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4 receptors: FGFR1-4 22 ligands: FGF1-23

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. ………LATER CAME TBX.

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4 receptors: FGFR1-4 22 ligands: FGF1-23

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  1. 9. MECHANISMS OF DEVELOPMENT I – REGULATION OF LIMB DEVELOPMENT BY FIBROBLAST GROWTH FACTORS (FGF)

  2. 4 receptors: FGFR1-4 22 ligands: FGF1-23

  3. AT THE BEGINNING THERE WAS VITAMIN A…….. RALDH2- retinaldehyde dehydrogenase 2

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

  5. Tbx5 Tbx5 Tbx4 Tbx4 Limb bud induced by implantation of FGF-bead

  6. ……..FOLLOWED BY FGF FGF10 proliferation in the mesoderm limb bud growth Fgf10-/- Fgf10-/- wt wt

  7. Fgf8

  8. Fgf8/Fgf4-/- Fgf8/Fgf4-/- wt wt

  9. How do the limbs grow? age resting cartilage proliferating cartilage bone proliferating cartilage resting cartilage

  10. FGFR3

  11. Fgfr3+/- Fgfr3-/- Fgfr3+/- Fgfr3-/- Tail

  12. FGFR3-related skeletal dysplasia STATURE I AC Hypochondroplasia Achondroplasia SADDAN Thanatophoric Dysplasia II FGF binds here III TM TK

  13. FGFR3-related skeletal dysplasia Achondroplasia

  14. Thanatophoric Dysplasia healthy - short long bones - brachydactyly - macrocephaly - low nasal bridge - spinal stenosis - temporal lobe malformations TD

  15. FGF4 FGF4 normal

  16. Fgfr3+/- Fgfr3+/+ Fgfr3-/- Fgfr3TD/+ Loss-of-function vs. Gain-of-function

  17. resting proliferating hypertrophic bone healthy TD

  18. FGFR3 inhibitschondrocyte proliferation by arresting their cell cycle in G1 phase 3H-thymidine (cpm x 103) % of cells FGF2 (h) FGF concentration (ng/ml)

  19. FGFR3 alters the cartilage-like phenotype of chondrocytes FGF (h) control FGF - 0.5 1 4 8 12 24 48 72 aggrecan collagen II collagen I GAPDH

  20. ……via MMP-mediated degradation of extracellular matrix control FGF2 control FGF2 MMP2 proMMP9 matureMMP9 MMP3 proMMP2 proMMP2 MMP9 intermediate MMP10 matureMMP2 MMP13 pro MMP3/10 mature MMP3/10 GAPDH proMMP13 matureMMP13

  21. FGFR3inhibits cartilage growth via Erk pathway FGF2 Ras RasN17 C1 C2 1’ 5’ 10’ 30’ 1h 2h 4h 8h P-Erk1/2 Erk1/2 cell colonies/100 cells (%) P-p38 - F1 F10 F100 p38 U0126 80 P-PLCg1 60 PLCg1 40 cells/wellx103 20 0 .001 .01 .1 1 10 100 Inhibitor concentration (mM)

  22. FGF2 FGFR3 FRS2 Ras Raf-1 MEK Erk

  23. C-type Natriuretic Peptide (CNP) over-expression results in skeleton overgrowth in mice wild-type CNP CNP over-expression??? wild-type CNP

  24. CNP rescues dwarfism caused by ACH mutation in FGFR3 wild-type Fgfr3Ach Fgfr3Ach/CNP

  25. CNP counteracts FGF2-mediated chondrocyte growth arrest through cGMP-dependent pathway 50 control 40 30 cells per well [x104] control 20 FGF2 10 FGF2 0 _ 0.1 0.2 0.5 1_ _ _ _CNP [mM] _ _ _ _ _ 10 100 200 500pCPT-cGMP [mM]

  26. CNP antagonizes FGF2-mediated loss of cartilage extracellular matrix in chondrocytes unstimulated FGF2 control CNP pCPT-cGMP

  27. CNP inhibits Erk MAP kinase module at the Raf level FGF2 CNP FGFR3 NP-R FRS2 cGMP STOP Ras PKG Raf-1 Growth arrest MEK Erk Matrix degradation

  28. FGFR3-K508M FGFR3-wt FGFR3-K650M GFP FLAG actin IP: FLAG FLAG WB STAT1 FGFR3-K650E FGFR3-wt substrate: STAT1++ ++ ++ SU5402(mM)_ +_ _ +_ ATP++_++_ FGFR3 associates with STAT1 and acts as STAT1-kinase in chondrocytes FGFR3 ? STAT1 ? ? CKI P-Y701-STAT1 Growth arrest STAT1 FGFR3

  29. 150kDa STAT3-YFP FGF2 100 IL6 IL11, LIF STAT3 75 FGFR3 IL6RA STAT3-YFP DAPI merge DIC/merge Shp2 Frs2 control gp130 FGF2 2h Cish Socs1 Socs3 STAT3 FGF2 48h nucleus IL6 30m FGF2 48h STAT3 IL6 30m Chronic FGF stimulus inhibits cytokine/STAT signaling in chondrocytes

  30. control FGF2 FGF2 [ng/ml] - 5 10 20 48 36 24 12 0 SA-b-gal FGFR3 causes premature senescence in chondrocytes SA-b-gal positive cells per 50 cells [n=10]

  31. FGFR3 signals towards the cytoskeleton in chondrocytes Phalloidin DAPI merge control FGF2

  32. FGFR3 signals via Wnt pathway in chondrocytes FGF2: C1 15‘ 1h 3h 6h 12h 24h 48h 72h C2 C3 b-catenin actin

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