DEVELOPMENT OF LIMB

Dr Anggraini Barlian

Learning objectives

The derivatives of mesoderm

The limb bud formation
The embryological development of upper limb
The embryological development of lower limb
The development of the vertebral column
The limb defects
To describe the vertebral column defects

Derivatives of
mesoderm:

* Paraxial/
somite:
- Myotome
- Dermatome
- Schleretome
* Intermediate
* Lateral
* Head

Classification of mesoderm :
Notochord: axial mesoderm
in adult substituted by columna vertebrate
schlerotome from somite
Somite : paraxial mesoderm
Dermatome Dermis
Myotome skeletal muscle
Schlerotome vertebrae, ribs

Mesoderm intermediate : Urogenital system

Mesoderm lateral :
mesoderm splancnic : circulatory system
mesoderm somatic : limbs

Stages development of
somites:
A. Mesoderm cells are
arranged around a
small cavity
B. Cells of ventral &
medial walls of
somites lose their
epithelial arrangement
& migrates
notochord
Cells
at dorsolateral
sclerotome
migrate limb &
body wall
musculature.
Dorsomedial cells
migrate myotome
C.Myotome continue
to extend
D. Dermatome

Expression patterns of genes that regulates somite differentiation. Sonic

hedgehog (Shh), secreted by notochord & floor plate of neural tube ventral
part of somite form sclerotome & expressed PAX1 control chondrogenesis &
vertebral formation. Wnt proteinactivate PAX3 demarcates dermomyotome.
Wnt prot dorsomedial portion of somite form epaxial muscle & express
muscle specific gene Myf5. Middorsal portion of somite dermis by
neurotropin 3 (NT-3) expressed by dorsal neural tube. Hypaxial musculature is
derived from the dorsolateral portion of somite under activating Wnt prot &
BMP-4 prot together activate MyoD expression

LIMB BUD FORMATION

Limb Bud Formation

The distal tip of the limb bud is covered with a transient
structure known as the apical ectodermal ridge (AER).
Limb outgrowth is dependent upon interactions between
the AER and the underlying mesoderm.
If the AER is removed, the mesoderm stops dividing.

3/24/2009

Limb Development
If a supernumerary AER is grafted adjacent to a
developing limb, a supernumerary limb results.
Prospective limb mesoderm grafted under flank
ectoderm will promote formation of an AER and a
supernumerary limb.

If limb bud mesoderm is removed from an early

limb bud, the AER regresses and the mesoderm
ceases proliferation.
Therefore, limb outgrowth involves reciprocal
interactions between the AER and underlying
mesoderm.
3/24/2009

What Controls Establishment of Limb Axes?

The limb has three axes: Proximo-distal; Anteriorposterior and Dorsal-ventral.
Proximo-distal
Removal of the AER at progressively later stages
results in a truncated limb with progressively more
distal elements. Thus, these elements are laid down in
a proximal-to-distal direction.
The AER does not regulate P-D polarity. Fates of cells
are determined by the length of time they spend in the
progress zone. Those residing there the longest
become the most distal elements.

Anterior-Posterior Axis
This axis is determined by the zone of
polarizing activity (ZPA), which is located at
the junction between the limb bud and the
body wall.
Grafts of the ZPA to the anterior margin of a
host limb bud causes duplication of digits in
mirror-image symmetry.
A diffusible morphogen (retinoic acid?) is
released from the ZPA.
3/24/2009

Which axis forms first?

1. anterior/posterior (1st to 5th digits)
2. ventral/dorsal (palm/ dorsum of hand)
3. proximal/distal (base of limb / digits)

Upper Limb
Lower

Pelvis
Scapula
Femur
Humerus
Tibia,
fibula
Radius,
ulna
Tarsals
Carpals
Digits
Digits

Metatarsals
Metacarpals
Phalanges
Phalanges

Upper Limb

Scapula
Humerus
Radius, ulna
Carpals
Digits
Metacarpals
Phalanges

Why Study Limb

Development?
Fundamental Understanding of the
process
Clinical relevance
Some statistics:
o Frequency of abnormality depends on severity
o Duplication of digits is fairly common at ~1 in
300 births
o Major failures of formation is less common, at
~ 1 in 100,000 births
(Indiana Hand Center)

How do these abnormalities

arise?
THROUGH IMPROPER
DEVELOPMENT!!!
The goal of limb development research is to
understand how cartilage, muscle and tendons
are formed in the right place and how they
make the right connections with each other to
form a normal functional limb

Limb Development is A Part

of Organogenesis
The process of organogenesis involves
using the basic body plan (organized
embryo) to develop specific organs (limbs,
heart, eyes) in specific regions that leads
to the fully functional organism, capable of
independent survival
Involves: pattern formation, specification of
positional information, induction,
morphogenesis, and cellular differentiation

How are these processes

involved?
Pattern formation directs cell identity and leads
to ordered spatial pattern of cell activity
Positional information directs where organs
will form
Induction direction of specific cell fate
Morphogenesis changing the form of cells
Differentiation acquire functional and structural
identity distinct from their surrounding cells

Model System
Development of the Chick
Limb I
Chick limbs begin to
develop on the 3rd day
after laying
The limb develop
from limb buds on the
body wall of the
embryo

Development of the Chick Limb

II

Development of the Chick Limb

III

As the limb bid grows outward, cells in the progress zone

proliferate and acquire a positional value

When cells leave the progress zone, cartilage may begin to
differentiate, and other elements gain their positional
information

Embryonic Chick Wing (day

10)

What Directs Limb Development?

The APICAL ECTODERMAL RIDGE (AER) is
responsible for the proximo-distal patterning of the
limb, which occurs over time through the progress
zone
Signals are sent both from the AER to the progress
zone, and from the progress zone to the AER

What Directs Limb

Development?
(contd)

The ZONE OF POLARIZING ACTIVITY

(ZPA) is responsible for antero-posterior
patterning of the limb
The ECTODERM controls patterning
along the dorso-ventral axis
Differences in the activation of genes
that control the interpretation of
positional information is responsible
for development of different limb
structures (i.e. upper or lower)

What Molecules Direct Limb

Development?
Sonic Hedgehog (Shh)
The Shh gene encodes
inductive signaling
molecules that function
during embryogenesis
Involved in establishing
cell fates
Key signal in patterning
of the antero-posterior
limb axis
Alx-4 represses Shh
expression

Fibroblast Growth
Factor 4 (FGF-4)
Member of FGF family
Mesodermal cell
stimulator and directs
developmental
signaling
FGF-4 -/- die on E 5.0
Transcriptionally
inactive in adult
tissues

Retinoic Acid : A Developmental

Morphogen
Retinoic acid is the active form of Vitamin A
Both excess retinoic acid and vitamin A
deficiency each affect left-right symmetry
(e.g., abnormal formation of the mammalian
heart) through alterations in gene expression.

3/24/2009

Cell Death and Digit Formation

Without apoptosis, the fingers would be webbed.:

syndactyly
3/24/2009

Limb malformation

These are clenched hands resulting

from camptodactyly (fingers bent
over) and clinodactyly (fingers
inclined to one side or the other). This Deformation from constraint with
particular appearance is very
oligohydramnios in utero can result in
suggestive of trisomy 18.
the appearance of the club feet
(varus deformity) seen here. The feet
are turned inward.

Example of abnormality absent of

hands (+ legs)
(amelia/ phocomelia) caused by
thalidomide (anti-vomiting)

Syndactyly represents fusion of two or

more digits. It can be an isolated
finding or part of syndromes that
define patterns of anomalies. Most of
these syndromes do not have a
specific genetic defect yet defined,
though some do. Seen above are the
digits of the hand fused together.
Below, the foot has rudimentary,

This is sirenomelia. The term comes from "siren"

or "mermaid" because of the characteristic fusion
of the lower extremities that results from a failure
in the development of a normal vascular supply
to lower extremities from the lower aorta in
utero.

Sirenomelia (caudal
dysgenesis) gastrulation
disruption. Loss of
mesoderm in the
lumbosacral region has
resulted in fusion of limb

Regulative
ability of Limb
field

What are these

abnormalities?

Polydactyly the
duplication of a digit

Caution: aspirin; streptomycin

(deafness); antidepressant (limb
deformities); tetracyclin (bone and
tooth anomalies) etc
Cocaine, LSD, PCP (CNS, limb
abnormalities, hypoxia)

Child with unilateral amelia

MALFORMATION IN CRANIAL DEVELOPMENT

VERTEBRA COLUMN DEVELOPMENT

Development of vertebrae