Fig. 4

Craniofacial development and gene expression in pitx2^ex4/5 morphants.

Developmental patterns of foxd3, dlx2a and dlx4a, and Tg(fli1:gfp) expression in pitx2 ^ex4/5 embryos. In situ hybridization was performed with foxd3, dlx2a, dlx4a or gfp-specific antisense riboprobe in control (A–P) or morphant (A2–P2) zebrafish embryos. Please note similar expression patterns in migrating neural crest cells (foxd3) and pharyngeal arch primordial regions (dlx2a) at 24-hpf (arrows in control (A–C) and morphant (A2–C2) embryos). Starting at 32-hpf, expression of dlx2a, dlx4a and Tg(fli1:gfp) in the posterior arches appears to be reduced in pitx2^ex4/5 fish (arrowheads in control (D–G) and morphant (D2–G2) embryos) while expression in the anterior arches (arrows in control (D–G) and morphant (D2–G2) embryos) is not significantly affected. In 48–72-hpf embryos, an increased expression of dlx4a in the anterior arches is detected (arrows in control (H–N) and morphant (H2–N2) embryos) while expression in the posterior arches remains reduced (arrowheads in control (H–N) and morphant (H2–N2) embryos); dlx4a expression around the oral cavity in 48-hpf embryos (arrow with asterisk in K and K2) as well as a frontward extension of the anterior arches at 72-hpf (arrows in L–N for controls and L2–N2 for morphants) are not observed in pitx2^ex4/5 fish. Analysis of sections at 72-hpf also shows broadened expression of dlx4a in the craniofacial region of morphant embryos (O2, P2) in comparison to controls (O, P).