notch2, aad29607, sort1, gstm3, atp5f1, mps1, and ghb1) on LG8 have orthologs located on human chromosome 1 (Fig. 2C). All 4 zebrafish igf genes are functional and they exhibit divergent expression patterns To determine whether all 4 zebrafish igf genes are functional, capped mRNA encoding each of the zebrafish IGF genes was generated and delivered to zebrafish eggs by microinjection. Their effects on Akt phosphorylation were analyzed by Western blot. Akt is a major downstream effector of the IGF1R in mammals and in zebrafish [19]. Western blot analysis indicated a marked increase in the levels of phosphorylated Akt in all IGF mRNA-injected groups compared to the GFP mRNA-injected controls at both shield and 15 somite stages (Fig. 3A). When quantified and expressed as the ratio of phospho-Akt/total Akt, all of the 4 IGF mRNAs caused significant increases (Fig. 3B), indicating that they are all capable of interacting with IGF-1R and activating the intracellular signaling cascade. Next, the temporal and spatial expression patterns of the 4 zebrafish igf genes were examined. As shown in Fig. 4A, IGF-1a mRNA was not detectable by RT-PCR in early embryogenesis. IGF-1a mRNA became detectable at the gastrulation stage (around 90%-epiboly). Its level gradually increased after 48 hpf. Likewise, IGF-2b mRNA was detected at 50%-epiboly and was maintained at relatively high levels thereafter (Fig. 4A). In comparison, both IGF-1b and IGF-2a mRNAs were easily Figure 3. All four zebrafish igf genes are functional. (A) Microinjection of each of the 4 zebrafish IGF mRNA into zebrafish embryo resulted in elevated Akt phospholylation. GFP, IGF-1a, -1b, -2a, and -2b mRNA was injected into zebrafish embryos at 1-2 cell stage. The injected embryos were raised to shield and 15 somite stages. Lysates were prepared and subjected to SDS-PAGE followed by immunoblot analysis using antibodies against total and phosphorylated Akt (Akt-P). (B) Densitometric analysis result of (A). The phosphorylated Akt/total Akt ratio was calculated and expressed as % of the control (GFP mRNA injected) group. Grey bars represent the shield stage groups, and dark bars represent the 15 somite stage groups. Values are means6SE (n = 3). *p,0.01 compared with the control group. doi:10.1371/journal.pone.0007026.g003 Multiple IGF-1 and -2 Genes Figure 4. Temporal and spatial expression patterns of the duplicated zebrafish IGF genes. (A) RT-PCR analysis of IGF mRNAs in zebrafish embryos at the indicated stages. Numbers are the different embryo developmental stages (1, 1–2 cell; 2, 32-cell; 3, 512-cell; 4, oblong; 5, 30%-epiboly; 6, 50%-epiboly; 7, 90%-epiboly; 8, tailbud; 9, 2-somite; 10, 12-somite; 11, 18-somite; 12, 24–26 somite; 13, Prim-6; 14, Prim-22; 15, 48hpf; 16, 72hpf; 17, 96hpf). (B) RT-PCR analysis of IGF mRNAs in female and male adult zebrafish tissues. doi:10.1371/journal.pone.0007026.g004 detected in all stages of embryogenesis, ranging from the 1-cell stage to 4 dpf larvae (Fig. 4A). These data suggest that IGF-1b and -2a transcripts are maternally deposited, while IGF-1a and -2b mRNAs are the products of the zygotic genes activated after the mid-blastula transition. In the adult stage, IGF-1a mRNA was easily detectable in the liver, brain, and eyes in both male and female fish, while IGF-1b mRNA was only detected in the gonad (Fig. 4B). IGF-2a mRNA was easily detected in all the tissues examined, although its levels in the muscle and gills were very low (Fig. 4B). In contrast, IGF-2b mRNA was exclusively expressed in the liver (Fig. 4B). There was no apparent gender difference. In agreement with the RT-PCR data, whole mount in situ RNA hybridization analysis showed that IGF-1a and IGF-2b mRNA was very low at 10 hpf, became detectable at 24 hpf, and highly expressed at 48 hpf, most abundantly in the anterior part of a embryo (data not shown). In comparison, IGF-1b and IGF-2a mRNA was detected in all stages in a wide range of tissues (data not shown). Forced expression of zebrafish IGFs causes similar defects in midline formation and notochord development but with different potencies The phenotypes of the IGF-2a mRNA (500 pg)-injected group are shown in Fig. 5A–L. Injection of IGF-2a mRNA (500 pg) PLoS ONE | www.plosone.org 4 September 2009 | Volume 4 | Issue 9 | e7026