1The Nephron2Yu et al., Curr. Opin. Gene. Dev. 14:550-7, 20043Shah, M. M. et al. Development 2004;131:1449-14624HoxB7:GFP(ureteric budexpression)YesYesCombinationNoN.A.MetanephricMesenchymeAloneN.A.NoUreteric BudAloneTubules?BranchingDuct ?Grobstein, 1955Kidney Induction in Vitro5Many heterologous tissues can induce metanephricmesenchyme to form tubulesBut only the metanephric mesenchyme can formtubules in response to inducersHence, the metanephric mesenchyme is highlypatterned before being induced to form tubules6Induction of the Ureteric Bud: C-ret and GDNFC-ret: TK receptor expressed in the nephric duct and ureteric budC-ret -/- mice: No metanephros (although mesonephros isnormal)Branching of the ureteric bud in tissue recombinations of c-ret -/-and wild type tissues:-+-/--++/+-/-+/+BudMesenchymeSchuchardt et al.Development 122,1919-1929 (1996)GDNF is a ligand for c-retGDNF -/- mice have no kidneys orureterGDNF is expressed in the kidneymesenchyme around the ureteric budbranchpoints, and can induce uretericbud branching and outgrowth in vitro(Sainio et al, Development 124, 4077-4087, 1997)7Tissue-specific genes are expressed in the kidney mesenchymeprior to its ineteraction with the duct8PronephrosMesonephrosUreteric budMetanephrosDuctTubuleGlomusFiltrationBarrierThe Vertebrate Kidneysom imThe Intermediate MesodermPartial List of Intermediate Mesoderm DerivativesKidney, Somatic Gonad, Hematopoietic Tissue, Aorta, AdrenalGlands9cdcoesomlpnntB C DnpsmimlpnpnsomimlpAsom im1011Phase I:Osr1St. 9 St. 12Phase II:Pax2, lim1, wt1Gene expression during intermediate mesoderm formationA B C D0h 5h 10h 15hFate Map of the Avian Intermediate MesodermSt. 5 St. 7 St. 912im imsomsomsomsomntsomimimlplpADBCEFquailpax-2quailpax-2IM is not specified in the primitive streaksomntpax-2quailimntsom*A CBpax-2quailimlppax-2quailimlplp*imlp*DIHFEGIM becomes progressively specified as it migratesfrom the primitive streakIM is determinedwith respect totransplantation intoparaxial tissues bySt. 8IM is NOTdetermined withrespect totransplantation intolateral tissues at St.8, but becomesdetermined by St. 913Paraxis(Mesogenin, Meox,Tbx6)Pax-2(Lim1, Wt1)FoxC2 Osr1 CytokeratinParaxial Paraxial/IM IM IM/LP LPimdcoeSom(paraxial)lpnntTranscription Factorsin the mesodermModels of Intermediate Mesoderm Patterning:The Medio-lateral AxisMediolateral gradientInteraction between two already specified tissuesVertical signaling14pax2lim1pax2quailQuail somiteQuail somite+ Chicklateral platemesodermA property in the lateral plate which can induceIM genes in the somiteBmp signaling at the time of initial IntermediateMesoderm formationBmp-2 Bmp-4 P-Smad1P-Smad1 from Faure et al., Dev. Biol. 244:44 (2002)15NormalBmpNogginosr1 lim1 wt1bbbnnAlterations in IM gene expression by manipulation ofBmp signaling in vivobnbnccccBmp-2 can generate IM from paraxial mesodermin a dose-responsive mannerosr1 lim1 cytokeratinSt. 5-6anteriorprimitivestreakSt. 8paraxialmesodermparaxis16Does BMP regulate IM gene expression cell-autonomously?Bmp signaling can induce IM gene expressioncell-autonomouslypCS2-c.a.Alk3-IRES-GFPGfp Scl/TalControlGfpGfpPax2Osr1psm17Direct responders to Bmp signaling ?Msx2Osr1Tbx6No RxBmp2CHXBmp2 +CHXSt. 8 PSMcultured for 3hoursDirectlyactivatedNot directly activated;Under negativeregulation?Not directlyrepressedParaxis(Mesogenin, Meox,Tbx6)Pax-2(Lim1, Wt1)FoxC2 Osr1 CytokeratinParaxial Paraxial/IM IM IM/LP LPimdcoeSom(paraxial)lpnntTranscription Factorsin the mesoderm18Intermediate Mesoderm markers are expandedmedially in FoxC1/C2 mutantsOsr1Osr1Lim1FoxC2 represses Pax-2 in the IMpMES-GFP(Control)pMES-FoxC2-GFPpMES-FoxC2-GFPDapiPax-2 GfpsomimsomimimimimimimimPax-2 GfpPax-2 Gfp19UntransfectedpMES-FoxC2-GFPDapiDapiPax-7Pax-7FoxC2 induces expression of the somite marker Pax-7 in the IMGfpsssimimimimimssosr1,pax,lim,etcFoxCPax7Somite IM Lateral PlateFoxCPax7BmpFoxCPax7BmpHighBmpParaxis Pax-2 Osr1