Evolution of melanopsin photoreceptors: discovery and characterization of a new melanopsin in nonmammalian vertebrates

Abstract

In mammals, the melanopsin gene (Opn4) encodes a sensory photopigment that underpins newly discovered inner retinal photoreceptors. Since its first discovery in Xenopus laevis and subsequent description in humans and mice, melanopsin genes have been described in all vertebrate classes. Until now, all of these sequences have been considered representatives of a single orthologous gene (albeit with duplications in the teleost fish). Here, we describe the discovery and functional characterisation of a new melanopsin gene in fish, bird, and amphibian genomes, demonstrating that, in fact, the vertebrates have evolved two quite separate melanopsins. On the basis of sequence similarity, chromosomal localisation, and phylogeny, we identify our new melanopsins as the true orthologs of the melanopsin gene previously described in mammals and term this grouping Opn4m. By contrast, the previously published melanopsin genes in nonmammalian vertebrates represent a separate branch of the melanopsin family which we term Opn4x. RT-PCR analysis in chicken, zebrafish, and Xenopus identifies expression of both Opn4m and Opn4x genes in tissues known to be photosensitive (eye, brain, and skin). In the day-14 chicken eye, Opn4m mRNA is found in a subset of cells in the outer nuclear, inner nuclear, and ganglion cell layers, the vast majority of which also express Opn4x. Importantly, we show that a representative of the new melanopsins (chicken Opn4m) encodes a photosensory pigment capable of activating G protein signalling cascades in a light- and retinaldehyde-dependent manner under heterologous expression in Neuro-2a cells. A comprehensive in silico analysis of vertebrate genomes indicates that while most vertebrate species have both Opn4m and Opn4x genes, the latter is absent from eutherian and, possibly, marsupial mammals, lost in the course of their evolution as a result of chromosomal reorganisation. Thus, our findings show for the first time that nonmammalian vertebrates retain two quite separate melanopsin genes, while mammals have just one. These data raise important questions regarding the functional differences between Opn4x and Opn4m pigments, the associated adaptive advantages for most vertebrate species in retaining both melanopsins, and the implications for mammalian biology of lacking Opn4x.

Figure 1. The Deduced Amino Acid Sequence of the New Chicken Melanopsin (Opn4m) Aligned against Human Melanopsin (OPN4) and the Previously Published Chicken Melanopsin Sequence (Opn4x)

The “core” region that contains the seven probable transmembrane (TM) domains (blue bars) as predicted by the rod opsin model of Palczewski et al. [ 56] is defined by the red arrows. Numbered diamonds indicate the retinal attachment site, K300 (1); potential Schiff base counterions, Y106 and E175 (2 and 3, respectively); and D127/R128/Y129 tripeptide (4); see text for more details. Shaded residues indicate residue conservation between at least two of the three melanopsins.

Figure 2. Heterologous Expression of Chicken Opn4m Indicates that It Is a Sensory Photopigment

(A) Representative whole-cell patch-clamp recordings from Neuro-2a cells transfected with chicken Opn4m or Opn4x, in the presence of 9- cis retinal exposed to a 420-nm light stimulus at the time indicated by the arrow. (B) Further analysis of the cOpn4m responses revealed that they were abolished by incubation with 100 μM suramin and absent in cells not exposed to retinal (paired t-tests, ** p < 0.01, *** p < 0.0001).

Figure 3. A Revised Phylogeny and Nomenclature of the Vertebrate Melanopsin Family

A maximum parsimony phylogenetic tree (derived from amino acid sequences 310 sites, 151 informative, rooted with Amphioxus Opn4) showing the relationship between the novel nonmammalian Opn4m melanopsins and previously published Opn4x melanopsin sequences. Branch confidence levels (% based on 500 bootstrap replicates) reveal an evolutionarily ancient split into the Opn4m and Opn4x branches. The teleost melanopsin nomenclature previously suggested by Drivenes et al. [ 17] is shown in parenthesis; see text for more details.

Figure 4. Schematic Diagrams Detailing the Chromosomal Regions Surrounding the Human and Chicken Melanopsin Loci

(A) Comparison of syntenic regions encompassing the OPN4 locus on human chromosome 10 with that of the Opn4m locus on chicken Chromosome 4. Gene order has been conserved. (B) Comparison of representative loci between the PTGD2 and SEC24B genes on human Chromosome 4q22.3-q25 (approximately 1 Mb apart) and the orthologous loci on chicken Chromosome 4. Note the intrachromosomal rearrangements and lack of an OPN4X locus in humans.

Figure 5. Localisation of Opn4m and Opn4x Expression in the Chicken Retina

Dual in situ hybridisation histochemistry using probes for both cOpn4x (fluorescein) and cOpn4m (digoxigenin) reveals extensive cOpn4x expression [green; (A)], a more restricted pattern for cOpn4m [red; (B)], and widespread colocalisation of mRNA for the two genes [yellow; (C), merged image] in sections from 2-wk-old chicken retina. Control sense probes showed no nonspecific labelling [(D), cOpn4x; panel E, cOpn4m; (F), merged image]. ONL = outer nuclear; INL = inner nuclear layer; GCL = ganglion cell layer. Scale bar = 50 μm.