Limb Development
Overview of Limb Formation
Initiation of Limb Development
Limb Field
Limb Bud
Outgrowth of the Limb Bud
Apical Ectodermal Ridge (AER)
Limb Bud Mesoderm
Morphogenetic Signaling
Development of Limb Tissues
Skeleton
Musculature
Innervation
Vasculature
Anatomy
Forelimb = arm – humerus, radius, ulna,
carpels, metacarpals, phalanges
Hindlimb = leg – Femur, tibia, fibula,
tarsals, metatarsals, phalanges
Generalized
Stylopod (humerus/femur)
Zeugopod (radius,ulna/tibia,fibula)
Autopod (hand/foot)
Limb Development
Limb Bud
Lateral Plate Mesoderm
Growth Reduction in the Flank
Limb Axes
Anterior
Posterior
Apical Ectodermal Ridge (AER)
The AER is an inducer of limb outgrowth
AER Removal
AER removal results in limb
truncation
Limbless mutant – the AER fails
to form
AER signal is Fibroblast Growth
Factors – FGF2, FGF4,
FGF8
AER Addition
Inducing an extra AER
can result in the
formation of a
Supernumerary limb
Eudiplopodia – mutant
(chick) with an
extra AER
AER grafting induces
supernumerary
limbs
Mesoderm Control of Limb
Pattern
Supernumerary Limbs
Zone of Polarizing Activity - ZPA
ZPA
ZPA grafts to the anterior induces supernumerary limbs
Supernumerary limbs are mirror symmetrical with normal
limb
Stimulates mesenchymal cell proliferation
Induces changes in the AER
ZPA signal is conserved – e.g. human ZPA induces chick
supernumerary limbs
RA induces the ZPA
The ZPA signal is Sonic Hedgehog
Clinical Terms
• Meromelia - Absence of part of the limb
• Amelia, Ectromelia - Absence of 1 or more limbs
• Phocomelia - Short, ill-formed limb (flipper limb)
• Hemimelia - Stunted distal limb
• Acrodolichomelia - Enlarged autopod (hand,foot)
• Adactyly - Absence of all digits
• Ectrodactyly - Absence of digits (one or more)
• Polydactyly - Extra digits
• Syndactyly - Fusion of digits
Limb Development
Overview of Limb Formation
Initiation of Limb Development
Limb Field
Limb Bud
Outgrowth of the Limb Bud
Apical Ectodermal Ridge (AER)
Limb Bud Mesoderm
Morphogenetic Signaling
Development of Limb Tissues
Skeleton
Musculature
Innervation
Vasculature
Initiation of Limb Development
Limb Morphogenetic field = population of cells
committed to give rise to a particular organ when
transplanted to a neutral site.
Fate maps – Cell marking to identify the cells that
participate in limb formation
Regulation - Cells within a field can modify their fates to
make up for deficiencies.
Specification – Cells fix their fate - Determination
FGFs Initiate Limb Formation
Establishment of the limb field involves of growth
factors particularly FGFs
Assay – Microcarrier bead implantation to flank (limb
competent tissue)
Endogenous gene expression
FGF10 (mesenchyme) Æ FGF8 (ectoderm)
FGF10 Knockout causes a limbless phenotype
FGF4/FGF8 double mutant Æ Limbless
Fgf10 – Mesenchymal Expression
Fgf10 KO is
Limbless
Fgf8
Fgf4; Fgf8 Double Knockout
Fgf10
FGF Induces Supernumerary
Limbs
FGF10 FGF8
#1 #2
RA is Involved in Limb Initiation
RA bead implantation mimics ZPA grafts – induces ZPA
Inhibiting RA synthesis Æ Limbless phenotype
Retinaldehyde Dehydrogenase Knockout Æ Limbless
phenotype
RA Æ Hoxb8 Æ Shh Æ Bmp2 Æ limb formation
RA
* *
*
Arms or Legs??
Question: what makes hind and fore limbs different?
In general the cells are similar and they behave
identically
Tbx genes are expressed early and distinguish fore
from hind limb.
Tbx–4 is hind limb-specific; Tbx-5 is forelimb-specific
Ptx1 gene controls Tbx4 - hindlimb – Changing Ptx1
expression can change a wing into a leg.
Ptx1 and Tbx genes encode for transcription factors
Tbx-5
Tbx-4
*
* *
Limb Development
Overview of Limb Formation
Initiation of Limb Development
Limb Field
Limb Bud
Outgrowth of the Limb Bud
Apical Ectodermal Ridge (AER)
Limb Bud Mesoderm
Morphogenetic Signaling
Development of Limb Tissues
Skeleton
Musculature
Innervation
Vasculature
Limb Bud
Early limb bud is composed of lateral plate mesoderm
Migratory cells invade the limb bud:
Myoblasts from somites
Pigment cells and Schwann cells from neural crest
Axons innervate the limb bud
Angioblasts
Migrating cells do not invade the growing limb apex
Limb Axes
Anterior
Posterior
Apical Ectodermal Ridge (AER)
Transient embryonic structure
Multilayered Ectoderm
Connected by Gap Junctions
Basal lamina separates AER
from underlying mesenchyme
Fibroblast Growth Factors
FGF-2, FGF-4, FGF-8 and FGF-9 are produced by the AER
FGF family is large
Heparan sulfate binding
Heparan sulfate is required
FGFR – FGF receptor
Transmembrane receptor tyrosine kinase
FGFs will stimulate limb outgrowth after AER removal
FGFs will induce regeneration of amputated limb buds
FGFs will induce flank supernumerary limbs
FGFs Expressed in the Limb
FGF 8 - AER
FGF 4 - AER
FGF 10 - Mesenchyme
AER removal and FGF beads
FGFs can replace the activity of the AER
FGFs Induce Limb Bud
Regeneration
Proximal Distal Axis
FGF2, FGF4, FGF8 produced by the AER can
replace AER function
FGF8/FGF4 - considered to be the endogenous
signal
The AER and FGFs function to maintain gene
expression at the tip of the limb bud (Shh,
Fgf10, Msx1, Hoxd13, many more)
Limb Axes
Anterior
Posterior
Sonic Hedgehog (SHH)
SHH is the ZPA signal
SHH is a secreted cholesterol linked-
protein – a Morphogen
SHH receptor is PATCHED – a
transmembrane protein
SHH signaling pathway is responsible
for some types of cancers
Other Hedgehog related genes
Indian Hedgehog
Desert Hedgehog
Sonic Hedgehog
RA Induces ZPA/SHH
RA Induces Shh
Shh KO
Cyclopia, absence of spinal column, absence of ribs
Forelimb truncations
Hindlimb with a single digit
Shh is required to maintain Fgf8
Polydactylous Mutants and Shh
Repressor Activity
Gli3
The Hedgehog Signal Transduction
Pathway
The Hedgehog Signal Transduction
Pathway
Greig Cephalopolysyndactyly Syndrome
Gli3 Gene - Autosomal Dominant
Large head, high prominent forehead, broad nose, wide-set eyes
Mild retardation
Multiple deformities of hands and feet – polydactyly, syndactyly
Pallister-Hall Syndrome
Gli3 - Autosomal Dominant
Syndactyly
Polydactyly – postaxial
Short digits
Shh / Gli3
Double
Mutant
Polarizing Zone Model
SHH Diffuses as a Complex
Pallister-Hall Syndrome (predicted)
Grieg
Cephalopolysyndactyly (predicted)
Syndrome
Limb Axes
Anterior
Posterior
Ectoderm Controls D/V Pattern
The AER separates Dorsal Ectoderm from Ventral Ectoderm
AER position is controlled by the expression of Radical Fringe
(RFng)
Dorsal Mesenchyme Pattern is controlled by the dorsal
ectoderm - production of Wnt7a – a secreted factor.
Induces Lmx1 (a homeobox containing gene) and
ventral to dorsal transformation. Wnt-7a knockout
displays dorsal to ventral transformation.
Ventral Ectoderm expresses Engrailed-1 (En-1) – a
transcription regulator. En-1 knockout mice display
ventral to dorsal transformation
Wnt7a and Lmx1
Expression
Wnt 7a (a secreted factor) is
expressed in the dorsal
ectoderm
Lmx1 - transcriptional regulator -
cysteine-histidine-rich LIM
domain and a homeodomain
Lmx1 is expressed in the dorsal
mesenchyme
Wt Wt
Ventral Dorsal Adult
Wnt7a -/- Wnt7a -/-
Ventral pads Ectopic Dorsal pads
Wnt7a Knockout
Wt Wt
Ventral tendon
Wt
Ventral sesamoid bone
Ectopic Lmx1b Expression
Results in Dorsal Pattern
Dorsal specification pathway – WNT7a Æ LMX1b Æ Dorsal pattern
Lmx1b – Nail Patella Syndrome
Autosomal dominant
Nails, Knees, Elbows, Ilium,
Scapula
Delayed ossification of
secondary centers
Renal insufficiency
Engrailed-1 (En-1)
• En–1 is expressed in the Ventral Ectoderm
• En-1 is a transcription factor
En-1 Wnt-7a Fgf-8
wt En-1-/-
En-1 Knockout
Circumferential
nails
Ectopic ventral
digits
Absence of ventral
tendon
Dorsal flexion
Absent Sesamoid
bone
Wnt7a/En1 Double Mutant Digit
Genetics of Limb Pattern
Hoxd13 and Polysyndactyly
3 pedigrees each with a polyalanine insertion in the DNA binding
region of the HOXD13 gene.
Limb Development
Overview of Limb Formation
Initiation of Limb Development
Limb Field
Limb Bud
Outgrowth of the Limb Bud
Apical Ectodermal Ridge (AER)
Limb Bud Mesoderm
Morphogenetic Signaling
Development of Limb Tissues
Skeleton
Musculature
Innervation
Vasculature
Cell Death and Digit Formation
Cell Death – Apoptosis is a normal Developmental
Pathway
Interdigital Cell Death – Paddle to Individual Digits
BMP signaling controls Interdigital Cell Death
Msx genes and RA also play a role in cell death
Absence of cell death results in syndactyly
Differentiation – Skeleton
Limb Skeleton – derived from Lateral Plate
Mesoderm
Differentiation is Proximal to Distal, Posterior to
Anterior
Endochondral Bone
Ectoderm inhibits Chondrogenesis
Important factors
BMPs
Indian Hedgehog (IHH)
Growth/differentiation factor-5 (Gdf-5)
Joint Formation
Joints (articulations) - junction between bones
3 Classes of fibrous joints
Dense fibrous tissue - little or no movement, e.g.
sutures of skull
Synchondroidal joint – interzone cells differentiate
into fibrocartilage, e.g. between pelvic bones
Snynovial joint, complex differentiation of
interzonal mesenchyme, e.g. knee and elbow
Synovial Joint
Precartilage rod – transverse splitting
1) Interzone mesenchyme differentiating into
fibroblastic tissue
2) Fibroblasts differentiate into 3 layers
2 cartilage layers with a dense connective
tissue in between
3) Central region forms menisci and ligament
surrounded by the joint capsule
4) Vacuoles form and coalesce to from the synovial
cavity.
Synovial Joint
Joint – induced by Wnt14 Æ Gdf-5 (Growth Differentiation Factor-5)
Noggin functions to exclude BMPs – without Noggin joints fail to
form
Differentiation – Musculature
Migration of myoblasts from
somites
Dorsal and Ventral muscle
masses
Tendons form from limb bud
mesenchyme interacting
with myotubes
Tendons form in the absence
of muscle
Differentiation – Innervation
Motor Axon from the
Spinal cord innervate
limb tissues
Local cues guide axons
Axons can regulate for
small changes
Sensory axons use motor
axons for guidance
Differentiation – Vasculature
Angioblast - Endothelial cell precursor
Fine capillary network Æ large central artery
Maginal sinus under the
AER – accumulates
blood and drains the
limb via peripheral
veins
Ectoderm inhibits
vasculature