Supplementary Figure 3: Injection of lkt/abcc4 splicing morpholinos phenocopies lkt mutants.

(a) Schematic diagram depicting zebrafish lkt/abcc4 gene structure, including exon 9, 10, 11, and intron 9, 10. Red bar: abcc4 morpholinos target site. Black arrows: primer sites for RT-PCR. (b) RT-PCR analyses showed that wild-type lkt/abcc4 transcripts contained fused exons 9, 10 and 11, whereas mis-spliced transcripts contained only fused exons 9 and 11 in embryos injected with different doses of lkt/abcc4-MO. Total RNAs were isolated from morphants and controls, and subjected to RT-PCR using primers located in exon 9 and 11. (c–e) Acetylated tubulin immunostaining showing a reduction of KV cilia (c), a loss of OV short cilia (d) and lack of some ear kinocilia in lkt/abcc4 morphants (e), similar to those of lkt mutants. (f–h) lkt/abcc4 morphants showing a curved body axis (f; 81%; n = 123), hydrocephalus (f; 78%, n = 123; red arrow), three otoliths (g; 80%; n = 123; red arrows) and reversed (left)-looped heart (h; 41%; n = 93). (i,j) In situ hybridization showing expression of abcc4 (e; arrowhead) and insulin, a pancreas marker (f; arrowhead). (k) Double in situ hybridization with abcc4 and insulin probes showing expression of both genes in the pancreas (arrowhead). Inset displays a high-magnification image of abcc4 and insulin expression patterns, which largely overlap. (l,m) In situ hybridization showing expression of abcc4 (l; arrowhead) and wt1b, a glomerulus marker (m; arrows). (n) Double in situ with abcc4 (arrowhead) and wt1b (arrows) probes showing expression in the pancreas and glomerulus, respectively. Inset shows an enlarged view of abcc4 and wt1b expression. Double in situ hybridization analyses were conducted using digoxingenin-labeled lkt antisense probe (i,k,l,n). insulin (j,k) and wt1b antisense probes (m,n) were fluorescein-labeled. Scale bar: 10 μm (c–e); 100 μm (f–h; i–n).