Fig. 3

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

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