Biomedical subjects

R T Moon

Publications and source records attributed to R T Moon.

At least 37 records · Page 2Linked to original sources

PubMed1999

Activation of a frizzled-2/beta-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Galphao and Galphat.

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G protein-linked receptors, though no loss-of-function studies have demonstrated a requirement for G proteins for Frizzled signaling. We engineered a Frizzled-2 chimera responsive to beta-adrenergic agonist by using the ligand-binding domains of the beta(2)-adrenergic receptor. The expectation was that the chimera would be sensitive both to drug-mediated activation and blockade, thereby circumventing the problem of purifying soluble and active Wnt ligand to activate Frizzled. Expression of the chimera in zebrafish embryos demonstrated isoproterenol (ISO)-stimulated, propranolol-sensitive calcium transients, thereby confirming the beta-adrenergic nature of Wnt signaling by the chimeric receptor. Because F9 embryonic teratocarcinoma cells form primitive endoderm after stable transfection of Frizzled-2 chimera and stimulation with ISO, they were subject to depletion of G protein subunits. ISO stimulation of endoderm formation of F9 stem cells expressing the chimeric receptor was blocked by pertussis toxin and by oligodeoxynucleotide antisense to Galphao, Galphat2, and Gbeta2. Our results demonstrate the requirement of two pertussis toxin-sensitive G proteins, Galphao and Galphat, for signaling by the Frizzled-2 receptor.

Amino Acid Sequence↗

PubMed1999

Activation of rat frizzled-1 promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via pathways that require Galpha(q) and Galpha(o) function.

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembrane-spanning motif characteristic of G-protein-linked receptors, although no loss-of-function studies have demonstrated a requirement for G-proteins for Wnt signaling by the gene product of frizzled-1. Medium conditioned by mouse F9 teratocarcinoma stem cells stably transfected to express either Xenopus Wnt-5a or Wnt-8 was used to test primitive endoderm formation of F9 stem cells. F9 stem cells expressing the rat Frizzled-1 receptors demonstrated endoderm formation in response to conditioned medium containing Wnt-8 but not to medium containing Wnt-5a. Primitive endoderm formation stimulated by Wnt-8 acting on the rat Frizzled-1 receptor was blocked by treatment with pertussis toxin by depletion of either Galpha(o) or Galpha(q) via antisense oligodeoxynucleotides, as well as by inhibitors of protein kinase C (bisindoylmaleimide) and of mitogen-activated protein kinase kinase (PD98059). Our results demonstrate the requirement for G-protein subunits Galpha(o) (a pertussis toxin substrate) and Galpha(q) for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Galpha(q)) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1999

A role for xGCNF in midbrain-hindbrain patterning in Xenopus laevis.

Cells in the presumptive neural ectoderm of Xenopus are committed to neural fate through a process called neural induction, which may involve proteins that antagonize BMP signaling pathways. To identify genes that are induced by the BMP antagonists and that may be involved in subsequent neural patterning, we used a suppression PCR-based subtraction screen. Here we investigate the prospective activities and functions of one of the genes, a nuclear orphan receptor previously described as xGCNF. In animal cap assays, xGCNF synergizes with ectopic chordin to induce the midbrain-hindbrain marker engrailed-2 (En-2). In Keller explants, which rely on endogenous factors for neural induction, similar increases in En-2 are observed. Expression in embryos of a dominant interfering form of xGCNF reduces the expression of endogenous En-2 and Krox-20. These gain-of-function and prospective loss-of-function experiments, taken with the observation that xGCNF is expressed in the early neural plate and is elevated in the prospective midbrain-hindbrain region, which subsequently expresses En-2, suggest that xGCNF may play a role in regulating En-2 and thus midbrain-hindbrain identity.

Animals↗

PubMed1999

Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.

Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner.

In studies of developmental signaling pathways stimulated by the Wnt proteins and their receptors, Xenopus Wnt-5A (Xwnt-5A) and a prospective Wnt receptor, rat Frizzled 2 (Rfz2), have been shown to stimulate inositol signaling and Ca2+ fluxes in zebrafish [1] [2] [3]. As protein kinase C (PKC) isoforms can respond to Ca2+ signals [4], we asked whether expression of different Wnt and Frizzled homologs modulates PKC. Expression of Rfz2 and Xwnt-5A resulted in translocation of PKC to the plasma membrane, whereas expression of rat Frizzled 1 (Rfz1), which activates a Wnt pathway using beta-catenin but not Ca2+ fluxes [5], did not. Rfz2 and Xwnt-5A were also able to stimulate PKC activity in an in vitro kinase assay. Agents that inhibit Rfz2-induced signaling through G-protein subunits blocked Rfz2-induced translocation of PKC. To determine if other Frizzled homologs differentially stimulate PKC, we tested mouse Frizzled (Mfz) homologs for their ability to induce PKC translocation relative to their ability to induce the expression of two target genes of beta-catenin, siamois and Xnr3. Mfz7 and Mfz8 stimulated siamois and Xnr3 expression but not PKC activation, whereas Mfz3, Mfz4 and Mfz6 reciprocally stimulated PKC activation but not expression of siamois or Xnr3. These results demonstrate that some but not all Wnt and Frizzled signals modulate PKC localization and stimulate PKC activity via a G-protein-dependent mechanism. In agreement with other studies [1] [2] [3]. [6] [7] these data support the existence of multiple Wnt and Frizzled signaling pathways in vertebrates.

Animals↗

PubMed1999

Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A.

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacted with APC in the yeast two-hybrid system. Expression of B56 reduced the abundance of beta-catenin and inhibited transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin was blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling.

Adenomatous Polyposis Coli Protein↗

PubMed1999

Maternal and embryonic expression of zebrafish lef1.

Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

Amino Acid Sequence↗

PubMed1999

Direct regulation of the Xenopus engrailed-2 promoter by the Wnt signaling pathway, and a molecular screen for Wnt-responsive genes, confirm a role for Wnt signaling during neural patterning in Xenopus.

The co-activation of Wnt signaling and concomitant inhibition of BMP signaling has previously been implicated in vertebrate neural patterning, as evidenced by the combinatorial induction of engrailed-2 and krox-20 in Xenopus. However, screens have not previously been conducted to identify additional potential target genes. Using a PCR-based screening method we determined that XA-1, xCRISP, UVS.2, two UVS.2-related genes, and xONR1 are induced in response to Xwnt-3a and a BMP-antagonist, noggin. Two additional genes, connexin 30 and retinoic acid receptor gamma were induced by Xwnt-3a alone. To determine whether any of the induced genes are direct targets of Wnt signaling, we focussed on engrailed-2. In the present study we show that the Xenopus engrailed-2 promoter contains three consensus binding sites for LEF/TCF, which are HMG box transcription factors which bind to beta-catenin in response to activation of the Wnt- 1 signaling pathway. An engrailed-2 promoter luciferase reporter construct containing these LEF/TCF sites is induced in embryo explant assays by the combination of Xwnt-3a or beta-catenin and noggin. These LEF/TCF sites are required for expression of engrailed-2, as a dominant negative Xtcf-3 blocks expression of endogenous engrailed-2 as well as expression of the reporter construct. Moreover, mutation of these three LEF/TCF sites abrogates expression of the reporter construct in response to noggin and Xwnt-3a or beta-catenin. We conclude that the engrailed-2 gene is a direct target of the Wnt signaling pathway, and that Wnt signaling works with BMP antagonists to regulate gene expression during patterning of the developing nervous system of Xenopus.

Animals↗

PubMed1999

Regulation of ribosomal S6 protein kinase-p90(rsk), glycogen synthase kinase 3, and beta-catenin in early Xenopus development.

beta-Catenin is a multifunctional protein that binds cadherins at the plasma membrane, HMG box transcription factors in the nucleus, and several cytoplasmic proteins that are involved in regulating its stability. In developing embryos and in some human cancers, the accumulation of beta-catenin in the cytoplasm and subsequently the nuclei of cells may be regulated by the Wnt-1 signaling cascade and by glycogen synthase kinase 3 (GSK-3). This has increased interest in regulators of both GSK-3 and beta-catenin. Searching for kinase activities able to phosphorylate the conserved, inhibitory-regulatory GSK-3 residue serine 9, we found p90(rsk) to be a potential upstream regulator of GSK-3. Overexpression of p90(rsk) in Xenopus embryos leads to increased steady-state levels of total beta-catenin but not of the free soluble protein. Instead, p90(rsk) overexpression increases the levels of beta-catenin in a cell fraction containing membrane-associated cadherins. Consistent with the lack of elevation of free beta-catenin levels, ectopic p90(rsk) was unable to rescue dorsal cell fate in embryos ventralized by UV irradiation. We show that p90(rsk) is a downstream target of fibroblast growth factor (FGF) signaling during early Xenopus development, since ectopic FGF signaling activates both endogenous and overexpressed p90(rsk). Moreover, overexpression of a dominant negative FGF receptor, which blocks endogenous FGF signaling, leads to decreased p90(rsk) kinase activity. Finally, we report that FGF inhibits endogenous GSK-3 activity in Xenopus embryos. We hypothesize that FGF and p90(rsk) play heretofore unsuspected roles in modulating GSK-3 and beta-catenin.

Animals↗

PubMed1999

XCtBP is a XTcf-3 co-repressor with roles throughout Xenopus development.

XTcf-3 is an HMG box transcription factor that mediates Xenopus dorsal-ventral axis formation. As a Wnt pathway effector, XTcf-3 interacts with beta-catenin and activates the expression of the dorsal organizing gene siamois, while in the absence of beta-catenin, XTcf-3 functions as a transcriptional repressor. We show that XTcf-3 contains amino- and carboxy-terminal repressor domains and have identified a Xenopus member of the C-terminal Binding Protein family of transcriptional co-repressors (XCtBP) as the C-terminal co-repressor. We show that two XCtBP binding sites near the XTcf-3 carboxy-terminus are required for the interaction of XTcf-3 and XCtBP and for the transcriptional repression mediated by the XTcf-3 carboxy-terminal domain. By fusing the GAL4 activation domain to XCtBP we have generated an antimorphic protein, XCtBP/G4A, that activates siamois transcription through an interaction with endogenous XTcf-3. Ectopic expression of XCtBP/G4A demonstrates that XCtBP functions in the regulation of head and notochord development. Our data support a role for XCtBP as a co-repressor throughout Xenopus development and indicate that XCtBP/G4A will be a useful tool in determining how XCtBP functions in various developmental processes.

Amino Acid Sequence↗

PubMed1999

Membrane-tethered Drosophila Armadillo cannot transduce Wingless signal on its own.

Drosophila Armadillo and its vertebrate homolog beta-catenin are key effectors of Wingless/Wnt signaling. In the current model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin, which then accumulates in nuclei and binds TCF/LEF family proteins, forming bipartite transcription factors which activate transcription of Wingless/Wnt responsive genes. This model was recently challenged. Overexpression in Xenopus of membrane-tethered beta-catenin or its paralog plakoglobin activates Wnt signaling, suggesting that nuclear localization of Armadillo/beta-catenin is not essential for signaling. Tethered plakoglobin or beta-catenin might signal on their own or might act indirectly by elevating levels of endogenous beta-catenin. We tested these hypotheses in Drosophila by removing endogenous Armadillo. We generated a series of mutant Armadillo proteins with altered intracellular localizations, and expressed these in wild-type and armadillo mutant backgrounds. We found that membrane-tethered Armadillo cannot signal on its own; however it can function in adherens junctions. We also created mutant forms of Armadillo carrying heterologous nuclear localization or nuclear export signals. Although these signals alter the subcellular localization of Arm when overexpressed in Xenopus, in Drosophila they have little effect on localization and only subtle effects on signaling. This supports a model in which Armadillo's nuclear localization is key for signaling, but in which Armadillo intracellular localization is controlled by the availability and affinity of its binding partners.

Animals↗

PubMed1998

Control of neural crest cell fate by the Wnt signalling pathway.

Environmental signals are important in the development of neural crest, during which process multipotent progenitor must choose from several fates. However, the nature of these environmental signals is unknown. A previous fate map of zebrafish cranial neural crest showed that lineage-restricted clones of pigment cells arise from medial cells near the neural keel, and that clones of neurons arise from lateral cells farther from the neural keel. Wnt-1 and Wnt-3a are candidate genes for influencing neural crest fate, as they are expressed next to medial, but not lateral, crest cells. Here we determine the role of Wnt signals in modulating the fate of neural crest by injecting messenger RNAs into single, premigratory neural crest cells of zebrafish. Lineage analysis of injected cells shows that activation of Wnt signalling by injection of mRNA encoding cytoplasmic beta-catenin promotes pigment-cell formation at the expense of neurons and glia. Conversely, inhibition of the Wnt pathway, by injection of mRNAs encoding either a truncated form of the transcription factor Tcf-3 or a dominant-negative Wnt, promotes neuronal fates at the expense of pigment cells. We conclude that endogenous Wnt signalling normally promotes pigment-cell formation by medial crest cells and thereby contributes to the diversity of neural crest cell fates.

Animals↗

PubMed1998

Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways.

In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. Using a heterologous system, we show that Dsh is recruited selectively to the membrane by Fz but not DFz2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. We propose that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation.

Adaptor Proteins, Signal Transducing↗

PubMed1998

From cortical rotation to organizer gene expression: toward a molecular explanation of axis specification in Xenopus.

After fertilization of Xenopus eggs, the cortex rotates relative to the cytoplasm, resulting in the formation of a cytoplasmic and transplantable dorsal-determining activity opposite the sperm entry point. This activity induces the dorsal expression of regulatory genes, which in turn establishes the Spemann organizer at the start of gastrulation. There has been considerable debate as to whether Vg1, or components of the Wnt-1 signaling pathway, normally function as this early dorsal determinant. Experiments now support the hypothesis that beta-catenin, a component of the Wnt pathway, provides the initial dorsoventral polarity to the embryo, and that Vg1 functions at a subsequent step in development. Specifically, beta-catenin is required for formation of the endogenous axes, and it is expressed at greater levels in dorsal cells during the early cleavage stages. Moreover, on the dorsal side of the embryo, complexes of beta-catenin and Tcf-3 directly bind the promoter of the dorsal regulatory genes siamois and twin and facilitate their expression, thereby contributing to the subsequent formation of the Spemann organizer. On the ventral side of the embryo, Tcf-3 likely works in the absence of beta-catenin as a transcriptional repressor of siamois. These and other data are considered in the context of how the initial polarization of the fertilized egg by the localized accumulation of beta-catenin establishes a range of subsequent dorsoventral asymmetries in the embryo.

Animals↗

PubMed1998

BMP-2/-4 and Wnt-8 cooperatively pattern the Xenopus mesoderm.

Establishment of the dorsoventral axis is central to animal embryonic organization. In Xenopus two different classes of signaling molecules function in the dorsoventral patterning of the mesoderm. Both the TGF-beta-related products of the BMP-2 and BMP-4 genes and the Wnt molecule encoded by Xenopus Wnt-8 specify ventral fate and appear to inhibit dorsal mesodermal development. The similar functions of these molecularly very different classes of signaling molecules prompted us to study their mutual regulation and to closely compare their roles in mesoderm patterning. We find that Wnt-8 and BMP-4 are indistinguishable in their abilities to induce expression of ventral genes. Although BMP-2/-4 signaling regulates Wnt-8 expression, these genes do not function in a linear pathway because Wnt-8 overexpression cannot compensate for an inhibition of BMP-2/-4 function, but rather BMP-4 overexpression rescues ventral gene expression in embryos with inhibited Wnt-8 function. We further find that Wnt-8 and BMP-2/-4 differ in their abilities to regulate dorsal gene expression. While BMP-4 appears to generally inhibit the expression of dorsal genes, XenopusWnt-8 only inhibits the expression of the notochord marker Xnot. Whereas the inhibitory effect of BMP-2/-4 localizes dorsal mesodermal fate, our results suggest that Xenopus Wnt-8 functions in the further patterning of the dorsal mesoderm into the most dorsal sector from which the notochord develops and the dorsolateral sector from where the somites differentiate.

Animals↗

PubMed1998

Wnt signaling: why is everything so negative?

The Wnt proteins constitute a family of secreted glycoproteins the members of which have essential signaling roles during embryogenesis. The recent identification of several new regulators of this signal transduction pathway have revealed unexpectedly intricate levels of constraint on Wnt-dependent gene activation, and studies in developing embryos and in cell culture systems have allowed a more complete understanding of the functional and biochemical interactions between components of this evolutionarily conserved pathway.

Animals↗

PubMed1998

Production of androgenetic haploids in zebrafish with ultraviolet light.

We describe a simple method for producing androgenetic haploid embryos in zebrafish. Mature eggs are expressed, and the maternal genome is inactivated by irradiation with short-wave ultraviolet light prior to in vitro fertilization. We demonstrate the absence of maternal genome in the resultant embryos using assays based on morphology and polymerase chain reaction. This technique for the production of androgenetic haploids will be extremely useful for identifying and recovering mutations as a complement to, or in place of, generating gynogenetic haploids.

Animals↗

PubMed1997

Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signalling.

In Drosophila, members of the frizzled family of tissue-polarity genes encode proteins that are likely to function as cell-surface receptors of the type known as Wnt receptors, and to initiate signal transduction across the cell membrane, although how they do this is unclear. We show here that the rat protein Frizzled-2 causes an increase in the release of intracellular calcium which is enhanced by Xwnt-5a, a member of the Wnt family. This release of intracellular calcium is suppressed by an inhibitor of the enzyme inositol monophosphatase and hence of the phosphatidylinositol signalling pathway; this suppression can be rescued by injection of the compound myo-inositol, which overcomes the decrease in this intermediate caused by the inhibitor. Agents that inhibit specific G-protein subunits, pertussis toxin, GDP-beta-S and alpha-transducin also inhibit the calcium release triggered by Xwnt-5a and rat Frizzled-2. Our results indicate that some Wnt proteins work through specific Frizzled homologues to stimulate the phosphatidylinositol signalling pathway via heterotrimeric G-protein subunits.

Animals↗