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BMC Biol
2004 May 07;2:6. doi: 10.1186/1741-7007-2-6.
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Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene.
Coffman JA
,
Dickey-Sims C
,
Haug JS
,
McCarthy JJ
,
Robertson AJ
.
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BACKGROUND: Runx transcription factors are important regulators of metazoan development. The sea urchin Runx gene SpRunt was previously identified as a trans-activator of the CyIIIa actin gene, a differentiation marker of larval aboral ectoderm. Here we extend the functional analysis of SpRunt, using morpholino antisense oligonucleotides (morpholinos) to interfere with SpRunt expression in the embryo.
RESULTS: The developmental effects of four different SpRunt-specific morpholinos were evaluated. The two morpholinos most effective at knocking down SpRunt produce an identical mitotic catastrophe phenotype at late cleavage stage that is an artifact of coincidental mis-targeting to histone mRNA, providing a cautionary example of the insufficiency of two different morpholinos as a control for specificity. The other two morpholinos produce gastrula stage proliferation and differentiation defects that are rescued by exogenous SpRunt mRNA. The expression of 22 genes involved in cell proliferation and differentiation was analyzed in the latter embryos by quantitative polymerase chain reaction. Knockdown of SpRunt was found to perturb the expression of differentiation markers in all of the major tissue territories as well as the expression of cell cycle control genes, including cyclin B and cyclin D.
CONCLUSIONS: SpRunt is essential for embryonic development, and is required globally to coordinate cell proliferation and differentiation.
Figure 1. Morpholino antisense targeting of SpRunt. (A) Schematic of SpRunt, showing the relative locations of sequences targeted by each of the five morpholinos used in this study. Exons 1–3 are depicted as boxes, with coding regions shaded. The Runt domain is black. Horizontal arrows indicate the positions of primers used for the RT-PCR shown in (C). (B) Immunoblots of protein translated in vivo from injected SpRunt mRNA co-injected with control morpholino (mC), m1, m2, or m3. As a loading control, the same blots were probed with an antibody to the sea urchin stem-loop binding protein (SLBP). (C) RT-PCR of SpRunt from total RNA extracted from early blastula stage embryos injected with mC, m4 or m5 (two separate experiments). RT-PCR of ubiquitin was used to control for RNA levels.
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.
Figure 3. Phenotype of late gastrula stage embryos injected with anti-SpRunt m2 and m5. DIC images (A-F) and cytometric analysis (G-I) of 48 hour embryos. (A) Control morpholino (mC)-injected embryo, cross section, with the oral side to the left and the gut indicated by an arrowhead. Bar = 20 μm. (B) mC-injected embryo, oral view, with the skeletal spicules indicated by an arrow (sp). (C) m2-injected embryo. (D) m5-injected embryo. (E-F) Embryos co-injected with m5 and SpRunt FL mRNA, showing the rescued development of gut (E) and spicule rudiments (F). (G) DNA histogram of mC-injected embryos. (H) DNA histogram of m5-injected embryos; note the accumulation of cells in G2/M. (I) DNA histogram of embryos co-injected with m5 and SpRunt mRNA. Note the reduced accumulation of cells in G2/M compared to the embryos injected with m5 alone.
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