Convergent extension and axial defects in Zic3 morphant zebrafish. (A–D) WISH of no tail expression in the notochord at 90% epiboly (A, B) and tailbud (C, D) stages. Control (CTRL) embyos (A, C) showed normal notochord development, whereas Zic3 morphants (B, D) exhibited a broader notochord. (E, F) WISH of myod and dlx3b at the 4-somite stage to label somites and the neural plate border, respectively. The red lines mark the width of the neural plate and the white lines indicate somite width. (G) Average width of the neural plate (measured at the red line) in control and Zic3 morphant embryos. (H,I) Live control (H) and Zic3 morphant (I) embryos at the 12-somite stage. The red lines indicate head-to-tail distance. (J) Average head-to-tail distances in control and Zic3 morphants. Live control (K) and Zic3 morphant (L) embryos at 2 days post-fertilization. Error bars represent SEM. **p < 0.01 by t-test.

Xenopus zic3 rescues axial defects in zebrafish Zic3 morphants. (A–D) Live embryos at 5 days post-fertilization. (A) Uninjected control embryos. (B) Xzic3 mRNA-injected embryos showed subtle phenotypes including a curled tail tip. (C) Zic3 morphant embryos had a shortened and curved A–P axis and edema. (D) Embryos co-injected with Zic3 morpholino and Xzic3 mRNA. (E) Percentage of embryos with a curved or shortened axis. Embryos with minor curled tail tips were scored to have normal body elongation.

Abnormal heart looping and gut formation in zebrafish Zic3 morphants. (A–C) Direction of cardiac looping (arrows) visualized by WISH of cardiac myosin light chain-2 (cmlc2) in 2 dpf embryos. Control embryos showed normal looping (A), whereas Zic3 morphants often showed mirror reversed (B) or unlooped (C) hearts. (D) Rescue of cardiac looping defects. Quantification of cardiac looping defects in control embryos is shown compared with embryos injected with Zic3 morpholino, Xenopus zic3 mRNA, or co-injected with Zic3 morpholino and Xenopus zic3 mRNA. (E–H) Analysis of gut looping by WISH with foxa3 in 2 dpf embryos. Control embryos (E) showed normal asymmetric positioning of the liver (L) and pancreas (P). Organ position (F), patterning (G), and number (H) were abnormal in Zic3 morphants. **p < 0.01 by Fisher′s exact test; statistical significance was determined through comparison of Zic3 MO + zic3 mRNA with Zic3 MO to demonstrate phenotypic rescue.

Abnormal L–R molecular marker expression in Xenopus and zebrafish Zic3 morphants. (A) Pitx2 expression in Xenopus Zic3 morphant embryos at stage 30. Embryos were photographed from both left and right sides. Red arrows highlight pitx2 expression. (B) Quantitative results of pitx2 expression in non-injected control (CTRL), control morpholino (CTRL MO) and Zic3 splice site morpholino (SS MO). (C) Southpaw (spaw) expression in zebrafish Zic3 morphant embryos. Red arrows highlight gene expression as left, right, bilateral or absent. (D) Quantitative results of spaw expression in control and Zic3 morphants.