Molecular Mechanisms Of Bone Development

Front 1

Limb development

Back 1

- ext. of lat. plate mesoderm → skeletal tissues

- somites → nerves, blood vessel, muscle

- Limb patterning:
*stylopod → humerus
*zeugopod → radius + ulna
*autopod → carpals, metacarpal, phalanges

Front 2

Bone formation's phase

Back 2

1. Migration of mesenchymal cells into place

2. cell interaction between mesenchymal cell and epithelial cell & BM

3. condensation of mesenchymal cells in cluster (start organogenesis)

4. differentiation into osteoblasts & chondrocytes

Front 3

2 mechanisms of bone formation

Back 3

1. Intramembranous
mesenchymal cell condense/differentiate →
cells for bone synthesis →
craniofacial skeleton + clavicle

2. Endochondral
mesenchymal cells condense/differentiate →
cartilage cell →
cartilage template of future bone undergo cartilage replacement, BV invasion, mineral deposition, bone synthesis →
axial & appendicular bones

Front 4

Describe RUNX2/CBPFA1 gene [3]
- what happens in mutation [4]

Back 4

- TF controls osteoblast differentiation
- For Intramembranous (craniofacial, clavicle)
- regulates osteoblast specific genes
- RUNX2/Cbfa1 deficient mice ~ normal cartilage skeleton develops + no bone tissue formed

2. Cleidocranial dysplasia (CCD)
- mutation in RUNX2/CBFA1 causing haploinsufficiency
- delayed closure of cranial suture/fontanelles
- hypoplasia/aplastic clavicles
- Dental abnormalities (delayed eruption of milk/permanet teeth, supernumerary teeth in permanent set)

Front 5

What is SOX9 gene? [3]
- what happens in mutation? [6]

Back 5

- SRY-related (sex-determining region Y gene) HMG (high-mobility-group) box (DNA-binding region)
- controls chondrocyte/early osteochondroprogenitor differentiation
- for endochondral (axial & appendicular)

2. Campomelic dysplasia
- bowing of long bones
- hypoplasia of scapula & pelvis
- abnormality of vertebral column
- ↓no of ribs
- small cranium due to severe craniofacial anomalies
- freqeunt XY sex reversal

Front 6

How is osteochondroprogenitor regulated? [5]

Back 6

1. Mesenchymal cell → osteochondro progenitor
*regulated by SOX9

2. Osteochondro progenitor → chondrocyte
*Sox9, sox5, sox6

3. Osteochondro progenitor → osteoblast
*Runx2, Osx (Runx2 also stimulates Osx)

4. Chondrocyte → hypertophic chondrocyte
*Runx2

5. Osteoblast → functional osteoblast
*ATF4

Front 7

pattern of limb formation [3]

Back 7

1. Proximal-distal (apical ectodermal ridge)
- FGF signaling

2. Anterior-posterior (zone of polarizing activity)
- Hedgehog signaling

3. Dorsal-ventral
- WNT signaling

Front 8

Describe FGF signaling
- features [4]
- action [4]
- pathway

Back 8

Fibroblast growth factors:
- neg. regulator of chondrocytes
- normally induce cell proliferation/differentiation in tissues
- interacts with HSPG (essential for signal transduction)
- at least 18 FGF and 4 FGFR are known

Action
- ↓chondrocyte proliferation
- ↑prod. of Ihh (↑Ptc-1 & PTHrP)
- ↑diff. of hypertrophic chondrocytes to terminally diff. chondrocytes (express osteopontin & other char. markers)
- ***BMP act in opposite direction

Pathway:
FGF → FGFR dimerization → STAT1 → cell cycle inhibitor → ↓proliferation

Front 9

Receptors for FGF [3]
- what do mutations in FGFR result in? [5]

Back 9

FGFR is receptor tyrosine kinase

Receptor FGFR1:
- expressed in pre & hypertrophic chondrocytes, perichondrium
- FGF18 delays diff. of hypertrophic chondrocytes

Receptor FGFR2:
- express in perichondrium, periosteum, primary spongiosa
- FGF18 delays osteoblast development

Receptor FGFR3:
- express in proliferating chondrocytes
- FGF18 ↓chondrocyte proliferation

Mutations in FGFR result in?
- Mutation in FGFR → activate tyrosine kinase domain
- phosphorylation of STAT1
- upregulation of cell cycle inhibitors
- block chondrocyte proliferation
- RETARD BONE GROWTH

Front 10

Mutations in FGFr3 [3]
- proposed effects

Back 10

Mutation in FGFR3: osteochondrodysplasia
- achondroplasia
- hypochondroplasia
- thanatophoric dysplasia

Effects of FGFR3
1. Mutations in TM → stabilization of dimer
2. Mutation resulting in forming disulphide bonds → ligand independent activation of receptors
3. Mutation in kinase domain → ligand independent activation of receptors

Front 11

Gain-of-function mutation effects [2]

Back 11

- stabilization of dimer
- ligand-independent activation of receptor

Front 12

Describe the Hedgehog signaling process [2]
- mechanism

Back 12

Morphogen concept
- diffuse through embryonic tissue
- diff. positional value gives diff cell fates
e.g. sonic hedgehog protein from ZPA governs AP development of limb

Mechanism:
- Hh → Ptc → (-) Smo → Gli complex → activator

Front 13

Indian hedgehog/PTH-related peptide (PTHrP)
- feedback system

Back 13

PTH-related protein (PTHfP)
- secreted from perichondrial cells & chondrocytes at ends of long bones
- action via PTH1R (G protein-coupled receptor)
- PTHrP/PTH1R have role in normal endochondral bone formation & bone elongation
(+) chondrocyte proliferation
(-) chondrocyte terminal differentiation
(-) Ihh production

Indian Hedgehog (IHH)
- secreted by prehypertrophic chondrocytes when PTHrP production is low
- action:
(+) chondrocyte proliferation
(+) convert perichondral cell → osteoblast
(+) chondrocyte terminal differentiation
(+) PTHrP production

Front 14

Mechanism of feedback loop between IHH and PTHrP

Back 14

- regulating relative proportion of proliferation & hypertrophic chondrocytes in growth plates

- Inactivation of PTH1R → retards growth:
*↓PTHrP to ↓proliferation
*↑hypertrophic differentiation

- Activation of PTH1R to retard growth
*impair hypertrophic differentiation, retard growth

1. Initial differentation
(+) Sox9

2. Proliferation
(+) Ihh, PTH-rP
(-) FGFR3

3. Terminal diff
(+) Ihh
(-) PTHrP