Injection of <i>pitx3</i> morpholino results in abnormal splicing of <i>pitx3</i> transcript and small lens phenotype.

Abstract

<p><b>A.</b> Schematic drawing of <i>pitx3</i> gene; position of initiation codon (ATG) and RT-PCR primers (1F and 3R) are indicated, exons are numbered. <b>B.</b> Sequencing of the <i>pitx3-mo</i> transcript generated with primers located in the first and third <i>pitx3</i> exons demonstrates absence of exon 2 in the resultant product. <b>C.</b> RT-PCR results obtained with <i>pitx3</i> 1F/3R primers using RNA extracted from embryos injected with control or <i>pitx3</i> morpholino. Please note a strong decrease in normal 276-bp product (black arrowhead) and presence of abnormal 148-bp product (red arrowhead) in <i>pitx3-mo</i> samples; hpf- hours post fertilization of analyzed embryos. <b>D–G.</b> Morphological phenotypes of zebrafish <i>pitx3-mo</i> embryos. In comparison to control embryos, a smaller head can be observed in <i>pitx3</i> morphants (72-hpf embryo is shown; D) as well as an obvious reduction in lens size (black arrow) at later stages (96-hpf embryos are shown; E–G). <b>H</b> and <b>I.</b> Expression of <i>pitx3</i> in the developing lens in 24-hpf embryos. Please note robust expression in the lens vesicle (arrows) as demonstrated by both whole mount (H) and section (I) in situ.</p