Figure 8. A proposed model for the evolution of multiple IGF genes in zebrafish. It is postulated that an insulin-like gene (ILP) identified in Amphioxus is the likely ancestor of modern vertebrate insulin/IGFs [2]. This ancestral gene subsequently duplicated to form distinct insulin (INS) and IGF, as found in early agnathan vertebrates [3]. The second round (2R) of whole-genome duplication gives rise to distinct IGF-1 and IGF-2 genes found in all gnathosomes [4]. The ‘‘fish specific’’ third round (3R) whole-genome duplication results in four distinct IGF genes, as found in zebrafish in this study. The duplicated IGF genes have evolved overlapping yet distinct functions by attaining differential regulatory mechanisms of gene expression, different peptide structure, and different biological activities. doi:10.1371/journal.pone.0007026.g008 expressed in most adult tissues. During embryogenesis, IGF-1b and IGF-2a mRNAs are detected from the 1-cell stage and throughout embryogenesis, whereas IGF-1a and IGF-2b mRNAs become detectable after the gastrula stage. In mammals, IGF-1 mRNA is highly expressed in the adult liver under the control of growth hormone (GH). IGF-1 mRNA is also expressed in many non-hepatic tissues and that extra-hepatic sources of IGF-1 can compensate for the absence of hepatic IGF-1 production in mice [39]. In juvenile and young salmon and trout, detectable amounts of IGF-1 mRNA are found in virtually all tissues examined, but the liver has the highest levels, suggesting that the liver is the major site of IGF-I mRNA production, as is the case in mammals and birds [40,41,42]. There is good evidence that GH regulates IGF-1 expression in teleost fish [43,44,45,46]. In mammals, IGF-2 is an important fetal growth factor and the IGF-2 gene is highly expressed in the mammalian fetus [47]. In rodents, the IGF-2 gene is highly expressed in the fetus [47]. Similarly, abundant IGF-2 mRNA is detected in fish embryos and larva [48]. Considerable interspecies variations are found during postnatal or posthatching regulation of IGF-2 gene expression. In the rat, the IGF-II gene expression declines after birth in all tissues except for choroid plexus and leptomenings, whereas in humans and guinea pigs levels of IGF-II mRNA remain high in many adult tissues [47]. Among the 4 zebrafish IGF genes, IGF-1a and IGF-2a are expressed in multiple adult tissues in zebrafish, resembling the profiles of their mammalian orthologs. In contrast, zebrafish IGF-1b mRNA is detected only in the gonad and IGF-2b mRNA in the liver. These data suggest that the duplicated IGF genes have undergone subfunction partitioning in their expression. The finding that igf-2b is exclusively expressed in the liver of adult zebrafish is intriguing in light of the previous reports that Multiple IGF-1 and -2 Genes IGF-2 mRNA levels are high in the adult liver and other tissues in rainbow trout and seabream [42,48] and that GH treatment significantly increases the IGF-2 mRNA levels in the liver in rainbow trout [46]. This raises the interesting possibility that IGF- 2a may be a major form of IGF ligand in the circulation in adult zebrafish. The gonad-specific expression of igf-1b in zebrafish is in good agreement with a recent report in tilapia [13]. IGF-1 has been shown to act in a paracrine fashion to regulate ovarian steroidogenesis in humans [49]. IGF-1 plays an important role in mammalian testicular function. Fish ovaries express IGF-1 mRNA [44,50] and have specific binding sites for IGF-1[51]. IGF-I binding to fish ovaries activates receptor tyrosine kinase activity and increases gonodotropin II-stimulated steroidogenesis in salmon granulosa cells [51]. A role of IGF-1 in inducing final oocyte maturation has also been demonstrated in fish [52]. The discovery of a gonad-specific IGF gene in teleost fish highlights the importance of local IGFs in the gonad and opens new avenues for future studies. IGF signaling is widely considered a key growth regulator in fetal and postnatal stages [1,53]. This dogma is primarily derived from mouse genetic studies. Overexpression of IGF-1 in mice increases the body weight by 30% [54]. IGF-I or IGF-II knockout mice had birth weights about 60% that of their littermates, while IGF-I/IGF-II double knockout mice were even smaller, weighing only 30% of their littermates [55,56]. The IGF-IR knockout mice had a birth weight 45% of their littermates [55,56]. The importance of IGFs in growth is also supported by the growth- retarded phenotype resulting from mutations in human IGF-I or IGF-IR genes. A homozygous partial deletion in the human IGF-I gene is associated with fetal and postnatal growth retardation [57]. Patients with point mutations in the IGF-IR gene exhibited severe intrauterine growth restriction and poor postnatal growth [58]. Recent studies in lower vertebrates suggested that IGF signaling may play additional roles during early development. In Xenopus, ectopic expression of IGF-2 was shown to induce anterior neural tissues [59]. Inhibition of IGF signaling using a dominant-negative IGF-IR or antisense morpholino reduced anterior tissues in Xenopus [59,60]. Contradictory evidence has been reported on this issue in zebrafish. Eivers et al. (2004) reported that overexpression of a secreted form of IGF1R (a truncated IGF-1R lacking both the transmembrane and intracellular domains) in zebrafish resulted in defects in anterior neural structure and overexpression of IGF-1 resulted in expansion in anterior structures at the expense of trunk and tail [61]. Loss-of-function studies in zebrafish, however, showed that suppression of IGF signaling by a dominant-negative IGF-IR or antisense morpholinos did not result in any anterior/ neural patterning defects [16,36]. Likewise, knockdown of zebrafish igf-2a and/or igf-2b did not cause defects in anterior neural patterning [12]. In this study, we have shown that an excess of IGF signaling did not cause notable anterior defects in zebrafish. These mRNAs encoding various IGF ligands were clearly functional because they induced Akt phosphorylation and caused morphological changes in developing zebrafish embryos. The lack of notable anterior/neural patterning defects in zebrafish revealed by previous loss-of-function studies [12,16,36] and the current gain-of-function study strongly argue against a critical role for IGF signaling in anterior/neural induction in zebrafish. A major finding made in this study is that IGF signaling plays a critical role in midline and notochord development. When tested in zebrafish embryos by mRNA injection experiments, all 4 zebrafish IGFs resulted in delayed mesoderm involution, poorly formed notochords and widened distance between somites. Our results indicate that ectopic expression of zebrafish IGF-2a or any IGF ligands, resulted in defects in the midline formation and an PLoS ONE | www.plosone.org 9 September 2009 | Volume 4 | Issue 9 | e7026