Professional Documents
Culture Documents
MANOJ RAMACHANDRAN
Consultant Paediatric and Young Adult Orthopaedic Surgeon,
Barts Health NHS Trust, London, England
Objectives
Embryonic limb development
Patterning and differentiation
Limb growth
Embryonic limb
development
Molecular level
Cell produces secreted protein
Protein binds to specific receptor
Intracellular changes transcription factors to nucleus
Bind to DNA expression of new target genes
Examples
Origins
Notochord/neural tube (3
rd
week)
Paraxial mesoderm segments
into blocks (somites)
Somites differentiate into:
Sclerotome (vertebrae and ribs)
Dermomyotome (dermis and
primordial muscle cells)
Mesoderm forms loosely woven
tissue, mesenchyme
(embryonic connective tissue),
which has bone-forming
capacity (4
th
week)
Limb bud 4
th
week
Limb bud = mesenchymal core of
mesoderm covered by a layer of
ectoderm (with leading bulge)
Bulging ectoderm = apical
ectodermal ridge
Core mainly lateral plate mesoderm
Limb bud 4
th
week
Migratory cells invade limb bud:
Myoblasts, angioblasts and
motor axons from somatic
mesoderm
Sensory nerves, pigment
cells and Schwann cells from
neural crest
Migratory cells do not invade
growing apex
How does it start?
Early embryo homeobox (HOX) genes
set up segmental body plan along
cranial-caudal axis
Upper limb fields established 4
th
week,
triggering expression of TBX5, WNT
and FGF that initiate limb bud
outgrowth
Tetramelia = failure of limb induction
associated with WNT3 and FGF10
mutations
Nick Vujicic, Life Without Limbs
Arms vs legs
Cells similar and behave identically
T-box (Tbx) genes expressed early and
differentiate fore from hind limb
Tbx-4 hind limb-specific
Tbx-5 fore limb-specific
Ptx1 gene controls Tbx4 (hind limb)
change wing into leg by changing Ptx1
expression
AER
Thickened ridge of ectoderm at tip
of limb bud
Exerts inductive influence on the
limb mesenchyme initiating
growth of limbs in proximal-distal
axis
Majority of cell division occurs deep
to AER in the progress zone (PZ)
Molecular control via FGF
FGF
Implant FGF (microcarrier beads) =
supernumerary limbs
FGF-10 knockout = limbless
Molecular control via FGF
Mesodermal FGF10, in conjunction
with ectodermal radical fringe (R-
FNG), induces ectodermal thickening
to form AER
AER maintains FGF10 expression in
underlying mesoderm (via WNTs)
FGF10 also sustains proliferation of PZ
Reciprocal loop of ectodermal and
mesodermal FGF/WNT proteins
maintains proximaldistal outgrowth
Control of limb bud
Within the progress
zone, the fate of
mesodermal cells is
determined by these
multiple signaling
centers
Proximodistal AER FGF-2, -4 and -8
Anteroposterior ZPA Shh
Dorsoventral Dorsal ectoderm Wnt-7a/LMX1B(dorsal)
Ventral ectoderm En-1(ventral)
ZPA
ZPA controls radial-ulnar axis from the
posterior (ulnar) limb mesoderm
ZPA expands limb width and posteriorizes
(ulnarizes) limb through secreted sonic
hedgehog (SHH)
AER and ZPA closely linked by reciprocal
feedback loop that maintains SHH
expression at the distal posterior (ulnar)
border of the AER during progressive
outgrowth
ZPA
ZPA grafts to anterior induces duplicated
mirror digits
ZPA
stimulates mesenchymal cell proliferation
induces changes in AER
acts via SHH to produce BMP-2 and -7
which pattern digits
Dorsal-ventral axis
WNT7a expressed in dorsal limb ectoderm
WNT7a activates expression of LMX1b,
which determines dorsal limb bud identity
Lack of LMX1b = nail-patella syndrome
Ventral ectoderm expresses en-1, which
has inhibitory effect on WNT7a,
restricting it to dorsal ectoderm
Lack of en-1 = relatively more WNT7a
leads to congenital palmar nail syndrome
The three axes
AER/FGF =
transverse arrest
FGF = radial
arrest
SHH = ulnar
arrest
LMX1B = dorsal
arrest
Growth of limb bud
Patterning and
differentiation
Formation of digits
Programmed cell death (apoptosis)
plays a role in sculpting the limb
Regions that are sculpted by cell
death:
Interdigital zone
Interior necrotic zone
(separates radius and ulna)
Anterior & posterior necrotic
zones (shape the end of the
limb)
BMPs
Signals for apoptosis are BMP2,
BMP4 and BMP7
Expressed in the interdigital
mesenchyme
Paddle to individual digits
Blocking BMP signaling prevents
interdigital apoptosis
NOGGIN
Absence of apoptosis - syndactyly
Skeletal differentiation
Differentiation into bones and joints is
from proximal to distal, posterior to
anterior
Endochondral bone formation
Important factors
BMPs
Indian Hedgehog (IHH)
GDF-5
Muscle differentiation
Migration of myoblasts from somites
Dorsal and ventral muscle masses
Tendons from limb bud mesenchyme
interact with myotubes
Nerve differentiation
Motor axon from the spinal cord
innervate dorsal and ventral muscle
masses from fifth week
Sensory axons use motor axons for
guidance
Dermatomal patterning
Vascular differentiation
Fine capillary network branches from
aortic intersegmental arteries -> large
central artery (primary axial artery)
Drain into marginal sinus under AER
accumulates blood and drains the limb
via peripheral veins
Primary axial artery becomes brachial
artery in arm and common
interosseous artery in forearm
Limb growth
How do bones form?
Primary ossification centre develops in
mid-diaphysis (chondrocyte
hypertrophy and vascular invasion)
Endochondral ossification at physes
Secondary centres near end of fetal
development
Subsequent limb growth
Longitudinal
Physeal endochondral ossification
Circumferential
Zone of Ranvier (osteoblasts,
fibroblasts and chondrocytes)
Periosteal appositional ossification
The physis
Cells
Matrix
Vascular invasion and
programmed cell death
Indian hedgehog protein main
mitogen in proliferative layer
Oxygen
Upper limb growth
First 5 years are characterized by rapid growth
followed by plateau till puberty
Proportions amongst various bones established
by 5 years of age
Ulna is 80% of the length of the humerus,
whereas the humerus represents 70% of the
length of the femur
Proximal humeral physis contributes to 80% of
humeral length
Objectives
Embryonic limb development
Patterning and differentiation
Limb growth
Thank you!
MANOJ RAMACHANDRAN
Consultant Paediatric and Young Adult Orthopaedic Surgeon,
Barts Health NHS Trust, London, England