Fig. S1

appa morpholino (MO) injection leads to a dose-dependent disruption in appa mRNA processing. A. Zebrafish embryos were injected with increasing doses of appa MO and scored for malformations and CNS cell death. B. Same experiments as panel A revealed doses that were toxic to the developing fish. C. appa splice block MO is efficacious, as it leads to disruption of appa mRNA. RNA was isolated from fish injected with appa MO, an equivalent dose of control MO, and subjected to RT-PCR. Fish injected with appa MO show a band at ~300 bp corresponding to mRNA with intron 2 retained. This band is absent in when fish are injected with the control MO, or when standard Taq is used in place of reverse-transcriptase. D. Sequencing of the aforementioned ~300 bp band confirms the retention of intron 2–3 in mature mRNA, and confirms our MO produces a truncated protein. Our sequence (top) was an exact match to zebrafish genomic clone NW_003336735 (bottom). Translation of the sequence, immediately 5′ of appa exon 3 (annotated in yellow at bottom right), predicts two termination codons (black, *). E–J. A second MO reagent against appa was used to test specificity of the phenotypes observed. Designed against a disparate portion of the gene, the 5′UTR and thus is a translation blocking (TB) MO. The efficacy of this MO is demonstrated in Fig. 1M and 1N. It produced mild and severe phenotypes (G and G′) indistinguishable from the splice blocking appa MO we primarily use in this work. The low dose of appa TB MO also showed a genetic interaction with the low dose of prp1 MO, equivalent to results in Fig. 3 (J, J′). MO dose is indicated on panels. K. Quantification of appa-TB MO demonstrates a dose-dependant effect by itself and an additive effect with prp1 MO during concerted delivery at sub-effective doses. Colour coding in the histogram is as per Fig. 2.