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Abstract
BackgroundThe 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 FindingsDepletion 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/SignificanceThese results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.
Highlights
Multicellular development requires that the basic processes of cell growth and proliferation be subjugated to a higher level ontogenetic program
We find that SpRunt-1 protein levels are regulated by the activity of glycogen synthase kinase 3 (GSK-3), suggesting that Runx expression and canonical Wnt signaling are mutually linked
To ask whether cell cycle transit is defective in SpRunt-1 morphants at late blastula stage, embryos were pulse-labeled with bromodeoxyuridine (BrdU) from 18–24 hours post-fertilization, fixed, and stained with a fluorescent anti-BrdU antibody
Summary
Multicellular development requires that the basic processes of cell growth and proliferation be subjugated to a higher level ontogenetic program. We extend our investigation of Runx function in sea urchin embryogenesis, showing that the earliest developmental defects associated with blockade of SpRunt-1 expression include deficits in blastula stage cell proliferation and wnt gene expression.
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