Topography of different photoreceptor cell types in the larval retina of Atlantic halibut (Hippoglossus hippoglossus)
1 Department of Molecular Biology, University of Bergen, PO Box 7800, N-5020 Bergen, Norway and
2 Institute of Marine Research, Austevoll Aquaculture Research Station, N-5392 Storebø, Norway
Present address: Sars Centre for Molecular Marine Biology, Thormøhlensgt. 55, N-5008 Bergen, Norway
*Author for correspondence (e-mail: Vidar.Helvik{at}mbi.uib.no)
Accepted April 19, 2001
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Summary |
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Key words: opsin, cone, rod, mosaic, photoreceptor, flatfish, Hippoglossus hippoglossus.
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Introduction |
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In the marine environment, there are examples of vertical migration during ontogeny that are associated with changes in the photic environment. Flatfish provide a very good example of such visual adaptation (Beaudet and Hawryshyn, 1999). The early stages of flatfish are consistent with a pelagic, surface-dwelling life style. The bilaterally symmetrical larvae have one eye on each side of the body and have typical retinas adapted to a bright light environment; the retina consists of single-cone cells only (Blaxter, 1968; Evans and Fernald, 1993; Pankhurst and Butler, 1996)
The larvae of many fish hatch with, or rapidly develop, a retina that consists entirely of single cones. This includes fishes such as herring Clupea harengus (Blaxter and Jones, 1967), perch Perca fluviatilis (Ahlbert, 1973), greenback flounder Rhombosolea tapirina (Pankhurst and Butler, 1996), black bream Acanthopagrus butcheri (Shand et al., 1999), red sea bream Pagrus major (Kawamura et al., 1984), New Zealand snapper Pagrus auratus (Pankhurst and Eagar, 1996) and Atlantic halibut Hippoglossus hippoglossus (Kvenseth et al., 1996). These fish are examples of species showing indirect development with a larval stage in which the retina consists of only cone cells; the rod cells appear during metamorphosis (Evans and Fernald, 1990).
The larval period in flatfish ends with metamorphosis, during which the pelagic larva is remodelled to a juvenile form adapted to a benthic life style. This remodelling is associated with changes in the general body plan: one eye migrates to the contralateral side of the head adjacent to the other eye, such that both eyes can scan the surroundings above the body plane. Microspectrophotometric analysis of the single cone cells in winter flounder (Pseudopleuronectes americanus) revealed only one kind of opsin (wavelength of maximum sensitivity, max=519nm) (Evans et al., 1993), suggesting that these larvae are not capable of wavelength discrimination and that prey detection is probably based on differences in brightness contrast. They further showed that the visual system in post-metamorphic winter flounder gains three new types of cone opsins (with
max values of 457nm, 532nm and 547nm) and one rod opsin (
max=506nm), whereas the larval visual pigment disappears. These results indicate that, in addition to the induction of rod opsin expression, there is a major change in cone opsin expression between the pre- and post-metamorphic phases, suggesting a regulation of opsin expression within existing cone photoreceptor cells.
Two species of fish, zebrafish (Danio rerio) and goldfish (Carassius auratus), are commonly used as models for retinal development in vertebrates, and most of the work related to gene expression and mosaic formation has been performed in these two closely related freshwater fishes. Retinal development in zebrafish and goldfish, in which both cones and rods appear simultaneously (Raymond et al., 1995; Stenkamp et al., 1996), is more closely related to that in species showing direct development (Evans and Fernald, 1990). Recently, attention has been focused on elucidating whether the rod photoreceptors contribute to cone mosaic patterning in these species with direct retina development (Wan and Stenkamp, 2000). These studies have included manipulations to examine the development of cone mosaics in retinas that lack rod photoreceptors. An examination of these events in a naturally developing retina would be useful.
The halibut is a typical example of a marine species with a pelagic larval stage occurring in a bright light environment. Concomitant with ontogenetic transformation to a juvenile life form, the halibut performs a vertical migration to a benthic life style. It is not known how the retinas of marine fish larvae are adapted to the pelagic environment and how they change during development to adjust to the benthic life style with a reduced light intensity and spectrum. We have used in situ hybridisation to analyse the photoreceptor cells and visual pigments in the larval halibut retina adapted to the pelagic marine environment.
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Materials and methods |
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In situ hybridisation
Halibut larvae were fixed in 4% paraformaldehyde-buffered phosphate-buffered saline (PBS), pH7.2, for 48h at 4°C. After a brief wash in PBS, the specimens were kept overnight in a 25% sucrose PBS solution with 30% Tissue Tek (O.C.T. Sakura Finetek Europe, Netherlands). Several larval heads were placed in a mould containing 100% Tissue Tek and orientated with the anteriorposterior axis perpendicular (transverse) or parallel (sagittal) to bottom of the mould before rapid freezing on an iron block pre-cooled in liquid nitrogen. Transverse or radial sections (10µm) of the eye were cut in a cryostat (Leitz Cryostat 1720,Wetzlar, Germany), air-dried and stored at 80°C until use.
Digoxigenin (DIG)-labelled RNA probes were prepared from the five halibut opsins (J. V. Helvik, Ø. Drivenes, T. H. Næss, A. Fjose and H. C. Seo, unpublished results) following the manufacturers instructions (Boehringer Mannheim, Germany), and the probe concentration was determined using spot test. In situ hybridisation was carried out according to the method of Barthel and Raymond (Barthel and Raymond, 1993) with some modifications. Briefly, the tissues were rehydrated, treated with proteinase K (10mgml-1) for 5min at 37°C, followed by postfixation in 4% paraformaldehyde in PBS, treated with acetic anhydride and dehydrated. Approximately 100µl of hybridisation mixture containing 100ng of DIG-labelled RNA probes was applied directly to each air-dried section, and the sections were incubated in a humidity chamber without coverslips for 15h at 60°C. The probes were detected using an anti-DIG antibody coupled to alkaline phosphatase. Labelling was visualized with chromogen substrates. For each of the labels, sense probes were applied as a negative control to the tissue to confirm the specificity of labelling.
Photography and computer graphics
A Leica DMLB microscope with a digital camera (Hitachi kP-160 CCD, Japan) was used to take images of serial sections. Adobe Photoshop (version 5.0) was used to process the images (the functions auto levels and sharpen were used) before printing on a Hewlett Packard laser jet printer. Regions of interest were copied from the printed images onto transparent paper. A Nikon microscope (Microphot-FXA, Tokyo, Japan) with Nomarski optics was used to take colour slides (Kodak Ektachrome 160 T), which were then digitised using a Nikon 35mm film scanner (Nikon LS-2000, Tokyo, Japan).
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Results |
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Topography of cone cells
Expression of ultraviolet-sensitive opsin is restricted to photoreceptor cells in the ventral part of the retina (Fig.2A,B, Fig.3A). Ultraviolet-sensitive opsin-expressing cells are located both nasal and temporal to the choroid fissure and cover approximately one-third of the retinal hemisphere. Within this region, the ultraviolet-sensitive opsin-expressing cells are intermingled with photoreceptor cells that do not express ultraviolet-sensitive opsin. The blue-sensitive opsin-expressing photoreceptor cells are evenly distributed over the entire retina with no apparent regional distribution (Fig.2C, Fig.3B). The distribution of green-sensitive opsin-expressing cells also covers the entire retina without any major regional specificity (Fig.2D, Fig.3C). The density of green-sensitive opsin expressing cells is too high to see the single cells in the 10µm thick cryosection (Fig.1E, Fig.2D). The distribution pattern of red-sensitive opsin-expressing cells is similar to that for blue-sensitive opsin, both with respect to distribution over the entire retina and also to the number of photoreceptor cells (Fig.2E, Fig.3D). Analysis of the retina at the first feeding and end of the yolk-sac stage revealed no photoreceptor cells with expressing rod-type opsin (Fig.2F).
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Discussion |
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The spectral characteristics of photoreceptors examined to date have been closely related to their morphological characteristics (Bowmaker, 1995). Thus, in fish larvae whose retinas contain only a single type of cone, one might assume that all photoreceptors contain the same visual pigment. In the winter flounder (P. americanus), which is closely related to the halibut, it has been shown that this single visual pigment absorbs in the green region of the spectrum (Evans et al., 1993). This result indicates that fish larvae have visual systems based on one cone type and that their visual system is transformed into one based on three or four cone types during metamorphosis. This study shows for the first time that photoreceptor cells that look identical at the morphological level may express different visual pigments. Furthermore, different cone opsins are present even though the classic morphological characteristics are not yet fully developed. Microspectrophotometry characterises photoreceptors according to their absorbance spectrum. However, the disadvantage with this method is that one can easily miss rare classes of photoreceptors or their regional distribution. This is especially problematic in larval retina, in which the absence of morphological characteristics in the single cones complicates the collection of cones for study.
The early life history of many marine teleosts includes a prolonged larval period, such that they follow an indirect developmental programme with delayed appearance of rod cells (Evans and Fernald, 1990; Balon, 1999). On the other hand, fishes with a more direct type developmental program (e.g. goldfish and zebrafish) develop a retina in which rod-type opsin is expressed first, followed by cone-type opsins red-, green-, blue- and lastly ultraviolet-sensitive opsin, during a restricted period in the late embryonic or early larval phase (Raymond et al., 1995; Stenkamp et al., 1996). Since our results also show that all different cone type opsins are expressed in the larval retina of fish with any indirect developmental program, the main difference between fish with these two different developmental programs seems to be related to the timing of expression of rod-type opsins, rather than the pattern of expression of cone-type opsin.
The retina of juvenile halibut is organised into a square mosaic, which first appears during metamorphosis (Kvenseth et al., 1996). In situ hybridisation staining of the photoreceptor cells in larval retina clearly shows an expression pattern that indicates some type of pattern of mosaic formation at this early stage of retinal development, when the retina consists only of single cones organised into a hexagonal row array. The identical morphology of the single cone cells makes it impossible to determine whether a green-sensitive opsin-expressing cell is a precursor for a double cone, although it is clear that the majority of the photoreceptor cells express green-sensitive opsin (see Fig.1E,G).
In a typical square mosaic pattern, there are two double cones for each single cone. Since green- and red-sensitive opsins are expressed in equal numbers in double cones while blue-sensitive opsin is expressed in single cones, the ratio should be 2:2:1 for green:red:blue cones. Likewise, if all double cones express green-sensitive opsin, the ratio should be 4:1 (green:blue cones). In Atlantic halibut larvae, our results indicate a ratio of green:red:blue cones of 25:1:1 in the central retina. This result, a transformation from a green-sensitive opsin-dominated retina at the larval stage to a square mosaic expression pattern later in development, implies changes in opsin expression within individual cones. A similar result was obtained using microspectrophotometry data in winter flounder (Evans et al., 1993). Further studies such as calculations of the number of cells and their location in the retina during larval stages and metamorphosis are needed to elucidate the pattern of mosaic formation in a flatfish like halibut. Nonetheless it is very interesting that the early mosaic pattern of opsin expression that we report here is present in a retina that lacks rod photoreceptors. These data, obtained from a naturally developing retina, support recent data suggesting that rod cells may not have an inductive role in the development of the cone mosaic (Wan and Stenkamp, 2000).
Ultraviolet-sensitive opsins are expressed in halibut larvae at the time of first feeding, implying that marine fish larvae living in the pelagic environment may be able to detect ultraviolet light. The ventral distribution of the ultraviolet photoreceptor cells shows that it is the downwelling ultraviolet light that is important for the larvae. The implications for the visual system are not yet clear, but it has been suggested that detection of ultraviolet light could increase the contrast of a zooplankton prey against the water background (Loew et al., 1993; Browman et al., 1994). In the case of halibut, the location of ultraviolet opsin in the ventral retina suggests that halibut see the prey as dark objects against a bright ultraviolet background.
The present in situ hybridisation study shows that green cones dominate the retina of larval halibut. Photoreceptor cells expressing blue- and red-sensitive opsins are also distributed over the entire retina, but much less extensively. In the pelagic marine environment, the spectral surroundings are also dominated by green light, so the domination of green-sensitive opsin-expressing photoreceptor cells correlates with the spectral profile of the surroundings. Spectral absorption analysis (i.e. microspectrophotometry) of visual pigments is needed to confirm the significance of this finding. It is worth noting that the halibut retina probably has higher resolution in the green region of the spectrum as a result of the high numbers of green-sensitive opsin-expressing cells compared with red- and blue-sensitive opsin-expressing cells. The implication of these finding for visual function remains unclear at this time.
Further molecular identification of the visual pigments and in situ hybridisation studies on the photoreceptor cells in other marine species are needed to verify if marine pelagic fish larvae have a green-sensitive cone-dominated retina and ultraviolet-sensitive cones distributed only in the ventral part of the retina.
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Acknowledgments |
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References |
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