L8 Limb Development of Muscoskeletal components of the limb

The limbs

Muscles with tissue junctions and tendons

Bones with joints

BONE DEVELOPMENT

What are the two types of bone formation in the limbs

Endochondral ossification (formation of cartilage template that is replaced by bone)

AND

Intramembranous ossification (bones forms directly from mesenchymal cells)

What forms by intramembranous ossification

Mainly flat bones

How do long bones of the skeleton form

By endochondral ossification where mesenchymal cells first condense and differentiate into chondrocytes to create a cartilage model that is later replaced by bone.

Endochondral ossification diagram, slide 6

What initiates chondrocyte differentiation during endochondral ossification

Expression of SOX9 initiates condensation of mesenchymal cells and Indian hedgehog (Ihh) regulated their differentiation into chondrocytes

What are the two types of bone formation in the limbs?

The two types are endochondral ossification and intramembranous ossification

How do long bones of the limbs form?

Long bones form by endochondral ossification where a cartilage template is replaced by bone

What initiates condensation of mesenchymal cells during bone development?

SOX9 initiates mesenchymal condensation and chondrocyte differentiation

What regulates chondrocyte differentiation during endochondral ossification?

Indian hedgehog (Ihh) regulates chondrocyte differentiation

What are the main stages of endochondral ossification?,

Endochondral ossification begins with SOX9-driven condensation of mesenchymal cells which differentiate into chondrocytes under control of Ihh. Chondrocytes produce collagen type II and form a cartilage template. These cells become hypertrophic, express collagen type X and the matrix becomes calcified

How does ossification begin in the cartilage model?

Ossification begins when blood vessels invade the centre of the cartilage model forming the primary ossification centre in the diaphysis. Pre-osteoblasts differentiate into osteoblasts and begin producing collagen type I to form mineralised bone

How does the long bone continue to grow after the primary ossification centre forms?

Ossification spreads from the diaphysis toward the epiphyses. A bone collar forms around the diaphysis which thickens to form a cortical bone layer. Proliferating chondrocytes remain organised in the growth plate allowing longitudinal growth (bone lengthens)

what do osteoclasts do during enchondrol ossification

brought in via the vasculature. They function to digest bone tissue as part of ongoing growth and remodelling throughout life

What is the function of RUNX2 in bone development?

RUNX2 is required for osteoblast differentiation and bone mineralisation. it allows cells to become osteoblasrts and is necessary for bone mineralisation

Where does the primary ossification centre form?

It forms in the diaphysis of long bones and is the first site where cartilage is replaced by bone

Where do secondary ossification centres develop?

They develop in the epiphyses after birth

What is the function of the growth plate?

The growth plate allows longitudinal bone growth through organised chondrocyte proliferation.

At what age do growth plates usually fuse?

Growth plates usually fuse around 20 years of age

What happens at birth and after in endochondral ossification?

At birth the diaphysis is ossified but the epiphyses remain cartilaginous. Secondary ossification centres form in the epiphyses after birth and growth continues at the epiphyseal plate until it fuses around 20 years of age

Which molecules regulate chondrocyte differentiation and growth plate activity?

Indian hedgehog, PTHrP and FGF18 regulate chondrocyte proliferation and differentiation within the growth plate

Why is knowledge of ossification centres clinically important?

It helps determine skeletal age and distinguish fractures from growth plates. Assists in forensic age estimation

Primary ossification centres are present at birth

visible on x-ray from birth

- metacarpal diaphyses: 9 weeks in utero

- phalangeal diaphyses: 9-12 weeks in utero

primary ossification centres developing after birth

Ossification of the carpal bones occurs in a predictable sequence,starting with the capitate and ending with the pisiform.​

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At birth, there is no calcification in the carpal bones. Although there isgreat individual variabilit

diagram of bone epiphysis, growth plate, metaphysis, diaphysis

MUSCLE DEVELOPMENT

From which embryological structure do limb skeletal muscles arise?

Limb skeletal muscles arise from the somites of the paraxial mesoderm. Form blocks in a regulated pattern

Cells of the somite are subdivided into the sclertotome and the dermamytome

What does the dermomyotome give rise to?

It gives rise to skeletal muscle and dermis

myo = muscle, derma = dermis

Figure of muscle development

When do muscle precursor cells migrate into the limb bud?

Migration begins during the 5th week of development

How do muscle precursor cells reach the limb bud

Limb musculator muscle group mpves ventrallt into the limb buds. Muscle precursor cells delaminate from the dermomyotome and migrate into the limb bud during the 5th week of development, a process that requires expression of the transcription factor LBX1

How is limb musculature initially organised within the limb bud?

Once inside the limb bud, migrating myogenic cells form two condensations called the dorsal and ventral muscle masses, which will later split into specific muscle groups

Dorsal – extensors and supinators (UL)extensors and abductors (LL)​

Ventral - flexors and pronators (UL) flexors andadductors (LL)

What does the dorsal muscle mass form?

It forms extensors and supinators in the upper limb and extensors and abductors in the lower limb

What does the ventral muscle mass form?

It forms flexors and pronators in the upper limb and flexors and adductors in the lower limb

Which transcription factor is required for migration of muscle precursors?

LBX1 is required for migration

ventral and dorsal muscle mass (upper limb)

ventral: anterior compartment of arm and forearm

dorsal: posterior compartment of muscles of arm and forearm

ventral and dorsal muscle mass (lower limb)

ventral: medial compartment muscles of thigh, posterior compartment of muscles of thigh, posterior compartments of leg

dorsal: anterior compartment muslces of thigh and leg

How does limb rotation affect final muscle orientation?

The upper limb rotates laterally and the lower limb rotates medially during development, which explains why extensors are posterior in the upper limb but anterior in the lower limb and why the great toe lies medially in anatomical position

specific surfaces of the hands and feet used in anatomical

Dorsal of the foot is the top - this is because of the rotation which has occurred

Dorsiflexion and plantarflexion

muscle development process

1. primary myogenesis

2. secondary myogenesis

3. postnatal muscle growth

What is primary myogenesis?

Primary myogenesis is the embryonic stage where myoblasts proliferate and differentiation to form myocytes then fuse to form multinucleated muscle fibres (myotubes)

What is secondary myogenesis?

Secondary myogenesis is the fetal stage where most muscle fibres are formed as primary muscle fibres fuse

What cells are responsible for postnatal muscle growth?

Satellite cells (quiescent cells in muscles) are responsible for postnatal muscle growth and repair. Respond to exercise/damage and form myocytes

molecular regulators of limb muscle development

lbx1 AND others

TENDON DEVELOPMENT

major structural component of tendon

Collagen type I, found in many other body tissues. Until recently lack of suitable markers avaialble for studying tendon develpment, now we have Scx

From which embryological tissue do limb tendons arise?

Limb tendons arise from the lateral plate mesoderm

What is Scleraxis (Scx)?

Scx is an early marker of tendon development that regulates collagen type I expression

what does Scx do

Regulates type I collagen in mouse tendons

BUT not thought out as the master regulator since Scx mutant mice still have tendons that attach muscle to bone so likely works with othermarkers

Loss of Scx activity will severely disrupt force-anchoring tendons (such as those in the limb)but only affect other tendons moderately.

other genes involved in tendon development

Tenomodulin (Tnmd) – marker for differentiated tendon cells​

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Mohawk (Mkx) – can reduce collagen typeIa expression​

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Early growth response 1 (Egr1) – KO shows reduced collagen fibrilorganisation and mechanical weakness in tendons​

where do tendons originate from

Tendons of different body regions originate from different embryological origins.

- Limb tendonsoriginate from the lateral platemesoderm (differsfrom skeletalmuscle)​

Do limb tendons require muscle for full maturation?

Yes, limb tendons require interaction with muscle to fully mature but this varies with anatomical location

- e.g. Limb tendons will initially form without muscle, but their maturation depends onmuscle-interaction

- Axial tendons will not form if axial muscle isabsent

JOINT DEVELOPMENT

joint development diagram

synovial joint formation process

1. Interzone forms (area between future bondes becomes fibrous tissue)

2. Articular cartilage forms on proximal and distal bone ends

3. Connective tossie condenses to form synovial tissue lining, ligaments, menisci, etc.

4. Cavitation creates a fluid filled joint cavity

What is the interzone in joint development?

The interzone is fibroblastic tissue that marks the future site of a synovial joint

Which growth factor regulates interzone formation?

GDF5 regulates interzone and joint development

What is cavitation in joint development?

Cavitation is the formation of the synovial joint cavity

molecular regulation of joint development control

- Growth differentiation factors (GDF) 5/6

- Noggin

- Wnts

TISSUE JUNCTIONS

two interfaces of the tissue junctions

bone-tendon interface and muscle-tendon interface

What is the enthesis? (bone tendon interfaces)

The enthesis is the mechanical graded interface between tendon and bone. It transfers force from compliant tendon to stuff bone

What is the structural organisation of the enthesis from tendon to bone?

The enthesis transitions from tendon to unmineralised fibrocartilage to mineralised fibrocartilage and finally to bone, creating a gradual change in stiffness

Plug of Fibrocartilage, has a lot of water so is a shock absorber

How does the enthesis develop during embryonic and postnatal life?

Bone and tendon initially develop as separate tissues in the embryo and a direct attachment forms after birth, with a mature graded enthesis developing postnatally in response to mechanical loading.

Mature enthesis not observed until 21 days postnatally.

what happens to enthesis if you paralyse a limb

Evidence that if you paralyse a limb you don't get the mature graded enthesis, important that normal forces are at play

What is the myotendinous junction?

It is the specialised interface between muscle and tendon that increases surface area for force transmission

An important transition site between the contractile muscle tissue and the strong, elastic tendon

How is the myotendinous junction structurally adapted for force transmission?

The myotendinous junction increases surface area through structural folding, allowing stronger attachment and efficient force transfer

Two different tissue masses

(myoblasts and the coyotes), these will fuse together but there is still separation between each other

What is the significance of muscle-tendon interaction during development?

Muscle presence maintains expression of tendon markers such as Scleraxis and is necessary for proper organisation and mechanical strength of the tendon

- seen in varies species; mouse, zebrafish, chick

- In drosophila, tendon cells do not need muscle to form but needinteraction with muscle cells to fully differentiate and mature.

MECHANOBIOLOGY IN LIMB BIOLOGY

What is mechanobiology in limb development?

Mechanobiology is the study of how mechanical forces such as tension, compression and muscle contraction influence bone, tendon, ligament and joint development

Why is muscle contraction important in limb development?

Muscle contraction provides mechanical forces that influence bone development growth, joint formation and tissue maturation

- without it we would not have fully formed tissues

mechanobiology in. limb development Bone development and growth ​

growth generated strain Wolpert proposed that radial stress from the perichondrium restricts circumferential growth and promotes longitudinal bone growth, although the exact mechanisms are still not fully understood

Muscular loading​ Directional strain on bone from musclecontraction​. Paralysis results in skeletal malformations ​. Generally accepted view that musclecontraction influences bone development andremodelling​

mechanobiology in limb development: Enthesis Maturation ​

At birth the enthesis is a simple attachment but mechanical input from muscle contraction is required for it to mature into a graded and well-organised interface

mechanobiology in limb development: Joint Formation ​

Muscle contraction helps drive joint cavitation and shapes developing articular surfaces, and absence of muscle activity can prevent proper joint cavity formation

What happens to joint development in muscle-less or paralysed embryos?,

In the absence of muscle contraction, joint cavitation fails to occur and cartilage markers such as Sox9 and Col2a1 remain highly expressed

Why is mechanobiology clinically important

Understanding mechanobiology helps explain skeletal deformities caused by paralysis or immobilisation and guides rehabilitation, tissue engineering and regenerative medicine

What happens if muscle contraction is absent during development?

Absence of muscle contraction leads to skeletal malformations and failure of proper joint formation

What is phocomelia?

Phocomelia is a congenital condition where long bones are absent or severely shortened

What is polydactyly?

Polydactyly is duplication of digits

What is syndactyly?

Syndactyly is failure of separation of digits

WORKSHOP QUESTIONS

Which limb bone forms by endochondral ossification?

The femur forms by endochondral ossification

Which bone forms by intramembranous ossification?

The occipital bone forms by intramembranous ossification

Which signalling molecule controls differentiation of mesenchymal cells into chondrocytes?

Indian hedgehog (Ihh) controls chondrocyte differentiation

What is the function of RUNX2 in bone development?

RUNX2 is required for osteogenic differentiation and bone mineralisation

Which stain colours mineralised bone red?

Alizarin red stains mineralised tissue red

Around what age do the last growth plates fuse in humans?

Growth plates fuse at around 20 years of age

At what age does the secondary ossification centre appear in the patella?

The patella secondary ossification centre appears around 3 to 5 years of age

Why is knowledge of primary and secondary ossification centres important?

It helps identify normal growth patterns and distinguish growth plates from fractures

Give one clinical use of knowing ossification centre timing?

It is used for skeletal age estimation in radiology and forensics

From which embryological structure do limb muscles originate?

Limb muscles originate from somites

Into which two parts do somites subdivide?

Somites subdivide into the sclerotome and dermomyotome

What else does the dermomyotome form besides skeletal muscle?

The dermomyotome also forms the dermis of the skin

During which week do muscle precursors migrate into the limb bud?

Muscle precursors migrate during the 5th week of development

Which muscle mass gives rise to the anterior compartment of the upper limb?

The ventral muscle mass forms the anterior compartment of the upper limb

How does limb rotation explain final lower limb orientation?

Medial rotation of the lower limb causes the great toe to lie medially in anatomical position

Which transcription factor is required for migration of muscle precursors?

LBX1 is required for migration

Do all muscles develop at the same time or via the same signals?

No, different muscles develop at different times and use different signals