Figure 3. appa interacts with prp1, but appb does not.

Panels A–E: Sub-effective doses of appa and prp1 gene knockdown synergize to produce an overt phenotype in the fish. Fish injected with a control morpholino (MO) (A), a sub-effective dose of appa (B) or prp1 (C) MO fail to display any signs of CNS cell death or disruptions in development, i.e. no severe phenotypes. D. When sub-effective doses of appa and prp1 are combined a severe phenotype emerges comprised of prominent morphological disruptions and an overt appearance of cell death within the CNS. E. 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 appa and prp1 MOs are combined. ** = P<0.01. Panels F–J present a similar experimental design to panels A–E, but represent appb knockdown instead of appa. When a sub-effective doses of appb and prp1 MOs are combined there is no significant increase in the number of fish showing developmental abnormalities or cell death within the CNS. K. Despite Appa and Appb being largely redundant during normal development (Fig. 2), they cannot replace each other when PrP1 abundance is reduced. appa mRNA is able to alleviate the phenotype caused by co-injection of sub-effective doses of appa and prp1 MOs. appa mRNA significantly reduced the percentage of fish displaying a severe phenotype. appb mRNA at an equivalent dose failed to reduce the percentage of fish displaying a phenotype. ** = P<0.01. L. app mRNAs with stop codon mutations are not able to rescue the app or appa+prp1 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 appb was carried out in other knockdown backgrounds (Fig. S5).