fu knockdown, overexpression in medaka, and fu expression pattern in Zebrafish. (A) Knockdown of fu was performed using the morpholino-oligonucleotide (MO) for splice blocking (5′-CAACCACCTTATTGACGACAAAACA-3′). Diagram of altered fu splicing in morphants of fu-i1e2 inserts intron 1 (+In. 1), resulting in an out-of-frame truncation of the fu protein, and splices exon 2 to a cryptic acceptor in exon 3 (- Ex. 2), causing an out-frame mutation of fu. The effect of the splice-blocking MO was verified by RT-PCR from 20 embryos total RNA (16-somite stage). Primers for checking the effect of MO were indicated in A (arrows). MO caused splice-blocking effectively. (B) fu mRNA injection rescued nkx2.2 expression in fu morpholino injected embryos. (C) fu overexpression induced ectopic nkx6.1 and olig2 expression (D) fu expressed in Hedgehog-dependent fashion also in zebrafish. The embryos treated with cyclopamine did not express fused or nkx2.2a. Scale bar: 500 µm in lateral view in B, C, D; 20 µm in cross-section in D.