Investigating Tonic Wnt Signaling Throughout the Adult CNS and in the Hippocampal Neurogenic Niche of BatGal and Ins-TopGal Mice

Wnt/β-catenin signaling has a well-established role in the development of the central nervous system (CNS), and recent evidence is extending this role to include the regulation of adult hippocampal function, including neurogenesis within the dentate gyrus. While the neuroanatomical expression pattern of many canonical Wnt signaling components have been investigated, the sites of signal integration and functional downstream β-catenin activation remain comparatively less characterized in the adult CNS. Using two independent transgenic β-catenin-activated LacZ reporter mouse lines (BatGal and ins-TopGal), we demonstrate that Wnt/β-catenin signaling is active in discrete regions of the adult mouse CNS. Intriguingly, BatGal mice exhibit a broad pattern of reporter expression in the CNS, while expression in ins-TopGal mice is more restricted. Further investigation of these two lines reveals temporal differences in β-catenin-activated reporter expression during neurogenesis within the adult hippocampus. Ins-TopGal mice display peaks of Wnt/β-catenin-activated reporter expression during early and later stages of neurogenesis suggesting Wnt/β-catenin signaling plays an important role during both progenitor cell amplification as well as neuronal maturation, integration, and/or maintenance; however, results from BatGal mice are not as convincing. Thus our data using ins-TopGal mice are consistent with the idea that Wnt signaling plays diverse roles during adult hippocampal neurogenesis and support the idea that multiple transgenic reporter lines must be rigorously compared during scientific investigations.     

Nuclear factor of activated T cells (NFAT) proteins repress canonical Wnt signaling via its interaction with Dishevelled (Dvl) protein and participate in regulating neural progenitor cell proliferation and differentiation

The Ca(2+) signaling pathway appears to regulate the processes of the early development through its antagonism of canonical Wnt/β-catenin signaling pathway. However, the underlying mechanism is still poorly understood. Here, we show that nuclear factor of activated T cells (NFAT), a component of Ca(2+) signaling, interacts directly with Dishevelled (Dvl) in a Ca(2+)-dependent manner. A dominant negative form of NFAT rescued the inhibition of the Wnt/β-catenin pathway triggered by the Ca(2+) signal. NFAT functioned downstream of β-catenin without interfering with its stability, but influencing the interaction of β-catenin with Dvl by its competitively binding to Dvl. Furthermore, we demonstrate that NFAT is a regulator in the proliferation and differentiation of neural progenitor cells by modulating canonical Wnt/β-catenin signaling pathway in the neural tube of chick embryo. Our findings suggest that NFAT negatively regulates canonical Wnt/β-catenin signaling by binding to Dvl, thereby participating in vertebrate neurogenesis.     

WNT protein-independent constitutive nuclear localization of beta-catenin protein and its low degradation rate in thalamic neurons

Nuclear localization of β-catenin is a hallmark of canonical Wnt signaling, a pathway that plays a crucial role in brain development and the neurogenesis of the adult brain. We recently showed that β-catenin accumulates specifically in mature thalamic neurons, where it regulates the expression of the Ca(v)3.1 voltage-gated calcium channel gene. Here, we investigated the mechanisms underlying β-catenin accumulation in thalamic neurons. We report that a lack of soluble factors produced either by glia or cortical neurons does not impair nuclear β-catenin accumulation in thalamic neurons. We next found that the number of thalamic neurons with β-catenin nuclear localization did not change when the Wnt/Dishevelled signaling pathway was inhibited by Dickkopf1 or a dominant negative mutant of Dishevelled3. These results suggest a WNT-independent cell-autonomous mechanism. We found that the protein levels of APC, AXIN1, and GSK3β, components of the β-catenin degradation complex, were lower in the thalamus than in the cortex of the adult rat brain. Reduced levels of these proteins were also observed in cultured thalamic neurons compared with cortical cultures. Finally, pulse-chase experiments confirmed that cytoplasmic β-catenin turnover was slower in thalamic neurons than in cortical neurons. Altogether, our data indicate that the nuclear localization of β-catenin in thalamic neurons is their cell-intrinsic feature, which was WNT-independent but associated with low levels of proteins involved in β-catenin labeling for ubiquitination and subsequent degradation.     

The Niche Factor Syndecan-1 Regulates the Maintenance and Proliferation of Neural Progenitor Cells during Mammalian Cortical Development

Neural progenitor cells (NPCs) divide and differentiate in a precisely regulated manner over time to achieve the remarkable expansion and assembly of the layered mammalian cerebral cortex. Both intrinsic signaling pathways and environmental factors control the behavior of NPCs during cortical development. Heparan sulphate proteoglycans (HSPG) are critical environmental regulators that help modulate and integrate environmental cues and downstream intracellular signals. Syndecan-1 (Sdc1), a major transmembrane HSPG, is highly enriched in the early neural germinal zone, but its function in modulating NPC behavior and cortical development has not been explored. In this study we investigate the expression pattern and function of Sdc1 in the developing mouse cerebral cortex. We found that Sdc1 is highly expressed by cortical NPCs. Knockdown of Sdc1 in vivo by in utero electroporation reduces NPC proliferation and causes their premature differentiation, corroborated in isolated cells in vitro. We found that Sdc1 knockdown leads to reduced levels of β-catenin, indicating reduced canonical Wnt signaling. Consistent with this, GSK3β inhibition helps rescue the Sdc1 knockdown phenotype, partially restoring NPC number and proliferation. Moreover, exogenous Wnt protein promotes cortical NPC proliferation, but this is prevented by Sdc1 knockdown. Thus, Sdc1 in the germinal niche is a key HSPG regulating the maintenance and proliferation of NPCs during cortical neurogenesis, in part by modulating the ability of NPCs to respond to Wnt ligands.     

Study of Wnt2 secreted by A-549 cells in paracrine activation of β-catenin in co-cultured mesenchymal stem cells

The canonical Wnt signal pathway is a key regulator of self-renewal and cell fate determination in various types of stem cells. The total pool of β-catenin consists of two different forms: the signaling form of the protein transmits the Wnt signals from the cell membrane to the target genes, whereas the membrane β-catenin is involved in formation of cell-to-cell contact at cadherin junctions. Earlier we developed an in vitro model of epithelial differentiation of mesenchymal stem cells (MSCs) co-cultured with epithelial A-549 cells. The purpose of the present work was to study the role of Wnt2 secreted by the A-549 cells in paracrine induction of β-catenin in co-cultured MSCs. Using the somatic gene knockdown technique, we obtained A-549 cell cultures with down-regulated WNT2. The MSCs co-cultured with the control A-549 cells displayed an increase in the levels of total cellular and signaling β-catenin and transactivation of a reporter construction containing the Lef/Tcf protein family binding sites. In contrast, β-catenin was not induced in the MSCs co-cultured with the A-549 cells with down-regulated WNT2 expression, but the total protein level was increased. We suggest that Wnt2 secreted by A-549 cells induces in co-cultured MSCs the Wnt/β-catenin signaling pathway, whereas the associated increase in total β-catenin level should be due to another mechanism.     

Bcl-2 increases stroke-induced striatal neurogenesis in adult brains by inhibiting BMP-4 function via activation of β-catenin signaling

Our previous experiments suggest that treatment with Bcl-2 increases proliferation and differentiation of neuronal progenitors induced by ischemic injury and ameliorates neurological functional deficits after stroke. However, in addition to its traditional anti-apoptotic effect, little is known about the concrete molecular modulation mechanism. In this study, Bcl-2-expressing plasmids were injected into the lateral ventricle of rat brains immediately following a 30-min occlusion of the middle cerebral artery to determine the role of Bcl-2 in adult neurogenesis. Bcl-2 overexpression reduced ischemic infarct and astrogenesis, and enhanced ischemia-induced striatal neurogenesis. We further found that Bcl-2 increased β-catenin, a key mediator of canonical Wnt/β-catenin signaling pathway, and reduced bone morphogenetic proteins-4 (BMP-4) expression in the ipsilateral striatum following ischemia. Treatment of stroke with β-catenin siRNA (i.c.v.) showed that β-catenin siRNA antagonized Bcl-2 neuroprotection against ischemic brain injury. More interestingly, β-catenin siRNA simultaneously abolished Bcl-2-mediated reduction of BMP-4 expression and enhancement of neurogenesis in the ipsilateral striatum. This effect is independent of Noggin, the known BMP antagonist. These findings highlight a new regulatory mechanism that Bcl-2 elevates ischemia-induced striatal neurogenesis by down-regulating expression of BMP-4 via activation of the Wnt/β-catenin signaling pathway in adult rat brains.     

Leucine-rich amelogenin peptide induces osteogenesis by activation of the Wnt pathway

We previously showed that one of the amelogenin splicing isoforms, Leucine-rich amelogenin peptide (LRAP), induced osteogenic differentiation of mouse embryonic stem cells; however, the signaling pathway(s) activated by LRAP remained unknown. Here, we demonstrated that the canonical Wnt/β-catenin signaling is activated upon LRAP treatment, as evidenced by elevated β-catenin level and increased Wnt reporter gene activity. Furthermore, a specific Wnt inhibitor sFRP-1 completely blocks the LRAP-mediated Wnt signaling. However, exogenous recombinant Wnt3a alone was less effective at osteogenic induction of mouse ES cells in comparison to LRAP. Using a quantitative real-time PCR array, we discovered that LRAP treatment up-regulated the expression of Wnt agonists and down-regulated the expression of Wnt antagonists. We conclude that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists.     

Effects of canonical Wnt signaling on dorso-ventral specification of the mouse telencephalon

Wnt signaling is involved in numerous processes during vertebrate CNS development. In this study, we used conditional Cre/loxP system in mouse to ablate or activate beta-catenin in the telencephalon in two time windows: before and after the onset of neurogenesis. We show that beta-catenin mediated Wnt signals are required to maintain the molecular identity of the pallium. Inactivation of beta-catenin in the telencephalon before neurogenesis results in downregulated expression of dorsal markers Emx1, Emx2 and Ngn2, and in ectopic up-regulation of ventral markers Gsh2, Mash1 and Dlx2 in the pallium. In contrast, ablation of ss-catenin after the onset of cortical neurogenesis (E11.5) does not result in a dorso-ventral fate shift. In addition, activation of canonical Wnt signaling in the subpallium leads to a repression of ventral telencephalic cell identities as shown by the down-regulation of subpallial markers Dlx2, Nkx2.1, Gsh2, Olig2 and Mash1. This was accompanied with an expansion of dorsal identities ventrally as shown by the expanded expression domains of pallial markers Pax6 and Ngn2. Thus, our data suggest that canonical Wnt signals are involved in maintaining the identity of the pallium by controlling expression of dorsal markers and by suppressing ventral programs from being activated in pallial progenitor cells.     

Polyethylenimine-cationized β-catenin protein transduction activates the Wnt canonical signaling pathway more effectively than cationic lipid-based transduction

The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by β-catenin protein transduction. Constitutively active β-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active β-catenin protein was added to HEK-293 cells, and induction of several Wnt/β-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active β-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active β-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.     

Beta-Catenin Signaling Negatively Regulates Intermediate Progenitor Population Numbers in the Developing Cortex

Intermediate progenitor cells constitute a second proliferative cell type in the developing mammalian cerebral cortex. Little is known about the factors that govern the production of intermediate progenitors. Although persistent expression of stabilized β-catenin was found to delay the maturation of radial glial progenitors into intermediate progenitors, the relationship between β-catenin signaling and intermediate progenitors remains poorly understood. Using a transgenic reporter mouse for Axin2, a direct target of Wnt/β-catenin signaling, we observed that β-catenin signaling is decreased in intermediate progenitor cells relative to radial glial progenitors. Conditional deletion of β-catenin from mouse cortical neural progenitors increased intermediate progenitor numbers, while conditional expression of stabilized β-catenin reduced the intermediate progenitor population. Together, these findings provide evidence that β-catenin signaling in radial progenitors negatively regulates intermediate progenitor cell number during cortical development.