9. MECHANISMS OF DEVELOPMENT I – REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH

FACTORS (FGF)

4 receptors: FGFR1-422 ligands: FGF1-23

AT THE BEGINNING THERE WAS VITAMIN A……..

RALDH2- retinaldehyde dehydrogenase 2

DNA binding domain derived from the prototype gene called transcription factor T. Limb identity factors Tbx4 (hindlimb) and Tbx5 (forelimb)

………LATER CAME TBX

Tbx5

Tbx4

Limb bud induced by implantation of FGF-bead

Tbx5

Tbx4

wt Fgf10-/- wt Fgf10-/-

FGF10 proliferation in the mesoderm limb bud growth……..FOLLOWED BY FGF

Fgf8

Fgf8/Fgf4-/-wt Fgf8/Fgf4-/-wt

How do the limbs grow?

resting cartilage

bone

proliferating cartilage

proliferating cartilage

age

resting cartilage

FGFR3

Fgfr3+/-

Fgfr3+/-

Fgfr3-/-

Tail

Fgfr3-/-

FGFR3-related skeletal dysplasia

HypochondroplasiaAchondroplasiaSADDANThanatophoric Dysplasia

STATURE AC

TM

TK

I

II

III

FGF binds here

FGFR3-related skeletal dysplasia

Achondroplasia

Thanatophoric Dysplasia

- short long bones

- brachydactyly

- macrocephaly

- low nasal bridge

- spinal stenosis

- temporal lobe malformations

healthy

TD

FGF4 FGF4 normal

Fgfr3+/-

Loss-of-function vs. Gain-of-function

Fgfr3-/-

Fgfr3+/+

Fgfr3TD/+

healthy TD

resting

proliferating

hypertrophic

bone

0.01 0.1 1 10 1000

5e+4

1e+5

2e+5

2e+5FGF1 FGF1 + HeparinFGF2FGF9ControlHeparin

3 H-t

hym

idin

e (

cpm

x 1

03 )

0 10 20 30 400

20

40

60

80

100

G0-G1SG2-M

FGF2 (h)

% o

f ce

lls

FGF concentration (ng/ml)

FGFR3 inhibits chondrocyte proliferation by arresting

their cell cycle in G1 phase

control FGF

FGFR3 alters the cartilage-like phenotype of chondrocytes

FGF (h)

- 0.5 1 4 8 12 24 48 72

aggrecan

collagen II

collagen I

GAPDH

proMMP2

……via MMP-mediated degradation of extracellular matrix

MMP2

MMP3

MMP9

MMP10

MMP13

GAPDH

con

trol

FG

F2

proMMP9 matureMMP9

intermediate matureMMP2

proMMP13 matureMMP13

con

trol

FG

F2

proMMP2

mature MMP3/10

pro MMP3/10

FGFR3 inhibits cartilage growth via Erk pathway

C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h

FGF2

P-Erk1/2

Erk1/2

P-p38

p38

P-PLC1 PLC1

0.001 0.01 0.1 1 10 100

0

20

40

60

80

.001 .01 .1 1 10 100Inhibitor concentration (M)

U0126

cel

ls/w

ellx

103

80

60

40

20

0

0

20

40

60

80

control f1 f10 f100

Series1Series2

cel

l co

lon

ies/

100

cells

(%

)

RasRasN17

- F1 F10 F100

FGFR3

Ras

Raf-1

MEK

Erk

FRS2

FGF2

C-type Natriuretic Peptide (CNP) over-expression results in skeleton overgrowth in mice

CNP

wild-type

CNP over-expression??? CNPwild-type

wild-type

Fgfr3Ach

Fgfr3Ach/CNP

CNP rescues dwarfism caused by ACH mutation in FGFR3

Series2

Series3

control

FGF2

FGF2

control

_ 0.1 0.2 0.5 1 _ _ _ _ CNP [M] _ _ _ _ _ 10 100 200 500 pCPT-cGMP [M]

50

40

30

20

10

0

cells

per

wel

l [x1

04]

CNP counteracts FGF2-mediated chondrocyte growth arrest through cGMP-dependent pathway

CNP antagonizes FGF2-mediated loss of cartilage extracellular matrix in chondrocytes

control

CNP

pCPT-cGMP

unstimulated FGF2

CNP inhibits Erk MAP kinase module at the Raf level

FGFR3

Ras

CNP

cGMP

PKG Raf-1

MEK

Erk

NP-R FRS2

STOP

FGF2

Growth arrest

Matrix degradation

FGFR3 associates with STAT1 and acts as STAT1-kinase in chondrocytes

CKI

FGFR3

STAT1

Growth arrest

?

?

?

FLAG

actin

FLAG IP: FLAG

WB STAT1

FG

FR

3-w

t

FG

FR

3-K

650M

FG

FR

3-K

508M

GF

P

FGFR3-wt

substrate: STAT1 + + + + + +

SU5402(M) _ + _ _ + _

ATP + + _ + + _

FGFR3-K650E

P-Y701-STAT1

STAT1

FGFR3

Chronic FGF stimulus inhibits cytokine/STAT signaling in

chondrocytes

STAT3-YFP DAPI merge DIC/merge

control

IL6 30mFGF2 48h

FGF2 2h

IL6 30m

FGF2 48h

STAT3

STAT3-YFP

75

150kDa

100 IL11, LIF

CishSocs1Socs3

FGF2

IL6

IL6RA Shp2

gp130

STAT3

STAT3

nucleus

FGFR3

Frs2

FGFR3 causes premature senescence in

chondrocytes

SA--gal

control FGF2 FGF2 [ng/ml] - 5 10 20

0

12

24

36

48

SA

--g

al p

osit

ive c

ells

per

50 c

ells [

n=

10]

FGFR3 signals towards the cytoskeleton in

chondrocytes

Phalloidin DAPI merge

control

FGF2

actin

FGF2: C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3

-catenin

FGFR3 signals via Wnt pathway in chondrocytes