Unexpected diversity and photoperiod dependence of the zebrafish melanopsin system.
ABSTRACT: Animals have evolved specialized photoreceptors in the retina and in extraocular tissues that allow them to measure light changes in their environment. In mammals, the retina is the only structure that detects light and relays this information to the brain. The classical photoreceptors, rods and cones, are responsible for vision through activation of rhodopsin and cone opsins. Melanopsin, another photopigment first discovered in Xenopus melanophores (Opn4x), is expressed in a small subset of retinal ganglion cells (RGCs) in the mammalian retina, where it mediates non-image forming functions such as circadian photoentrainment and sleep. While mammals have a single melanopsin gene (opn4), zebrafish show remarkable diversity with two opn4x-related and three opn4-related genes expressed in distinct patterns in multiple neuronal cell types of the developing retina, including bipolar interneurons. The intronless opn4.1 gene is transcribed in photoreceptors as well as in horizontal cells and produces functional photopigment. Four genes are also expressed in the zebrafish embryonic brain, but not in the photoreceptive pineal gland. We discovered that photoperiod length influences expression of two of the opn4-related genes in retinal layers involved in signaling light information to RGCs. Moreover, both genes are expressed in a robust diurnal rhythm but with different phases in relation to the light-dark cycle. The results suggest that melanopsin has an expanded role in modulating the retinal circuitry of fish.
Project description:Melanopsin is the photopigment that confers photosensitivity to a subset of retinal ganglion cells (pRGCs) that regulate many non-image-forming tasks such as the detection of light for circadian entrainment. Recent studies have begun to subdivide the pRGCs on the basis of morphology and function, but the origin of these differences is not yet fully understood. Here we report the identification of two isoforms of melanopsin from the mouse Opn4 locus, a previously described long isoform (Opn4L) and a novel short isoform (Opn4S) that more closely resembles the sequence and structure of rat and human melanopsins. Both isoforms, Opn4L and Opn4S, are expressed in the ganglion cell layer of the retina, traffic to the plasma membrane and form a functional photopigment in vitro. Quantitative PCR revealed that Opn4S is 40 times more abundant than Opn4L. The two variants encode predicted proteins of 521 and 466 aa and only differ in the length of their C-terminal tails. Antibodies raised to isoform-specific epitopes identified two discrete populations of melanopsin-expressing RGCs, those that coexpress Opn4L and Opn4S and those that express Opn4L only. Recent evidence suggests that pRGCs show a range of anatomical subtypes, which may reflect the functional diversity reported for mouse Opn4-mediated light responses. The distinct isoforms of Opn4 described in this study provide a potential molecular basis for generating this diversity, and it seems likely that their differential expression plays a role in generating the variety of pRGC light responses found in the mammalian retina.
Project description: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.
Project description:The master circadian pacemaker in mammals resides in the hypothalamic suprachiasmatic nuclei (SCN) and is synchronized to ambient light/dark cycles (i.e., photoentrainment). Melanopsin (Opn4) and classical rod-cone photoreceptors are believed to provide all the photic input necessary for circadian photoentrainment. Although the UVA-sensitive photopigment Opn5 is known to be expressed in retinal ganglion cells, its physiological role remains unclear and a potential role for Opn5 in the photoentrainment of the master clock has not been addressed. Here we report impaired photoentrainment and phase shifting to UVA light in Opn5-null mice. However, triple-knockout mice lacking all known functional circadian photoreceptors (i.e., rods, cones, and melanopsin) failed to entrain to UVA-light/dark cycles, despite the presence of Opn5, demonstrating that Opn5 alone is not sufficient for photoentrainment of the SCN clock. Since Opn5 is involved in the regulation of the retinal circadian clock, disrupted retinal function may cause impaired circadian photoentrainment in Opn5-null mice.
Project description:Melanopsin (Opn4) is a photopigment found in a subset of retinal ganglion cells (RGCs) that project to various brain areas. These neurons are intrinsically photosensitive (ipRGCs) and are implicated in nonimage-forming responses to environmental light such as the pupillary light reflex and circadian entrainment. Recent evidence indicates that ipRGCs respond to light at birth, but questions remain as to whether and when they undergo significant functional changes. We used bacterial artificial chromosome transgenesis to engineer a mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of the melanopsin promoter. Double immunolabeling for EGFP and melanopsin demonstrates their colocalization in ganglion cells of mutant mouse retinas. Electrophysiological recordings of ipRGCs in neonatal mice (postnatal day 0 [P0] to P7) demonstrated that these cells responded to light with small and sluggish depolarization. However, starting at P11 we observed ipRGCs that responded to light with a larger and faster onset (<1 s) and offset (<1 s) depolarization. These faster, larger depolarizations were observed in most ipRGCs by early adult ages. However, on application of a cocktail of synaptic blockers, we found that all cells responded to light with slow onset (>2.5 s) and offset (>10 s) depolarization, revealing the intrinsic, melanopsin-mediated light responses. The extrinsic, cone/rod influence on ipRGCs correlates with their extensive dendritic stratification in the inner plexiform layer. Collectively, these results demonstrate that ipRGCs make use of melanopsin for phototransduction before eye opening and that these cells further integrate signals derived from the outer retina as the retina matures.
Project description:In mammals, melanopsin is exclusively expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), which play an important role in circadian photoentrainment and other nonimage-forming functions. These ipRGCs reside in the inner retina, far removed from the pigment epithelium, which synthesizes the 11-cis retinal chromophore used by rod and cone photoreceptors to regenerate opsin for light detection. There has been considerable interest in the identification of the melanopsin chromophore and in understanding the process of photopigment regeneration in photoreceptors that are not in proximity to the classical visual cycle. We have devised an immuno-magnetic purification protocol that allows melanopsin-expressing retinal ganglion cells to be isolated and collected from multiple mouse retinas. Using this technique, we have demonstrated that native melanopsin in vivo exclusively binds 11-cis retinal in the dark and that illumination causes isomerization to the all-trans isoform. Furthermore, spectral analysis of the melanopsin photoproduct shows the formation of a protonated metarhodopsin with a maximum absorbance between 520 and 540 nm. These results indicate that even if melanopsin functions as a bistable photopigment with photo-regenerative activity native melanopsin must also use some other light-independent retinoid regeneration mechanism to return to the dark state, where all of the retinal is observed to be in the 11-cis form.
Project description:Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.
Project description:A unique class of intrinsically photosensitive retinal ganglion cells in mammalian retinae has been recently discovered and characterized. These neurons can generate visual signals in the absence of inputs from rods and cones, the conventional photoreceptors in the visual system. These light sensitive ganglion cells (mRGCs) express the non-rod, non-cone photopigment melanopsin and play well documented roles in modulating pupil responses to light, photoentrainment of circadian rhythms, mood, sleep and other adaptive light functions. While most research efforts in mammals have focused on mRGCs in retina, recent studies reveal that melanopsin is expressed in non-retinal tissues. For example, light-evoked melanopsin activation in extra retinal tissue regulates pupil constriction in the iris and vasodilation in the vasculature of the heart and tail. As another example of nonretinal melanopsin expression we report here the previously unrecognized localization of this photopigment in nerve fibers within the cornea. Surprisingly, we were unable to detect light responses in the melanopsin-expressing corneal fibers in spite of our histological evidence based on genetically driven markers and antibody staining. We tested further for melanopsin localization in cell bodies of the trigeminal ganglia (TG), the principal nuclei of the peripheral nervous system that project sensory fibers to the cornea, and found expression of melanopsin mRNA in a subset of TG neurons. However, neither electrophysiological recordings nor calcium imaging revealed any light responsiveness in the melanopsin positive TG neurons. Given that we found no light-evoked activation of melanopsin-expressing fibers in cornea or in cell bodies in the TG, we propose that melanopsin protein might serve other sensory functions in the cornea. One justification for this idea is that melanopsin expressed in Drosophila photoreceptors can serve as a temperature sensor.
Project description:Mammals contain 1 melanopsin (Opn4) gene that is expressed in a subset of retinal ganglion cells to serve as a photopigment involved in non-image-forming vision such as photoentrainment of circadian rhythms. In contrast, most nonmammalian vertebrates possess multiple melanopsins that are distributed in various types of retinal cells; however, their functions remain unclear. We previously found that the lamprey has only 1 type of mammalian-like melanopsin gene, which is similar to that observed in mammals. Here we investigated the molecular properties and localization of melanopsin in the lamprey and other cyclostome hagfish retinas, which contribute to visual functions including image-forming vision and mainly to non-image-forming vision, respectively. We isolated 1 type of mammalian-like melanopsin cDNA from the eyes of each species. We showed that the recombinant lamprey melanopsin was a blue light-sensitive pigment and that both the lamprey and hagfish melanopsins caused light-dependent increases in calcium ion concentration in cultured cells in a manner that was similar to that observed for mammalian melanopsins. We observed that melanopsin was distributed in several types of retinal cells, including horizontal cells and ganglion cells, in the lamprey retina, despite the existence of only 1 melanopsin gene in the lamprey. In contrast, melanopsin was almost specifically distributed to retinal ganglion cells in the hagfish retina. Furthermore, we found that the melanopsin-expressing horizontal cells connected to the rhodopsin-containing short photoreceptor cells in the lamprey. Taken together, our findings suggest that in cyclostomes, the global distribution of melanopsin in retinal cells might not be related to the melanopsin gene number but to the extent of retinal contribution to visual function.
Project description:Synchronization of the mammalian master circadian pacemaker to the daily light/dark cycle is mediated exclusively through retinal photoreceptors. The mammalian retina itself is also a self-sustained circadian oscillator. Here we report that the retinal molecular circadian clock can be entrained by lighting cycles in vitro, but that rods, cones, and melanopsin (Opn4) are not required for this entrainment. In vivo, retinas of Opn4(-/-);rd1/rd1 mice synchronize to light/dark cycles regardless of the phase of the master circadian pacemakers of the suprachiasmatic nuclei or the behavior of the animal. These data demonstrate that the retina uses a separate mechanism for local entrainment of its circadian clock than for entrainment of organism-level rhythmicity.
Project description:The molecular circadian clocks in the mammalian retina are locally synchronized by environmental light cycles independent of the suprachiasmatic nuclei (SCN) in the brain. Unexpectedly, this entrainment does not require rods, cones, or melanopsin (OPN4), possibly suggesting the involvement of another retinal photopigment. Here, we show that the ex vivo mouse retinal rhythm is most sensitive to short-wavelength light but that this photoentrainment requires neither the short-wavelength-sensitive cone pigment [S-pigment or cone opsin (OPN1SW)] nor encephalopsin (OPN3). However, retinas lacking neuropsin (OPN5) fail to photoentrain, even though other visual functions appear largely normal. Initial evidence suggests that OPN5 is expressed in select retinal ganglion cells. Remarkably, the mouse corneal circadian rhythm is also photoentrainable ex vivo, and this photoentrainment likewise requires OPN5. Our findings reveal a light-sensing function for mammalian OPN5, until now an orphan opsin.