Melanophores of the isolated tail fin of the Xenopus tadpole respond to light, resulting in melanin aggregation in the melanophores.
Western blot analysis showed that a protein in the Xenopus tail fins, in which photosensitive melanophores exist, had reacted with the antibody against bovine rhodopsin.
RT-PCR and nested-PCR using rhodopsin-specific primers showed the expression of rhodopsin mRNA in the tail fins. The amino acid sequences deduced from the PCR products were completely identical to those of rhodopsin.
We also detected the mRNA of melanopsin in the tail fin, another opsin originally described in cultured melanophores of Xenopus.
These results indicate that these two types of opsin molecules exist in Xenopus tail fin and may take part in the photo-response in melanophores of the Xenopus tadpole.
INTRODUCTION
Many lower vertebrates are able to change body color to adapt to their ambient coloration. These changes depend upon the activities of chromatophores in the body skin. Generally, the motility of chromatophores is controlled by the nervous and/or hormonal system on the basis of visual information, but some of the chromatophores are directly controlled by light. The photo-response of melanophores in the tail fin of Xenopus tadpole is well known (Bagnara, 1957; Lythgoe and Thompson, 1984; Daniolos et al., 1990; Moriya et al., 1996). The tail fins isolated from the Xenopus tadpole change skin color in response to light without the aid of vision and/or a central nervous system. Under illumination, the fin becomes pale as a result of melanosome aggregation in melanophores; in contrast, the fin becomes dark after interruption of light, due to melanosome dispersion (Moriya et al., 1996). These responses to light were observed in tad-poles of the later-stage (stages 51–56). Interestingly, quite the opposite responses occur in melanophores derived from the Xenopus embryo; that is, melanosomes disperse under illumination and aggregate in darkness (Bagnara and Obika, 1967; Daniolos et al., 1990; Rollag, 1996). The mechanism and the switch of the photo-response during the development are not yet clearly understood. Our previous observations using the later-stage of tadpole suggest that the photo-response in the Xenopus melanophores seems to be close to the visual response in retinal cells, based on the following arguments: 1) The action spectrum (maximum 500 nm) of the response is similar to the absorption spectrum of rhodopsin (Moriya et al., 1996). 2) The involvement of Gi/Gt and small G protein Rho in the photo-response is suggested, since this response was blocked by pertussis toxin or botulinum exoenzyme C3 and since the ADP-ribosylation of the G proteins was dependent on light (Miyashita et al., 1996). 3) Cyclic-GMP caused dispersion of melanophores under illumination (Moriya et al., 1996).
We demonstrate here that in addition to melanopsin, which has been reported in cultured melanophores derived from Xenopus embryo (Provencio et al., 1998), a rhodopsin-like molecule exists in Xenopus tadpole tail fin, where photosensitive melanophores exist.
MATERIALS AND METHODS
Materials
Tadpoles at stages 45–54 of Xenopus laevis were used. They were purchased from a commercial source or were bred in our laboratory and kept at 25°C under natural light conditions.
The tail fins (stage 54) were isolated from body trunks and were separated into the anterior and the posterior parts, since melanophores are found only in the posterior part. In the case of younger tadpole (stage 45), the whole tail fin was used.
Western blotting
The whole protein extract was prepared from the Xenopus tail fin according to the method described by Kikuchi et al. (1994). After treatment with 10% (V/V) trichloroacetic acid for 60 min on ice, the isolated tail fins (n=30) were homogenized in 160 μl of solubilizing solution (9 M urea, 2% (W/V) Triton X-100, and 1%(W/V) dithiothreitol) with a small metal homogenizer (Physcotoron, Tokyo, Japan). After adding 40 μl of 10%(W/V) lithium dodecyl sulfate, the homogenate was sonicated again. The solubilized proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and then transblotted onto nitrocellulose membranes (Pharmacia, Uppsala, Sweden). After blocking with 5% skim milk and 0.5% Tween-20 in PBS at 37°C for 1 hr and with Block Ace (Dainippon Pharmaceutical, Osaka, Japan) overnight at 5°C, the membranes were probed with polyclonal antibodies against both bovine rhodopsin (1:5000) and octopus rhodopsin (1:500). Normal rabbit serum was used as control. Subsequent visualization of antibody binding was carried out with an ABC peroxidase system (VECTASTAIN ABC kit) (Vector Laboratories, Burlingame, CA).
Reverse transcription (RT)-PCR
Total RNA of the Xenopus tail fin was extracted using ISOGEN (NIPPON GENE, Toyama, Japan). After treatment with deoxyribonuclease (NIPPON GENE, Toyama, Japan), 5 μg of total RNA was reverse transcribed using oligo (dT) as a primer and a SuperScript™ Preamplification System (GIBCO-BRL, Life Technologies, Grand Island, NY). PCR was performed using an Expand™ High Fidelity PCR System (Boehringer, Indianapolis, IN) with 2 μl of the cDNA corresponding to 0.5 μg of total RNA and 10 pmol each of the appropriate sense and antisense primers. The final reaction volume was 25 μl. The primers used for detection of rhodopsin mRNA were synthesized (ESPEC OLIGO SERVICE CORP, Tsukuba, Japan) to correspond to the nucleotide position (N.P.) of rhodopsin gene DNA of Xenopus, as previously described (Batni et al, 1996), R1: 5′primer, sense 5′-CAGCACAAGAAACTCAGAACA (N.P.289–319), R2: 3′primer, anti-sense 3′-CGACTCTTTCTCCAGTGGTCT (N.P.1944-1964), R3:3′primer, anti-sense 3′-CGATAGATGTTAGGACAGTAGATG (N.P. 2103–2126) and R4: 5′primer, sense 5′-GCTGAGAAAGAGGTCACCAGA (N.P.1944–1964). Primers (R1, R2 and R3) were chosen so that the expected PCR products would span introns 1,2 and 3, thereby eliminating the possibility of genomic DNA contamination. For detection of melanopsin mRNA, we referred to the report of Provencio et al. (1998) on melanopsin and designed the primers; M1: 5′primer, sense 5′-TTGGGCTGGGCATGGTAAATCTTT (N.P. 957-980) and M2: 3′primer, anti-sense 3′-ACGATGGAATGGAGCTTAACAGTA (N.P. 1530–1553).
The cycling protocol used was 94°C for 1 min, 60.5°C for 1 min and 72°C for 2 min for the designated number of cycles.
Sequences were compared with the GenBank database by using BLAST and BLASTIN algorithms (Altschul et al., 1990)
RESULTS AND DISCUSSION
Anti-bovine rhodopsin antibody reacted with a protein of about 35 kDa in the extract of Xenopus tail fin (Fig. 1, lane 2). The size of the protein was identical to bovine rhodopsin and was compatible with amphibian rhodopsin reported previously (Okano et al., 2000). We detected another immunoreacted protein with a higher molecular mass (about 78 kDa), shown in lane 2, which conceivably corresponds to a dimmer of rhodopsin.
Fig. 1
Western blotting of the posterior tail fin extract with anti-bovine rhodopsin antibody. Lane 1: Bovine ROS membrane (ñ: bovine rhodopsin as standard), Lane 2: Posterior tail fin, Lane 3: Control. The numbers indicate the size (kDa) of standard protein (left) or of the immunoreacted protein (right).
On the other hand, anti-octopus rhodopsin antibody reacted with a protein of 50 kDa in the fin extract (Fig. 2). This 50 kDa protein seems to be different from rhodopsin from the bovine eye or the Xenopus tail fin and its size is similar to that of octopus rhodopsin. This protein is likely to be melanopsin, since melanopsin is strongly related to octopus rhodopsin (Provencio et al., 1998). These results suggest the existence of two kinds of rhodopsin family protein in the tail fin. To confirm this possibility, expression of mRNAs for rhodopsin and melanopsin was examined.
Fig. 2
Western blotting of the posterior tail fin extract with anti-octopus rhodopsin antibody. Lane 1: octopus rhodopsin (ñ: octopus rhodopsin as standard), Lane 2 : posterior tail fin. The numbers indicate the size (kDa) of standard protein (left) or of the immunore-acted protein (right).
We performed RT-PCR in Xenopus tail fin using a variety of primers for rhodopsin. Fig. 3A shows the results of RT-PCR, where three sets of the primers, R1/R3, R1/R2 and R4/R3, were used to amplify cDNA obtained from the tail fin. All three sets of primers synthesized the expected sizes of DNAs, namely: 183bp for R4/R3, 567bp for R1/R2 and 729bp for R1/R3. Since the band of the R1/R3 product was very faint, a second (semi-nested) PCR analysis was performed using the first PCR product as the template. Two predicted PCR products were detected using R1/R2 and R4/R3 respectively as the primer sets (Fig. 3B).
Fig. 3
Agarose gel electrophoresis of the PCR products amplified with the primers for rhodopsin mRNA.
A: The first PCR analyses using cDNAs of the posterior tail fin as the template. 1: R4/R3, 2: R1/R2, 3: R1/R3, M: marker, 60.5°C, 37 cycles. The numbers indicate the size (bp) of standard DNA (right). The arrows indicate the PCR products (expected sizes; 729, 567 and 183 bp, respectively).
B: The second (semi-nested) PCR analyses. The template was the first PCR product (R1/R3) in A. 1: R4/R3, 2: R1/R2, M: Marker, 60.5°C, 35 cycles. The numbers indicate the size (bp) of standard DNA (right). The arrows indicate the PCR products (expected sizes; 567 and 183 bp, respectively).
C: The second (semi-nested) PCR analyses using R1/R2 primer set. The template was the first PCR product (R1/R3). M: marker, 1: brain, 2:Posterior tail fin, 3: Anterior tail fin, 60° C, 35 cycles. The numbers indicate the size (bp) of standard DNA (left). The arrow indicates the PCR product (expected size; 567 bp).
The tail fin was separated into two parts before extraction of RNA, since the anterior part contains no melanophores while the posterior part does. Using the primers described above, the PCR products were found only in the posterior part and not in the anterior; as was expected (Fig. 3C). This result suggests that rhodopsin mRNA may be expressed within melanophore cells. This concurred with results showing that melanophores in the isolated tail fin aggregated in response to a local application of light (Moriya et al., 1996). The PCR products were also detected in the brain of Xenopus tadpole (Fig. 3C). In previous studies rhodopsin molecules were detected in extraretinal tissues including in the brain of non mammalian vertebrates (Wada et al., 1998; Okano et al., 2000).
The deduced amino acid sequence of the PCR products (R1/R2) and (R4/R3) were completely identical to Gln64-Arg252 and Ala246-Tyr306, their corresponding parts of Xenopus rhodopsin, respectively; on the other hand, these sequences showed a low identity with the corresponding parts (Arg54-Lys250 and Asn244-Tyr304) of melanopsin (30% and 45%, respectively). Thus the RT-PCR results showing rhodopsin mRNA expression, in combination with the results of the Western blotting (Fig. 1), strongly support the existence of rhodopsin molecules in the Xenopus tail fin. This is coincident with our former results showing that the photo-response of the melanophores in the tail fin is likely to be closely related to that of the visual system in eyes (Moriya et al., 1996; Miyashita et al., 1996).
To better understand the expression of opsin molecules in Xenopus tail fin, RT-PCR was performed using melanopsin specific primers. When RT-PCR was performed on mRNA extracted from the posterior part of the tail fin of later stage tadpoles (stage 54), a very faint band was obtained (data not shown). The RNA obtained from the tail of younger tadpole (stage 45) produced two clear bands (Fig. 4). One of them (band 1) was close in size to the expected size of 597 bp. The deduced amino acid sequence of band 1 was identical with a corresponding part (Gly276-Arg469) of Xenopus melanopsin. The deduced amino acid sequence of band 2 was identical to melanopsin except for an additional 47 amino acids between Gln396 and Asp397 of melanopsin. It is possible that band 2 is a variant mRNA caused by alternative RNA splicing.
Fig. 4
Agarose gel electrophoresis of the PCR products amplified with the primers for melanopsin mRNA using cDNAs of the Xenopus skin as the template.
1: M1/M2, 2: R4/R3, 3: R1/R2, 4: R1/R3, M: Marker, 60.5°C, 35 cycles. The products in lanes 2–4 show the expression of rhodopsin mRNA as described in Fig. 3. The numbers indicate the size (bp) of standard DNA (right).
These results showing that melanopsin mRNA is expressed in the tail fin where melanophores are present concur with the results shown by Provencio et al. (1998). The combined results suggest that the Xenopus tail fin contains both rhodopsin and melanopsin. Melanopsin was isolated from cultured melanophores, which were originally obtained from the early stages of embryo (stage 30-35) and responded to light by activating melanosome dispersion (Daniolos et al., 1990; Rollag, 1996; Provencio et al., 1998). On the other hand, cultured melanophores derived from tad-poles at stage 50–54 aggregated melanosomes on illumination (Seldenrijk et al., 1979). We consider that melanopsin and rhodopsin play different roles in the process of photo-response: rhodopsin is probably involved in the aggregation of melanosomes whereas melanopsin is active in the dispersion. In fact, Rollag et al. (2000) reported recently that the melanophores expressing the melanopsin transgene was more sensitive to light and induced remarkable melanin dispersion. During embryonic development, rhodopsin, was first detectable as a visual pigment at stage 35, but the level was very low (Saha and Grainger, 1993). It is likely that in the early stage of development of Xenopus, there is a quantitative difference in the expression of the two opsins. The expression of rhodopsin increases as development takes place and this may explain the reversal of the photo-response in the Xenopus melanophores.
We found that vertebrate type rhodopsin mRNA was expressed in cultured murine melanocytes (Miyashita et al., 1999). We are interested to know whether all pigment cells inherently carry the two types of opsin and are able to respond to light through these photoreceptor molecules.
Acknowledgments
This research was supported by a grant from the Ministry of Education, Science and Culture of Japan (No.11640683).
