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


RESULTS
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
These results suggest that Runx expression and Wnt signaling are mutually linked in a feedback circuit that controls cell proliferation during development.
Robertson
Anthony J
AJ
Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, USA.
Coluccio
Alison
A
Knowlton
Peter
P
Dickey-Sims
Carrie
C
Coffman
James A
JA
D000818:Animals; D036703:Blastula / Q000196:embryology*; D002454:Cell Differentiation; D002470:Cell Survival; D050676:Core Binding Factor Alpha 2 Subunit / Q000378:metabolism; D050657:Core Binding Factor alpha Subunits / Q000096:biosynthesis* / Q000502:physiology; D056741:Cyclin D; D016213:Cyclins / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012617:Sea Urchins; D014158:Transcription, Genetic; D051153:Wnt Proteins / Q000378:metabolism*
D050676:Core Binding Factor Alpha 2 Subunit; D050657:Core Binding Factor alpha Subunits; D056741:Cyclin D; D016213:Cyclins; D051153:Wnt Proteins; D038362:Glycogen Synthase Kinase 3