Feiner N , Meyer A , Kuraku S .

	Fig. 1.—. Domain structure of vertebrate Pax proteins and phylogenetic distribution of Pax4, -6, and -10 genes across jawed vertebrates. (A) Presences of paired domains (PD), homeodomains (HD), and octapeptides (O) for all vertebrate Pax subtypes. 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+Γ4 model (shape parameter of the gamma distribution α = 0.57). The scale bar on the upper left indicates 0.2 substitutions per site.
	Fig. 3.—. Evolutionary origins of pax6-neighboring genes in teleost genomes. (A) Conserved synteny between the genomic regions of the gar Pax6 ortholog and the two teleost pax6 subtypes. Co-orthologs to the spotted gar genes located within 1 Mb flanking the pax6 gene that are harbored in the vicinity of pax6a and/or -6b of selected teleosts are shown in the same color (see supplementary table S9, Supplementary Material online, for accession IDs). Asterisks mark names of the selected gene families that retained both duplicates derived from the TSGD. (B) Molecular phylogenetic trees of selected gene families. ML trees of three gene families are shown, and statistical support values (P values) of alternative scenarios assuming a more ancient origin of the pax6a-linked genes (compatible with Hypothesis 1 in fig. 2A) are given in the grey box on the lower left as inset. The Depdc7 phylogeny is based on 321 amino acid residues and the JTT+Γ4 model (shape parameter of the gamma distribution α = 1.39) was assumed. The ML tree of Wt1 genes was inferred from 331 amino acid residues assuming the JTT+F+Γ4 model (shape parameter of the gamma distribution α = 1.45). The Kiaa1549l phylogenetic analysis used an alignment of 295 amino acid residues and the JTT+Γ4 model (shape parameter of the gamma distribution α = 1.30). Bootstrap probabilities are provided for each node. The scale bars on the upper left of each phylogenetic tree indicate 0.2 substitutions per site.
	Fig. 4.—. Intragenomic conserved synteny between Pax6 and -10 containing regions in the green anole lizard. Outer grey bars represent chromosomes 1 and 6 of the green anole that harbor Pax6 and -10 (shown in red), respectively, and their paralogous regions on chromosomes 4 and 5. Magnifications of the genomic regions indicated on the chromosomes (gray bars) are shown in the center. Gene-by-gene paralogies among the four members of the quartet are highlighted with diagonal lines: Gray lines for paralogy of gene families with two paralogs, blue lines for three paralogs, and green line for four paralogs.
	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.
	Fig. 6.—. Expression profiles of Pax4, -6, and -10 in zebrafish, Xenopus, and green anole. (A) Expression levels of pax10a in a developmental series of zebrafish with semiquantitative RT-PCRs. Heat map indicates upregulation of pax10a in late developmental stages reaching a plateau at 5 dpf. Color scale at the right indicates the relative expression levels normalized to values between 0 and 1 for no expression (blue) and for the highest observed expression level (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 µm.
	Fig. 7.—. Evolutionary scenario focusing on the functional diversification of the Pax4/6/10 class of genes. Expression domains identified in this study are mapped with those previously described onto a simplified gene tree of the Pax4/6/10 class. Based on parsimonious reconstruction, a proto-Pax4/6/10 gene at the last common ancestor of protostomes and deuterostomes, the so-called Urbilateria, was most likely expressed in photoreceptors, olfactory placode, developing the eye and CNS. Secondary modification, such as the gain of a pancreatic expression before the 2R-WGD or the loss of several Pax4 and -10 expression domains, led to the functional differentiation among Pax4, -6, and -10.

Amemiya, The African coelacanth genome provides insights into tetrapod evolution. 2013, Pubmed

Amemiya, The African coelacanth genome provides insights into tetrapod evolution. 2013, Pubmed
Amores, Genome evolution and meiotic maps by massively parallel DNA sequencing: spotted gar, an outgroup for the teleost genome duplication. 2011, Pubmed
Amores, Zebrafish hox clusters and vertebrate genome evolution. 1998, Pubmed
Anisimova, State-of the art methodologies dictate new standards for phylogenetic analysis. 2013, Pubmed
Begemann, The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain. 2001, Pubmed
Belecky-Adams, Pax-6, Prox 1, and Chx10 homeobox gene expression correlates with phenotypic fate of retinal precursor cells. 1997, Pubmed
Breitling, Origin of the paired domain. 2000, Pubmed
Carriere, Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina. 1993, Pubmed
Castoe, Sequencing the genome of the Burmese python (Python molurus bivittatus) as a model for studying extreme adaptations in snakes. 2011, Pubmed
Chi, Getting your Pax straight: Pax proteins in development and disease. 2002, Pubmed
Chiari, Phylogenomic analyses support the position of turtles as the sister group of birds and crocodiles (Archosauria). 2012, Pubmed
Crawford, More than 1000 ultraconserved elements provide evidence that turtles are the sister group of archosaurs. 2012, Pubmed
Crow, The "fish-specific" Hox cluster duplication is coincident with the origin of teleosts. 2006, Pubmed
Delporte, Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors. 2008, Pubmed
Edgar, MUSCLE: a multiple sequence alignment method with reduced time and space complexity. 2004, Pubmed
Feiner, The origin of bmp16, a novel Bmp2/4 relative, retained in teleost fish genomes. 2009, Pubmed
Ferreiro-Galve, Pax6 expression during retinogenesis in sharks: comparison with markers of cell proliferation and neuronal differentiation. 2012, Pubmed
Gawantka, Antagonizing the Spemann organizer: role of the homeobox gene Xvent-1. 1995, Pubmed , Xenbase
Glardon, Isolation and developmental expression of the amphioxus Pax-6 gene (AmphiPax-6): insights into eye and photoreceptor evolution. 1998, Pubmed
Halder, Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila. 1995, Pubmed
Hoegg, Phylogenetic timing of the fish-specific genome duplication correlates with the diversification of teleost fish. 2004, Pubmed
Holland, AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest. 1999, Pubmed
Holland, Gene duplications and the origins of vertebrate development. 1994, Pubmed
Hoshiyama, Evolution of the gene families forming the Pax/Six regulatory network: isolation of genes from primitive animals and molecular phylogenetic analyses. 2007, Pubmed
Hubbard, Ensembl 2009. 2009, Pubmed
Huelsenbeck, MRBAYES: Bayesian inference of phylogenetic trees. 2001, Pubmed
Hurley, A new time-scale for ray-finned fish evolution. 2007, Pubmed
Iwabe, Sister group relationship of turtles to the bird-crocodilian clade revealed by nuclear DNA-coded proteins. 2005, Pubmed
Kaessmann, Origins, evolution, and phenotypic impact of new genes. 2010, Pubmed
Kammandel, Distinct cis-essential modules direct the time-space pattern of the Pax6 gene activity. 1999, Pubmed
Klüver, Regulatory back-up circuit of medaka Wt1 co-orthologs ensures PGC maintenance. 2009, Pubmed
Kuraku, Impact of asymmetric gene repertoire between cyclostomes and gnathostomes. 2013, Pubmed
Kuraku, Timing of genome duplications relative to the origin of the vertebrates: did cyclostomes diverge before or after? 2009, Pubmed
Kuraku, Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolution. 2005, Pubmed
Kuraku, Detection and phylogenetic assessment of conserved synteny derived from whole genome duplications. 2012, Pubmed
Lakowski, Mechanisms controlling Pax6 isoform expression in the retina have been conserved between teleosts and mammals. 2007, Pubmed
Lundin, Evolution of the vertebrate genome as reflected in paralogous chromosomal regions in man and the house mouse. 1993, Pubmed
Lynch, The probability of preservation of a newly arisen gene duplicate. 2001, Pubmed
Macdonald, Distribution of Pax6 protein during eye development suggests discrete roles in proliferative and differentiated visual cells. 1997, Pubmed
Manousaki, Co-orthology of Pax4 and Pax6 to the fly eyeless gene: molecular phylogenetic, comparative genomic, and embryological analyses. 2011, Pubmed
Matus, Expression of Pax gene family members in the anthozoan cnidarian, Nematostella vectensis. 2007, Pubmed
Mehta, Evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum). 2013, Pubmed
Meyer, From 2R to 3R: evidence for a fish-specific genome duplication (FSGD). 2005, Pubmed
Mishra, PAX6, paired domain influences sequence recognition by the homeodomain. 2002, Pubmed
Moreno, Spatio-temporal expression of Pax6 in Xenopus forebrain. 2008, Pubmed , Xenbase
Mulley, Parallel retention of Pdx2 genes in cartilaginous fish and coelacanths. 2010, Pubmed
Nakatani, Reconstruction of the vertebrate ancestral genome reveals dynamic genome reorganization in early vertebrates. 2007, Pubmed
Nornes, Zebrafish contains two pax6 genes involved in eye development. 1998, Pubmed
Ogasawara, Developmental expression of Pax1/9 genes in urochordate and hemichordate gills: insight into function and evolution of the pharyngeal epithelium. 1999, Pubmed
Osumi, Concise review: Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator. 2008, Pubmed
Pilz, Mapping of the human homologs of the murine paired-box-containing genes. 1993, Pubmed
Prince, Splitting pairs: the diverging fates of duplicated genes. 2002, Pubmed
Qiu, Unresolved orthology and peculiar coding sequence properties of lamprey genes: the KCNA gene family as test case. 2011, Pubmed
Rath, Developmental and daily expression of the Pax4 and Pax6 homeobox genes in the rat retina: localization of Pax4 in photoreceptor cells. 2009, Pubmed
Ravi, Sequencing of Pax6 loci from the elephant shark reveals a family of Pax6 genes in vertebrate genomes, forged by ancient duplications and divergences. 2013, Pubmed
Reinecke, Immunohistochemical localization of polypeptide hormones in endocrine cells of the digestive tract of Branchiostoma lanceolatum. 1981, Pubmed
Rest, Molecular systematics of primary reptilian lineages and the tuatara mitochondrial genome. 2003, Pubmed
Sanger, A developmental staging series for the lizard genus Anolis: a new system for the integration of evolution, development, and ecology. 2008, Pubmed
Schmidt, TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. 2002, Pubmed
Shaffer, The western painted turtle genome, a model for the evolution of extreme physiological adaptations in a slowly evolving lineage. 2013, Pubmed
Sheng, Direct regulation of rhodopsin 1 by Pax-6/eyeless in Drosophila: evidence for a conserved function in photoreceptors. 1997, Pubmed
Shimodaira, CONSEL: for assessing the confidence of phylogenetic tree selection. 2001, Pubmed
Sosa-Pineda, The Pax4 gene is essential for differentiation of insulin-producing beta cells in the mammalian pancreas. 1997, Pubmed
St John, Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes. 2012, Pubmed
St-Onge, Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas. 1997, Pubmed
Stanke, AUGUSTUS: a web server for gene finding in eukaryotes. 2004, Pubmed
Sun, Identification and expression of an elastase homologue in Branchiostoma belcheri with implications to the origin of vertebrate pancreas. 2010, Pubmed
Tamura, MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. 2011, Pubmed
Tamura, Assignment of the human PAX4 gene to chromosome band 7q32 by fluorescence in situ hybridization. 1994, Pubmed
Turque, Pax-QNR/Pax-6, a paired box- and homeobox-containing gene expressed in neurons, is also expressed in pancreatic endocrine cells. 1994, Pubmed
Underhill, PAX proteins and fables of their reconstruction. 2012, Pubmed
Wada, Tripartite organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx genes. 1998, Pubmed
Walther, Pax-6, a murine paired box gene, is expressed in the developing CNS. 1991, Pubmed
Wang, The draft genomes of soft-shell turtle and green sea turtle yield insights into the development and evolution of the turtle-specific body plan. 2013, Pubmed
Wehr, Pax and vertebrate development. 1996, Pubmed
Wullimann, Detailed immunohistology of Pax6 protein and tyrosine hydroxylase in the early zebrafish brain suggests role of Pax6 gene in development of dopaminergic diencephalic neurons. 2001, Pubmed
Zardoya, Complete mitochondrial genome suggests diapsid affinities of turtles. 1998, Pubmed
Zhang, Specification of sense-organ identity by a Caenorhabditis elegans Pax-6 homologue. 1995, Pubmed