Prenatal ß-catenin/Brn2/Tbr2 transcriptional cascade regulates adult social and stereotypic behaviors.

Social interaction is a fundamental behavior in all animal species, but the developmental timing of the social neural circuit formation and the cellular and molecular mechanisms governing its formation are poorly understood. We generated a mouse model with mutations in two Disheveled genes, Dvl1 and Dvl3, that displays adult social and repetitive behavioral abnormalities associated with transient embryonic brain enlargement during deep layer cortical neuron formation. These phenotypes were mediated by the embryonic expansion of basal neural progenitor cells (NPCs) via deregulation of a ß-catenin/Brn2/Tbr2 transcriptional cascade. Transient pharmacological activation of the canonical Wnt pathway during this period of early corticogenesis rescued the ß-catenin/Brn2/Tbr2 transcriptional cascade and the embryonic brain phenotypes. Remarkably, this embryonic treatment prevented adult behavioral deficits and partially rescued abnormal brain structure in Dvl mutant mice. Our findings define a mechanism that links fetal brain development and adult behavior, demonstrating a fetal origin for social and repetitive behavior deficits seen in disorders such as autism.

Regulation of endodermal differentiation of human embryonic stem cells through integrin-ECM interactions.

Many cellular responses during development are regulated by interactions between integrin receptors and extracellular matrix proteins (ECMPs). Although the majority of recent studies in human embryonic stem cell (hESC) differentiation have focused on the role of growth factors, such as FGF, TGFß, and WNT, relatively little is known about the role of ECMP-integrin signaling in this process. Moreover, current strategies to direct hESC differentiation into various lineages are inefficient and have yet to produce functionally mature cells in vitro. This suggests that additional factors, such as ECMPs, are required for the efficient differentiation of hESCs. Using a high-throughput multifactorial cellular array technology, we investigated the effect of hundreds of ECMP combinations and concentrations on differentiation of several hPSC lines to definitive endoderm (DE), an early embryonic cell population fated to give rise to internal organs such as the lung, liver, pancreas, stomach, and intestine. From this screen we identified fibronectin (FN) and vitronectin (VTN) as ECMP components that promoted DE differentiation. Analysis of integrin expression revealed that differentiation toward DE led to an increase in FN-binding integrin α5 (ITGA5) and VTN-binding integrin αV (ITGAV). Conditional short hairpin RNA-mediated knockdown of ITGA5 and ITGAV disrupted hESC differentiation toward DE. Finally, fluorescence-based cell sorting for ITGA5 and ITGAV significantly enriched cells with gene expression signatures associated with DE, demonstrating that these cell surface proteins permit isolation and enrichment of DE from hESCs. These data provide evidence that FN and VTN promote endoderm differentiation of hESCs through interaction with ITGA5 and ITGAV, and that ECMP-integrin interactions are required for hESC differentiation into functionally mature cells.

The status of Wnt signalling regulates neural and epidermal fates in the chick embryo.

The acquisition of neural fate by embryonic ectodermal cells is a fundamental step in the formation of the vertebrate nervous system. Neural induction seems to involve signalling by fibroblast growth factors (FGFs) and attenuation of the activity of bone morphogenetic protein (BMP). But FGFs, either alone or in combination with BMP antagonists, are not sufficient to induce neural fate in prospective epidermal ectoderm of amniote embryos. These findings suggest that additional signals are involved in the specification of neural fate. Here we show that the state of Wnt signalling is a critical determinant of neural and epidermal fates in the chick embryo. Continual Wnt signalling blocks the response of epiblast cells to FGF signals, permitting the expression and signalling of BMP to direct an epidermal fate. Conversely, a lack of exposure of epiblast cells to Wnt signals permits FGFs to induce a neural fate.

Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism.

Lymphocyte enhancer factor-1 (LEF-1) is a member of the LEF-1/TCF family of transcription factors, which have been implicated in Wnt signaling and tumorigenesis. LEF-1 was originally identified in pre-B and T cells, but its function in B lymphocyte development remains unknown. Here we report that LEF-1-deficient mice exhibit defects in pro-B cell proliferation and survival in vitro and in vivo. We further show that Lef1-/- pro-B cells display elevated levels of fas and c-myc transcription, providing a potential mechanism for their increased sensitivity to apoptosis. Finally, we establish a link between Wnt signaling and normal B cell development by demonstrating that Wnt proteins are mitogenic for pro-B cells and that this effect is mediated by LEF-1.

A Drosophila Axin homolog, Daxin, inhibits Wnt signaling.

The vertebrate Axin protein, the product of the mouse fused gene, binds to beta-catenin to inhibit Wnt signaling. We have identified a homolog of Axin in Drosophila, Daxin. Using double-stranded RNA interference, we generated loss-of-function phenotypes that are similar to overexpression of the Drosophila Wnt gene wingless (wg). Overexpression of Daxin produces phenotypes similar to loss of wg. In addition, we show that Daxin overexpression can modify phenotypes elicited by wg and another Drosophila Wnt gene, DWnt-2. Using immunoprecipitation of endogenous Daxin protein from embryos we show that Daxin interacts with Armadillo and Zeste-white 3. The loss-of-function and overexpression phenotypes show that Daxin, like its mammalian counterpart, acts as a negative regulator of wg/Wnt signaling.

Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex.

The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it was not clear whether and how Wnt signaling regulates the beta-catenin complex. Here we show that Axin is dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery.

WNT signaling in the control of hair growth and structure.

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.

Beta-catenin: a key mediator of Wnt signaling.

Beta-catenin is a pivotal player in the signaling pathway initiated by Wnt proteins, mediators of several developmental processes. beta-catenin activity is controlled by a large number of binding partners that affect the stability and the localization of beta-catenin and is thereby able to participate in such varying processes as gene expression and cell adhesion. Activating mutations in beta-catenin and in components regulating its stability can contribute to the formation of certain tumors.

Two members of the Tcf family implicated in Wnt/beta-catenin signaling during embryogenesis in the mouse.

Tcf transcription factors interact with beta-catenin and Armadillo to mediate Wnt/Wingless signaling. We now report the characterization of genes encoding two murine members of the Tcf family, mTcf-3 and mTcf-4. mTcf-3 mRNA is ubiquitously present in embryonic day 6.5 (E6.5) mouse embryos but gradually disappears over the next 3 to 4 days. mTcf-4 expression occurs first at E10.5 and is restricted to di- and mesencephalon and the intestinal epithelium during embryogenesis. The mTcf-3 and mTcf-4 proteins bind a canonical Tcf DNA motif and can complex with the transcriptional coactivator beta-catenin. Overexpression of Wnt-1 in a mammary epithelial cell line leads to the formation of a nuclear complex between beta-catenin and Tcf proteins and to Tcf reporter gene transcription. These data demonstrate a direct link between Wnt stimulation and beta-catenin/Tcf transcriptional activation and imply a role for mTcf-3 and -4 in early Wnt-driven developmental decisions in the mouse embryo.

Casein kinase 2 associates with and phosphorylates dishevelled.

The dishevelled (dsh) gene of Drosophila melanogaster encodes a phosphoprotein whose phosphorylation state is elevated by Wingless stimulation, suggesting that the phosphorylation of Dsh and the kinase(s) responsible for this phosphorylation are integral parts of the Wg signaling pathway. We found that immunoprecipitated Dsh protein from embryos and from cells in tissue culture is associated with a kinase activity that phosphorylates Dsh in vitro. Purification and peptide sequencing of a 38 kDa protein co-purifying with this kinase activity showed it to be identical to Drosophila Casein Kinase 2 (CK2). Tryptic phosphopeptide mapping indicates that identical peptides are phosphorylated by CK2 in vitro and in vivo, suggesting that CK2 is at least one of the kinases that phosphorylates Dsh. Overexpression of Dfz2, a Wingless receptor, also stimulated phosphorylation of Dsh, Dsh-associated kinase activity, and association of CK2 with Dsh, thus suggesting a role for CK2 in the transduction of the Wg signal.

The Wnt-1 proto-oncogene induces changes in morphology, gene expression, and growth factor responsiveness in PC12 cells.

The product of the Wnt-1 proto-oncogene is a secreted glycoprotein that is normally produced in regions of the embryonic neural tube. We show here that expression of mouse Wnt-1 cDNA in the rat PC12 pheochromocytoma cell line causes a dramatic conversion from a round to a flat cell morphology. In addition, PC12 cells expressing Wnt-1 (PC12/Wnt-1) fail to extend neurites after treatment with NGF, despite the presence and activation of high affinity NGF receptors encoded by the trk gene and the induction of early response genes. Furthermore, PC12/Wnt-1 cells fail to express several neuron- and chromaffin-specific genes, indicating that PC12/Wnt-1 cells have assumed a new phenotype. Although NGF and FGF utilize similar signal transduction pathways in PC12 cells, only FGF is capable of inducing a morphological response and synthesis of transin mRNA in PC12/Wnt-1 cells.

Efficient incorporation of human CD4 protein into avian leukosis virus particles.

Virus envelope (Env) proteins are thought to contain specific signals for selective uptake by virus particles. In the course of attempting to define these signals by testing virus incorporation of CD4-Env chimeric proteins, normal human CD4 was found to be efficiently and selectively assembled into avian leukosis virus particles in quail cells. Viruses bearing CD4 at their surface may be useful reagents in the design of retrovirus-mediated gene therapy for the acquired immune deficiency syndrome.

Microautoradiographic investigations of sulfate uptake by glands and epidermal cells of water lily (Nymphaea) leaves with special reference to the effect of poly-L-lysine.

The uptake of(35)S-labelled sulfate ions into hydropote cells (densely cytoplasmic gland cells) and into epidermal cells (highly vacuolated cells) ofNymphaea leaves is dependent on metabolic energy. Only a very small fraction of the accumulated(35)S is incorporated into organic macromolecules during the experimental period. Both cell types exhibit a hyperbolic isotherm for(35)S uptake from labelled K2SO4 solutions over an external concentration range of 0 to 0.5MM. Although the gland and epidermal cells behave qualitatively similarly, the glands generally absorb about twice as much(35)S per unit area of sections of the cells as do the epidermal cells. At 3 °C, poly-L-lysine concentrations of 10(-8) M and up to 10(-7) M enhance(35)S uptake by the epidermal and gland cells for the first 7.5 hr after application of the poly-L-lysine. Samples treated with 5×10(-7) M poly-L-lysine are indistinguishable from the controls over the same period. After longer periods of treatment with poly-L-lysine (7.5 to 24 hr), the rates of(35)S uptake were reduced by all poly-L-lysine concentrations between the range 10(-8) to 5×10(-7) M. After 7.5 hr of(35)S uptake, the control samples contained the smallest amount of label, but after an uptake period of 24 hr the amount of label in the controls is considerably larger than in samples treated with poly-L-lysine. The results suggest that poly-L-lysine increases the membrane permeability and alters the metabolic uptake of sulfate in both hydropotes and epidermal cells.

[Phytochrome dependent variation of grouth and ion uptake of leaves and internodes of etiolated pea (pisum sativum) seedlings]. / Phytochromabhängige Veränderungen des Wachstums und der Ionenaufnahme etiolierter Erbsenkeimlinge.

Inhibition of internodial growth of pea seedlings by light is compensated for by increased growth of leaves. At a given time the sum of fresh weight of internodes plus the product of fresh weight of leaves times a certain factor is constant in darkness or with different periods of light. This correlation may reflect a competition of internodes and leaves for materials delivered at a lightindependent rate from the cotyledons. This hypothesis was tested by immersing roots of pea seedlings into (86)Rb labelled K-solutions for one day in darkness, removing the plants from the solutions, exposing the seedlings to near or far red light and measuring the radioactivity and fresh weights of leaves and internodes separately. Radioactivity and fresh-weight were both dependent on phytochrome; i.e. inhibition of ion uptake and of growth in internodes and promotion of both processes in leaves by near red light as compared to dark or far red controls are mediated by phytochrome.Short time experiments of ion uptake by the roots show that K transport into the shoot organs is promoted by light after a lag phase of somewhat more than one hour. This interval corresponds well to the lag phase of the light induced growth inhibition of internodes.Seedlings deprived of cotyledons and roots grow well in water but exhibit no difference in growth rate of leaves and internodes in light and darkness. Light dependence is restored if the seedlings are submersed in approximately 3% sucrose solutions. This result seems to indicate that the influence of light on growth rates of leaves and internodes is dependent on the uptake of material by the cell. It seems possible that in the etiolated pea seedling light promotes growth of leaves by promoting uptake and hampers growth of internodes by inhibiting uptake of essential growth material delivered from the cotyledons.