Absorption of a photon by visual pigments induces isomerization of 11-cis-retinaldehyde (RAL) chromophore to all-trans-RAL. Since the opsins lacking 11-cis-RAL lose light sensitivity, sustained vision requires continuous regeneration of 11-cis-RAL via the process called 'visual cycle'. Protostomes and vertebrates use essentially different machinery of visual pigment regeneration, and the origin and early evolution of the vertebrate visual cycle is an unsolved mystery. Here we compare visual retinoid cycles between different photoreceptors of vertebrates, including rods, cones and non-visual photoreceptors, as well as between vertebrates and invertebrates. The visual cycle systems in ascidians, the closest living relatives of vertebrates, show an intermediate state between vertebrates and non-chordate invertebrates. The ascidian larva may use retinochrome-like opsin as the major isomerase. The entire process of the visual cycle can occur inside the photoreceptor cells with distinct subcellular compartmentalization, although the visual cycle components are also present in surrounding non-photoreceptor cells. The adult ascidian probably uses RPE65 isomerase, and trans-to-cis isomerization may occur in distinct cellular compartments, which is similar to the vertebrate situation. The complete transition to the sophisticated retinoid cycle of vertebrates may have required acquisition of new genes, such as interphotoreceptor retinoid-binding protein, and functional evolution of the visual cycle genes.

In contrast to mammals, adult teleost fish exhibit an enormous capacity to replace damaged neurons with newly generated ones after injuries in the central nervous system. In the present study, the role of microglia/macrophages, identified by tomato lectin binding, was examined in this process of neuronal regeneration in the corpus cerebelli of the teleost fish Apteronotus leptorhynchus. In the intact corpus cerebelli, or after short survival times following application of a mechanical lesion to this cerebellar subdivision, microglia/macrophages were virtually absent. Conversely, approximately 3 days after application of the lesion, the areal density of microglia/macrophages started to increase at and near the lesion site in the ipsilateral hemisphere, as well as in the contralateral hemisphere, and reached maximum levels at approximately 10 days post lesion. The density remained elevated until it reached background levels approximately one month after the injury. By comparing the time course of the appearance of microglia/macrophages with that of other regenerative events occurring within the first few weeks of wound healing in this model system, we hypothesize that one possible function of microglia/macrophages might be to remove debris of cells that have undergone apoptotic cell death at the lesion site.

We previously identified three genes that encode putative visual cycle proteins that are homologues of retinal G-protein coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP) and beta-carotene 15,15'-monooxygenase (Ci-BCO) in the ascidian Ciona intestinalis. Ci-opsin3 and Ci-CRALBP are localized in both ocellus photoreceptor cells and surrounding non-photoreceptor cells in the brain vesicle of the larva. In the present study, we investigated the possible role and evolutionary origin of the BCO/RPE65 family in the visual cycle by analyzing Ci-BCO localization by immunohistochemistry and by identifying a novel gene that encodes a homologue of retinal pigment epithelium-specific 65 kDa protein (Ci-RPE65) in C. intestinalis. In situ hybridization and expressed sequence tag (EST) profiles consistently suggest that Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable to that of Ci-opsin3 and Ci-CRALBP. Ci-RPE65 is also expressed in various adult tissues, including the gill, body wall and intestine, suggesting that Ci-RPE65 plays a role in addition to that in the visual cycle. In contrast, Ci-BCO is predominantly localized in ocellus photoreceptor cells of the larva. The larval visual cycle seems to use Ci-opsin3 as a photo-isomerase. Our results also suggest that the RPE65-dependent visual cycle is used in the adult photoreceptors of a primitive chordate.

In vertebrates, the absorption of light by rhodopsin leads to the isomerization of 11-cis-retinal chromophore to its all-trans form. In the visual cycle, all-trans retinal is converted back to 11-cis retinal. Mammalian visual cycle takes place in photoreceptor cells and retinal pigment epithelial (RPE) cells, while that of cephalopods is completed within a photoreceptor cell. To identify visual cycle system in the primitive chordate ascidians, we studied the localization of the ascidian visual cycle genes and proteins by in situ hybridization and whole-mount immunohistochemistry, respectively. We identified four genes encoding putative visual cycle proteins, homologs of retinal G protein-coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP), beta-carotene 15,15'monooxygenase (Ci-BCO) and RPE-specific 65 kDa protein (Ci-RPE65) in the ascidian, Ciona intestinalis. In contrast to Ci-BCO, which is predominantly localized in ocellus photoreceptor cells of the larva, Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable with that of Ci-opsin3 and Ci-CRALBP. Proteins of Ci-opsin3, Ci-CRALBP and Ci-BCO are localized in photoreceptor cells. These results suggest that the larval visual cycle uses Ci-opsin3 as a photoisomerase, while the visual cycle of the adult photoreceptors is RPE65-dependent. The colocalization of visual cycle proteins in the photoreceptor cells suggest that ascidian visual cycle takes place in a photoreceptor cell as seen in the cephalopod visual cycle.