zebrafish <i>pitx2</i> knockdown and associated phenotype.

Abstract

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

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