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.
Table 1.
Amino Acid Identity (Percent) across the “Core” Region of Representative Vertebrate 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.
Table 2.
Comparison of the Tissue Expression Pattern of Opn4m and Opn4x in Various Vertebrate Species as Detected by RT-PCR in This Study or from Previously Published Data
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.