A non-mammalian type opsin 5 functions dually in the photoreceptive and non-photoreceptive organs of birds

Abstract

A mammalian type opsin 5 (neuropsin) is a recently identified ultraviolet (UV)-sensitive pigment of the retina and other photosensitive organs in birds. Two other opsin 5-related molecules have been found in the genomes of non-mammalian vertebrates. However, their functions have not been examined as yet. Here, we identify the molecular properties of a second avian opsin 5, cOpn5L2 (chicken opsin 5-like 2), and its localization in the post-hatch chicken. Spectrophotometric analysis and radionucleotide-binding assay have revealed that cOpn5L2 is a UV-sensitive bistable pigment that couples with the Gi subtype of guanine nucleotide-binding protein (G protein). As a bistable pigment, it also shows the direct binding ability to agonist all-trans-retinal to activate G protein. The absorption maxima of UV-light-absorbing and visible light-absorbing forms were 350 and 521 nm, respectively. Expression analysis showed relatively high expression of cOpn5L2 mRNA in the adrenal gland, which is not photoreceptive but an endocrine organ, while lower expression was found in the brain and retina. At the protein level, cOpn5L2 immunoreactive cells were present in the chromaffin cells of the adrenal gland. In the brain, cOpn5L2 immunoreactive cells were found in the paraventricular and supraoptic nuclei of the anterior hypothalamus, known for photoreceptive deep brain areas. In the retina, cOpn5L2 protein was localized to subsets of cells in the ganglion cell layer and the inner nuclear layer. These results suggest that the non-mammalian type opsin 5 (Opn5L2) functions as a second UV sensor in the photoreceptive organs, while it might function as chemosensor using its direct binding ability to agonist all-trans-retinal in non-photoreceptive organs such as the adrenal gland of birds.

Figure 1. Molecular properties of cOpn5L2.

Absorption spectra, retinal configurations and G protein activation of cOpn5L2. A, Absorption spectra of cOpn5L2 purified after incubation with 11-cis-retinal. Spectra were recorded in the dark (curve 1), after UV light irradiation (curve 2), after subsequent yellow light (>500 nm) irradiation (curve 3), and after UV light re-irradiation (curve 4). (inset) The calculated absorption spectra of cOpn5L2 in the dark (curve 1) and after UV light irradiation (curve 2). The calculation procedures are described in the text. B, Spectral changes caused by UV light irradiation (curve 1), subsequent yellow light irradiation (curve 2), and UV light re-irradiation (curve 3). C, Absorption spectra of cOpn5L2 purified after incubation with all-trans-retinal. Spectra were recorded in the dark (curve 1), after yellow light irradiation (curve 2), after subsequent UV light irradiation (curve 3), after yellow light re-irradiation (curve 4), and after UV light re-irradiation (curve 5). D, Spectral changes caused by yellow light irradiation (curve 1), subsequent UV light irradiation (curve 2), yellow light re-irradiation (curve 3), and UV light re-irradiation (curve 4). E, Retinal configurations in cOpn5L2 purified after incubation with all-trans-retinal. (left-hand panel) The retinal isomers before irradiation, after yellow light irradiation, and after subsequent UV light irradiation were analyzed with HPLC after extraction of the chromophore as retinal oximes (syn and anti forms of 9-cis-, 11-cis-, 13-cis-, and all-trans-retinal oximes). (right-hand panel) Isomeric compositions of retinal before and after light irradiation of cOpn5L2. F, Gi-type G protein activation ability by cOpn5L2 purified after incubation with 11-cis-retinal. The time-dependent change of the activity was measured in the dark (open circle), after UV light irradiation (open square), after subsequent yellow light irradiation (open triangle), and after UV light re-irradiation (open diamond). G, The Gi activation ability of cOpn5L2 purified after incubation with all-trans-retinal. The activity was measured in the dark (open circle), after yellow light irradiation (open square), after subsequent UV light irradiation (open triangle), and after yellow light re-irradiation (open diamond). G protein activation assay in (F) and (G) was performed at 0°C, and data are presented as the means ± S.D. of three independent experiments.

Figure 2. The expression pattern of cOpn5L2.

Revealed by quantitative PCR (A), in situ hybridization (B, C), western blot analysis (D), and immunohistochemistry (E, F). A, Quantitative PCR analysis of ten tissues (as shown) from post-hatching chick (2 weeks). cOpn5m mRNA level in retina is referred to as 1. B, C, In situ hybridization of the chick adrenal gland at post-hatching day 30 (P30). A sense probe for cOpn5L2 shows no staining in the consecutive section (C). D, (Left and middle) Western blot analysis using a cOpn5L2 (N-term) or cOpn5L2 (C-term) antibody. Cell lysates of mock or cOpn5L2-expressing cells were loaded. (Right) cOpn5L2 cDNA is tagged with C-terminal amino acids of bovine rhodopsin and its expression was confirmed using an anti-bovine rhodopsin antibody (rho1D4). E, F, Immunohistochemistory of the brain using cOpn5L2 (N-term) (E) or cOpn5L2 (C-term) (F) antibody. The lateral hypothalamic area (P10) is shown. Nuclei are stained with DAPI (blue). cOpn5L2 immunoreactive (IR) cells are shown in green (arrowheads). Scale bars, 100 µm (B, C), and 50 µm (E, F).

Figure 3. Immunohistochemistry of the adrenal gland.

cOpn5L2 immunoreactivity is shown in green, cell nuclei in blue, and TH (B, C, D′) or F-actin (E) in magenta. A–C, cOpn5L2 IR cells are localized to the TH-producing cells in the adrenal gland at P15. D, D′, High magnification of the adrenal medullary cells at P30. E, to reveal the epithelial cell shape, filamentous actin is visualized by staining with rhodamine-phalloidin. cOpn5L2-immunoreactivity is observed in the cytoplasm. F, The xz and yz views of a selected point in the image are shown, supporting the cOpn5L2 localization in the cytoplasm. Scale bars, 50 µm (A–C), 10 µm (D, D′, E), and 7.5 µm (F).

Figure 4. Distribution of cOpn5L2 immunoreactive neurons in chicken brain at P10.

Three coronal levels, from posterior hypothalamus to preoptic regions, are illustrated. The approximate location of these regions is shown on the sagittal schema of chicken brain on the left. Red boxes indicate the areas of the confocal images. cOpn5L2 IR neurons and perikarya are indicated in green. The scale bars represent 20 µm. In all panels except (D, I), the sections were co-stained by two antibodies. The marker antibodies (magenta) used are indicated in each panel. For all images the DAPI stain is blue. A–D, Coronal sections through the posterior hypothalamus. A, Pineal gland (PG), in which there are no cOpn5L2 IR cells. As a positive control, PG was stained with an anti-serotonin antibody. B, Paraventricular organ (PVO), in which there are no cOpn5L2 IR cells. As a control, the PVO was stained with an anti-serotonin antibody. C, The posterior hypothalamic nucleus (PH), located laterally to the PVO. Weak but distinct immunoreactivity for cOpn5L2 is observed. D, High magnification of cOpn5L2 IR cells in the lateral hypothalamic area (LH), shown in Figure 2F. In these cells, thread-like immunoreactive signals are seen. E–H, Coronal sections through the anterior hypothalamus. E, Paraventricular nucleus (Pa) and nucleus anterior medialis hypothalami (AM). cOpn5L2 IR cells are found in the Pa, dorsal to the TH IR cells in the AM. F, High magnification of a cOpn5L2 IR cell shown in (E). The large soma is immunoreactive for cOpn5L2, and TH IR fibers are seen in the vicinity of the cOpn5L2 IR cell. G, Paraventricular nucleus, in which cOpn5L2 IR cells and GnRH IR fibers (yellow arrows) are scattered. H, High magnification of two cOpn5L2 IR cells shown in (G). A yellow arrow indicates a GnRH IR fiber. I–K, Coronal sections through the preoptic region. I, Supraoptic nucleus (SO), in which cOpn5L2 IR somata and long fibers are seen. The cOpn5L2 IR neuron is bipolar. J, In the SO, cOpn5L2 IR and GnRH IR cells are observed, shown in white. High magnification of a representative cell (arrow) is shown in the inset. K, In the SO, some vasotocin (vaso) IR cells are also positive for cOpn5L2 (arrows).

Figure 5. Distribution of cOpn5L2 immunoreactive neurons in the forebrain.

A–C, Coronal sections through the anterior hypothalamus at P10. A, The dorsal part of the paraventricular nucleus, in which many vasotocin-positive cells are seen, but no cOpn5L2 IR cells are observed. B, Paraventricular nucleus, ventral to the region shown in (A), in which cOpn5L2 IR cells are seen. C, High magnification of paraventricular nucleus. A different section from that shown in (B). Some vasotocin IR cells are also positive for cOpn5L2 (arrows). D, E, Coronal sections through the anterior hypothalamus. Images are focused on the telencephalon. D, cOpn5L2 IR cells are found in the bed nucleus of the stria terminalis, lateral part (BstL), in which TH IR fibers are prominent. E, cOpn5L2 IR cells are also scattered in the lateral region of the telencephalon. Co, optic chiasm; IIIv, third ventricle; Lv, lateral ventricle. The scale bars represent 20 µm.

Figure 6. Immunohistochemistry of the chick retina at P10.

A, cOpn5L2 IR cells in the inner nuclear layer (inl) and ganglion cell layer (gcl) (green, arrowheads). Parvalbumin is visualized in magenta to reveal subsets of amacrine cells in the inner nuclear layer (inl) and sublamina I and V in the inner plexiform layer (ipl) . B, A representative cOpn5L2 IR cell in the INL. C, A representative cOpn5L2 IR cell in the GCL. D, A cOpn5L2 IR cell in the INL after two-color ABC immunostaining. E, A cOpn5m IR cell in the INL of the same retinal section as shown in (D). F-F″, The cOpn5L2 IR cell (green in F, F″) in the GCL is not positive for ChAT (magenta in F′, F″). G-G″, The cOpn5L2 IR cell (arrow) in the GCL (G, G″) is positive for Islet1 (G′, G″). H-H″, cOpn5L2 IR cells in the INL (H, H″) are positive for Meis (H′, H″). I-I″, The cOpn5L2 IR cell in the INL (I, I″) is positive for GAD65/67 (I′, I″). J, J′, cOpn5L2 IR cells in the INL (green, arrowheads in J) are not positive for ChAT. Some cOpn5L2 IR cells are adjacent to ChAT IR cells (J), and others are separate from the ChAT IR cells (J′). K, One cOpn5L2 IR cell in the vicinity of a serotonin IR cell, while the other is located apart from it. L, cOpn5L2 IR cells are not positive for VIP. M, A cOpn5L2 IR cell adjacent to a TH IR cell. For all images, DAPI is blue. Scale bars, 50 µm (A), 10 µm (B–E, G-I″, K–M), and 20 µm (F-F″, J, J′).

Figure 7. Functions of cOpn5L2 may depend on how much light is available to the cOpn5L2-expressing tissues.

Availability of retinoids and interactions with cOpn5L2 may also influence whether photons or ligands transduce signals.