Protein Kinase a Signalling Via CREB Controls Myogenesis Induced By

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...... staining was found in the dorsal somitic cells (Fig. 1a), where Pax3 expression was first upregulated (Fig. 1b). As somites are patterned Protein kinase A signalling via along the dorso-ventral axis to form the dermomyotome and CREB controls myogenesis sclerotome, we found strong P-CREB (Fig. 1c) and Pax3 (Fig. 1d) staining in the entire dermomyotome. As the myotome forms, high induced by Wnt proteins levels of P-CREB were found in cells at the medial and lateral dermomyotomal edges (Fig. 1e, f), where Myf5 (Fig. 1h) and MyoD Alice E. Chen1, David D. Ginty2 & Chen-Ming Fan1 (Fig. 1g) expression was initiated. As cells move into the myotome proper, where MyoD and Myf5 continue to be expressed, P-CREB 1Department of Embryology, Carnegie Institution of Washington, 115 West was downregulated to a level slightly above that of the sclerotome University Parkway, Baltimore, Maryland 21210, USA 2 (Fig.1e).ThelevelofP-CREBwasalsolowerinthemore Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins mature dermomyotome compared with earlier stages (compare University School of Medicine, Baltimore, Maryland 21205, USA ...... Fig. 1c and e). Thus, the dynamic patterns of P-CREB in the somite Select members of the Wnt family of secreted glycoproteins have coincide with the patterns of myogenic gene induction. been implicated in inducing the myogenic determinant genes These results led us to examine whether CREB null mutants14 2/2 2/2 Pax3, MyoD and Myf5 during mammalian embryogenesis1,2, but (CREB ) exhibit myogenic defects. CREB mice are born at the mechanism of induction has not been defined. We describe an reduced mendelian frequency14 (15%). We found that CREB2/2 unexpected role for protein kinase A (PKA) signalling via CREB mice were present at the expected 25% ratio between E9.5–E11.5, in this induction. Using a combination of in vitro explant assays, mutant analysis and gene delivery into mouse embryos cultured ex vivo, we demonstrate that adenylyl cyclase signalling via PKA and its target transcription factor CREB are required for Wnt-directed myogenic gene expression. Wnt proteins can also stimulate CREB-mediated transcription, providing evidence for a Wnt signalling pathway involving PKA and CREB. Our findings raise the possibility that PKA/CREB signalling may also contribute to other Wnt-regulated processes in embryonic patterning, stem cell renewal and cancer3. Mammalian trunk skeletal muscle is derived from the somites, which are mesodermal segments flanking the embryonic midline. Upon receiving inductive signals from the neural tube and surface ectoderm, myogenic precursors in the dorsal somite (the dermomyotome) first express Pax3. Subsequently, cells at the medial and lateral edges of the dermomyotome initiate expression of Myf5 and MyoD, respectively. These cells delaminate from the dermomyotome to form the myotome. The medial myotome gives rise to the deep back muscles and the lateral myotome gives rise to the limb and body wall muscles4. Pax3, Myf5 and MyoD are essential for initiation of the muscle programme5,6. Wnt proteins such as Wnt1 (expressed in the dorsal neural tube) and Wnt7a (expressed in the lateral surface ectoderm) can induce these myogenic determinant genes in vitro1,2,7. Although the canonical Wnt/b-catenin pathway is typically implicated in cell fate-specific gene activation, neither an activated form of b-catenin8 nor LiCl treatment (not shown), which mimics b-catenin activation, can initiate myogenesis. Non-canonical Wnt pathways, identified based on homology between the Frizzled family of Wnt receptors and heterotrimeric G protein- coupled receptors9, use G protein effectors including protein kinase C, Ca2þ and cyclic GMP. However, these effectors exert effects primarily on cell morphology and behaviour10–12 rather than gene expression. We established the in vivo relevance of adenylyl cyclase (AC) pathway activity in myogenesis by assaying for a downstream effector of this cascade, CREB, in embryonic regions undergoing myogenesis. CREB is the founding member of the CREB family of basic leucine zipper transcription factors (including ATF-1 and Figure 1 P-CREB staining patterns coincide with myogenic induction. CREM), which bind the cyclic AMP-responsive element (CRE) as Immunohistochemistry was performed on transverse sections of mouse embryos at E9.5 homodimers or heterodimers and activate transcription upon (a–d) and E10.5 (e–h). a, c, P-CREB and (b, d) Pax3 staining in the first forming (sI) and phosphorylation by PKA13. Embryonic regions where CREB is fifth (sV) somites, dorsal somitic cells (dsom) and dermomyotome (dm). e–h, Staining at phosphorylated on its transcriptional regulatory site, Ser 133 (P- forelimb (fl) level. e, P-CREB correlates with regions patterned by Wnt proteins, for CREB), may represent potential areas of PKA activity. We per- example, the dermomyotome edges, dorsal neural tube (nt), neural crest (nc), dorsal root formed immunohistochemistry on mouse sections at embryonic ganglion (drg) and limb mesenchyme (lm). P-CREB (magnified in f) overlaps with MyoD day 9.5 (E9.5) and E10.5 using antibodies against CREB and expression (g) at the lateral (lat) edge and Myf5 expression (h) at the medial (med) edge. P-CREB. In contrast to the ubiquitous expression of CREB (see Open and black arrowheads indicate reduced P-CREB levels in the myotome (my) and Supplementary Fig. S1), P-CREB was detected in myogenic regions dermomyotome, respectively. Red blood cells (rbc) and a few sclerotome (scl) cells are of the somite (Fig. 1). At the time of somite formation, P-CREB P-CREB-positive (c), significance unknown.

letters to nature but by E12.5 approximately half of them died, indicating that Pax3 expression was found in the dermomyotome of all somites lethality occurs between E11.5 and E12.5. At E10.5, CREB2/2 (CREB2/2M, n ¼ 3; CREB2/2S, n ¼ 3). Strong expression of embryos existed as two populations, distinguishable by gross MyoD in tightly clustered cells was found in the lateral myotome morphology: mild ‘CREB2/2M’ (11%) and severe ‘CREB2/2S’ of wild-type embryos (n ¼ 6; Fig. 2h). Mutants had reduced and (15%) mutants (n ¼ 88). CREB2/2M embryos were comparable punctate MyoD expression, which was restricted to the more mature in size to wild-type littermates, whereas CREB2/2S embryos were anterior somites (CREB2/2M, n ¼ 5; CREB2/2S, n ¼ 4; Fig. 2j, l). ,35% smaller. In CREB2/2S somites, the rate of cell proliferation In wild-type embryos, the Myf5 expression domain formed a well was decreased by ,30%, but no increase in the rate of apoptosis was defined flat medial edge (n ¼ 3; Fig. 2n). In the mutant embryos, observed (see Supplementary Fig. S2). Myf5 expression at the medial edge appeared disorganized and Whole-mount in situ hybridization (WISH) was employed to pointed in shape (CREB2/2M, n ¼ 3; CREB2/2S, n ¼ 3; Fig. 2p, r). analyse wild-type, CREB2/2M and CREB2/2S embryos at E10.5 The myotome proper also had many fewer Myf5-positive cells. (embryos of similar size and stage were chosen for normalization of Interestingly, the Pax3 and Myf5 defects observed in the CREB analyses, Fig. 2a–t). The domain of Pax3 expression appeared mutants are reminiscent of those found in Wnt1 2/2 Wnt3a 2/2 smaller in the mutants compared with the wild type (n ¼ 3; embryos15. We also noted that Myf5 expression was not found at the compare square brackets in Fig. 2b, d, f). Despite its reduced levels, cervical somites of CREB mutants, suggesting an anterior-specific

Figure 2 CREB mutants have myogenic defects. a–t, WISH analysis of Pax3 (a–f, trunk adjacent transverse sections at the forelimb level of E10.5 wild-type (upper row) and enlarged in b, d, f), MyoD (g–l, trunk enlarged in h, j, l), Myf5 (m–r, trunk enlarged in n, CREB2/2M (lower row) mice. Brackets in u, v denote dermomyotome (dm) size, p, r) and Pax1 (s, t) in E10.5 wild-type (þ/þ) (top row), CREB2/2M (middle row) and arrowheads in w, x show MyoD-positive cells; myotome (my), neural tube (nt). a 0 , CREB2/2S (bottom row) mice. Box in a shows the enlarged area. Square brackets (b, d, f) Northern analysis using ATF-1 and CREM probes on total RNA of wild-type, CREBþ/2, indicate Pax3 domain size. Arrowheads (h, j, l) indicate MyoD expression. Square bracket CREB2/2M and CREB2/2S mice. 28S RNA reflects sample loads. Signals are shown (n) and angled brackets (p, r) compare the shape of Myf5-positive medial edges (enlarged as an X-ray film image (bottom); quantified and plotted as fold differences relative to wild- in insets). Black lines indicate expression boundaries along the anterior-posterior axis, type levels (top). forelimb (fl), hindlimb (hl). u–z, Immunohistochemistry of Pax3, MyoD and Myf5 on

letters to nature requirement for CREB function. Importantly, the defects of MyoD ATF-1 and CREM contributes to the milder myogenic defects and Myf5 expression were more pronounced in CREB2/2S than in observed in CREB2/2M embryos. CREB2/2M embryos. These defects were not due to alterations in We therefore asked whether inhibition of all CREB family Wnt1 or Wnt7a, as their expression was normal in CREB mutants members in the somite would block myogensis in vivo. To do this, (see Supplementary Fig. S3). Finally, we observed no obvious we generated an adenovirus (Ad) harbouring an expression cassette change in Pax1 (a sclerotome marker) expression in the somite of for a dominant-negative inhibitor of CREB, acidic-CREB (ACREB). CREB2/2S embryos (n ¼ 3; compare Fig. 2s and t), indicating that ACREB contains an acidic amphipathic extension fused to the the somitic defects are specific to the myogenic lineages. amino terminus of the CREB leucine zipper16 andactsasa These defects were substantiated by analysing Pax3, MyoD and dominant-negative for all CREB family members by interfering Myf5 expression by immunohistochemistry on sectioned E10.5 with their binding to the CRE13. We employed microinjection to wild-type and CREB mutant embryos (Fig. 2u–z). At the forelimb deliver control and ACREB-expressing adenoviruses, both of level of CREB2/2M embryos, the size of the Pax3-positive domain which carry a green fluorescent protein (GFP) expression cassette was reduced. Only a few weakly MyoD-positive cells at the lateral (Ad-GFP and Ad-ACREB, respectively), into E9.75 somites between edges were detected. Myf5-positive cells in the medial edge and the forelimb and hindlimb on the right side of the embryo, leaving myotome proper were reduced and disorganized. Even fewer Pax3-, the left side as an uninjected internal control (Fig. 3a, see Sup- MyoD- and Myf5-positive cells were found in CREB2/2S embryos plementary Video S4). Embryos were then cultured overnight in a (not shown). Together, these studies reveal that CREB mutants have rotating bottle culturing unit. defects in the medial and lateral edges as well as in the myotome Healthy embryos with robust adenoviral infection in the somite proper. (assessed by GFP, Fig. 3b) were selected for WISH analysis. Control The differential severity of myogenic defects in CREB2/2M and Ad-GFP injected into the somites did not affect Pax3 (n ¼ 6), MyoD CREB2/2S embryos suggests the possibility of differential compen- (n ¼ 7) or Myf5 (n ¼ 6) expression (Fig. 3c, e, g). In contrast, sation by ATF-1 and/or CREM. To test this, we examined ATF-1 myogenesis was inhibited in embryos injected with Ad-ACREB and CREM expression in wild-type, CREBþ/2, CREB2/2M and (Fig. 3d, f, h) as indicated by the severe reduction of Pax3 CREB2/2S mice by northern blot analysis (Fig. 2a 0 ). Compared (n ¼ 11), MyoD (n ¼ 10) and Myf5 (n ¼ 13) expression. Somites with wild-type embryos, both CREBþ/2 and CREB2/2S embryos anterior and posterior to the injected region showed normal expressed twofold higher levels of CREM, whereas CREB2/2M expression of all three genes. The myogenic defects in Ad-ACREB embryos expressed ,2.5-fold higher levels of ATF-1 and about infected somites were more severe than those in the CREB mutant, fourfold higher levels of CREM. We propose that upregulation of supporting a redundant function among CREB family members. As Pax3 and Myf5 are already expressed at E9.75 (refs 17, 18), we suggest that CREB participates in their maintenance. Pax1 expression was not altered by ACREB (see Supplementary Fig. S5). Together, these data indicate that CREB-related function is required selectively for myogenic gene expression in vivo. We next addressed how CREB fits into the existing model of myogenic induction. In addition to Wnt1 and Wnt7a, Shh has also been implicated in promoting Myf5 expression1,19.However, because CREB mutants do not have defects in Pax1,whose expression depends on Shh signalling, it seems unlikely that CREB mediates Shh signalling. We therefore tested whether CREB mediates Wnt1- and Wnt7a-induced myogenesis by taking advan- tage of an in vitro system in which myogenic induction by Wnt can be directly assayed. Explants of E9.5 mouse presomitic mesoderm (psm) were co-cultured with control RatB1a cells (B1a), or B1a cells stably expressing Wnt1 (Wnt1-B1a) or Wnt7a (Wnt7a-B1a) in collagen gels2. Expression of Pax3, MyoD and Myf5 was assayed 48 h after culture by radioactive in situ hybridization (RISH) (Fig. 4a) or semiquantitative polymerase chain reaction with reverse transcription (RT–PCR, Fig. 4b). Although both Wnt proteins activated all three myogenic genes, Wnt1 preferentially activated Myf5 and Wnt7a preferentially activated MyoD, consistent with previous reports1. Infection of explants with Ad-ACREB repressed Wnt1- and Wnt7a-mediated myogenesis (Fig. 4c). At low doses, downregula- tion of MyoD was observed. At higher doses, Myf5 expression was repressed and Pax3 expression was reduced. Infection with the control Ad-GFP had no effect. These results demonstrate that CREB-related activity is required for Wnt-induced myogenesis in vitro. Furthermore, different thresholds of CREB activity differ- entially affect Pax3, MyoD and Myf5 expression. ACREB did not Figure 3 Functional elimination of the CREB family inhibits myogenesis in vivo. affect targets of the canonical WNT/b-catenin pathway (Tcf1 and a, GFP-tagged adenoviruses were microinjected into trunk somites at E9.75. b, GFP mTroy20), indicating the specificity of myogenic inhibition. expression (enlarged in inset) in somites following culture (right); brightfield image of To address whether CREB acts downstream of Wnt, we used same embryo (left). WISH analysis of Pax3 (c, d), MyoD (e, f) and Myf5 (g, h) in embryos immunohistochemistry to identify whether CREB is phosphoryl- injected with Ad-GFP (GFP) (c, e, g) or Ad-ACREB (ACREB) (d, f, h). Somites on the right ated in psm co-cultured with Wnt1-B1a and Wnt7a-B1a cells. We side were injected (region of injection located between arrowheads). Uninjected sides and observed moderate levels of P-CREB staining in psm cells from somites anterior and posterior to the injected regions (open arrowheads) retain normal Wnt1-B1a co-cultures (n ¼ 5, Fig. 4f) and high levels of P-CREB in gene expression. cells from Wnt7a-B1a co-cultures (n ¼ 5, Fig. 4g). In contrast, only

letters to nature a few scattered P-CREB-positive cells were present in psm alone infection of 10T1/2/CRE-Luc cells with Ad-ACREB but not (n ¼ 5, Fig. 4d) and psm/B1a co-cultures (n ¼ 5, Fig. 4e). These Ad-GFP blocked reporter induction by both Wnt proteins. In data suggest that Wnt1 and Wnt7a can activate CREB phosphoryl- contrast, inhibitors of PKC, calcium/calmodulin-dependent protein ation in psm cells in vitro. kinase II or MAP kinase (also known to converge on CREB) had We then used cultured cell lines to test if Wnt could induce mild or no effect. Wnt-induction of CRE-Luc activity was CREB-mediated transcription. C57MG (known to be Wnt-respon- also independent of other Wnt pathways involving activation sive21) and 10T1/2 (known to have myogenic potential22) cells of b-catenin or JNK (see Supplementary Fig. S6). Furthermore, carrying a CRE-luciferase (CRE-Luc) reporter were co-cultured CRE-Luc reporter activity was not induced by Shh in either of the with B1a, Wnt1-B1a or Wnt7a-B1a cells (Fig. 4h). The reporter cell lines (not shown). Finally, a Wnt11-B1a cell line, which cannot activity of C57MG/CRE-Luc cells was induced ,3.7-fold by activate MyoD and Myf5 in the psm but can activate the Rho Wnt1-B1a cells and ,4.3-fold by Wnt7a-B1a cells relative to pathway (not shown), failed to induce reporter activity in 10T1/2/ B1a cells (n ¼ 3, P , 0.005). Similarly, the reporter activity of CRE-Luc cells (n ¼ 3, P . 0.5, Fig. 4h). Together, these results 10T1/2/CRE-Luc cells was induced ,2.2-fold by Wnt1-B1a cells demonstrate that Wnt1 and Wnt7a activate CREB-mediated and ,3.7-fold by Wnt7a-B1a cells (n ¼ 3, P , 0.005). Activation of transcription via the AC pathway. the CRE-Luc reporter by both Wnt proteins appeared to be To address whether Wnt activates CREB directly, we treated psm primarily mediated by the AC cascade, because an inhibitor of cells with recombinant Wnt3a (rWnt3a) and assayed for P-CREB. AC, 2 0 -5 0 dideoxyadenosine (ddA), dramatically reduced reporter We observed CREB phosphorylation within 10 min of application. activity induced by Wnt1 and Wnt7a (Fig. 4h). Furthermore, This induction was dependent on AC and cAMP-dependent PKA

Figure 4 CREB acts downstream of Wnt signalling. a, RISH analysis of Pax3, MyoD and positive cells (open arrowheads). Wnt-B1a (f) or Wnt7a-B1a (g) co-cultures induce Myf5 in sections of psm co-cultured with B1a, Wnt1-B1a (Wnt1), or Wnt7a-B1a (Wnt7a) moderate and strong P-CREB staining, respectively (square brackets). Red dashed lines cells. Dashed lines represent the boundary between psm (right) and cell lines (left), pink outline borders (determined by morphology) between psm (right) and cell lines (left), black granules show the RISH signal. b, Lower panel shows RT–PCR of Pax3, MyoD and Myf5 in dashed line in f shows the outer edge of psm. P-CREB was also detected in cell lines. co-cultures, cDNA from E10.5 embryos (þC, positive control), no cDNA (-C, negative h, CRE-Luc reporter activities in co-cultures of 10T1/2/CRE-Luc or C57MG/CRE-Luc cells control) and Actb (normalization control). Pixel intensities of typical PCR products are with B1a, Wnt1-B1a, Wnt7a-B1a or Wnt11-B1a cells, with (þ) or without (2)30mM ddA within the linear range of ampliﬁcation (30 cycles) at noted amounts of cDNA inputs (upper treatment. Luciferase activities shown as fold induction relative to B1a co-cultures panel). Bars represent range of values (n ¼ 3). c, RT–PCR of Pax3, Myf5, MyoD, mTroy (mean ^ s.d.). (i) P-CREB staining of psm cells after 10 min treatments: control and Tcf1 in psm co-cultures infected with Ad-GFP (GFP) or increasing amounts of Ad- (carrier media alone), rWnt3a (100 ng ml21), rWnt3a plus ddA (25 mM) or rWnt3a plus ACREB (ACREB, relative amounts shown by open triangles). d–g, Immunohistochemistry Ad-dnP (dnP). of P-CREB in psm explants. Psm alone (d) and B1a cell co-cultures (e) have few P-CREB

letters to nature activity, because psm cells lacked P-CREB staining when treated ,2.3-fold and ,5.7-fold higher in forskolin-treated samples than with ddA or when infected with an adenovirus carrying a domi- in untreated samples. Pax3 expression was unaffected by forskolin nant-negative form of the PKA RIa regulatory subunit (dnP, which treatment. We observed similar results when we increased levels of cannot bind cAMP23; adenovirus Ad-dnP) (n ¼ 7, Fig. 4i). The cAMP (the downstream effector of AC) using either dibutryl-cAMP rapid kinetics of CREB phosphorylation by rWnt3a strongly suggest (dbc), a membrane-permeable analogue (n ¼ 3, Fig. 5d), or 3-iso- that CREB is a direct downstream effector of Wnt signalling via AC butyl-1-methyl-xanthine (IBMX), an inhibitor of cAMP and PKA in somitic cells. phosphodiesterase (not shown). In contrast, activation of PKC by On the basis of these ﬁndings, we propose that the AC signalling 12-O-tetradecanoylphorbol-13-acetate (TPA) (n ¼ 3, Fig. 5d) had cascade leading to CREB activation is required for Wnt-induced no effect. Together, these data show that although both Wnt myogenesis. To test this, we assessed the effects of pharmacological proteins require AC activity, they may differ in their capacity to reagents and adenoviruses that modulate components of this path- activate AC in the psm (Fig. 4f, g), providing a possible explanation way on psm/Wnt-B1a co-cultures. AC activity is regulated by for their preferential myogenic-inducing properties. two classes of G proteins: Gsa, an AC stimulator, and Gia,anAC We next asked whether blocking PKA activity would block inhibitor24. Increasing AC activity using either pertussis toxin (PTX, myogenesis. Infection of explant co-cultures with Ad-dnP, but not aGia inhibitor) or cholera toxin (CTX, a Gsa stimulator), enhanced control Ad-GFP, repressed both Wnt1- and Wnt7a-directed myo- Myf5 and MyoD expression induced by Wnt1 (n ¼ 5) but not by genic gene expression (Fig. 5e). Wnt1 and Wnt7a protein pro- Wnt7a (n ¼ 5) (Fig. 5a). Pax3 levels were relatively unaffected. duction and secretion by Wnt1-B1a and Wnt7a-B1a cells were not Conversely, decreasing AC activity using Mas7 (which is 30-fold affected by dnP, as veriﬁed by normal myogenic induction in co- more potent at activating Gia compared to Gsa, ref. 25) inhibited cultures of uninfected psm with Ad-dnP infected Wnt1-B1a or both Wnt1- and Wnt7a-induced myogenesis. This repression was Wnt7a-B1a cells (not shown). We did not detect Pax1 expression in not observed in co-cultures exposed to an inactive analogue, Mas17 these cultures, indicating that unlike what has been reported in the (Fig. 5b). Wnt1 and Wnt7a protein production was unchanged by neural tube26, the Shh pathway is not activated by dnP in the mouse Mas7 (not shown). These data imply that modulation of AC activity somitic mesoderm. Finally, consistent with Ad-ACREB injections, via Ga proteins (which probably couple to Frizzled receptors) may embryos injected with Ad-dnP also exhibited loss of Pax3, MyoD be a mechanism by which Wnt proteins signal. and Myf5 expression (n ¼ 12 each, Fig. 5f–h). These results together To test the contribution of AC directly, we applied ddA to the indicate that PKA activity is required for Wnt-mediated myogenesis psm explant co-cultures. ddA blocked Wnt1- (n ¼ 3) and Wnt7a- in vitro and in vivo. induced (n ¼ 3) myogenic gene expression (Fig. 5c). ddA did not Our data support a model in which Wnt-mediated myogenic affect Wnt1 and Wnt7a protein production (not shown). In con- gene expression is dependent on the activation of AC, PKA and trast, forskolin (an activator of AC) enhanced Wnt1- (Fig. 5d) but CREB. Consistent with our model, core CRE sites (CGTCA) are not Wnt7a- (not shown) mediated myogenesis. In psm/Wnt1-B1a present in the promoter regions of Myf5 and Pax3 genes. Whether co-cultures (n ¼ 6), Myf5 and MyoD expression were respectively CREB directly activates the myogenic genes remains to be deter-

Figure 5 G proteins, AC, cAMP and PKA mediate Wnt-induced myogenesis. a–e, RT–PCR untreated cells (2). e, Pax1 expression is not activated by dnP. These treatments have no analysis of Pax3, MyoD and Myf5 in psm co-cultures with B1a, Wnt1-B1a (Wnt1) and effect on B1a co-cultures. f–h, WISH analysis of Pax3, MyoD and Myf5 in Ad-dnP injected Wnt7a-B1a (Wnt7a) cells treated with (a) CTX (50 ng ml21) or PTX (75 ng ml21), (b) Mas7 embryos. Black arrowheads bracket targeted somites. Uninjected sides and somites (25 mM) or Mas17 (25 mM), (c) ddA (25 mM), (d) forskolin (fsk, 5 mM), dbc (1 mM) or TPA anterior and posterior to the injected regions (open arrowheads) retain normal gene (100 ng ml21) and (e) Ad-GFP (GFP) or Ad-dnP (dnP); control samples as in Fig. 4, expression.

letters to nature mined. The new link between Wnt and PKA/CREB signalling Reporter cell lines broadens the repertoire of downstream components used by Wnt 10T1/2 and C57MG cell lines each carrying a stably integrated CRE-Luc reporter were 21 proteins. As certain Wnt proteins are also known to participate in clonally selected with hygromycin (200 mgml ). For co-cultures, 10T1/2 or C57MG cells 3 were mixed (at 1:2 and 2:1 ratios, respectively) with B1a or Wnt-B1a cells. Luciferase cell proliferation and survival , they may utilize the PKA/CREB activity was determined as described28. The protein concentration used for normalization pathway to mediate these processes because these roles are well was determined using the BCA protein assay reagent (Pierce). 27 described for CREB . Given the diverse functions of the Wnt family Received 5 August; accepted 20 October 2004; doi:10.1038/nature03126. in vertebrate and invertebrate embryogenesis, our data provide the Published online 28 November 2004. basis for investigating a general role of PKA/CREB in Wnt-directed 1. Tajbakhsh, S. et al. Differential activation of Myf5 and MyoD by different Wnts in explants of mouse developmental programs. Our discovery may also encourage the paraxial mesoderm and the later activation of myogenesis in the absence of Myf5. Development 125, manipulation of PKA/CREB pathway components as a means to 4155–4162 (1998). modulate or re-direct Wnt-mediated stem cell renewal and 2. Fan, C. M., Lee, C. & Tessier-Lavigne, M. A role for WNT proteins in induction of dermomyotome. cancer. A Dev. Biol. 191, 160–165 (1997). 3. Moon, R., Bowerman, B., Boutros, M. & Perrimon, N. The promise and perils of Wnt signaling through b-catenin. Science 296, 1644–1646 (2002). Methods 4. Ordahl, C. (ed.) Somitogenesis Part 2 129–268 (Academic, Toronto, 2000). Explant culture 5. Rudnicki, M. et al. MyoD or Myf5 is required for the formation of skeletal muscle. Cell 75, 1351–1359 (1993). E9.5 CD1 mouse psm explants were cultured in collagen gel alone, or in combination with 6. Tajbakhsh, S., Rocancourt, D., Cossu, G. & Buckingham, M. Redefining the genetic hierarchies B1a, Wnt1-B1a or Wnt7a-B1a cells as described2, except that 2.5 ng ml21 basic fibroblast controlling skeletal myogenesis: Pax3 and Myf5 act upstream of MyoD. Cell 89, 127–138 (1997). growth factor (bFGF, Promega) was used. All cultures were 48 h. For rWNT3a application, 7. Parr, B., Shea, M., Vassileva, G. & McMahon, A. Mouse Wnt genes exhibit discrete domains of psm explants were plated on 8-chamber slides (Falcon) for 18 h in the presence or absence expression in the early embryonic CNS and limb buds. Development 119, 247–261 (1993). of pharmacological reagents or adenovirus, followed by 10 min of treatment with rWNT3a 8. Capdevila, J., Tabin, C. & Johnson, R. Control of dorsoventral somite patterning by Wnt-1 and (R&D Systems) or carrier media (control). Mas7, Mas17, CTX, PTX, dbc, IBMX, TPA and b-catenin. Dev. Biol. 193, 182–194 (1998). ddA were obtained from Calbiochem. Forskolin was obtained from Sigma. All reagents 9. Barnes, M., Duckworth, D. & Beeley, L. Frizzled proteins constitute a novel family of G protein- were directly applied to cultures. coupled receptors, most closely related to the secretin family. Trends Pharmacol. Sci. 19, 399–400 (1998). Adenovirus production þ 10. Kuhl, M., Sheldahl, L., Park, M., Miller, J. & Moon, R. The Wnt/Ca2 pathway: a new vertebrate Wnt 28 16 23 GFP adenovirus (Ad-GFP) was previously described . FLAG-tagged-ACREB and dnP signaling pathway takes shape. Trends Genet. 16, 279–283 (2000). expressing adenoviruses (Ad-ACREB and Ad-dnP, respectively) were produced using the 11. Huelsken, J. & Behrens, J. The Wnt signalling pathway. J. Cell Sci. 115, 3977–3978 (2002). AdenoEasy System (Qbiogene). Purified Ad-dnP and Ad-ACREB were verified to be 12. Wang, H. Y. & Malbon, C. Wnt-frizzled signaling to G-protein-coupled effectors. Cell. Mol. Life Sci. 8 10 functional. 10 –10 plaque-forming units were added to explant culture media to achieve 61, 69–75 (2004). ,100% infection, as visualized by GFP fluorescence. 13. Shaywitz, A. & Greenberg, M. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821–861 (1999). Semi-quantitative RT–PCR 14. Rudolph, D. et al. Impaired fetal T cell development and perinatal lethality in mice lacking the cAMP Total RNA from the explants was isolated using RNasol and analysed by RT–PCR with response element binding protein. Proc. Natl Acad. Sci. USA 95, 4481–4486 (1998). 30–32 cycles of amplification. For primer sequences, see khttp://www.ciwemb.edu/labs/ 15. Ikeya, M. & Takada, S. Wnt signaling from the dorsal neural tube is required for the formation of the fan/index.htmll. PCR products were resolved on 2% agarose gels and visualized by medial dermomyotome. Development 125, 4969–4976 (1998). ethidium bromide staining. Images were captured using a UVP 7500 Gel Documentation 16. Ahn, S. et al. A dominant-negative inhibitor of CREB reveals that it is a general mediator of stimulus- System and quantified using Image-Quant v.1.2 (Amersham Pharmacia). Each marker dependent transcription of c-fos. Mol. Cell. Biol. 18, 967–977 (1998). was assayed in an independent PCR reaction per cDNA sample. Quantified PCR products 17. Ott, M., Bober, E., Lyons, G., Arnold, H. & Buckingham, M. 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Supplementary Information accompanies the paper on www.nature.com/nature. Hybridization signals were quantified using a phosphoimager and ImageQuant software.

Embryo injection and culture Acknowledgements We thank D. Koshland, Y. Zheng, A. Fire, L. Buttitta and M. Tessier-Lavigne for critical reading of this manuscript and C. Pratt for graphics assistance. D.D.G. is an Whole E9.75 CD1 embryos were collected in L-15/5% heat inactivated horse serum investigator of the Howard Hughes Medical Institute. (Invitrogen) for injection. Purified adenoviruses were injected into somites (4.6–9.2 nl per somite) using a Nanoinjector (Drummond Scientific). Following injection, embryos were transferred to a rotating bottle culture unit (BTC Engineering) and cultured overnight in Competing interests statement The authors declare that they have no competing financial media (2 ml per embryo) consisting of 100 units ml21 penicillin, 100 mgml21 interests. streptomycin, 50% DMEM/F12 (Invitrogen) and 50% freshly isolated, sterile-filtered rat serum, under humidified conditions at 40% O2,5%CO2, 55% N2,378C. Correspondence and requests for materials should be addressed to C.M.F. ([email protected]).