Altered regulation of left–right asymmetry genes in pegasus morphants. (A) Expression profiles of zebrafish pitx2. Fold change in expression of pitx2 in embryos injected with Pegasus morpholino (MO) or RNA (over-expression) as determined by microarray analysis and qRT-PCR, as indicated in Fig. 3F. (B) Analysis of the zebrafish pitx2 gene promoter. Sequence of the 500 bp immediately upstream from the transcriptional start site of zebrafish pitx2 revealing several potential ‘atypical’ Pegasus (GNNNGNNG) binding sites (arrows above). (C–H, J–Q, T–U) WISH analysis of pitx2, lefty2 and spaw. Embryos injected with scrambled morpholino (ScrambleMO: C–E, J–K, T) or pegasus ATG morpholino (PegATGMO: F–H, L–Q, U–V) were subjected to WISH using antisense probes targeting pitx2 (C–H) and lefty2 (J–Q) at 22 hpf and spaw (T–V) at 16 hpf. Embryos are of lateral (C, F) or oblique dorsal (D–E, G–H, J–Q, T–V) view, with anterior to the left. (I, R, S, W) Asymmetry of pitx2, lefty2 and spaw. Quantitation of the percentage of embryos expressing pitx2 (I), lefty2 (R, S) or spaw (W) specifically on the left, right or both, when injected with Scramble morpholino (Scr), pegasus ATG morpholino (Peg) or pegasus ATG and p53 morpholinos (Peg+p53).
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