Suppression of tgfβ3 expression in the zebrafish embryo. (A) Gene knockdown via antisense Morpholino™ oligonucleotide mediated translation blocking (T-MO), and pre-mRNA splice inhibition (e1i1-MO and i1e2-MO). Morpholino™ annealing sites are indicated as horizontal black bars. Opposing gray and black arrows and arrowheads indicate positions of primer pairs used to detect presence of aberrantly-spliced and normally-spliced tgfβ3 mature transcripts, respectively. Introns are not drawn to scale. (B) Except for STD-MO injected embryos, all MO-injected larvae (96 hpf) have a smaller head, malformed lower jaw (arrows), and smaller heart with enlarged pericardial space (arrowheads). (C, D) RT-PCR of i1e2-MO and e1i1-MO injected embryos detects the presence of 362 bp and a 311 bp splice-modified tgfβ3 transcript fragments, respectively, which are not observed in uninjected controls (U). β-actin (561 bp) was used at a loading control. (E) Real-time relative quantitative PCR reveals a ∼50% reduction in normally-spliced tgfβ3 mRNA in i1e2-MO injected embryos compared to uninjected controls at 1 dpf and 4 dpf. (F) Co-injection of e1i1-MO with i1e2-MO suppresses correctly-spliced tgfβ3 mRNA by 93% compared to uninjected controls at 1 dpf. Standard deviations are based on three biological replicates. (G) Normal expression of tgfβ3 in a subpopulation of migrating neural crest cells at the 18-somite and 24 hpf stages, and in the ethmoid plate (ep), pharyngeal arch (pa), pectoral fin (pf), lens (le) and heart (ht) at 48 hpf. Red staining marks dlx2a-positive neural crest streams S1–S3.
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