Runx Expression Is Mitogenic and Mutually Linked to Wnt Activity in Blastula-Stage Sea Urchin Embryos 英文参考文献.docVIP

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Runx Expression Is Mitogenic and Mutually Linked to Wnt Activity in Blastula-Stage Sea Urchin Embryos 英文参考文献.doc

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Runx Expression Is Mitogenic and Mutually Linked to Wnt Activity in Blastula-Stage Sea Urchin Embryos 英文参考文献

RunxExpressionIsMitogenicandMutuallyLinkedto WntActivityinBlastula-StageSeaUrchinEmbryos AnthonyJ.Robertson1,AlisonColuccio1,PeterKnowlton1,CarrieDickey-Sims2,JamesA.Coffman1* 1MountDesertIslandBiologicalLaboratory,SalisburyCove,Maine,UnitedStatesofAmerica,2StowersInstituteforMedicalResearch,KansasCity,Missouri,UnitedStates ofAmerica Abstract Background:TheRunthomologydomain(Runx)definesametazoanfamilyofsequence-specifictranscriptionalregulatory 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 embryogenesisforcellsurvivalanddifferentiation. Methodology/PrincipalFindings:DepletionofSpRunt-1bymorpholinoantisense-mediatedknockdowncausesablastula stage deficit in cell proliferation, as shown by bromodeoxyuridine (BrdU) incorporation and direct cell counts. Reverse transcriptioncoupledpolymerasechainreaction(RT-PCR)studiesshowthatthecellproliferationdeficitispresagedbya deficitintheexpressionofseveralzygoticwntgenes,includingwnt8,akeyregulatorofendomesodermdevelopment.In addition, SpRunt-1-depleted blastulae underexpress cyclinD, an effector of mitogenic Wnt signaling. Blastula stage cell proliferationisalsoimpededbyknockdownofeitherwnt8orcyclinD.Chromatinimmunoprecipitation(ChIP)indicatesthat Runx target sites within 59 sequences flanking cyclinD, wnt6 and wnt8 are directly bound by SpRunt-1 protein at late blastulastage.Furthermore,experimentsusingagreenfluorescentprotein(GFP)reportertransgeneshowthattheblastula- stageoperationofacis-regulatorymodulepreviouslyshowntoberequiredforwnt8expression(Minokawaetal.,Dev.Biol. 288:545–558,2005)is dependenton itsdirect sequence-specific interactionwithSpRunt-1.Finally,inhibitor studiesand immunoblotanalysisshowthatSpRunt-1proteinlevelsarenegativelyregulatedbyglycogensynthasekinase(GSK)-3. Conclusions/Significance:TheseresultssuggestthatRunxexpressio