Expression of pineal ultraviolet- and green-like opsins in the pineal organ and retina of teleosts
1 Department of Zoology, University of Lund, Lund, Sweden,
2 Institute of Marine Research, Austevoll Aquaculture Research Station, N-5392 Storebø, Norway,
3 Department of Pathology, University of Lund, Sölvegatan 25, 22185, Lund, Sweden and
4 Department of Molecular Biology, University of Bergen, Bergen, Norway
* Present address: Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6
Author for correspondence at address 3 (e-mail: bo.holmqvist{at}pat.lu.se)
Accepted April 19, 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: pineal organ, retina, fish, cloning, mRNA, in situ hybridisation.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although photoreceptors in the pineal organ and the retina are employed in different photosensory functions, they display many structural, functional and biochemical similarities (see Meissl, 1997). Extraretinal photoreceptors typically exhibit maximal sensitivities in the short (blue) to middle (green) wavelengths (Shand and Foster, 1999), as do several cone types in the retina (Levine and MacNichol, 1979; Hárosi, 1994). The presence of retinal ultraviolet-sensitive cones is well established (see Tovée, 1995), whereas evidence for extraretinal ultraviolet photoreceptors is scarce (Uchida and Morita, 1990). Several studies suggest that the pineal organ of adult teleosts utilises two or more photopigments. Immunocytochemical detection of opsin proteins in the pineal organ of various species indicates that pineal photoreceptors contain retinal-like opsins, possibly structurally similar to both cone opsin(s) and rod opsin (Ekström and Meissl, 1997; Forsell et al., 1997; Meyer-Rochow et al., 1999). Intracellular recordings and microspectrophotometric studies of the pineal organ in rainbow trout Oncorhynchus mykiss have demonstrated photoreceptors with maximal spectral sensitivities in the short to middle wavelengths (Meissl and Ekström, 1988; Kusmic et al., 1993). In the pike Esox lucius, ultraviolet light causes a long-lasting inhibition of the action potential discharge of pineal chromaticity neurons (Falcon and Meissl, 1981), although the implications of this finding for the number of pineal photoreceptor types in this animal are unknown.
Recent molecular studies in teleosts have identified gene sequences encoding a rod-like opsin expressed in the brain and the pineal organ (Mano et al., 1999; Philp et al., 2000a) and a vertebrate ancient (VA) opsin expressed in various extraretinal tissues including the pineal organ (Soni and Foster, 1997; Kojima et al., 2000; Moutsaki et al., 2000; Philp et al., 2000b). Other novel opsin molecules (e.g. pinopsins) have been identified in the pineal organ/complex of birds, lampreys and lizards (Okano et al., 1994; Kawamura and Yokoyama, 1997; Yokoyama and Zhang, 1997). Both pinopsin (in chicken) and VA opsin (in salmon) are short-wavelength-sensitive when reconstituted with vitamin A1 (the wavelength of maximum sensitivity, max, is 470nm in the chicken and 451nm in the salmon) (Okano et al., 1994; Soni et al., 1998).
In many vertebrates, the retina contains cone photoreceptors with maximum absorption in the ultraviolet region of the spectrum (max in the range 355400nm) (Hárosi, 1994; Tovée, 1995). Microspectrophotometric investigations have demonstrated the presence of ultraviolet-sensitive retinal photopigments in a large number of teleost species (Hárosi and Hashimoto, 1983; Bowmaker et al., 1991; Hárosi, 1994; McFarland and Loew, 1994; Carleton et al., 2000; Novales Flamarique and Hárosi, 2000). Gene sequences encoding different opsins, including opsins in the ultraviolet class (SWS-1, short-wavelength-sensitive class 1 opsins) (see Yokoyama and Yokoyama, 1996), have been identified in goldfish Carassius auratus, zebrafish Danio rerio, killifish Oryzias latipes and Lake Malawi cichlids (Johnson et al., 1993; Hisatomi et al., 1996; Hisatomi et al., 1997; Vihtelic et al., 1999; Carleton et al., 2000). In the retina of adult goldfish and killifish, ultraviolet opsin mRNA is expressed in miniature single cones (Hisatomi et al., 1996) and in short single cones (Hisatomi et al., 1997), respectively. A corresponding distribution of ultraviolet opsin protein in the zebrafish retina has been demonstrated using immunocytochemistry (Vihtelic et al., 1999). All single cones are ultraviolet-sensitive in the Lake Malawi cichlid Metriaclima zebra (Carleton et al., 2000), whereas only the so-called corner cones appear to be ultraviolet-sensitive in salmonids (Bowmaker and Kunz, 1987; Beaudet et al., 1993; Hawryshyn and Hárosi, 1994).
Immunocytochemical studies of developing teleosts have demonstrated that opsin proteins and other phototransduction molecules are present in the pineal organ during embryonic development (Östholm et al., 1987; Forsell et al., 1997), a time when the retina is much less, or not at all, differentiated. The temporal pattern of opsin mRNA expression has been studied in the retina of goldfish and zebrafish. In these animals, rod opsin is expressed first, followed by green, red and ultraviolet opsins (Raymond et al., 1995; Stenkamp et al., 1996). Although the pineal organ is probably involved in early photosensory processes, only a small number of pineal opsin sequences have been identified and, consequently, little is known about the temporal correlation of opsin mRNA expression between the pineal organ and the retina.
Our work focuses on the development of the pineal organ and the retina of different teleosts, with special emphasis on the differentiation of photoreceptor cells and second-order neurons. This research combines molecular and histochemical methods including reverse transcriptase/polymerase chain reaction (RT-PCR) and gene cloning techniques, non-radioactive in situ hybridisation and immunocytochemistry. Here, we review and expand upon data from a marine flatfish, the Atlantic halibut (Hippoglossus hippoglossus), and two other marine species, the Atlantic herring (Clupea harengus) and the Atlantic cod (Gadus morhua), concerning the cellular and molecular differentiation of pineal and retinal photoreceptors (Holmqvist et al., 1996; Forsell et al., 1997; Forsell et al., 1998; J. Forsell, P. Ekström, W. J. DeGrip, J. V. Helvik and B. Holmqvist, in preparation). In addition, we present new data from recent in situ hybridisation studies on seven other teleost species.
![]() |
Early differentiation of the pineal organ |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
Molecular identification of ultraviolet and green opsins in the halibut |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In situ hybridisation was performed using digoxigenin-labelled RNA sense and anti-sense probes prepared from whole cloned cDNA fragments (see Holmqvist et al., 2000) of HPO1 and HPO4. Both anti-sense probes revealed expression in pineal photoreceptors of halibut embryos, larvae and adults (Fig.1E,F), and our immunocytochemical data suggest corresponding distributions of opsin proteins (J. Forsell, P. Ekström and B. Holmqvist, in preparation). Furthermore, mRNAs for both ultraviolet- and green-like opsins were expressed in the retinal photoreceptors of halibut larvae (Fig.1H). The sense probes did not produce any labelling. In halibut, pineal sensitivity to both short and middle wavelengths may thus be essential for light-influenced events during embryonic and initial larval development.
![]() |
Expression of ultraviolet- and green-like opsins in other teleosts |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Labelling with the HPO1 probe met all the criteria detailed above. Labelling with the HPO4 probe also fulfilled all the criteria, but at a lower hybridisation temperature (57°C instead of 65°C for HPO1), indicating relatively lower sequence similarities of labelled ultraviolet-like mRNAs (when compared with green-like mRNAs). The endogenous mRNA transcripts that hybridised with the HPO1 and HPO4 probes were not identified in this study. However, the high sequence homologies between teleost opsins, the fulfilled stringency criteria of the in situ hybridisations and the good correlation between the distribution of in situ hybridisation labelling and the morphological and microspectrophotometric descriptions of photoreceptors and visual pigments together provide evidence that the observed hybridisations represent expression of putative ultraviolet- and green-like opsins in the pineal organ and retina of the teleost species examined. The results obtained for each species detailing the expression patterns in the pineal organ and/or the retina are summarised in Table1.
|
|
|
In the Central American cichlid Archocentarus nigrofasciatus and the two Lake Malawi cichlid species, HPO4 hybridisation in the retina was restricted to single cones (Fig.3G). These findings are consistent with microspectrophotometric-derived absorbance spectra from the Lake Malawi cichlid Metriaclima zebra; in this species, all single cones are ultraviolet-sensitive, with max at 368nm (Carleton et al., 2000). The specificity of the HPO4 labelling in cichlids is further supported by its high structural identity (83%) with the ultraviolet opsins of different Lake Malawi cichlids (Carleton et al., 2000). In all the cichlid species, HPO1 hybridisation was localised in double cones and some rods.
Both HPO1 and HPO4 hybridisation was detected in the retina of larval turbot. The HPO4-positive photoreceptors were restricted to the ventral retina (Fig.3H); this localisation and the morphological appearance of photoreceptors were similar to those observed for HPO4-labelled photoreceptors in the retina of larval halibut (Fig.1H). The presence of putative green-like opsins in this animal is in accordance with behavioural responses to middle-wavelength light observed at the larval stage (H. I. Browman, personal communication). In the herring and in the gadid species (cod and haddock), the expression of green opsin mRNAs and the absence of expression of ultraviolet opsin mRNAs (Fig.3IK) are consistent with results from spectral sensitivity studies of larval herring (Blaxter, 1968) and with preliminary microspectrophotometric observations of retinal visual pigments in cod and haddock larvae (F. I. Hárosi, personal communication), respectively.
Together, our results suggest that various teleost species express both green- and ultraviolet-like opsins in retinal photoreceptors and green-like opsins in pineal photoreceptors. In the species studied here, the expression of these opsins is similar in the larval and adult stages. Only the halibut appears to express a pineal ultraviolet opsin similar to HPO4. It is possible that the halibut compensates for what appears to be delayed retinal differentiation with a functional pineal organ possessing green and ultraviolet opsins from the early stages of development. Further studies with different fish species at various life stages are required to assess the extent of ultraviolet opsin expression among teleost phyla. Such investigations may, in turn, provide valuable clues to the origin and evolution of ultraviolet photoreceptors in teleost fishes and to the various functions of ultraviolet sensitivity.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ali, M. A., Klyne, M. A., Park, E. H. and Lee, S. H. (1988). Pineal and retinal photoreceptors in embryonic Rivulus marmoratus Poey. Anat. Anz. 167, 359369.[Medline]
Beaudet, L., Browman, H. I. and Hawryshyn, C. W. (1993). Optic nerve response and retinal structure in rainbow trout of different sizes. Vision Res. 33, 17391746.[Medline]
Blackshaw, S. and Snyder, S. (1997). Parapinopsin, a novel catfish opsin localized in the parapineal organ, defines a new gene family. J. Neurosci. 17, 80838092.
Blaxter, J. H. S. (1968). Visual thresholds and spectral sensitivity of herring larvae. J. Exp. Biol. 48, 3953.
Bowmaker, J. K. and Hunt, D. M. (1999). Molecular biology of photoreceptor spectral sensitivity. In Adaptive Mechanisms in the Ecology of Vision (ed. S. N. Archer, M. Djamgoz and E. Loew), pp. 439462. Dordrecht, Boston, London: Kluwer Academic Publisher.
Bowmaker, J. K. and Kunz, Y. W. (1987). Ultraviolet receptors, tetrachromatic colour vision and retinal mosaics in the brown trout (Salmo trutta): Age-dependent changes. Vision Res. 27, 21012108.[Medline]
Bowmaker, J. K., Thorpe, A. and Douglas, R. H. (1991). Ultraviolet-sensitive cones in goldfish. Vision Res. 31, 349352.[Medline]
Carleton, K. L., Harosi, F. I. and Kocher, T. D. (2000). Visual pigments of African cichlid fishes: evidence for ultraviolet vision from microspectrophotometric and DNA sequences. Vision Res. 40, 879890.[Medline]
Chang, B. S. W., Crandall, K. A., Carulli, J. P. and Hartl, D. L. (1995). Opsin phylogeny and evolution: a model for blue shifts in wavelength regulation. Mol. Phylogenet. Evol. 4, 3143.[Medline]
Ekström, P., Borg, B. and van Veen, Th. (1983). Ontogenetic development of the pineal organ, parapineal organ and retina of three-spined stickleback, Gasterosteus aculeatus L. (Teleostei). Cell Tissue Res. 233, 593609.[Medline]
Ekström, P. and Meissl, H. (1997). The pineal organ of teleost fishes. Rev. Fish Biol. Fish. 7, 199284.
Falcon, J. and Meissl, H. (1981). The photosensory function of the pineal organ of the pike (Esox lucius L.). Correlation between structure and function. J. Comp. Physiol. A 144, 127137.
Forsell, J., Ekström, P., Helvik, J. V., Blackshaw, S., Kallesö, T., Degrip, W. J. and Holmqvist, B. (1998). Development and molecular identity of extraretinal photoreceptors in early life stages of marine teleosts. Soc. Neurosci. Abstr. 24, 810.
Forsell, J., Holmqvist, B., Helvik, J. V. and Ekström, P. (1997). Role of the pineal organ in the photoregulated hatching of the Atlantic halibut. Int. J. Devl. Biol. 41, 591595.
Hárosi, F. I. (1994). An analysis of two spectral properties of vertebrate visual pigments. Vision Res. 34, 13591367.[Medline]
Hárosi, F. I. and Hashimoto, Y. (1983). Ultraviolet visual pigment in a vertebrate: a tetrachromatic cone system in the Japanese dace. Science 222, 10211023.[Medline]
Hawryshyn, C. W. and Hárosi, F. I. (1994). Spectral characteristics of visual pigments in rainbow trout (Oncorhynchus mykiss). Vision Res. 34, 13851392.[Medline]
Helvik, J. V. and Walther, B. T. (1992). Photo-regulation of the hatching process of halibut (Hippoglossus hippoglossus) eggs. J. Exp. Zool. 263, 204209.
Hisatomi, O., Satoh, T., Barthel, L. K., Stenkamp, D. L., Raymond, P. A. and Tokunaga, F. (1996). Molecular cloning and characterisation of the putative ultraviolet-sensitive visual pigment of goldfish. Vision Res. 36, 933939.[Medline]
Hisatomi, O., Satoh, T. and Tokunaga, F. (1997). The primary structure and distribution of killifish visual pigments. Vision Res. 37, 30893096.[Medline]
Holmqvist, B. I., Ellingsen, B., Alm, P., Forsell, J., Öyan, A.-M., Göksöyr, A., Fjose, A. and Seo, H.-C. (2000). Identification and distribution of nitric oxide synthase in the brain of adult zebrafish. Neurosci. Lett. 292, 119122.[Medline]
Holmqvist, B. I., Forsell, J. and Helvik, J. V. (1996). Patterns of embryonic development of the brain and sensory organs studied in three marine teleost species. Soc. Neurosci. Abstr. 22, 991.
Johnson, R. L., Grant, K. B., Zankel, T. C., Boehm, M. F., Merbs, S. L., Nathans, J. and Nakanishi, K. (1993). Cloning and expression of goldfish opsin sequences. Biochemistry 32, 208214.[Medline]
Kawamura, S. and Yokoyama, S. (1997). Expression of visual and nonvisual opsins in American chameleon. Vision Res. 37, 18671871.[Medline]
Kojima, D., Mano, H. and Fukada, Y. (2000). Vertebrate ancient-long opsin: A green-sensitive photoreceptor molecule present in zebrafish deep brain retinal horizontal cells. J. Neurosci. 20, 28452851.
Kunz, Y. W., Wildenbourg, G., Goodrich, L. and Callaghan, E. (1994). The fate of ultraviolet receptors in the retina of the Atlantic salmon (Salmon salar). Vision Res. 34, 13751383.[Medline]
Kusmic, C., Barsanti, L., Passarelli, V. and Gualtieri, P. (1993). Photoreceptor morphology and visual pigment content in the pineal organ and in the retina of the juvenile and adult trout Salmo irideus. Micron 24, 279286.
Kvenseth, A. M., Pittman, K. and Helvik, J. V. (1996). Eye development in Atlantic halibut (Hippoglossus hippoglossus): differentiation and development of the retina from early yolk sac stages through metamorphosis. Can. J. Aquat. Sci. 53, 25242534.
Levine, J. S. and MacNichol, E. F., Jr (1979). Visual pigments in teleost fishes: Effects of habitat, microhabitat and behaviour on visual pigment evolution. Sensory Proc. 3, 95131.
Mangor-Jensen, A. and Waiwood, K. G. (1995). The effect of light exposure on buoyancy of halibut eggs. J. Fish Biol. 47, 1825.
Mano, H., Kojima, D. and Fukada, Y. (1999). Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland. Mol. Brain Res. 73, 110118.[Medline]
McFarland, W. N. and Loew, E. R. (1994). Ultraviolet visual pigments in marine fishes of the family Pomacentridae. Vision Res. 34, 13931396.[Medline]
Meissl, H. (1997). Photic regulation of pineal function. Analogies between retinal and pineal photoreception. Biol. Cell 89, 549554.[Medline]
Meissl, H. and Ekström, P. (1988). Photoreceptor responses to light in the isolated pineal organ of the trout, Salmo gairdneri. Neurosci. 24, 10711076.[Medline]
Meyer-Rochow, V. B., Morita, Y. and Tamotsu, S. (1999). Immunocytochemical observations of the pineal organ and retina of the Antartic teleosts Pagothenia borchgrevinki and Trematomus bernacchii. J. Neurocytol. 28, 125130.[Medline]
Moutsaki, P., Bellingham, J., Soni, B. G., David-Gray, Z. K. and Foster, R. G. (2000). Sequence, genomic structure and tissue expression of carp (Cyprinus carpio L.) vertebrate ancient (VA) opsin. FEBS Lett. 473, 316322.[Medline]
Negishi, K. and Wagner, H.-J. (1995). Differentiation of photoreceptors, glia and neurons in the retina of the cichlid fish Aequidens pulcher, an immunohistochemical study. Devl. Brain Res. 89, 87102.[Medline]
Novales Flamarique, I. (2000). The ontogeny of ultraviolet sensitivity, cone disappearance and regeneration in the sockeye salmon, Oncorhynchus nerka. J. Exp. Biol. 203, 11611172.
Novales Flamarique, I. and Hárosi, F.I. (2000). Photoreceptors, visual pigments and ellipsosomes in the killifish, Fundulus heteroclitus: A microspectrophotometric and histological study. Visual Neurosci. 17, 403420.[Medline]
Okano, T., Yoshikawa, T. and Fukada, Y. (1994). Pinopsin is a chicken pineal photoreceptive molecule. Nature 372, 9497.[Medline]
Omura, Y. and Oguri, M. (1993). Early development of the pineal photoreceptors prior to retinal differentiation in the embryonic rainbow trout, Oncorhynchys mykiss (Teleostei). Arch. Histol. Cytol. 56, 283291.[Medline]
Östholm, T., Brännäs, E. and van Veen, Th. (1987). The pineal organ is the first differentiated light receptor in the embryonic salmon, Salmo salar L. Cell Tissue Res. 249, 641646.[Medline]
Philp, A. R., Bellingham, J., Garcia-Fernandez, J. M. and Foster, R. G. (2000a). A novel rod-like opsin isolated from the extra-retinal photoreceptors of teleost fish. FEBS Lett. 468, 181188.[Medline]
Philp, A. R., Garcia-Fernandez, J. M., Soni, B. G., Lucas, R. J., Bellingham, J. and Foster, R. G. (2000b). Vertebrate ancient (VA) opsin and extraretinal photoreception in the Atlantic salmon (Salmo salar). J. Exp. Biol. 203, 19251936.
Raymond, P. A., Barthel, L. K. and Curran, G. A. (1995). Developmental patterning of rod and cone photoreceptors in embryonic zebrafish. J. Comp. Neurol. 359, 537550.[Medline]
Raymond, P. A., Barthel, L. K., Rounsifer, M. E., Sullivan, S. A. and Knight, J. K. (1993). Expression of rod and cone visual pigments in goldfish and zebrafish: A rhodopsin-like gene is expressed in cones. Neuron 10, 11611174.[Medline]
Robinson, J,, Schmitt, E. A., Harosi, F. I., Reece, R. J. and Dowling, J. E. (1993). Zebrafish ultraviolet visual pigment: Absorption spectrum, sequence and localization. Proc. Natl. Acad. Sci. USA 90, 60096012.[Abstract]
Shand, J. and Foster, R. G. (1999). The extraretinal photoreceptors of non-mammalian vertebrates. In Adaptive Mechanisms in the Ecology of Vision (ed. S. N. Archer, M. Djamgoz and E. Loew), pp. 197222. Dordrecht, Boston, London: Kluwer Academic Publishers.
Soni, B. G. and Foster, R. G. (1997). A novel and ancient vertebrate opsin. FEBS Lett. 406, 279283.[Medline]
Soni, B. G., Philp, A., Knox, B. E. and Foster, R. G. (1998). Novel retinal photoreceptors. Nature 394, 2728.[Medline]
Stenkamp, D. L., Hisatomi, O., Barthel, L. K., Tokunaga, F. and Raymond, P. A. (1996). Temporal expression of rod and cone opsins in embryonic goldfish retina predicts the spatial organization of the cone mosaic. Invest. Ophthalmol. Visual Sci. 37, 363376.[Abstract]
Tovée, M. J. (1995). Ultra-violet photoreceptors in the animal kingdom: their distribution and function. Trend. Ecol. Evol. 10, 455460.
Uchida, K. and Morita, Y. (1990). Intracellular responses from UV-sensitive cells in the photosensory pineal organ. Brain Res. 534, 237242.[Medline]
Vigh, B., Vigh-Teichmann, I., Reinhard, I., Szél, A. and van Veen Th. (1986) Opsin immunoreactions in the developing and adult pineal organ. In The Pineal Gland during Development from Fetus to Adult (ed. D. Gupta and R. G. Foster), pp. 3142. London, Sydney: Croom Helm.
Vihtelic, T. S., Doro, C. and Hyde, D. R. (1999). Cloning and characterisation of six zebrafish photoreceptor opsin cDNAs and immunolocalization of their corresponding proteins. Visual Neurosci. 16, 571585.[Medline]
Yokoyama, S. and Yokoyama, R. (1996). Adaptive evolution of photoreceptors and visual pigments in vertebrates. Annu. Rev. Ecol. Syst. 27, 543567.
Yokoyama, S. and Zhang, H. (1997). Cloning and characterisation of the pineal gland-specific opsin gene of marine lamprey (Petromyzon marinus). Gene 202, 8993.[Medline]