Reactive oxygen species (ROS) [e.g., superoxide anion (O₂^-), hydrogen peroxide (H₂O₂)] are thought of as cytotoxic and mutagenic; however, recent data point to important biological roles for ROS [1-4]. Phagocytes generate large amounts of O₂^- as part of their microbicidal activity, which results from activation of a membrane-associated NADPH oxidase. The key redox component of the oxidase is flavocytochrome b₅₅₈, which is comprised of an O₂^--generating catalytic subunit, gp91phox (a.k.a., Nox2), and a non-catalytic subunit, p22phox [5-7]. Recent studies indicate that similar NADPH oxidase systems are present in a wide variety of non-phagocytic cells. While the nature of these non-phagocyte NADPH oxidases is still being defined, it is clear that they are functionally and structurally distinct from the phagocyte oxidases.

Nox2 has four conserved histidines that ligate two hemes between the 3^rd and 5^th of 6 transmembrane (TM) α-helices and subregions that fold to provide binding cavities for FAD and NADPH [2,5,7-10] (referred to here as the "Nox domain" as shown in Figure 1A). These canonical regions occupy about 35% of the Nox2 sequence; however, little is known about essential roles for the other regions. Recently, mammalian homologues of Nox2 were identified and now constitute the Nox/Duox family [2]. In humans, there are five Noxes (Nox1-5) plus Duox1 and Duox2 (Figure 1B). The activation of Nox2 has been extensively studied and requires the association of essential cofactors. Nox2 alone is inactive and must associate with p22phox to form a non-covalent heterodimer known as flavocytochrome b₅₅₈. Additional regulatory subunits consist of p67phox, p47phox, p40phox and the small GTPase Rac [2,5,6]. Upon cell activation, these subunits translocate from the cytosol, assembling with and activating flavocytochrome b₅₅₈ at the cell or phagosomal membrane. Respective homologues of p47phox and p67phox, "Nox Organizer 1" (NOXO1) and "Nox Activator 1" (NOXA1), regulate Nox1 [11-15], and NOXO1 also activates Nox3 [16-18]. In comparison, Nox4 requires p22phox but no other subunits [19-21]. All members of the human Nox/Duox family contain a flavocytochrome moiety, which we refer to as the "Nox domain". Nox5 consists of a Nox domain plus an N-terminal EF-hand-containing calcium-binding domain [22,23]. Duox1/2 build on the Nox5 structure by adding an N-terminal peroxidase domain [24,25]. Nox/Duox family members are reported in mouse, rat, cow, guinea pig, Takifugu rubripes (T. rubripes), Caenorhabditis elegans (C. elegans), Ciona intestinalis (C. intestinalis), sea urchin, fungi, cellular slime mold amoeba Dictyostelium discoideum (D. discoideum), red alga Chondus crispus (C. crispus) and Porphyra yezoensis (P. yezoensis), and green plants, including Arabidopsis thaliana (A. thaliana) [22,24,26-41]. Plant Noxes are similar in domain structure to Nox5 [26,42]. The EF-hands of human Nox5, human Duox2, and an A. thaliana Nox are essential for calcium-stimulated activity [23,25,26]. Red alga C. crispus and P. yezoensis possess an unusual Nox enzyme [31]. The algal Nox lacks an N-terminal EF-hand region and no regulatory subunit homologs are present; however, the algal Nox has 4 additional predicted transmembrane α-helices that are situated between the 1^st and 2^nd NADPH-binding sub-regions [31].

Figure 1 Schematic domain structures of Nox family. (A) The Nox domain possesses 6 transmembrane α-helices (I through VI in boxes), two hemes ("heme" in diamonds), and predicted sub-regions that provide binding cavities for a co-enzyme FAD (FAD1 and FAD2) (more ...)

Genetic approaches have implicated Nox/Duox-derived ROS in biological roles and pathological conditions, including hypertension (Nox1), innate immunity (Nox2, Duox), suppression of pathogen-induced cell death (plant Nox), stomatal closure (plant Nox), otoconia formation in the inner ear (Nox3), biosynthesis of extracellular matrix (Duox), and thyroid hormone biosynthesis (Duox1/2) [18,24,29,43-48]. Although widely expressed, little is known about evolutionary relationships among Nox proteins.

Herein, we analyzed Nox/Duox protein sequences from 14 vertebrates, one urochordate, one echinodermate, three insects, one nematode, four fungi, two red algae, one amoeba, and one green plant. Using this large data set, we report (i) a novel molecular taxonomy and phylogeny of Nox/Duox proteins, (ii) synteny of vertebrate Nox/Duox genes by genome annotation, (iii) evolutionary substitution rates of vertebrate Nox proteins and regulatory subunits, and (iv) identification of key amino acid residues and regions conserved among all Nox proteins.

A molecular taxonomy, constructed by aligning the deduced amino acid sequences of the Nox domains (omitting other domains and unique features, such as the extra transmembrane domains of algae NoxD) revealed seven subfamilies (Figure 2). The subgroup consisting of Nox1-3 includes Noxes that are regulated by regulatory subunits, including Nox2, the classical phagocyte oxidase catalytic subunit (numbers 11–20 in Figure 2). Nox2 of the urochordate C. intestinalis (# 26 in Figure 2) branched from a root common to vertebrate Nox1-3 and conserves features common to all Nox1-3 proteins. Sea urchin S. purpuratus belongs to Echinodermata, which diverged early from an ancestor common to urochordates and vertebrates [49], and the S. purpuratus genome had two Nox2-like proteins, Nox2A and Nox2B (#27 and #28 in Figure 2), which branched from a root common to all chordate Noxes 1–3. The taxonomy indicates that the S. purpuratus Nox2 isologs represent a branch closest to the primordial ancestor of all isologs of vertebrate Nox1, Nox2 and Nox3. The Nox4 subfamily includes a urochordate isolog and branched from a root close to the Nox1-3 subfamily. Both the Nox4 and the Nox1-3 subfamilies originated from a common branch, and together form the two subgroups that are known to require a p22phox subunit for activity and/or stability [19,20,50]. Neither of these subgroups occurs in green plants, fungi, or nematodes. For example, the S. purpuratus genome did not contain a distinct Nox4 ortholog. Although the fruit fly Drosophila melanogaster (D. melanogaster) and honeybee Apis mellifera (A. mellifera) did not possess an ortholog of Nox1-3 or Nox4, the malaria mosquito A. gambiae possessed one unique Nox gene, NoxM (mosquito-Ag NoxM, #38 in Figure 2). We also searched for the gene in another mosquito genome Aedes aegypti (A. aegypti), the principal vector of yellow and dengue fevers, using the NCBI BLAST server [51]. The A. aegypti genome contained one NoxM ortholog (GenBank™ No. EAT37894) similar to A. gambiae NoxM, and also had Nox5 and Duox orthologs (GenBank™ No. EAT46816 and EAT40728, respectively). The A. gambiae NoxM protein branched from a root common to all chordate Nox 4 (Figure 2); however, the bootstrap value of the branch was only 60%, making it unclear whether NoxM belongs to the p22phox-regulated subgroups (Nox4 and Nox1-3) or to the NoxA/NoxB subgroup. The A. gambiae genome did not encode a distinct p22phox ortholog, suggesting that the NoxA/NoxB subgroup (which is p22phox-independent) may be a more appropriate assignment for this NoxM.

Figure 2 Molecular taxonomy of the Nox domains of Nox/Duox proteins. Amino acid sequences of the following species were trimmed to the length corresponding to human Nox2 and were aligned: human-Hs, H. sapiens; Cow-Bt, B. Taurus; dog-Cf, C. familliaris; rat-Rn, (more ...)

The Nox5 subgroup was composed of the orthologs (#'s 39–52 in Figure 2) present in vertebrates [except for Mus muscles (M. muscles) and Rattus norvegicus (R. norvegicus)], echinoderm, and insects; however, Nox5 was not found in the urochordate C. intestinalis, or the nematode C. elegans. A Nox5 ortholog of a green-spotted pufferfish Tetraodon nigroviridis (T. nigroviridis) was classified in the Nox5 subgroup, as shown in Figure 2 (# 47), but the predicted protein does not contain the N-terminal extension with the calcium-binding domain that is present in other Nox5 orthologs (amino acid sequence is shown in Additional file 4). However, the nucleotide sequence around the presumed start codon (gaggcaugc, methinone codon underlined) also did not match to a consensus Kozak sequence [cc(a/g)ccaugg] [52], suggesting that the reported sequence is likely to be incomplete. A Nox5 ortholog (# 45) of zebrafish Danio rerio (D. rerio) also did not contain the presumed start codon and the calcium-binding domain (sequence is shown in Additional file 4), suggesting that the sequence of this Nox5 ortholog is also incomplete. The genome of sea urchin S. purpuratus encodes two Nox5 isologs, Nox5A and Nox5B (Figure 2). The Duox subgroup showed broad expression in Bilateria, such as vertebrates, urochordates, echinoderms, nematodes and insects, but was not found in amoeba, fungi, or plants. Plant Nox homologs, previously termed "respiratory burst oxidase homologues (rboh)" [26], formed a distinct subgroup (Figure 2), and A. thaliana had 10 rboh homologues, suggesting specialized functions or tissue expression.

Noxes representing the NoxA/NoxB subgroup and the NoxC subgroup were present in fungi, and all fungi examined contained both NoxA and NoxB, except for Aspergillus nidulans (A. nidulans), which possessed only a NoxA gene. Yeast [Schizosaccharomyces pombe (S. pombe) and Saccharomyces cerevisiae (S. cerevisiae)] did not possess any Noxes. The domain structure of the NoxA ortholog is similar to that of Nox1-Nox4; whereas, NoxB proteins also have a short N-terminal extension that does not contain any recognizable domains or motifs (Figure 1B). Fungal NoxC, present in M. grisea and Fusarium graminearum (F. graminearum), has an N-terminal EF-hand domain (Figure 1B). The slime mold amoeba D. discoideum, a protozoan that straddles the boundary between animals and plants [53], contained three Nox isologs, NoxA, NoxB, and NoxC (Figure 1B). Amoeba NoxC has an EF-hand domain as well as an N-terminal extension containing an asparagine-rich region ("N", Figure 1B); however, NoxA and NoxB both lack the EF-hand domain (Figure 1B). The EF-hand-containing subfamilies (Nox5, Duox, NoxC, and plant Nox) were the most abundant of the Noxes, comprising well over half of the taxonomic tree (Figure 2). Unlike other members of the NoxC/NoxD family, algal Noxes (#s 100 and 101 in Figure 2) do not contain an EF-hand domain (Figure 1B), but branched from a root shared by amoeba and fungal NoxC (Figure 2). Therefore, we refer to these algal Nox proteins that lack an EF hand domain as NoxD, which together with NoxC form a distinct subgroup. Because they share structural features common to EF hand-containing Noxes, we speculate that that these Noxes may be regulated by an as-yet unknown calcium-binding protein.

Figure 3 Syntenies of Nox/Duox genes. (A) Summary of the occurrence of Nox/Duox genes within eukaryotes. The number indicates the orthologs in each organism. Superscripted letters "a" and "b" represent incomplete amino acid sequences predicted from nucleotide (more ...)

The human Nox3 gene is positioned following TIAM2, TFBIM and CLDN20 on chromosome 6 (Figure 3D), a synteny that was conserved in mammals and chickens Gallus gallus (G. gallus). Puffer fish, fugu T. rubripes and tetraodon T. nigroviridis lacked a Nox3 ortholog, and TIAM2, TFBIM and CLDN20 were followed instead by FILIP1, TMEM30A and COL12A1. In the genome of X. tropicalis, there was greater variation in synteny: TFBIM was present, but neither Nox3 nor other linked markers were seen. A Nox3 gene was not found in the genome of zebrafish D. rerio, but nucleotide fragments encoding these marker genes of D. rerio were too short to demonstrate the absence of Nox3 by syntenic analysis. Thus, Nox3 emerged during evolution sometime after the emergence of fish and amphibians from a common ancestor of birds and mammals.

The synteny of genetic markers surrounding Nox5 was highly conserved in human, dog Canis familliaris (C. familliaris), mouse, rat, chicken, cow Bos taurus (B. taurus), opossum Monodelphis domestica, (M. domestica, a Nox5 ortholog sequence DDBJ™ accession No. BR000304) and frog (Figure 3F). We also found Nox5 gene fragments in rabbit Oryctolagus cuniculus (O. cuniculus) and armadillo Dasypus novemcinctus (D. novemcinctus) draft-sequenced genomes (DDBJ accession No. BR000301 and BR000302, respectively). However, rodents (mouse and rat in this study) lacked Nox5, clearly demonstrating that this gene had been lost. Interestingly, pufferfish T. rubripes and T. nigroviridis had Nox5-like genes, but the gene markers were present on a different scaffold fragment (Figure 3F). Mammals and frog X. tropicalis each had two paralogs of Duox (Figure 3G), while chicken had only one. Fish genomes possessed a single Duox gene that followed a single NIP gene (Figure 3G). Like Duox, the NIP gene has also undergone gene duplication to form NIP1 and NIP2. Interestingly, NIP1 and NIP2 in Figure 3G are identical to DuoxA1 and DuoxA2, respectively, proteins that were recently described to participate in the activation and maturation of Duoxes [56]. Due to the complexity of the tetraploid genomes of zebrafish and incomplete genomic sequence, however, we cannot rule out a second Duox in another chromosomal location.

Figure 4 Loop and segment sizes joining transmembrane regions and canonical NADPH-oxidase domains. After alignment of canonical and TM domains, the numbers of amino acids linking these regions were counted. I to VI in boxes indicate the six predicted TM α-helices, (more ...)

Figure 5 Comparison of molecular clocks of Nox domains.(A) Amino acid sequences were trimmed to the length of human Nox2 as Nox domains, and the numbers of amino acid substitutions per site relative to the human isolog were counted. Each data point represents (more ...)

Figure 6 Identification of amino acid residues conserved in all Nox/Duox proteins. The single letter amino acid code is used, and consensus amino acid residues and locations are identified based on the alignment in Additional file 5. In the column labeled "consensus", (more ...)

Figure 7 Effects of mutations of conserved amino acids on Nox enzymatic activity and formation of the Nox2-p22phox complex. (A) Conserved amino acids (circles) are indicated on a schematic of the Nox domain, and residue numbers corresponding to the human Nox2 (more ...)

Figure 8 Sequences of EF-hand motifs in Nox5, Duox, plant Nox, and NoxC. (A) EF-hand containing Nox/Duox family members are listed and demonstrate the widespread occurrence of these Noxes. Parentheses represent the presence of Nox5 ortholog in the T. nigroviridis (more ...)

Figure 9 Occurrence of Noxes in the phylogenetic tree. A schematic phylogeny of organisms was created from genomic information [49, 53, 67, 91]. Branch lengths are not proportional to divergence. "Mammals" in this figure include H. sapiens, C. familliaris, R. (more ...)

This report provides the first extensive analysis of Nox sequences and synteny throughout evolution and provides a conceptual framework for future structure/enzymatic function studies and for understanding the diversity of biological functions of these enzymes. Molecular taxonomy (Figure 2) revealed seven Nox/Duox subfamilies rather than the three that were previously identified based on the presence or absence of calcium-binding and peroxidase domains [2]. Significantly, Noxes are not present in prokaryotes. One can speculate that while defense enzymes, such as superoxide dismutase, evolved very early to protect aerobic organisms to protect against accidentally-generated ROS, later organisms subsequently developed the capacity to generate ROS in a regulated, "deliberate" manner, with specific regulatory subunits co-evolving with specific Noxes. The earliest Nox2 ortholog seems to have appeared in the sea urchin S. purpuratus. A number of investigators have suggested that sea urchin has phagocytic cells that express an ortholog of the complement component C3 and can phagocytose against invading microbes [68-70]. Although it is unclear whether these cells produce ROS to kill microbes, the taxonomy shown in Figure 2 implies that the sea urchin expresses a Nox2 ortholog that might play a role in the innate immune response.

The synteny of each Nox/Duox member raises new questions about Nox/Duox evolution. For example, Nox3 in mouse inner ear is essential for formation of otoconia, mineralized structures that participate in the vestibular system in perception of gravity [44]. Fish and amphibians also have otoconia (called otoliths in fish) but do not express Nox3 (Figure 3). This may implicate another Nox, for example Nox1 ortholog, in otoconia formation prior to Nox3 appearance in land vertebrates, or it may point to a unique function of Nox3 in land vertebrates. Kiss et al. have suggested that lactoperoxidase (LPO) functions in peroxidation of the lipid envelope of globular substance in the inner ear together with Nox3 [18]. Interestingly, molecular taxonomy of animal heme peroxidases demonstrates that LPO orthologs emerged in birds and mammals, but not in fish (T. Kawahara and J. D. Lambeth, unpublished observation). It implies that Noxes and their physiological partners evolved simultaneously, resulting in gaining a new function. Mosquito has a unique Nox gene, NoxM. Although a physiological function of NoxM is completely unknown, a unique appearance of NoxM gene in the species imply a possible relationship between NoxM and sucking of blood or a playing role as a principal vector of the pathogen. While the function of Nox5 is not yet understood, its loss in rodents suggests that another Nox may compensate in these species, implying a certain degree of plasticity of Nox isoform function. Alternatively, Nox5 may not perform an essential function, at least in short-lived species.

It is of interest to compare residue substitution rates among Nox isologs, since all members possess fundamentally similar structures in their flavocytochrome domains, including a high degree of conservation of binding sites for prosthetic groups. Substitution rates vary among different proteins, such as EGF (~ 2.5), NGF (~ 1.0), lactate dehydrogenase (~ 0.5), cytochrome c (~ 0.3), and histone H3 (~ 0.014) [71]. The substitution rates of Nox/Duox subfamilies ranged from 0.3 ~ 0.7 (Figure 5); whereas, those of p22phox, organizer proteins (p47phox and NOXO1), and activator proteins (p67phox and NOXA1) were 0.5 ~ 1.2. The substitution rates of Nox2 and its regulatory subunits, p47phox and p67phox, were relatively low, implying evolutionary changes in these proteins are more poorly tolerated. Such a result may be explained by the importance of the biological function of Nox2 in host defense and by the stringent regulation of this enzyme system to prevent inappropriate activation leading tissue damage [5,58,59]. In addition, the Nox2 system requires multiple protein interactions among catalytic and regulatory subunits with upstream regulatory subunits and lipids, and these undoubtedly impose strict limitations on the number of tolerated mutations. Although incompletely understood, substitution data point to the critical nature of Duox functions, since changes in the Duox sequence are also poorly tolerated over evolutionary time (Figure 5). Duoxes are implicated in thyroid hormone biosynthesis [43] and innate immunity in lung [72], and are distributed in a variety of other tissues where they perform unknown functions. Duox is also implicated in fertilization in sea urchin, where H₂O₂-supported cross-linking of fertilization envelope proteins prevents polyspermy [73].

In contrast to other eukaryotes, yeast (S. pombe and S. cerevisiae) did not possess Noxes. Yeast ferric reductase (Fre) has a domain structure similar to Noxes but participates in iron uptake rather than oxygen reduction [74]. Alignment between human Nox2 and Fre proteins demonstrates that the sequence of Fre proteins is very different from the Noxes except for the VXGPFG-motif and NADPH-binding site residues (see Additional file 9). This suggests that this distant homolog has evolved in yeast to carry out an entirely different function, and it is debatable whether it should even be classified with the Nox family.

In addition to binding residues for prosthetic groups, the present study has identified four additional regions (B-loop, TM6-FAD, VXGPFG-motif, and C-terminus) as critical for function in all Noxes. The specific functions of these regions are not yet fully understood; however, mutational analysis demonstrates their importance (Figure 7B). The B-loop (Arg-80) and TM6-FAD (Gly-322) regions of Nox2 appear to participate directly or indirectly in binding to p22phox (Figure 7C), since their mutation prevented co-immunoprecipitation of Nox2 and p22phox. In addition, these mutations prevented glycosylation of Nox2 to form the mature 91 kDa form of the protein, supporting the concept that association with p22phox is a necessary pre-requisite for glycosylation [75]. Nevertheless, these two amino acid residues are also conserved in Noxes that do not require p22phox (e.g., human Nox5). Thus, these residues might mediate another important interaction in Nox5 that is analogous to that with p22phox, and ongoing studies are investigating the roles of these residues in Nox5 function. The presence of non-canonical, but highly conserved residues and regions that are shown in Figure 7A suggests that Noxes might have an unknown common feature relevant to the mechanism of activation or a common biological function. Moreover, these conserved regions may also provide a key to identifying a novel common molecule that interacts and co-operates with all Nox/Duox proteins.

In summary, we report herein an exhaustive analysis of Nox/Duox protein family. The present studies provide a new molecular classification system in which Nox and Duox proteins are organized into seven distinct subfamilies. These studies also identify Nox3 as the most recently emerged Nox. Calcium-regulated, EF-hand-containing Noxes appeared very early during the evolution of eukaryotes. Two mosquitoes possess a unique Nox gene, NoxM, but not fruit fly or ant. Consistent with the physiological importance of Nox2 in innate immunity and Duox for hormone synthesis and host defense, these two Nox proteins are more stringently conserved of all Noxes. By comparison of amino acid sequences, 68 residues were identified as highly conserved among all Nox/Duox orthologs, and the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity. Thus, this report provides a conceptual basis for understanding the evolutionary history of Noxes and Duoxes and provides key structural information relevant to the activation mechanisms of modern Nox/Duox proteins.

In addition to gene accession numbers that have been published in papers cited above [26,28,30,31,33,39,41,42], existing homologues/orthologs of Nox/Duox were searched using NCBI HomoloGene [76]. Human Nox1-Nox5, Duox1, and Duox2; dog Nox1, Nox4, Duox1, and Duox2; mouse Nox1-Nox4, Duox1, and Duox2; rat Nox1-Nox4, Duox1, and Duox2; chicken Nox3; and fruit fly Nox5 and Duox amino acid sequences were obtained from GenBank, and accession numbers are shown in Additional file 4. BLASTP searches were performed for amino acid sequences predicted computationally from genomes of C. familliaris, R. norvegicus, M. musculus, D. novemcinctus, O. cuniculus, G. gallus, X. tropicalis, D. rerio, T. rubripes, T. nigroviridis, O. latipes, C. intestinalis and C. elegans [55]. Sequences that had >50% identity to the sequence of the closest template were selected.

To identify the Nox/Duox orthologs of sea urchin S. purpuratus and insects (D. melanogaster, A. mellifera, A. gambiae), BLASTP searches were performed using the NCBI sea urchin protein database [77] and the NCBI Eukaryotic Genome Database [78], respectively. Fungi Genome BLAST and NCBI BLASTP [51] and DictyBase BLASTP [79] were used to search for Nox homologs in fungi (P. anserina, A. nidulans, M. grisea, and F. graminearum) and amoeba (D. discoideum), respectively. The presence of 10 genes encoding A. thaliana Nox homologs has been predicted [42], which was confirmed by searching the TIGR A. thaliana Protein Database [80]. Sequences and accession numbers of assembled Nox orthologs are listed in Additional file 4, and information on additional database searching is described in Additional file 12. Assembled sequences, including newly defined or previously mis-annotated sequences, were annotated based on molecular taxonomy analysis of the Nox domain (Figure 2), and these sequences were deposited as third party annotation (TPA) sequences in GenBank/EMBL/DDBJ database (accession No. BR000261–BR000304). All amino acid sequences analyzed in this study are shown in Additional file 4 (Nox/Duox proteins) and Additional file 9 (Fre proteins).

To estimate evolutionary substitution rates, we screened orthologs of the regulatory subunits, p22phox, p47phox, NOXO1, p67phox, and NOXA1 in vertebrates (H. sapiens, C. familliaris, R. norvegicus, M. musculus, G. gallus, X. tropicalis, D. rerio, T. rubripes, T. nigroviridis) using servers described above. In this study, partial Nox/Duox or regulatory subunit genes, which lacked either a presumed start codon or a stop codon, were assumed to be intact if they showed >50% identity to the closest homolog. All amino acid sequences of the regulatory subunits analyzed in this study are shown in Additional file 6.

Nox, NADPH oxidase: Duox, dual oxidase; ROS, reactive oxygen species; rboh, respiratory burst oxidase homologue; TM, transmembrane; CGD, Chronic Granulomatous Disease; HLH, helix-loop-helix; Myr, 10⁶ years; PMA, phorbol 12-myristate 13-acetate; WB, Western blotting; IP, immunoprecipitation; mAb, monoclonal antibody.

The majority of this work here described was planned and carried out by TK in collaboration with MTQ. JDL critically revised the manuscript for important intellectual content and data analysis. All authors read and approved the final manuscript.

We express our appreciation to Dr. Al Jesaitis for the generous gift of monoclonal antibody against human p22phox and to Dr. Yasushi Okamura for suggesting of sea urchin genome research. We are grateful to Drs. Dirk Roos, Eunice Laurent and Mrs. Heather Jackson for reading and commenting on the manuscript. We thank Dr. Andreas Fritz and Mr. Robert Esternberg for their advice and help to search for zebrafish orthologs. We also thank Mrs. Asami Kawahara for great cooperation and patience during long nights and weekends of computational analysis at home. This work was supported by NIH grants CA105116, GM067717, AR42426, and RR020185.