3. DEVELOPMENT OF MUSCLE, CARTILAGE AND BONE



cartilage/bone
/ myotome                                    muscle


   Limb bud (myotome cells in purple)



      AT THE BEGINNING THERE WAS VITAMIN A……..
Nature. 1992 Jan 23;355(6358):352-3.
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

AER
RA           Hox6 (limb field)           Tbx (limb identity)           limb growth/patterning


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

Fgf8



………FOLLOWED BY SHH
SHH function:  normal     attenuated          lost
Nature 418, 979-983 (29 August 2002)




Fgf8/Fgf4-/-
wt
Fgf8/Fgf4-/-
wt
………FOLLOWED BY MORE FGF
  Gail Martin




MyoD+/- Myf-5+/-
MUSCLE DIFFERENTIATION BY BASIC HELIX-LOOP-HELIX (bHLH) FACTORS
MyoD-/- Myf-5-/-
a-actin IHC
(smooth and striated muscle fibers)
desmin IHC
(skeletal muscle fibers, myoblast-like cells)

                 CARTILAGE DIFFERENTIATION BY SOX9 (Sry-Box9)
wt
Sox9+/-
Sox9-/-
  alcian blue
collagen type II

BONE DIFFERENTIATION BY CBFA1 (RUNX2)
           Runx2 (Runt-related transcription factor 2)
Runx2+/-
Runx2-/-
 wt

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




wt
Ihh-/-
wt
Ihh-/-
Indian hedgehog (Ihh)

Coll type X in situ
Parathyroid hormone-related peptide (PTHrP)
wt
Pthrp-/-
wt
Pthrp-/-
wt
Pthrp receptor
             Sternal cartilage







Vegfa-/-
wt


uORF
Neor inserted here
Vegf transcription
uORF
Vegf ORF
uORF
Neor
Vegf         OK
 uORF
Vegf ORF

           C-type Natriuretic Peptide (CNP)
CNP
wild-type
CNP over-expression???
CNP
wild-type

Fgfr3+/-
Fgfr3-/-
 Chuxia Deng

WHEN SOMETHING GOES WRONG WITH FGF4
FGF4
FGF4 normal

Hypochondroplasia
Achondroplasia
SADDAN
Thanatophoric Dysplasia
STATURE
AC
TM
TK
I
II
III
FGF binds here
Leslie Thompson      William Wilcox
WHEN SOMETHING GOES WRONG WITH FGFR3

Achondroplasia
TD
normal
Thanatophoric dysplasia

wild-type
Fgfr3Ach
Fgfr3Ach/CNP
CNP rescues dwarfism caused by ACH
Kazuwa Nakao
Nature Medicine  10, 80 - 86 (2004)

CNP and FGFR3 pathways interact to maintain normal growth
      FGFR3
    Ras
CNP
      cGMP
      PKG
      Raf-1
    MEK
    Erk
NP-R
    FRS2
STOP
          FGF4
     GROWTH




























XY
CAMPOMELIC DYSPLASIA
   Sox9 haploinsufficiency
WHEN SOMETHING GOES WRONG WITH SOX9

………. OR WITH SHH
POLYDACTYLY TYPE-A
Loss-of-function mutation in
Gli3 (negative regulator of SHH)
POLYDACTYLY TYPE-II
SHH upregulation via transcriptional enhancer mutation

DD
normal
perlecan-/-
wt
………. OR WITH PERLECAN (Dyssegmental dysplasia - perlecan loss-of-function)

………. OR WITH ANOTHER 140 OR SO GENES