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Fig. S2 etv5b overexpression rescues the 5-HT phenotype seen in etv5b splice morphants. (A) RT-PCR on total RNA extracted from 48 or 72 hpf pooled (n=6), uninjected or mismatch controls as well as embryos injected with increasing concentrations of etv5b splice MO. Injection of the splice MO results in almost complete loss of the wild-type band (wt) and the presence of a morphant band (mo) corresponding to an insertion of the entire intron 10 (79 bp), which in the amino acid sequence results in a premature stop codon. (B-E) Confocal maximum intensity projections showing embryos after RNA rescue of the etv5b splice MO phenotype. Embryos were subjected to the indicated treatments and processed for 5-HT immunohistochemistry (dissected brains). The intermediate/posterior (i./p.) clusters of the hypothalamus are shown. Ventral views, anterior left. Scale bar: 50 μm. (F) The number of 5-HT cells obtained in the rescue experiment in the i./p. and anterior raphe (r.) clusters at 72 hpf after the indicated treatments expressed as percentage of control. etv5b splice MO alone results in a significant decrease in the number of 5-HT-expressing cells as compared with uninjected control siblings (92.3±1.5%, P=6.6×10–4), whereas etv5b RNA injections led to a significant increase (108.3±2.1%, P=2.07×10–3). n, total number of embryos analysed for each experiment. *Pd0.05; n.s., not significant.