Such feature. Our experimental evidence supports Albalat’s [18] view that the

Such feature. Our experimental evidence supports Albalat’s [18] view that the cephalochordate (Branchiostoma) and tunicate (Ciona) proteins related to SC1 vertebrate visual cycle components are probably not involved in chromophore regeneration. This, together with the phylogenetic analysis of RPE65 and the absence of LRAT makes us conclude that Ciona intestinalis does not have a visual cycle comparable to vertebrates. As we do not have any indications that a prototype of the vertebrate visual cycle was secondarily lost in Ciona intestinalis, it likely never evolved. In fact, Nakashima et al. [43] conclude that 11-cis retinal in the Ciona larval ocellus is supplied from Ci-opsin3, a photoisomerase opsin, suggesting reliance on the more primitive pathway. Larval Ciona also expresses Ci-opsin1, a ciliary-type opsin as its visual pigment [13,44]. Furthermore, the expression of Ciona BCMOa/CiRPE65 occurs in the sessile adult stage where non-visual roles, such as phototropism, siphon contraction and gamete release [13] have been proposed for photoreception, and not in the freeswimming larval stage. Ci-opsin3 is also expressed in the adult neural complex along with Ci-CRALBP and the BCMOa/CiRPE65. If BCMOa/Ci-RPE65 is not capable of isomerizing retinol, as we have found, then this role can be accomplished by the photoisomerase RGR opsin homolog Ci-opsin3 in both developmental stages. (Conversely, we could not detect any photosiomerases (peropsin or RGR opsin homologs) in the lamprey genome that could potentially accomplish RPE65independent visual chromophore regeneration.) The presence of a CRALBP-like protein in Ciona 1480666 (Ci-CRALBP) suggests that trapping and transport of 11-cis retinal derived from Ci-opsin3 could occur in both the larva and adult stages. Though CiCRALBP clusters with vertebrate CRALBP (unlike Branchiostoma CRALBP-like homolog [13], which clusters with a-tocopherol transfer protein (aTTP; data not shown), another member of the CRAL-TRIO family 1676428 [45]), it has not yet been shown experimentally to actually bind 11-cis retinal. The absence of a robust LRATOrigin and Evolution of Vertebrate Visual CycleFigure 5. Alignment of Human and Lamprey RPE65. CLUSTAL W (1.83) alignment of Human RPE65 and Lamprey RPE65. GenBank/EBI accession numbers are as follows: human RPE65, NP_000320, lamprey RPE65 JX115001. Red, conserved residues around catalytic cysteine. doi:10.1371/journal.pone.0049975.gortholog (other than a SULT1-ST7-like homolog) in Ciona further weakens the case for a patent visual cycle in ascidians. Thus, we conclude that Ciona does not GNF-7 possess a coherent retinoid metabolic pathway that is comparable with the vertebrate visual cycle. To reiterate, our main rationale for this view is the absence of functional RPE65 and LRAT orthologs. Given the evident absence of a vertebrate-like visual cycle in the pre-vertebrate chordates, it was important to establish its earliest origins in the most primitive vertebrates. Various components of the vertebrate retina phototransduction system have been found inthe lamprey including opsins [39,42], photoreceptor-specific transducins [46], and photoreceptor-specific cyclic nucleotide phosphodiesterase 6 (PDE6) [47]. However, until now, components of the vertebrate visual cycle had not been identified in lamprey. We find that lamprey RPE65 is remarkably similar to mammalian RPE65 (72 identity/92 similarity), indicating that most, if not all, of the transition to a functional isomerohydrolase from a.Such feature. Our experimental evidence supports Albalat’s [18] view that the cephalochordate (Branchiostoma) and tunicate (Ciona) proteins related to vertebrate visual cycle components are probably not involved in chromophore regeneration. This, together with the phylogenetic analysis of RPE65 and the absence of LRAT makes us conclude that Ciona intestinalis does not have a visual cycle comparable to vertebrates. As we do not have any indications that a prototype of the vertebrate visual cycle was secondarily lost in Ciona intestinalis, it likely never evolved. In fact, Nakashima et al. [43] conclude that 11-cis retinal in the Ciona larval ocellus is supplied from Ci-opsin3, a photoisomerase opsin, suggesting reliance on the more primitive pathway. Larval Ciona also expresses Ci-opsin1, a ciliary-type opsin as its visual pigment [13,44]. Furthermore, the expression of Ciona BCMOa/CiRPE65 occurs in the sessile adult stage where non-visual roles, such as phototropism, siphon contraction and gamete release [13] have been proposed for photoreception, and not in the freeswimming larval stage. Ci-opsin3 is also expressed in the adult neural complex along with Ci-CRALBP and the BCMOa/CiRPE65. If BCMOa/Ci-RPE65 is not capable of isomerizing retinol, as we have found, then this role can be accomplished by the photoisomerase RGR opsin homolog Ci-opsin3 in both developmental stages. (Conversely, we could not detect any photosiomerases (peropsin or RGR opsin homologs) in the lamprey genome that could potentially accomplish RPE65independent visual chromophore regeneration.) The presence of a CRALBP-like protein in Ciona 1480666 (Ci-CRALBP) suggests that trapping and transport of 11-cis retinal derived from Ci-opsin3 could occur in both the larva and adult stages. Though CiCRALBP clusters with vertebrate CRALBP (unlike Branchiostoma CRALBP-like homolog [13], which clusters with a-tocopherol transfer protein (aTTP; data not shown), another member of the CRAL-TRIO family 1676428 [45]), it has not yet been shown experimentally to actually bind 11-cis retinal. The absence of a robust LRATOrigin and Evolution of Vertebrate Visual CycleFigure 5. Alignment of Human and Lamprey RPE65. CLUSTAL W (1.83) alignment of Human RPE65 and Lamprey RPE65. GenBank/EBI accession numbers are as follows: human RPE65, NP_000320, lamprey RPE65 JX115001. Red, conserved residues around catalytic cysteine. doi:10.1371/journal.pone.0049975.gortholog (other than a SULT1-ST7-like homolog) in Ciona further weakens the case for a patent visual cycle in ascidians. Thus, we conclude that Ciona does not possess a coherent retinoid metabolic pathway that is comparable with the vertebrate visual cycle. To reiterate, our main rationale for this view is the absence of functional RPE65 and LRAT orthologs. Given the evident absence of a vertebrate-like visual cycle in the pre-vertebrate chordates, it was important to establish its earliest origins in the most primitive vertebrates. Various components of the vertebrate retina phototransduction system have been found inthe lamprey including opsins [39,42], photoreceptor-specific transducins [46], and photoreceptor-specific cyclic nucleotide phosphodiesterase 6 (PDE6) [47]. However, until now, components of the vertebrate visual cycle had not been identified in lamprey. We find that lamprey RPE65 is remarkably similar to mammalian RPE65 (72 identity/92 similarity), indicating that most, if not all, of the transition to a functional isomerohydrolase from a.