1Development of Vertebrate LimbsThe modelsChickMouseSkeletal Pattern of the Chick Wing2Limb Bud FormationMartin, IJDB, 34:323-336.Outgrowth and patterning of mouse limb budsIntroduction• Limb buds develop fromlateral plate mesoderm(somatic layer)3 developmental axes:• proximo-distal,• antero-posterior,• dorso-ventralPattern is organized by:• progress zone• apical ectodermal ridge• polarizing region,• dorsal ectoderm.PosteriorAnteriorProximal DistalDorsalventral3Limbs have three signaling centers• AER: Apical Ectodermal Ridge (the AER overlies progress zone @ distal tip of bud• ZPA: Zone of Polarizing ActivityThe proximodistal axisThalidomideGiven for morning sickness in 1950’s-1960’sNow used for leprosymultiple myeloma andlymphoma4Apical Ectodermal Ridge (AER)maintains proximo-distal outgrowth of limb bud.The Apical Ectodermal Ridge (AER) develops along thedistal margin of the limb budAERRemoval of AER causes stage-dependentloss of distal structures5Effect of the AER on the underlying mesenchymeRemoval of the AER at different time points results instage-dependent truncation of the limb skeletonearlylateFGF8 is expressed in the Apical Ectodermal Ridge(other Fgfs in the AER are Fgf4, Fgf9 and Fgf17)6ChickWhat does the AER removal tell us about specificationof proximodistal pattern?Proximal skeletal elements are specified before distalProgress zone model7Early progress zone grafted to late wing budLate progress zone grafted to early wing budTwo Models for the Mesodermal Specification of theProximal-Distal Axis of the LimbThe anteroposterior axis8Polydactyly: additional digitsPreaxial polydactyly: additional digits at the anteriorPostaxial polydactyly: additional digits at the posterioranteriorposteriorZPA Grafted to Anterior Limb Bud MesodermPolydactyly in Hemmingway’s Cat,Key West, FL9Shh mediates the signaling activity of the ZPAFeedback loop between the AER & the ZPA in the limb budHox Gene Expression is Dynamic During the Formation of theTetrapod Limb10Control of Limb Bone Elements by Paralogous Hox GenesDeletion of Limb Bone Elements by the Deletion of Paralogous Hox GenesNormal mouseforelimbLoss of HoxA11 & HoxD11 deletes zeugopod (ulna/radius)Loss of HoxD13 affects digitpatterning (polysyndactyly)Loss of HoxA13 & HoxD13deletes digits Decreased dosage Hoxd13/Hoxa13 causes coordinated reductionof digits and external genitalia11Initiation of limb buddingInitiation of limb buddingFgf10 is expressed in pre-limblateral plate mesodermFGF Bead placed in flank between normal limbs24 hr 36 hr 48 hrEctopic limb bud has reversed a-p polarity12Why does ectopic limb bud have reversed a-p polarity?WingFlankLegShh is expressed at anterior ofectopic limb bud.Polarizing potential is graded in flankWingFlankLegHoxB8 specifies the potential to express Shh inforelimbFgf activates polarizing potentialWingFlankLeg?LPMHand2 encodes potential to express Shh in both limbs13Ectopic expression of Hand2 inducesectopic ZPA/Shh expression and digit duplicationPrx1 promoter drivesexpression throughout both limb buds WT Prx1-Hand2Model for induction of ZPAHand2Weak Hand2Dorsal-Ventral Polarity14Positioning the AEREngrailed- Wnt7a is expressed in dorsal ectoderm- Engrailed1 is in ventral ectodermHow Wnt7a induces “dorsal” Identity:Wnt7a induces Lmx1b in dorsal mesenchymeEngrailed KO limb is double dorsal, becauseWnt7a (& Lmx1b) is activated on ventral side.Thus, Engrailed1 normally represses Wnt7a ventrally, thereby preventing dorsalization of ventral side of the limb.When Wnt7a is knocked out, the dorsal side is ventralized (hair is lost & pads form).Engrailed1, however, is not activateddorsally (En is not needed for ventralization).Thus, the “default” is for the limb to develop with ventral identity.Wnt7a expression actively dorsalizes, but thismust be repressed ventrally to establish D-Vpolarity.+/+Wnt7a -/-Wnt7aWnt3aR-FringeEn1Lmx1bWhat would you expect to result from removal of both Wnt7a & En1?(Dorsal views of paw)15The AER forms in the ectoderm at boundary between dorsal and ventralDistal limb mesenchyme sends AER-inducing signal, but only ectodermalcells at D-V boundary can respondShifting the D-V boundary causes shifting of AER position.Specification of limb positionLateral plate mesoderm is regionalized bydifferential expression of Hox9 genes16Respecification of flank cells towards wing or leg identity isassociated with respecification of the LPM Hox codeAnteriorFGF beadPosteriorFGF beadHox9 genes are differentially expressed along LPMand are associated with limb position and identityFgfFgfFlank Hox code is altered followingFgf application to flank. Consequently, flank cells develop in context of Hox gene expression normally found at wing or leg level, and differentiate into ectopic wings or legs.Lateral plate mesoderm cells at wing, flank and leg levelsexpress different combinations and levels of Hox9 genes.Patterns of Cell Death in Leg Primordia of Duckand Chick Embryos17Inhibition of Cell Death by Inhibiting