Cubozoan genome illuminates functional diversification of opsins and photoreceptor evolution

Abstract

Animals sense light primarily by an opsin-based photopigment present in a photoreceptor cell. Cnidaria are arguably the most basal phylum containing a well-developed visual system. The evolutionary history of opsins in the animal kingdom has not yet been resolved. Here, we study the evolution of animal opsins by genome-wide analysis of the cubozoan jellyfish Tripedalia cystophora, a cnidarian possessing complex lens-containing eyes and minor photoreceptors. A large number of opsin genes with distinct tissue- and stage-specific expression were identified. Our phylogenetic analysis unequivocally classifies cubozoan opsins as a sister group to c-opsins and documents lineage-specific expansion of the opsin gene repertoire in the cubozoan genome. Functional analyses provided evidence for the use of the Gs-cAMP signaling pathway in a small set of cubozoan opsins, indicating the possibility that the majority of other cubozoan opsins signal via distinct pathways. Additionally, these tests uncovered subtle differences among individual opsins, suggesting possible fine-tuning for specific photoreceptor tasks. Based on phylogenetic, expression and biochemical analysis we propose that rapid lineage- and species-specific duplications of the intron-less opsin genes and their subsequent functional diversification promoted evolution of a large repertoire of both visual and extraocular photoreceptors in cubozoans.

Figure 1. Schematic representation of the opsin phylogenetic analysis of a large set of opsin genes including the cubozoan dataset.

A) Phylogenetic analysis performed in this study recovered previously described four major opsin lineages – r-opsins, c-opsins, group 4 opsins and cnidopsins. Herein the C-opsins and cnidopsins form sister groups. (For details see Fig. S3), B) Detailed inspection of cnidopsin branch indicates extensive gene duplication and lineage-specific expansion of cnidarian opsins. (For details see Fig. S4).

Figure 2. Opsin-Gs-cAMP assay.

Light activation of opsin-Gs-cAMP pathway by selected opsins. GloSensor™-20F cAMP HEK293 cells (Promega) were transfected with expression vectors encoding genes for different opsins, treated and stimulated with light, as described in Materials and Methods. Arrows represent simple light pulses, multiple arrowheads represent repeated stimulation. Each graph represents a mean of triplicates for every sample. A) Previously reported Gs-cAMP pathway stimulating opsin from C. rastonii (Caryb) showed ability to increase the cAMP level in our setup (visualized with cAMP-dependent luciferase activity). The exact homolog of Caryb from T. cystophora Tcop13 showed a highly similar response in our assay. Opsin RH1 from medaka, expected to signal via Gt leading to cGMP decrease, showed no change in luciferase activity. B)–E) Examples of different Tcop light responses. Tcop5 showed faster and weaker activation of the Gs-cAMP pathway than Tcop13. Tcop18 did not activate the Gs-cAMP pathway. F) Analysis of tripeptide activity in Tcop13 was performed. Tcop13 tripeptide HKQ was replaced with tripeptides NKQ, SKS and NRS (originally found in opsins Tcop1 or bovine rhodopsin, Tcop14 and Tcop18 – none of which activated the Gs cascade). Tripeptide mutation did not disrupt Gs activation by Tcop13, but influenced length or sensitivity of Tcop13 response to light stimulation. NT – non-transfected cells used as negative control; Caryb – signal for cells transfected with a vector expressing opsin from C. rastonii, used as positive control; RH1 – signal for cells transfected with a vector expressing opsin RH1 from medaka fish Oryzias latipes, used as negative control; Tcop5, Tcop13, Tcop18 – signal for cells transfected with vectors expressing opsins from T. cystophora - Tcop5, Tcop13 or Tcop18, respectively; NKQ, SKS, NRS – Tcop13 original tripeptide HKQ replaced with tripeptides NKQ, SKS or NRS.

Figure 3. Test of T. cystophora medusa phototaxis after NF449 treatment.

A) Schematic representation of the testing chamber. B) Statistical analysis of the light response of T. cystophora after NF449 (Gαs inhibitor) treatments (0 μM, 100 μM, 1 mM). Bars represent the percentage of phototactic medusae in given time point.

Figure 4. mRNA expression levels of T. cystophora opsins in dissected body parts of adult jellyfish.

A) For real-time PCR analysis, medusae were dissected into eight body parts: tentacles (tentac.), manubrium (manb.), male gonads, gastric pouch (gastric p.), bell, outer umbrella (outer um.), sub-umbrella (sub-um.) and rhopalia. B–S) mRNA expression level of opsins for each dissected body part relative to the rhopalium expression (1.0 – red line). y – axis: relative mRNA expression level, x – axis: analyzed body parts.

Figure 5. mRNA expression levels of T. cystophora opsins in different life stages.

A) For real-time PCR analysis, animals of nine subsequent life stages were collected: non-pigmented larva (np. larva), pigmented larva (p. larva), vegetative polyp (veg.polyp), three polyp-to-medusa metamorphosing stages (metam1, 2–3, 4), juvenile medusa (juv. med.), adult female (ad. female) and adult male. B-S) mRNA expression levels of opsins for each life stage relative to the juvenile medusa expression (1.0 – red line). x – axis: analyzed stages, y – axis: relative mRNA expression level.

Figure 6. Visual organs of T. cystophora and immunohistochemical localization of Tcop13 and Tcop18.

A) Schematic diagram of the rhopalium. The large (LLE) and small (ULE) complex eyes lie along the medial line, while the pit and slit ocelli are paired laterally. B) Schematic diagram of rhopalium sagittal plane (adapted from O´Connor 2009). C) Sagittal section through the rhopalium. Upper (ULE) and lower (LLE) lens eyes contain the typical components of camera-type eyes: a cornea (C), a lens (L), and a retina consisting of a ciliary layer (CL), a pigment layer (PL) and a neural layer (NL). St – statocyst, S – stalk. D) Schematic representation of the lens eye retina. The ciliary layer (CL) is dominated by the ciliary segments of type-B receptor cells (red). Scattered among the type-B receptor cells are the cone-shaped projections of type-A photoreceptor cells (green). In the neural layer (NL), both receptors types have their cell bodies with nuclei (dark blue); only type-A receptor cell bodies are positive for opsin signal. Projections of type-A photoreceptor cell bodies create a compact layer (LA) surrounding the whole retina. E–H) Confocal images of immuno-histochemical staining for Tcop13 (red), Tcop18 (green), DAPI (blue) in the upper lens eye (ULE). I-L) Large camera-type eye (LLE) retina longitudinal section. M–P) Large camera-type eye retina transverse section. (Scale bars: 50 μm).

Figure 7. Schematic representation of opsin expression patterns according to their phylogenetic relationship.

T. cystophora opsins can be classified into two groups, a probable more ancient Tc-group 1 opsin, with a broader expression pattern, and Tc-group 2 – rhopalium-specific opsins. The size and shade intensity of the symbols corresponds with the level of expression. Green coloured box and branches represent rhopalia specific Tc group 1A opsins. Purple coloured box and branches represent male specific Tc group 1B opsins. Red coloured box and branches represent rhopalia specific Tc-group 2 opsins.

Figure 8. Possible scenario for expansion and functional diversification of opsins in T. cystophora.

Our data and data from other studies show that Cnidarian intron-less opsins might have been derived from an ancient eumetazoan ciliary-like opsin containing introns by retro-transposition. Once anchored in the genome the ancient cnidopsin gene underwent several rounds of duplication, diversification and sensitivity tuning. Individual opsins were thus accommodated for distinct functions in diverse tissue photoreceptors - ocular, extraocular and larval. These opsins differ in stage- or tissue-expression, primary structure and also in subsequent cellular signaling – either via Gs-cAMP pathway or other G-protein pathways. For further information see Discussion.