L13 - Chondrogenesis

Question 1

What is the embryonic origin of craniofacial skeleton?

Answer 1

Cranial neural crest cells

Question 2

What is the embryonic origin of axial skeleton?

Answer 2

Somites/paraxial mesoderm

Question 3

What is the embryonic origin of limb/appendicular skeleton?

Answer 3

Lateral mesoderm

Question 4

What do Hox genes control in somites?

Answer 4

Anterior-posterior patterning

Question 5

How do hox genes work?

Answer 5

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

Question 6

Example of hox gene use in chick and mouse

Answer 6

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

Question 7

How can hox genes control vertebrate size?

Answer 7

Hox genes control genes involved in cell proliferation

Can couple the positional info to the amount of proliferation

Question 8

What are the 3 steps leading to axial skeleton formation?

Answer 8

1. Sclerotome induction - in ventral part of somites
2. Cartilage formation – chondrogenesis
3. Ossification of axial skeleton – osteogenesis

Question 9

Chondrogenesis method

Answer 9

1. Stem cells – undergoes specification
2. Sclerotomal cells – undergoes determination
3. Chondroblasts – undergoes differentiation
4. Chondrocytes – undergoes maturation
5. Hypertrophic chondrocytes

Question 10

What pax genes are involved in chondrogenesis?

Answer 10

Pax1/9

Question 11

What pax genes are involved in myogenesis?

Answer 11

Pax3/7

Question 12

What genes are expressed during sclerotome formation?

Answer 12

Ventral part of somite undergoes epithelial-mesenchymal transition
Pax 1 and Pax 9 genes are exclusively expressed by sclerotome cells

Question 13

In sclerotome cells is Pax1 expressed more medially or laterally?

Answer 13

Medially

Question 14

In sclerotome cells is Pax9 expressed more medially or laterally?

Answer 14

Laterally

Question 15

Pax1 knockout phenotype?

Answer 15

Mice are viable

Have abnormalities in the vertebral column, the sternum and the scapula

Question 16

Pax9 knockout phenotype?

Answer 16

Mice die shortly after birth

Have abnormal craniofacial, visceral and limb skeletogenesis

Question 17

Pax1/9 knockout phenotype?

Answer 17

Mice completely lack derivatives of medial sclerotome - vertebral bodies, intervertebral discs, proximal ribs
Distal part of ribs not affected

Question 18

What did the Pax1/9 knockout show?

Answer 18

Pax1 or Pax9 is required for medial sclerotome development

Demonstrated the essential role in axial skeleton formation

Question 19

Where are Pax1/9 expressed in the sclerotome and what does it form?

Answer 19

Medial sclerotome –> vertebral bodies

Question 20

What does the lateral sclerotome form?

Answer 20

Proximal rib

Question 21

What does the dorsal sclerotome form?

Answer 21

Neural arches

Question 22

What does the dermomyotome form?

Answer 22

Distal vertebral rib and sternal rib

Question 23

How were the derivatives of the sclerotome discovered?

Answer 23

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

Question 24

What signals control sclerotome formation?

Answer 24

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

Question 25

What process occur in the sclerotomal cell to convert in to a chondroblast?

Answer 25

1. Migration of cells around notochord 2. Down regulation of Pax1 and Pax9 3. Condensation of cells – extracellular matrix proteins

Question 26

What process occur in the chondroblast cell to convert in to a chondrocyte?

Answer 26

1. Proliferation induced by BMP2, BMP4 and BMP5 | 2. Production of cartilage matrix – requires Sox9

Question 27

What happens to the sclerotome cells around the notochord?

Answer 27

Condensation

Question 28

What are the two models of osteogenesis?

Answer 28

Intramembranous ossification | Endochondral ossification

Question 29

Intramembranous ossification method

Answer 29

Used for ossification of bones from skull 1. Mesenchymal cells 2. Nodules 3. Osteoblasts 4. Osteocytes 5. Bone

Question 30

Endochondral ossification

Answer 30

Used for ossification of most limb bones | Bone developed by replacement of cartilage model

Question 31

Endochondral ossification method

Answer 31

1. Chondrogenesis – formation of a cartilaginous model of bone 2. Chondrocytes stop dividing and become hypertrophic 3. Chondrocytes die by apoptosis - blood vessels and osteoblasts enter this space which will become bone marrow 4. Osteoblasts replace the disappearing cartilage and form primary ossification centre 5. Blood vessels enter the ends 6. Secondary ossification centres and formed in epiphyses leaving a cartilage plate between epiphysis and diaphysis

Question 32

What is CD?

Answer 32

Campomelic dysplasia

Question 33

What is CCD?

Answer 33

Cleidocranial dysplasia

Question 34

What causes CD?

Answer 34

Loss of function mutation in Sox9

Question 35

What causes CCD?

Answer 35

Mutation in Runx2

Question 36

What is the phenotype of CD?

Answer 36

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

Question 37

What is the phenotype of CCD?

Answer 37

Leads to defects in formation of bones and in formation of hypertrophic chondrocytes

Question 38

Runx2 studies in CDD

Answer 38

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

Question 39

Sox9 studies in CD

Answer 39

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

Question 40

Condition knockout sox9 studies in mice

Answer 40

In mice – conditional knockouts Allows you to control when and where the gene is knocked out Knock out Sox9 at different positions and timing

Question 41

What happens if you knockout out Sox9 immediately after mesenchymal condensation?

Answer 41

Deficit in the formation of proliferative chondrocytes and therefore bones

Question 42

What happens if you knockout out Sox9 after proliferative chondrocytes have formed?

Answer 42

Reduction in the maturation of chondrocytes into hypertrophic chondrocytes

Question 43

What is special about the region in between epiphysis and diaphysis that has not been ossified?

Answer 43

``` 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 ```

Question 44

What does the negative feedback loop between Ihh and PTHrP control?

Answer 44

Chondrocyte differentiation and maturation

Question 45

What is PTHrP?

Answer 45

Parathyroid hormone-related protein

Question 46

What do gain of function mutations in FGFR3 do?

Answer 46

Can introduce these mutations in mouse – gives the same phenotype - Dwarfism They promote the proliferation of chondroblasts Make receptor constitutively active

Question 47

FGFR3 block?

Answer 47

Ihh

Question 48

Ihh activates?

Answer 48

PTHrP

Question 49

As cells progress and differentiate and mature the pre-hypertrophic chondrocytes secrete?

Answer 49

Ihh

Question 50

What is the role of Ihh?

Answer 50

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