neural plate marker at the bud stage and midbrain marker at the 15-somite stage [22]; 5) pax6b, an anterior neural plate marker at the bud stage [23]; 6) pax2a, a midbrain hindbrain boundary and otic vesicle marker [24]; and 7) krox20, a third and fifth rhombomere marker in the hindbrain [25]. Ectopic expression of IGF-2a did not alter the anterior neural plate patterning at the bud stage, as indicated by the similar expression patterns of otx2 (Fig. 6, panels A, B, E, and F) and pax6b (Fig. 6, panels C, D, G, and H) between the GFP mRNA-injected control and IGF-2a mRNA injected embryos. Nor did IGF-2a expression alter the forebrain patterning, as indicated by the similar emx1 expression patterns (Fig. 6, panels M and N). Expression of IGF-2a caused marked changes in pax2a expression patterns. As shown in Fig. 6, panel I, in the control embryos, pax2a mRNA was detected in the midbrain and hindbrain boundary (a V shaped domain) at the bud stage. In the IGF-2a mRNA-injected embryos, however, pax2a mRNA expressing cells remained as two separate domains at both stages (Fig. 6, panels J and V), suggesting a defect in the midline/ notochord development. This effect appeared to be specific to the anterior as the pax2a expression in the otic vesicles was normal (Fig. 6, panel V). There were also notable changes in krox20 mRNA expression at the bug stage. While the krox20 mRNA expression in the third and fifth rhombomere already fused in the midline in the control embryos (Fig. 6, panel K), they remained as two separate domains in the IGF-2a mRNA-injected embryos (Fig. 6, panel L). At the 15-somite stage, no significant difference was seen with emx1 expression or otx2 mRNA expressing, although the otx2 mRNA expressing domain tended to be smaller (Fig. 6, panels M–P). Likewise, IGF-2a overexpression did not alter the expression patterns of rx1 or rx2 mRNA expressing pattern (Fig. 6, panels Q–T). At this stage, pax2a mRNA is expressed in both in the midbrain and hindbrain boundary and the otic vesicles [24 and Fig. 6, panel U]. Interestingly, at this advanced stage, pax2a mRNA expressing cells remained as two separate domains in the IGF-2a mRNA-injected embryos at both stages. This effect appeared to be specific to the brain region as the pax2a expression in the otic vesicles was normal (Fig. 6, panel V). At this stage, the krox20 mRNA expressing cells in the third and fifth rhombomere of the hind brain already fused in the midline in the control embryos (Fig. 6, panel W), while they remained separated domains in the IGF-2a mRNA-injected embryos. Additionally, the distance between the third and fifth rhombomere was shortened in the IGF-2a mRNA-injected embryos (Fig. 6, panel X). These results suggest a possible defect in midline patterning in the IGF-2a mRNA-injected embryos. To further investigate the possible defects in midline patterning, we performed whole mount in situ hybridization analysis using two midline specific markers, shh [26] and ntl [27], as well as two skeletal muscle markers, myoD [28]and myogenin [29]. As shown in Fig. 7, panels A–D, the expression domain of ntl mRNA at the bud stage was markedly expanded in the IGF-2a mRNA-injected embryos compared to the controls. Among the IGF-2a mRNA- injected embryos, 65% (n = 17) showed marked enlargement in the ntl mRNA expression domain. At the 15-somite stage, a large number of the IGF-2a mRNA-injected embryos showed abnormal ntl mRNA expression patterns (Fig. 7, panel F) and some even had double or partial double notochords (Fig. 7, panel G). Likewise, 71% (n = 21) of the IGF-2a mRNA-injected embryos had enlarged shh mRNA expression domains (Fig. 7, panel M) and 24% had double or partial double notochords (Fig. 7, panel N). In agreement with the enlarged notochord, we found that overex- pression of IGF-2a increased the space between the myoD (Fig. 7, panels J and K) and myogenin (Fig. 7, panels O and P) expressing Multiple IGF-1 and -2 Genes somites at the 15-somite stage. This is most evident in the anterior region of an embryo. Taken together, these results suggest that an excess of IGF signaling causes defects in the midline formation and an expansion of the notochord during early embryogenesis. Discussion In this study, we have provided evidence suggesting that there are four distinct IGF genes in the zebrafish genome and all four IGF genes are expressed and functional. Although a number of partial or full-length cDNAs encoding several zebrafish IGF peptides have been documented recently, to our knowledge, our study is the first to characterize the structure of these zebrafish IGF genes, map their chromosomal loci, and identify their major transcripts. We show that zebrafish igf-1a has only 1 type of E- domain sequence (Ea like) but two distinct 39UTRs. All other zebrafish igf genes have one 39UTR. Both zebrafish igf-2a and igf- 1b have two distinct 59-UTR sequences, which result in two different transcription initiation sites and different signal peptides. Alternative splicing has been previously reported for mammalian IGF-1 and IGF-2 genes [30,31]. For example human IGF-1 has 3 distinct transcripts, which result in 3 distinct types of E-domains (Ea, Eb and Ec) [10,32]. Among the four IGF genes, igf-2a and -2b are clearly the orthologous to human IGF-2 and igf-1a is the IGF-1 ortholog. This conclusion is supported by phylogenetic analysis, detailed structural analysis, and synteny analysis results. The identity of the fourth IGF gene, however, is more enigmatic. This IGF peptide is the same to the so-called IGF-3 recently cloned from Tilapia by Wang et al. [13] recently. Zebrafish IGF-1b/IGF-3 is considerably different from the other 3 zebrafish IGF peptides. Its sequence identity with human IGF-1 is considerably lower (52%) compared to that of zebrafish IGF-1a (82%). Phylogentic analysis has placed zebrafish IGF-1b in a distinct group, independent from the IGF-1 and IGF-2 subgroups. The synteny relationship of zebrafish igf-1b to the human IGF-1 is also fairly weak. We considered two possible hypothetical scenarios accounting for the origin of this particular zebrafish igf. One is that it represents one of the IGF-1 duplicates but it has diverged significantly from other igf genes after gene duplication due to its restricted expression in the gonads. The other is that it is a novel IGF as proposed by Wang et al. (2008) [13]. We favor the first scenario because it is the simplest way to interpret the data. This would mean that zebarfish igf-1b has diverged at a faster rate than the other IGF paralogs. Indeed, recent comparative and evolutionary biology studies using large numbers of duplicated genes in T. rubripes, D. rerio, T. nigroviridis and O. latipes have shown several cases of asymmetrical acceleration of evolutionary rate among the duplicates after the duplication [33,34,35]. If the second scenario were true, it would mean that one of the IGF-1 duplicates was lost after the genome duplication event and another novel IGF gene was born. Our conclusion is also in agreement with the notion that ray-finned fish have experienced a third whole-genome duplication (3R) approx- imately 350 million years ago. Therefore, we propose to term this gene as igf-1b and the encoded peptide IGF-1b. Our finding that there are 4 igf genes in zebrafish and that they each encode a functional IGF ligand is interesting when considering the evolution of the insulin/IGF gene family. There are a number of published studies on the evolution of this important family. Chan et al. (1990) has characterized an insulin-like molecule from the protochordate amphioxus [2]. This molecule contains features characteristic of both vertebrate insulin and IGF, and therefore may represent a modern version of the ancestral molecule for the insulin/IGF gene family [2]. In the Atlantic hagfish, an agnathns, 2 PLoS ONE | www.plosone.org 6 September 2009 | Volume 4 | Issue 9 | e7026