Limb developmentEd Laufer elaufer@columbia.eduLimb DevelopmentWhat signals control initiation of limb bud formation ?What signals transform the embryonic limb bud into a mature limb comprised of a precisely interconnected array of many different tissues?What we will cover...The developing limb is a model environment to study developmentcell fate specification - eg bone vs muscle vs tendoncell migration and pathfinding - muscle precursors, innervationcell proliferation/programmed cell death (apoptosis)- correct tissue morphology, interdigital cell deathlimb abnormalities are associatedwith many human congenital syndromes tissue patterning - organization of structures in 3D spaceEARLY LIMB PATTERNING:Limb formation initiates during the fourth week of development (E9.0 in mouse) as the primary axis (AP) is still elongating. First the forelimb and then the hindlimb begin as protrusions from the lateral plate mesoderm at the sides of the embryo. Limb buds consist of a core of mesenchyme and an outer covering of ectoderm.1 in 200 live human births display limb defects.Forelimb budHindlimb budHuman Limb Development5 weeks6 weeks8 weeksLimbs rotate inwardDay 33: hand plate, forearm, shoulderDay 37: Carpal region, digital plateDay 38: Finger rays, necrotic zonesDay 44: toe raysDay 47: horizontal flexionDay 52: tactile padsA-P (fingers); D-V (palm); P-D (length)5 weeks6 weeks8 weeksLimb skeletal elements:ChickenMouseStylopod: The proximal element of a limb. The humerus in the forelimb and femur in the hindlimbZeugopod:The intermediate element of a limb. The radius and ulna in the forelimb and the tibia and fibula in the hindlimbAutopod: The distal elements of a limb. The wrist and the fingers in the forelimb and the ankle and toes in the hindlimbDorsal: top of hand/pawVentral: palmHow is limb initiation controlled?Secreted signaling molecules, fibroblast growth factors (FGFs) can induce ectopic limb formation from cells in the flank of the chick embryoPre-limb bud stage chick embryo(H+H st15)After 9 further days of incubation (st36)-a single molecule (FGF) is capable of inducing cascade leading to limb formation-cells in the flank are competent to respond to this secreted signalKawakami et al., Cell 2001Members of another family of secreted proteins, Wnts, can also induceformation of ectopic limbs from the inter-limb lateral plate mesodermLimb initiationAxial cueWnt?LPMFgf10EctodermWnt3a, Fgf8Transfers information from the body to the limb axislpm: lateral plate mesodermLimb-type specificationMorphologically uniform limb buds developto form morphologically distinct limb elementsCell identity and plasticity in the developing limb budSaunders et al., 1957, 1959Limb-type specification: candidate geneschick st.29Tbx5Tbx4Pitx1A source of FGF applied to the interlimb flank induces ectopic limb formation in the chick embryo: Tbx expression correlates with morphologyConditional knock-out of Tbx5 in the limbsleads to the absence of the forelimbsTbx5lox/lox;Prx1creControl no Tbx5 in limbTbx4 for Tbx5: RescueReplacement of Tbx5 with Tbx4 in the limb rescues limb outgrowth. The limb remains a forelimb.Correlation is not causality: causality must be determined experimentallyLimb abnormalities are associatedwith many human congenital syndromes Mutations in Human TBX5 are associated with Holt-Oram Syndrome (HOS)Mutations in human TBX3 are associated withUlnar-Mammary Syndrome(UMS)Tbx3Tbx5second most-common congenital abnormality in human live birthsalso common abnormality following environmental insult (eg Thalidomide)3 signaling centers pattern the 3 primary axes of the limbApical ectodermal ridge (AER) - proximal-distalZone of polarizing activity (ZPA) - anterior-posteriorDorsal ectoderm - dorsal-ventralApical ectodermal ridge (AER) - proximal-distal axisSignals from the AER maintain limb outgrowthAER manipulations give insight into AER functionrequiredpromotes outgrowthnot instructivenot instructiveFGF sourceConclusions from ‘classical’ embryological studies- the AER is required for normal proximal-distal patternEarly stage limb budLater stage limb budSaunders, 1948- required for outgrowth, full P-D pattern- elements laid down in proximal to distal progressiontimeThe progress zone (PZ) modelSummerbell et al. Nature 1973The length of time a cell spends in the PZ may determineproximal-distal identityEvidence supporting a role for FGFs in proximal-distal patterning - Fgf8 is expressed in the apical ectodermal ridge (AER)i.e. present at right time, in right placeWhole mount RNA in situ hybridization - chick (st23)What factors are regulating outgrowth?Fgf8/Fgf4 double knock-out mouse: a genetic equivalent to AER removal in the chickcontrolmutantSun et al., Nature 2002Limb outgrowth disruptedbut distal structures doform. This would not bepredicted by the progresszone modelAlternative model: early allocation followed by expansionprogenitor pools are specified early during limb outgrowthA signal (FGF) from the AER progressively expands these preexisting populationsAER defects give rise to proximodistal outgrowth phenotypesAm J Hum Genet. 2000 July; 67(1): 59–66.Published online 2000 June 5.Split-hand/split-foot malformation (SHFM) caused by p63 mutation: reduced AER maintenanceDiplopodia: ectopic AER?Eudiplopodia: ectopic AER (chicken)Pediatric Radiology 200310.1007/s00247-003-1017-3The axes of the limbZone of polarizing activity (ZPA) - anterior-posteriorSaunders and Gasseling 1968A region of cells in the posterior limb bud, the zone of polarizing activity (ZPA)Is important for patterning the anterior-posterior axis of the limb Again, an importantsignaling center in the limb was initially identified in ‘classical’embryological experimentsEctopic digits are not derived from the ZPA graft itself. They are induced in the host tissue. This is a non cell-autonomous phenotype.Morphogen model: cell identity via threshold responses to a gradient of signaling moleculecirca 1969Sonic Hedgehog (Shh), a vertebrate homolog of the Drosophila (fruit fly) gene hedgehog, is expressed in the ZPADirect evidence for a role of Shh in anterior-posterior patterningThe complementary approach:Deletion or ‘knock-out’ of the Shh gene leads to a disruption in anterior-posterior patterning of the limbChiang et al., Nature 1996Kraus et al., Mech Dev. 2001Biochemistry: Gli3 transcription factor mediates Shh functionGli3 loss-of-function results in polydactyly.Surprisingly, Shh/Gli3 double mutants look identical to Gli3 nullsShh modulates inherent polydactylous limb ‘ground state.’Shh inhibits an inhibitor of digit formation and imposes polarityAcheiropodia phenotype in manAcheiropodia: deletion of the SHH limb enhancerpreaxial polydactyly: ectopic anterior SHH activitymousehumanJ Anat. 2003 January; 202(1): 13–20.doi: 10.1046/j.1469-7580.2003.00148.x.Human Figure 2 Affected individual (IV-18) from family 4. Note the small fingerlike appendages at the end of the arm, which are present bilaterally (A and B). C, Radiograph showing tapered amputation of the distal tibia. The proximal tibial epiphysis is well preserved. D, Radiograph showing dysplastic distal humerus articulating with a rudimentary forearm composed of three dysplastic long bones.Region is in intron 5 of human LMBR1 locus, 1MB away from Shh coding sequenceAm J Hum Genet. 2001 January; 68(1): 38–45.Published online 2000 November 22.Copyright © 2001 by The American Society of Human Genetics. All rights reserved.SHH function in generating the amniote limb skeletonRos, M. A. et al. Development 2003;130:527-537SHH function in generating the amniote limb skeletonThe Dorsal-Ventral AxisDorsal-ventral limb asymmetryDorsalDorsal-ventral asymmetry is required for coordinated limb movementSignals from the dorsal ectoderm play an important role inpatterning the dorsal-ventral axisInitial observations in‘classical’ embryologicalexperimentsCandidates identifiedMisexpression of Wnt7a in the ventral ectoderm leads to the ectopic induction of Lmx-1 in the ventral mesenchymeGenetic deletion of Lmx1b in the mouse leads to a loss of dorsal limb patternA cascade of factors in the ectoderm controls dorsal-ventral polarity in the mesodermHuman LMX1B mutation: Nail Patella SyndromeSignaling center crosstalk:Signals from the AER (FGFs) are required to maintain expression of ShhDorsal ectodermZPAAERSignaling centersWnt7aShhFGFPtc(receptor)Shh targetseg Bmp2 HoxD genesPATTERNlmx1cellproliferationLimb patterning occurs through the coordination of signals from three signaling centersSZASZAHINDLIMBSFORELIMBSHoxa13/Hoxd13Hoxa13/Hoxd13Hoxa11/Hoxd11Hox9PHox10P()Patterning of the limb elements: Hox9 through Hox13 paralogous groups are responsible for establishing morphological patternHox10Hox11Hox13SZAFromental-Ramain, et al, 1996ZHoxa11/Hoxc11/Hoxd11Hoxa10/Hoxc10/Hoxd10hlhlflfl & hlWellik, et al, 2003Apoptosis regulates interdigit formationBMP signaling regulates interdigital apoptosisDominant-negative BMP receptors inhibit interdigit cell death Tendons develop in situ, while muscle progenitors migrate in from the somites and follow a prepattern established in the limb mesenchymeTendon MuscleSummaryInitiation: localized FGF activityOutgrowth: FGFs produced by the AERAnterior-posterior patterning: ZPA/Sonic hedgehogDorsal-ventral patterning: engrailed-wnt7a-lmx1bIntegration of signaling centersTbx genes are required for limb outgrowthHox genes are essential regulators of limb development and control segmental developmentApoptosis sculpts interdigital regions.Summary