2012 limb devlopment

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    Limb devlopment

    M.Sc. 2012

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    Human embryo -32 days

    Placodes sensory placodes, lens pit, otocyst, nasal placode,primary/secondary vesicles, fourth ventricle of brain Mesoderm continuedsegmentation of paraxial mesoderm (somite pairs), heart prominence

    Head 1st, 2nd and 3rd pharyngeal arch, forebrain, site of lens placode, siteof otic placode, stomodeum

    Body - heart, liver, umbilical cord, mesonephric ridge visible externally as

    bulges. Limb upper and lower limb buds growing

    Neural first appearance of the future cerebral hemispheres. Cerebellar platedifferentiated to an intermediate layer, and future rhombic lip identifiable

    Ventricular System Subarachnoid space initially as irregular spaces on theventral surface of the spinal cord.

    Liverhepatic gland and its vascular channels enlarge, hematopoieticfunction appears

    Eye - Lens the lens placode is indented by the lens pit

    http://php.med.unsw.edu.au/embryology/index.php?title=Placodeshttp://php.med.unsw.edu.au/embryology/index.php?title=Mesodermhttp://php.med.unsw.edu.au/embryology/index.php?title=Head_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Gastrointestinal_Tract_-_Liver_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Limb_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Neural_System_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Neural_-_Ventricular_System_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Gastrointestinal_Tract_-_Liver_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Vision_-_Lens_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Vision_-_Lens_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Vision_-_Lens_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Vision_-_Lens_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Vision_-_Lens_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Vision_-_Lens_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Gastrointestinal_Tract_-_Liver_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Gastrointestinal_Tract_-_Liver_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Neural_-_Ventricular_System_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Neural_-_Ventricular_System_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Neural_System_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Neural_System_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Limb_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Limb_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Gastrointestinal_Tract_-_Liver_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Head_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Mesodermhttp://php.med.unsw.edu.au/embryology/index.php?title=Placodes
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    LIMB DEVELOPMENT Each limb results from a developmental field. The

    developmental fields are determined during gastrulation. These limb fields are established by the expression of

    HOX genes. The expression ofTbx-4 causes the limb todevelop into a forelimb and expression of the factorTbx-5 causes the limb to develop into a hind limb.

    Beginning from the fourth week from fertilization, overa period of 25 days, a complex of genetic signals controlthe intricate pathways that result in a limb with thecorrect orientation, size, and number of digits.

    Limb development is a continuous process divided into

    four stages:1. bud stage (initial outgrowth),

    2. paddle stage (dorsoventral flattening),

    3. the plate stage (relative expansion of the distal end),

    4. rotation stage (rotation around the proximodistal axis).

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    1. LIMB BUD FORMATION

    34 day old human embryo(5mm)

    34 pairs of somites

    Forelimb (lower left) started to

    develop Hindlimb just beginning (rightside)

    By day 37,In upper limb bud :

    1. nerves: median nerve, radial

    nerve and ulnar nerve enteredinto hand plate,

    2. myoblasts: spindle shapedand oriented parallel to limbbud axis

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    Limb Bud formation(contd.)

    The limbs of the embryo develop from buds that protrudefrom the side of the main body axis.

    Limb buds arise on the lateral body at the level ofsclerotomes as ectoderm and mesoderm (somite)proliferations.

    Each limb bud consists of a mesenchymal core ofmesoderm covered by an ectodermic cap.

    Limb buds will become the early arms and legs of theembryo. The upper limbs appear before the lower limbs

    that are delayed about two days in respect to the upperlimbs.

    At the early stages of embryonic development, theforelimb and hind limb buds look like paddles on eitherside of the embryo and are indistinguishable from one

    another.

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    2. LIMB PADDLE FORMATION

    The limb buds continue their formation by the migrationand proliferation of the differentiating mesenchymaltissues.

    The ectoderm at the tip of the bud thickens to form aspecialized structure, called the apical ectodermal ridge.This structure is the signaling center that allows propergrowth along the proximodistal axis (shoulders to digits).

    Along with it , the limb becomes flattened along thedorsoventral axis (back of hand to palm) and asymmetricalong the anteroposterior axis (thumb to little finger).

    Proximodistal, dorsoventral, and anteroposterior axesrepresent the routes of the normal limb growth.

    The most proximal structure (stylopod) begins todifferentiate first, followed by the progressivedifferentiation of more distal structures (zeugopod andautopod).

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    Limb paddle (contd.)

    This outgrowth and patterning depends on theestablishment and maintenance of other signalingcenters within the limb bud, named the zone of polarizingactivity located in the mesenchyme at the posteriormargin of the bud, and the non ridge ectoderm of the

    bud. These developmental components areinterdependent and, through a series of reciprocalsignals and feedback systems, yield the correct tissuepatterning and growth.

    Each bud develops to form a complex of interconnected

    limb elements comprised of bone, muscle, and tendoncharacteristic of either the fore limb or hind limb. Theactual trigger for limb bud initiation is still unknown,although likely candidates have been identified asFibroblast Growth Factor 8 and 10.

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    3.PLATE STAGE

    The plate stage is characterized by the formation of flattened plate-like areas on the distal ends of the limbs called the hand plates andfoot plates

    They are flattened along the dorsoventral axis. Within these distalplates, some structure is noticeable.

    There are radially arranged thickenings called digital rays(precursors of the digits). Between the digital rays are thin areaswhere cells begin to undergo apoptosis (programmed cell death)that allow the separating of the digits. The thin areas are calledinterdigital grooves. This arrangement gives rise to free digits.

    A constriction on the limb just proximal to the hand and footplates,called primary constrictions of the limb, is evident in this stage.

    These constrictions will develop into wrists and ankles. At approximately seven weeks, the longitudinal axes of the upper

    and lower limb buds are parallel.

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    4. ROTATION

    In the rotation stage, the position of the limb buds relative to thetrunk change in a predetermined manner not related to muscleactivity or inherent osseous torsion.

    During this stage, the rotation of the limbs creates a threedimensional structure. Because of the differential growth of thecartilage model that continue to elongate the limb, different parts

    grow at different rates. This causes a twisting or rotation of eachlimb around its proximodistal axis.

    Upper limbs rotate one way (laterally or externally), while lowerlimbs rotate the other way (medially or internally) bringing the greattoe to the midline from its initial postaxial position. This creates thecharacteristic positions of the limbs with the point of the elbowfacing caudally and dorsally, and the knee facing cranially andventrally.

    Consequently, the equivalent bones and muscles of the upper andlower limbs are oriented 180 degrees apart. This means that in thestructural organization of the upper and lower limbs, their flexorsand extensors are positioned on opposite sides and themovements at equivalentjoints are in opposite directions

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    Carnegie stage 12 to 23 Human Forelimb Development:

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    Summary

    In the 5th week hand and footplates appear at the ends oflimb buds and ridges form digital rays. Cells between the

    digital rays are removed by programmed cell death

    (apoptosis).

    Late in Carnegie stage embryogenesis (Stage 20-23, 8th

    week) limb rotation occurs. Forelimbs and hindlimbs rotate

    in different directions, upper limb rotates dorsally, lower

    limb rotates ventrally, thumb and toe rostral, knee and

    elbow face outward.

    Bones within the limb form by endochondrial ossification(begins Carnegie stage 18) throughout embryo. This

    process is the replacement of cartilage with bone (week 5-

    12).

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    Timeline limb development

    By day 44,LimbBone forms by endochondrialossification and throughout embryo replacementof cartilage with bone (week 5-12).

    By day 50, upper limbs begin to rotate ventrally

    By day 53, fingers and toes lengthen By day 56, upper limbs longer and bent at

    elbow, hands and feet turned inward, foot withseparated digits, wrist, hand with separated

    digits By day 64, fingernails appear

    By week 14,toenails appear

    http://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Limb_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Bone_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Bone_Developmenthttp://php.med.unsw.edu.au/embryology/index.php?title=Musculoskeletal_System_-_Limb_Development
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    Molecular control of limbformation

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    Limb bud

    Limb bud: Mesoderm &

    Epithelial Ectoderm

    Ectoderm over

    mesoderm

    Ectoderm thickened as

    Apical Ectodermal Ridge

    (AER)

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    Cells that Contribute to Mesoderm

    of the Limb Bud Limb mesoderm (mesenchyme) comes

    from the somite and lateral platemesoderm

    The Lateral Plate Mesoderm contributesto the skeleton, blood vessels &connective tissue

    The Somite Mesoderm contributes tothe Musculature

    Nerve cells & Neural crest cells migratein as well

    Motor Axons from spinal cord willinnervate limb

    Neural Crest gives rise to sensorynerves, schwann cells, pigment cells

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    Apical ectodermal ridge (AER).

    Secretes fibroblast growth factor (FGF)

    proteins. Required for limb growth and patterning along

    the proximal-distal axis.

    Required for

    pattern formationalong the

    dorsal-ventral

    axis.

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    Early limb dvelopment

    Limb grows & develops proximo-distallyZone of Cell Division: Region of actively dividing cells

    Zone of Differentiation: Region of cell specialization

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    Organizer

    regions.

    Zone of polarizing activity (ZPA).

    Secretes Sonic hedgehog, a protein growth factor.Required for pattern formation of the limb along the

    anterior-posterior axis.

    Homeobox-containing (Hox) genes play a role in

    specifying the identity of regions of the limb, as well as the

    body as a whole.

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    The Organization and Polarity of the Developing Limb Bud

    The limb bud has a strict pattern and polarity. Development is organized around the A-P, D-V and P-V

    axes. The tissues of the limb will differentiate in a specific pattern that is defined in part by the existing

    embryonic regions: the Apical Ectodermal Ridge (AER), the Zone of Polarizing Activity (ZPA) and the

    Progress Zone (PZ).

    The AER acts as the organizing region for the proximodistal axis of the limb. The ZPA organizes the limb

    along the A-P axis. It does this in part through the expression ofSonic hedgehog resulting in theproduction of the soluble sonic hedgehog protein. Sonic hedgehog mediates many developmental events.

    In the limb it not only meditates A-P Axis formation but also the maintenance of the AER.

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    EPITHELIAL-MESENCHYMAL

    INTERACTIONS DURING LIMB

    DEVELOPMENTExperiments originally done in chickens

    Modified here to show how results might apply to

    human limb

    Removal of AER stops limb development

    Addition of AER causes formation of 2nd limbSplitting AER leads to 2nd limb

    Inferences :AER controls limb developmentLimb mesoderm dictates limb development;

    almost any epithelial ectoderm can replace

    normal limb epithelium

    Type of limb depends on type of mesoderm

    Not species specific: Inter-specific grafts show

    same induction

    Inducer may be universal

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    Pattern Formation in theVertebrate Limb is a chainof events involving cellsignaling anddifferentiation. .

    Induction plays a majorrole in pattern formation.

    Positional

    information, suppliedby molecular cues,tells a cell where it isrelative to the animalsbody axes

    e.g.Limb development in chicks as

    a model of pattern formation.Wings and legs begin as limb buds.Each component of the limb is orientedwith regard to three axes:Proximal-distalAnterior-posteriorDorsal-ventral.

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    Transcription factor code for developmental

    identities (particular region)

    HOM

    HOX

    Hox: homeotic selector-fly mutant: transform one part of the body into another

    Homeodomain-bind to DNA-TF( regulate a large set of downstream genes)

    Structure and action-

    conserved

    limb

    Rostral-caudal axis

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    Homeotic transformation of the wing and

    haltere

    Homeotic genes

    mutated into homeosis transformation

    Bithorax-haltere into wing

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    Mutation in HoxD13

    synpolydactylyExtra digits & interphalangeal webbing (hetero)

    Similar but more severe & bony malformation

    of hands, wrists (Homo)

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    Organizers in animal embryos: Spemannorganizerand ZPA

    Spemann organizer

    Transplantation experiment

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    Action of morphogen (paracrine signal)

    Human Sonic headehog

    (notochord secreted to induce brain

    and spinal cord development)

    High conc.-neural tube

    Low conc.motor neurons

    In limb (asymmetrical pattern of

    Digits)-zone of polarizing activity

    HedgehogDrosophila mutant (alter epidermal bristles)

    Different concentrations to different fates

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    Signal Transduction & Limb Formation

    During limb development the limb bud grows away from the body in a proximo-

    (close) distal (away from) fashion.

    Developmentally, as the limb bud lengthens and limb components are

    specified and start to differentiate, what were once distal regions become

    proximal as new distal regions form. This continues until the limb is fully

    developed and the final relationship of limb components is defined.

    For exampe , in early embryogenesis, the humerus as it forms is initially the most

    distal component but once the radius and ulna and subsequent components form, it

    becomes proximal to them.PROGRESS-ZONE MODEL OF LIMB DEVELOPMENT,

    1. The AER secretes FGF that influences the closest cells (those in the progress zone)

    to develop into distal structures. FGF is a distalizing factor in limb development.

    Those cells that are not within range of the AERs influence remain proximal in nature.

    2. As the AER extends out due to the continued division of cells in the progress zone, it

    continues to affect the closest cells by causing them to be specified as distal structurecells.

    3. Those that fall out of the range of influence of the AER are no longer influenced by

    the effects of FGF and retain their previously defined status (i.e., are now proximal

    components not influenced by the distalizing effect of FGF).

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    Events of Signal Transduction & Limb

    Formation

    1. Fibroblast Growth Factor (FGF) family of factors islinked to the initiation of bud formation, maintaining budoutgrowth, and the induction of a regeneration

    2. FGFs are secreted primarily by AER

    3. Tyrosine kinase FGF receptor is expressed on the

    surface mesenchyme cells4. FGF Released by AER binds to FGF Receptor (areceptor tyosine kinase or RTK) & activates It

    5. RTK then phosphorylates critical proteins

    6. This causes the mesenchyme cells to release retinoic

    acid (RA)7. RA induces Hox Gene Expression in target cells

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    FGFs

    FGFs produced in the AER serve at least two major functions.

    1. to stimulate the proliferation of cells in the progress zone due to their mitogenic

    activities for limb bud mesenchyme and thus produce the new cells required for limb

    outgrowth.

    2. to maintain Shh expression in the ZPA.

    FGF 4 although not required to induce Shh expression is largely responsible for

    maintenance of its expression as the limb elongates. The regulatory interaction

    between FGF4 and Shh could be reciprocal as Shh produced in the ZPA induces and

    maintains FGF 4 expression in the AER. This positive feedback loop between FGF 4

    and Shh could be one of the mechanisms by which outgrowth and patterning of limb

    would be coordinately regulated, although additional molecules such as Wnt7a are

    likely to play a role in regulating Shh expression.

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    FGFs

    One of the target for FGF signaling from the AER is FGF 10 which is expressed in the

    distal limb bud mesenchyme. This factor is able to interact with FGF 8 and there

    might be a positive feed-back loop between FGF 10 and FGF 8. This reciprocal

    regulation is likely to be mediated by two isoforms of FGFR 2, FGFR 2b (that binds

    FGF 10 exclusively) and FGFR 2c (that binds FGF 8).

    A recent model has been proposed in which FGF 10 made in the mesenchyme of the

    limb field diffuses in the ectoderm where it binds FGFR 2b and induces FGF 8 in theectoderm. The FGF 8 in turn diffuses into the mesoderm and activates FGFR 2c

    which causes the upregulation of FGF 10. The FGF 10 then continues the loop and

    results in limb bud induction.

    Hence FGFR 2 appears to be essential for limb bud initiation whereas FGFR 1 seems

    to play an essential role at several stages of limb development. This assertion is

    based on the study of mouse models and expression patterns which have revealedan important function of FGFR 1 in specification of P-D axis formation.

    FGFR 1-mediated signals are required for maintaining ZPA and progress zone

    activities.

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    Thalidomide-induced embryopathy

    Teratology-teratogens

    Thalidomidedamaging tissue within the proliferating center

    Proximal-distal axis

    PZ: progress zone, AER: apical ectodermal ridge, FGF: fibroblast growth factor

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    Role of HOX ,BMP

    Some of the FGF in conjonction with Shh can affect expression of the bone

    morphogenetic protein (Bmp-2 and 7) and Hox genes, mostly Hoxd-12 and

    Hoxd-13. These latter genes are members of the Hoxdcomplex and are

    expressed within the distal wrist (Hoxd 12) and within the hand and fingers

    (Hoxd 12 and 13).

    The role of the Hoxd 13 gene in the proximodistal differentiation of limbsegments has been illustrated by the demonstration that mutations in the

    human gene transforms the metacarpals to carpals and metatarsals to

    tarsals. Likewise, overexpression of Hoxd13 in chick limb bud resulted in

    the transcriptional repression in the proximal part of the limb ofMeis, the

    vertebrate ortholog of an homeo-box containing gene in drosophila called

    homothorax(hth) that is required for proximal leg development.

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    BMP antagonists in signaling networks.

    (a) Gremlin in limb-bud development. The predicted interactions between Gremlin, BMP4, FGF4

    and Shh : Gremlin maintains FGF/Shh positive-feedback signaling during limb outgrowth by

    preventing BMP4 inhibition of this loop. Maintenance of this loop is essential for correct limb-bud

    development.

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    skeleton of the limb

    The skeleton of the limb arises from somatic mesodermby means of endochondral ossification.

    Formation of the intermediate segment (forearm)involves programmed apoptosis to separate a single

    mesenchymal condensation into two cartilage models(one for the radius and one for the ulna).

    In addition, separation of the digits depends onapoptosis within the interdigital grooves.

    Cartilage breaks down to form the joints in specific

    points. Periosteum, ligaments, tendons, andintramuscular connective tissues form from the non-condensed mesenchyme.

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    Skeleton of limb

    In addition to somatic mesoderm, there are cells that migrate

    into the limb bud from the body wall. These cells are identified

    into three groups:

    (1) somitic components (somitic myotomes in particular) that

    migrate into the limb buds and give rise to all of the

    musculature of the limb

    (2) spinal nerves from the brachial plexus that go to the upper

    limb and from the lumbosacral plexus that go to the lower limb,

    and

    (3) blood vessel precursors going into the limb to provide the

    vasculature.

    By the end of the eighth week, the limb is perfectly formed. From

    there on out, the only remaining development is growth that is

    synchronized with that of the fetal body.

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    Skeleton formation

    The skeletal elements of the limb developfrom a column-like mesodermal

    condensation that appears along the long

    axis of the limb bud during the fifth week of

    gestation in human.

    With the exception of clavicle, the bones

    of the limbs form by ossification of a

    cartilaginous precursor according to aprocess called endochondral

    ossification.

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    Skeleton formation

    Mesenchymal cells from the lateral plate condense to form prechondrogenic

    elements in the proximal region of the limb, giving rise to the anlagen of the humerus

    (or femur). Distal extension of the process results in the formation of the ulna and

    radius (or fibula and tibia) which further branches and segments to form the posterior

    proximal carpal (tarsal) element as well as the digital rays of digits IV-II.

    Prechondrocytes in the prechondrogenic condensations differentiate into

    chondrocytes in response to growth factors and secrete molecules characteristic ofthe extracellular matrix such as collagen type II and aggrecan (a large

    proteoglycan). The initial phase of chondrification results in the formation of a

    cartilaginous envelope, the perichondrium.

    This perichondrium in which bone morphogenetic protein 2, 4 and 7 (BMP 2, 4 and 7)

    and parathyroid hormone/ parathyroid hormone-related peptide receptor

    (PTH/PTHrPR) are expressed, inhibits chondrocyte proliferation and maturationthereby helping to control the growth and differentiation of the forming cartilage

    elements.

    As the cartilage elements grow different zones can be distinguished that demarcate

    the progressive differentiation of the chondrocytes. Cells at the ends of the elements

    are immature and undergo rapid proliferation.

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    Skeleton formation

    Adjacent to the proliferation zone are the larger and more sparsely distributed pre-

    hypertrophic chondrocytes that express Indian hedgehog(Ihh), PTH/PTHrPR,

    BMP 6 and BMP receptor IA (BMPRIA). The terminally differentiated hypertrophic

    cells express a unique form of collagen, type X collagen, and eventually undergo

    programmed cell death and are replaced by osteoblasts.

    Defective cartilage growth occurs in a wide spectrum of disorders called

    chondrodysplasias that usually result in dwarfisms of variable severity. The mostcommon of these disorders is achondroplasia , a dominant genetic disease caused

    by a recurrent activating mutation in the transmembrane domain of FGFR3 affecting

    chondrocyte proliferation and differentiation.

    The process ofbone ossification begins in a region called the primary ossification

    center. Mesenchymal cells in the perichondrium differentiate into osteoblasts that

    secrete the calcium salt matrix of mineralized bone and form a primary bone collararound the bone which thickens as osteoblasts differentiate. In addition to

    chondrocytes and osteoblasts, a third cell type of hematopoietic origin, the

    osteoclasts contribute to skeletal remodeling throughout development. Indeed, the

    function of osteoblasts and osteoclasts is intimately linked since osteoblast

    synthesize and secrete molecules that control osteoclast differentiation.

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    (C) Schematic showing the

    contribution of the neural

    crest, lateral plate

    mesoderm, paraxial

    mesoderm, and notochord tothe three major parts of the

    skeleton. (D) Mid-sagittal

    sections through thenotochord of mouse

    embryos at the gestation

    days 12.5 (E12.5, top) and

    E15.5 (bottom). The E12.5

    notochord is a rod-like

    structure that becomes

    surrounded by themesenchymal cell

    condensations of theprospective vertebral bodies

    (VB) and IVD. E15.5 VB are

    cartilaginous and notochord

    cells have migrated into the

    intervertebral disc spaces,

    where they have formed NP.

    Sections are stained withnuclear fast red and with

    Alcian blue, which is specific

    of the notochord and

    cartilage extracellular matrix.

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    Fate and molecular control of skeletogenic mesenchymal cells.

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    hondrocyte early differentiation and development of cartilage primordia. (A)

    Alcian blue staining of a mouse embryo at E14.5 demonstrates that

    chondrocyte differentiation of skeletogenic cells leads to the formation of a

    primary skeleton that is entirely cartilaginous. (B) Sections through the

    developing paws of mouse embryos illustrate the major steps of early

    chondrogenesis. At E10.5, the limb bud is filled with skeletogenic cells. By

    E12.5, some of these cells have formed precartilaginous condensations that

    prefigure the future digits. By E14.5, condensed prechondrocytes have

    undergone chondrocyte early differentiation.

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    Chondrocyte maturation and development of cartilage growth plates.(A) Sections through a mouse embryo tibia (T) illustrate the development of growth plates andendochondral bone. At E13.5, early chondrocytes in the center of cartilage primordia undergo

    prehypertrophic and hypertrophic maturation. They reach terminal maturation and are replaced by

    endochondral bone by E15.5. Later on, growth plates maintain themselves and elongate developing bones.

    Chondrocytes keep proliferating and give rise, layer by layer, to maturing chondrocytes. These cells, which

    eventually die, are replaced by bone. The sections are stained with Alcian blue and nuclear fast red. (B)

    Schematic of the molecular control of GP chondrocytes.

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    Osteoblast differentiation

    and intramembranous and

    endochondral ossification.(A) Sections through an endochondral

    bone in a newborn mouse show the

    replacement of cartilage by bone. The left

    section is stained with Alcian blue and the

    right one with the von Kossa reagent,

    which leaves a brown precipitate on the

    mineralized bone matrix. (B) Schematic

    showing how GP chondrocytes and bone-

    forming cells interact with each other toachieve endochondral ossification. (C)

    Coronal sections of a newborn mouse

    head. In the suture linking the two frontal

    bones (top panel), osteoblast precursors

    are surrounded by an abundant

    collagenous matrix. Further away (bottom

    panel), osteoblasts mature and deposit a

    mineralized bone matrix. This matrix is

    stained with the von Kossa reagent. (D)

    Schematic of the molecular control of

    osteoblast differentiation.

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    Synovial joint development.(A) Sections through the mouse knee joint at various stages of development. At E12, the presumptive joint region

    (arrow) is not distinguishable from the femur (F) and T precartilaginous condensations. At E13.5, this region becomes

    distinguishable as surrounding cartilage primordia are overtly developing. At E16.5, joint morphogenesis is well

    advanced. The joint cavity has formed between the patella (P) and F. Cruciate ligaments and FP, lined with synovial

    tissue, are developed. At the postnatal day 19, the knee joint is mature. The AC is separated from the epiphyseal GP

    by a secondary center of ossification. The sections are stained with Alcian blue and nuclear fast red. (B) Schematic of

    the molecular control of synovial joint cell differentiation.

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    Models for the development of sexually

    dimorphic digit proportions

    AR (blue circles) and ER (pink circles) are

    present in the digit condensations of

    male and female embryos, with higher

    levels found in 4D

    . (A) In males, digits are exposed to high

    levels of circulating androgen and lowlevels of circulating estrogen, which

    results in preferential binding and

    activation of AR

    (ARA represents the androgen bound to

    the AR). High AR activity and low ER

    activity (ARA/er) in males leads to

    differential gene expression profiles in4D relative to 2D (green indicates genes

    higher in 4D, and red indicates genes

    higher in 2D). In turn, chondrocyteproliferation is increased in the proximal

    phalanx of 4D, which results in elongation

    of 4D relative to 2D,

    leading to a lower 2D:4D ratio.

    (B) In females, digits are exposed to high

    levels of estrogen and low levels of

    androgen, leading to preferential binding

    and activation of ER (ERE). Low AR

    activity and high ER activity (ar/ ERE)induces an opposite shift in the

    skeletogenic gene expression profile

    of 4D relative to 2D (indicated by gene

    names in green and red, as above).Higher levels of activated ER cause

    decreased chondrocyte proliferation in the

    middle phalanx of 4D, which reduces its

    growth relative to 2D and results in a

    higher 2D:4D ratio.

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    Summary molecular control

    Limb Initiation

    FGF

    FGF10 , FGF8 (lateral plate intermediate mesoderm)

    prior to bud formation

    FGF8 (limb ectoderm) FGFR2

    FGF can respecify Hox gene expression (Hox9- limb position)

    Hox could activate FGF expression Limb Specification (Fore- Hind-)

    regulated by T-box genes (transcription factor)

    Tbx5- forelimb

    Tbx4 leg

    Limb Axes

    Limb Patterning- Axes Signals give positional information which is interpreted by Hox gene expression establishing

    programs of differentiation.

    Proximodistal Axis

    Dorsoventral Axis

    Anteroposterior Axis

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    Summary

    Limb Patterning- Axes :Wing has been used as Model of limb development as chick wingeasy to manipulate: removal, grafting, additional ARER, ZPA etc

    Limb Patterning- Axes

    Proximodistal Axis

    AER formed by Wnt7a

    then AER secretes FGF2, 4, 8

    stimulates proliferation and outgrowth Dorsoventral Axis

    somite provides dorsal signal to mesenchyme

    which dorsalizes ectoderm

    ectoderm then signals back (Wnt7a) to mesenchyme to pattern limb

    Anteroposterior Axis

    ZPA zone of polarizing activity

    mesenchymal posterior region of limb

    addition of extra ZPA duplicated digits

    signal is Shh