Figure 3.

Zebrafish studies. (A) The predominant ahi1 transcript in zebrafish embryos consists of 27 exons. The position of the previously published splice-blocking MO, SPL8, is indicated with a dark blue arrowhead at the splice acceptor site of exon 13. The e23i23 morpholino used in this study is indicated with a light blue arrowhead at the splice donor site of exon 23. (B) A reduction in the normal splice form, visualized as a 438 bp fragment in the control lane, is observed in e23i23MO-injected embryos at 2 dpf. A smaller running band containing an in-frame 63 bp deletion of the 23rd exon is observed in the e23i23 samples (red arrow). (C) The SPL8 morpholino strongly reduces the normal splice product of 517 bp at 2 dpf. No mis-spliced product was observed in our experiments. (D) The structure of the Ahi1 protein is conserved between human and zebrafish, with intact coiled-coil, WD-repeat and SH3 domains. Uninjected control shows normally elongated body axis, normal brain formation and an otic vesicle with two distinct otoliths (red brackets). (E) The predominant splice form in e23i23 morpholino-injected embryos encodes a protein lacking 21 amino acids of the N-terminal region of the SH3 domain (black arrowhead). e23i23MO-injected embryo is morphologically indistinguishable from controls. (F) Embryos injected with the previously described SPL8 morpholino (11) are depleted of the normal ahi1 transcript and exhibit a phenotype consistent with a severe ciliopathy, including hydrocephaly (asterisk), curved body axis and otic vesicle abnormalities. Scale bars: 250 µm.