In situ hybridization with <i>pitx2-exon 5</i> antisense riboprobe in control and morphant embryos.

Abstract

<p>The <i>pitx2</i> antisense riboprobe comprising exon 5 sequence detects wild-type and abnormally spliced <i>pitx2^ex4/5</i> transcripts. Images of whole mount embryos (<b>A–F</b>, <b>J–M</b> and <b>Q–U</b>) and sections (<b>G–I</b>, <b>N–P</b> and <b>V, W</b>) are shown for embryonic stages of 24-hpf (<b>A–D</b>), 48-hpf (<b>E–I</b>), 72-hpf (<b>J–P</b>) and 120-hpf (<b>Q–W</b>). Please note abnormal <i>pitx2</i> transcripts around the developing eye (arrowheads in <b>B, D</b>) and pharyngeal arches (arrows in <b>B</b>) in morphants at 24-hpf, which is similar to <i>pitx2</i> expression in control embryos (<b>A</b>, <b>C</b>). Staining for <i>pitx2</i> positive cells in morphant embryos at 48-, 72- and 120-hpf identifies abnormal patterns during ocular and craniofacial development in comparison to <i>pitx2</i> expression in control embryos (<b>E–W</b>). In terms of ocular patterns, some 48- and 72-hpf morphants demonstrate an accumulation of <i>pitx2</i> transcriptionally active cells in the anterior segment of the eye (arrowheads; images for whole mount and sections from two different <i>pitx2^ex4/5</i> morphant embryos at 48-hpf (<b>F, H, I</b>) and 72-hpf (<b>K, M, O, P</b>) in comparison to control 48-hpf (<b>E,G</b>) and 72-hpf (<b>J, L, N</b>) are shown). In 120-hpf eyes, a disorganized pattern of <i>pitx2</i> positive cells continues to be observed in <i>pitx2^ex4/5</i> morphant embryos (arrowheads in <b>T</b>, <b>U</b>, two different morphant embryos are shown, and <b>W</b>) in comparison to <i>pitx2</i> expression in control embryos (<b>S, V</b>); in addition to the abnormal pattern in the anterior structures, an increased signal behind the lens corresponding to the hyaloid vasculature is also observed (asterisks in <b>W</b>). With regards to craniofacial development, in 72-hpf morphant embryos, strong staining is observed around the malformed oral cavity and arches with the level of expression similar to control <i>pitx2</i> expression (arrows in <b>J–M</b>); at 120-hpf, <i>pitx2</i> transcripts continue to be strongly expressed in the malformed pharyngeal arches (arrows in <b>R</b> and <b>W</b>) while expression of wild-type <i>pitx2</i> is downregulated in control 120-hpf embryos (arrows in <b>Q</b> and <b>V</b>).</p