Protein kinase A signalling via CREB controls myogenesis

induced by Wnt proteins

Chen et al, 2005, Nature

Myogenesis

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Sclerotome → vertebrae, ribs

Myotome → skeletal muscles

Medial myotome → dorsal trunk muscles

Lateral myotome → ventral limb muscles

Dermatome → dermis (skin)

(V)

(D)

Pax3

The EMBO Journal, review, 1999

Wnt & Myogenesis

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

CRE : cAMP Responsive Element

PKA : cAMP-dependent Protein Kinase A

CREB : CRE Binding protein (basic leucine zipper protein)

E 9.5 day

Psm : Presomitic mesoderm sI : first forming somite sV : fifth forming somitent : neural tube dsom : dorsal somitic cells scl : sclerotome

Immunohistochemistry

E9.5 mouse embryo.

Psm : Presomitic mesodermtb : tail bud.

E 10.5 day drg : dorsal root ganglion nc : notochordmy : myotome dm : dermomyotome

CREB null mutant : Wnt1-/-, Wnt3a-/- embroys

Whole-mount In Situ Hybridization

Shh

Pax1

ATF-1 (activating transcription factor-1), CREM (CRE modulator) : CREB family members

Immunohistochemistry Northern analysis

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

ddA : adenylyl cyclase inhibitordnP : dominant-negative form of PKA

10T1/2 : myogenic potentialC57MG : Wnt-responsive

CRE-Luc reporter assay

P-CREB staining

psm cells

wnt

CREBP

CRE Luc

CTX, PTX : AC stimulator

M7(Mas7) : Gαi(inhibitory subunit) stimulatorM17(Mas17) : M7 inactive analogue

ddA : AC(adenylyl cyclase) inhibitor

dnP : PKA inhibitor

fsk(forskolin) : AC activator

dbc : cAMP activator

TPA : PKC activator

co-culture : psm/ B1a → RT-PCR

PKA inhibitor

Microinjection (in vivo)

PKA activity is required for Wnt-mediated myogenesis in vitro & in vivo

Discussion

Pourquié et al, 2005, nature <news and views>

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