Fig. 3

A. B. Schematic representation shows the genomic organization of the lin-28a and lin-28b genes in zebrafish. Regions targeted by splice-blocking morpholinos are shown. Arrows depict the location of the two primer sets to amplify exon and intron sequences, which were utilized in the RT-PCR analysis. C. D. The efficacy of MO was validated by RT-PCR using primers as indicated in A, B. Total RNA was isolated from lin-28a, lin-28b or control MO-injected embryos at 5 hpf and analyzed by using specific primers for lin-28a and lin-28b. Upper panel shows a reduced expression of lin-28a and lin-28b mRNA, and an increase of an aberrantly spliced lin-28b transcripts corresponding to the presence of intron 1. Lower panel shows RT-PCR products with gapdh primers used as internal control. E. Representative images of severe phenotype of lin-28a and lin-28b morphants at 5 hpf. F. Vegetal view of control, lin-28a and lin-28b morphants at 8 hpf. Arrow shows the similar extent of prechordal plate extension in control, lin-28a and lin-28b morphant embryos; bracket shows a reduced extent in epiboly in lin-28a and lin-28b morphant embryos compared with controls. G. Representatives of mild and severe phenotypes in lin-28a and lin-28b morphants at 30 hpf. Both lin-28a and lin-28b morphants develop morphological phenotypes displaying shorten body axis, small anterior structures and aberrant tail morphology ranging from mild (Right) to severe (Left) compare to control MO injected embryos (Top). Arrowheads indicate the smaller head. H. Quantification of the efficiency of rescue from gastrulation defects following co-injection of lin-28a MO, lin-28b MO and mRNAs. More than half of the lin-28a MO or lin-28b MO injected embryos had died by 30 hpf. The frequencies of the phenotypes were similar between lin-28a and lin-28b morphants. The frequency of the dead, mild and severe phenotypes decreased with a rescue experiment in which 300 pg of lin-28a and lin-28b mRNAs were co-injected with the MO. (p<0.0001, lin-28a MO vs. lin-28a MO+lin-28a mRNA, lin-28b MO vs. lin-28b MO+lin-28b mRNA, Fisher’s exact test). The total number of embryos is noted above each bar.