Eye development in the four-eyed fish Anableps anableps: cranial and retinal adaptations to simultaneous aerial and aquatic vision

Abstract

The unique eyes of the four-eyed fish Anableps anableps have long intrigued biologists. Key features associated with the bulging eye of Anableps include the expanded frontal bone and the duplicated pupils and cornea. Furthermore, the Anableps retina expresses different photoreceptor genes in dorsal and ventral regions, potentially associated with distinct aerial and aquatic stimuli. To gain insight into the developmental basis of the Anableps unique eye, we examined neurocranium and eye ontogeny, as well as photoreceptor gene expression during larval stages. First, we described six larval stages during which duplication of eye structures occurs. Our osteological analysis of neurocranium ontogeny revealed another distinctive Anablepid feature: an ossified interorbital septum partially separating the orbital cavities. Furthermore, we identified the onset of differences in cell proliferation and cell layer density between dorsal and ventral regions of the retina. Finally, we show that differential photoreceptor gene expression in the retina initiates during development, suggesting that it is inherited and not environmentally determined. In sum, our results shed light on the ontogenetic steps leading to the highly derived Anableps eye.

Keywords: Anableps; eye duplication; four-eyed fish; novelty; opsin.

Figure 1.

The Anableps eye. (a) Anableps anableps adult female specimen. (b) Close-up view of the A. anableps eye; dorsal and ventral corneas and pupils are visible. (c) Schematic of the visual aerial and aquatic inputs (sagittal view of the eye). RPE, retinal pigmented epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; ON, optic nerve; DR, dorsal retina; DP, dorsal pupil; VR, ventral retina; VP, ventral pupil; Ch, Choroid; L, lens; Ir, iris; PS, pigmented strip; DC, dorsal cornea; VC, ventral cornea; Do, dorsal; Ve, ventral; Di, distal; Pr, proximal. Scale bars: 1 cm (a), 0.5 cm (b).

Figure 2.

Eye development during larval stages. Anableps anableps larvae at stage 1 (a,b), stage 2 (c,d), stage 3 (e,f), stage 4 (g,h), stage 5 (i,j) and stage 6 (k,l). Figures to the right are higher magnification images of ocular region. Scale bars: 5 mm (a,c,e,g,i,k), 500 µm (b,d,f,h,j,l).

Figure 3.

Neurocranium ontogeny. Anableps anableps neurocranium at stage 3 (a,b), stage 4 (c,d), stage 5 (e,f) stage 6 (g,h) and in the adult (i,j). Panels show dorsal (left) and lateral (right) views. Boc, basioccipital; EpBar, epiphyseal bar; Epo, epiotic; EpoPr, epiotic process; EthPla, ethmoid plate; Exoc, exoccipital; Fro, frontal; IntSp, interorbital septum; LamOrb, lamina orbitonasalis; LEt, lateral ethmoid; MFl, membranous flap; OtCap, otic capsule; Par, parietal; Pro, prootic; Pto, pterotic; Psph, parasphenoid; Soc, supraoccipital; Sph, sphenotic; TM, taenia marginalis; TrCom, trabecula communis; the frontal and the interorbital septum are denoted in bold. Scale bars: 1 mm.

Figure 4.

Differences in retinal cell thickness and proliferation. Haematoxylin and eosin stained A. anableps eye at stage 6 (a), panels show dorsal (b) and ventral (c) retina. Measurements of thickness of the dorsal and ventral retinal layers ONL (d), INL (e) and IPL (f) using ImageJ software (n = 5, mean and standard deviation were calculated for a total of 27 eye sections). (g) Assessment of area (μm²) of PH3-positive cells using ImageJ software (n = 2; biological replicates for each larval stage of development). DR, dorsal retina; VR, ventral retina; L, lens; Ir, iris; PS, pigmented strip; DC, dorsal cornea; VC, ventral cornea; RPE, retinal pigmented epithelium; IS, inner segments; OS, outer segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 200 µm.

Figure 5.

Asymmetric opsin gene expression in the larval retina. (a) Analysis of the pattern of expression of five visual opsins in ventral retina, relative to the dorsal retina, as determined by qPCR. The relative expression values were determined using the 2^−ΔΔCT method. Bars represent mean in log₂ scale ±standard deviation of biological and technical replicates (n = 9; three biological and three technical replicates). Student's t-test was used to assess statistical significance between samples (**p < 0.01 and ***p < 0.001). (b–k) In situ hybridizations of visual opsins in eye sections of larvae of A. anableps: rh1 (b,c), rh2-1 (d,e), rh2-2 (f,g), sws2b (h,i) and lws (j,k). Left column shows expression pattern in portions of the dorsal retina, as well right column in the ventral retina. Scale bars: 0.05 mm. (l–o) Immunofluorescence assay showing opsin expression on the dorsal and ventral retina in A. anableps at stage 6. (l,m) Immunostaining showed that rhodopsin is expressed equally in the dorsal and ventral retina. (n) Opsin L/M monoclonal is only expressed in the dorsal retina. (o) Opsin L/M monoclonal expression is not detected on the ventral retina. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Cryosections are 20 µm thick. Scale bars: 50 µm (b–k) and 200 µm (l–o).