1. Protein kinase A signalling via CREB controls myogenesis
induced by Wnt proteins
Chen et al, 2005, Nature

2. Myogenesis (D) (V) Sclerotome → vertebrae, ribs
Myotome → skeletal muscles
Medial myotome → dorsal trunk muscles
Lateral myotome → ventral limb muscles
Dermatome → dermis (skin)
Pax3

3. Wnt & Myogenesis
The EMBO Journal, review, 1999

4. The EMBO Journal, review, 1999
Delivery of an activated form of beta-catenin to somitic mesoderm mimics the effects of Wnt-1, demonstrating that Wnt-1 likely acts directly on somitic mesoderm, and not through adjacent tissues via an indirect signal relay mechanism. (Capdevila et al, 1998, Dev Biol.)

5. CREB signaling PKA : cAMP-dependent Protein Kinase A
CRE : cAMP Responsive Element
CREB : CRE Binding protein (basic leucine zipper protein)

7. Immunohistochemistry
E 9.5 day
Immunohistochemistry
Psm : Presomitic mesoderm sI : first forming somite sV : fifth forming somite
nt : neural tube dsom : dorsal somitic cells scl : sclerotome

8. Psm : Presomitic mesoderm
tb : tail bud.
E9.5 mouse embryo.

9. E 10.5 day drg : dorsal root ganglion nc : notochord my : myotome
dm : dermomyotome

10. Whole-mount In Situ Hybridization
Shh
Pax1
CREB null mutant : Wnt1-/-, Wnt3a-/- embroys

11. Immunohistochemistry Northern analysis
ATF-1 (activating transcription factor-1), CREM (CRE modulator)
: CREB family members

12. Microinjection ACREB(acidic-CREB) : dominant-negative inhibitor of
all CREB family members
Microinjection
: GFP(control) &
ACREB-expressing adenovirus
fl : forelimb

13. co-culture RT-PCR : psm(presomitic mesoderm) with B1a, Wnt1&7a-B1a
ACREB
: inhibitor of CREB
C : positive control
cDNA from
E10.5 embryos
Actb(ß-actin)
: normalization control
Canonical wnt
target gene

14. Immunohistochemistry : P-CREB CRE-Luc reporter assay
10T1/2 : myogenic potential
C57MG : Wnt-responsive
ddA : adenylyl cyclase inhibitor
dnP : dominant-negative form of PKA
wnt
CREB P
CRE
Luc
psm cells
P-CREB staining

15. co-culture : psm/ B1a → RT-PCR
CTX, PTX : AC stimulator
M7(Mas7) : Gαi(inhibitory subunit) stimulator
M17(Mas17) : M7 inactive analogue
ddA : AC(adenylyl cyclase) inhibitor
fsk(forskolin) : AC activator
dbc : cAMP activator
TPA : PKC activator
dnP : PKA inhibitor

16. Microinjection (in vivo)
PKA inhibitor
PKA activity is required for Wnt-mediated myogenesis in vitro & in vivo

17. (Wnt-1 induced secreted protein)
Discussion
ß-catenin
Cytoplasmic localization
cAMP accumulation
PKA activation
CREB phosphorylation
WISP-1 transcription ↑
(Wnt-1 induced secreted protein)
cf)
Lifeng Xu et al, 2000, Genes & Dev
Pourquié et al, 2005, nature <news and views>