Fig. 3

Disruption of msx. (A–F) Lateral views of live embryos at 28 hpf showing the whole embryo and an enlargement of the midbrain–hindbrain region. (A) Wild-type, (B) x8/x8 mutant, (C) msxB morphant, (D) msxC morphant, (E) msxE morphant, and (F) msxBCE morphant. Regions of the brain with a darkened granular appearance are indicative of elevated cell death (cd). (G) Schematic of general intron–exon structure of msx genes and mature mRNAs. Splice-blocking morpholinos were designed to bind to splice donor sites as shown (msx-MO). For each gene, PCR primers (arrows) were designed to amplify exon1 plus the 52 end of exon2 (primers 1 and 2) or the entire coding region (primers 1 and 3). (H) Analysis of morpholino efficacy. Primers for the indicated genes (left) were used to amplify RT-PCR products from lysates of embryos injected with the indicated morpholinos (top). All morpholinos are highly specific and affect only the targeted transcript. The effects of msxE-MO were the strongest, with no detectable PCR products from either primer set. In the case of msxC-MO, amplification of the entire coding region (c1/c3 primers) yielded no signal corresponding to the mature mRNA, although a weak band was detected at a higher molecular weight corresponding to unspliced message (not shown). This residual msxC transcript presumably accounts for the appearance of a weak band corresponding to exon1 (c1/c2 primers). msxB-MO gave a more complex pattern: Primers b1 and b2 gave only a faint band of the expected size whereas amplification of the entire coding region (primers b1 and b3) gave three faint bands, one corresponding to mature mRNA and two with lower molecular weight (not shown). The latter presumably reflect activation of cryptic splice sites. odc expression was used as a constitutive control (Draper et al., 2001).