LIMB

DEVELOPMENT
Palacios ~Ramirez~Regilme~Ricafrente
Initiation of Limb Development
 The limb arises as a condensation of cells from the
lateral plate mesoderm and its ectodermal origin
 The limbs do not begin to form until the primordia of
most other organs are laid down and the musculature
of the body wall is already differentiated
 Limb disk – flat surfaced limb primordium and is
highly regulative system
 Requirement: SOMITES
 Prime mover: MESODERM
Regulative properties of the early limb
primordium

 Limb primordium – self-differentiating system

 When part of the limb disk is removed, the psrt that is left
continues to develop into a perfectly normal limb
 When the limb disk is split into two halves which are
prevented from fusing, each half gives rise to a complete
limb
 When two harmonious limb disk are superimposed, the
cells become reorganized so that they form only a single
limb
 Disaggregated limb mesoderm that is grafted to the flank
reorganizes and often forms a normal limb
Axial determination of the limbs
 Limb is an asymmetric structure
 Three axes:
 Proximodistal (P-D)
 Anteroposterior (A-P)
 Dorsoventral (D-V)
 Ross Harrison’s experiment:
 Demonstrated that the individual axes of the limb are fixed at different times
of the development
 Development of the A-P axis proceeded on the basis on the information
intrinsic to the rotated limb disk
 Development of the D-V axis was dictated by the body of the host

 Limb sequence of the axial determination:

 A-P >> D-V >> P-D
Homebox Genes and the Developing Limb

 Hoxa and Hoxd complexes and other homebox-containing

gens are expressed during limb development
 Hoxd genes are expressed in a strict spatial sequence that
reflects their organization on the chromosomes
 3’ gens expressed more anteriorly that 5’ genes

 The gens are expressed in a 3’ to 5’ temporal sequence

 Combination of Hoxd genes expressed within a given region
of limb bud mesoderm may determine the type of digit that is
formed
 Homebox-containing genes are expressed in developing limbs
and they play important roles in limb morphogenesis
Outgrowth of the limb bud
 As soon as the limb primordium begins o project from the
surface of the embryo, it is called a limb bud
 Limb bud consists of a mesodermal core of homegenous-
appearing mesenchymal cells
 This is covered by a sheet of ectoderm
 Saunders experiment:
 Role of the band of thickened ectoderm (apical ectodermal
ridge)
 Demonstrated a critical interaction between the apical
ectodermal rodge and the underlying mesodermal core during
growth of the wing bud (chick embryo)
APICAL ECTODERMAL
RIDGE
 STRUCTURE OF THE RIDGE
 Apical Ectodermal Ridge
 Terminal thickening at the appendage buds of most vertebrates
 Runs along the anteroposterior margin of the bud
 Takes shape shortly after the limb bud begins to project from
the lateral surface of the embryo
 Typically attains greatest degree of development when the limb
is at the paddle shaped stage.
 As the digits begins to differentiate , the ridge begins to regress
 Presence of numerous gap junctions which connect the cells of
the ridge with one another.
 STRUCTURE OF THE RIDGE
 Birds , Mammals and Anuran Amphibian Embryos
 Nipple-shaped structure in cross section
 Fishes and some Reptiles
 Apical epidermis seems to be thrown into a tight fold
 Urodeles
 Don’t have an apical thickening on the limb buds
 STRUCTURE OF THE RIDGE
 Two Principal Histological Configurations
1. In Birds
-arrangement is pseudostratified columnar
epithelium

2. In Mammals
-arranged in stratified cuboidal or squamous
epithelium
PROPERTIES OF THE APICAL
ECTODERMAL RIDGE

 Apical ectodermal ridge is considered by most

researchers to act as stimulator of outgrowth of the
limb bud.
 Surgical exchange experiments have shown that the
apical ridge of the chick and mouse embryo can
promote outgrowth of limb mesoderm.
PROPERTIES OF THE APICAL
ECTODERMAL RIDGE

 Experiment by Rubin and Saunders (1972)

 Grafted apical ridges from older chick leg buds unto
young leg buds
 Normal development ensued despite differences in age
and proximodistal level
 FGF (Fibroblasts Growth Factor)- 2 and FGF-4
 Can be a substitute for apical ectodermal ridge in
promoting outgrowth and morphogenesis of limb
MESODERM OF THE
EARLY LIMB BUD
STRUCTURE
 Mesoderm of Early Limb Bud
 Consist of homogenous appearing mound of mesenchymal
cells embedded in a loose matrix of collagen fibers and
mucopolysaccharide ground substance
 Cells: with large nuclei and prominent nucleoli and scanty
basophilic cytoplasm
 Progress Zone
 Rich in ground substance
 Role in growth and morphogenesis of the limb

• Limb Bud is well vascularized and has a prominent marginal

sinus.
ROLE OF THE MESODERM
 Mesoderm of the early limb bud stimulates the
early ectoderm to form the apical ectodermal
ridge .
 In wingless mutant, development of wing bud is
normal but apical ridge soon degenerates.
 In polydactyl, normal mesoderm and mutant
ectoderm give rise to normal limbs, while mutant
mesoderm plus normal ectoderm produces
polydactylous appenadages.
CELL DEATH IN DEVELOPING
LIMB
 Appears in the forelimb,
first in the region of the
future maxilla(armpit),
patches of dying cells are
later seen in the elbow
region and between the
developing digits.
 Posterior Necrotic Zone
 Area of cell death in the
future axilla of the chick
CELL DEATH IN DEVELOPING LIMB

 The persistence of webs of soft tissue between digit

is associated with reduced amounts of cell death.
 Interdigital cell death is prominent in all major
groups of amniote embryos.
 Apoptosis or programmed cell death is the result of
the activation of a variety of genes which trigger
and promote the process.
CELL DEATH IN DEVELOPING LIMB

 Grafting experiment shows that death clock is set at

approximately stage 17 of development
 Up to stage 22, reversibility is possible if the cells
of the posterior necrotic zone are grafted to the
dorsal side of wing bud
 After stage 22 death is irreversible
MORPHOGENETIC
CONTROL OF
DEVELOPING LIMB
Anteroposterior axis - Proximodistal axis - Dorsoventral axis
ANTEROPOSTERIOR AXIS

Zone of Polarizing Activity (ZPA)

 located at the posterior edge of the limb bud near
its junction to the body wall
 responsible for ANTEROPOSTERIOR
PATTERNING of the limb
ANTEROPOSTERIOR AXIS
Zone of Polarizing Activity (ZPA)

 Retinoic Acid  Sonic Hedgehog (Shh)

 Derivative of Vitamin A
 Tickle and colleagues
(1982) application of
retinoic cid to the
anterior part of the avian
wing results in the
duplications like those
caused by ZPA grafts
 Secondary gradient of
the HoxD expression
 Avian equivalent to
segment polarity gene
hedgehog in Drosophila
 Expresses an RNA
product that is localized
to the region of the ZPA
 ANTEROPOSTERIOR AXIS
Zone of Polarizing Activity (ZPA)

 Modes of transmission of the ZPA signal

1. Pure diffusion
2. Cell-to-cell transfer of the morphogen or downstream
messengers through gap junctions
PROXIMODISTAL AXIS

Apical Ectodermal Ridge (AER)

 Stimulates limb growth along the PROXIMODISTAL
AXIS through the mediation of fibroblast growth
factors
 influences the expression of the Msx-1(Ghox-7) gene
 PROXIMODISTAL AXIS

Progress Zone (PZ)

 lies beneath AER
 Cells remain undifferentiated
 Region where patterning appears to take place
 cells begin to differentiate only after leaving the progress zone.
DORSOVENTRAL AXIS

 Least well-understood aspects of limb

development
 No molecular information
 Ectoderm plays a crucial role
Differentiation of the Skeleton

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DEVELOPMENT OF THE
VASCULAR SUPPLY
Development of the Vascular Supply
 The earliest limb bud is supplied by a fine capillary
network arising from the several segmental
branches of the aorta and from the endogenous
angioblasts.
 Primary central artery is one of the first to take
shape.
 Blood from the central artery travels through a
capillary network and then collects in a marginal
sinus.
 Experiments by Feinberg and Noden (1991)
demonstrated an inhibitory influence of the
ectoderm on angionesis in the adjacent mesoderm.
 The capillary networks and the marginal sinus itself
respond to growth of the limb bud by sending out
new vascular sprouts.
 Blood first drains from the marginal venous channels
into superficial venous plexus of the body.
 As digital rays become established, the apical
marginal sinus begins to break up, but the proximal
portions persists in mammals as the basilic and
cephalic veins.
 The formation of the major arteries of the extremities
is a complex process.
Innervation of the Embryonic Limb

 Early limb bud forms in the absence of nerves, and it

remains in the early period of outgrowth.
 Number of grafting experiments shown the
interrelationships betwwen nerves and many
components of the limb.
 limb buds placed in etopic locations on the body or, in
birds, on the chorioallontoic membrane,underwent normal
development.
 Another type of experiment, aneurogenic limbs have been
surgically removing the neural tube of early embryos.
 Factors that determine the pattern of nerves within a
limb;
 That the pattern of innervation is determined entirely by the
configuration of the other tissue components of the limb,
with the ingrowing nerves passively following
environmental cues;
 That the ingrowing nerves themselves are uniquely
specified, allowing them to home in upon specific
predeterminedstructures within the limb.
LIMB REGENERATION
LIMB REGENERATION
 The limbs of some vertebrates are endowed with
the capacity to regenerate after they have been
amputated.
 Amputation is followed by epidermal wound healing –
 within few days, the limb stump enters the period of
dedifferentation
 those cells are agregate and proliferate to form
blastema
 As the blastema grows, it forms a new limb.
 Three conditions are normally required for the
initiation of the limb regeneration.
 A wound epidermis
 Mesodermal damage
 An adequate nerve supply
 The regeneration of extremities is most successful
in fish, salamanders and anuran tadpoles.
 The limbs of mammals and birds do not regenerate.
 A number of factors are likely to be involved in the
loss of regenerative power, and the stimulation of
partial regenerations of limb.