Fig. S2

pwp2h is the mutated gene in tti^s450. (A) Sequence of pwp2h in WT and tti^s450 cDNA reveals that tti^s450 larvae utilize a cryptic splice site in exon 10 due to a mutation in the splice acceptor site in intron 9. This results in an 11 bp deletion (bracket) which causes a frame-shift in the pwp2h coding sequence resulting in 13 aberrant amino acids and a premature stop codon in exon 10. (B, C) Upon microinjection into the yolk of 1–4 cell WT zebrafish embryos, a pwp2h-targeted MO (15 ng) produces a robust tti^s450 phenotype at 120 hpf (C). Vehicle-injected controls appear WT (B). (D–G) Non-complementation of 2 independent pwph2 alleles confirms that pwph2 is the mutated gene in tti^s450. Heterozygous tti^s450 carriers were crossed with heterozygous carriers of s927, an independent pwph2 allele identified in the 2-CLIP screen [30]. One quarter of the offspring are compound tti^s450;tti^s927 mutants (E) and exhibit the tti^s450 phenotype (F) at 120 hpf including impaired development of the digestive organs, eye and craniofacial structures. Other panels show WT (D) and tti^s927 mutant (G) larvae at 120 hpf. These data indicate that both alleles correspond to the same genetic locus. e, eye; ib, intestinal bulb; sb, swim bladder; y, yolk. (H) The nucleotide sequence of pwp2h cDNA generated from tti^s927 larvae contains a T→A transversion (arrow). (I) The base change in tti^s927 results in a highly conserved branched amino acid (valine, shaded blue) being replaced by glutamic acid. Alignment was performed using ClustalW.