Multiple IGF-1 and -2 Genes Figure 2. Structural and phylogenic analysis of four zebrafish IGFs. (A) Sequence alignment of mature zebrafish (zf) IGF-1a, IGF-1b, IGF-2a, IGF-2b and mature human (h) IGF-1 and IGF-2. The B–C–A–D domains are labeled. Star indicates conserved cysteine. (B) Phylogenetic analysis of the vertebrate insulin/IGF family members. Amino acid sequences in the B and A domains were analyzed by the neighbor-joining method using the MEGA3 program [64]. Gaps were removed from the alignment. Bootstrap values derived from 1,000 runs are shown. Relaxins are used as an outgroup. Accession numbers for the sequences used are: human (IGF-1, NM_000618; IGF-2, NM_000612; insulin, NM_000207; relaxin, NM_080864), mouse (IGF-1, NM_010512; IGF-2, NM_010514, insulin 1, NM_008386; insulin 2, NM_008387; relaxin, NM_173184), Xenopus (IGF-1, M29857; IGF-2, BC070545), zebrafish (insulin a, NM_131056; insulin b, NM_001039064), common carp (IGF-1, EF536889; IGF-2, AF402958; insulin, X00989), trout (IGF-1, EF432852; IGF-2, EF432854; insulin, M21170), tilapia (IGF-1, AF033796; IGF-2, AF033801; IGF-3, EU272147; insulin, AF038123 ), shark (IGF-1, Z50081; IGF-2, Z50082), hagfish (IGF, M57735; insulin, V00649 ), sea lamprey (IGF, AB081462), Amphioxus insulin-like peptide mRNA (ILP, M55302). Sequence for Tetraodon, fugu and stickleback IGF-1, IGF2 and insulin were obtained by searching the Tetraodon genome (http://www.genoscope.cns.fr/externe/English/Projets/ Projet_C/C.html), fugu genome (http://fugu.hgmp.mrc.ac.uk/), and stickleback genome (http://www.ensembl.org/ Gasterosteus_aculeatus/). Sequence of sea lamprey insulin was obtained by searching sea lamprey genome (http://pre.ensembl.org/Petromyzon_marinus/). (C) Conserved synteny between human (Hs) and zebrafish (Zf) IGF loci. Vertical gray lines indicate a group of genes on the same chromosome, with order ignored to facilitate the comparison of orthologs and paralogs. Horizontal gray lines connect presumed orthologs within chromosome groups as well as paralogs between chromosome groups. doi:10.1371/journal.pone.0007026.g002 human IGFs, all 4 zebrafish IGF peptides consisted of 4 domains (B–C–A–D). They all possess 6 conserved cysteine residues: 2 in the B domain and 4 in the A domain region. Phylogenetic analyses grouped all four zebrafish IGF peptides into the IGF clade, indicating that they are IGF like molecules rather than insulin or relaxin related peptides (Fig. 2B). Zebrafish IGF-2a and IGF-2b are clearly more closely related to human IGF-2 because they share equally high sequence identities with that of human IGF-2 (76% and 75%, respectively) and a 70% sequence identity with each other. Phylogenetic analysis also grouped them into the IGF-2 subgroup. Zebrafish IGF-1a and IGF-1b are more divergent because they are only 50% identical with each other. While zebrafish IGF-1a shares 81% sequence identity to that of human IGF-1, the sequence identity between zebrafish IGF-1b and human IGF-1 is much lower (52%). Phylogenetic analysis also placed IGF- 1b in a separate group - together with the recently reported tilapia IGF-3 [13] (Fig. 2B). The duplicated zebrafish igf-2 genes share strong synteny with the human IGF-2. As shown in Fig. 2C, zebrafish igf-2a and igf-2b are located on LG 25 and 7, respectively. There are a number of genes on LG25 and 7 whose orthologs are located on human chromosome 11, where the human IGF-2 is found. Zebrafish igf-1a is located on LG4. There are five other zebrafish genes (rpl18a, mdm2, impo, idhb, and znf10) in this region whose orthologs are located on human chromosome 12, where the human IGF-1 resides. The zebrafish igf- 1b gene, on the other hand, is located on LG8. Although we were able to find 1 gene (COL2a1) in this region whose orthologous gene is located on human chromosome 12, 8 other zebrafish genes (nras, PLoS ONE | www.plosone.org 3 September 2009 | Volume 4 | Issue 9 | e7026