Petra Pandur

Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos

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.

Six1 promotes a placodal fate within the lateral neurogenic ectoderm by functioning as both a transcriptional activator and repressor

Cranial placodes, which give rise to sensory organs in the vertebrate head, are important embryonic structures whose development has not been well studied because of their transient nature and paucity of molecular markers. We have used markers of pre-placodal ectoderm (PPE) (six1, eya1) to determine that gradients of both neural inducers and anteroposterior signals are necessary to induce and appropriately position the PPE. Overexpression of six1 expands the PPE at the expense of neural crest and epidermis, whereas knock-down of Six1 results in reduction of the PPE domain and expansion of the neural plate, neural crest and epidermis. Using expression of activator and repressor constructs of six1 or co-expression of wild-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.

Non-canonical Wnt Signaling Enhances Differentiation of Human Circulating Progenitor Cells to Cardiomyogenic Cells

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 α-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-3β, we further determined whether the glycogen synthase kinase-3β 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.

The LIM-only protein FHL2 interacts with β-catenin and promotes differentiation of mouse myoblasts

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.

Xenopus Six1 gene is expressed in neurogenic cranial placodes and maintained in the differentiating lateral lines

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.

The Drosophila homolog of vertebrate Islet1 is a key component in early cardiogenesis.

In mouse, the LIM-homeodomain transcription factor Islet1 (Isl1) has been shown to demarcate a separate cardiac cell population that is essential for the formation of the right ventricle and the outflow tract of the heart. Whether Isl1 plays a crucial role in the early regulatory network of transcription factors that establishes a cardiac fate in mesodermal cells has not been fully resolved. We have analyzed the role of the Drosophila homolog of Isl1, tailup (tup), in cardiac specification and formation of the dorsal vessel. The early expression of Tup in the cardiac mesoderm suggests that Tup functions in cardiac specification. Indeed, tup mutants are characterized by a reduction of the essential early cardiac transcription factors Tin, Pnr and Dorsocross1-3 (Doc). Conversely, Tup expression depends on each of these cardiac factors, as well as on the early inductive signals Dpp and Wg. Genetic interactions show that tup cooperates with tin, pnr and Doc in heart cell specification. Germ layer-specific loss-of-function and rescue experiments reveal that Tup also functions in the ectoderm to regulate cardiogenesis and implicate the involvement of different LIM-domain-interacting proteins in the mesoderm and ectoderm. Gain-of-function analyses for tup and pnr suggest that a proper balance of these factors is also required for the specification of Eve-expressing pericardial cells. Since tup is required for proper cardiogenesis in an invertebrate organism, we believe it is appropriate to include tup/Isl1 in the core set of ancestral cardiac transcription factors that govern a cardiac fate.

What does it take to make a heart

Ever increasing advances are being made in our quest to understand what it takes to direct pluripotent precursor cells to adopt a specific developmental fate. Eventually, the obvious goal is that targeted manipulation of these precursor cells will result in an efficient and reliable production of tissue‐specific cells, which can be safely employed for therapeutic purposes. We have gained an incredible insight as to which molecular pathways are involved in governing neural, skeletal and cardiac muscle fate decisions. However, we still face the challenge of how to direct, for example, a cardiac fate in stem cells in the amounts needed to be employed for regenerative means. Equally importantly, we need to resolve critical questions such as: can the in vitro generated cardiomyocytes actually functionally replace damaged heart tissue? Here I will provide an overview of the molecules and signalling pathways that have first been demonstrated in embryological studies to function in cardiogenesis, and summarize how this knowledge is being applied to differentiate mouse and human embryonic stem cells into cardiomyocytes.

Islet1-expressing cardiac progenitor cells: a comparison across species.

Adult mammalian cardiac stem cells express the LIM-homeodomain transcription factor Islet1 (Isl1). They are considered remnants of Isl1-positive embryonic cardiac progenitor cells. During amniote heart development, Isl1-positive progenitor cells give rise mainly to the outflow tract, the right ventricle, and parts of the atria. This led to the hypothesis that the development of the right ventricle of the amniote heart depends on the recruitment of additional cells to the primary heart tube. The region from which these additional, Isl1-positive cells originate is called second heart field, as opposed to the first heart field whose cells form the primary heart tube. Here, we review the available data about Isl1 in different species, demonstrating that Isl1 is an important component of the core transcription factor network driving early cardiogenesis in animals of the two clades, deuterostomes, and protostomes. The data support the view of a single cardiac progenitor cell population that includes Isl1-expressing cells and which differentiates into the various cardiac lineages during embryonic development in vertebrates but not in other phyla of the animal kingdom.

DM-GRASP/ALCAM/CD166 is required for cardiac morphogenesis and maintenance of cardiac identity in first heart field derived cells.

Vertebrate heart development requires specification of cardiac precursor cells, migration of cardiac progenitors as well as coordinated cell movements during looping and septation. DM-GRASP/ALCAM/CD166 is a member of the neuronal immunoglobulin domain superfamily of cell adhesion molecules and was recently suggested to be a target gene of non-canonical Wnt signalling. Loss of DM-GRASP function did not affect specification of cardiac progenitor cells. Later during development, expression of cardiac marker genes in the first heart field of Xenopus laevis such as Tbx20 and TnIc was reduced, whereas expression of the second heart field marker genes Isl-1 and BMP-4 was unaffected. Furthermore, loss of DM-GRASP function resulted in defective cell adhesion and cardiac morphogenesis. Additionally, expression of DM-GRASP can rescue the phenotype that results from the loss of non-canonical Wnt11-R signalling suggesting that DM-GRASP and non-canonical Wnt signalling are functionally coupled during cardiac development.

Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.

Molecular asymmetries in the animal–vegetal axis of the Xenopus oocyte are well known to regulate the formation of gametes and germ layers. Likewise, many transplantation and explant studies demonstrate that maternal dorsalizing activities are localized to the future dorsal side of the embryo after fertilization, but to date only a few of the molecules involved in this process have been shown to be asymmetrically distributed. In this report, we identify two new aspects of the maternal regulation of dorsal–ventral fate asymmetry in Xenopus blastomeres: cytoplasmic polyadenylation of dorsal maternal mRNAs and localized Wnt8b signaling. Previous studies demonstrated that there are maternal, dorsal axis‐inducing RNAs localized to dorsal animal blastomeres that become activated between the 8‐ and 16‐cell stage (Hainski and Moody [ 1992 ] Development 116:347–355; Hainski and Moody [ 1996 ] Dev. Genet. 19:210–221). We report herein that the activation of these axis‐inducing dorsal mRNAs is regulated by cytoplasmic polyadenylation. We also show that maternal wnt8b mRNA is concentrated in ventral animal blastomeres. These ventral cells and exogenous Wnt8b both inhibit the dorsal fate of neighboring blastomeres in culture, indicating that a maternal Wnt signal also contributes to segregating dorsal and ventral fates.