Origin and evolution of the Rax homeobox gene by comprehensive evolutionary analysis

Abstract

Rax is one of the key transcription factors crucial for vertebrate eye development. In this study, we conducted comprehensive evolutionary analysis of Rax. We found that Bilateria and Cnidaria possess Rax, but Placozoa, Porifera, and Ctenophora do not, implying that the origin of the Rax gene dates back to the common ancestor of Cnidaria and Bilateria. The results of molecular phylogenetic and synteny analyses on Rax loci between jawed and jawless vertebrates indicate that segmental duplication of the Rax locus occurred in an early common ancestor of jawed vertebrates, resulting in two Rax paralogs in jawed vertebrates, Rax and Rax2. By analyzing 86 mammalian genomes from all four major groups of mammals, we found that at least five independent Rax2 gene loss events occurred in mammals. This study may provide novel insights into the evolution of the eye.

Keywords: Pax6; Rax; eye; homeobox gene; molecular evolution; retina.

Fig. 1

Phylogeny of animal species and their Rax genes. (A) Cladogram of representative animals and domain organizations of their Rax genes. Note the absence of Rax in Ctenophora, Porifera, and Placozoa. Blue boxes indicate octapeptides, magenta boxes indicate homeodomains, and yellow boxes indicate OAR motifs. The cladogram topology is derived from previous studies [54, 55, 56, 57]. It should be noted that the Metazoan phylogeny is controversial. The arrow indicates the presumed origin of the Rax gene. The scale bar indicates 100 amino acid residues. (B) Sequence alignments of the octapeptide, homeodomain, and OAR motif in Rax orthologs. These three domains/motifs are well conserved in Cnidaria and Bilateria. Each residue is colored according to the Clustal X residue code [58].

Fig. 2

Evolution of Rax and Rax2 gene structures in vertebrates. Synteny around Rax and Rax2 genes in basal vertebrates. While jawed vertebrates possess both Rax and Rax2, jawless vertebrates possess a single Rax. Black lines indicate paralogous or orthologous relationships. Scaffold names are indicated on the right of the panel.

Fig. 3

Multiple sequence alignments of Rax orthologs. (A) Multiple sequence alignment of Rax orthologs using clustal omega. The 22 protein sequences of Rax were aligned by clustal omega [28]. (B) Multiple sequence alignment of Rax orthologs using muscle. The same set of protein sequences in (A) were aligned by muscle [30]. Each residue is colored according to the Clustal X residue code [58].

Fig. 4

Molecular phylogenetic analysis of Rax and Rax2 in various animal species. Multiple sequence alignments were used to construct maximum‐likelihood trees from (A) clustal omega or (C) muscle and neighbor‐joining trees from (B) clustal omega or (D) muscle. In all these analyses, the JTT model was used as the amino acid substitution model. Jawed vertebrate Rax sequences are colored in red. Jawed vertebrate Rax2 sequences are colored in blue. Lamprey and hagfish Rax sequences are colored in magenta. The scale bars represent 0.2 amino acid substitutions per site. Bootstrap values are given on each node.

Fig. 5

Rax and Rax2 gene structures. (A) Rax and Rax2 gene structures of seven vertebrates. All Rax and Rax2 genes are composed of three exons. Protein‐coding sequences of Rax or Rax2 are colored blue or green, respectively. In shark, spotted gar, and coelacanth, start codons of Rax2 (red line) are located on the first exon. In Xenopus, zebra finch, opossum, and human, start codons of Rax2 are shifted to the second exon (arrowhead). The arrow indicates the presumed point of Rax2 octapeptide loss. The scale bar indicates 1 kbp. (B) Rax and Rax2 gene structures of spotted gar and human. Rax and Rax2 gene structures and their respective protein domain/motif architectures are shown. Note that human RAX2 has its start codon at the second exon and lacks the octapeptide. Blue boxes indicate octapeptides, magenta boxes indicate homeodomains, and yellow boxes indicate OAR motifs. The scale bar indicates 1 kbp.

Fig. 6

Rax2 gene losses in mammals. (A) Phylogeny of mammals and Rax2 gene loss. Mammals are divided into four major groups. Numbers of Rax2 gene loss events are given in parentheses. The topology of this cladogram is based on previous reports [59, 60, 61]. (B) Synteny of the Rax2 locus in Euarchontoglires. Rock rabbit and a subgroup of rodents, including mouse, rat, and degu, show independent Rax2 gene loss (blue lines). (C) Synteny of Rax2 locus in Laurasiatheria. There are two independent Rax2 gene loss events in Laurasiatheria (blue lines). (D) Synteny of Rax2 locus in Afrotheria. Elephant lacks the Rax2 gene (blue line). Rax2 genes are colored red. Black lines indicate orthologous relationships. Scaffold name is shown below each taxon name.

Fig. 7

Phylogenetic analysis of mammalian Rax and Rax2. (A) A maximum‐likelihood tree of mammalian Rax and Rax2. A maximum‐likelihood tree was constructed from the amino acid sequence alignment containing Rax (red) and Rax2 (blue) from 86 placental mammals and opossum (Monodelphis domestica), a marsupial. The scale bars represent 0.1 amino acid substitutions per site. Bootstrap values > 0.6 are given on each node. (B) Ka/Ks ratio distributions of mammalian Rax (upper panel) or Rax2 (lower panel). Ka/Ks ratios comparing human RAX and RAX2 with respective mammalian Rax and Rax2 were calculated. The average Ka/Ks values are indicated by dotted lines.