which includes the
splice site of zebrafish VA- and VAL-opsin, has been shown to play a
critical role in the proper folding and stability of bovine rhodopsin
(Weiss et al., 1994).
Consistent with the previous study on salmon VA-opsin (Soni et al.,
1998), we also detected the expression of VAL-opsin in a class of
horizontal cells of the zebrafish retina. What is the role of VAL-opsin
in the retinal horizontal cells? In spite of no evidence for the supply
of 11-cis-retinal to retinal horizontal cells, the spatially
well ordered distribution of VAL-positive cells in the inner nuclear
layer seems to indicate its functional contribution to retinal
physiology. In general, gap junctions between neighboring horizontal
cells enable electrocoupling among the cells to form a large receptive
field (Murakami et al., 1995). The pores of gap junctions in the
horizontal cells are closed in the presence of background illumination,
making the receptive field narrower (Piccolino, 1986). It has been
accepted that such an effect of light on the teleost horizontal cell is
mediated by dopamine released from a class of interplexiform cells, but the presence of an alternative pathway (dopamine independent) is also
suggested (Baldridge and Ball, 1991; Umino et al., 1991). We can
speculate that VAL-opsin in the horizontal cells may participate in the
light-sensitive regulation of gap junctions. It is equally possible
that VAL-opsin plays a role in resetting the phase of the retinal
circadian clock, which regulates the rhythmic production of melatonin
in the zebrafish retina (Cahill, 1996). Recent studies on transgenic
mice lacking rods and cones (Freedman et al., 1999; Lucas et al., 1999)
have demonstrated that non-rod/non-cone photoreceptors in the eyes can
reset the circadian clock phase. Just like cryptochromes (Miyamoto and
Sancar, 1998) and melanopsin (Provencio et al., 2000) found in the
ganglion and inner nuclear layers, VAL-opsin present in the horizontal
cells is a candidate for the circadian photoreceptor resetting the
clock phase. In addition to VAL-opsin, VA-opsin also showed highly
specific distribution in the zebrafish retina (Fig. 5), but its
physiological role is totally unclear at present. The failure to
reconstitute VA-opsin-based photosensitive pigment with
11-cis-retinal in vitro (Fig. 2) might indicate a nonphotoreceptive function in the zebrafish.
In summary, functionally active VAL-opsin, a novel variant of VA-opsin,
is present in the diencephalic cells and in the retinal horizontal
cells, suggesting its multiple roles in visual and nonvisual
photoreceptive physiology. The identification of functional opsin in
the zebrafish brain opens a new way to the molecular and genetic
characterization of the deep brain photoreception system.
| |
FOOTNOTES |
Received Dec. 7, 1999; revised Jan. 24, 2000; accepted Jan. 31, 2000.
This work was supported in part by Grants-in-Aid from the Japanese
Ministry of Education, Science, Sports and Culture. H. M. is
supported by a research fellowship of the Japanese Society for the
Promotion of Science for Young Scientists. We thank Prof. J. Nathans
(Johns Hopkins University School of Medicine) for providing pRSV-TAg
and 293S cells, Prof. M. Tachibana (The University of Tokyo) and Dr. M. Murata (National Institute for Physiological Sciences) for valuable
discussion about VAL-positive cells, and Drs. T. Okano, K. Sanada, and
T. Yoshikawa, and F. Shimizu (this laboratory) for helpful comments and
providing PCR primers and other materials.
Correspondence should be addressed to: Dr. Yoshitaka Fukada, Department
of Biophysics and Biochemistry, Graduate School of Science, The
University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
E-mail: sfukada{at}mail.ecc.u-tokyo.ac.jp.
| |
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ABSTRACT
INTRODUCTION
