Fig. 2

rbm47 knockdown in developing zebrafish embryos disrupts head formation. A: Two morpholino knockdown strategies were used to disrupt rbm47 function. MO-rbm47-ATG hybridizes to the start codon, preventing translation initiation. MO-rbm47-E1I1 hybridizes to the exon-1/intron-1 splice donor site, blocking splicing to exon 2. Schematic drawing is not to scale. B: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to show interrupted splicing of RNA extracted from MO-rbm47-E1I1 zebrafish, which amplifies a band of approximately 3.5 kb due to retention of intron 1. PCR cycles: lane 1 (34 cycles); lane 2 (30 cycles); lane 3 (30 cycles). C: Upper panels demonstrate typical loss of head phenotype in rbm47 knockdown zebrafish (b and c) and a control zebrafish injected with control morpholino (a). The lower panels depict typical small head phenotypes in MO-rbm47 injected fish (d and e). Scale bar = 200 µm. D: The incidence of total loss of head development in morpholino-injected zebrafish. Injection of morpholinos targeting rbm47 resulted in a 9-16% incidence of the headless phenotype. Co-injection of rbm47 mRNA or wnt8a blocking morpholino with rbm47 blocking morpholino resulted in a decreased incidence of headlessness (for detailed numbers and statistics, see Table 1).