Chondrogenesis Flashcards
What are the 3 embryonic origins of BONES?
1) Cranial neural crest cells
2) Somites
3) Lateral mesoderm
What bones do the cranial neural crest cells give rise to?
The craniofacial skeleton
What bones do the somites give rise to?
The axial skeleton
What is the axial skeleton?
Trunk - ribs and vertebrae
What does the lateral mesoderm give rise to?
The limb (appendicular) skeleton
What do Hox genes do?
How?
Control the anterior-posterior patterning of somites
How?
- Organised in sequential expression in the embryo in AP fashion
- Determines a CODE that is read by cells - provides positional identity to cells
- Tells the cells what type of vertebrae to become
What does the hox code dictate?
The TRANSITIONS between the different types of vertebrae
What is the transition between the cervical and thoracic vertebrae determined by in the mouse/chick?
Hox c5
Hox c6
Why does the chick have more cervical vertebrae than the mouse?
Boundary between Hox c5 and Hox c6 is shifted more anteriorly in the mouse
How many cervical vertebraes do mammals have?
7
How do mammal vertebrae differ?
How is this controlled?
Differ in size
Controlled by hox genes:
- Control the genes that are involved in cell proliferation
What genes do hox genes control?
- Genes that control positional identity
- Genes that control proliferation
What are the 3 steps leading to AXIAL skeleton formation?
1) Scelerotome induction in the somite
2) Cartilage formation
3) Ossification of axial skeleton
Where are the sclerotome precursors in the somite?
In the VENTRAL part
What does the sclerotome form?
Cartilage and bone
What is chondrogenesis?
The formation of cartiladge
What is osteogenesis?
The formation of bone (ossification)
What are the stages of chondrogensis?
1) Start with a MULTIPOTENT cell in the somite
2) Cell becomes SPECIFIED to become a SCLEROTOMAL cell
3) Cell DETERMINED to specific lineage - CHONDROBLASTS
4) Cell DIFFERENTIATES to CHONDROCYTES
5) Cell undergoes MATURATION - to form HYPERTROPHIC CHONDROCYTES
What are the first cell types of cartilage?
Chondroblasts
What are pax genes?
Paired-box genes
What pax genes are involved in the formation of cartilage?
How is this different to muscle?
Cartilage:
- Pax 1
- Pax 9
Muscle:
- Pax 3
In sclerotomal tissue, where is pax 1 and pax9 expressed?
Where is pax1 more strongly expressed?
Pax 9?
In BOTH the medial and lateral sclerotome
Pax 1 - more medial
Pax 9 - more lateral
What is the result of a Pax 1 KO?
Abnormalities in the vertebral column, sternum and scapula
What is the result of a Pax9 KO?
Abnormalities in the skeleton of:
- Craniofacial
- Visceral
- Limb
Die shortly after birth
Where is pax9 expressed?
- Cranial region
- Limbs
- Somites
What is the result of Pax 1/9 KO?
What does this show?
COMPLETELY lack derivatives of the MEDIAL sclerotome
Shows:
- Functional redundancy
- Need at least one of Pax1 or Pax9 to go through axial skeleton formation
What are the derivatives of the MEDIAL scelerotome?
- Vertebral bodies
- Intravertebral discs
- Proximal ribs
What structures are unaffected in the Pax 1/9 KO?
- Distal ribs
- Neural arches
What lead scientists to fate map where distinct regions of the axial skeleton arise from?
Examples?
The phenotype from the Pax1/Pax9 KO
Example:
- Loss of vertebral bodies, intravertebral discs and proximal ribs –> mean these structures are derived from the medial and lateral sclerotome
- Remains of neural arches and distal ribs –> these structures do NOT arise from the medial or lateral sclerotome
Where do the neural arches arise from?
Why?
From the DORSAL sclerotome
Doesn’t express pax1/9
Where do the proximal part of the ribs derive from?
Lateral sclerotome
Where does the vertebral bodies arise from?
Medial scelrotome
Which parts of the ribs have a dermomyotomal origin?
- Distal parts of the rib
- Sternal ribs
What parts have sclerotomal origin?
- Vertebral body
- Neural arches
- Proximal rib
What are the signals that control sclerotome formation?
Extrinsic cues:
- Shh release from the notochord
- Diffuses into the ventral parts of the somite
- Induces Pax1 and Pax9 expression
How do we know that shh induces the expression of Pax1 and Pax9 in the ventral somite?
KOs of shh cause the same defects as KOs in Pax1/Pax9
How is transcription restricted to the sclerotome?
What else does this do?
- BMP4 INHIBITORS released from the lateral mesoderm, prevents the expansion of the pax1/9 domain
- Also contributes to the medial/lateral boundary by restricting pax1 diffusion into the lateral part of the somite
What is the next step in the formation of bone, once have specification to sclerotome?
Cells must undergo determination and differentiation to form CARTILAGE
How does a sclerotmal cell become determined and form a condroblast cell?
1) Migration of sclerotomal cells to around the notochord
2) Down regulation of Pax1 and Pax9
3) Up regulation of ECM proteins that are specific to cartilage and help with CONDENSATION of the cells
Why is Pax1 and Pax9 down regulated when the sclerotomal cells are undergoing determination?
They are no longer needed as the scelrotomal cells are already specified
(Pax1 and Pax9 are only needed to SPECIFY)
How is a chondroblast different to a chondrocyte?
Chondroblast - un-differnetiated cartilage cell
Chondrocyte - differentiate cartilage cell
How go from a chondroblast –> chondrocyte?
1) Proliferation induced by BMP2, BMP4, BMP5
2) Production of a cartilage matrix (collagen)
What does the production and secretion of a cartilage matrix require?
Sox9
What is Sox9 proteins?
A HMG-box TRANSCRIPTION FACTOR
What are the cartilage matrix proteins?
Collagen II
Collagen IX
Collagen XI
Why does the sclerotome condense around the notochord?
In order to form a vertebrae
What is osteogenesis/ossification?
The formation of bone
What are the 2 main models for osteogenesis?
1) Intramembranous ossification
2) Endochondral ossification
What is intramembranous ossification used for?
To form the bones of the skull
Where does endochondral ossification occur?
In the SOMITES and LIMB skeleton
What is the main difference between intramembranous ossification and endochondral ossification?
Intramembranous:
- FOLLOWING condensation of cells - don’t have differentiation into cartilage
- Differentiation STRAIGHT into osteoblasts
Endochondral:
- Cartilage forms
- Cartilage later REPLACED by bone
What is the model for intramembranous ossification?
- Mesenchymal cells condense (NOT around the notochord)
- Nodules
- Osteoblasts
- Osteocytes
- Bone
What is the process of endochondral ossification?
1) Start with tissue made of mesenchymal cells
2) Forms a CARTILAGE MODEL of the bone
3) Chondrocytes stop dividing and start becoming hypertrophic (increase in size)
4) Hypertrophic chondrocytes then DIE by APOPTOSIS - leaving a SPACE
5) Space left is invaded by BLOOD VESSES from the PERICHONDRIUM
6) Blood vessels bring in OSTEOBLASTS
7) SECONDARY OSSIFICATION in the epiphysis
What is the pericondrium?
The connective tissue that surrounds the cartilage when it is not a joint
What is the function of the osteoblasts that the blood vessel brings in?
Degrade the matrix (collagen)
AND
Deposit bone matrix
What do the osteoblasts become after they have deposited bone matrix?
Bone marrow
What is the epiphysis?
The end part of the bone
What is the diaphysis?
The long part of the bone
What is left after secondary ossification?
What is this structure known as?
A cartilage plate between the epiphysis and the diaphysis - that hasn’t been modified
Known as the GROWTH PLATE
What is the growth plate important in?
Important in the POSTNATAL growth of bones
What occurs during secondary ossification?
The same process as primary ossification:
- Chondrocytes –> hypertrophic chondrocytes
- Hypertrophic chondrocytes DIE
- Leave gap
- Gap invaded by blood vessels
- Blood vessels bring in osteoblasts
- Osteoblasts degrade the cartilage matrix and deposit bone matrix
What studies helped us to understand how you maintain a balance between producing osteoblasts or chondrocytes from chondrocytes and how maintain balance between intramembranous or endochondral ossification?
Studies of human syndromes and studies in mice:
- CD
- CCD
What is CD?
Campomelic dsplasia
What is CCD?
Cleidocranial dysplasia
What is CD a result of?
What does it cause?
What does this show?
Mutation in Sox9
Leads to the death of children at a young age:
- Defect in formation of cartilage and long bones
- Defect in intramembranous ossification
Shows:
- Sox9 occurs EARLY on in the COMMON STEP between intramembranous ossification and endocondral ossification (condensation of mesenchyme)
What does the phenotype of So9 KO resemble?
What does this show?
Resembles mice with a KO of collagen II and collagen XI
Shows:
Sox9 is needed to induce the transcription of cartilage matrix
Using conditional KO, what happens if KO Sox9 IMMEDIATELY AFTER the condensation?
What does this show?
Deficit in the formation of proliferative chondrocytes and bones
Shows:
Sox9 is required for the differentiation of the sclerotomal mesenchymal cells –> Chondrocytes
What type of cells are chondroblasts?
Mesenchymal cells
Using conditional KO, what happens if KO Sox9 immediately AFTER the formation of chondrocytes?
What does this show?
Perturbations in the maturation of chondrocytes into hypertrophic chondrocytes
Shows:
- Sox9 has a role by LIMITING how much MATURATION of chondrocytes occurs
What causes CCD?
Mutation in transcription factor Runx2
What do mutations of Runx2 cause in the human?
- Deficits in the formation of BONES and HYPERTROPHIC CHONDROCYTES
- But there is presence of CHONDROCYTES (not able to mature)
What is Runx2 required for in the mouse?
The expression of the transcription factor Osterix
What happens if KO Osterix in the mouse?
- NO defects in the formation of cartilage
- Defects in the formation of BONE
What is the function of Runx2?
2 functions:
1) Controls the formation of cartilage by allowing chondrocytes to mature to hypertrophic chondrocytes
2) Controls ossification through the control of Osterix transcription factor (controls the formation of osteoblasts and osteocytes)
What transcription factor is used to balance cartilage formation vs ossification?
Runx2
What is the structure of the growth plate?
Layered organisation containing ALL the cell types:
- Proliferative chondrocytes
- Differentiated chondrocytes
- Pre-hypertrophic chondrocytes
- Hypertrophic chondrocytes
- Zone of ossification
What ensures that bones will grow postnatally in a continuous manner?
The layered organisation of the growth plate
AND
How many cells are present in the growth plate
How are the different cell types int he growth plate controlled and maintained as we grow postnatally?
How do these molecules interact?
Using 3 factors:
1) PTHrP - Parathyroid hormone-related protein
2) Ihh - Indian hedgehog
3) FGFR3 - Fibroblast growth receptor 3
FGFR3 —| Ihh —> PTHrP
What do gain-of-function mutations in FGFR3 cause?
How?
Dwarfism
Promote the proliferation of chondroblasts
(Activate Ihh, which activates PTHrP, which activates the proliferation of cells and prevents the differentiation)
What do mutations in PTHrP cause?
How?
Dwarfism
Failure to control MATURATION of chondrocytes into hypertrophic chondrocytes
Due to cells differentiating too quickly (loss of the progenitor pool)
How do FGFR3, Ihh and PTHrP control the growth of bones?
1) Postnatally, as cells progress in the growth plate - PRE-HYPERTROPHIC CHONDROCYTES begin to express Ihh
2) Ihh acts on the pericondrium and promotes the release of PTHrP from the pericondrial cells
3) PTHrP diffuses into the area of the CHONDROBLASTS - preventing them from progressing and differentiating into CHONDROCYTES
4) Feedback loop promotes the PROLIFERATION of chondroblasts and PREVENTS their differentiation
Why is it important to maintain the proliferation of chondroblasts and prevent their differentiation?
To maintain a POOL of PROGENITOR cells in the growth plate
How do mutations in FGFR3 and PTHrP cause dwarfism?
- Drive cells to differentiate too quickly - prevent the maintenance of the progenitor pool
OR
- Get an excess of progenitors that are unable to differentiate
