Pax2a and Pax8 have overlapping functions in ear development. (A) Schematic map showing the genomic structure of pax8 (after Pfeffer et al., 1998), the protein domain structure and the positions of the splice-blocking morpholinos. Black arrows indicate MO E5/I5 and E9/I9, the most efficacious combination used in this study. The white arrows E2/I2, E3/I3 and E4/I4 show positions of the other three spliceblocking MOs that yielded only weak or no phenotype. The sequence of the first exon (?) is possibly incomplete. The efficacy of E5/I5 and E9/I9 pax8-MOs combination is visualized by in situ hybridization (B,C). (B) In wild-type embryos at the one-somite stage, pax8 transcripts in the preotic region are localized primarily in the cytoplasm leaving the nuclei relatively clear. (C) In E5/I5 and E9/I9 pax8-MOs injected embryos, pax8 transcripts are localized mostly in nuclei leaving the cytoplasm free of signal. (D-S) Knockdown of Pax8 in pax2a– mutants has a severe otic phenotype. Wild-type embryos (D,H,L,P), pax2a– mutants (E,I,M,Q) and pax8-MO-injected wild-type embryos (F,J,N,R) are similar, but, in contrast to pax2a– mutants, are depleted of Pax8 (G,K,O,S). The otic vesicle is absent in pax8-MO-injected pax2a– mutant embryos and cannot be detected at 22 h (G) or 50 h (K). However, Dlx3b (L-O) and cldna (P-S) are present in pax2a– mutants depleted of Pax8. (D-O) Side views, anterior towards the left, dorsal towards the top; (B,C,P-S) dorsal views, anterior towards the top. Scale bar: 30 μm for B,C; 200 μm for D-G; 75 μm for H-K; 60 μm for L-O; 100 μm for P-S.
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