Synteny analysis of rhodopsin and pinopsin genes in the Actinopterygii

In a previous study, we isolated the rhodopsin genes from non-teleost fishes in the Actinopterygii [13]. In this study, we first searched for the rhodopsin genes in the genomes of teleost fishes in the Osteoglossomorpha and Elopomorpha and compared the syntenies in the Actinopterygii (Fig. 1 and Supplementary Data 1). An intron-containing rhodopsin gene, an orthologue of the tetrapod rhodopsin gene, is found in Asian arowana (S. formosus) and the mormyrid fish (Brienomyrus brachyistius) in the Osteoglossomorpha, and in Atlantic tarpon, Atlantic bonefish (Albula goreensis) and Japanese eel (A. japonica) in the Elopomorpha. The synteny block around this intron-containing rhodopsin gene in the Teleostei is generally conserved with that in gray bichir in the Polypteriformes and spotted gar in the Holostei. This rhodopsin gene is missing in the corresponding synteny block of sterlet (Acipenser ruthenus). Another rhodopsin gene, an intron-less rhodopsin gene, is found in sterlet and spotted gar, but not in gray bichir in the Polypteriformes, among non-teleost fishes in the Actinopterygii. Thus, it can be speculated that the intron-less rhodopsin gene emerged by retroduplication after branching of the Polypteriformes in the Actinopterygii, which is consistent with our previous molecular phylogenetic analysis of the rhodopsin genes found in non-teleost fishes [13]. This intron-less rhodopsin gene is found in the conserved synteny block of Asian arowana and the mormyrid fish in the Osteoglossomorpha, and in Atlantic tarpon, Atlantic bonefish, and Japanese eel in the Elopomorpha. Among these teleost fishes, Japanese eel has two intron-less rhodopsin genes, freshwater type (fw-rho) and deep-sea type (ds-rho), which are thought to have been duplicated by teleost-specific whole genome duplication [24]. Thus, it is supposed that other fishes lost one intron-less rhodopsin gene during the evolutionary process [7]. A detailed comparison of the syntenies of the two intron-less rhodopsin genes showed that Atlantic bonefish lost the gene flanking adamts9, whereas Asian arowana, the mormyrid fish and Atlantic tarpon lost the other gene. This suggests that a loss of an intron-less rhodopsin gene occurred independently and randomly in the Osteoglossomorpha and Elopomorpha lineages.

Fig. 1

The synteny analysis of rhodopsin and pinopsin genes in the Actinopterygii. The synteny block of orthologous genes flanking the intron-containing rhodopsin gene (blue triangle with vertical lines), the intron-less rhodopsin gene (pink triangle), and the pinopsin gene (light green triangle) in actinopterygian species. The genes flanking the opsin loci are shown by white triangles. Gene names are indicated above the coelacanth (Latimeria chalumnae) genes, intraflagellar transport 122 (ift122) (gene number 1 shown by white triangle), H1.8 linker histone (h1-8) (gene number 2), plexin D1 (plxnd1) (gene number 3), prickle planar cell polarity protein 2 (prickle2) (gene number 4), ADAM metallopeptidase with thrombospondin type 1 motif 9 (adamts9) (gene number 5) and membrane-associated guanylate kinase, WW and PDZ domain containing 1 (magi1) (gene number 6), double C2 domain beta (doc2b) (gene number 7), L-asparaginase (L-ASNase) (gene number 8), and cytosolic arginine sensor for mTORC1 subunit 2 (castor2) (gene number 9). In Atlantic tarpon (M. atlanticus), regarding the two intron-less rhodopsin genes, the gene flanking adamts9 is found in the conserved synteny block, whereas the genome region including the other gene is deleted. Detailed gene information is shown in Supplementary file 2

In previous studies, we identified a pinopsin gene from non-teleost fishes (spotted gar, Siberian sturgeon (Acipenser baerii) and gray bichir) and showed its abundant expression in the pineal gland [13, 17]. However, the pinopsin gene has not been identified in the genomes of the Teleostei [20]. In this study, we searched for the pinopsin gene in the genomes of the Osteoglossomorpha and Elopomorpha. We successfully identified a pinopsin gene in the genomes of two species, Atlantic tarpon and Indo-Pacific tarpon (Megalops cyprinoides), in the Elopiformes of the Elopomorpha. The synteny block around the pinopsin gene in Atlantic tarpon is generally conserved with that of non-teleost fishes. By contrast, the pinopsin gene is missing in the corresponding synteny block of other teleost fishes.

Analysis of expression patterns of rhodopsin and pinopsin genes

Analyses of rhodopsin genes in some teleost fishes showed that the intron-less rhodopsin gene is utilized in the retina, whereas the intron-containing rhodopsin gene is abundantly and exclusively expressed in the pineal gland [10, 25]. On the other hand, our previous study detected the abundant expression of both the intron-less and intron-containing rhodopsin genes in the retina, not in the pineal gland, of spotted gar [13]. These findings indicate that the intron-containing rhodopsin gene changed its distribution pattern to exclusive and abundant expression in the pineal gland after branching of the Holostei. Thus, to compare the distribution patterns of the intron-less and intron-containing rhodopsin genes of teleost fishes in the Osteoglossomorpha and Elopomorpha, we conducted in situ hybridization analysis in the retina and pineal gland of these fishes (Figs. 2, 3, 4). In the retina of Australian bonytongue (S. jardinii), which belongs to the same genus as Asian arowana in the Osteoglossomorpha, we observed the expression of mRNA of the intron-less rhodopsin gene in the outer nuclear layer (Figs. 2A, C). However, we could not detect the expression signals of the intron-containing rhodopsin gene in the retina (Figs. 2B, D). By contrast, the Australian bonytongue pineal gland abundantly expressed mRNA of the intron-containing rhodopsin gene, but not mRNA of the intron-less rhodopsin gene (Figs. 2E–H). In addition, analysis of the rhodopsin genes in Japanese eel in the Elopomorpha revealed that the photoreceptor cells in the retina expressed mRNAs of two intron-less rhodopsin genes (fw-rho and ds-rho), but not mRNA of the intron-containing rhodopsin gene (Figs. 3A–F). We also observed that the Japanese eel pineal gland expressed mRNA of the intron-containing rhodopsin gene, but not mRNAs of the intron-less rhodopsin genes (Figs. 3G–L).

Fig. 2

Distribution of mRNA of the intron-less and intron-containing rhodopsin genes in the retina and the pineal gland of Australian bonytongue. A-D, Distribution of the transcripts of Australian bonytongue intron-less rhodopsin gene (A, C) and intron-containing rhodopsin gene (B, D) in the retina. These sections were hybridized with antisense probes (A, B) or corresponding sense probes (C, D). Scale bar: 50 μm. Abbreviations: RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. E–H, Distribution of the transcripts of Australian bonytongue intron-less rhodopsin gene (E, G) and intron-containing rhodopsin gene (F, H) in the transverse sections of the pineal gland. These sections were hybridized with antisense probes (E, F) or corresponding sense probes (G, H). Scale bar: 100 μm

Fig. 3

Distribution of mRNA of the intron-less and intron-containing rhodopsin genes in the retina and the pineal gland of Japanese eel. A–F, Distribution of the transcripts of Japanese eel intron-less rhodopsin genes, namely freshwater type rhodopsin gene (fw-rho) (A, D) and deep-sea type rhodopsin gene (ds-rho) (B, E), and intron-containing rhodopsin gene (C, F) in the retina. These sections were hybridized with antisense probes (A–C) or corresponding sense probes (D–F). Scale bar: 50 μm. Abbreviations: RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. G–L, Distribution of the transcripts of Japanese eel fw-rho gene (G, J), ds-rho gene (H, K), and intron-containing rhodopsin gene (I, L) in the transverse sections of the pineal gland. These sections were hybridized with antisense probes (G–I) or corresponding sense probes (J–L). Scale bar: 50 μm

Fig. 4

Distribution of mRNA of the intron-less and intron-containing rhodopsin genes and pinopsin gene in the retina and the pineal gland of Atlantic tarpon. A–F, Distribution of the transcripts of Atlantic tarpon intron-less rhodopsin gene (A, D), intron-containing rhodopsin gene (B, E), and pinopsin gene (C, F) in the retina. These sections were hybridized with antisense probes (A–C) or corresponding sense probes (D–F). Scale bar: 50 μm. Abbreviations: RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. G–L, Distribution of the transcripts of Atlantic tarpon intron-less rhodopsin gene (G, J), intron-containing rhodopsin gene (H, K), and pinopsin gene (I, L) in the transverse sections of the pineal gland. These sections were hybridized with antisense probes (G–I) or corresponding sense probes (J–L). Scale bar: 100 μm. M–P, Distribution of the transcripts of Atlantic tarpon intron-containing rhodopsin gene (M, O) and pinopsin gene (N, P) in the sagittal sections of the pineal gland. Rostral is to the left, and dorsal is up. These sections were hybridized with antisense probes (M, N) or corresponding sense probes (O, P). Scale bar: 100 μm

Among the fishes in the Teleostei we investigated, Atlantic tarpon and Indo-Pacific tarpon in the Elopomorpha are the only species that have the pinopsin gene in their genomes. Thus, we analyzed the distribution patterns of transcripts of rhodopsin and pinopsin genes in the retina and pineal gland of Atlantic tarpon. The expression signals of the intron-less rhodopsin gene were strongly detected in the photoreceptor cells of the retina, but not in the pineal gland (Figs. 4A, D, G and J). By contrast, the transcript of the intron-containing rhodopsin gene was abundantly expressed in the pineal gland, but not in the retina (Figs. 4B, E, H and K). In addition, we observed that the transcript of the pinopsin gene was also expressed in the pineal gland, but not in the retina (Figs. 4C, F, I and L). These findings in Australian bonytongue, Japanese eel and Atlantic tarpon indicate the common expression patterns of rhodopsin genes in these teleost fishes, namely expression of the intron-less rhodopsin gene in the retina and expression of the intron-containing rhodopsin gene in the pineal gland. This expression pattern of the intron-containing rhodopsin gene is different from that in spotted gar [13]. This comparison suggests that the intron-containing rhodopsin gene changed its expression pattern to one restricted to the pineal gland in the common ancestor of the Teleostei. On the other hand, the abundant expression of the pinopsin gene in the pineal gland is conserved between non-teleost fishes (spotted gar, Siberian sturgeon and gray bichir) and a teleost fish (Atlantic tarpon).

Next, we compared the expression patterns of the intron-containing rhodopsin gene and the pinopsin gene on the Atlantic tarpon pineal gland. We conducted in situ hybridization analysis in the sagittal sections of the Atlantic tarpon pineal gland (Figs. 4M–P). Our analysis showed that the expression signals of the intron-containing rhodopsin gene were broadly detected from the rostral to caudal region but were weaker in the caudal region. By contrast, the expression signals of the pinopsin gene were restricted to the caudal region. This shows that the regions where the intron-containing rhodopsin gene and the pinopsin gene mainly function are different in the Atlantic tarpon pineal gland.

In the analysis of the expression patterns of the rhodopsin and pinopsin genes in Atlantic tarpon, we noticed an anatomical characteristic of the cranium. From the dorsal side of Atlantic tarpon (body length: ~ 10 cm), we could observe a small dusky-red spot in the center of the brain without removing the skin and cranium (Fig. S1A). To analyze the morphology of the cranium, we conducted Alcian Blue staining on sagittal sections of the head including the cranium (Fig. S1B). A pit-like structure of the chondrocranium was formed under the bony cranium and corresponded to the dusky-red spot observed from the dorsal side. Moreover, the pineal gland was located under this pit-like structure. This cranium structure is similar to those of lampreys and cartilaginous fishes [26]. However, this structure is not common in the Teleostei and has only been reported in several species such as rainbow trout (Oncorhynchus mykiss) and gilthead seabream (Sparus aurata) [27,28,29]. This morphological characteristic of the cranium is considered to contribute to the improvement of light transmission to the pineal gland in Atlantic tarpon.

Spectral analysis of rhodopsin and pinopsin proteins

Next, we analyzed the molecular properties of rhodopsin and pinopsin proteins of Australian bonytongue, Japanese eel and Atlantic tarpon. We prepared the recombinant proteins of rhodopsin and pinopsin in cultured cells and purified the photo-pigments after the addition of 11-cis retinal (Fig. S2). All of the rhodopsin proteins encoded by the intron-less rhodopsin genes and the exo-rhodopsin proteins encoded by the intron-containing rhodopsin genes had λmax at around 500 nm, with the exception of Japanese eel deep-sea type rhodopsin protein. The λmax of Japanese eel deep-sea type rhodopsin protein was located at 483 nm and this blue-shift was previously predicted by the mutations at positions 83 and 292 (D83N/A292S) (in the bovine rhodopsin numbering system) [30, 31]. In addition, the analysis of the Atlantic tarpon pinopsin protein unveiled an interesting spectral shift. Atlantic tarpon pinopsin protein had λmax at 499 nm, which is comparable with that of Atlantic tarpon exo-rhodopsin protein (Figs. S2G, H). The previous reports showed that pinopsin proteins are blue-sensitive opsins with λmax located at 465 ~ 480 nm [16, 17]. Thus, Atlantic tarpon uniquely has green-sensitive pinopsin protein in the pineal gland. We searched for the amino acid residue(s) responsible for this red-shift of Atlantic tarpon pinopsin protein by comparing the amino acid sequences among blue-sensitive Xenopus tropicalis and spotted gar pinopsin proteins and green-sensitive Atlantic tarpon pinopsin protein and found that Xenopus and spotted gar pinopsin proteins have Ala269 and Ser292, whereas Atlantic tarpon and Indo-Pacific tarpon pinopsin proteins have Thr269 and Ala292 (Fig. S3). We then performed mutational analysis at positions 269 and 292 of Atlantic tarpon pinopsin protein. The λmax of T269A and A292S single mutant proteins were blue-shifted (~ 10 nm) compared to λmax of the wild-type protein. In addition, T269A/A292S double mutant protein showed a further blue-shift (18 nm) of λmax (481 nm), which is in close agreement with λmax of spotted gar pinopsin protein (478 nm) (Fig. 5A). It is well known that the residues at positions 269 and 292 are responsible for the spectral tuning in vertebrate red-sensitive cone visual pigments and, especially, the mutation at position 269 (alanine or threonine) contributes to the spectral difference between human red- and green-sensitive cone visual pigments [32, 33]. These results indicate that Atlantic tarpon acquired the green-sensitive pinopsin protein through a molecular mechanism which is common with that underlying vertebrate color vision. In addition, we performed G protein activation analysis of Atlantic tarpon pinopsin protein. Previous studies reported that the pinopsin proteins from several species activate transducin in a light-dependent manner [16, 17]. Our analysis also confirmed the coupling of the green-sensitive Atlantic tarpon pinopsin protein with transducin after light irradiation (Fig. 5B). This means that Atlantic tarpon pinopsin protein changed its absorption spectrum without losing its G protein coupling function.

Fig. 5

Molecular properties of rhodopsin and pinopsin proteins from fishes in the Actinopterygii. A, Comparison of the absorption spectra of wild-type and mutants of Atlantic tarpon pinopsin. Absorption spectra of wild-type (curve 1, λmax = 499 nm), T269A mutant (curve 2, 489 nm), A292S mutant (curve 3, 490 nm) and T269A/A292S mutant (curve 4, 481 nm) were normalized to be ~ 1.0 at λmax. Absorption spectrum of spotted gar pinopsin (curve 5, 478 nm) is also shown. B, Activation of transducin by Atlantic tarpon pinopsin protein. The transducin activation ability was measured using the GTPγS binding assay in the dark (closed circle) and after yellow light (> 500 nm) irradiation (open circles). Data were obtained at 15 ºC and are presented as the means ± S.E.M of three independent experiments. C, Comparison of the decay of meta II of rhodopsin proteins encoded by the intron-containing gene (Exorh, blue trace) and intron-less gene (Rho, red trace) of Australian bonytongue. D, Comparison of the decay of meta II of rhodopsin proteins encoded by the intron-containing gene (Exorh, blue trace) and intron-less genes, fw-rho (Fw-Rho, orange trace) and ds-rho (Ds-Rho, red trace), of Japanese eel. E, Comparison of the decay of meta II of rhodopsin proteins encoded by the intron-containing gene (Exorh, blue trace) and intron-less gene (Rho, red trace) of Atlantic tarpon. The traces in C–E indicate the average calculated based on three independent measurements with standard errors shown by shaded region. The data in C–E were fitted by a single exponential function to estimate the decay time constant as follows; Australian bonytongue rhodopsin encoded by the intron-less gene, 740 s; Australian bonytongue rhodopsin encoded by the intron-containing gene, 98 s; Japanese eel rhodopsin encoded by the intron-less gene fw-rho, 223 s; Japanese eel rhodopsin encoded by the intron-less gene ds-rho, 817 s; Japanese eel rhodopsin encoded by the intron-containing gene, 56 s; Atlantic tarpon rhodopsin encoded by the intron-less gene, 709 s; Atlantic tarpon rhodopsin encoded by the intron-containing gene, 70 s

Analysis of meta II of rhodopsin and exo-rhodopsin proteins

Next, we compared the molecular properties of meta II of rhodopsin and exo-rhodopsin proteins. It has been reported that meta II of exo-rhodopsin protein in the pineal gland decays faster than that of the rhodopsin protein in rod cells of the retina [11, 12]. However, our previous study showed that the intron-containing and intron-less rhodopsin genes of non-teleost fishes in the Actinopterygii work exclusively in rod cells of the retina and meta II of rhodopsin proteins encoded by these genes decays slowly [13]. These results suggest that the short lifetime of meta II of exo-rhodopsin protein was acquired during the early evolutionary process of the Teleostei, and can contribute to the optimization of the exo-rhodopsin protein for the pineal photoreception under bright conditions by facilitating bleach recovery of photo-pigment. Thus, in this study, we compared the lifetime of meta II among rhodopsin and exo-rhodopsin proteins of teleost fishes in the Osteoglossomorpha and Elopomorpha. To estimate the lifetime of meta II, we measured the release of the retinal from meta II by monitoring the intrinsic tryptophan fluorescence emission. Our analysis showed that meta II of exo-rhodopsin proteins found in the pineal gland of Australian bonytongue, Japanese eel and Atlantic tarpon decays faster than that of rhodopsin proteins in the retina of the teleost fishes (Figs. 5C–E). These results indicate that exo-rhodopsin proteins of teleost fishes in the Osteoglossomorpha and Elopomorpha have meta II with a lifetime shorter than that of rhodopsin proteins of these fishes.

Finally, we searched for the amino acid residue(s) whose mutation(s) results in the short lifetime of meta II of exo-rhodopsin. We compared the sequences of rhodopsin and exo-rhodopsin proteins in the Actinopterygii and picked up nine candidate residues (positions 88, 98, 107, 151, 194, 201, 210, 224, and 277) (Fig. S4). We replaced each residue of Atlantic tarpon intron-containing rhodopsin (exo-rhodopsin) gene with the corresponding residue encoded by the gray bichir intron-containing rhodopsin gene (V88F, A98S, V107E, K151N, P194L, T201E, L210V, S224G, and A277T) and prepared these mutant proteins of Atlantic tarpon exo-rhodopsin after reconstitution with 11-cis retinal. All of the mutant proteins had the λmax at around 500 nm (Fig. S5 inset). We measured the decay rate of meta II of these mutant proteins (Fig. S5). Among them, the P194L mutant protein showed a slightly prolonged lifetime of meta II (Fig. S5E), which is consistent with a previous report about rhodopsin proteins of deep-diving vertebrates [34]. However, the other mutant proteins maintained a short lifetime of meta II. Thus, we speculate that not a single mutation but the combination of several mutations contributes to shortening the lifetime of meta II of exo-rhodopsin protein.