What genes tell us about pinhole- and camera-eye evolution in cephalopods

By Nolan Lassiter (Pitzer College) and Ryan Madden (Pitzer College) [Edited by Lars Schmitz, as part of BIOL 167 “Sensory Evolution”, an upper division class at the W.M. Keck Science Department. Written for educational purposes only].

Imagine how difficult it would be to climb the evolutionary ladder without being able to see! Eyes are often considered to have first begun to evolve from organisms with single photosensitive cells during the “Cambrian explosion” of life. In studying the rise and evolution of eyes, we often see a strong evolutionary selection for complex eyes with stronger acuity and clarity, especially among predators. This is because better eyes presumably allow the predator a greater chance to catch prey and may result in better fitness. Thus, it is always curious to see when a specific type of predator in a large predatory clade has seemingly weaker and morphologically different eyes than the others.

Figure 1. The Nautilus has a remarkable pinhole eye. Seawater can enter the eye chamber because there is no cornea or lens (!). [from http://species.wikimedia.org/wiki/File:Nautilus_pompilius_%28head%29.jpg ]

Figure 1. The Nautilus has a remarkable pinhole eye. Seawater can enter the eye chamber because there is no cornea or lens (!). [from http://species.wikimedia.org/wiki/File:Nautilus_pompilius_%28head%29.jpg ]

Figure 2. Schematic cross section of a pinhole eye. The aperture must remain small in order to generate an image of reasonable quality. [from http://upload.wikimedia.org/wikipedia/commons/thumb/a/a3/Pinhole_eye.svg/500px-Pinhole_eye.svg.png ]

Figure 2. Schematic cross section of a pinhole eye. The aperture must remain small in order to generate an image of reasonable quality. [from http://upload.wikimedia.org/wikipedia/commons/thumb/a/a3/Pinhole_eye.svg/500px-Pinhole_eye.svg.png ]

The Nautilus is the only cephalopod that does not have a lens-bearing camera-type eye (Figure 1). It is, however, still a predator that forages and feeds on smaller organisms in the sand/dirt in the bottom of the ocean. It utilizes a pinhole eye (Figure 2) that doesn’t protect the retina from the water of its environment and lacks a refracting apparatus, and as a result is not considered to have strong vision. While it is able to differentiate basic objects, the Nautilus does not have as strong of eyesight as its cousin’s, the squids, which have lens-bearing camera-like eyes (Figure 3). How did the eyes of cephalopods evolve?

Figure 3. Cephalopod eye anatomy (Bathyteuthis). Cephalopds have eyes that are convergent to vertebrate eyes --- both have camera-type eyes with a single lens. [from http://en.wikipedia.org/wiki/File:Eye_squid.jpg ]

Figure 3. Cephalopod eye anatomy (Bathyteuthis). Cephalopds have eyes that are convergent to vertebrate eyes — both have camera-type eyes with a single lens. [from http://en.wikipedia.org/wiki/File:Eye_squid.jpg ]

To explore the genetic control of development of pinhole and camera-type eye in cephalopods, Sousounis et al. (2013) utilized next-generation RNA sequencing, targeting Nautilus and the Pygmy squid. RNA sequencing has revolutionized the exploration of gene analysis and its application in the study of evolution, by allowing researchers to examine phylogenetic relationships at a molecular level. Previous research had demonstrated that there are several common genes related to lens and photoreceptor development found in both Drosophila and human genomes (Halder et al. 1995) that potentially could also be similar to those found in molluscs. Essentially, by identifying genes responsible for eye development one can reconstruct the complex evolutionary path to vertebrate and invertebrate eyes.

Sousounis et al. examined the RNA transcriptomes that were “marked” in developing Nautilus and Pygmy squid eyes. Then the contigs, or overlaps between sliced RNA fragments of various length, were compared to examine what genes are responsible for developing the Nautilus pinhole-type eye as opposed to a camera-type eye in squid. Basically, the researchers were able to enrich the RNA and examine exactly what sections of the genome specifically guide the sequencing and assembly of the eye. This examination also allowed for comparison of the contigs to that of Drosophila and human eyes. There were many interesting findings in the study that led to a more directed hypothesis and remained in line with previous discoveries regarding the differences in vertebrate and invertebrate photoreceptors (Fernald, 2006).

Genes involved in nucleic-acid binding proteins were overexpressed in squid and not Nautilus. Genes involved in metabolic and catalytic function were overexpressed in Nautilus early eye development (Figure 4). Sousounis et al. think that this is a consequence of the faster and more complex morphogenesis of the squid eye.

Figure 4. This figure displays the genetic components that are over-expressed in Nautilus eyes compared to that of the Pygmy Squid. [Figure 2 of Sousounis et al. 2013]

Figure 4. This figure displays the genetic components that are over-expressed in Nautilus eyes compared to that of the Pygmy Squid. [Figure 2 of Sousounis et al. 2013]

Many genes that are used in assembling the cephalopod eyes are similar to human homologues, whereas genes that are part of photoreceptor assembly show a mix of similarities to both humans and flies. Interestingly, the Sousounis team observed a lot of crossover in the genes involved in eye development between all four species, suggesting that eye morphology is largely conserved in beginning stages of development, but then has a few very important genes that help in differentiation and ultimately give rise to the diversity of eye types we have today.

The Sousounis team may also have identified a gene that is centrally important for developing the lens (Figure 5 shows their general approach to this problem). Pygmy squid have CAP1 gene while the Nautilus expresses a slightly different version, called capt. Capt has been found to be involved in morphogenesis in Drosophila, an organism with a compound eye (but not with a single, big lens like in vertebrates and squid). One hypothesis is that the presence and absence of capt and CAP1 might help determining if the eye will have a lens or not. Another gene that may potentially be involved in lens formation is NF1/Nf1, which shows similarities to both human and fly genes.

Figure 5. Nautilus and Pygmy Squid transcriptomes were compared with human and Drosophila proteomes in order to find candidate genes for lens development. Contigs of Nautilus and Pygmy Squid that had a homologue in either human or fly were further examined. [Figure 4 of Sousounis et al. 2013]

Figure 5. Nautilus and Pygmy Squid transcriptomes were compared with human and Drosophila proteomes in order to find candidate genes for lens development. Contigs of Nautilus and Pygmy Squid that had a homologue in either human or fly were further examined. [Figure 4 of Sousounis et al. 2013]

For future investigations, it would be very interesting to utilize either gene add-in or gene knockout methodology to further explore gene functions in the morphogenesis of eyes. For example, one could inhibit or alter the function of the NF1/Nf1 gene, thought to be involved in lens development. Through this analysis the development and exact role of the NF1/Nf1 gene could be further understood. For more insight into early lens development the same approach could be used but instead inhibit the CAP1 gene. Sousounis et al. provide an important piece to the puzzle of eye evolution, but many more pieces remain to be discovered.

References

Fernald, R. D. 2006. Casting a genetic light on the evolution of eyes. Science 313: 1914-1918. (DOI:10.1126/science.1127889).

Halder, G., Callaerts, P., Gehring, W. J. 1995. Induction of ectopic eyes by targeted expression of the eyeless gene in DrosophilaScience 267: 1788-1792. (DOI: 10.1126/science.7892602).

Halder, G., Callaerts, P., Gehring, W. J. 1995. New perspectives on eye evolution. Current Opinion in Genetics & Development 5: 602-609. (DOI: 10.1016/0959-437X(95)80029-8).

Sousounis, K., Ogura, A., Tsonis, P.A. 2013. Transcriptome analysis of Nautilus and Pygmy squid developing eye provides insights in lens and eye evolution. PLoS ONE 8: E78054. (DOI: DOI: 10.1371/journal.pone.0078054).

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