Lecture 13: chondrogenesis

Question 1

Describe the general pathway of chondrogenesis.

Answer 1

Stem cells → specification → sclerotomal cells → determination → chondroblasts → differentiation → chondrocytes → ossification → hypertrophic chondrocytes

1. Mesodermal (stem) cells need to be specified to restrict the cell fate
2. Determination to become stable = chondroblast. Cells proliferating.
3. Further restrict cell fate i.e., give them specific properties = chondrocytes. Expresses lots of collagen and ECM
4. Maturation i.e., cells become hypertrophic

Question 2

What are Pax genes? State which are expressed in muscle progenitor cells and explain the experiments that support their function.

Answer 2

Bones come from Pax1 and Pax9+ cells which are paired-domain transcription factors. The timing of their expression syncs with specification and determination of a skeletal cell fate (can be tested with a KO).

LOF = is this gene NECESSARY for a particular effect?

GOF = is this gene SUFFICIENT for a particular effect?

LOF of Pax1 = subtle abnormalities

LOF of Pax9 = mice die after birth because they have severe craniofacial defects.

So far, no dramatic effect on axial skeleton – not expected => functional redundancy!

To test redundancy, perform a double KO =>

LOF of Pax1/9 = severe defects in axial skeleton => one or the other is absolutely necessary for axial skeleton. No defects in neural arch => Pax1/9 doesn’t define cells that give rise to neural arches => neural arches must sit in the dorsal part of sclerotome, outside of Pax1/9 expression domain.

Question 3

Explain the process of chondrogenesis.

Answer 3

1. SHH diffuses to the adjacent parts of mesoderm and regulates D-V patterning of somites. High SHH signalling = expression of Pax1/9 in VENTRAL part of somites -> coordinates formation of muscle and bone.
2. Negative regulation by BMP4 which creates a boundary to prevent Pax1 expanding too laterally. Cells are now specialised to form cartilage in the sclerotome (chondroblast)
3. Migration and condensation of cells around the notochord.
4. Downregulation of cell-fate specific genes and upregulation of SOX9, a gene important for cartilage.
5. BMP2, 4 and 5 initiate proliferation.
6. Cells now chondrocytes and are characterised by a progressive increase in ECM genes.
7. Endochondral ossification occurs (intramembranous ossification occurs for skull)

Question 4

Explain the process of endochondral ossification.

Answer 4

• Chondrocytes stop dividing and become hypertrophic (size increases as more matrix protein is made and deposited around the cell for nutrients)
• Chondrocytes die by apoptosis
• Blood vessels and osteoblasts take over this space (become bone marrow)
• Osteoblasts replace the disappearing cartilage and form the primary ossification centre
• Blood vessels enter the ends
• Secondary ossification centres form around the ends leaving a growth plate (where ossification doesn’t occur)

Must control balance of cartilage- and bone-producing cells through a set of feedback loops

Sox9 regulates cartilage matrix gene expression but also controls transition from chondrocytes to hypertrophic chondrocytes (cartilage).

Runx2 = TF that drives differentiation of bone from cells but also coordinates WHEN these events take place. Runx2 expression = stimulate bone pathway and block cartilage formation.

Question 5

Describe the role of Hox genes in A-P patterning.

Answer 5

Mammals have a fixed number of cervical vertebrae. How did mammals evolve to have similar number but different size of vertebrae?

One hypothesis is that Hox genes, in addition of control A-P identity, control also cell proliferation.

HOX gene = homeobox containing TFs, highly conserved in evolution. They have a particular arrangement so that genes in 5’ form posterior structures and 3’ genes are more anterior. This allows us to establish a HOX code i.e., certain combos of HOX gene expression encodes a certain positional identity of the cells.

HOX code gives positional identity to cells in the axial skeleton. Distinguish different vertebrates via their HOX code. The spinal cord region boundaries are important for determining identity because they are conserved through evolution. HOX genes can also control cell proliferation which can be linked to positional identity => link size.

Question 6

Explain the steps leading to bone growth and the implications of mutations in the genes involved.

Answer 6

Regulating bone growth is important because many mutations here result in dwarfism.

The growth plate is located between primary and secondary ossification centres:

It contains all cell types implicated in the formation of bone and maintains a population of progenitors which can contribute to more bone cells. All due to two important genes: IHH and PTHrP.of which there is a negative feedback loop between.

When cells become hypertrophic, they start producing IHH which acts on chondroblasts and perichondrium cells, stimulating them to produce PTHrP hormone.

Receptor for hormone is in low conc in chondroblasts but in high conc in pre-hypertrophic and hypertrophic chondrocytes. They respond to PTHrP by preventing the differentiation of hypertrophic chondrocytes and promote proliferation of chondroblasts i.e., maintain a pool of progenitor cells which give rise to the future bone.

Mutations in IHH or PTHrT = dwarfism because run out of progenitors => short bones.