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Fig. 2
Knockdown of smyd1a splicing by E8I8-MO and E9I9-MO.
A. Location of the E8I8-MO and E9I9-MO splicing blockers at the junction of exon8/intron8 and exon 9/intron 9, respectively. Defective splicing using a cryptic splicing site in exon 8 in E8I8-MO injected embryos resulted in a deletion of 14. Defective splicing using a cryptic splicing site in exon 9 in E9I9-MO injected embryos resulted in a deletion of 58 bp. B. RT-PCR showing the defective splicing induced by the E8I8-MO or E9I9-MO or both MOs. Compared with the PCR results from the wild type (wt) control embryos where a single band was generated, two bands were observed in the E8I8-MO injected embryos. Sequence analysis revealed that the smaller band contained a 14 bp deletion at the end of exon 8. The upper band represented the heteroduplex formed by the normal and defectively spliced products. Similarly, three bands were detected in E9I9-MO injected embryos. Sequence analysis revealed that the smaller band contained a 58 bp deletion at the end of exon 9. The middle band was the normal spliced product, whereas the upper band represented the heteroduplex formed by the normal and defectively spliced products. Two major bands were detected in E8I8-MO and E9I9-MO co-injected embryos. Sequence analysis revealed that the smaller band resulted from defective splicing. C–H. Morphology of smyd1a knockdown embryos at 48 hpf. Zebrafish embryos injected with control MO (C, D), E8I8-MO (E, F) or E9I9-MO (G, H) at 48 hpf. I–N. Knockdown of smyd1a expression had no effect on myoblast specification and early differentiation of slow and fast myofibers. In situ hybridization showing normal MyoD expression in control-MO (I) or smyd1a MO (L) injected embryos at 14 hpf. Cross views of slow (J, M) or fast (K, N) MHC expression in control (J, K) or smyd1a MO (M, N) injected embryos at 24 hpf.