molecular and functional analyses suggest that these IGF genes have undergone subfunctionalization partitioning at the levels of gene expression, protein structure, and biological activities. Furthermore, taking advantage of the amenability of the zebrafish model, we unraveled a previously unrecognized role of IGF in regulating midline and notochord development during embryogenesis in vivo. Results Identification and characterization of 4 genes encoding distinct IGF peptides in zebrafish By searching available zebrafish databases and performing 59 and 39RACE experiments, we have identified 4 genes and a number of full-length cDNAs encoding 4 structurally distinct IGF- like peptides (IGF-1a, -1b, -2a, and -2b). The structure of these zebrafish igf genes and their transcripts are shown in Fig. 1. Zebrafish igf-1a has 5 exons and 4 introns and it spans approximately 17 kb. Two distinct igf-1a mRNA transcripts (T1, 1509 bp and T2, 2008 bp) were found (Fig. 1A). These igf1a transcripts have an identical open reading frame of 483 bp encoding a polypeptide of 161 amino acids (a.a.). This peptide can be divided into a 44 a.a. putative signal peptide, a 29 a.a. B domain, a 12 a.a. C domain, a 21 a.a. A domain, a 8 a.a. D domain, and a 47 a.a. E domain. T1 and T2, likely resulted from alternative splicing, have distinct 39 UTR of 823 and 1322 bp, Multiple IGF-1 and -2 Genes respectively (Fig. 1A). Zebrafish igf-1b (9.1 kb and 5.9 kb) has 4 exons and it has 2 different IGF-1b transcripts (T1, 1269 bp and T2, 1209 bp). The full-length igf-1b T1 includes an open reading frame of 513 bp, which encodes a polypeptide of 171 a.a. (containing a 25 a.a. signal peptide, a 76 a.a. E domain, and the B–C–A–D domain). The igf-1b T2 differs from T1 only in the 59 UTR and a portion of the signal peptide (Fig. 1A). Zebrafish igf-2a has 4 exons and spans 5.9,70.8 kb DNA (Fig. 1B). Two igf-2a transcripts (1727 and 1723 bp) were identified. igf-2a T1 and T2, likely resulted from alternative splicing sites in the exon 1 and exon 2, differ in the 59 UTR and part of the signal peptide region. The open reading frame for igf-2a T1 and T2 are 636 bp and 669 bp, respectively. They encode polypeptides of 212 and 223 a.a., which containing a 48/59 a.a. signal peptide, a 29 a.a. B domain, an 11 a.a. C domain, a 21 a.a. A domain, a 7 a.a. D domain, and a 96 a.a. E domain (Fig. 1B). Zebrafish igf-2b has 4 exons and spans about 5.9 kb. Only one transcript was found for this gene. The full-length cDNA is 2066 bp. It contains an open reading frame of 591 bp encoding a polypeptide of 197 a.a., including a 40 a.a. signal peptide, a 28 a.a. B domain, a 9 a.a. C domain, a 21 a.a. A domain, a 7 a.a. D domain, and a 92 a.a. E domain (Fig. 1B). The 4 zebrafish IGF genes and their various mRNAs encode 4 distinct mature IGF peptides, IGF-1a (70 a.a.), IGF-1b (70 a.a.), IGF-2a (68 a.a.), and IGF-2b (65 a.a.). These peptides are highly similar to human IGFs in their primary structure (Fig. 2A). Like Figure 1. Molecular characterization of four zebrafish igf genes and their multiple transcripts. (A) Structure of the zebrafish igf-1a and -1b genes. Signal peptide and E domain regions are shown in grey boxes and the B–C–A–D domains of the mature peptide are shown in dark boxes with the number of amino acids indicated. 39 and 59-UTRs are shown in bold lines with the number of base pairs shown (bp). Introns sequences are shown in dashed lines and are indicated in kb. Two alternative splicing transcripts (T1 and T2) are found in the 39-UTR of igf-1a. Two alternative splicing transcripts are also found in the 59-UTR and signal peptide region of igf-1b. (B) Structure of the zebrafish igf-2a and igf-2b. Two alternative splicing transcripts are found in the 59-UTR and signal peptide region of igf-2a. doi:10.1371/journal.pone.0007026.g001 PLoS ONE | www.plosone.org 2 September 2009 | Volume 4 | Issue 9 | e7026