Fig. 2

zebrafish pitx2 knockdown and associated phenotype.

A. Schematic drawing of pitx2 genomic structure. Exons are shown as numbered boxes, sizes are indicated at the top (for exons) or at the bottom (for introns). The positions of primers to amplify pitx2 transcripts are shown and numbered 1–3; primers 1 and 3 are used for pitx2a and 2 and 3 for pitx2c. The position of antisense morpholino oligonucleotides, pitx2a^ATG, pitx2c^ATG and pitx2^ex4/5sp, are shown with red lines. B. RT-PCR of pitx2 expression in pitx2^ex4/5 morphants. Please note a complete absence of normal pitx2 transcripts (indicated with black arrows) and the presence of an abnormal large PCR product (indicated with red arrowheads) in mRNA extracted from pitx2^ex4/5 embryos at 24–48-hpf, the presence of both normal (diminished) and abnormal products at 72–96-hpf pitx2^ex4/5, and normal levels of pitx2 by 120-hpf due to weakening of morpholino effects. C. DNA sequencing of the abnormal PCR product observed in pitx2^ex4/5 morphant embryos identified the presence of the 902-bp intron 4 in the pitx2^ex4/5 transcript consistent with aberrant splicing (forward sequence is shown and the beginning of the intron is indicated with a red arrow; exon 4 sequence is shown in upper case while intron 4 is in lower case letters; the exon-intron junction sequence corresponding to the pitx2^ex4/5 antisense oligomer is indicated in red). Therefore, the pitx2^ex4/5 protein is predicted to contain partial pitx2 sequence (lacking amino acids encoded by exon 5) followed by 10 erroneous amino acids (pitx2^ex4/5 stop codon is indicated with red box). D. Representative images of pitx2^ex4/5 morphants, control morpholino-injected embryos and larvae developed from uninjected eggs at 48-hpf, 96-hpf and 7-dpf. E. Bar graph showing the distribution of observed embryonic phenotypes following pitx2^ex4/5 morpholino injections into p53/ and wild-type zebrafish eggs. e- eye, j- jaw, pe- pericardial edema.