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Protein kinase A signalling via CREB
controls myogenesis
induced by Wnt proteins
Chen et al, 2005, Nature
Myogenesis
(D)
(V)
Sclerotome → vertebrae, ribs
Myotome →
Pax3
skeletal muscles
Medial myotome → dorsal trunk muscles
Lateral myotome → ventral limb muscles
Dermatome → dermis (skin)
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Wnt & Myogenesis
The EMBO Journal, review, 1999
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.)
CREB signaling
PKA : cAMP-dependent Protein Kinase A
CRE : cAMP Responsive Element
CREB : CRE Binding protein (basic leucine zipper protein)
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
Psm : Presomitic mesoderm
tb
: tail bud.
E9.5 mouse embryo.
E 10.5 day
drg : dorsal root ganglion
nc : notochord
my : myotome
dm : dermomyotome
Whole-mount In Situ Hybridization
Shh
Pax1
CREB null mutant : Wnt1-/-, Wnt3a-/- embroys
Immunohistochemistry
Northern analysis
ATF-1 (activating transcription factor-1),
CREM (CRE modulator)
: CREB family members
ACREB(acidic-CREB)
: dominant-negative inhibitor of
all CREB family members
Microinjection
: GFP(control) &
ACREB-expressing adenovirus
fl : forelimb
co-culture
: psm(presomitic mesoderm) with B1a, Wnt1&7a-B1a
RT-PCR
ACREB
: inhibitor of CREB
C : positive control
cDNA from
E10.5 embryos
Actb(ß-actin)
: normalization control
Canonical wnt
target gene
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
P
CREB
CRE
psm cells
P-CREB staining
Luc
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
dnP
: PKC activator
: PKA inhibitor
Microinjection (in vivo)
PKA inhibitor
PKA activity is required for Wnt-mediated myogenesis in vitro & in vivo
Discussion
cf)
ß-catenin
Cytoplasmic localization
cAMP accumulation
PKA activation
CREB phosphorylation
WISP-1 transcription ↑
(Wnt-1 induced secreted protein)
Lifeng Xu et al, 2000, Genes & Dev
Pourquié et al, 2005, nature <news and views>