Upper limb embryology and growth

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