Developmental dynamics of cone photoreceptors in the eel

Abstract

Background: Many fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins?

Results: The Rh2 and Sws2 opsin sequences from the European Eel were isolated, sequenced and expressed in vitro for an accurate measurement of their lambdamax values. In situ hybridisation revealed that glass eels express only rh2 opsin in their cone photoreceptors, while larger yellow eels continue to express rh2 opsin in the majority of their cones, but also have <5% of cones which express sws2 opsin. Silver eels showed the same expression pattern as the larger yellow eels. This observation was confirmed by qPCR (quantitative polymerase chain reaction).

Conclusions: Larger yellow and silver European eels express two different cone opsins, rh2 and sws2. This work demonstrates that only the Rh2 cone opsin is present in younger fish (smaller yellow and glass), the sws2 opsin being expressed additionally only by older fish and only in <5% of cone cells.

Figure 1

Phylogenetic tree for visual opsin gene sequences. The tree was generated by the neighbour-joining method [35], using amino acid sequences aligned by ClustalW [36]. The degree of support for internal branching was assessed by bootstrapping with 1000 replicates using the MEGA2 computer package [37]. The calibration bar is equivalent to 0.1 substitutions per site. GenBank accession numbers for the sequences (from top to bottom) are Eel rh1 "freshwater", L78007; Eel rh1 "deep sea", L78008; Goldfish rh1, L11863; Killifish rh1, AY296738; Salmon rh2, AF201470; Eel rh2, FJ515778; Goldfish rh2/1, L11866, Goldfish rh2/2, L11865; Ayu rh2/2, AB098704; Ayu rh2/1, AB098703; Salmon rh2, AY214132; Metriaclima zebra rh2B, DQ0088652; Killifish rh2, AY296739; Metriaclima zebra rh2Aβ, DQ088650; Metriaclima zebra rh2Aa, DQ088651; Eel sws2, FJ515779; Salmon sws2, AY214134; Metriaclima zebra sws2B, AF247118; Killifish sws2B, AY296736; Killifish sws2A, AY296737; Metriaclima zebra sws2A, AF247114; Goldfish sws2, L11864; Goldfish sws1, D85863; Killifish sws1, AY296735; Salmon sws1, AY214133; Goldfish lws, L11867; Killifish lws, AY296740; Salmon lws, AY214131; Drosophila rh4, NM_057353.

Figure 2

In vitro expression of eel cone opsins. A. Expression of the eel sws2 opsin protein gave a calculated λ_max of 445.6 ± 0.1 nm, in agreement with the previously measured λ_max of 435 nm allowing for the differences in chromophores. B. Expression of the eel rh2 opsin protein gave a calculated λ_max of 524.5 ± 0.0 nm, which is almost identical with the measured λ_max of 525 nm. The close agreement between the MSP measurements and those of the in vitro expressed protein confirms that the isolated opsin sequences are those expressed by the cones.

Figure 3

Distribution of opsin-expressing cone cells in eel retina. Transverse sections of eel retina from different developmental stages showing localisation of cone opsin expression as detected by in situ hybridisation of the 5' end of rh2 or sws2 opsin mRNA. Cone cell bodies in the eel are arranged in a single layer between the inner nuclear layer (INL) and the rod outer segments (ROS). Glass eel: (A) the layer of cone cells expresses rh2 opsin (horizontal arrowhead), (B) sws2 opsin expression was not detected in the glass eel retina. Yellow eel: (C) the layer of cone cells expressing rh2 opsin (horizontal arrowhead), (D) occasional cone cells expressing sws2 opsin (vertical arrowheads). Silver eel: (E) monolayer of cone cells expressing rh2 opsin (horizontal arrowhead), (F) occasional cone cells express sws2 opsin (vertical arrowheads). Scale bar is 100μm, all panels are to same scale. RPE, retinal pigment epithelium; ROS, rod outer segments; ONL, outer nuclear layer; INL, inner nuclear layer.

Figure 4

Changes in expression levels of opsins at different life stages of the eel. A. The expression level of rh2 and sws2 cone opsin transcripts as copy numbers per 50 ng total RNA. The level of green opsin is approximately equivalent in the different life stages, but the amount of blue opsin doubles between the glass and silver stage. B. The expression of the two rod (rh1) opsin transcripts as copy numbers per 50 ng total RNA. The level of fwo transcript appears to increase slightly between the glass and later stages, but the amount of dso transcript increases markedly between the glass and silver life stages. Error bars show standard deviations which are necessarily large for the non-glass eels as each sample represents a point on a continuum.

Figure 5

Changes in the retina in different life stages of the eel. 2 μm thick plastic-embedded sections of eel retina from different life stages were stained with Richardson's Stain. It can be clearly seen that the thickness of the retina, and especially of the outer nuclear layer (rod cell nuclei), doubles between the glass and the silver stages of the life cycle. The size of the eye itself changes dramatically between the different stages, as indicated in the bottom panel, increasing ten-fold in diameter. Such changes must necessitate large scale remodelling of the retinal components. RPE, retinal pigment epithelium; ROS, rod outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer. Scale bar = 50 μm, all histology panels are to the same scale.