Lecture 15 - chondrogenesis (axial skeleton formation)

Question 1

What are the embryonic origins of bones?

Answer 1

• somites gives rise to axial skeleton (skeleton in body
• origin of limb skeleton derives from lateral mesoderm not somites
• craniofacial skeleton derives from cranial neural crest

All using the same molecular pathways to undergo specification, differentiation & maturation

Question 2

Where does the axial skeleton (vertebral column, ribs) form from?

Answer 2

paraxial mesoderm

Question 3

Where does the appendicular skeleton form from?

Answer 3

the lateral mesoderm

Question 4

Where does the cranio-facial form from?

Answer 4

neural crest cells

Question 5

What has a mouse embryo with histological stain that allows visualisation of elements of the developing skeleton showed?

Answer 5

Along A/P axis, bones that form from somites do not look the same, but differences in shape & size. Segmented mesoderm allows some pathways to be made, but slight tweaks allows specifics to be created

Question 6

How are Hox Genes involved in patterning of A/P axis in an embryo?

Answer 6

HOX genes expressed in a co-linear manner - co-linearity of gene expression spatially in the embryo that correlates to how those genes are found in their position in the genome.

Different series of HOX genes, ordered in sequential manner that represents how they are expressed in the embryo.

HOX code - expression of a particular HOX gene will confer a particular A/P identity to cells in that part of axis.

Question 7

How do somites relate to HOX gene expression?

Answer 7

There are different numbers of certain vertebrae in different animals. This correlates to a different expanse of HOX code. Despite these differences (e.g. number of cervical vertebrae), there are features that are conserved that allows somites to know whether they will become a cervical or thoracic & where that boundary will lie. One side of boundary HOXc5 & the other side HOXc6.

Expressed at different positions in A/P axis, this will drive change from cervical to thoracic.

HOX transcription factors can also control proliferation, allowing to tie positional identity to proliferation of bones, which allows correlation to A/P axis.

Question 8

What are the 3 steps leading to axial skeletal formation?

Answer 8

1. Sclerotome induction
2. Cartilage formation = chrondrogenesis
3. Ossification of axial skeleton = osteogenesis

Question 9

What is sclerotome induction?

Answer 9

Take cells that are living in soites as epithelial cells & tell them to make some sclerotome.

Question 10

What occurs in chrondrogenesis (cartilage formation)?

Answer 10

(recap of myofiber creation)
Pluripotent cells that live in somite that undergo determination to decide what tissue they will make.

This leads to formation of muscle progenitor cells - myoblasts.

After differentiation they create differentiate muscle cells - myotubes (ALL FROM DORSAL PART OF SOMITE). They maturation

Question 11

What occurs in the ventral part of somite?

Answer 11

Similar except instead of muscle, cartilage is created

Question 12

Describe the process of chrondrogenesis

Answer 12

Pluripotent stem cells (specification) - sclerotomal cells (determination) - chrondroblasts (differentiation) - chrondrocytes (maturation) - hypertrophic chrondrocytes

Question 13

How does the axial skeleton originate from the sclerotome?

Answer 13

While the dorsal somite remains epithelial & forms the dermomyotome, precursor of the myotome & dermatome, the ventral somitic cells undergo an epithelial-to-mesenchymal transition and form the sclerotome, then cartilage

The sclerotome contains progenitor cells for the axial skeleton

Sclerotomal cells express the paired-box TFs - Pax1 & Pax0

Question 14

What is the role of Pax Genes = Paired-box genes?

Answer 14

Pax3 expressed in dorsal & important pre-determinator for muscle determination. Due to a genome duplication event, there are 2 forms of pax3 - pax3/7

Pax1/9 - chondrogenesis (drive formation of cartilage in ventral somite. Important for sclerotome induction/specification)

Pax3/7 - drive myogenesis

DNA binding domain but lost homeodomain - only bind domain through their paired-domain.

Question 15

What genes are expressed during sclerotome formation?

Answer 15

Medial lateral patterning to where Pax1 & Pax9 are expressed. Pax1 expressed in ventral somite cells undergoing EMT, but expressed in medial position, close to neural tube. More lateral somite cells express Pax9.

Genes are expressed in the right place.

Question 16

What are the affects of gene targeting inactivation of Pax genes?

Answer 16

Pax1 KO: mice are viable, have abnormalities in the vertebral column, the sternum & the scapula.

Pax9 KO: mice die shortly after birth. Have abnormal craniofacial, visceral & limb skeletogenesis

Pax1/9 KO: mice completely lack derivatives of medial sclerotome: vertebral bodies, intervertebral discs, proximal ribs.

Pax1 or Pax9 is required for medial sclerotome development.

Some redundancy but both clearly important.

Question 17

What do functional experiments of Pax1/9 KOs allow?

Answer 17

allow mapping of phenotypes of gene expression & allocation of gene function to specific structures of interest - e.g. axial skeleton

Question 18

What is derived from dorsal part of somite?

Answer 18

Distal rib & sternal rib

Question 19

What are the signals controlling sclerotome formation?

Answer 19

Notochord, neural tube, ectoderm & lateral plate mesoderm all provide signals that are spatially organized around the sclerotome that help provide localised signals that allow different parts of somite to develop in different ways.

Shh expression in the notochord is also important in inducing expression of Myf5. This doesn’t happen in the ventral part of the somite, as Wnt signalling isn’t produced (only produced dorsal part of neural tube & close to notochord). This means only the areas close to these areas will produce Myf5, whereas this isn’t true for ventral part of somite.

Question 20

Summarise chondrogenesis

Answer 20

• 3 distinct embryonic tissues participate to the formation of the skeleton. Somite generate axial skeleton.
• Pax1 & Pax9 are essential for axial skeleton formation. Scleraxis also plays a role in sclerotome formation.
• Shh & BMP4 are involved in the induction & patterning of Pax1 & Pax9 expression.

Question 21

What are the different stages of chondrogenesis?

Answer 21

Sclerotomal cell –> chondroblast –> chondrocyte

Question 22

Sclerotomal cell –> chondroblast

Answer 22

1) migration of cells around notochord
2) down-regulation of Pax1 & Pax9
3) condensation of cells - ECM proteins

Question 23

Chrondroblast –> chondrocyte (differentiated cartilage cells)

Answer 23

1) proliferation induced by BMP2, BMP4 & BMP5
2) production of cartilage matrix, requires Sox9

Question 24

Describe the process of creating chondrocytes

Answer 24

Production of cartilage from sclerotomal cells

Sclerotomal cells in ventral part of somite migrate around the notochord & downregulate Pax1&Pax9 (TFs which are involved in telling cell to be sclerotomal). They then condense, changing their gene expression profile, & make ECM proteins, an important part of bine & cartilage. These are now chondroblasts.

The chondroblasts then proliferate, induced by several different of BMP family (bone morphogenic protein) - BMP2, BMP4 & BMP5. This leads production of cartilage matrix in the chondroblasts, which requires Sox9. They are then differentiating into chondrocytes.

Question 25

What are the 2 main modes of ossification?

Answer 25

Intramembranous ossification: - mainly used for ossification of bones from skull (mesenchymal cells --> nodules --> osteoblasts --> osteocytes --> bone. Endochondral ossification: - used for ossification of most bones (limb). Bone develops by replacement of a cartilage model.

Question 26

What are the stages of endochondral ossification?

Answer 26

1. Chondrogenesis - formation of a cartilagenous model of the bone 2.Chondrocytes stop dividing & become hypertonic 3. Chondrocytes die by apoptosis, blood vessels & osteoblasts enter this space, which will become bone marrow. 4. Osteoblast replace the disappearing cartilage & form primary ossification center. 5. Blood vessels enter the epiphyses (ends). 6. Secondary ossification centres are formed in epiphyses, leaving a cartilage place (growth plate) between epiphysis & diaphysis.

Question 27

Describe the ossification centres

Answer 27

Primary ossification in the centre, with secondary ossification at the end, separated by plate of cartilage (growth plate) - important for post-natal bone elongation Ossification centres at the end of bone - epiphyses Ossification centres in the middle of bone - diaphysis

Question 28

How do we regulate the balance the development of cartilage & bone?

Answer 28

Mesenchymal cells can turn into cartilage or bone Axial skeleton - produce cartilage & hypertrophic chondricytes Once cartilage made, osteoblasts need to be made. Requires temporal control - Wnt activation activates osteocyte development. - Wnt inhibition activates hypertrophic chondrocyte development.

Question 29

How are genes involved in regulating this process?

Answer 29

Found in mutations in bone formation CCD - cannot form cartilage scaffold Runx2 mutation = cartilage formation but problems with ossification This means Runx2 is required to drive mesenchymal condensates to form osteoblasts instead of chondrocytes. Runx2 is also important for driving expression of Osterix.

Question 30

How to control balance of genes?

Answer 30

Sox9 is a short-lived factor, but drives cartilage expression. Lack of Sox9 leads to expression of Runx2 & creates osteoblasts - quick switch between 2 pathways. Some tissues are able to directly create - Wnt signalling is active & can inhibit the cartilaginous pathway.

Question 31

What controls chondrocyte differentiation & maturation?

Answer 31

A negative feedback loop between Ihh & PTHrP Ihh - Indian hedgehog PTHrP - Parathyroid hormone-related protein FGFR3 - inhibits Ihh Between the epiphysis & diaphysis centres Satellite cells drive growth in muscles. A number of things involved in driving bone growth.

Question 32

What do mutations in Ihh & PTHrP lead to?

Answer 32

Mutations in these genes cause dwarfism. These genes interact to maintain a balance which allows them to keep a pool of precursor cells in the growth plate, which allowed continued to future growth of the bone. A cells progress from hypertrophic chondroblasts to chondrocytes, they express Ihh. This triggers a signalling cascade, leading to activation of expression of PTHrP. Feedback mechanism of signals is important to keep having the ability of making new bones & elongate your bones. Mutations will affect bone elongation.