9. MECHANISMS OF DEVELOPMENT I – REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH FACTORS (FGF) 4 receptors: FGFR1-4 22 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 Hypochondroplasia Achondroplasia SADDAN Thanatophoric 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 FGF2 (h) FGF concentration (ng/ml) FGFR3 inhibits chondrocyte proliferation by arresting their cell cycle in G1 phase Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. 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 Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. proMMP2 ……via MMP-mediated degradation of extracellular matrix MMP2 MMP3 MMP9 MMP10 MMP13 GAPDH proMMP9 matureMMP9 intermediate matureMMP2 proMMP13 matureMMP13 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-PLCg1 PLCg1 .001 .01 .1 1 10 100 Inhibitor concentration (mM) U0126 80 60 40 20 0 Ras RasN17 - F1 F10 F100 Lower expression levels of FGFR3 than FGFR1 but all zones express same amounts. 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??? CNP wild-type wild-type Fgfr3Ach Fgfr3Ach/CNP CNP rescues dwarfism caused by ACH mutation in FGFR3 control FGF2 FGF2 control _ 0.1 0.2 0.5 1 _ _ _ _ CNP [mM] _ _ _ _ _ 10 100 200 500 pCPT-cGMP [mM] 50 40 30 20 10 0 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 FGFR3-wt substrate: STAT1 + + + + + + SU5402(mM) _ + _ _ + _ 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 30m FGF2 48h FGF2 2h IL6 30m FGF2 48h STAT3 STAT3-YFP 75 150kDa 100 IL11, LIF Cish Socs1 Socs3 FGF2 IL6 IL6RA Shp2 gp130 STAT3 STAT3 nucleus FGFR3 Frs2 FGFR3 causes premature senescence in chondrocytes SA-b-gal control FGF2 FGF2 [ng/ml] - 5 10 20 0 12 24 36 48 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 b-catenin FGFR3 signals via Wnt pathway in chondrocytes