Robertson AJ et al. (2008), Runx expression is mitogenic and mutually linke...

ECB-ART-40955

PLoS One 2008 Jan 01;311:e3770. doi: 10.1371/journal.pone.0003770.

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Runx expression is mitogenic and mutually linked to Wnt activity in blastula-stage sea urchin embryos.

Robertson AJ , Coluccio A , Knowlton P , Dickey-Sims C , Coffman JA .

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BACKGROUND: The Runt homology domain (Runx) defines a metazoan family of sequence-specific transcriptional regulatory proteins that are critical for animal development and causally associated with a variety of mammalian cancers. The sea urchin Runx gene SpRunt-1 is expressed throughout the blastula stage embryo, and is required globally during embryogenesis for cell survival and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of SpRunt-1 by morpholino antisense-mediated knockdown causes a blastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcription coupled polymerase chain reaction (RT-PCR) studies show that the cell proliferation deficit is presaged by a deficit in the expression of several zygotic wnt genes, including wnt8, a key regulator of endomesoderm development. In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferation is also impeded by knockdown of either wnt8 or cyclinD. Chromatin immunoprecipitation (ChIP) indicates that Runx target sites within 5'' sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastula stage. Furthermore, experiments using a green fluorescent protein (GFP) reporter transgene show that the blastula-stage operation of a cis-regulatory module previously shown to be required for wnt8 expression (Minokawa et al., Dev. Biol. 288: 545-558, 2005) is dependent on its direct sequence-specific interaction with SpRunt-1. Finally, inhibitor studies and immunoblot analysis show that SpRunt-1 protein levels are negatively regulated by glycogen synthase kinase (GSK)-3. CONCLUSIONS/SIGNIFICANCE: These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.

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Species referenced: Echinodermata
Genes referenced: gsk3a irak1bp1 LOC100887844 LOC100893907 LOC105444658 LOC115919910 LOC115922213 LOC578305 LOC583082 LOC590297 polr3a runx2 wnt6 wnt8a
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???displayArticle.morpholinos??? LOC115927903 MO1 wnt6 MO1 wnt8a MO1

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	Figure 1. Blocking Runx expression causes a cell proliferation deficit in blastula stage sea urchin embryos.(A) Immunofluorescence labeling of BrdU incorporated from 18–24 hpf in control and SpRunt-1MASO-injected embryos. (B) Average cell numbers from four control-injected and four SpRunt-1 morphants at multiple time points from hatching to mesenchyme blastula stage. The error bars show the standard deviations.
	Figure 2. RT-PCR analysis of blastula stage wnt gene expression.(A) RT-PCR products obtained from control and SpRunt-1 morphants at 16 and 24 hpf using primer sets specific to several zygotically-expressed wnt genes, displayed by agarose gel electrophoresis. The intensity of the bands gives a rough indication of the relative levels of expression. The RT-PCR product for ubiquitin shows that approximately equivalent amounts of RNA were used in each sample. (B) Quantitative RT-PCR showing the ubiquitin-normalized difference in cycle number needed to achieve threshold fluorescence (ΔCt) in real-time RT-PCR of wnt6, wnt8, and cyclinD at 16 and 20 hpf. The ΔCt corresponding to a 3-fold difference in transcript abundance is indicated. Each bar represents the average of three or more separate measurements, except in the case of wnt6, which represents two measurements for the 16 hr sample. The number of biological replicates used to obtain each average was as follows: for wnt6, one at 16 hrs and two at 20 hrs (two and three measurements, respectively); for wnt8, two per time point (three measurements each); and for cyclinD, one per time point (three measurements each). The error bars show the standard deviations. Statistical significance calculated using a t-test is indicated by asterisks: P = .0049; P = .0005; **P<.0001.
	Figure 3. The effect of MASO-mediated knockdown of wnt8, wnt6, wnt8+wnt6, cyclinD, and PKC1 on cell proliferation in blastula stage embryos.Each bar represents the average number of cells per embryo. The error bars show the standard errors of the mean. Significance was calculated using a z-test; z>3, P<0.01, *z>4, P<0.001. The total number of embryos scored for each control/injected set is indicated under each heading on the x axis; the number of experimental repetitions for each set is in parenthesis.
	Figure 4. SpRunt-1 is bound to DNA in the 5′ flanking regions of cyclinD, wnt6, and wnt8 in 20 hr blastula stage nuclei, and is required for blastula-stage activity of wnt8 cis-regulatory module C.(A) Schematic representation of cyclinD, wnt6, and wnt8. Exons are shown as black bars. The previously-characterized wnt8 cis-regulatory modules [48] are shown as open bars. Locations of the consensus Runx binding motif (TGT/CGGT) are indicated by vertical lines. Arrows show approximate primer locations for ChIP analysis. (B) PCR amplicons of cyclinD, wnt6, and wnt8 obtained from ChIP of 20 hr embryo chromatin using anti-SpRunt-1 polyclonal IgG, or an equivalent quantity of non-immune IgG. In initial experiments, real-time PCR was used to determine a threshold number of cycles needed to obtain non-saturating signals from both the input DNA and SpRunt-1 ChIP DNA; this cycle number was then used as an end point in the experiments depicted here. Since an equivalent quantity of input DNA was used as template in each PCR, the relative band intensities give a rough indication of the enrichment obtained for each sequence. Thus, the wnt6 amplicon (which centers on Runx target site) is shown to be substantially enriched by ChIP compared to the cyclinD amplicon (which does not center on a Runx target site). (C) Schematic of modC-EpGFP (not to scale). (D) Examples of modC-EpGFP expression in hatched blastulae. (E) RT-PCR analysis comparing modC-EpGFP expression in control and SpRunt-1 morphants, and to expression of modCΔRunx-EpGFP. The PCR products obtained without reverse transcriptase (RT) shows the relative levels of transgene incorporation for each experiment.
	Figure 5. SpRunt-1 expression is negatively regulated by GSK-3.(A) Examples of SpRunt-1 morphants developed in the absence or presence of the GSK-3 inhibitor SB216763 beginning at blastula stage. The embryo on the left is an untreated three day old morphant; the one on the right is a three day old SB216763-treated morphant from the same group of injected embryos. (B) Quantitation of phenotypes obtained in the experiment shown in A. “Arrested” refers to a phenotype similar that on the left in A; “Full pluteus” refers to a phenotype similar to that on the right. “Stunted pluteus” refers to a phenotype intermediate between the two. (C) Immunoblot showing SpRunt-1 protein levels in equivalent numbers of normal blastulae and blastulae cultured from 20–24 hpf in the presence of SB216763. Actin serves as a loading control.
	Figure 6. A hypothetical pan-metazoan regulatory circuit linking Runx expression to wnt activity and the developmental control of cell proliferation.Positive (activating) interactions are indicated by lines terminating in arrows; negative (inhibiting) interactions are indicated by lines terminating in bars. Both protein-protein and protein-DNA (cis-regulatory) interactions are shown; the latter are depicted by standard gene symbols (horizontal lines bearing a bent arrow). Interactions revealed in this work are shown in color; the others are gleaned from the literature (see text for supporting references).

References [+] :

Aberg, Runx2 mediates FGF signaling from epithelium to mesenchyme during tooth morphogenesis. 2004, Pubmed