MO-mediated depletion of map9 leads to early defects and/or embryo death and growth failure. (A) Injection of splice-blocking MOex5 leads to the production of a mis-spliced map9 mRNA. RT-PCR was performed using map9 specific primers and RNA from embryos injected at the 1-cell stage with MOex5 (5 and 8 hpf), translation-blocking MO-ATG (8 hpf), or the control MOmis (8 hpf). PCR products were separated on 0.8% agarose gels. MOex5 PCR products show 2 bands that differ by ~300 bp in size. (B) Top: subcloning and sequencing of these 2 fragments revealed that the upper band corresponds to wild-type (wt) map 9, whereas the lower band corresponds to an mRNA in which exon 5 and 6 are deleted. mRNA from MO-ATG and MOmis-injected embryos show only wt map9. The position of MOex5 is indicated by an arrow on the acceptor splice junction of exon 5. Exons 5 and 6 are shaded in gray, and black boxes represent untranslated regions. Bottom: measure by qPCR of the expression of the 2 map9 mRNA forms in 8 hpf MOex5 and MO-ATG morphants. In MOex5 morphants the expression of map9 is ~5 times lower than in MO-ATG morphants, and the misspliced forms represents 11% of map9 mRNA vs. background level < 1% in MO-ATG morphants (inset). (C-F) One-cell stage zebrafish embryos were injected with 1 pmol of anti-map9 MOex5 or control MOmis and imaged at mid-epiboly. Growth and epiboly in MOex5 morphants are arrested very early. (G-L) Embryos at 24 and 48 hpf after injection of 0.25 pmol of the 2 MOs. Map9 morphants show complex malformations, including underdeveloped nervous system, absence of the eyes, abnormal yolk, notochord (I and J, arrow in the insets), somites and tail (J, arrowheads), and pericardial edema (L, arrowhead). In (I and J), the insets are enlargements of the dotted boxes.
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