Multiple IGF-1 and -2 Genes Figure 7. Overexpression of IGF-2a results in abnormal notochord development. ntl expression in the notochord at the bud stage in a control embryo (panels A and C) and an IGF-2a mRNA injected embryo (panels B and D). ntl expression in the notochord at the 15-somite stage in a control embryo (panel E) and IGF-2a mRNA injected embryos (panels F and G). Among the IGF-2a mRNA injected embryos, 65% showed the expression pattern shown in panel F and 35% showed that in panel G (n = 17). myoD expression in the somite at the bud stage in a control embryo (panel H) and an IGF-2a mRNA injected embryo (panel I). myoD expression in the somite at the 15-somite stage in a control embryo (panel J) and an IGF-2a mRNA injected embryo (panel K). shh expression in the notochord at the 15-somite stage in a control embryo (panel L) and IGF-2a mRNA injected embryos (panels M and N). Among the IGF-2a mRNA injected embryos, 71% showed expression pattern shown in panel M and 24% showed that in panel N (n = 21). myogenin expression in the somite at the 15-somite stage in a control embryo (panel O) and an IGF-2a mRNA injected embryo (panel P). Panels A, B, E–G, and L–N are dorsal view with head up; panels C and D are vegetal view with dorsal the the right; panels H–K, O, and P are dorsal views with head to the left. doi:10.1371/journal.pone.0007026.g007 members of the IGF/insulin gene family have been identified - one is an insulin ortholog and the other is IGF-like. The hagfish IGF is equally related to human IGF-1 and IGF-2, and may therefore be a prototypical IGF molecule [3]. Duguay et al. (1995) have shown that the spiny dogfish, an elasmobranch species, has an IGF-1-like molecule and an IGF-2-like molecule, suggesting the prototypical IGF molecule diverged in an ancestor of the extant ganathostomes [4]. A large number of studies have shown that two distinct IGF genes (IGF-1 and IGF-2) and an insulin gene are present in most vertebrates, including birds, and mammals [1]. Based on the previous studies by others and the current study, we speculate that a single ancestral insulin/IGF molecule is present in protochordate animals. The first genome duplication (1R) that presumably occurred 500 million years ago in metazoans, might have resulted in the appearance of a distinct insulin molecule and an IGF molecule in agnathans. The second round whole-genome duplica- tion (2R) that occurred 400 million years ago, might have given the rise to separate IGF-1 and IGF-2 genes. The ‘‘fish specific’’ or third round whole-genome duplication (3R) that occurred approximately 350 million years ago might have resulted in two distinct igf genes in zebrafish and probably other teleost fishes (Fig. 8). The retention of these IGF genes in the zebrafish genome suggests that each of them may have evolved unique and indispensable roles. This notion is supported by a recent study reporting distinct embryonic roles of zebrafish IGF-2a and -2b [12]. In this study, we provide further evidence that these zebrafish IGF genes have evolved overlapping yet distinct biological properties by attaining distinct temporal expression patterns, different peptide structure, and different biological activities (Fig. 8). The duplicated igf-1 and duplicated igf-2 appear to have diversified in several ways. First, each of these IGF ligand genes exhibits distinct spatial and temporal expression patterns. During early development, IGF-1b and IGF-2a mRNAs are detected throughout the embryogenesis, while IGF-1a and IGF- 2b mRNAs are detectable only after the gastrula stage. In the adult, IGF-2a mRNA is found in all tissues examined with the exception of the muscle and gill, while IGF-2b mRNA is only expressed in the liver. In comparison, IGF-1a mRNA is detected in the liver, brain, and eyes. IGF-1b mRNA, on the other hand, is exclusively expressed in the ovary and testes. Furthermore, these 4 IGF peptides have clear differences in their primary structure and biological activities. When tested in zebrafish embryos by mRNA injection experiment, all resulted in the activation of Akt signaling and had biological effects in developing zebrafish embryos. These findings make an important advance in our understanding of the structural and functional evolution of the IGF/insulin family. We have previously reported that zebrafish contain two functional igf-1r genes [5,16,36], two functional igfbp-1, two igfbp-2, and two igfbp-5 genes in zebrafish [37,38]. In this study, we have shown that there are two functional igf-1 and two igf-2 genes in zebrafish. Therefore, it appears that most, if not all, components in the IGF signaling pathway are retained as duplicates in the zebrafish genome. The retention of these IGF genes indicates that each of them may have evolved unique and indispensable roles. One intriguing finding made in this study is that after duplication each pair has evolved very distinct expression profiles. While zebrafish IGF-1a mRNA is easily detected in multiple adult tissues in zebrafish, IGF-1b mRNA is only found in the gonad. Likewise, IGF- 2b mRNA is only detected in the liver whereas IGF-2a mRNA is PLoS ONE | www.plosone.org 8 September 2009 | Volume 4 | Issue 9 | e7026