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Figure 2 | BMC Biology

Figure 2

From: Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene

Figure 2

Embryos injected with anti-SpRunt m1 and m4 undergo a mitotic catastrophe caused by antisense mis-targeting to histone mRNA. (A-C) Confocal images of fixed 15 hr embryos stained with DAPI (blue) and rhodamine phalloidin (red). (A) Control (mC)-injected embryo, (B) m1-injected embryo, and (C) m4-injected embryo. The arrow indicates a pair of 'cut' cells. Bar = 10 μm. (D) SDS PAGE of whole nuclei containing equivalent amounts of DNA (0.5 μg) from 14-hour control (mC) and m1-injected samples, stained with SYPRO Ruby protein stain. The positions of core histones, obtained from the mobility of calf thymus histone standards run on the same gel, are shown on the left of the gel, while the positions of molecular weight standards are indicated on the right. (E) Total protein from 600 mC-, m1-, or m4-injected embryos labeled metabolically with 35S-Met/Cys from 4 to 8 hours post-fertilization (hpf). Histones are easily identified by their characteristic size and stoichiometry, and by the fact that at this stage they represent ~5–10% of the total protein synthesized in the embryo [27]. The positions of histones H3 and H4 are indicated. (F, G) Sequence alignment between (F) the target sequence for m1 and α-histone H3 mRNA, and (G) the target sequence for m4 and α-histone H4 mRNA. Sequence identities are highlighted in black. The start codons are underlined in each sequence. (H) In vivo translation of synthetic histone H3 mRNA co-injected with m1. The injected zygotes were labeled metabolically with 35S-Met/Cys for 2 hours, during the first cleavage cycle. (I) 15-hr blastula stage embryo, stained as in A-C, showing rescue of m1-induced cell division defects by co-injection of 1 μg/μl H3 mRNA; bar = 10 μm.

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