Vertebrate Limb Patterning Chris Campbell cc59@ 829-3462

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  • Vertebrate Limb PatterningChris Campbellcc59@buffalo.edu 829-3462

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  • Origin of the limbDorsal view of early embryo showingSomites (arrows) and neural folds *. Somites form starting near the head region and progressing towards the tail. Counting somites indicates the developmental stage of an embryo

    *

  • Basic limb buds structures are conserved among vertebratesVertebrate limbs originate as twopairs of limb buds that appear atspecific levels of the embryonic flank. Forelimbbuds appear first (~E9 in mouse) followed byhindlimb buds ~ 0.5 -1day later. Each limb bud consists of lateral platemesoderm (LPM) covered by ectoderm.

    E10

  • The parts of the vertebrate limbstylopod zeugopodDuboc (2011) Dev Dyn 240, 1017

  • Bat wingsnake

  • Techniques to Study the Regulation of Differentiation During DevelopmentObservation of normal development in animals of different speciesReporter genes or other markers (such as fluoresceinated lectins or antibodies) to delineate particular cell typesAblation and transplantation experimentsUse classical genetics to identify genes in which mutations affect limb development. (phenotype to genotype) Loss or gain of function mutations (knockouts, transgenics, injection of sense, antisense or small inhibitory RNAs) (genotype> phenotype)In vitro organ and cell culture

  • What makes chick a good model system for studying limb development?Ease of physical manipulation

  • The developing limb bud is patterned along three axes : Proximal-Distal Anterior-PosteriorDorsal-Ventral

  • The apical ectodermal ridge (AER) isa specialized epithelium (derived from theectoderm) that controls limb bud outgrowth.The AER is a transient structure that does notitself contribute cells to the mature limb.Proximal Distal PatterningE-cadherin

  • What regulates limb bud outgrowth?AER is required forlimb bud outgrowthLimb mesenchyme is required for and specifies type of limb.AER can be replacedby FGF bead. **Inductive interactions between AER and limb mesenchyme similar to the inductive interactions that take placebetween metanephric mesenchyme and ureteric bud.

  • Cohn (1999 ) Nature 399, 474

  • What regulates limb bud outgrowth?Studies in the early 90s indicated that placing a bead soaked in one of several FGFs adjacentto chick flank between stage 13 and stage 17 of development could induce ectopic limbs.

    These limbs were frequently quite well developed and tended to be more wing like if the beadwas placed anteriorly (i.e. near where wing normally forms) and more leg like if placed posteriorly.

    Digit patterns were almost always reversed (indicating a defect in AP patterning) as were Shh (signaling molecule) and hoxd13 (transcription factor) expression patterns. These genes arenormally expressed in the posterior part of the bud but were expressed anteriorly in the ectopic buds.Cohn et al. (1995)Cell 80, 739ShhAER functions by providing an FGFand also is involved in AP patterning.

  • Proximal elements are patterned prior to distal elements and patterning is dependent on signals(FGFs) from the AER

  • Fgfs (fibroblast growth factors) are soluble secreted molecules that bind to their receptors and activate kinase signaling pathways leading to the modulation of transcription of specific genes. Fgf4AER ck/o no effectFgf8AER ck/o limb deformitiesFgf9AER k/o no effectFgf17AER k/o no effect

    Fgf10LPM k/o limb deformities

    FGFs and Their Mechanism of ActionFGF10in situSun (2000) Nat Gen 25, 83

  • Fgf10 and limb budFgf10 is expressed in the mesenchyme. In its absence:H&EE10.5Sekine (1999) Nat Gen 21, 139

  • Lewandoski (2000) NatureGenetics 26, 460Fgf8Fgf8-/- is an early embryonic lethal and therefore two different conditional knockouts were made*RAR-cre;Fgf8-/flox*RARCre is expressed in forelimbAER but not in hindlimb AER.Msx-Cre is expressed in both FL and HL butexpression is turned on after Fgf8 in FL (see b-i).Fgf4expressedfor longerperiod oftime inFgf8 koContrary to the predictions of the progress zone model, it is the zeugopod that is most affected in RAR-cre;Fgf8 mutants, while the stylopod and more distal autopod is relatively normal.

  • Fgf4/Fgf8 conditional double knockouts have a more severe phenotype than Fgf8 single knockoutsMsx2-Cre;Fgf4-/flox;Fgf8-/floxSun (2002) Nature 418, 501Dt = deltoid tuberosityDt = deltoid tuberosity

  • AER forms in mutant hindlimb buds (unlike what happens in Fgf10-/- mice) but at ~E10.5 expression of many mesenchyme markers is perturbed.By ~E11.25 the AER will begin to degenerate.Knocking out Fgf4 & Fgf8 in the AER causes altered gene expression in the mesenchyme including expansion of a normally proximally expressed gene.Fgf4-/-Fgf8-/-

  • Mercader (2000) Dev 127, 3961

  • Sun (2002) Nature 418, 501

  • Zeller (2010) Curr Op Gen & Dev 20, 384

  • Patterning of the vertebrate limbSignaling by the non AER ectoderm controls dorsal ventral patterningThe developing limb bud is patterned along three axes : Proximal-DistalDorsal-Ventral Anterior-PosteriorDorsalproximaldistal

  • BMPs and Dorsoventral Patterningnormally expressed in ventral ectodermEn-1Bmp4 & Bmp7

    Normally expressed in dorsal ectodermWnt7-a & r-fng

    Normally expressed in dorsal mesenchymeLmx-1 & Shh

    Normally expressed in AERFgf8Bmp4Bmp7ShhFgf8dorsalventralIn situ for Shh (molecular marker for ZPA) & Fgf8(molecular marker for the AER)Pizette et al. (2001) Dev.128,4463

  • BMPs signal by binding to BMPreceptors. This binding can beinhibited by BMP antagonistssuch as Noggin or GremlinCanalis Endocrine Reviews 24, 218 (2001)

  • Bmp4Bmp7Fgf8Downregulate BMP(v) signaling by misexpression of Noggin (BMP antagonist) by retroviral injections to the ectoderm leads to loss of en1(ve) and expansion of Wnt-7a (de), Lmx1 (dm) & Shh (dm) into ventral region of bud (i.e. Dorsalization of bud). Also leads to misexpression of ShhAnd Fgf8Upregulate BMP(v) signaling by misexpression of a constitutively active Bmp receptor leads to expansion of en1, and loss of Wnt-7a & Lmx1(d) (i.e.Ventralization of bud). Also leads to loss of AERPizette et al. (2001) Dev.128,4463Shh

  • EnWnt7aFgf8Bmp2Engrailed knockout mouseLoomis (1996) Nature 382, 360

  • Li (2010) Dev 137, 1181

  • Patterning of the vertebrate limbAnterior-posterior patterning iscontrolled by signals from the mesenchymal cells of the ZPA(zone of polarizing activity)The developing limb bud is patterned along three axes : Proximal-DistalDorsal-Ventral Anterior-Posteriorproximaldistal

  • The ZPA and Shh signalingZPA** notochord andfloorplate organizingcenters possess similaractivity when graftedonto limb buds spinal cord will inducemetanephric mesenchymeanteriorposteriorDuplication of posterior digits

  • Riddle et al. (1993) Cell 75, 1401Tickle et al. (1982) demonstrated that applying RA can result in similar mirror-imageduplications to what is seen with ZPA. However, a series of experiments suggested that RA does not act as a morphogen in this system but acts by inducing an ectopic AER. The evidencefor this was 1) required more RA to induce limb bud duplications than is normally seen in the ZPA.2) it is the tissue DISTAL to an RA implant that acquires ZPA activity not the area immediatelyadjacent.

    Based on the ability of drosophila hedgehog to act as a morphogen in flies, Riddle et al. soughtto clone a hedgehog related gene(s) expressed in chicken limb buds in the ZPA. Found Shh.Evidence that Shh is the morphogen that mediates ZPA polarizing activityExpressed in correct temporal and spatial pattern during limb bud development. (ZPA activityhad been mapped by assaying small blocks of limb tissue at different stages of developmentfor their ability to induce digit duplication.)

    Shh is also expressed in other tissues that can induce digit duplications when grafted onto anterior chick limb buds (i.e. notochord).

    RA induces Shh.

    Shh induces digit duplications and Hox gene expression repatterning similar to ZPA grafts.

  • Cohn (1999 ) Nature 399, 474

  • Shh-/- MiceChiang et al. (2001) Dev. Biol. 236,421Mutant mouse limbscontain a single digitBased on gene expressionpattern and morphology itis digit 1. Thereforedigit 1 doesnt requireShh signaling. Shh-/- mice form truncated limbs with single digits and show inappropriate patterning of Hoxgenes (early) and gdf5 & Msx1 (markers expressed in digits). Unlike mesenchyme from wt mouselimb buds, Shh-/- mesenchyme has no polarizing activity when grafted onto chick limbbuds. Several AER markers were expressed early but later disappeared (eg Fgf8). Bmp2&4 are expressed at low levels, Gremlin(known target of Shh signaling and inhibitor ofBMP signaling) is turned on but rapidly downregulated & Gli3 expression (negatively regulated by Shh) is extended.

    In the absence of SHH, AER formation is initiated but doesnt persist as long as in wt.

  • Shh:GFPCre/+;R26R/+Harfe (2004) Cell 118, 517Shh:GFPCre/+;R26R/+Shh:CreERT/+;R26R/+

  • Xu (2011) Dev Dyn 240, 1303

  • T-box genes and limb patterning FL or HL?Tbx2Tbx3Tbx4Tbx5Ancient T-box gene2/34/5Tbx2Tbx4Tbx3Tbx5amphioxis

  • T-box genes & Limb specificationGibson Brown (1996) Mech. Dev.56, 93 reported that Tbx4 was expressed primarilyin hindlimbs while Tbx5 is expressed primarily in forelimbs and proposed that these genesmay specify limb identity.

    Tbx4Tbx5chick embryoszebrafishTamura (1999) Mech Dev 87, 181

  • RCAS-EnTbx5RCAS-EnTbx4Tbx4 and Tbx5 Specify Legs and Wings Respectively in ChicksTakeuchi (2003) Dev.