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Evolution of the Vertebrate Pax4/6 Class of Genes with Focus on Its Novel Member, the Pax10 Gene

The members of the paired box (Pax) family regulate key developmental pathways in many metazoans as tissue-specific transcription factors. Vertebrate genomes typically possess nine Pax genes (Pax1–9), which are derived from four proto-Pax genes in the vertebrate ancestor that were later expanded through the so-called two-round (2R) whole-genome duplication. A recent study proposed that pax6a genes of a subset of teleost fishes (namely, acanthopterygians) are remnants of a paralog generated in the 2R genome duplication, to be renamed pax6.3, and reported one more group of vertebrate Pax genes (Pax6.2), most closely related to the Pax4/6 class. We propose to designate this new member Pax10 instead and reconstruct the evolutionary history of the Pax4/6/10 class with solid phylogenetic evidence. Our synteny analysis showed that Pax4, -6, and -10 originated in the 2R genome duplications early in vertebrate evolution. The phylogenetic analyses of relationships between teleost pax6a and other Pax4, -6, and -10 genes, however, do not support the proposed hypothesis of an ancient origin of the acanthopterygian pax6a genes in the 2R genome duplication. Instead, we confirmed the traditional scenario that the acanthopterygian pax6a is derived from the more recent teleost-specific genome duplication. Notably, Pax6 is present in all vertebrates surveyed to date, whereas Pax4 and -10 were lost multiple times in independent vertebrate lineages, likely because of their restricted expression patterns: Among Pax6-positive domains, Pax10 has retained expression in the adult retina alone, which we documented through in situ hybridization and quantitative reverse transcription polymerase chain reaction experiments on zebrafish, Xenopus, and anole lizard.


Introduction
The vertebrate gene repertoire was shaped by two rounds (2R) of whole-genome duplications (WGD) early in vertebrate evolution (Ohno 1970;Lundin 1993;Holland et al. 1994). These events initially generated four paralogs in vertebrates corresponding to a single invertebrate ortholog, and subsequent processes such as neofunctionalization, subfunctionalization, or complete loss of function modified this initial four-fold genetic abundance of evolutionary raw material. Genes that play crucial roles in development tend to be highly conserved and are therefore present in the genomes of diverse vertebrates. In contrast, genes that are less crucial for development and survival experience less selective pressure in the form of balancing selection, and are hence permitted to be differentially lost in vertebrate lineages (Lynch et al. 2001). Examples are the Bmp16 gene, a sister gene of the highly conserved Bmp2 and -4 genes (Feiner et al. 2009), which has only been found in teleosts so far, and the Pdx2 gene, a duplicate of the pancreatic key regulator Pdx1, which is only retained in cartilaginous fish and coelacanths (Mulley and Holland 2010).
Members of the Pax gene family encode transcription factors that play crucial regulatory roles in metazoan development (reviewed in Wehr and Gruss 1996). All vertebrate Pax proteins identified to date are characterized by the possession of a paired domain (Breitling and Gerber 2000;Underhill 2012). They are divided into four classes, namely Pax1/9, Pax3/7, Pax2/5/8, and Pax4/6, based on the completeness of a homeodomain and the presence of an octapeptide motif ( fig. 1A; Wehr and Gruss 1996;Chi and Epstein 2002). The last common ancestor of bilaterians, the so-called "Urbilateria," already possessed proto-orthologs of these four Pax classes, plus an additional class, the PaxA/Pox neuro class, restricted to invertebrates (Matus et al. 2007). Preceding the radiation of vertebrates, each of the four classes was quadruplicated by the 2R-WGD (Wada et al. 1998;Holland et al. 1999;Ogasawara et al. 1999;Manousaki et al. 2011).
The Pax4/6 class of genes consists of Pax4 and -6 genes as well as a recently identified gene named Pax6.2 (Ravi et al. 2013). This novel gene has only been identified in a chondrichthyan (the elephant shark, Callorhinchus milii), several teleost fish, a reptile (the green anole, Anolis carolinensis), and an amphibian (the frog, Xenopus tropicalis; fig. 1B; Ravi et al. 2013). Pax4, the other close relative of Pax6 (Manousaki et al. 2011), also shows a mosaic pattern of phylogenetic distribution, confined to mammals (Pilz et al. 1993;Tamura et al. 1994) and teleost fish (Hoshiyama et al. 2007;Manousaki et al. 2011), whereas the Pax6 gene is identified in every vertebrate genome sequenced to date (fig. 1B). Ravi et al. (2013) also recently called the phylogeny of teleost fish pax6 genes into question. Acanthopterygii is a group of teleost fish, and among the typical laboratory animals with the sequenced genomes, it includes medaka, Fugu, and stickleback, but not zebrafish. They concluded that acanthopterygian pax6a and -6b genes did not originate in the so-called "third-round" WGD in the teleost lineage (teleost-specific genome duplication, TSGD; Amores et al. 1998;Wittbrodt et al. 1998;Meyer and Van de Peer 2005). Instead, they proposed that acanthopterygian pax6a genes originated in a more ancient event, namely the 2R-WGD at the base of vertebrate evolution ( fig. 2A; Ravi et al. 2013). Importantly, this would imply that the acanthopterygian pax6a genes are not orthologous to all other vertebrate Pax6 genes. To demarcate acanthopterygian pax6a genes and emphasize their ancient origin, Ravi et al. (2013) therefore proposed to rename them pax6.3.
The Pax6 gene is famous for its essential role as "master control gene" for eye development. Studies in the 1990s revealed the ability of an ectopically expressed mouse Pax6 gene to induce ectopic eyes in Drosophila (Halder et al. 1995). Apart from this inductive role in eye development, the vertebrate Pax6 gene is involved in the development of the central nervous system (CNS), including fore-and hindbrain, the neural tube, the pituitary, the nasal epithelium, and the endocrine part of the pancreas (Walther and Gruss 1991;St-Onge et al. 1997). In zebrafish, pax6b, but not pax6a ("pax6.3" in Ravi et al. 2013), is expressed in the endocrine pancreas (Delporte et al. 2008). The vertebrate paralog of Pax6, the Pax4 gene, is necessary for the differentiation of insulin-producing b-cells in the endocrine part of the mammalian pancreas (Sosa-Pineda No paired box has been identified in any of the Pax10 genes, and thus, mature Pax10 proteins presumably lack a paired domain. (B) Phylogeny of major vertebrate taxa with indicated patterns of presence and presumed absence of Pax4, -6, and -10 genes. The presence of these genes was investigated using exhaustive Blast searches in publicly available whole-genome sequences (see supplementary table S1, Supplementary Material online, for details). The chondrichthyan Pax10 gene was reported by Ravi et al. (2013). Inferred secondary gene losses are indicated with red and blue crosses and mapped onto the generally accepted jawed vertebrate phylogeny. Question marks indicate uncertainties about the absence of genes because of insufficient sequence information of the respective taxa. The phylogenetic position of turtles is based on molecular phylogenetic studies (Zardoya and Meyer 1998;Rest et al. 2003;Iwabe et al. 2005;Chiari et al. 2012;Crawford et al. 2012;Wang et al. 2013).
FIG. 2.-Phylogenetic relationships within the Pax4/6/10 class of genes. (A) Schematic presentations of two scenarios of the evolution of the Pax4/6/10 class of genes. Hypothesis 1, proposed by Ravi et al. (2013), assumes an ancient origin of one group of acanthopterygian pax6 genes, namely pax6.3. In addition, this hypothesis does not take the group of Pax4 genes into account. Hypothesis 2 is proposed based on our phylogenetic analysis, and it postulates the origin of both groups of teleost pax6 genes, namely pax6a and -6b, in the TSGD. The gene nomenclature in Hypothesis 1 is adopted from Ravi et al. (2013). (B) ML tree showing phylogenetic relationships among vertebrate genes belonging to the Pax4/6/10 class, with Pax3 and -7 genes as outgroup. Exact names of the genes are included only when they experienced additional lineage-specific duplications. Support values are shown for each node in order, bootstrap probabilities in the ML and Bayesian posterior probabilities. Only bootstrap probabilities above 50 are shown. The inference is based on 99 amino acid residues, assuming the JTT+À 4 model (shape parameter of the gamma distribution = 0.57). The scale bar on the upper left indicates 0.2 substitutions per site. et al. 1997), but not of the zebrafish pancreas (Djiotsa et al. 2012). This indicates evolutionarily conserved roles of Pax6 and lineage-specific differences in the roles of Pax4 during pancreas development among vertebrates. The Pax4 genes of mammals and teleost fishes are not implicated in the development of eyes (Rath et al. 2009;Manousaki et al. 2011). However, mammalian Pax4 genes are expressed in photoreceptors in the outer nuclear layer of the adult mammalian retina in a diurnal rhythm (Rath et al. 2009). The Pax6 gene is expressed in other neuronal layers of the mature retina, namely the inner nuclear layer, the ganglion cell layer, and in several species including a shark, zebrafish, chicken, and mouse, and also in the horizontal cell layer (Belecky-Adams et al. 1997;Macdonald and Wilson 1997;Wullimann and Rink 2001;Ferreiro-Galve et al. 2011). The teleost pax4 gene has an expression domain that has not been attributed to any other Pax gene, namely the stomodeum which corresponds to the developing lip (Manousaki et al. 2011).
Information on the developmental roles of the novel Pax6 relative, Pax6.2, is limited because it was found only very recently (Ravi et al. 2013) and also because it is absent from the genomes of traditional model species. This recent study revealed the expression of the elephant shark Pax6.2 gene in its adult eye, shared with Pax4 and -6 genes, and the adult kidney in which none of the other Pax6 relatives have been shown to be expressed (Ravi et al. 2013). Its zebrafish ortholog is expressed in the head region during early developmental stages, and becomes restricted to the inner nuclear layer of the retina during late developmental stages (Ravi et al. 2013).
In this study, we assessed the molecular phylogeny of the recently identified relative of the Pax6 gene (Pax6.2 in Ravi et al. 2013) and propose to call it Pax10 instead. By conducting rigorous molecular phylogenetic analyses and considering conserved synteny, we demonstrate that Pax4, -6, and -10 originated in the 2R-WGD, and that the two pax6 genes of teleost fishes (pax6a and pax6b) were duplicated in the TSGD. Our gene expression analyses in the zebrafish, a frog, and the green anole lizard revealed the Pax10 expression in the adult retina and brain and suggested that Pax10 presumably plays no role during early development. Our reanalysis provides a synthetic understanding of the evolution of the vertebrate Pax4/6/10 gene repertoire and the functional partitioning of these genes.

Materials and Methods
In Silico Identification of Novel Pax Genes Using the green anole Pax10 and human Pax4 nucleotide sequences as queries, we performed BLASTn searches in the NCBI dbEST (National Center for Biotechnology Information) as well as in the nr/nt database and in nucleotide genomic sequences of species included in the Ensembl genome browser. Similarly, local BLASTn searches were performed in downloaded genome-wide or transcriptomic sequence resources of three cyclostomes (inshore hagfish, sea lamprey, and Japanese lamprey), three chondrichthyans (elephant shark, little skate, and cloudy catshark), a basal actinopterygian (spotted gar), the African coelacanth, reptiles (American alligator, Burmese python, Chinese soft-shell turtle, painted turtle, and saltwater crocodile), and birds (budgerigar, collared flycatcher, mallard, medium ground finch, turkey, and zebra finch; see supplementary table S1, Supplementary Material online, for details). Candidate sequences with E value of less than 1e-05 in these BLASTn searches were subjected to preliminary phylogenetic tree inferences. Sequences placed inside the Pax4/6/10 class were selected, and their longest open reading frames (ORFs) were curated either manually or by using the gene prediction program AUGUSTUS (Stanke et al. 2004). All sequences identified in this in silico screen, except for saltwater crocodile Pax10 and Japanese lamprey Pax6B (supplementary tables S2 and S3, Supplementary Material online), are deposited in EMBL under accession numbers HF567444-HF567455.

Animals
Wild-type embryos and albino adults of the zebrafish (Danio rerio) and embryos (staging according to Nieuwkoop and Faber 1994) and adults of Xenopus laevis were obtained from captive breeding colonies. Eggs of the green anole lizard (A. carolinensis) were harvested from in-house captive breeding colonies and incubated at 28 C and approximately 70% humidity until they reached required stages determined after Sanger et al. (2008). Animals that were subjected to sectioning or whole-mount in situ hybridizations were stored in methanol after fixation in 4% paraformaldehyde or Serra's fixative, respectively. All experiments were conducted in accordance with the animal use protocols of the University of Konstanz.

Reverse Transcription Polymerase Chain Reaction
Total RNA of embryos of zebrafish (24 hpf), Xenopus (11 individuals for each of stages 22, 30, and 40), and the green anole (stage 8.5) were extracted and reverse transcribed into cDNA using SuperScript III (Invitrogen), following the instructions of the 3 0 -RACE System (Invitrogen). Oligonucleotide primers were designed based on Pax10 transcript sequences of zebrafish (ENSDART00000075395), X. tropicalis (ENSXETT00000065934), and the green anole (ENSACAT00000013868) to amplify full-length cDNAs of zebrafish pax10a, X. laevis Pax10a and -10b, and the green anole Pax10 genes. 3 0 -ends of these four fragments were isolated by applying the 3 0 -RACE System (Invitrogen). 5 0 -ends of the green anole Pax10 cDNA were obtained using the GeneRacer Kit (Invitrogen) and 5 0 -ends of the zebrafish pax10a and X. laevis Pax10a and -10b were obtained using the 5 0 -RACE System (Invitrogen). Full-length Pax10 cDNA sequences of zebrafish, X. laevis, and green anole were assembled from two fragments and are deposited in EMBL under accession numbers HF567440-HF567443.
In order to detect Pax6 expression in the eyes of adult zebrafish, Xenopus, and the green anole, cDNA fragments covering the respective 3 0 -ends were isolated using the 3 0 -RACE System (Invitrogen). Primers were designed based on Pax6 transcript sequences of zebrafish (Ensembl ID: ENSDART00000148420 for pax6a and ENSDART00000145946 for pax6b), X. laevis (NCBI: NM_001085944 for Pax6a and NM_001172195 for Pax6b), and the green anole (Ensembl ID: ENSACAT00000002377). Zebrafish pax4 probe synthesis employed the cDNA fragment previously reported (Manousaki et al. 2011).
In order to analyze differential expression of Pax6 and -10 genes between various organs of adult zebrafish, Xenopus and the green anole, and between embryonic stages of developing zebrafish, the animals were dissected. Total RNA was extracted using TRIzol (Invitrogen) and treated with DNase I. The integrity of the extracted RNA was monitored using the Bioanalyzer 2100 (Agilent). Gene-specific primers to amplify approximately 200-bp-long cDNA fragments of Pax6, -10, and GAPDH were designed (supplementary table S4, Supplementary Material online). It should be noted that primers for the amplification of X. laevis Pax6 and -10 were designed to capture both "a" and "b" paralogs. The specificity of each primer pair was determined in a preliminary pilot polymerase chain reaction, and the amplification of comparable amounts of GAPDH cDNA fragments was used as proxy for similar quantity of cDNAs between samples. Semiquantitative reverse transcription (RT)-PCR runs were conducted using the DreamTaq TM DNA Polymerase (Fermentas). A predenaturing step at 95 C for 3 min was followed by 35-50 cycles of three steps (95 C for 30 s, 58 C for 1 min, and 72 C for 1 min). The amount of PCR product was visualized using standard agarose gel electrophoresis (supplementary fig. S1, Supplementary Material online). The intensity of individual bands was quantified using GelQuant.NET software (http://bioche mlabsolutions.com, last accessed June 26, 2014). Expression levels of Pax6 and -10 genes were normalized to relative expression levels of GAPDH. The resulting values were further normalized to tissues with the highest expression level for the respective gene that was defined as 1. These values were shown in a heat map generated by CIMMiner (http://discover.nci.nih.gov/cimminer, last accessed June 26, 2014), which was also used for the clustering of genes based on expression patterns with an Euclidean distance algorithm.

In Situ Hybridization
The aforementioned 3 0 -and 5 0 -cDNA fragments of zebrafish, Xenopus, and green anole Pax10, 3 0 -fragments of zebrafish, Xenopus, and green anole Pax6, and 3 0 -fragment of zebrafish pax4 were used as templates for riboprobe synthesis. Wholemount in situ hybridizations for two Pax10 probes and a Pax6 probe as positive control were performed using embryonic zebrafish (Begemann et al. 2001), Xenopus (Gawantka et al. 1995), and green anole (Di-Poï N, personal communication). In situ hybridizations on paraffin-embedded sections for adult eyes of zebrafish, Xenopus, and green anole were performed using the aforementioned riboprobes as previously described (Kuraku et al. 2005).

Molecular Phylogenetic Analysis
An optimal multiple alignment of all retrieved cDNA sequences was constructed using MEGA5 (Tamura et al. 2011), in which the MUSCLE program (Edgar 2004) is implemented. Molecular phylogenetic trees were inferred using regions of selected vertebrate Pax amino acid sequences that were unambiguously aligned with no gaps (supplementary table S5, Supplementary Material online). Several sequence fragments of previously unidentified Pax4/6/10 genes (supplementary table S6, Supplementary Material online) were excluded from the phylogenetic analysis depicted in figure 2B, but their affiliation to individual Pax subtypes was verified in separate phylogenetic analyses (data not shown). The human, Cio. intestinalis, and fly genes belonging to the Pax3/7 gene class served as outgroup because of the lack and truncation of the homeodomains of Pax1/9 and Pax2/5/8 class of genes, respectively. MEGA5 was used to determine the optimal amino acid substitution model and to reconstruct maximum likelihood (ML). Bayesian tree inference was performed using MrBayes 3.1 (Huelsenbeck and Ronquist 2001), with which we ran two independent chains with 5,000,000 generations for each, sampled every 100 generations, and excluded 25% of the samples as burnin. Convergence of the two chains was diagnosed using the Tracer v1.5 software (http://tree.bio.ed.ac.uk/software/tracer/, last accessed June 26, 2014). The main data set consisted of a broad selection of metazoan genes belonging to the Pax4/6/10 class of genes ( fig. 2B). In addition, we performed a phylogenetic analysis focusing on vertebrate Pax6 genes in which amphioxus Pax6 genes served as outgroup (supplementary fig. S2, Supplementary Material online). To assess the statistical support for alternative hypotheses ( fig. 2A), per site log likelihoods of two constrained tree topologies and the ML tree were calculated using Tree-Puzzle (Schmidt et al. 2002) and statistically assessed using CONSEL (Shimodaira and Hasegawa 2001).

Identification of Conserved Synteny
Using the Ensembl database, we identified genes within a 1-Mb region flanking pax6 in the genome of the spotted gar. Orthologs of these genes in zebrafish, stickleback, and medaka were downloaded via the BioMart interface and plotted against the focal region of the spotted gar ( fig. 3A). The three gene families that possess members both in the vicinity of pax6a and -6b genes were further analyzed. ML trees were reconstructed as described above to infer the evolutionary origin of the teleost duplicates ( fig. 3B). The search for conserved intragenomic synteny (Kuraku and Meyer 2012) in the green anole focused on up to 10-Mb genomic regions flanking the Pax6 and -10 genes. Referring to the Ensembl "Gene Tree," the duplication patterns of neighboring gene families were investigated. We identified two additional chromosomal regions that harbor a similar gene array. A second search for paralogous relationships between these two identified chromosomal regions was conducted. The identified pairs, triplets, and quartets of paralogs located on the four chromosomes were plotted ( fig. 4).
In order to analyze the mode of the putative loss of Pax10 in the mammalian and avian lineages, we downloaded a list of Ensembl IDs of genes harbored in the 500-kb genomic regions flanking the green anole Pax10 gene, together with IDs of opossum, human, and chicken orthologs of those genes via the BioMart interface. Opossum, human, and chicken orthologs located on chromosomes 4, 19, and 27, respectively, were plotted along the orthologous region on anole chromosome 6 (fig. 5 and supplementary tables S7 and S8, Supplementary Material online).

Identification of Novel Pax4/6/10 Genes in Diverse Vertebrates Except for Mammals and Birds
We identified a group of protein-coding genes closely related to Pax4 and -6 in the Gene Tree view in release 54 of the Ensembl genome database (http://www.ensembl.org, last accessed June 26, 2014; released in spring 2009; Hubbard et al. 2009). In this tree at Ensembl, these uncharacterized genes possessed by zebrafish (ENSDARG00000053364) and stickleback (Gasterosteus aculeatus; ENSGACG00000007835) were placed basal to the vertebrate Pax6 group of genes. More recent Ensembl releases (e.g., release 66) suggest that this gene is also possessed by X. tropicalis (ENSXETG00000032534), the green anole lizard (A. carolinensis; ENSACAG00000013797), Atlantic cod (Gadus morhua; ENSGMOG00000007260), and Nile tilapia (Oreochromis niloticus; ENSONIG00000020082). Following the conventional nomenclature of the Pax gene family (Pax1-9) and our intensive phylogenetic analysis (see below), we designated this group of genes as Pax10. Our survey of publicly available genome-wide sequence resources (supplementary  table S1 Ravi et al. 2013). Moreover, in All. mississippiensis, Chr. picta bellii, and L. chalumnae, we identified orthologs of the Pax4 gene which was previously identified only in teleosts and mammals (see supplementary tables S1, S5, and S6, Supplementary Material online, for details). The Pax4 genes of the Chinese soft-shell turtle (Pelodiscus sinensis; ENSPSIG00000006191) and the spotted gar (Lepisosteus oculatus; ENSGACP00000026154_1 at Ensembl Pre) are available in the Ensembl genome database since release 68 and in Pre-Ensembl, respectively. So far, no Pax4 ortholog has been identified in transcriptomes and genomes of birds, lepidosaurs, amphibians, and chondrichthyans, whereas Pax10 is most likely absent from the genomes of mammals and birds ( fig. 1B). In addition to the previously described Japanese lamprey Pax6 gene (AB061220 in NCBI nucleotide), we identified another Pax6-like sequence in the genome assembly of this species (Mehta et al. 2013) that we designated Pax6B. The putative ORF of this sequence is disrupted by a stretch of N's and thus is partial. Therefore, this Japanese lamprey Pax6B gene was not included in the phylogenetic analysis of the entire Pax4/6/10 class of genes ( fig. 2), whereas it was included in the phylogenetic analysis of its subset, Pax6 (supplementary fig. S2, Supplementary Material online). We would like to alert our readers to potential misidentification of these genes, as the novel relatives of Pax6, Pax10 genes, are sometimes annotated as Pax6 or Pax6-like in Ensembl.

Domain Structure of the Deduced Pax10 Protein Sequences
The deduced amino acid sequences of the Pax10 genes, identified above in silico, contain a complete homeodomain preceded by a putative start codon, but lack the characteristic paired domain common to all other Pax proteins ( fig. 1A). To rule out the possibility that incomplete annotations of genome assemblies led to the nonidentification of the paired domain, we performed extensive searches with tBLASTn in nucleotide sequences of the selected genomic contigs containing Pax10 using peptide sequences of the paired domain as queries. However, this approach did not reveal any potential paired domain upstream of the identified Pax10 ORFs. By means of RT-PCR, we isolated full-length cDNAs of a single Pax10 member in the zebrafish (designated pax10a by Ravi et al. 2013 based on a comparison of syntenic relationships among teleosts) and A. carolinensis. We also isolated two full-length cDNAs of X. laevis (designated Pax10a and -10b)-evidence of transcription of these genes. In silico translation of these cDNA sequences confirmed the lack of a paired domain in the products of these Pax10 genes.
Phylogenetic Relationships among Pax4, -6, and -10 We performed a molecular phylogenetic analysis employing a data set containing the members of the Pax4/6/10 class covering major metazoan taxa as well as human, Cio. intestinalis, and fly Pax3/7 (paired) genes as outgroup (see supplementary table S5, Supplementary Material online). The inferred ML tree ( fig. 2B) placed the putative Nematostella vectensis Pax6 ortholog outside the monophyletic group containing bilaterian Pax4, -6, and -10 genes. Within this group, gnathostome Pax10 genes formed a monophyletic group (bootstrap probability in the ML analysis, 98). Except for the opossum Pax6, gnathostome Pax6 genes formed a monophyletic group that was inferred to be a sister group of the Pax10 group of genes (bootstrap probability in the ML analysis, 30). The two previously identified cyclostome Pax genes, namely Pax6 of the inshore hagfish (Eptatretus burgeri) and Pax6 of the Japanese lamprey (Lethenteron japonicum, also referred to as Arctic lamprey Lethenteron camtschaticum), form an exclusive cluster that is placed at the base of the gnathostome Pax6 and -10 subgroups ( fig. 2B). It should be noted that the newly  2B). The group of Pax4 genes forms a monophyletic cluster that is placed outside all other bilaterian Pax6 and -10 genes. Support values for this inferred tree are generally low. This poorly resolved tree topology prevents us from deriving more clear-cut conclusions about the phylogenetic relationships among the individual Pax4, -6, and -10 subgroups. In particular, the phylogenetic position of the acanthopterygian pax6a (pax6.3 in Ravi et al. 2013) genes in relation to other vertebrate Pax6 genes could not be reliably inferred by this analysis. Although the ML analyses suggest an origin of acanthopterygian pax6a and -6b genes through the TSGD ( fig. 2B), the support for this duplication node (bootstrap probability in the ML analysis, 18) is poor.
To test the hypothesis postulating an ancient origin of acanthopterygian pax6a (pax6.3) genes ( fig. 2A; Ravi et al. 2013), we complemented the heuristic ML tree reconstruction with statistical tests. We simulated tree topologies for alternative scenarios supporting an early (Hypothesis 1; Ravi et al. 2013) or late (Hypothesis 2) origin of acanthopterygian pax6a (pax6.3) genes ( fig. 2A) and computed likelihoods for them. Although the latter hypothesis yielded larger likelihood values, no significant difference was observed in the levels of support among the two hypotheses: The P values of Hypothesis 1 are 0.12 (approximately unbiased [AU] test) and 0.42 (Shimodaira-Hasegawa [SH] test), and those of Hypothesis 2 are 0.38 (AU test) and 0.44 (SH test). Thus, the question about the evolutionary origin of acanthopterygian pax6a (pax6.3) genes cannot be solely answered with a molecular phylogenetic approach. Therefore, we investigated gene orders in the genomic regions flanking teleost pax6 genes.
We detected conserved synteny between the genomic region containing the single Pax6 gene in the spotted gar FIG. 5.-Conserved synteny between the Pax10-containing region in the green anole and its orthologous regions in mammals and birds. A 1-Mb region flanking the green anole Pax10 gene, shown in red, was analyzed and gene-by-gene orthologies are indicated with gray lines. (A) Conserved synteny between the green anole Pax10-containing region and its orthologous regions in the opossum and human genomes. The dense pattern of one-to-one orthologies suggests a small-scale deletion of Pax10 in the lineage leading to eutherians, before the split of the marsupial lineage. (B) Synteny between the green anole Pax10-containing region and the chicken genome. The lack of one-to-one orthologies in the region around the green anole Pax10 gene is best explained by a large-scale deletion in the avian lineage. Green anole genes whose orthologs are located elsewhere in the opossum, human, or chicken genome are indicated with blue bars, whereas green anole genes lacking orthologs in these genomes are shown with grey bars. Exact genomic locations and accession IDs of the identified orthologs are included in supplementary tables S7 and S8, Supplementary Material online. chr., chromosome. and those containing pax6a and -6b genes in medaka, stickleback and zebrafish ( fig. 3A and supplementary table S9, Supplementary Material online). The TSGD presumably occurred after the split of teleost fish from the lineage of Lepisosteiformes, including the spotted gar (Hoegg et al. 2004;Crow et al. 2006;Hurley et al. 2007;Amores et al. 2011). Thus, this one-to-two relationship between the spotted gar and teleost fish matches the pattern resulting from the TSGD. We also investigated the molecular phylogeny of the neighboring gene families (fig. 3B). We sought to determine the timing of duplication that gave rise to pairs of genes flanking acanthopterygian pax6a and -6b, namely in the Depdc7, Wt1, and Kiaa1549l gene families. The inferred ML trees suggested a duplication that gave rise to the teleost paralogs in question in the actinopterygian fish lineage ( fig. 3B). Two of the gene families (Depdc7 and Kiaa1549l) indicated duplications after the split of the spotted gar from the teleost stem lineage, coinciding with the TSGD. To further test this hypothesis, we computed likelihoods of alternative, non-ML tree topologies supporting the ancient origin of pax6a-linked (pax6.3) genes at the base of vertebrate evolution. This analysis rejected an ancient origin of pax6a-linked genes at the base of vertebrate evolution for two of the three gene families (Depdc7 and Kiaa1549l) with P values below 0.1 in the AU and SH tests. For the Wt1 gene family, this hypothesis was not significantly rejected (P value of 0.29 in the AU test and 0.28 in the SH test), but was less likely than a more recent duplication in the TSGD ( fig. 3B). Although not unambiguously inferred by our analysis, the timing of duplication of Wt1 genes in the TSGD has been demonstrated previously (Kluever et al. 2009). The synchrony of the duplications of these flanking genes suggests that they were duplicated in a large-scale event, probably in the TSGD. Hence, the paralogy between pax6a and -6b genes, embedded in this region, should have originated through the TSGD (see fig. 2A).
Because our molecular phylogenetic analysis did not yield high confidence about the timing of diversification of the Pax4/6/ 10 gene class, we employed a synteny analysis (see Materials and Methods). An intragenomic synteny analysis of the green anole lizard showed a dense pattern of gene-by-gene paralogies among four chromosomal regions including those containing Pax6 and -10 ( fig. 4). The gene families whose duplications coincide with the 2R-WGD show a dense pattern of gene-by-gene paralogy among the identified regions on chromosomes 1, 4, 5, and 6 ( fig. 4). In addition, five of the identified gene families were previously shown to have diversified early in vertebrate evolution (Manousaki et al. 2011). Interestingly, the orthologous regions in the human and chicken genomes were suggested to be derived from a single ancestral vertebrate chromosome, namely "D," reported by Nakatani et al. (2007). These results suggest that the quartet of the identified regions, on chromosomes 1, 4, 5, and 6 in the green anole genome, originated from a largescale quadruplication, probably as a result of the 2R-WGD.

Mode of Secondary Loss of Pax10 in Mammals
Our search for Pax10 genes in public databases and subsequent phylogenetic analyses suggested the absence of this gene in mammals and birds (see figs. 1B and 2). We employed intergenomic synteny analyses in order to determine whether the absence was caused by a large-scale deletion of a chromosomal segment or a small-scale gene loss. If it is caused by a major deletion, simultaneous loss of multiple neighboring genes should be observed. Our comparison between the 1-Mb genomic region flanking the Pax10 gene in the green anole, the human, and opossum genomes revealed conserved synteny ( fig. 5A). Of the 19 protein-coding genes contained in this region in the green anole, 15 and 14 genes, respectively, have orthologs in particular regions on human chromosome 19 and opossum chromosome 4 (see supplementary table S7, Supplementary Material online, for exact positions of the orthologs). Notably, orthologs of one immediate neighbor of Pax10 (an uncharacterized gene encoding 776 amino acidlong putative peptide; ENSACAG00000013620) are also absent from mammalian genomes. Thus, the absence of the Pax10 gene from mammalian genomes was presumably caused by a small-scale deletion early in mammalian evolution before the divergence between the marsupial and eutherian lineages. If there was no massive chromosomal rearrangement that could have hindered our synteny analyses, this deletion event in the mammalian lineage could have involved as few as two genes.
We also performed a synteny comparison between the green anole and the chicken. Of the 19 genes in the Pax10containing region in the green anole genome, only two were unambiguously shown to have orthologs in the chicken genome (see supplementary table S8, Supplementary Material online, for positions of the orthologs). Those two anole genes are more than 230 kb away from the Pax10 gene ( fig. 5B). The absence of the chicken orthologs of Pax10 and other genes in this genomic region in anole suggests that its counterpart region got lost in the lineage leading to chicken. Compared with a small-scale loss in the mammalian lineage, the loss in the avian lineage could have been larger in size, possibly involving more than ten genes.
Expression Patterns of Pax10 in Zebrafish, Xenopus, and Green Anole We conducted semiquantitative RT-PCR using a developmental series of zebrafish to characterize the temporal expression profile of pax10a. This experiment suggested an onset of pax10a expression at 25 h postfertilization (hpf) with the maximal expression level at 5 days postfertilization (dpf; fig. 6A  and supplementary fig. S1, Supplementary Material online). This result prompted us to examine whether Pax10 is expressed in adult animals. Therefore, we analyzed Pax10 expression levels, as well as those of Pax6, in individual organs of zebrafish, Xenopus, and green anoles. Our comparison revealed strong Pax10 expressions in the eyes of all three examined animals and also subtle expression in testes of Xenopus and brain tissue of zebrafish and the green anole ( fig. 6B). It also revealed intensive Pax6 expressions in the eyes and weaker expressions in the brain, pancreas, and testis, which was common to all the animals examined ( fig. 6B). We performed clustering of the analyzed genes based on their similarity in expression levels in the examined organs ( fig. 6B). It partly recovered their phylogenetic relationship$ closely related gene pairs (e.g., zebrafish pax6a and -6b, as well as Xenopus Pax10 and zebrafish pax10a) also showed the highest similarity in expression patterns.
In order to scrutinize the expression patterns of these genes, we performed in situ hybridizations on sectioned eyes of adult zebrafish, Xenopus, and green anoles. We performed expression analyses of pax4, -6a, -6b, and -10a genes in zebrafish, Pax6 and -10 in the green anole, and Pax6a and -10b in X. laevis and identified distinct expression signals of all investigated genes in specific layers of the mature retina, namely, intensively in the inner nuclear layer and weakly in the ganglion cell layer ( fig. 6C-R). In addition, we identified transcripts of zebrafish pax6a, -6b, and -10a in the horizontal cell layer ( fig. 6D, F, and H) and those of zebrafish pax4 in the outer nuclear layer ( fig. 6J). In the retina of the green anole, the expression domain of Pax6 in the inner nuclear layer was revealed to be nested within the broader area of Pax10-expressing cells ( fig. 6L and N).

Discussion
Phylogeny of the Pax4/6/10 Class of Genes Only recently, a novel relative of Pax6, designated Pax6.2 (Pax10 in this study), was reported (Ravi et al. 2013). This study ignored Pax4, the other relative previously revealed to have been split from Pax6 in the 2R-WGD (Manousaki et al. 2011). Therefore, in this study, we included Pax4 genes in the data set and sought to reconstruct the evolutionary history of the entire Pax4/6/10 gene class. The group of Pax4 genes, with long branches, was placed outside the clade containing vertebrate and invertebrate Pax6 genes as well as Pax10 genes ( fig. 2B). Monophyly of Pax6 could not be unambiguously inferred ( fig. 2B), and the group of Pax10 genes was placed inside the vertebrate Pax6 genes. As shown for many other genes (Qiu et al. 2011;Kuraku 2013), the phylogenetic positions of the cyclostome Pax6 homologs were not unambiguously determined ( fig. 2B). Still, Japanese lamprey Pax6B, newly reported in this study, was suggested to be an ortholog of the previously reported hagfish Pax6 gene (supplementary fig. S2, Supplementary Material online). The previously reported Japanese lamprey Pax6 was instead suggested to be orthologous to gnathostome Pax6 genes (supplementary fig.  S2, Supplementary Material online). As for the position of acanthopterygian pax6b genes questioned by Ravi et al. ( fig. 2A), our phylogenetic analysis ( fig. 2B) supported the scenario that they originated in a duplication in the teleost fish lineage (Hypothesis 2) instead of the scenario that it originated in the 2R-WGD (Hypothesis 1).
Synteny analyses have been widely used as reliable tool to identify traces of WGD (Kuraku and Meyer 2012 and references therein). It is particularly useful when molecular phylogenies of the involved genes cannot be reliably reconstructed. Our analysis demonstrated conserved synteny among four chromosomal regions including the two containing Pax6 and -10, respectively ( fig. 4). The duplications giving rise to this quartet, inferred by the diversification patterns of neighboring gene families (Manousaki et al. 2011), occurred after the split of the tunicate and cephalochordate lineages but before the cyclostome-gnathostome split. This timing coincides with the 2R-WGD early in vertebrate evolution ).

Origin of Acanthopterygian pax6a Genes
The study by Ravi et al. suggested the origin of acanthopterygian pax6a (pax6.3) genes in the 2R-WGD (Hypothesis 1 in   FIG. 6.-Continued (red), respectively. (B) Heat map visualizing expression levels of Pax6 and -10 in individual organs of adult zebrafish, Xenopus, and green anole. In adult zebrafish, pax6a, -6b, and -10a transcripts were detected in the brain, testis, and eye. High levels of Pax6 expression were detected in Xenopus laevis brain and eye, and low levels in pancreas and intestine, whereas Pax10 expression signals were found in the eye and testis. In green anole, Pax6 transcripts were detected in the eye, brain, and testis and at low concentrations in pancreas, and Pax10 expression was detected in the eye and brain. It should be noted that the zebrafish pancreas was not analyzed (NA) because its anatomical structure was not precisely identified. The phylogram on the left reflects the phylogenetic relationships of the genes inferred in figure 2B, whereas that on the right shows the clustering based on their expression levels in various organs. (C-R) In situ hybridizations of Pax4, -6, and -10 orthologs in the retinas of adult zebrafish, Xenopus, and the green anole. C-J show expression patterns in the retinas of an albino zebrafish, K-N of a green anole, and O-R of a X. laevis. D, F, H, J, L, N, P, and R are magnifications of the rectangles in A, C, E, G, I, K, M, and O, respectively. All investigated genes were strongly expressed in the inner nuclear layer (i) and weakly in the ganglion cell layer (g). Zebrafish pax6a, -6b, and -10a also showed weak expressions in the horizontal cell layer (h in D, F, and H) and zebrafish pax4 transcripts were detected in the outer nuclear layer (o in J). It was evident from the results for Anolis carolinensis that the Pax6 expression is nested within that of Pax10 in the inner nuclear layer of the retina (arrows in L and N). Scale bar: 200 mm. fig. 2A). Evidence for this hypothesis was: 1) "Subpartitioning" of conserved noncoding elements, 2) the absence of exon 5a from pax6a (pax6.3) genes, and 3) a simple molecular phylogenetic analysis with the neighbor-joining method and excluding Pax4 genes (Ravi et al. 2013). The first evidence, that is, the divergence or loss of cis-elements between acanthopterygian pax6a (pax6.3) and other gnathostome Pax6 genes, can be explained by subfunctionalization and asymmetric retention of expression domains after the gene duplication. It is a recurrent pattern in gene family evolution that one duplicate, here the acanthopterygian pax6b, retains the ancestral role, whereas the other duplicate, pax6a (pax6.3) in this case, is more prone to changes in their functions (reviewed in Prince and Pickett 2002). The sole reliance on presence and absence of conserved noncoding elements, without any tree-based approach, might therefore mislead the reconstruction of phylogenetic relationships. Regarding the second evidence, Ravi et al. regarded the lack of exon 5a in acanthopterygian pax6a (pax6.3) genes as the evidence of exclusion of those genes from the canonical Pax6 genes (Ravi et al. 2013). On the other hand, however, they report "N-terminal protein extension" of all teleost pax6 genes (including both pax6a and -6b), which can be regarded as evidence of phylogenetic proximity between pax6a and -6b. Thus, the comparison of gene structures does not add to our understanding of the evolution of Pax6 genes. As for the third evidence mentioned above, we reanalyzed the phylogeny of the entire Pax4/6/10 class by employing a more rigorous methodology in light of the standard procedure of molecular phylogenetics (Anisimova et al. 2013). Although our molecular phylogenetic analysis focusing on the Pax gene family did not unambiguously solve the question about the timing of the acanthopterygian pax6a-6b split, expansion of the scope of our study to a broader genomic region enhanced the power of the analysis ( fig. 3). Two of the three gene families whose members reside near pax6a and -6b (Depdc7 and Kiaa1549l) unambiguously rejected their more ancient origins in the 2R-WGD, and one gene family (WT1) also strongly favored their duplication in the TSGD ( fig. 3B). This strongly suggests that these genomic regions, including pax6a and -6b genes, originated in the TSGD (Hypothesis 2 in fig. 2A). Ravi et al. also analyzed conserved synteny (fig. 6B and C of Ravi et al. 2013) but did not recognize the copresence of the Depdc7, Wt1, and Dnajc24 family members as the evidence of the orthology between the zebrafish pax6a (pax6.3 in their hypothesis) and the pax6b of the other teleost fishes. Another argument supporting Hypothesis 2 is the phylogenetic distribution of teleost pax6 genes. According to Ravi et al., acanthopterygian lost one of the TSGD-derived duplicates orthologous to Pax6 and is the only taxon retaining the 2R-derived pax6.3 gene (Hypothesis 1 in fig. 2A). This hypothesis assumes independent losses of its orthologs in all other vertebrate lineages (cyclostomes, chondrichthyans, nonteleost actinopterygians, coelacanth, and tetrapods as depicted in fig.  9 in Ravi et al. 2013). Taken together, we strongly argue that the genes designated as pax6.3 by Ravi et al. are indeed the second TSGD-derived gene, orthologous to Pax6, which should continue to be called pax6a (Hypothesis 2 in fig. 2A).
Asymmetric Gene Retention Rates between Pax6 and Pax4/-10 In the course of vertebrate evolution, Pax4, -6, and -10 genes experienced different patterns of retention between vertebrate lineages. Although the Pax6 gene is present in all vertebrates investigated to date, Pax4 and -10 were lost multiple times secondarily in independent lineages ( fig. 1B). This asymmetry of gene retention was also manifested after the TSGDin each teleost genome that we analyzed, we did not identify more than one pax10 gene ( fig. 2B). Using a synteny-based approach, it was suggested that the zebrafish pax10a gene is a TSGD-derived paralog of the identified acanthopterygian pax10b genes (Ravi et al. 2013). The tetraploid X. laevis is the only species possessing two copies of both Pax6 and -10 genes (see fig. 2B).
Although the general trend of unequal patterns of gene retention is clear, the exact number of secondary gene losses of Pax4 and -10 remains difficult to determine. The certainty with which one can infer a secondary gene loss in a given lineage depends on the quality of available sequence resources and the density of taxon sampling. In Ensembl release 71, the mammalian lineage is represented with 40 genomes, including those sequenced with low coverage, whereas only one amphibian and six sauropsid genomes are available. However, the fact that we identified a Pax6 gene in every genome we investigated suggests that the absences of Pax4 and -10 genes are not caused by insufficient sequence information, but rather by secondary gene losses. In contrast, the absence of Pax10 genes from the mammalian lineage can be inferred with a high degree of confidence. By investigating synteny between the anole Pax10-containing genomic region and orthologous regions in mammalian and avian genomes, we demonstrated that different modes of gene loss caused the absence of Pax10 in these two lineages. Although we identified a trace of small-scale loss in mammalian genomes, loss of a larger genomic segment was suggested in the avian lineage ( fig. 5).
Functional Diversification of Pax4, -6, and -10 Genes An explanation for the asymmetric gene retention between Pax6 and the other two paralogs, Pax4 and -10 genes, might be rapid subfunctionalization soon after the 2R-WGD in which only Pax6 retained the essential role as master control gene for eye development. This crucial role could have imposed a high selection pressure on the Pax6 gene making it indispensable. Comparison of Pax10 expression patterns among a teleost fish (zebrafish), an amphibian (X. laevis), and a reptile (the green anole lizard) allowed us to corroborate this hypothesis and infer the ancestral expression profile of the euteleostome Pax10 gene. We did not identify significant Pax10 expression in early embryos of the three examined species, and quantification of Pax10 transcripts of a developmental series of zebrafish suggested the onset of pax10a expression during later developmental stages ( fig. 6A). This finding is in accordance with a previous study reporting an increasing expression level of zebrafish pax10a from 1 dpf to 5 dpf (Ravi et al. 2013). Morpholino depletion for pax10a in zebrafish causes a reduction in eye size (Ravi et al. 2013), indicating a restricted role of pax10a to eye growth. The crucial role in the initiation of eye development is presumably retained solely by the Pax6 gene.
Our semiquantitative RT-PCR of adult zebrafish, Xenopus, and green anoles confirmed the Pax6 expression in the eye, pancreas, and brain ( fig. 6B), as described previously (Nornes et al. 1998;Moreno et al. 2008). Exceptions are the identified expression signals in the testes of Pax6 as well as of Pax10. This expression domain has not been described for any Pax4 or -6 gene before. Excessive gene expression in testes can be explained by a permissive state of chromatin in testes owing to the peculiar properties of transcription in meiotic and postmeiotic cells ("out of testis" hypothesis; Kaessmann 2010). In light of this hypothesis, these identified transcripts might merely be caused by leaky transcription. Results of our semiquantitative RT-PCR revealed expressions of Pax10 genes in the eyes of all three investigated species and also in the brain tissue of zebrafish and the green anole, but not X. laevis ( fig. 6B). Our inference based on the maximum parsimony principle suggests that the Pax10 gene in the ancestral euteleostome was expressed in the eyes, testes, and brains of adults. The elephant shark Pax10 gene also shows expression in mature eyes (Ravi et al. 2013), indicating that the Pax10 expression in the adult eyes was established before the split between the euteleostome and chondrichthyan lineages. On the other hand, we observed among-lineage differences in the Pax10 expression: Expression in the adult kidney is unique to the elephant shark, whereas those in the adult brains and testes are confined to euteleostomes ( fig. 6B; Ravi et al. 2013).
The expression signals of Pax4, -6, and -10 in the adult eyes are detected in specific layers in the retina ( fig. 6C-R). The retinal expression of Pax6 genes in the inner and the ganglion cell layer identified in this study is equivalent to that in other vertebrate species (reviewed in Osumi et al. 2008). However, we did not detect Anolis and Xenopus Pax6 transcripts in the horizontal cell layer ( fig. 6K-R), an expression domain previously reported for several vertebrates, including an elasmobranch shark, zebrafish, chicken, and mouse (Belecky-Adams et al. 1997;Macdonald and Wilson 1997;Wullimann and Rink 2001;Ferreiro-Galve et al. 2011). We report for the first time expression of a Pax4 gene, namely zebrafish pax4, in the inner nuclear and the ganglion cell layer in addition to the outer nuclear layer ( fig. 6J) as reported in mammals (Rath et al. 2009). The expression of Pax10 is almost identical to Pax6, except that the Pax6 expression is nested within the broader area of Pax10-positive cells in Anolis ( fig. 6L-N). The expression of zebrafish pax10a appears to alter in the course of development. Although the pax10a expression is restricted to the inner nuclear layer at 2 dpf (Ravi et al. 2013), it expands to the horizontal and ganglion cell layer in the adult retina ( fig. 6C and D). A parsimonious reconstruction of the nondevelopmental expression profile indicates a role of Pax10 in the mature retina and brain of the euteleostome ancestor ( fig. 7).
Although the proto-Pax4/6/10 gene in the vertebrate ancestor had a dual role in the visual system, namely an early one in eye morphogenesis and a late one in mature photoreceptors, these roles could have been subdivided during vertebrate evolution. The vertebrate Pax6 gene retained the crucial role in eye morphogenesis, whereas Pax4 and -10 as well as Pax6 are coexpressed in the mature vertebrate retina ( fig. 7).
The redundancy resulting from the quadruplication of a proto-Pax4/6/10 gene in the 2R-WGD presumably led to a functional partitioning among the four paralogs early in vertebrate evolution. In order to understand these secondary changes in associated developmental pathways, the ancestral expression profile of the proto-Pax4/6/10 gene needs to be reconstructed and compared with those of individual Pax4, -6, and -10 genes in extant vertebrates. This reconstruction of the ancestral state should be reinforced by comparing expression patterns of their orthologs of nonvertebrate deuterostomes with those of protostomes. So far, sparse information of expression patterns in adults for most of invertebrates does not allow a reliable reconstruction of nondevelopmental roles of the ancestral Pax4/6/10 gene. However, expression of an eyeless isoform in adult Drosophila eyes, more precisely in photoreceptors, has been reported (Sheng et al. 1997). The reconstruction of the ancestral expression profile of Urbilateria suggests a role of the proto-Pax4/6/10 gene in the developing visual and olfactory systems, the CNS, and mature photoreceptors ( fig. 7).
Pancreatic expression of Pax4 and -6 was reported in mammals (Turque et al. 1994;Sosa-Pineda et al. 1997) and teleost fish (Thisse and Thisse 2004;Manousaki et al. 2011). The single Pax4/6/10 proto-ortholog in amphioxus lacks expression in the possible pancreas homolog (Reinecke 1981;Glardon et al. 1998;Sun et al. 2010). After the split of cephalochordate and tunicate lineages, the proto-Pax4/6/10 should have gained the pancreatic expression domain in the vertebrate ancestor before the 2R-WGD. Thus, expression of Pax4 and -6 in the pancreas presumably represents a synapomorphy of vertebrates. On the other hand, the vertebrate Pax10 gene, the third paralog, presumably did not retain the pancreatic expression domain (fig. 7).
Another interesting feature of the newly identified Pax10 gene is the lack of a paired domain in the deduced protein sequence ( fig. 1A; Ravi et al. 2013). Although no other Pax gene has ever been shown to completely lack the eponymous paired box, there are reports of "paired-less" Pax6 isoforms through alternative splicing in quail (Carriere et al. 1993), mouse (Kammandel et al. 1999), and zebrafish (Lakowski et al. 2007), as well as the ortholog in Caenorhabditis elegans, vab-3 (Zhang and Emmons 1995). It was shown in mice that paired-less Pax6 is one of the three Pax6 isoforms whose transcriptions are initiated from three alternative promoters (Kammandel et al. 1999) and is expressed in a tissue-specific manner (Mishra et al. 2002). The structural similarity between paired-less Pax6 isoforms and the Pax10 gene is intriguing. It will be interesting to disentangle the functional interplay on the cellular level among the paired-less Pax10 gene, isoforms of Pax6 and also Pax4. Teleosts, in particular zebrafish, would be an ideal system as they are amenable to genetic experiments and have retained orthologs of Pax4, -6, and -10. Chromatin immunoprecipitation combined with sequencing (ChIP-seq) experiments could potentially reveal differences and overlaps in the target DNA of these transcription factors. Knockout (or -down) experiments of pax10 could reveal to what extent its sister genes might compensate for its loss of function and thereby reveal potential redundancy. This experiment might allude to possible consequences that a secondary gene loss of Pax4 and/or -10 might have had on associated regulatory pathways.