Development of the Limbs

During the 4th week of gestation, limb buds form on the sides of the developing embryo. The tips of these buds condense
into the apical ectodermal ridge (AER). The AER continues the elongation of the limb buds and maintains its growth by
continuously producing fibroblast growth factor 8 (FGF8). As the AER grows away from the body, tissues differentiate. After
the cartilage models are formed in the developing limbs, arteries invade central and peripheral areas, giving rise to primary
and secondary centers of ossification. The process of endochondral ossification is completed when those centers meet and
the epiphyseal plate is no longer present.

Last updated: March 25, 2024

CONTENTS

Limb Formation
Rotation of the Limbs
Formation of Musculature
Development of Vasculature
Development of Limb Bones
and Joints
Genetic and Molecular
Patterning of Limbs
Clinical Relevance
References

Limb Formation
Day 24: appearance of upper limb bud
Day 26: appearance of lower limb bud
Week 5: tail segment present between lower limbs → gradually disappears
Limb bud:
Location: ventrolateral aspect of the body
Coming from lateral plate mesoderm initiated by fibroblast growth factor 10
(FGF10)
Covered by ectoderm
Has a core of denser condensed mesenchyme
At the tip: apical ectodermal ridge (AER)
Position:
Upper limb bud: opposite to caudal cervical segment
Lower limb bud: opposite to lumbar and sacral segments
AER:
Part of the ectoderm that underlies the limb bud
Produces FGF8 to maintain elongation of the limbs
AER exerts influence on surrounding tissue to keep it undifferentiated:
As AER migrates away, more proximal tissue differentiates into cartilage and
muscle.
Limbs form proximally to distally in segments:
Stylopod: humerus and femur
Zeugopod: radius/ulna and tibia/fibula
Autopod: carpals, metacarpals, digits/tarsals, digits/metatarsals
Hand and foot plates form as upper and lower limbs extend:
Initially appear like fused paddles around 6th week
Digital rays: appear as webbed tissue between fingers begins to undergo
apoptosis
Day 52–56: Fingers and toes appear.
 Apical ectodermal ridge:
This ridge is located at the tip of the limb bud. Its function is the production of fibroblast growth factor,
which induces proliferation of ectodermal cells to maintain limb elongation.
Image by Lecturio.

Mnemonic:

To quickly recall what happens during the 4th week, remember: 4 weeks → 4 limbs, 4
heart chambers

Rotation of the Limbs

As the embryo changes from a flat disk to a 3-dimensional structure, the limbs
rotate.
Upper limb:
Week 4: Limb buds emerge from coronal plane.
Week 6: Limb buds move toward sagittal plane.
Weeks 6–8: lateral 90-degree rotation:
Flexor compartments shifted anteriorly
Elbows located posteriorly
Proper allocation of dermatome innervation
Lower limb:
Week 4: Limb buds emerge from coronal plane.
Week 6: Limb buds move toward sagittal plane.
Weeks 6–8: medial 90-degree rotation:
Flexor compartments located posteriorly
Extensor compartments located anteriorly
 Rotation of the limbs:
As upper and lower limbs grow, they bend at the elbow and knee with the palms and soles of the feet
facing medially. During 90-degree opposite rotation, the elbow points toward the posterior side, whereas
the knee points anteriorly.
Image by Lecturio.

Formation of Musculature
Overview
Muscle comes from myotome: ventrolateral cells originating from somites located on
either side of neural tube that migrate to form muscles. As myotomes migrate, they form:

Epimere (epiaxial):
Cluster of developing muscle from the myotome
Goes into the back → future intrinsic muscles of the back
Hypomere (hypoaxial):
Travels into the torso → anterior thoracic wall → abdominal wall → limbs
Extends ventrolaterally
Forms body-wall and limb muscle
After migration to the limb buds, they separate again:
Posterior mass (dorsal) → extensor muscles
Anterior mass (ventral) → flexor muscles
Upper limbs
Somites give rise to the musculature of levels C4–C8, T1–T2.
Posterior condensation gives rise to extensor and supinator muscles.
Anterior condensation gives rise to flexor and pronator muscles.
Musculature of the upper limbs
Somites corresponding to C4–C8, T1–T2 give rise to posterior and anterior aspects of the musculature of
the upper limb. Flexor muscles arise from anterior aspects of these somites, while extensors arise from
the dorsal aspects.
Image by Lecturio.

Lower limbs
Somites that give rise to the musculature of levels L1–L5, S1–S2
Posterior condensation: gives rise to the flexor and abductor muscles
Anterior condensation: gives rise to extensor and adductor muscles

Nerve innervation
Somites give rise to the musculature of levels L1–L5, S1–S2.
Upper limbs: C4–T2
Lower limbs: L4–S3
Nerves get stretched from the spinal cord as upper and lower limbs develop:
Brachial and lumbosacral plexus are made.

Development of Vasculature
Blood vessels invade developing musculature around 35–36 days of gestation.
Upper limb: segmental artery → axillary artery
Lower limb: segmental artery → femoral artery
Initially, arteries go to the limb buds → enlarge but stay deep → capillaries form
Venous drainage is more superficial.
No superficial arteries at the core of the limbs

Development of Limb Bones and Joints

Limb bones
Limb buds condense → Models of hyaline cartilage begin to form.
Endochondral ossification: mesenchyme → cartilage → bone:
Mesenchyme: loose, undifferentiated connective tissue
Cartilage: tougher, avascular; relies on diffusion
Bone collar:
Layer of periosteum that surrounds the diaphysis
Formed by osteoblasts
Primary ossification center:
Forms diaphysis—shaft of the bone containing bone marrow cavity
Artery invades middle of the cartilage → brings osteoprogenitor cells
Bone formation proceeds outward
Secondary ossification center:
Forms epiphysis
At the bone extremities
Separated from primary ossification center by metaphysis
Alkaline phosphatase:
Produced by chondrocytes
Responsible for mineral deposition on bones
Primary and secondary ossification centers get closer → Epiphyseal growth plate
tries to proliferate and push them away.
Epiphyseal plates remain open until the early 20s → bone can no longer elongate,
and growth arrests
Medullary cavity:
Formed by the action of osteoclasts
Core of diaphysis
Major site of hematopoiesis
Joints
Mesenchyme between the bones stays as dense irregular connective tissue.
Synovial spaces form as the bones grow through the complex process of apoptosis.
Intracapsular ligaments (anterior cruciate ligament (ACL) and
posterior cruciate ligament (PCL)):
Remnants of connective tissue
Dense and regular
 Connective tissue:
As the bones grow, connective tissue remains between them as dense regular connective tissue or
fibrocartilage and accumulate synovial fluid.
Image by Lecturio.

Genetic and Molecular Patterning of Limbs

Positioning, orientation, and growth of limbs in the developing embryo is determined
genetically and by the expression of specific timed chemical signals.

Craniocaudal axis
Positioning of limbs along the craniocaudal axis is determined by the homeobox (HOX)
genes.

This segment of genes (60 amino acids long) encodes for DNA-binding proteins,
which regulate gene expression.
The HOX-containing genes regulate multiple aspects of cell growth and
differentiation.
These genes are arranged sequentially in a cranial-to-caudal manner, and they
determine the overall pattern and shape of the developing embryo.
For example:
HOXB8 expressed at cranial border of forelimb
Mutations in this gene alter the position of these limbs.
Forelimb positioning is determined by the transcription factor T-box
transcription factor 5 (TBX5); hindlimb positioning is determined by TBX4.

Proximodistal axis
 Proximodistal patterning is regulated by maintaining the AER distally as the embryo
develops.
Radical fringe is a signaling factor expressed in the dorsal ½ of the limb ectoderm:
Induces the expression SER2 at the border between cells that are expressing
radical fringe and those that are not
AER is localized to that border.
Once AER is developed:
Expresses multiple classes of FGF
Maintains the undifferentiated area of profusely replicating mesenchyme cells
immediately proximal to the AER
As the cells duplicate, the limb grows distally.
The further the mesenchymal cells are from the AER, the less FGF can
influence the mesenchymal cells, which can then differentiate thanks to the
influence of other signaling molecules:
Retinoic acid: synthesized by flank mesenchyme, causes differentiation
of tissue into stylopod
Sonic hedgehog (SHH): expression of this gene leads to differentiation of
the zeugopod and autopod

Anteroposterior axis
Regulated by zone of polarizing activity (ZPA)
A cluster of mesenchymal cells in the posterior portion of the limb, near the AER
Secrete the signaling factor SHH
The expression of SHH determines the correct anteroposterior orientation of limbs;
e.g., misexpression of SHH in the anterior portion of the hand can lead to digit
duplication.

Dorsoventral axis
Ventral ectoderm expresses the transcription factor EN1.
EN1 blocks the secretion of WNT7a:
WNT7a is therefore localized to the dorsal ectoderm.
Induces expression of LMX1 → a transcription factor that specifies “dorsal”
orientation

Clinical Relevance
Achondroplasia: mutation in FGFR3 gene and corresponding protein that has a
negative effect on endochondral ossification. Mutation causes overactivity of the
receptor signaling pathway, which results in impaired growth and proliferation of
chondrocytes. Affected individuals tend to have shortened limb bones, and facial
bones do not elongate.
Sirenomelia: also called mermaid syndrome. Sirenomelia is a rare congenital
deformity in which the legs are fused together. The disorder is mainly characterized
by the fusion of the legs with rotation of the fibula. Sirenomelia may also include
absence of the lower spine and abnormalities of the pelvis and renal organs.
Sirenomelia has a poor prognosis, with most infants not surviving the 1st year of life.
Syndactyly: Cutaneous syndactyly is webbing between the digits. Syndactyly
occurs because of the failure of apoptosis, which is responsible for the separation
of the digits. Osseous syndactyly consists of fusions of the bones of the digits.
Amelia: absence of 1 or both limbs, which occurs because of suspension in
development during the 4th week.
Meromelia: condition in which a part of the limb is missing. Meromelia occurs
because of a disturbance in development during the 5th week of gestation.
Examples include hemimelia (absence of fibula) and phocomelia (hands and feet
attached directly to the body).

References