Pinopsin evolved as the ancestral dim-light visual opsin in vertebrates

Abstract

Pinopsin is the opsin most closely related to vertebrate visual pigments on the phylogenetic tree. This opsin has been discovered among many vertebrates, except mammals and teleosts, and was thought to exclusively function in their brain for extraocular photoreception. Here, we show the possibility that pinopsin also contributes to scotopic vision in some vertebrate species. Pinopsin is distributed in the retina of non-teleost fishes and frogs, especially in their rod photoreceptor cells, in addition to their brain. Moreover, the retinal chromophore of pinopsin exhibits a thermal isomerization rate considerably lower than those of cone visual pigments, but comparable to that of rhodopsin. Therefore, pinopsin can function as a rhodopsin-like visual pigment in the retinas of these lower vertebrates. Since pinopsin diversified before the branching of rhodopsin on the phylogenetic tree, two-step adaptation to scotopic vision would have occurred through the independent acquisition of pinopsin and rhodopsin by the vertebrate lineage.

Fig. 1

RT-PCR analysis of pinopsin expression in eyes and brains of non-teleost fishes and amphibians. Pinopsin transcript was detected from brains of all the fishes and amphibians investigated. The transcript was also detected from eyes of non-teleost fishes (coral catshark, spotted gar, Siberian sturgeon, gray bichir, and spotted African lungfish) and anurans (X. tropicalis and American bullfrog), but not from eyes of urodelans (Japanese fire bellied newt and Mexican salamander). β-actin transcript was detected from all the samples as an internal standard. Sequences of PCR primers and amplified sizes of each PCR are shown in Supplementary Table 1. Full gel images are shown in Supplementary Figure 2

Fig. 2

Distribution of pinopsin in the retina of spotted gar and X. tropicalis. a–d Detection of spotted gar pinopsin mRNA in the retina by in situ hybridization analysis. Horizontal (a, b) or frontal (c, d) consecutive sections were hybridized with pinopsin antisense (a, c) and sense (b, d) probes. Arrows indicate the positions of hybridization signals. e–g Detection of X. tropicalis pinopsin mRNA in the retina by in situ hybridization analysis. Frontal consecutive sections were hybridized with pinopsin antisense (e, g) and sense (f) probes. Dorsal region (e, f) and ventral region (g) of retina are shown, respectively. All the sections shown in panels a–g were counterstained with nuclear fast red. h–j Double immunofluorescence staining in the spotted gar retina showing pinopsin (h, green), rhodopsin (l, magenta), and the merge image (j). k–m Double immunofluorescence staining in the spotted gar retina showing pinopsin (k, green), red-sensitive cone pigment (l, magenta), and the merge image (m). White arrows indicate the positions of the positive signals of anti-pinopsin antibody. n–p Double immunofluorescence staining in the X. tropicalis retina showing pinopsin (n, green), rhodopsin (o, magenta), and merge images (p). q–s Double immunofluorescence staining in the X. tropicalis retina showing pinopsin (q, green), anti-red-sensitive cone pigment (r, magenta), and merge images (s). White arrows and arrow heads indicate the positions of the positive signals of anti-pinopsin antibody in outer segments of rod and cone photoreceptor cells, respectively. Nuclei in the sections shown in panels (h–s) were counterstained with Hoechst33342 (blue). Scale bar: a, b, 100 μm; c–s, 50 μm

Fig. 3

Comparison of the thermal activation rate between pinopsin and visual pigments. a The v_dark, v_light, and k_d of bovine rhodopsin, X. tropicalis rhodopsin, spotted gar rhodopsin, X. tropicalis pinopsin, spotted gar pinopsin, and three cone pigments (mouse green-sensitive one, chicken green-sensitive one, and zebrafish blue-sensitive one). The values of bovine rhodopsin, spotted gar rhodopsin, X. tropicalis pinopsin, and spotted gar pinopsin were calculated from the results shown in Supplementary Figure 6. The values of X. tropicalis rhodopsin and three cone pigments were referred to our previous studies^,. b Comparison of k_th of rhodopsins, pinopsins, and cone pigments. The k_th values were estimated from data in panel a (see details in Methods). Error bars represent the standard error of the mean estimated based on more than three independent measurements. An asterisk (*) indicates a significant difference in relative rate constants between opsins and chicken green-sensitive cone pigment (P < 0.05; Student’s t test, two-tailed)

Fig. 4

Detailed analysis of phylogenetic relationship between pinopsin and visual pigments. a Molecular phylogenetic tree of pinopsin and visual pigments inferred by maximum likelihood (ML) method. The ML tree was inferred by using JTT-tm model and Yang’s discrete gamma model with an optimized shape parameter alpha of 0.93. A sequence alignment of 249 amino acids in length was used for the tree inference. The numbers at each branch are bootstrap probabilities^,. Partial tree topologies indicated by solid and broken ellipses were statistically tested in (b) and (c), respectively. RH1, RH2, SWS1, SWS2, LWS, P, VA, and Opn3 denote chicken rhodopsin, green-sensitive opsin, violet-sensitive opsin, blue-sensitive opsin, red-sensitive opsin (iodopsin), pinopsin, vertebrate ancient opsin, and Opsin3, respectively. b ML and other tree topologies of six opsins not statistically rejected by AU test. The ML tree of six opsins was inferred by the same method and models (alpha = 0.71) of (a). A highly reliable sequence alignment of 290 amino acids in length was used for the tree construction. Out of all the 105 tree topologies for six operational taxonomic units (OTUs), RH1, RH2, SWS1, SWS2, LWS, and P, the ML (tree number 1) and only one (tree number 2) topologies were not rejected by AU test at the 5% statistical significance level. The values of KH and SH statistical tests for these tree topologies are also represented in the list. c ML and other 14 tree topologies of eight opsins. Partial tree topology denoted by [[[RH1, RH2], SWS2], SWS1] was fixed in this analysis because the topology was contained in the ML tree of (a) and strongly supported by the statistical test of (b). Out of all the 15 tree topologies for five OTUs, [[[RH1, RH2], SWS2], SWS1], LWS, P, VA, and Opn3, the ML (tree number 1) and other nine (tree numbers 2 to 10) topologies were not rejected by AU test at the 5% level of significance. The values of KH and SH tests are also shown in the list

Fig. 5

Comparison of thermal stability among rhodopsin, pinopsin, and cone pigments. Bovine rhodopsin (a), Xenopus pinopsin (b), spotted gar pinopsin (c), chicken green-sensitive cone pigment (d), and mouse green-sensitive cone pigment (e) after purified in 0.02% DM were incubated at 37 °C and spectral changes were measured. After the incubation in the dark, the samples were mixed with 50 mM hydroxylamine and irradiated with yellow light (> 480 nm) to estimate the amount of the pigments. Difference spectra were obtained by subtracting the spectrum before incubation at 37 °C from those measured after incubation and irradiation as shown in Supplementary Figure 9. f The amounts of residual pigments were plotted against incubation time at 37 °C. The amounts of residual pigments were estimated from the difference absorbance at 540 nm in Supplementary Figure 9