Genetic mechanisms involved in the evolution of the cephalopod camera eye revealed by transcriptomic and developmental studies

Abstract

Background: Coleoid cephalopods (squids and octopuses) have evolved a camera eye, the structure of which is very similar to that found in vertebrates and which is considered a classic example of convergent evolution. Other molluscs, however, possess mirror, pin-hole, or compound eyes, all of which differ from the camera eye in the degree of complexity of the eye structures and neurons participating in the visual circuit. Therefore, genes expressed in the cephalopod eye after divergence from the common molluscan ancestor could be involved in eye evolution through association with the acquisition of new structural components. To clarify the genetic mechanisms that contributed to the evolution of the cephalopod camera eye, we applied comprehensive transcriptomic analysis and conducted developmental validation of candidate genes involved in coleoid cephalopod eye evolution.

Results: We compared gene expression in the eyes of 6 molluscan (3 cephalopod and 3 non-cephalopod) species and selected 5,707 genes as cephalopod camera eye-specific candidate genes on the basis of homology searches against 3 molluscan species without camera eyes. First, we confirmed the expression of these 5,707 genes in the cephalopod camera eye formation processes by developmental array analysis. Second, using molecular evolutionary (dN/dS) analysis to detect positive selection in the cephalopod lineage, we identified 156 of these genes in which functions appeared to have changed after the divergence of cephalopods from the molluscan ancestor and which contributed to structural and functional diversification. Third, we selected 1,571 genes, expressed in the camera eyes of both cephalopods and vertebrates, which could have independently acquired a function related to eye development at the expression level. Finally, as experimental validation, we identified three functionally novel cephalopod camera eye genes related to optic lobe formation in cephalopods by in situ hybridization analysis of embryonic pygmy squid.

Conclusion: We identified 156 genes positively selected in the cephalopod lineage and 1,571 genes commonly found in the cephalopod and vertebrate camera eyes from the analysis of cephalopod camera eye specificity at the expression level. Experimental validation showed that the cephalopod camera eye-specific candidate genes include those expressed in the outer part of the optic lobes, which unique to coleoid cephalopods. The results of this study suggest that changes in gene expression and in the primary structure of proteins (through positive selection) from those in the common molluscan ancestor could have contributed, at least in part, to cephalopod camera eye acquisition.

Figure 1

Phylogenetic view of molluscan eye diversification. Camera eyes were independently acquired in the coleoid cephalopod (squids and octopuses) and vertebrate lineages.

Figure 2

Estimation and analysis procedures for cephalopod camera eye-specific candidate genes.

Figure 3

Differential gene expression profiles of camera eye genes at different developmental stages. The Venn diagram indicates numbers of cephalopod camera eye-specific genes for three cephalopod species (PS, the pygmy squid; S, squid; O, octopus). Each box represent the number of genes expressed at the three embryonic stages of the pygmy squid found in the developmental array. In total, 3,075 genes (2,320, 235 and 519 from PS, S, and O, respectively) were positive against pygmy squid embryonic RNA.

Figure 4

Localization of camera eye-specific genes in the pygmy squid embryos. (A-C) Whole-mount in situ hybridization with probes for the Ets-4 homolog. (D, E) Whole-mount in situ hybridization with probes for the Hla-b associated transcript homolog. (F) Whole-mount in situ hybridization with probes for the HMGb3 homolog. (A) Tissue surrounding the eye primodia (E) and tips of the arms (A) at stage 20 expressed the Ets-4 homolog. The Ets-4 transcripts were not detected in the mantle (M). (B) The Ets-4 transcript localized in the external part of the optic lobes (OL) and the central part of brain at stage 22. The yolk sac (Y) was removed using forceps. (C) Specific staining was localized in the optic lobes and central part of the brain at stage 25. The Ets-4 homolog was also expressed in the funnel organ (F). (D) A horizontal cryosection at the dotted line in (C). The Ets-4 transcripts appeared to be localozed in the part of the brain (the middle esophageal mass). Up side showes dorsal of the body. (E) A horizontal cryosection at the solid line in (C). The Ets-4 transcripts apper to be restricted in glanular cell layer (GL) in the optic lobes. (F) The HMGb3 transcripts were restricted to the internal part of the optic lobes at stage 25. Non-specific staining was found in the shell sac (S), as shown in the control experiment (Figure 5c). (G) The specific staining of the Hla-b-associated transcript was found in the head region, including the optic lobes, at stage 22. (A), stage 20. (B), (G), stage 22. (C), (D), (E), (F), stage 25. Bar = 100 μm in (A)-(C), (F), (G); 50 μm in (D), (E).

Figure 5

Localization of positively selected genes within the cephalopod camera eye. (A, B) Whole-mount in situ hybridization with probes for the centaurin gamma homolog. (C) Control experiment using whole-mount in situ hybridization with sense probes for the centaurin gamma homolog. (A) The centaurin gamma transcripts were detected in the optic lobes (OL), but not in the mantle (M) or eyes (E) at stage 22. (B) Specific staining was found in the head region, including the optic lobes (OL), at stage 25. Non-specific staining was detected in the shell sac (S), as shown in (C). (C) Non-specific staining was detected in the shell sac, but not in the head region. (D) A horizontal cryosection at the solid line in (B). The Centaurin gamma transcripts appears to be restricted in the perikaryal part of the central brain and optic lobes (OL). (A), (C), stage 22. (B), stage 25. Bar = 100 μm in (A-C); 50 μm in (D).