Multiple IGF-1 and -2 Genes Figure 5. Effect of forced IGF expression in zebrafish embryos. (A, B) Lateral view of a GFP mRNA injected control embryo (A) and an IGF-2a mRNA injected embryo (B) at the bud stage. Note the delayed mesoderm involution associated with an open blastopore (.75%, n = 328) in the IGF-2a mRNA injected embryos (B). (C, D) Lateral views of a control embryo (C) or an IGF-2a mRNA injected embryo (D) at 2-somite stage. Note the shortened A-P axis and more posterior tissues in (D). (E–H) Dorsal view of a control (E, G) or an IGF-2a mRNA injected embryo (F, H) at the 5-somite (E and F) and 15-somite stages (G and H). All embryos are dorsal views with head up. Arrows indicate the width of the notochord. Black bars in panels E and F show the width of the somite. Scale bar = 200 mm. (I–L) Morphology of a GFP mRNA injected control embryo (I and K) and an IGF-2a mRNA injected embryo (J and L) at 24 hpf. I and J are lateral views with head to the left and K and L are dorsal views with head to the left. MB, mid brain; OtV, otic vesicle; NC, notochord. Scale bar = 200 mm. (M) Dose-dependent effects of various IGFs in zebrafish embryos. The results are means of 3–4 independent experiments. doi:10.1371/journal.pone.0007026.g005 resulted in delayed mesoderm involution as evident from an open blastopore at the bud stage (Fig. 5B). At the 2-somite stage, there was a notable reduction in the posterior tissues (Fig. 5D). At the 5- somite stage, the IGF-2a mRNA-injected embryos showed poorly formed notochords and widened distance between the two columns of somites (Fig. 5F). This defect became more evident at the 15-somite stage (Fig. 5H). Compared to the control (Fig. 5K), the IGF-2a mRNA-injected embryos showed marked expansion of dorsal neural tissues and axial domain (Fig. 5L). Additionally, these embryos showed a severe shortening body length (Fig. 5J). To determine whether these IGFs have similar biological activities, each IGF mRNA was injected at the doses of 300, 500, and 800 pg/embryo and percentages of embryos exhibiting the above phenotype were determined at the 2–5 somite stage and at 24 hpf. Zebrafish IGF-1a, -1b, -2a and -2b all caused similar defects in dose-dependent manners (Fig. 5M), but there were considerable differences in their effectiveness. Injection of IGF-2a at 300 pg/embryo resulted in a score value of 33%,35%; a score value of 75%,80% at 500 pg; and over 90% at 800 pg/embryo at the 2–5 somite and 24hpf stages. Similar results were obtained with the injection of IGF-1a mRNA and to a lesser degree with IGF-2b mRNA (Fig. 5M). In comparison, IGF-1b appeared to have a weaker effect. It only had a score of 20% at the dose of 300 pg/embryo, a score of ,40% at 500 pg, and ,60% at 800 pg/embryo (Fig. 5M). Forced expression of IGF-2a disrupts midline and notochord patterning We next analyzed the effect of IGF-2a ectopic expression on embryonic patterning by in situ hybridization using the following markers: 1) emx1, a telencephalon marker [20]; 2) rx1, a retina marker [21]; 3) rx2, a retina marker [21]; 4) otx2, an anterior PLoS ONE | www.plosone.org 5 September 2009 | Volume 4 | Issue 9 | e7026