L13 - Chondrogenesis Flashcards
What is the embryonic origin of craniofacial skeleton?
Cranial neural crest cells
What is the embryonic origin of axial skeleton?
Somites/paraxial mesoderm
What is the embryonic origin of limb/appendicular skeleton?
Lateral mesoderm
What do Hox genes control in somites?
Anterior-posterior patterning
How do hox genes work?
Encode identity in embryonic development
The sequential expression occurs in an anterior/posterior manor
- Determines a code that is read by cells, providing cells with a positional identity
Control of where the genes occurs is translated within the type of vertebrate
Example of hox gene use in chick and mouse
The chick has more cervical vertebra than the mouse
- Transition between cervical and thoracic vertebrae is determined and the position is conserved between birds and mouse
- Dictated by boundary from HoxC5 to HoxC6
- However mouse has fewer cervical vertebra than chick so boundary shifter anteriorly
How can hox genes control vertebrate size?
Hox genes control genes involved in cell proliferation
Can couple the positional info to the amount of proliferation
What are the 3 steps leading to axial skeleton formation?
- Sclerotome induction - in ventral part of somites
- Cartilage formation – chondrogenesis
- Ossification of axial skeleton – osteogenesis
Chondrogenesis method
- Stem cells – undergoes specification
- Sclerotomal cells – undergoes determination
- Chondroblasts – undergoes differentiation
- Chondrocytes – undergoes maturation
- Hypertrophic chondrocytes
What pax genes are involved in chondrogenesis?
Pax1/9
What pax genes are involved in myogenesis?
Pax3/7
What genes are expressed during sclerotome formation?
Ventral part of somite undergoes epithelial-mesenchymal transition
Pax 1 and Pax 9 genes are exclusively expressed by sclerotome cells
In sclerotome cells is Pax1 expressed more medially or laterally?
Medially
In sclerotome cells is Pax9 expressed more medially or laterally?
Laterally
Pax1 knockout phenotype?
Mice are viable
Have abnormalities in the vertebral column, the sternum and the scapula
Pax9 knockout phenotype?
Mice die shortly after birth
Have abnormal craniofacial, visceral and limb skeletogenesis
Pax1/9 knockout phenotype?
Mice completely lack derivatives of medial sclerotome - vertebral bodies, intervertebral discs, proximal ribs
Distal part of ribs not affected
What did the Pax1/9 knockout show?
Pax1 or Pax9 is required for medial sclerotome development
Demonstrated the essential role in axial skeleton formation
Where are Pax1/9 expressed in the sclerotome and what does it form?
Medial sclerotome –> vertebral bodies
What does the lateral sclerotome form?
Proximal rib
What does the dorsal sclerotome form?
Neural arches
What does the dermomyotome form?
Distal vertebral rib and sternal rib
How were the derivatives of the sclerotome discovered?
These were all identified using the double knockout
- Pax1 and Pax9 expressed in medial and lateral areas
- In double knockout neural arches not affected – know that neural arches arise from dorsal aspect not expressing Pax1/9
What signals control sclerotome formation?
Shh signalling from the notochord induces expression of Pax1 and Pax9
BMP signalling from the lateral plate inhibits Pax1 and Pax9
- Contributes to medial/lateral boundary
- Inhibits diffusion of Pax1 into lateral domain
What process occur in the sclerotomal cell to convert in to a chondroblast?
- Migration of cells around notochord
- Down regulation of Pax1 and Pax9
- Condensation of cells – extracellular matrix proteins
What process occur in the chondroblast cell to convert in to a chondrocyte?
- Proliferation induced by BMP2, BMP4 and BMP5
2. Production of cartilage matrix – requires Sox9
What happens to the sclerotome cells around the notochord?
Condensation
What are the two models of osteogenesis?
Intramembranous ossification
Endochondral ossification
Intramembranous ossification method
Used for ossification of bones from skull
- Mesenchymal cells
- Nodules
- Osteoblasts
- Osteocytes
- Bone
Endochondral ossification
Used for ossification of most limb bones
Bone developed by replacement of cartilage model
Endochondral ossification method
- Chondrogenesis – formation of a cartilaginous model of bone
- Chondrocytes stop dividing and become hypertrophic
- Chondrocytes die by apoptosis - blood vessels and osteoblasts enter this space which will become bone marrow
- Osteoblasts replace the disappearing cartilage and form primary ossification centre
- Blood vessels enter the ends
- Secondary ossification centres and formed in epiphyses leaving a cartilage plate between epiphysis and diaphysis
What is CD?
Campomelic dysplasia
What is CCD?
Cleidocranial dysplasia
What causes CD?
Loss of function mutation in Sox9
What causes CCD?
Mutation in Runx2
What is the phenotype of CD?
Leads to death of babies during the prenatal period or in early infancy
Die due to skeletal abnormalities suggesting a defect in cartilage
Many of the defects are similar to those caused by mutations in cartilage collagens II/XI
Heterozygous Sox9 null mice show most of the abnormalities seen in CD patients
What is the phenotype of CCD?
Leads to defects in formation of bones and in formation of hypertrophic chondrocytes
Runx2 studies in CDD
Proliferative chondrocytes not capable of proliferating
As with Sox9, Runx2 acts in two locations
Runx2 is required for the expression of osterix
- Osterix knockout only shows defects in osteogenesis not chondrogenesis
Allows you to balance osteogenesis and chondrogenesis
Sox9 studies in CD
Sox9 plays a role early on in mesenchymal condensations and bone formation
Forward genetics – identify gene responsible for human phenotype – Sox9
Reverse genetics – need to tell you how the gene is controlling this process
Condition knockout sox9 studies in mice
In mice – conditional knockouts
Allows you to control when and where the gene is knocked out
Knock out Sox9 at different positions and timing
What happens if you knockout out Sox9 immediately after mesenchymal condensation?
Deficit in the formation of proliferative chondrocytes and therefore bones
What happens if you knockout out Sox9 after proliferative chondrocytes have formed?
Reduction in the maturation of chondrocytes into hypertrophic chondrocytes
What is special about the region in between epiphysis and diaphysis that has not been ossified?
Layered organisation of cells Have every single cell type present - Proliferative chondroblasts - Differentiated chondrocytes - Pre-hypertrophic chondrocytes - Hypertrophic chondrocytes - Zone of specification You have cells of every single step of cartilage to bone formation
What does the negative feedback loop between Ihh and PTHrP control?
Chondrocyte differentiation and maturation
What is PTHrP?
Parathyroid hormone-related protein
What do gain of function mutations in FGFR3 do?
Can introduce these mutations in mouse – gives the same phenotype
- Dwarfism
They promote the proliferation of chondroblasts
Make receptor constitutively active
FGFR3 block?
Ihh
Ihh activates?
PTHrP
As cells progress and differentiate and mature the pre-hypertrophic chondrocytes secrete?
Ihh
What is the role of Ihh?
Induces production of PTHrP
Inhibits further progression to hypertrophic chondrocytes
Promotes differentiation of cells earlier on– induced chondroblast proliferation
- Negative feedback loop
Maintain a pool of progenitor cells in the growth plate for future muscle growth
