Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification, which indicates that Wnt-11 may be involved in heart development. Here we show, through loss- and gain-of-function experiments, that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of beta-catenin and involves protein kinase C and Jun amino-terminal kinase. Our results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction.

type six1 with activating or repressing co-factors (eya1 and groucho, respectively), we demonstrate that Six1 inhibits neural crest and epidermal genes via transcriptional repression and enhances PPE genes via transcriptional activation. Ectopic expression of neural plate, neural crest and epidermal genes in the PPE demonstrates that these factors mutually influence each other to establish the appropriate boundaries between these ectodermal domains.Key words: Pre-placodal ectoderm, Neural crest, foxD3, zic2, sox2, sox3, keratin, dlx5, dlx6 Research article 5872 Woda et al., 2003). Zic genes are initially expressed throughout the neural plate in response to anti-BMP factors, and as they become restricted to its lateral border they initiate neural crest fates (Nakata et al., 1997;Nakata et al., 1998; Brewster et al., 1998;Kuo et al., 1998;Mizuseki et al., 1998). The roles that border genes play to specify the fates of the different ectodermal subdomains remain to be elucidated. Although placodes have long been recognized as important embryonic structures, their transient nature and the lack of specific molecular markers have made it difficult to study the mechanisms by which they form. Recently, however, markers of the PPE during the initial induction of the placodes have been identified in Xenopus. six1 is homologous to Drosophila sine oculis; it is characterized by a homeobox DNA-binding domain and a protein-protein interaction domain called the Six domain. It is initially expressed in a band surrounding the anterior neural plate and later in all neurogenic placodes (Pandur and Moody, 2000). eya1 is homologous to Drosophila eyes absent (eya); it functions as a co-factor for Six genes of the Six1/2 and Six4/5 subfamilies (Pignoni et al., 1997;Ohto et al., 1999;Ikeda et al., 2002) and is expressed in a pattern very similar to that of six1 (David et al., 2001). We have used these markers to demonstrate that gradients of both neural inducer and anteroposterior signals are required for proper PPE formation. Moreover, we show that six1 expression is required for the establishment of the PPE, and it promotes the PPE at the expense of the neural crest and epidermis by both activating and repressing target gene expression. Finally, we demonstrate that several genes expressed in the embryonic ectoderm mutually influence each other to define its distinct subdomains. Materials and methods Expression constructsThe full open-reading frames of Xenopus six1 and Drosophila groucho (Dgroucho; LD33829, Berkeley Drosophila Genome Project) were cloned into expression vectors (pDH105, pCS2+). To generate a chimeric transactivating six1 construct, the Six domain plus the homeodomain (SDHD; amino acids 9-183) was amplified by PCR and ligated upstream of the VP16 activation domain in pCS2VP16 (from M. Whitman). To generate a chimeric repressive six1 construct, the SDHD region was ligated downstream of the Engrailed repressor (EnR) domain in pCS2EnR (from D. Kessler). RNA microinjectionTranscripts of six1 (400-600 pg), six1VP16 (100 ...

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt–β-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt–Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt–β-catenin and the PCP pathways, its potential involvement in the Wnt–Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshΔDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt–Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt–Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.

FHL2 is a LIM-domain protein expressed in myoblasts but down-regulated in malignant rhabdomyosarcoma cells, suggesting an important role of FHL2 in muscle development. To investigate the importance of FHL2 during myoblast differentiation, we performed a yeast two-hybrid screen using a cDNA library derived from myoblasts induced for differentiation. We identified β-catenin as a novel interaction partner of FHL2 and confirmed the specificity of association by direct in vitro binding tests and coimmunoprecipitation assays from cell lysates. Deletion analysis of both proteins revealed that the NH2-terminal part of β-catenin is sufficient for binding in yeast, but addition of the first armadillo repeat is necessary for binding FHL2 in mammalian cells, whereas the presence of all four LIM domains of FHL2 is needed for the interaction. Expression of FHL2 counteracts β-catenin–mediated activation of a TCF/LEF-dependent reporter gene in a dose-dependent and muscle cell–specific manner. After injection into Xenopus embryos, FHL2 inhibited the β-catenin–induced axis duplication. C2C12 mouse myoblasts stably expressing FHL2 show increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. These data imply that FHL2 is a muscle-specific repressor of LEF/TCF target genes and promotes myogenic differentiation by interacting with β-catenin.

Wnt proteins can activate different intracellular signaling cascades in various organisms by interacting with receptors of the Frizzled family. The first identified Wnt signaling pathway, the Wnt/beta-catenin pathway, has been studied in much detail and is highly conserved among species. As to non-canonical Wnt pathways, the current situation is more nebulous partly because the intracellular mediators of this pathway are not yet fully understood and, in some cases, even identified. However, there are increasing data that prove the existence of non-canonical Wnt signaling and demonstrate its involvement in different developmental processes. In vertebrates, Wnt-11 and Wnt-5A can activate the Wnt/JNK pathway, which resembles the planar cell polarity pathway in Drosophila. The Wnt/Ca(2+)-pathway has only been described in Xenopus and zebrafish so far and it is unclear whether it also exists in other organisms. Two recent papers provide us with new insight into non-canonical Wnt signaling by (1) presenting a new intracellular mediator of non-canonical signaling in Xenopus1 and (2) implicating the existence of an additional non-canonical Wnt signaling pathway in flies.

Human endothelial circulating progenitor cells (CPCs) can differentiate to cardiomyogenic cells during co-culture with neonatal rat cardiomyocytes. Wnt proteins induce myogenic specification and cardiac myogenesis. Here, we elucidated the effect of Wnts on differentiation of CPCs to cardiomyogenic cells. CPCs from peripheral blood mononuclear cells were isolated from healthy volunteers and co-cultured with neonatal rat cardiomyocytes. 6 -10 days after co-culture, cardiac differentiation was determined by ␣-sarcomeric actinin staining of human lymphocyte antigen-positive cells (fluorescence-activated cell-sorting analysis) and mRNA expression of human myosin heavy chain and atrial natriuretic peptide. Supplementation of co-cultures with Wnt11-conditioned medium significantly enhanced the differentiation of CPCs to cardiomyocytes (1.7 ؎ 0.3-fold), whereas Wnt3A-conditioned medium showed no effect. Cell fusion was not affected by Wnt11-conditioned medium. Because Wnts inhibit glycogen synthase kinase-3␤, we further determined whether the glycogen synthase kinase-3␤ inhibitor LiCl also enhanced cardiac differentiation of CPCs. However, LiCl (10 mM) did not affect CPC differentiation. In contrast, Wnt11-conditioned medium time-dependently activated protein kinase C (PKC). Moreover, the PKC inhibitors bisindolylmaleimide I and III significantly blocked differentiation of CPCs to cardiomyocytes. PKC activation by phorbol 12-myristate 13-acetate significantly increased CPC differentiation to a similar extent as compared with Wnt11-conditioned medium. Our data demonstrate that Wnt11, but not Wnt3A, augments cardiomyogenic differentiation of human CPCs. Wnt11 promotes cardiac differentiation via the non-canonical PKC-dependent signaling pathway.

Six genes are homeobox-containing transcription factors, many of which are expressed in head structures. We isolated a full-length cDNA of a previously unknown Xenopus member of this family. It shares a high sequence homology with mouse and human Six1, which during development are expressed in mesoderm and muscle. In contrast, XSix1 is prominently expressed in all neurogenic cephalic placodes and lateral line primordia from neurula to tadpole stages. The neurons derived from these placodes do not express XSix1, but the lateral line mechanoreceptors maintain expression. XSix1 is weakly expressed in muscle later in development.

Islet-1 is a LIM-homeodomain transcription factor that has been defined to label cardiac progenitor cells of the second heart field. Here we provide the first analysis of the expression pattern of Xenopus islet-1 (Xisl-1) in the context of cardiovascular development. During early stages of heart development Xisl-1 is co-expressed with Nkx2.5 in the cardiac crescent in Xenopus supporting the notion of an initially single heart field. At subsequent stages of cardiogenesis the expression domains of Xisl-1 and Nkx2.5 become more distinct with Xisl-1 being detected more anterior to Nkx2.5, however both factors continue to be co-expressed in the dorsal mesocardium and pericardial roof of the linear heart tube. The presence of a cardiac Xisl-1 progenitor pool in an amphibian whose heart lacks an anatomically separated right ventricle is intriguing. Functional analyses show that Xisl-1 is required for normal heart development. Inhibition of Xisl-1 results in defects in heart morphogenesis and in the downregulation of early cardiac markers implicating a role for Xisl-1 in cardiac specification. Additionally, Xisl-1 loss-of-function affects the expression of several vascular markers demonstrating the involvement of Xisl-1 in vasculogenesis.