Here we report the identification of a novel human opsin, melanopsin, that is expressed in cells of the mammalian inner retina. The human melanopsin gene consists of 10 exons and is mapped to chromosome 10q22. This chromosomal localization and gene structure differs significantly from that of other human opsins that typically have four to seven exons. A survey of 26 anatomical sites indicates that, in humans, melanopsin is expressed only in the eye. In situ hybridization histochemistry shows that melanopsin expression is restricted to cells within the ganglion and amacrine cell layers of the primate and murine retinas. Notably, expression is not observed in retinal photoreceptor cells, the opsin-containing cells of the outer retina that initiate vision. The unique inner retinal localization of melanopsin suggests that it is not involved in image formation but rather may mediate nonvisual photoreceptive tasks, such as the regulation of circadian rhythms and the acute suppression of pineal melatonin. The anatomical distribution of melanopsinpositive retinal cells is similar to the pattern of cells known to project from the retina to the suprachiasmatic nuclei of the hypothalamus, a primary circadian pacemaker.

We have identified an opsin, melanopsin, in photosensitive dermal melanophores of Xenopus laevis. Its deduced amino acid sequence shares greatest homology with cephalopod opsins. The predicted secondary structure of melanopsin indicates the presence of a long cytoplasmic tail with multiple putative phosphorylation sites, suggesting that this opsin's function may be finely regulated. Melanopsin mRNA is expressed in hypothalamic sites thought to contain deep brain photoreceptors and in the iris, a structure known to be directly photosensitive in amphibians. Melanopsin message is also localized in retinal cells residing in the outermost lamina of the inner nuclear layer where horizontal cells are typically found. Its expression in retinal and nonretinal tissues suggests a role in vision and nonvisual photoreceptive tasks, such as photic control of skin pigmentation, pupillary aperture, and circadian and photoperiodic physiology.Photopigments consist of an integral membrane apoprotein, opsin, that is covalently linked to a retinaldehyde chromophore. Upon illumination, the chromophore is photoisomerized, forcing a conformational change in the surrounding opsin and subsequently initiating an intracellular signaling cascade. The conversion of light into neural signals within photoreceptor cells of the retina is the best studied example of opsin-mediated signaling.Dermal melanophores of Xenopus laevis, like retinal photoreceptors, are photosensitive (1). When maintained in vitro, the melanosomes in dermal melanophores migrate to the cellular periphery in response to illumination (2) or the activation of a wide variety of G protein-coupled receptors (3). The melanophore response to these agents has led to the development of innovative systems for studying functional interactions between ligands and their receptors (4, 5). Melanophore photosensitivity is reversibly activated by retinaldehydes (6) and has an action spectrum characteristic of opsinlike photopigments (2). In an effort to identify an opsin that may regulate melanosome migration, we have analyzed protein extracts of cultured melanophores for opsin immunoreactivity and screened a melanophore cDNA library for opsin-like nucleotide sequences. MATERIALS AND METHODSWestern Blot. Approximately 10 5 cultured Xenopus laevis dermal melanophores were washed twice with 1ϫ calcium-and magnesium-free Dulbecco's phosphate buffer and lysed with lysis buffer [1% IGEPAL CA-630 (Sigma)͞0.2 M NaBH 3 CN͞1 mM phenylmethylsulfonyl fluoride͞aprotinin (0.1 trypsin inhibitor unit͞ml)͞5 mM EDTA͞0.086 M NaC 2 H 3 O 2 , pH 5.0, 4°C]. A homogenate of one early postmetamorphic adult eye was also extracted as above. Lysates were centrifuged and the supernatants subjected to SDS͞PAGE analysis and subsequent electroblotting onto a poly(vinylidene difluoride) membrane. The blot was probed with a 1:2,000 dilution of antisera (CERN 886) raised against bovine rhodopsin and detected by enhanced chemiluminescence.cDNA Library Screen. A X. laevis dermal melanophore oligo(dT) cDNA library was...

A full-length cDNA was isolated for a thyroid hormone response gene in the metamorphosing frog intestine and shown by sequence analysis to be the frog homolog of the mammalian extracellular matrix metalloproteinase stromelysin-3 (ST3). Northern hybridization indicated that ST3 gene expression is differentially activated in tadpole tissues during metamorphosis. In the small intestine, in situ hybridization localized high levels of ST3 mRNA to fibroblast-like cells during thyroid hormone-induced metamorphosis. ST3mRNA was undetectable in the intestine prior to metamorphosis, while high levels were present at the metamorphic climax. At this time, primary intestinal epithelial cells are known to undergo cell death and replacement by secondary epithelial cells, arguing that ST3 is involved in the modification of the extracellular matrix during apoptosis. ST3mRNA was also expressed at high levels during tadpole tail resorption, but not in premetamorphic tail or developing hindlimb, further supporting a role for ST3 when tissue remodeling is accompanied by large-scale cell death. Premetamorphic tadpoles treated with thyroid hormone showed a similar but compressed time course of ST3 gene regulation, suggesting that thyroid hormone controls ST3 gene expression during metamorphosis. In contrast, during embryogenesis, ST3 was expressed before endogenous thyroid hormone is detectable, indicating that ST3 can also be regulated independently of thyroid hormone. These findings implicate that ST3 participates in the modification of the extracellular matrix during matamorphic apoptosis, but Northern analyses using heterologous probes raise the possibility that additional matrix metalloproteinases may also be involved.