Tetrapod Limb DevelopmentBiology 4361 – Developmental BiologyJuly 29, 2009Tetrapod Limbs© Vicki Lockard and Paul Barry © Father Alejandro Sanchez © Merlin D. Tuttle© Anne FischerLimb Development - OverviewPatterningEarly developmentSpecificationEstablishing limb axesMorphogenic modelsCross-talk / regulationFrancesca V. Mariani and Gail R. Martin 2003 Nature 423:319-325 doi:10.1038/nature01655Limb PatterningHumerusRadiusUlnaCarpalsMetacarpalsPhalangesHuman armChicken wingChicken legShoulder FingerPinkieThumbKnucklePalmProximal DistalPosteriorAnteriorDorsalVentralLimb FieldLimb BudFormationVertebrate Limb BudsLimb budSomitesEctodermMesodermalmesenchymeLateral platemesodermHox expressiondetermines limbbud locationHox5Hox6Hoxc6 Specification of Limb BudsForelimb initiation:- anterior-most point ofHoxc6 expressionTbx Genes Specify Limb TypeTbx expression initiated by Wnts, FGFsNOTE – Tbx genes are not the first step in forelimb/hindlimb specification(initial step(s) unknown)Fgf/Wnt - Limb Bud InitiationLimb Bud AxesDistalProximalAnteriorPosteriorDorsalVentralApical Ectodermal RidgeApical Ectodermal Ridge (AER)Fgf10 initiates AER via Wnt3a, β-cateninAER expresses Fgf8, Fgf4; maintains Fgf10 expressionProximal-Distal AxisApical Ectodermal Ridge (AER) forms at boundarybetween dorsal and ventral ectodermFgf8lateralplatemesodermFgf10Fgf10Lateral plate mesoderm expresses Fgf10Apical Ectodermal Ridge ManipulationExtent of development dependson time of AER removalDistal structures are duplicated;- note structureDegree of “legness” of wing depends on placement of leg mesenchymeProgress ZoneProgress Zone – mesodermal mesenchyme; receives AER signals:- promotes proliferation (mitosis)- prevents differentiation into cartilage- maintains expression of A/P and D/V-related signalsProgress Zone(PZ)Apical Ectodermal Ridge (AER)Fgf8Fgf4PZFgf8Fgf10PZ mesenchyme specifies proximal-distal axis- transplantation experiments demonstrated that positional information was carried by PZ cells- PZs conveyed age-appropriate specification instructionsAER establishes Progress Zone~200 μmProximal-Distal Specification ModelsProgress zone model: Identity established by residence time in PZProximalDistalEarly allocation and progenitor expansion: Elements specified earlySpecifying mechanism - ??Anterior-Posterior SpecificationZone of PolarizingActivity (ZPA)Apical Ectodermal Ridge (AER)Progress Zone (PZ)MorphogenShhShh necessary and sufficient for establishing ZPA(NOTE – Shh not necessary for polarity of styolpod)Shh induced by dHAND and Hoxb8ZPA maintained by feedback loop with AERZPA/AER Feedback Loop Model1. dHAND - bHLH transcription factor and Fgf8 from AER stimulate Shh- Fgf8 (and Fgf4) maintains Shhexpression2. Shh up-regulates Gremlin1 in posterior mesenchyme- Grem1 antagonizes BMP ligands(BMPs repress Fgf expression in AER)3. Wnt7a maintains ShhWnt7a determines the size of AER Loss-of-function mutants (both Shh and Grem1) = syndactyly, loss of digitsZPA Morphogen GradientZPAShh gradient12345[Morphogen]HighLowPosterior Anterior5432ZPA TransplantationMirror-image duplication effects can be replicated by transplanting Shh bead Posterior tissue transplant to anterior = duplicated autopodRetinoic acid operates upstream of Shh- implant RA-soaked bead =mirror-image duplication- possible Hox gene involvementC. Tickle, Nature Molecular Biology 7(2006)45-53234432“new” posteriorDrosophila Hedgehog PathwayShh(vertebrates)/ Gli1, 2, 3(vertebrates)/ Gli1, 2, 3(vertebrates)Shh/Gli InteractionsGli3 – proteolytic fragment – acts as a transcriptional repressor (Gli3R)- represses e.g. dHAND, Gremlin, Fgf4, Hoxd13Without Shh:With Shh:Gli’s retained in long form – acts as a transcriptional activator (Gli3A)- e.g. Gli1 activates ShhZPAShh gradientGli3AGli3RShh/Gli InteractionsC. Tickle, Nature Molecular Biology 7(2006)45-53Shh main function may be to relieve Gli3R repression in posterior regionAnterior – high Gli3RPosterior – low Gli3R/ high Gli3AShh-/-= 1 digit;- Gli3R prevailsGli3-/-= polydactyly- Shh prevails~ 8 digits- unpatternedShh Specifies Digit Identitydigit 4 progenitor cellsdigit 5 precursors(paracrine)(autocrine & paracrine)(autocrine)BMPs Regulate Digital IdentityShh initiates BMP2 and BMP7 gradients- BMPs in interdigital mesoderm specifies identity of digits anteriorlyNoggin – BMP antagonistInsert BMP antagonistinto interdigital webbingNOTE – Fgfs from AER control phalange development;Shh bead inserted between digits can add phalange; Shhsustains Fgf signal; Fgf inhibitor = lack of phalange Remove interdigitalmesoderm- BMP targets unknownNOTE – BMP effects probably only on tissue “primed” by ShhHox Genes in Early Limb Bud5’ Hox Genes Pattern Limb ElementsForelimbHindlimbDorsal-Ventral SpecificationZone ofPolarizing Activity(ZPA)Apical Ectodermal Ridge (AER)Progress Zone (PZ)EctodermWnt7a – necessary and sufficient to dorsalize limb bud- induces Lmx1 in dorsal mesenchyme- Lmx1 knockouts = ventralized phenotype- Wnt7a knockouts = ventral footpads on both surfacesDorsalVentralApoptosis in Limb PrimordiaBMP
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