<p>Panels <b>A–E</b>: Sub-effective doses of <i>appa</i> and <i>prp1</i> gene knockdown synergize to produce an overt phenotype in the fish. Fish injected with a control morpholino (MO) (<b>A</b>), a sub-effective dose of <i>appa</i> (<b>B</b>) or <i>prp1</i> (<b>C</b>) MO fail to display any signs of CNS cell death or disruptions in development, i.e. no severe phenotypes. <b>D.</b> When sub-effective doses of <i>appa</i> and <i>prp1</i> are combined a severe phenotype emerges comprised of prominent morphological disruptions and an overt appearance of cell death within the CNS. <b>E.</b> The abundance of fish with normal morphology observed is significantly reduced, and the percentage of fish displaying cell death within the CNS is significantly increased when sub-effective doses of <i>appa</i> and <i>prp1</i> MOs are combined. ** = P<0.01. Panels <b>F–J</b> present a similar experimental design to panels A–E, but represent <i>appb</i> knockdown instead of <i>appa</i>. When a sub-effective doses of <i>appb</i> and <i>prp1</i> MOs are combined there is no significant increase in the number of fish showing developmental abnormalities or cell death within the CNS. <b>K</b>. Despite Appa and Appb being largely redundant during normal development (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone-0051305-g002" target="_blank">Fig. 2</a>), they cannot replace each other when PrP1 abundance is reduced. <i>appa</i> mRNA is able to alleviate the phenotype caused by co-injection of sub-effective doses of <i>appa</i> and <i>prp1</i> MOs. <i>appa</i> mRNA significantly reduced the percentage of fish displaying a severe phenotype. <i>appb</i> mRNA at an equivalent dose failed to reduce the percentage of fish displaying a phenotype. ** = P<0.01. <b>L. </b><i>app</i> mRNAs with stop codon mutations are not able to rescue the <i>app</i> or <i>appa</i>+<i>prp1</i> knockdown phenotypes. Data from the mutations S3X;E5X and 14_15 insT are shown (WT = wild type). Further analysis of these mRNAs and similar ones for <i>appb</i> was carried out in other knockdown backgrounds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051305#pone.0051305.s005" target="_blank">Fig. S5</a>).</p
