Multiple Functions of Cerberus Cooperate to Induce Heart Downstream of Nodal ⁎ Ann C

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Developmental Biology 303 (2007) 57–65 www.elsevier.com/locate/ydbio

Multiple functions of Cerberus cooperate to induce heart downstream of Nodal ⁎ Ann C. Foley, Oksana Korol, Anjuli M. Timmer, Mark Mercola

Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA Received for publication 13 July 2006; revised 5 October 2006; accepted 22 October 2006 Available online 26 October 2006

Abstract

The TGFβ family member Nodal has been implicated in heart induction through misexpression of a dominant negative version of the type I Nodal receptor (Alk4) and targeted deletion of the co-receptor Cripto in murine ESCs and mouse embryos; however, whether Nodal acts directly or indirectly to induce heart tissue or interacts with other signaling molecules or pathways remained unclear. Here we present Xenopus embryological studies demonstrating an unforeseen role for the DAN family protein Cerberus within presumptive foregut endoderm as essential for differentiation of cardiac mesoderm in response to Nodal. Ectopic activation of Nodal signaling in non-cardiogenic ventroposterior mesendoderm, either by misexpression of the Nodal homologue XNr1 together with Cripto or by a constitutively active Alk4 (caAlk4), induced both cardiac markers and Cerberus. Mosaic lineage tracing studies revealed that Nodal/Cripto and caAlk4 induced cardiac markers cell non-autonomously, thus supporting the idea that Cerberus or another diffusible factor is an essential mediator of Nodal-induced cardiogenesis. Cerberus alone was found sufficient to initiate cardiogenesis at a distance from its site of synthesis. Conversely, morpholino-mediated specific knockdown of Cerberus reduced both endogenous cardiomyogenesis and ectopic heart induction resulting from misactivation of Nodal/Cripto signaling. Since the specific knockdown of Cerberus did not abrogate heart induction by the Wnt antagonist Dkk1, Nodal/Cripto and Wnt antagonists appear to initiate cardiogenesis through distinct pathways. This idea was further supported by the combinatorial effect of morpholino-medicated knockdown of Cerberus and Hex, which is required for Dkk1-induced cardiogenesis, and the differential roles of essential downstream effectors: Nodal pathway activation did not induce the transcriptional repressor Hex while Dkk-1 did not induce Cerberus. These studies demonstrated that cardiogenesis in mesoderm depends on Nodal- mediated induction of Cerberus in underlying endoderm, and that this pathway functions in a pathway parallel to cardiogenesis initiated through the induction of Hex by Wnt antagonists. Both pathways operate in endoderm to initiate cardiogenesis in overlying mesoderm. © 2006 Elsevier Inc. All rights reserved.

Keywords: Cardiogenesis; Xenopus; Cerberus; Nodal; Hex; Dickkopf; Heart induction

Introduction regulation of downstream diffusible proteins (Foley and Mercola, 2005a). Nodal proteins are members of the TGFβ Heart induction in vertebrates is initiated during early superfamily of signaling molecules that are important for embryogenesis by specification of paired cardiac primordia numerous developmental processes, including the formation of within the mesoderm on either side of the dorsal midline. axial mesoderm [Xenopus,(Agius et al., 2000; Jones et al., Several classes of secreted proteins have been shown to be 1995) zebrafish (Toyama et al., 1995), mouse (Conlon et al., capable of inducing heart differentiation in non-cardiac 1994a; Zhou et al., 1993a) and chick (Bertocchini et al., 2004; mesoderm, including Dickkopf-1 and Crescent, both of which Bertocchini and Stern, 2002)] and induction and patterning of inhibit the canonical Wnt/β-catenin signaling pathway (Marvin the endoderm (Agius et al., 2000; Chang and Hemmati- et al., 2001; Schneider and Mercola, 2001) and act through the Brivanlou, 2000; David and Rosa, 2001; Henry et al., 1996). non-cell-autonomous induction of the homeodomain protein Nodal signaling is transduced to the cytoplasm through a Hex in the endoderm underlying the cardiac mesoderm and heteromeric receptor complex consisting of the activin type II receptor, the Alk4 type I receptor (Chen et al., 2004; Reissmann ⁎ Corresponding author. Fax: +1 858 713 6274. et al., 2001) and the EGF-CFC protein Cripto (Gritsman et al., E-mail address: [email protected] (M. Mercola). 1999; Yeo and Whitman, 2001). In Xenopus, overexpression of

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.10.033 58 A.C. Foley et al. / Developmental Biology 303 (2007) 57–65 several Nodal family members or a constitutively activated form (EcoRI, T7); pXαMhc (HindIII, T7); pGEM3Z-Nkx2.5 (XbaI, T7); pGemT- of Alk4 (caAlk4) has been shown to induce cardiac mesoderm Tbx5 (Not1, T7) and pXTnIc (Not1, T7). Following in situ hybridization, most explants were paraffin embedded and sectioned for analysis. (Reissmann et al., 2001; Takahashi et al., 2000). Disruption of this signaling complex by the use of dominant negative Alk Morpholino and mRNA injection receptors or the genetic disruption of the gene encoding Cripto blocks spontaneous cardiomyocyte differentiation in murine Synthetic, capped mRNA for injection was transcribed from plasmids pSP6- embryonic stem cells (ESCs) (Parisi et al., 2003; Sonntag et al., nls-βgal, pCS2-Dkk1, pCS2-XNr1, pCS2-CA-Alk4, and pCS2-XCer and pCS2- 2005; Xu et al., 1998a, 1999b), Xenopus embryos (Reissmann XCer-Short (kind gifts from Eddy deRobertis) using SP6 and T7 mMessage kits et al., 2001) and zebrafish (Griffin and Kimelman, 2002; Reiter (Ambion). All cDNAs used encode Xenopus proteins with the exception of the hAlk4-CAT206D construct that contains a mutated, constitutively active form of et al., 2001). Although these results suggested a potential role the human Alk4 gene. The antisense Cer morpholino oligonucleotide was for Nodal signaling in heart induction, a mechanistic under- designed against bases −35 to −11 upstream of the AUG (5′-CTAGACCCTG- standing was elusive in part because previous studies were not CAGTGTTTCTGAGCG-3′) as designed and validated by Silva et al. (2003) and designed to isolate a specific cardiogenic function from the 2 pmol was injected. The rescue was carried out using a pCS2-XCer, which lacks broader inductive and patterning effects of Nodal on mesendo- the sequence corresponding to the morpholino. The antisense Hex morpholino was 5′-GGTGCTGGTACTGCATGTCGATTCC-3′ (Foley and Mercola, 2005b; derm. In particular, whether Nodal signaling specifically affects Smithers and Jones, 2002) and injected at 1 pmol. The standard control cardiogenesis, potential downstream signaling mediators, and morpholino provided by Gene Tools was also injected at 2 pmol. Depending on the potential cooperation with other signaling pathways that pattern experiment, either a 10,000 MWAlexa 546 lysinated dextran (AD546, Molecular anterior mesendoderm all remained to be resolved. Probes) or mRNA encoding nls-β-gal were co-injected as lineage labels. Our study demonstrates that the Xenopus Nodal homologue XNr1 is sufficient to specify an ectopic heart field in non- Quantitative RT-PCR cardiac mesoderm. Most importantly, mosaic analysis of the 10–12 explants were pooled from each round of injections and RNA was induced heart tissue showed that cells expressing either XNr1 produced using Qiagen Rneasy Lipid Tissue Kit (Qiagen). First strand synthesis with its co-receptor Cripto or caAlk4 were precluded from was carried out using a poly-dT primer and Superscript II Reverse Transcriptase joining the heart field, suggesting that Nodal signaling cell- (Invitrogen). Real time PCR was performed on a Roche Light Cycler using the autonomously inhibits cardiogenesis while simultaneously Light Cycler FastStart DNA master SYBR Green I kit (Roche). Quantification was carried out by normalizing levels to amount of total cDNA using the stimulating production of a diffusible intermediary that induces ubiquitously expressed EF1α transcript. The EF1α primer spans an intron and cardiogenesis in adjacent cells. Gain and loss of function PCR in the absence of RT thus confirmed that RNA samples were free from interventions showed that the secreted Cerberus protein is genomic DNA contamination. Q-RT-PCR was performed on 6 biological produced by the cells that respond to Nodal and is essential to samples and run data shown as the average of two technical replicates. initiate cardiogenesis in adjacent cells but is not required for Primer Pairs were as below: heart induction by Wnt antagonists. Cerberus mRNA is induced Cerberus: (+) 5′-AAGCCTGGTGCCAAGAT-3′;(−)5′-ATTCAGCGT- directly by Nodal (Osada and Wright, 1999; Piccolo et al., 1999; CAGGTGGTT-3′ Yamamoto et al., 2003) in a spatiotemporal domain that EF1A: (+) 5′-TACCCTCCTCTTGGTCGTT-3′;(−)5′-GGTTTTCGCTGC- localizes precisely to the region of dorsoanterior endoderm TTTCTG-3′ required for cardiogenesis (Schneider and Mercola, 1999b). Our Hex: (+) 5′-GTG GCTACTTACCGGAC-3′;(−)5′-CCTTTCCGCTTG- ′ study, therefore, illuminates a complex genetic cascade for heart TGCA-3 specification that involves signaling through parallel pathways that antagonize Nodal and Wnt activity in the endoderm Results resulting in production of diffusible signals such as Cerberus. Xnr-1/cripto signaling pathway induces early and late markers of cardiac mesoderm Materials and methods

Embryo and explant culture Synthetic mRNAs encoding XNr1 and Cripto were co- injected at doses ranging from 5 pg each to 100 pg each into the Embryos were fertilized in vitro, dejellied in 2% cysteine–HCl, pH7.8, and non-cardiac ventroposterior mesoderm (VMZ) of 4–16 cell maintained in 0.1× MMR (Peng, 1991). Embryos were staged according to stage Xenopus embryos. When embryos reached early gastrula Nieuwkoop and Faber (1994). Dorsoanterior marginal zone (DMZ) or stage (Nieuwkoop and Faber st. 10.25–10.5), VMZ regions ventroposterior marginal zone (VMZ) explants were dissected at stage – were explanted and cultured in isolation until age-matched 10.25 10.5, when the blastopore was clearly discernible. Explant dissections – were performed in 0.75× MMR using a fine tungsten needle and processed siblings reached st. 23 25. 25 pg or higher of each co-injected immediately or cultured in 0.75× MMR until sibling controls had reached mRNA consistently yielded robust expression of the heart field appropriate stages. For gene expression analysis, tissues were flash frozen for markers Nkx2.5 and Tbx5 visualized by in situ hybridization subsequent RNA isolation or fixed in MEMPFA for in situ hybridization as below. (Nkx2.5, 51% of explants were positive, n=109; Tbx5, 49%, n=108) (Figs. 1B, D). In subsequent experiments, 50 pg of In situ hybridization and histology each mRNA was injected unless noted otherwise. When – In situ hybridization was performed according the protocol of Harland cultured to st. 30 32, similarly processed explants expressed (1991). Digoxygenin-labelled probes were transcribed from the following transcripts encoding the cardiac contractile proteins αMhc and linearized plasmid templates (restriction digest, polymerase): pBS-Cerberus TnIc (αMhc , 27%, n=26; TnIc, 48%, n=72) (Figs. 1C, E). A.C. Foley et al. / Developmental Biology 303 (2007) 57–65 59

Fig. 1. XNr1/Cripto and caAlk4 misexpression induce ectopic expression of cardiac genes Nkx2.5, Tbx5, TnIc and αMHC cell non-autonomously. (A) One ventral blastomere of 16–32 cell stage embryos were injected with mRNAs encoding XNr1 and Cripto or caAlk4 along with AD546 (red) as a lineage label. The normally non- cardiogenic VMZ mesendoderm was explanted at the onset of gastrulation (stage 10.25–10.5), cultured until cardiac tissue expressed Nkx2.5 and Tbx5 (stage 23–25) or Tnic and αMHC (stage 30), then stained for gene expression by in situ hybridization (blue). (B–G) Examples of cell non-autonomous gene induction on histological section. Injection of XNr1 and Cripto or caAlk4 mRNAs with AD 546 induced Nkx2.5 (B, F), Tbx5 (D) TnIc (E, G) and αMHC (C). Images represent a photo merging of brightfield and fluorescent images. Note that cardiac marker positive cells (blue) were localized at a distance from lineage-labeled cells (red). Scale bar in panels B, D–G represents 40 μM, scale bar in panel C represents 20 μM.

Although Xnr-1/Cripto induced robust expression of both early along with the fluorescent Alexa-dextran 546 (AD546) as a and late cardiac markers, beating heat tubes were rarely lineage label into single ventral blastomeres of 8–16 cell stage observed and, when examined in histological section, we did embryos (Fig. 1A). VMZ explants were prepared as above and not observe the characteristic architecture of tube with open cultured until age-matched siblings had reached either stage lumen that is typical of native myocardium or VMZ explants 25–28 or stage 30–32, when they were processed for in situ induced by Dkk1 (Schneider and Mercola, 2001) at this hybridization and examined for localization of heart field developmental stage (note absence of lumen in Figs. 1C, E, markers Nkx2.5 and Tbx5, or the cardiac contractile protein and data not shown). Similar results were obtained with caAlk4 genes αMhc and TnIc relative to the lineage label in histological (Nkx2.5, 14%, n=135; TNIc 19%, n=48) (Figs. 1F, G). section. In all cases, the lineage label, which marks the progeny Immunoprecipitation and chemical cross-linking experi- of the injected cells, was located adjacent to but not overlapping ments show that Cripto, the prototypic member of the epidermal the induced cardiac tissue (Figs. 1B–E). Similarly, caAlk4 growth factor-related Cripto-FRL1-Criptic (EGF-CFC) family, induced cardiac mesoderm cell non-autonomously, although in interacts with Nodal and Alk4, suggesting its role as a co- some explants we observed minor overlap indicating that receptor necessary to recruit Nodal to receptor complexes activation of the Nodal signaling pathway does not obligatorily (Gritsman et al., 1999; Yeo and Whitman, 2001). Cripto has also preclude heart induction (Figs. 1F, G). been reported to signal independently of Nodal and Alk4 to activate the MAP kinase and Akt pathway (Bianco et al., 2003; Nodal induces transient Cerberus transcripts in injected VMZs Yan et al., 2001). To determine if Cripto has Nodal-independent with normal spatiotemporal expression pattern cardiac-inducing activity, we injected Cripto mRNA alone as above and assessed the induction of cardiac markers. We The preceding results suggested that Nodal/Cripto and Alk4 injected 50 pg, which in our hands was the optimal dose for co- signaling initiates cardiogenesis indirectly by triggering pro- injection with Xnr-1, as well as two higher doses, 100 pg and duction of a diffusible inducing factor. Previous studies had 200 pg. None of the doses tested showed any induction of mapped the spatial expression of two genes, Cerberus (Cer) cardiac gene expression or morphological evidence of cardiac and the homeodomain transcription factor Hex, coincident with differentiation (n=76, data not shown); therefore, we conclude the region of deep dorsoanterior endoderm that stimulates heart that the cardiogenic function of Cripto is to potentiate Nodal/ induction (Schneider and Mercola, 1999b). Whereas the XNr1signaling. transcriptional repressive function of Hex is essential for heart induction by the Wnt antagonist Dkk1 (Foley and Mercola, Induction of cardiac markers by Xnr-1/cripto signaling is cell 2005a), Cerberus has not been placed in a heart-inducing non-autonomous cascade. To test if Cerberus is a component of the Nodal pathway, two ventral blastomeres of 4–8 cell stage embryos To test whether XNr1/Cripto signaling acts directly on the were injected with XNr1 and Cripto mRNAs or with activated cardiac mesoderm, we co-injected Xnr-1 and cripto mRNAs Alk4 mRNA and VMZ explants were isolated when embryos 60 A.C. Foley et al. / Developmental Biology 303 (2007) 57–65 reached early gastrula stages either immediately at stage 10.25– cells (Figs. 2B–E). Induction was however, not strictly cell- 10.5 or when age-matched siblings had reached stages 11, 13 or autonomous. We observed instances of cells adjacent to the 19. Both injected XNr1/Cripto and caAlk4 induced Cer lineage-labeled cells that also expressed Cer, as best visualized transcription (Fig. 2). Interestingly, the kinetics recapitulated using a nuclear localized lacZ to trace cell lineage (Figs. 2D, E). the transient pulse of expression seen during normal embryonic Interestingly, ectopic Cer never occurred in the deep endoderm development with maximal levels of induction at early gastrula (Figs. 2D, E red arrowheads) or in the superficial pigmented stages (st. 10.5–11), declining after gastrulation (st. 13) such layer (Fig. 2F), despite many instances of staining with the that ectopic transcripts were no longer detectable by early lineage label indicating descent from blastomeres injected with neurula stages (st. 19) (Figs. 2A and G–I). In a mosaic assay, the Nodal pathway activator reflecting, possibly, either a injection of single ventro-posterior blastomeres with XNr1/ requirement for additional mesendoderm-specific signaling or Cripto and either β-galactosidase mRNA or AD546 as a lineage the presence of signals that alter Nodal responsiveness as has label followed by isolation of VMZ explants at st. 10.25–10.5 been demonstrated by (Engleka et al., 2001). By contrast, and examination on histological section revealed that the induction of Cer by caAlk4 was almost entirely cell non- majority of ectopic Cer occurred in the progeny of the injected autonomous (Figs. 2G–I) suggesting responsiveness to Nodal

Fig. 2. XNr1/Cripto and caAlk4 induce endogenous Cer mRNA. Embryos were injected with synthetic mRNA for XNr1 and Cripto or caAlk4 into one ventral blastomere of a 4–8 cell stage embryo. Non-cardiogenic VMZ explants were dissected at onset of gastrulation (stage 10.25–10.5) then either frozen immediately for subsequent RNA isolation or grown in culture and frozen when age matched siblings had reached stages 10.5, 11, 13 and 19 and analyzed for mRNA levels by quantitative RT-PCR (data presented represent the average of two technical replicates each from two of the 6 biological replicates performed). (A) Plot depicts the fold induction of Cer in injected cells relative to uninjected controls, after normalization to levels of the ubiquitously expressed Ef1α transcript. (B–I) Examples of Cer induction detected by in situ hybridization relative to the AD546 (B, C, G, H, I) or β-galactosidase (D–F) lineage labels that mark the progeny of injected cells. XNr1 and Cripto induced expression of Cerberus in mesendodermal cells in a manner that was largely cell autonomous (note nearly complete overlap of Cerberus with AD546 in merged image (C) and large number of purple stained nuclei in Cerberus-expressing tissue (D, E, green arrows)). Neighboring cells that did not express the injected mRNAs often expressed Cer, as can be seen in panel E by the presence of nuclei visualized by DAPI but without β-galactosidase lineage label stain (blue arrows). In contrast, cells in the deep endoderm (D, E, red arrows) and cells in the pigmented epithelium (F) never expressed Cer despite expressing the injected β- galactosidase lineage label. Injection of caAlk4 also induced expression of Cerberus (G–I); however, induction was almost entirely cell non-autonomous [note lack of overlap between injected cells (red) and Cer-expressing cells (blue)], suggestive that chronic misactivation of the Nodal pathway is incompatible with Cer expression. Scale bars in panels B–F represents 20 μM and in panels G–I 100 μM. A.C. Foley et al. / Developmental Biology 303 (2007) 57–65 61 signaling may be required for the maintenance of Cerberus with the idea that localized production of the secreted Cerberus expression. Ectopic Cer-positive cells in the VMZ exhibited a protein induces cardiac mesoderm in nearby tissue. The distance remarkable involution behavior reminiscent of DMZ deep from lineage label to ectopic cardiac mesoderm was generally mesendoderm during gastrulation (Figs. 2G–I), indicating that further than that seen in VMZ explants injected with XNr1/ ectopic Nodal pathway activation elicited a normal develop- Cripto or caAlk4, perhaps suggesting a thresholded response to mental response. Cerberus or that the cells that produce Cerberus and cells that express early heart markers might migrate away from each other Cerberus is an essential mediator of XNr-1/cripto-induced during the time course of the experiment. cardiogenesis A requirement for Cerberus was evaluated by injecting the antisense morpholino oligonucleotide (Cer MO), (Silva et al., To test Cerberus involvement in heart induction, Cer mRNA 2003) with XNr1 and Cripto mRNAs into 1 ventral blastomere was co-injected with the lineage label AD546 into one ventral of the 4-cell stage embryo at doses that give robust cardiac blastomere at the 8–16 cell stage and explants were isolated at induction. Cer Mo significantly decreased the incidence of heart st. 10.25–10.5 and cultured until age-matched siblings had induction by XNr1/Cripto (7.4%, n=121, when co-injected reached st. 23–25 when they were analyzed for expression of with Cer MO versus 34.3% explants were positive in absence of the early cardiac markers Nkx2.5 and Tbx5. Injected explants Cer MO, n=137, Figs. 3C, G, G′). In separate experiments, Cer showed robust expression of early cardiac markers (Nkx2.5, Mo also decreased XNr1/Cripto induction of αMhc (6.3% in 69% of explants were positive, n=58; Tbx5, 72%, n=113). presence of Cer MO, n=16, versus 32.8% positive in absence, Interestingly, Cerberus-injected explants did not show expres- n=22), and Nkx2.5 (7.3% in presence of Cer MO, n=82, versus sion of markers for differentiated cardiac mesoderm (data not 44.6% positive in absence, n=148, Figs. 3F, F′). shown). The lineage label was most often distant from cells Having demonstrated that Cerberus is essential for ectopic expressing the induced Nkx2.5 or Tbx5 (Figs. 3A, B), consistent cardiogenesis induced by Nodal pathway activation, we then

Fig. 3. Cerberus is required for normal cardiogenesis and ectopic heart induction by XNr1 and Cripto but not for heart induction by the Wnt antagonist Dkk1. (A, B) Cerberus was co-injected into one ventral blastomere at the 4–8 cell stage with AD546. Explants of the non-cardiac VMZ were isolated at early gastrula stages 10.25 to 10.5 and grown until age-matched siblings had reached stage 23–35 and processed by in situ hybridization. Cerberus induced expression of both Nkx2.5 and Tbx5 (red arrowheads) cell non-autonomously. Scale bars represent 20 μM. (C) Morpholino mediated knockdown of Cerberus decreases the percentage of heart induction by XNr1 and Cripto but not by Dkk1. Asterisk indicates statistically significant (p<0.001, Student's T-test) difference between treatment classes for greater than 3 experimental trials. (D–H′) Examples of explants showing effects of Cerberus morpholino-mediated knockdown on endogenous heart induction and heart induction by XNr1 and Cripto versus that by Dkk1. DMZ explants express the early and late cardiac markers Nkx2.5 and TnIC (red arrowheads panels D, E) whereas morpholino-injected explants lacked heart gene expression (D′,E′) without effecting head development and loss of Nkx2.5 was rescued by co-injection of a plasmid that directed expression of Cerberus protein (panel D′ inset). VMZ explants injected with XNr1 and Cripto expressed early and late cardiac markers Nkx2.5 and TnIc, respectively (red arrowheads G, G′). Morpholino-knockdown of Cerberus caused loss of heart markers without reduction in axial structures (F′,G′). Dkk1 injected either alone (H) or with a standard control morpholino (panel H insert) induced expression of TnIc (red arrowhead). Cer MO did not effect induction of TnIc by Dkk1 (panel H′ red arrowhead). 62 A.C. Foley et al. / Developmental Biology 303 (2007) 57–65 tested for a role in endogenous heart development by injecting early cardiac induction (Fig. 4E; 84.1%, n=44, for Cer MO and 2 pmol of Cer MO into the 2 dorsal blastomeres of 4-cell stage 52.2%, n=46, for Hex MO as compared to 95.4%, n=109, for embryos. As for ectopic induction, 2 pmol doses were able to Control MO). Co-injection of Cer MO and Hex MO at these significantly reduce expression of each of the cardiac markers conditions more severely attenuated heart induction than either we examined relative to controls injected with either control alone (Fig. 4E; 34%, n=47), indicative of a genetic interaction. morpholino or injected with an inert mRNA, including: Together, these experiments demonstrate that Nodal pathway Nkx2.5 (58.5% positive in presence of Cer MO, n=41, versus activation does not overlap with Dkk1 induction of cardiogen- 94%, n=42, for controls, Figs. 3D, D′); Tbx5 (65.4% in esis, at least at the levels of the downstream effectors Cerberus presence of Cer MO, n =52, versus 78.9%, n=38, for and Hex. controls); TnIc (35.9%, n=39, in presence of Cer MO versus 75%, n=20, for controls, Figs. 3E, E′), and αMhc (59.8%, n=82, versus 68.8%, n=32, for controls). Consistent with the observations of Silva et al. (2003), head structures were essentially unaffected by morpholino injection (see Figs. 3D′, E′), supporting the idea that Cerberus plays specific roles in mesendoderm patterning despite potency as an ectopic head inducer. Co-injection of 25 pg of pCS2-XCer plasmid (which lacks the sequence targeted by Cer MO) with 2 pmol of the Cer MO rescued the expression of Nkx2.5 (Fig. 3D′ inset) (85.7%, n=42), confirming selectivity of Cer MO for the endogenous mRNA. Taken together, our results implicate Cerberus as an essential effector of the Nodal pathway for cardiogenesis.

Heart induction by XNr1 and Dkk1 are distinct at the levels of Cerberus and Hex

Since Dkk-1 also induces ectopic cardiac gene expression in VMZ explants (Schneider and Mercola, 2001), we next examined involvement of Cerberus in this pathway. Dkk-1 was injected into 1 ventral blastomere at the 4-cell stage either alone or in combination with 2 pmol of Cer Mo. As shown in Figs. 3C, H, H′, Cer MO did not interfere with the ability of Dkk-1 to induce cardiac marker expression (55.3%, n=38, of explants expressed TnIc when injected with Dkk-1 alone as opposed to 71%, n=62, when Dkk-1 was co-injected with Cer MO). This result suggests that the Nodal and Dkk1 pathways are – distinct at the level of Cerberus. We had shown that the Fig. 4. Cer-S inhibition of Nodal contributes to heart induction. (A E) Embryos were injected into two dorsal/vegetal blastomeres at the 8 cell stage with either homeodomain transcription factor Hex is an essential compo- 2 pmol control MO (A), 1 pmol Hex MO (B), 2 pmol Cer MO (C) or nent of heart induction by Dkk1 (Foley and Mercola, 2005b), combination of 2 pmol Cer MO and 1 pmol Hex MO (D). DMZ explants raising the possibility that cardiogenesis induced by Nodal isolated at early gastrula stages (10–10.25) and grown until age-matched pathway activation might also require Hex. Ectopic Hex siblings had reached stage 23–25 when they were processed by in situ transcripts were not induced in VMZ tissue by targeting hybridization for expression of Nkx2.5 (arrowhead in panel A). Incidence of Nkx2.5 expression is shown in in panel E. Asterisks indicate statistical injections of XNr-1/Cripto mRNAs, at doses ranging form significance (p<0.01, Student's T-test) between indicated conditions for greater 25 pg each up to 100 pg each, into single blastomeres of the than 3 independent experiments. (F–I) Embryos were injected into one ventral 4-cell stage embryo. VMZ explants were dissected at st. 10.5 blastomere at the 4–8 cell stage with either the truncated form of Cerberus, Cer- and snap frozen until analysis of Hex transcript levels by S (F), the truncated version of the BMP receptor, tBR (G), or the two constructs quantitative RT-PCR normalizing to that for EF1α. Induction of together (H). VMZ explants were isolated at early gastrula stages 10.25 to 10.5 and grown until age-matched siblings had reached stage 23–25 and processed Hex transcript was not seen in response to injections of XNr1/ by in situ hybridization for expression of either Nkx2.5 (arrowhead in in panel Cripto (data not shown). Similarly, Cerberus also did not induce H) or Tbx5. Neither Cer-S nor tBR alone induced expression of Nkx2,5 or Tbx5 Hex, and previous results had shown that Dkk1 does not induce whereas co-injection of Cer-S and tBR induced robust expression of both Cer (Foley and Mercola, 2005b). We then used the specific markers. The incidence of expression is plotted in the histogram (I). (J) The data morpholinos to verify that the pathways are distinct by loss-of- indicate that two pathways for specification of the heart field in mesoderm are independent at the level of Hex and Cerberus. Dkk1 (and other canonical Wnt function experiments. Embryos were injected into two dorso- antagonists) act through the homeodomain transcriptional repressor Hex to vegetal blastomeres at the 8 cell stage with either 2 pmol of Cer induce an as yet unidentified diffusible intermediate whereas activation of the Mo or 1 pmol of Hex MO, levels which submaximally abrogate Nodal pathway results in production of the secreted factor Cerberus. A.C. Foley et al. / Developmental Biology 303 (2007) 57–65 63

Multiple roles for Cerberus 2001; Meno et al., 1999; Norris et al., 2002), suggesting a model whereby pre-chordal and cardiac mesoderm, as well as Cerberus is a multifunctional protein that antagonizes Wnt, endoderm, require higher, earlier and/or more sustained levels Nodal and BMP signaling. In order to begin to understand the of Nodal signaling than does formation of more posterior basis for heart induction by Cerberus, we injected mRNA that mesoderm. Our results suggest a spatially and/or temporally encodes a truncated form of the protein known as Cerberus- complex model in which Nodal initiates cardiogenesis through Short (Cer-S), which does not antagonize Wnts or BMPs but induction of a diffusible signal that we showed to consist of retains the ability to block Nodal signaling. Cer-S was unable to Cerberus by both gain and loss of function experiments. initiate cardiogenesis, since only 1 explant out of 78 showed Cerberus is a multifunctional protein that inhibits Wnt, BMP ectopic Nkx2.5 expression and only 1 out of 52 expressed and Nodal activity. Consequently, the unexpected finding that ectopic Tbx5 when injected into single blastomeres of VMZ and the truncated Cer-S protein, which contains the Nodal inhibitory assayed as for Cerberus above. Similarly, VMZ explants activity, retained the ability to initiate ectopic cardiogenesis in injected with a truncated form of the BMP receptor (tBR), secondary body axes induced by tBR points to the possibility which blocks BMP signaling specifically (Graff et al., 1994) that Cerberus participates in a feedback mechanism to establish also did not show consistent ectopic Nkx2.5 (1.5%, n=65) or the appropriate level and/or timing of Nodal signaling needed Tbx5 (2.5%, n=40). Since neither a Nodal nor BMP antagonist for heart field specification (diagrammed in Fig. 4J). Moreover, induced Nkx25 or Tbx5, these results implicate Wnt antagonism the feedback inhibition of Nodal might account for the tight as a component of the cardiogenic function of Cerberus, but this control of endogenous Cer transcripts, which accumulate only hypothesis cannot be verified directly since the Wnt antagoniz- at the onset of gastrulation in a pattern that coincides precisely, ing activity is not known to be retained on a fragment that lacks both temporally and spatially, with the domain of heart- Nodal- and BMP-inhibiting activities. Cerberus within the inducing activity within the deep dorsoanterior endoderm embryo might function in combination with other signals and (Schneider and Mercola, 1999a). Tight control of endogenous these can be recapitulated in part by injection of tBR, which Cer expression is likely to be essential for heart induction induces secondary body axes that are both devoid of anterior because persistent expression adjacent to the heart field after structures (Graff et al., 1994; Suzuki et al., 1994) and lack early gastrulation would be expected to interfere with BMP, which is cardiac gene expression (Nkx2.5, 0%, n=18). In contrast to required for continued cardiogenesis in Xenopus (reviewed in injection of either alone, co-injection of Cer-S complemented Foley and Mercola, 2004), and this might explain why Cerberus tBR and robustly induced Nkx2.5 in 44.2% (n=52) and Tbx5 in injection induces only markers of the heart field (Tbx5 and 50% (n=32) of injected VMZ explants (Figs. 4F–I), implicating Nkx2.5) but not later genes encoding contractile proteins inhibition of Nodal signaling as a component of Cerberus indicative of cardiomyogenesis. cardiogenic activity. The contribution of multiple functions of The fact that Cer-S injected alone into non-cardiogenic Cerberus to heart field induction are discussed below. mesoderm did not induce ectopic cardiac tissue suggests that the truncated protein lacks an additional activity that contributes to Discussion heart induction. Since the BMP antagonist tBR alone did not initiate cardiogenesis while co-injection with Cer-S was Our results revealed multiple ways in which Cerberus sufficient, it seems likely that BMP antagonism is involved. interacts with the Nodal signaling pathway to induce cardio- BMP is known to be required for heart induction, but Xenopus genic mesoderm in Xenopus. Although Nodal family members studies place its function slightly later to maintain the induced have been implicated in heart induction, their role in this state (Shi et al., 2000). Finally, we cannot rule out the process has not been distinguished from their more general involvement of the Wnt antagonist activity of Cerberus. ability to induce mesoderm and endoderm. For example, high Importantly, the finding that Cerberus did not induce Hex doses of recombinant Activin, which has overlapping affinity expression, which is induced by inhibition of canonical Wnt with Nodal for cell surface receptors, can induce cardiogenesis signaling in VMZ, and that Dkk1 induction of cardiac gene in Xenopus animal cap and chick epiblast tissue (both primitive expression was not blocked by Cer MO indicate that the Dkk1/ ectoderm) (Logan and Mohun, 1993; Yatskievych et al., 1997); Wnt antagonism and Nodal pathways operate in parallel at least however, since both mesoderm and endoderm form in these at the level of these downstream effectors (Fig. 4J). experiments, whether cardiogenesis results from a direct effect Results from prior studies of mouse embryonic and ESC of receptor activation or by an interaction between these tissues cardiogenesis are consistent with the idea of a diffusible signal has not been resolved. One clue as to how this family of downstream of Nodal, but the nature of the factor was not molecules might specify specific mesodermal fates came from elucidated. Nodal−/− (Conlon et al., 1994b; Zhou et al., 1993b) the finding that Activin (Green and Smith, 1990; Symes et al., and Smad2−/− (Waldrip et al., 1998) mouse embryos fail to 1994), as well as Nodal and its homologues can act in a develop a primitive streak and die before anterior structures, concentration and/or duration-sensitive manner to specify fate including heart, form and they also do not express markers of and behavioral differences of induced mesendoderm in the anterior visceral endoderm (AVE), an extraembryonic tissue zebrafish (Gritsman et al., 1999; Reiter et al., 2001), Xenopus that shares gene expression profiles and inductive properties (Agius et al., 2000; Chang and Hemmati-Brivanlou, 2000; with the deep dorsoanterior endoderm in Xenopus. An elegant Faure et al., 2000; Lee et al., 2001) and mouse (Lowe et al., series of chimera experiments by Robertson and colleagues 64 A.C. Foley et al. / Developmental Biology 303 (2007) 57–65

(Brennan et al., 2001; Varlet et al., 1997) overcame the early pathway activated by Cripto-1 through Glypican-1 and c-Src. Cancer Res. – lethality and showed that Nodal signaling within the AVE and 63, 1192 1197. Biben, C., Stanley, E., Fabri, L., Kotecha, S., Rhinn, M., Drinkwater, C., Lah, epiblast is essential to specify anterior structures. Although M., Wang, C.-C., Nash, A., Hilton, D., Ang, S.-L., Mohun, T., Harvey, R.P., mouse embryos lacking Cripto produce mesoderm, they lack 1998. Murine cerberus homolgue mCer-1: a candidate anterior patterning anteroposterior axial development and do not form cardiomyo- molecule. Dev. Biol. 194, 135–151. cytes (Ding et al., 1998; Xu et al., 1999a). Similarly, Cripto−/− Brennan, J., Lu, C.C., Norris, D.P., Rodriguez, T.A., Beddington, R.S., ESCs also form mesoderm and cardiogenesis is blocked (Xu et Robertson, E.J., 2001. Nodal signalling in the epiblast patterns the early − − mouse embryo. Nature 411, 965–969. al., 1998b). Comparison of the phenotypes of Cripto / and −/− Chang, C., Hemmati-Brivanlou, A., 2000. A post-mid-blastula transition Nodal embryos indicates that Cripto is essential for Nodal to requirement for TGFbeta signaling in early endodermal specification. signal in both the epiblast and the AVE that overlies the epiblast. Mech. Dev. 90, 227–235. Moreover, considerable evidence shows that the AVE is Chen, Y., Mironova, E., Whitaker, L.L., Edwards, L., Yost, H.J., Ramsdell, A.F., required for induction of heart (Arai et al., 1997) and other 2004. ALK4 functions as a receptor for multiple TGF beta-related ligands to regulate left–right axis determination and mesoderm induction in Xenopus. anterior structures (Beddington and Robertson, 1999), sugges- Dev. Biol. 268, 280–294. tive that Nodal signaling from this tissue initiates a Cripto- Conlon, F.L., Lyons, F.M., Takaesu, N., Barth, K.S., Kispert, A., Herrmann, B., dependent cascade that is essential for heart induction. In favor Robertson, E.J., 1994a. A primary requirement for nodal in the formation of a diffusible signal downstream of Nodal, Cripto−/− mESCs and maintenance of the primitive streak in the mouse. Development 120, – introduced into wild type mouse blastocysts efficiently 1919 1928. Conlon, F.L., Lyons, K.M., Takaesu, N., Barth, K.S., Kispert, A., Herrmann, B., contribute to the heart (Xu et al., 1999a), demonstrating that Robertson, E.J., 1994b. A primary requirement for nodal in the formation secreted or membrane localized factors from the wild type cells and maintenance of the primitive streak in the mouse. Development 120, act on the mutant cells. Although a shed version of Cripto has 1919–1928. been postulated as a potential mediator (Parisi et al., 2003), the David, N.B., Rosa, F.M., 2001. Cell autonomous commitment to an endodermal murine data could also be explained by the involvement of the fate and behaviour by activation of Nodal signalling. 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