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Genome Res. 14:1555-1561, 2004 ©2004 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/04 $5.00 Letter Comparative Analysis of Gene Expression for Convergent Evolution of Camera Eye Between Octopus and HumanCenter for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, 411-8540, Japan
Although the camera eye of the octopus is very similar to that of humans, phylogenetic and embryological analyses have suggested that their camera eyes have been acquired independently. It has been known as a typical example of convergent evolution. To study the molecular basis of convergent evolution of camera eyes, we conducted a comparative analysis of gene expression in octopus and human camera eyes. We sequenced 16,432 ESTs of the octopus eye, leading to 1052 nonredundant genes that have matches in the protein database. Comparing these 1052 genes with 13,303 already-known ESTs of the human eye, 729 (69.3%) genes were commonly expressed between the human and octopus eyes. On the contrary, when we compared octopus eye ESTs with human connective tissue ESTs, the expression similarity was quite low. To trace the evolutionary changes that are potentially responsible for camera eye formation, we also compared octopus-eye ESTs with the completed genome sequences of other organisms. We found that 1019 out of the 1052 genes had already existed at the common ancestor of bilateria, and 875 genes were conserved between humans and octopuses. It suggests that a larger number of conserved genes and their similar gene expression may be responsible for the convergent evolution of the camera eye.
The evolution of the eye is one of the most complicated and interesting stories for molecular biologists and molecular evolutionists. Much is known about the photoreceptive organs of various animals (Salvini-Plawn and Mayr 1977
This view has been changed, however, by Gehring and Ikeo (1999  ), who maintain that the expression of the common master regulator Pax6 in both types of eyes indicates the divergence of these two types of eyes from a single prototype eye present in the common ancestor of cephalopods and vertebrates. It has previously been reported that Pax6, a "master control" gene for the development of the eye, is highly conserved across species. Within molluscs, it has been shown that the scallop, ear shell, and squid all express Pax6 (Tomarev et al. 1997). Pax6 expression has not yet been observed in the octopus. However, the expression of Pax6 in the camera eye of the squid, a member of the same phylum, supports the prediction that Pax6 controls the development of the octopus eye. However, there is no clear explanation of how the elaborate camera eyes of humans and octopuses evolved from the prototype eye. In other words, there is a gap between the evolution of genes expressed in the camera eye and the evolution of morphological structures of the camera eye. Studying gene expression can lead us to speculate on the biological and functional mechanisms. It is of particular interest to examine the convergent evolution of human and octopus camera eyes from the viewpoint of gene expression, in order to understand the diversification of these organs. Here, we address the question of what kind of gene expressions are maintained in their camera eyes during evolution. Thus, we focus on the gene expression profiles to explain the evolutionary process of camera eyes. For this purpose, we use two approaches. First, we conduct a comparative analysis of gene expression between human and octopus eyes to examine the similarity of gene expression. Second, we estimate the ancestral gene set of the camera eye to determine the number of genes that have been conserved since the divergence of these two lineages from the common ancestor. We then discuss the evolutionary mechanism of convergent evolution of the camera eye from the viewpoint of gene expression profiles.
Gene Expression Profile of the Octopus Eye We made a gene-expression profile based on the occurrence frequency of the mRNAs in the octopus eye. The expressed sequence tag (EST) assembly system was used to obtain 2824 nonredundant sequences from a total of 16,432 clones. To investigate the possible functions of these octopus-eye ESTs, we conducted a similarity search for identifying homologous sequences in the nonredundant protein database. Using the 2824 sequences as a query, we identified homologs for 1052 of the ESTs (Supplemental Table). This set of 1052 genes was used for the analyses described below, to determine the relationships of the genes expressed in the octopus eye to those that are involved in the developmental process and biological function of the eyes of other species (Table 1). The sequences of octopus-eye ESTs are available online at http://www.cib.nig.ac.jp/dda/database/octopus.htm.
Commonly Expressed Genes in Human and Octopus Camera Eyes Using the 1052 nonredundant gene sequences, a comparative analysis of gene expression in human and octopus camera eyes was performed to estimate the number of genes that are commonly expressed in these two lineages. A similarity search was conducted using the set of genes expressed in the octopus eye and the gene-expression data for the human eye obtained from the three databases of BodyMap, the Mammalian Gene Collection (MGC), and the National Eye Institute NEIBank. First, we created a single set of genes from the three distinct databases of gene expression for the human eye (see Methods section). Next, we obtained full-length gene sequences by comparing the gene-expression database with the human gene data from Ensembl (at the European Bioinformatics Institute) and UniGene (at the National Center for Biotechnology Information). We confirmed that 13,303 human-eye ESTs have matches in protein databases. Using the 1052 nonredundant sequences from the octopus eye as query sequences, we performed a BLAST search against the 13,303 human genes. As a result, we identified a total of 729 genes that were commonly expressed in both human and octopus eyes (Fig. 3). It follows that 69.3% of the 1052 nonredundant sequences from the octopus eye were commonly expressed in the human eye. To test whether these genes are significantly different when the same sequences from the octopus eye are compared with the genes expressed in human tissues other than eyes, we also performed a homology search between octopus-eye ESTs and human-connective-tissue ESTs. To be fair, we used 2430 human-connective-tissue ESTs as well as 3809 human-eye ESTs from the same database, BodyMap. We then found that the number of genes commonly expressed between the octopus eye and the human connective tissues was only 44, whereas that of genes between the octopus and human eyes was 162 (Fig. 3). Thus, the former was about four times less than the latter.
Moreover, to investigate the functional characteristics of the commonly expressed genes in human and octopus eyes, we conducted functional annotation of each gene (Table 2). A comprehensive survey of the literature showed that some of the genes have been previously reported to have functions that are related mainly to the eye. Besides, the homologous genes to six3, lhx2, retinal arrestin, retinal dehydrogenase,  -catenin, neuron-specific enolase, and human nuclear-transport receptor karyopherin/importin- were found to be expressed in the octopus eye. These genes are known to be important for the formation and function of the vertebrate camera eye. For example, Six3 is necessary for the patterning of anterior neuroectoderm including the retina (Carl et al. 2002), and Lhx2, which is regulated by Pax6, is also necessary for normal development of the eye, particularly the retina (Porter et al. 1997).
Ancestral Gene Set of the Camera Eye To discuss the molecular mechanism of convergent evolution, it is crucial to determine how many expressed genes in the camera eye of the octopus were present in the genome of the last shared ancestor of the two lineages, namely, the common ancestor of bilateria. The ancestral gene set can be estimated by comparing the genes that are expressed in the octopus eye with those that are present in the genomes of deuterostomes and out-group species of bilaterians. If octopus genes have homologs in the deuterostomes or out-group bilaterians, we can reasonably consider that such genes should have existed in the last common ancestor of protostomes and deuterostomes. To estimate the ancestral gene set for the camera eye, we conducted a similarity search by comparing the 1052 nonredundant sequences of octopus-eye ESTs with a total of 1,039,847 genes from the genomes of deuterostomes and out-group bilaterian species. As a result, we identified 1019 out of the 1052 sequences that can be considered as the putative ancestral gene set for the camera eye at the time of the divergence of human and octopus lineages (Fig. 4; Supplemental Table).
Conservation of Camera Eye-Related Genes in Extant Animals Because the genomes of modern bilaterians have diverged from their ancestral genome, the molecular changes that led to the evolution of the camera eye in humans and octopuses might be discernable in their genomes. It is possible to speculate on the evolutionary processes that led to the development of the camera eye by estimating the genetic changes that occurred in the ancestral gene set. The availability of partial or complete genome sequences for several species has made it possible to estimate whether or not a particular gene has been conserved during its evolution. In this study, we use the term "evolutionary conservation" in the meaning that the genes had existed in the common ancestor of the organisms compared. Thus, we estimated the number of genes in the putative ancestral gene set for the camera eye that have been evolutionarily conserved among the nematode, fly, and human lineages. First, using the 1019 genes in our proposed ancestral gene set for the camera eye, we conducted a similarity search to infer homology against all the genes in the complete genomes of several species. Then, we estimated the number of genes in the ancestral gene set that had been evolutionarily conserved in the genome. Among the protostomes, we found that 714, 769, and 777 of the 1019 genes in the ancestral gene set were evolutionarily conserved in the genomes of nematodes, mosquitos, and flies, respectively (Fig. 4). In the case of deuterostomes, 834 of the 1019 genes were evolutionarily conserved in the tunicate genome, and 867, 865, and 879 genes were evolutionarily conserved in the vertebrate genomes of Fugu, mouse, and human (Fig. 4). Therefore, it is clear that genes from the proposed ancestral gene set for the camera eye have been evolutionarily conserved for the vertebrate genome in a higher proportion than for the insect or nematode genomes.
The Loss of Genes Related to the Camera Eye
We further investigated the loss of genes from the putative ancestral gene set for the camera eye through the following analysis. Using the patterns of gene conservation described in the previous section, each of the gene-loss events was assigned to a particular branch of the phylogenetic tree. For example, genes that are conserved between humans and flies but not between humans and nematodes were considered to have been lost in nematodes after the divergence of nematode and human lineages. Applying this principle to all of the genes in the proposed ancestral gene set for the camera eye, we assigned all of the potential gene-loss events to the phylogeny. The results showed that 305, 250, and 242 of the 1019 genes in the ancestral gene set (29.9%, 24.5%, and 23.7%) were lost in nematodes, mosquitoes, and flies, respectively. In contrast, only 142, 144, and 130 of the 1019 genes (12.8%–14.1%) were lost in vertebrates (Fugu, mouse, and human; Fig. 4; Table 3).
The Function of Camera Eye-Specific Genes
Similarity in Gene Expression of Human and Octopus Eyes In spite of the evolutionary divergence between octopuses and humans, 69.3% of the genes examined (729 of the 1052 genes) were commonly expressed in the camera eyes of human and octopus. Moreover, comparison of octopus-eye ESTs with genes in the human connective tissue indicates that the similarity of gene expression between human and octopus eyes should be remarkable. Note that the increase of gene expression similarities between human and octopus eyes from 15% (162/1052) to 69.3% (729/1052) is caused by the increase of the EST data set of human eye from only 3809 ESTs in the database of BodyMap to 13,303 human-eye ESTs in the combined database of NEIbank, MGC, and BodyMap. This observation suggests that many more similarities of gene expression between human and octopus eyes will be observed when the EST data increase further. Although these 729 genes might contain housekeeping genes because the 44 genes were also found in the ESTs of human connective tissues in BodyMap, we found that many more genes (118 genes) were specific to the camera eye in this case. Therefore, we suggest that these 729 genes contain genes necessary for the developmental process and biological function of the camera eye. From the viewpoint of key genes in eye development, Pax6 has not been found in the octopus. However, a six3 homolog was present in the set of octopus-eye ESTs that were identified in this study. The six3 gene is involved in a downstream part of the developmental pathway of eye formation controlled by Pax6. Therefore, it is likely that the Pax6-pathway for eye formation is conserved in the octopus. These observations imply that the gene expression patterns in both the eyes of humans and octopuses are remarkably similar. In other words, the common ancestor of octopus and human had not only the common master regulator, Pax6, but also the ancestral gene set for the camera eyes. For several reasons, it is unlikely that all of the genes expressed in the octopus eye were identified in this analysis. It is difficult to detect genes that are expressed at a low level. Moreover, the octopus-eye ESTs were generated randomly from the cDNA libraries, which could have been incomplete as a result of mRNA degradation. Furthermore, mRNAs can only be obtained from cells at a particular developmental stage and time. Therefore, it is obvious that we cannot describe all of the genes related to the developmental process or biological function of the camera eye using a sample from an adult octopus. However, even though the genes used in the present study are only a subset of all the expressed genes, it is quite possible that the genes having important roles in the developmental process and biological function are identified through comparative studies of octopus eye ESTs.
Had the Origin of the Gene Set for the Camera Eye Been Formed in the Common Ancestor of Bilateria? Moreover, the numbers of conserved genes in insects and nematodes were less than those in vertebrates; nevertheless, insects and nematodes are more closely related to octopuses than are vertebrates. This indicates that insects and nematodes have lost many more genes in their lineages than humans have. On the other hand, the number of conserved genes in tunicates is larger than those in insects or worms, even though they also do not possess the camera eye structure. One of the possible reasons is that insects or nematodes have lost the genes possibly unimportant for their body plans, because these organisms are known to tend to lose the genes unless they are important (Fig. 3). As we estimated 1019 genes for ancestral genes for the camera eye, more than 760 genes were conserved in flies or worms. This conservation in flies or worms suggests that not all of these genes were specific to the camera eye. In the case of conserved genes in flies or worms, it is possible that these genes derived from the common ancestral gene set can be used in other organs and cells such as the photo-sensory system. To test whether ancestral genes for the camera eye include duplicated genes after octopus speciation and to evaluate the influence of such duplicated genes, if any, on the evolutionary process, we first estimated the number of lineage-specific duplications in octopus lineages. Then, we subtracted the number of the possible duplicated genes from the total number of the ancestral genes for the camera eye. As a result, we observed that the number of ancestral genes, the conserved genes in humans, the conserved genes in flies, and the conserved genes in worms decreased from 1019, 879, 777, and 714 to 943, 806, 708, and 660, respectively, indicating only a small number of decreased genes. Therefore, the duplicated genes might not affect our discussion much.
In this study, we assumed the phylogeny in which flies are closer to worms than vertebrates. However, there is an alternative hypothesis in which flies are closer to vertebrates rather than worms (Blair et al. 2002 Furthermore, it is also possible that a bias in the total number of genes in the database used in the present study would affect the estimated number of the ancestral gene set for the camera eye, because humans and mice have many more genes in the database than flies or worms. However, this possibility can be reasonably ruled out because we used all the genes deduced in the complete genome for the species examined in the present study.
The Evolutionary Role of Gene Loss
Molecular Mechanism of Convergent Evolution of the Camera Eye Our results indicate that most of the genes, including several gene pathways necessary for the evolution of the camera eye, might be shared between human and octopus lineages. Therefore, there is strong evidence that the evolutionary mechanisms for the camera eyes of humans and octopuses are subjected to similar gene expression profiles of the commonly conserved gene set, although the developmental processes of the human and octopus eyes are a bit different. We believe that these gene expression similarities could be the bridge of understanding from the genetic system of eye evolution to the developmental process of the camera eyes of humans and octopuses. In future studies, the functional validation of evolutionary events responsible for camera eye formation in both lineages will help us to understand how the elaborate camera eye evolved from the primitive eye that must have had the ancestral gene set for the camera eye.
Animals Octopus vulgaris, a Japanese madako captured from the Suruga Bay population, which belongs to the phylum Mollusca and class Cephalopoda, was used for the following studies. It was commercially obtained from the Numazu fish market.
Construction of cDNA Library for the Octopus Eye
Making Nonredundant Sequences and Assembling Sequences
Homology Search
Functional Annotation
Databases for Gene Expression in the Human Eye
We express our gratitude to Yumi Takezawa, Hiroaki Yamamoto, Chi-Chiu Wang, and Jung-Shan Hwang for their help, suggestions, and discussion. This study was supported, in part, by a grant to T.G. from MEXT (Ministry of Education, Sports, Culture, Science and Technology) of Japan. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.2268104.
1 Corresponding author. [Supplemental material is available online at www.genome.org.]
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http://www.cib.nig.ac.jp/dda/database/octopus.htm; sequences of octopus-eye ESTs.
Received December 14, 2003; accepted in revised format May 12, 2004.  CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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, which is expressed in the rod outer segment of the retina, was found in humans and octopuses, but not in the invertebrates examined except octopus (Stohr et al. 1999
1800 genes that were expressed in human eyes (retina, cornea, etc.). MGC, the Mammalian Gene Collection (Strausberg et al. 1999
