papp-a2 knockdown causes ventral curvature and notochord defects. (A,B) RT-PCR analysis of the effect of papp-a2-targeted morpholino ei1i on papp-a2 pre-mRNA splicing showing a fast-migrating band (LMW) (A) resulting from the use of a cryptic splice donor site in exon 1, and a slow-migrating band (HMW) (B) resulting from the retention of intron 1 as determined by sequencing. These mis-spliced transcripts encode amino acid residue 1–242 or 1–275 of the prepro-protein plus eight or 30 residues, respectively, encoded by intron 1 followed by a stop codon. Values for e1i1 are the amount of morpholino in ng per embryo. WT, wild type. (C) RT-PCR analysis of the effect of papp-a2-targeted morpholino i3e4 on papp-a2 pre-mRNA splicing. Values for i3e4 are the amount of morpholino in ng per embryo. (D) 24 hpf embryos injected with 5.0ng control morpholino (cMO), 5.0ng papp-a2-targeted splice inhibiting morpholino (e1i1), 2.5ng translation inhibiting morpholino (ATG), or 2.0ng splice-inhibiting morpholino (i3e4) with or without 5ng p53-targeted morpholino (p53). Necrosis of the central nervous system was observed from both e1i1 and ATG, indicating non-specific p53-activating activity (Ekker and Larson, 2001; Robu et al., 2007). Co-injection with p53-targeting morpholino (Robu et al., 2007) specifically diminished the observed necrosis, whereas the other phenotypes remained unaffected. p53-targeting morpholino was included in all subsequent knockdown experiments. Insets show a magnified in view from the area indicated by the arrow. Notochord outlines are indicated with dashed lines. (E) noto whole mount in situ hybridization of 9 hpf embryos. e1i1+P2 indicates co-injection of e1i1 morpholino with 500pg Papp-a2-encoding mRNA. Numbers indicate fractions of embryos displaying the depicted phenotype. Animal pole view, dorsal to the top.