L26 - Molecular Mechanisms of Bone Development

Question 1

Define the 2 main embryological origins for MSS system.

Answer 1

 Lateral plate mesoderm > skeletal tissues

 Somites > nerves, blood vessels, muscles

Question 2

Define the 4 phases of skeletal morphogenesis?

Answer 2

1. Migration of mesenchymal cells
2. Cell interaction between mesenchymal and epithelial cells across basement membrane
3. Condensation of mesenchymal cells
4. Differentiation of condensed mesenchymal cells via 𝑺𝒐𝒙𝟗 into osteochondroprogenitor

Question 3

Define the transcription factors that regulate differentiation from osterochondroprogenitor cells to Osteoblasts? Intramembranous ossification

Answer 3

RUNX2**/CBFA1: from osteochondroprogenitor to osteoblast

ATF4: from osteoblast to function osteoblasts

Form craniofacial skeleton and clavicles

Question 4

Name the disease caused by RUNX2 loss of function mutation/ haploinsufficiency?

Answer 4

cleidocranial dysplasia (CCD)

• Delayed closure of cranial suture, fontanelles
• Dental abnormalities: late teeth change
• Hypoplasia / aplastic clavicles

Question 5

Define the transcription factor that regulate differentiation from osteochondroprogenitor to chondrocytes? Endochondral ossification

Answer 5

Sox9**, Sox5, Sox6: osteochondroprogenitor to chondrocyte

Runx2: chondrocyte to hypertrophic chondrocyte

• expression of cartilage-specific extracellular matrix: Collagen II, IX, XI + Aggrecan
• form axial and appendicular bones

Question 6

Name the disease caused by Sox9 loss of function mutation?

Answer 6

campomelic dysplasia

• small, abnormal cranium
• Frequent XY sex reversal, e.g. male with female genitalia
• Scapular, pelvic hypoplasia, long bone and spine abnormality

Question 7

List the key signalling pathways that regulate linear bone growth?

Answer 7

1. Fibroblast growth factors (FGFs)

2. Indian Hedgehog-PThrP loop

Question 8

Which transcription factor regulating osteochondroprogenitor differentiation is sex determining?

Answer 8

SOX9

SRY - related = SRY must be activated for differentiation into male sex

Question 9

Define the transcription factor that drives mesenchymal cell differentiation in skeletal morphogenesis?

Answer 9

Sox9: drives mesenchymal cell&raquo_space; osteochondroprogenitor

Sox9 remaining turned on = form chondrocyte
Sox9 turned off + RUNX2 turned on = form osteoblast

Question 10

Define the signalling pathway for Proximal-distal bone growth. Describe the process?

Answer 10

FGF (Fgf 8)

• Cartilage are formed as continuous rods
• Selective Apoptosis
  produces bifurcations, segmentations&raquo_space; form joints

Question 11

Define the signalling pathway for Anterior- posterior patterning of digits in development?

Answer 11

Sonic Hedgehog (Shh) signalling

At the Zone of polarizing activity (ZPA)

Question 12

Describe how sonic-hedgehog determines the ant.-post. patterning of digits?

Answer 12

Shh = morphogen

Morphogen concentration gradient creates positional values / different thresholds

> > determine different cell fates along the gradient

Question 13

Describe how the Hedgehog protein is transported for signalling between cells?

Answer 13

1. Hh-sending cell: efflux Hh precursor bound to cholesterol (Hn-N)
2. Hh- receiving cell: Hh binds to Ptc (receptor for Hh)
3. Inhibited Ptc remove tonic inhibition on Smo (smoothen) receptor

> > Smo self-activate Gli complex and trigger signalling response

Question 14

Name the disease caused by abnormal anterior -posterior patterning of digits?

Answer 14

Greig cephalopolysyndactyly syndrome (many fingers)

Question 15

What pathway determines the dorsal-central positioning of limb formation?

Answer 15

WNT (Wnt7a > Lmx1b):

e.g. determine back of hand vs. palm

Question 16

List the sequence of events in endochondral bone formation?

Answer 16

1. Cartilage template
2. Chondrocyte hypertrophy
3. Cartilage mineralization
4. Vascular invasion
5. Cartilage replacement with bone
6. Growth plate cartilage
7. Linear growth of long bones

Question 17

Describe the overall action of FGF signalling on the growth of long bones?

Answer 17

Fibroblast growth factors (FGFs) = ligand, negative regulators/inhibitors of proliferating chondrocyte at Growth Plates**

not the usual role of FGF causing cell proliferation

Question 18

Define the type of receptor of FGF-R?

Answer 18

receptor tyrosine kinases

Question 19

Which type of FGF receptor is most important in regulating proliferation of growing long bones?

Answer 19

FGFR3 = most important in proliferating zone

Question 20

Which protein is required to act with FGF3 on growth plates of bones?

Answer 20

heparan sulphate proteoglycan (HSPG)

Question 21

Describe the FGF intracellular signalling events that occur in proliferative chondrochytes?

Answer 21

1. FGF bind to FGFR (tyrosine kinase receptor)
2. FGFR dimerize to activate kinase activity
3. Phosphorylate STAT1 and activate transcription of p21-CIP1
4. Binds CDK4, 2 complexes > inhibit cyclin D, E dependent kinases
5. Inhibit cell cycle porgression from G1 to S

Question 22

List the 3 different modes of FGFR3 mutation?

Answer 22

3 gain of function mutations:

1. Transmembrane domain mutation = over-stabilize dimerization of FGFR
2. Mutation forms permenant disulfide bond between 2 FGFR = Ligand independent activation
3. Mutation in kinase domain = self-activate, ligand-independent

Question 23

Summarize the bone developmental outcomes of FGFR3 mutation?

Answer 23

All gain of function mutations» increase cell cycle inhibition on proliferating chondrocyte:

• Retard bone growth
• more inhibition on hypertrophic differentiation = lower osteogenesis

Question 24

Define the exact mutation in FGFR that causes Achondroplasia?

Answer 24

G380R (>97%) mutation in the transmembrane domain of FGFR3

Question 25

List the factors that determine linear bone elongation rate in development?

Answer 25

Parathyroid hormone (PTH)

PTHrP (PTH-related protein)

Indian hedgehog (Ihh)

Question 26

Function of Indian hegdehog? Where is it expressed in growing bones?

Answer 26

 Stimulates chondrocyte proliferation
 Prevents chondrocyte hypertrophy

Expressed in growth plate (restricted to prehypertrophic cells) **

Question 27

Describe the short range effects of Indian hedgehog on bone growth?

Answer 27

Indian hedgehog = morphogen with concentration gradient

Short range: Highest concentration» bind to Ptc&raquo_space; activate Smoothen (Smo)&raquo_space; activate Gli complex for PROLIFERATION

Question 28

Describe the long range effects of Indian hedgehog on bone growth?

Answer 28

Long range: low concentration at perichondrium&raquo_space; activate PTHrP&raquo_space; PTHrP diffuse back to prehypertrophic cells (express PTHrP receptors)

> > SUPPRESS hypertrophic differentiation.

Negative feedback regulation on proliferating chondrocytes in growth plate

Question 29

Outcome of over-inactivation of PTHrP and PTHrP-R in prehypertrophic cells of growing bone?

Answer 29

Inactivate PTHrP-R, PTHrP:

Less negative feedback on hypertrophic differentiation

> > cells hypertrophy faster than rate of proliferation

> > Reduced number of proliferating cells

> > Bones become shorter

Question 30

Outcome of over-Activation of PTHrP and PTHrP-R in prehypertrophic cells of growing bone?

Answer 30

Activation = increase inhibition on hypertrophic differentiation at prehypertrophic cells on growing bone = less osteogenesis

= Retard growth