progenitor cells

Brain Research 1651 (2016) 73–87

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Manipulating Wnt signaling at different subcellular levels affects the fate of neonatal neural stem/progenitor cells Jan Kriska a,b, Pavel Honsa a, David Dzamba a, Olena Butenko a, Denisa Kolenicova a, Lucie Janeckova a,c, Zuzana Nahacka c, Ladislav Andera c, Zbynek Kozmik c, M. Mark Taketo d, Vladimir Korinek a,c, Miroslava Anderova a,b,n a

Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic 2nd Faculty of Medicine, Charles University in Prague, V Uvalu 84, 150 06 Prague 5, Czech Republic c Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic d Department of Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida Konoé-cho, Sakyo-ku, Kyoto 606-8501, Japan b

art ic l e i nf o

a b s t r a c t

Article history: Received 18 April 2016 Received in revised form 2 August 2016 Accepted 18 September 2016 Available online 19 September 2016

The canonical Wnt signaling pathway plays an important role in embryogenesis, and the establishment of neurogenic niches. It is involved in proliferation and differentiation of neural progenitors, since elevated Wnt/β-catenin signaling promotes differentiation of neural stem/progenitor cells (NS/PCs1) towards neuroblasts. Nevertheless, it remains elusive how the differentiation program of neural progenitors is influenced by the Wnt signaling output. Using transgenic mouse models, we found that in vitro activation of Wnt signaling resulted in higher expression of β-catenin protein and Wnt/β-catenin target genes, while Wnt signaling inhibition resulted in the reverse effect. Within differentiated cells, we identified three electrophysiologically and immunocytochemically distinct cell types, whose incidence was markedly affected by the Wnt signaling output. Activation of the pathway suppressed gliogenesis, and promoted differentiation of NS/PCs towards a neuronal phenotype, while its inhibition led to suppressed neurogenesis and increased counts of cells of glial phenotype. Moreover, Wnt signaling hyperactivation resulted in an increased incidence of cells expressing outwardly rectifying K þ currents, together with inwardly rectifying Na þ currents, a typical current pattern of immature neurons, while blocking the pathway led to the opposite effect. Taken together, our data indicate that the Wnt signaling pathway orchestrates neonatal NS/PCs differentiation towards cells with neuronal characteristics, which might be important for nervous tissue regeneration during central nervous system disorders. Furthermore, the transgenic mouse strains used in this study may serve as a convenient tool to manipulate βcatenin-dependent signaling in neural progenitors in the neonatal brain. & 2016 Published by Elsevier B.V.

Keywords: β-catenin signaling Neonatal mouse Neurogenesis Gliogenesis Patch-clamp technique Ion channel

1. Introduction The Wnt family constitutes a large group of secreted cysteinerich glycosylated proteins that are implicated in many cell processes, such as embryonic cell patterning, proliferation, differentiation, and programmed cell death (reviewed in Clevers et al.

n Correspondence to: Department of Cellular Neurophysiology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech Republic. E-mail addresses: [email protected] (J. Kriska), [email protected] (P. Honsa), [email protected] (D. Dzamba), [email protected] (O. Butenko), [email protected] (D. Kolenicova), [email protected] (L. Janeckova), [email protected] (Z. Nahacka), [email protected] (L. Andera), [email protected] (Z. Kozmik), [email protected] (M.M. Taketo), [email protected] (V. Korinek), [email protected] (M. Anderova). 1 NS/PCs, neural stem/progenitor cells.

http://dx.doi.org/10.1016/j.brainres.2016.09.026 0006-8993/& 2016 Published by Elsevier B.V.

(2014), Lim and Nusse (2013), Nusse and Varmus (2012)). Three major Wnt signaling pathways have been identified: the canonical Wnt (β-catenin) pathway (Fig. 1, right), and two non-canonical pathways – the planar cell polarity, and Wnt/Ca2 þ pathways (van Amerongen, 2012). The key element of canonical Wnt signaling, βcatenin, plays a crucial role in gene transcription and cell adhesion (Mosimann et al., 2009; Nelson and Nusse, 2004). In this pathway, the interaction between a Wnt ligand and its receptor Frizzled, triggers the recruitment of Axin to the plasma membrane, resulting in the inhibition of β-catenin phosphorylation by glycogen synthase kinase 3β (GSK-3β2), and the subsequent escape of nonphosphorylated β-catenin from degradation in the proteasome. Beta-catenin thus accumulates in the cytoplasm, and then translocates into the nucleus, where it forms a complex with the 2

GSK-3β, glycogen synthase kinase 3β.

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Fig. 1. Modulating the Wnt signaling pathway in transgenic mice. (A-D) Schemes depicting genetic modifications present in the mouse strains used in the study. (A) Suppression of Wnt signaling at the membrane level, due to the over-expression of a secreted Wnt inhibitor, Dkk1. (B) Activation of the pathway caused by the production of a stable variant of β-catenin. Cre-mediated excision of exon 3 (E3, red rectangle) removes the amino acid sequence involved in the degradation of the protein, and thus stable β-catenin aberrantly activates Wnt target genes. (C) The Wnt-responsive transcription in the nucleus blocked by dnTCF4 protein. (D) Cre deletor mouse, Rosa26CreERT2, carries the gene encoding tamoxifen-inducible Cre recombinase, fused with a modified form of the estrogen receptor (CreERT2). (Right) Simplified scheme of the canonical Wnt signaling pathway. Red arrows point to the interference site affected in the transgenic mice used in the study. Abbreviations: APC, Adenomatous Polyposis Coli; Cre, Cre recombinase; Dkk1, Dickkopf 1; dnTCF4, dominant negative T-Cell Factor 4; Dvl, Dishevelled; E2 – E10, exon 2 – 10; EGFP, enhanced green fluorescent protein; GSK-3β, Glycogen Synthase Kinase 3β; LRP5/6, Low-density Lipoprotein 5/6; P, phosphorylated site; pA, polyadenylation site; PGK-Neo, neomycin resistance cassette; TCF/ LEF, T-Cell Factor/Lymphoid Enhancer Factor; Wnt, Wingless/Integrated.

transcription factors; T-cell factor, and lymphoid enhancer factor (Tcf3/Lef4), which leads to the activation of target genes (Städeli et al., 2006). The canonical Wnt pathway is tightly regulated and can be suppressed at different cellular levels (reviewed in Varela-Nallar and Inestrosa (2013)). In the extracellular space, members of a multigene family of secreted Dickkopf (Dkk5) proteins can antagonize the pathway by binding to the Wnt co-receptor low-density lipoprotein 5/6 (LRP 5/66) (Glinka et al., 1998; Mao et al., 2002, 2001; Munji et al., 2011). In the nucleus, the inhibitor of β-catenin and Tcf4 (ICAT7) impairs Wnt signaling, by preventing the formation of the complex between β-catenin and Tcf4 (Daniels and Weis, 2002; Tago et al., 2000). In vitro studies using tissue explants from newborn mice have suggested the effect of Wnts on proliferation and differentiation of neural stem/progenitor cells (NS/PCs). These cells are fully competent to respond to Wnt stimulation, as the quantitative reversetranscription polymerase chain reaction (qRT-PCR8) analysis confirmed the expression of genes encoding different Wnt ligands (Hirsch et al., 2007; Wang et al., 2015). Furthermore, the canonical Wnt signaling pathway increases the neonatal hippocampal NS/ PCs proliferation induced by hypoxia (Cui et al., 2011). Recently, we have shown that Wnt7a over-expression significantly promotes 3 4 5 6 7 8

Tcf, T-cell factor. Lef, lymphoid enhancer factor. Dkk1, Dickkopf 1. LRP 5/6, low-density lipoprotein 5/6. ICAT, inhibitor of β-catenin and Tcf4. qRT-PCR, quantitative RT-PCR.

differentiation of neural progenitors towards neuroblasts, while gliogenesis is attenuated (Prajerova et al., 2010). Others have shown that in differentiating cell cultures, elevated canonical Wnt signaling reduces mammalian hairy and enhancer-of-split homologs (Hes9) 1 and Hes5 expression, suggesting that during neural development, β-catenin-mediated signaling enhances neurogenesis from progenitor cells by interfering with the Notch pathway activity (Hirsch et al., 2007). Additionally, in the cochlear nucleus, the first central relay of the auditory pathway, the survival of neurons hinges, to some extent, on neurogenesis within the population of progenitors that display active canonical Wnt signaling (Volkenstein et al., 2013). In vivo experiments have confirmed the Wnt signaling pathway contribution to regulation of neuronal survival and homeostasis, and to proliferation and differentiation of progenitor populations in the hippocampus, and the subventricular zone (SVZ10), two major germinal zones of the postnatal mouse brain (Bowman et al., 2013). It was also suggested that Wnt activation stimulates the proliferation of leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr511)-positive progenitors, which leads to augmented neurogenesis in early ontogenesis stages (Chen et al., 2014). In the adult SVZ, β-catenin signaling is activated in neural stem cells and transit amplifying progenitors. It was demonstrated that increased expression of stabilized β-catenin, and subsequent 9

Hes, hairy and enhancer-of-split. SVZ, subventricular zone. 11 Lgr5, leucine-rich repeat-containing G-protein coupled receptor 5. 10

J. Kriska et al. / Brain Research 1651 (2016) 73–87

inhibition of GSK-3β, increases the number of progenitors leading to increased amounts of newly generated neurons in the olfactory bulb (Adachi et al., 2007). Adult hippocampal neurogenesis is also controlled by canonical Wnt signaling. Blocking Wnt signaling in the rat hippocampus led to a decrease in the population of proliferating neuroblasts, as well as in the number of newly derived doublecortin (DCX12)-positive and maturating microtubule-associated protein 2 (MAP213)-positive neurons, while, simultaneously, the number of glial fibrillary acidic protein (GFAP14)-positive astrocytes was increased (Lie et al., 2005; Yu et al., 2006). Moreover, treatment with andrographolide, a competitive inhibitor of GSK3β, increased NS/PCs proliferation, and the number of newborn neurons in the hippocampus (Varela-Nallar et al., 2015). The above-mentioned data demonstrate the importance of canonical Wnt signaling for the NS/PCs formation and function throughout development as well as in neonatal and adult neurogenesis. Moreover, since NS/PCs possess the capacity of generating other cell types in the central nervous system (CNS15), manipulating Wnt signaling in these cells could provide new insights into neurogenesis/gliogenesis, in both the healthy and diseased CNS. Therefore, we employed three transgenic mouse strains that allow manipulation of the Wnt signaling pathway, and combined several methodical approaches, such as the patch-clamp technique, immunocytochemistry, calcium imaging, Western blot and qRT-PCR, to demonstrate the impact of Wnt signaling on NS/PCs differentiation in the early postnatal stages of mouse ontogenesis, and to introduce suitable animal models for experiments related to Wnt/β-catenin signaling in the neonatal brain.

2. Results 2.1. Expression of

β-catenin and Wnt signaling target genes

To confirm that our in vitro approach, using NS/PCs isolated from dominant negative (dn16) TCF4,17 Dkk1, and Ex3 transgenic mice (for more information see Section 5.1 Transgenic animals), represents a suitable tool for affecting the Wnt signaling pathway, we carried out immunocytochemical, and Western blot analyses of differentiated neonatal NS/PCs for β-catenin, the main effector of the canonical Wnt pathway (Fig. 2A-D and Fig. 3A). After inhibiting the pathway in the nucleus, i.e., in cells producing dnTCF4, there was no significant difference in the levels of β-catenin protein (Fig. 2A and Fig. 2D). However, Wnt signaling pathway suppression by Dkk1 resulted in a marked decrease in the amount of β-catenin (Fig. 2B and Fig. 2D). Moreover, stabilization of β-catenin was clearly detected in Ex3 cells (Fig. 2C and Fig. 2D). Higher total levels of truncated β-catenin protein in Ex3 cells were also detected on Western blots (Fig. 3A). Notice that β-catenin in Ex3 cultures migrates as a double-band, since the mutated variant lacks amino acids encoded by exon 23. As expected, in Ex3 cells, a lower quantity of the N-terminally phosphorylated protein was observed. Using Quantity One software (Bio-Rad, Hercules, CA, USA), we estimated the β-catenin-to-phosphorylated-β-catenin ratio of the peak heights in control and (Z)-4-hydroxytamoxifen (4OHT18)treated cells. In differentiated neonatal NS/PCs, derived from dnTCF4 and Dkk1 mice, the ratio changed only negligibly (from 1.27 to 0.84 in Dkk1, and from 1.00 to 0.85 in dnTCF4 mice). In

contrast, in differentiated neonatal NS/PCs derived from Ex3 mice, the ratio increased from 0.90 (control) to 7.46 (4OHT added). Additionally, we performed the densitometric analysis (Fig. S1), which confirmed a negligible decrease in β-catenin, and an increase in phosphorylated β-catenin, in Dkk1 derived cell cultures. Next, we analyzed the expression of three Wnt signaling target genes – Axin2, SP5 transcription factor (Sp519), and Tumor necrosis factor receptor superfamily member 19 (Troy20). In Fig. 3B, we show that suppression of Wnt signaling in the nucleus led to lower expression of Sp5 (average Ct value of 34.23 70.37 in controls changed to average Ct value of 36.13 70.13 in 4OHT-treated cultures) and Troy (28.11 70.06 to 28.47 70.11), while no changes in the expression of Wnt target genes were detected after inhibiting the pathway at the membrane level. Hyperactivation of the Wnt/ β-catenin pathway resulted in markedly higher expression of all examined target genes (32.74 70.14 to 27.49 70.10 in Axin2, 35.88 70.17 to 26.55 70.10 in Sp5, and 28.86 70.42 to 23.757 0.21 in Troy). Thus, our results indicate that the approach employing NS/PCs isolated from the neonatal frontal cortex of the three types of transgenic mice might represent a suitable in vitro system for manipulating the Wnt signaling pathway. Taken together, we have shown that activation of the pathway resulted in higher amounts of β-catenin protein, and an increased expression of Wnt target genes in differentiated NS/PCs. Inhibiting the pathway at the membrane level or in the nucleus, we showed lower quantities of β-catenin protein, and decreased expression of Wnt target genes, respectively. We attempted to analyze which Wnt ligands are involved in Wnt signaling activation. Our qRT-PCR analysis has revealed that only Wnt4, Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt9a and Wnt10b ligands were present in our cultures (Fig. S2). The expression of other Wnt ligands was very low, or they were not expressed at all. 2.2. The incidence of different cell types among differentiated neonatal neural stem/progenitor cells In order to estimate the impact of the Wnt signaling pathway inhibition/activation on neonatal NS/PC differentiation, we employed the patch-clamp technique in the whole-cell configuration to assess the current profiles of in vitro differentiated cells. Control (n¼ 347) and 4OHT-treated (n ¼380) cells were subsequently divided into three groups, according to their electrophysiological properties (Fig. 4A-G). Flat-shaped GFAP-positive cells (n ¼242; Fig. 4D) displayed passive time- and voltage-independent K þ currents (Fig. 4A), their average resting membrane potential (Vm21) was  86.38 70.26 mV, and their membrane resistance (IR22) was 81.62 71.91 MΩ. Round DCX/MAP2-positive cells (n¼ 289; Fig. 4F, G), expressing fast activating and inactivating outwardly rectifying K þ (KA23) currents, and delayed outwardly rectifying K þ (KDR24) currents (Fig. 4C), were characterized by Vm of  72.04 70.90 mV, and high values of IR (1649.79 744.05 MΩ). Branched plateletderived growth factor alpha receptor (PDGFαR25)-positive cells (n¼ 196; Fig. 4E), with a complex current pattern, expressed inwardly rectifying K þ (KIR26) currents, in addition to KDR and KA currents (Fig. 4B), their Vm was  85.78 70.45 mV, and IR was 218.56 7 8.24 MΩ. Post-recording immunocytochemical identification revealed that the majority of cells expressing passive 19

12 13 14 15 16 17 18

DCX, doublecortin. MAP2, microtubule-associated protein 2. GFAP, glial fibrillary acidic protein. CNS, central nervous system. dn, dominant negative. TCF4, human T-cell factor 4. 4OHT, (Z)-4-hydroxytamoxifen.

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20 21 22 23 24 25 26

Sp5, SP5 transcription factor. Troy, tumor necrosis factor receptor superfamily, member 19. Vm, resting membrane potential. IR, membrane resistance. KA, fast activating and inactivating outwardly rectifying K þ current. KDR, delayed outwardly rectifying K þ current. PDGFαR, platelet-derived growth factor alpha receptor. KIR, inwardly rectifying K þ current.

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Fig. 2. Beta-catenin staining in differentiated neonatal neural stem/progenitor cells. (A-C) Representative fluorescent images of DAPI and β-catenin staining in cells with inhibited (dnTCF4, Dkk1), or activated (Ex3) Wnt signaling. Cells were treated with ethanol (CTRL) or (Z)-4-hydroxytamoxifen (4OHT), and analyzed eight days after the onset of in vitro differentiation. (D) Quantification of β-catenin expression, showing the proportion of the area of positively-stained cells to the DAPI-positive area (n ¼12). The area of β-catenin fluorescence in control cells was arbitrarily set to 1. The values are represented as mean7 S.D. (standard deviation). Statistical significance was calculated using t-test,***, po 0.001. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Dkk1, neural stem/progenitor cells derived from neonatal Dkk1 mice; dnTCF4, neural stem/progenitor cells derived from neonatal dnTCF4 mice; Ex3, neural stem/progenitor cells derived from neonatal Ex3 mice.

currents were GFAP-positive, while most of the cells displaying outwardly rectifying currents were DCX/MAP2-positive. Complex currents were expressed mainly by PDGFαR-positive cells. The electrophysiological analyses also revealed that Wnt signaling inhibition at the nuclear, as well as membrane level (dnTCF4 and Dkk1, respectively) markedly lowered the incidence of cells displaying outwardly rectifying currents (Fig. 5A, out27), and also marginally raised the incidence of cells with a passive current profile (Fig. 5A, pas28). On the other hand, activation of the pathway (Ex3) led to a decreased number of cells with a passive

27 28

out, outwardly rectifying current profile. pas, passive current profile.

current profile (Fig. 5A, pas), and to an increased number of cells with an outwardly rectifying current pattern (Fig. 5A, out); however, neither of the changes in the cell incidence was significant. Both inhibition and activation of the pathway caused an increase in the incidence of cells displaying complex currents (Fig. 5A, com29); nevertheless, such increase was statistically significant only in dnTCF4 cells. Immunocytochemistry confirmed lower expression of DCX and MAP2, in the cells with the suppressed Wnt signaling pathway (Fig. 5B and Fig. 5C, dnTCF4 and Dkk1), and decreased expression of GFAP in the cells after Wnt signaling activation (Fig. 5B and Fig. 5C, Ex3). Furthermore, Western blot 29

com, complex current profile.

J. Kriska et al. / Brain Research 1651 (2016) 73–87

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Fig. 3. Beta-catenin protein and Wnt signaling target genes in differentiated neonatal neural stem/progenitor cells. (A) Western blot analysis of total and phosphorylated βcatenin, with intensity profiles representing the number and size of the respective bands. Arrowheads mark the molecular weight of 92 kDa, which is characteristic of βcatenin. (B) Quantitative RT-PCR analysis of Wnt signaling target genes. Together, four founder mice were used to derive NS/PCs for each mouse strain, and the analysis was performed in technical triplicates. The average Ct (cycle threshold) values were normalized to GAPDH, and two other housekeeping genes (ubiquitin and β-actin) are shown in the graph. Axin2, Sp5 and Troy are Wnt target genes. The expression level of a given gene in control cells was arbitrarily set to 1. The cells were analyzed eight days after the onset of in vitro differentiation. Statistical significance was calculated using t-test,*, p o0.05;**, po 0.01;***, p o 0.001. Abbreviations: 4OHT, tamoxifen-treated cultures; βact, β-actin; β-cat, total β-catenin; CTRL, control cultures; Dkk1, neural stem/progenitor cells derived from neonatal Dkk1 mice; dnTCF4, neural stem/progenitor cells derived from neonatal dnTCF4 mice; Ex3, neural stem/progenitor cells derived from neonatal Ex3 mice; pβ-cat, phosphorylated β-catenin; Sp5, SP5 transcription factor; Troy, Tumor necrosis factor receptor superfamily member 19; Ubb, Ubiquitin.

analysis revealed higher β III tubulin expression in the cells with activated pathway, when compared to the controls (Fig. 5C, Ex3, inset). Taken together, activation of the Wnt signaling pathway increases the expression of β III tubulin, a neuronal marker, and decreases the incidence of GFAP-positive glial cells displaying passive time- and voltage-independent K þ currents. In contrast, upon suppression of the Wnt pathway, the incidence of GFAPpositive cells is increased, and moreover, the number of DCX/ MAP2-positive neuronal cells expressing outwardly rectifying K þ currents is decreased. Thus, these findings imply that the approach may serve as a suitable tool to study the role of Wnt signaling in neonatal neurogenesis/gliogenesis. 2.3. Membrane properties of differentiated neonatal neural stem/ progenitor cells We analyzed passive and active electrophysiological properties of 1245 differentiated cells. In those displaying a passive current pattern (Table 1), the impact of Wnt signaling pathway manipulation on their electrophysiological properties was minimal. Blocking Wnt signaling, affected mainly passive electrophysiological properties, since we found that dnTCF4 and Dkk1 expression resulted in hyperpolarized cell membrane, and lower Cm values. However, Wnt/β-catenin signaling had a more profound effect on electrophysiological properties of differentiated progenitors, showing a complex current pattern (Table 2). Membrane

capacitance (Cm30) decreased after Wnt signaling inhibition, while it was not changed after its activation. Interestingly, current densities of KIR currents were higher, regardless of Wnt signaling inhibition/activation. Wnt signaling manipulation also affected electrophysiological properties of cells expressing outwardly rectifying currents (Table 3). Both inhibition and activation of the Wnt signaling pathway caused hyperpolarization of the cell membrane, but only Wnt signaling inhibition resulted in lower Cm values. Current densities of KDR currents were higher in cells with inhibited Wnt signaling. Besides the differences in the passive membrane properties, and the expression of K þ channels, we identified changes in the expression of voltage-dependent Na þ channels in DCX/MAP2positive neuron-like cells, with an outwardly rectifying current pattern (Fig. 6A), and also changes in the incidence of such cells (Fig. 6B), that were also capable of generating action potentials (Fig. 6C). Inhibition of the Wnt signaling pathway at the membrane and nuclear level led to the absence of cells expressing Na þ channels (Fig. 6B, dnTCF4, and Dkk1), while activation of the pathway caused an increase in the Na þ current densities, as well as in the cell incidence, when compared to the controls (Fig. 6B, Ex3). Measurements of intracellular calcium revealed that after inhibition of the pathway at the cell membrane level, the percentage of MAP2/DCX-positive cells responding to 100 mM glutamate 30

Cm, membrane capacitance.

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Fig. 4. Characteristics of differentiated neural stem/progenitor cells. (A, D) Cells with a passive current pattern are mostly GFAP-positive and flat-shaped. They display predominantly time- and voltage-independent K þ currents together with small amplitudes of delayed outwardly rectifying K þ currents (KDR) and inwardly rectifying K þ currents (KIR). (B, E) The majority of cells displaying a complex current profile are branched or bipolar, and PDGFαR-positive. They express fast activating and inactivating outwardly rectifying K þ currents (KA), as well as KDR and KIR currents. (C, F, G) Cells with an outwardly rectifying current pattern are DCX/MAP2-positive, with a round shape, and express KA and KDR currents. Current patterns were obtained by hyper-, and depolarizing the cell membrane from the holding potential of  70 mV to the values ranging from  160 to 40 mV at 10 mV intervals. Scale ¼ 50 mm. Abbreviations: DCX, doublecortin; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; PDGFαR, platelet-derived growth factor alpha receptor.

decreased significantly, from  90% in control, to  20%, and the average amplitude of the glutamate-evoked response decreased by 66% (Fig. 7A, left, black squares; Fig. 7A, right). Additionally, Dkk1 over-expression also resulted in a higher response to 50 mM adenosine 5′-triphosphate (ATP31) application, when compared to the controls (Fig. 7A, left, gray squares); however, the average amplitude of responding cells remained unchanged. On the other hand, activation of the pathway led to the lower average amplitude in response to ATP application (Fig. 7B, left, gray squares; Fig. 7B, right), while the Ca2 þ elevations in response to glutamate application were comparable with those observed in controls (Fig. 7B, left, black squares). Inhibition of the pathway in the nucleus (dnTCF4) showed no significant changes in the cell response to glutamate/ATP application. These results show that the alterations in β-catenin signaling influence the distribution of distinct K þ channels, as well as the ability of cells to transport Ca2 þ after the application of glutamate and ATP. Furthermore, the activation of the Wnt signaling pathway increases the incidence of cells expressing outwardly rectifying K þ currents, together with inwardly rectifying Na þ currents.

31

ATP, adenosine 5′-triphosphate.

3. Discussion In this study, we showed that blocking Wnt signaling by Dkk1 resulted in decreased amounts of β-catenin, while its stabilization was detected in Ex3 cells, producing a truncated variant of the protein. Additionally, Wnt/β-catenin signaling activation increased the expression of β III tubulin, and decreased the incidence of GFAP-positive cells displaying passive time- and voltage-independent K þ currents. On the contrary, by suppressing the Wnt pathway, the incidence of GFAP-positive cells increased marginally, and the number of DCX/MAP2-positive cells expressing outwardly rectifying K þ currents decreased significantly. Furthermore, Wnt signaling activation increased the incidence of cells expressing outwardly rectifying K þ currents, together with inwardly rectifying Na þ currents, and influenced the ability of cells to transport Ca2 þ after glutamate and ATP application. 3.1. Manipulating the Wnt/β-catenin signaling pathway using transgenic mice We employed three transgenic mouse strains, enabling tamoxifen-inducible Cre32-mediated DNA recombination. Using this approach, we were able to inhibit Wnt signaling either at the nuclear or membrane level or, alternatively, to activate the Wnt 32

Cre, Cre recombinase.

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Fig. 5. Inhibition/activation of the Wnt signaling pathway changes the incidence of different cell types. (A) The incidence of cells displaying passive (pas), complex (com) and outwardly rectifying (out) current profiles. Four (Dkk1 mice) or three (dnTCF4 and Ex3 mice) founder mice were used to derive NS/PCs, and the incidence was quantified from the following total number of cells (in brackets): dnTCF4-CTRL (149), dnTCF4-4OHT (184), Dkk1-CTRL (97), Dkk1-4OHT (101), Ex3-CTRL (101), and Ex3-4OHT (95). The relative incidence of cells in controls was arbitrarily set to 1. (B) Representative images of GFAP (top), DCX (middle) and MAP2 (bottom) staining. Scale ¼ 50 mm. (C) Quantification of GFAP, DCX and MAP2 expression showing the area of positively stained cells, within 318.2 mm  318.2 mm large inspected region (n¼ 6). The area of GFAP/DCX/MAP2 fluorescence in control cells was arbitrarily set to 1. Western blot analysis of β III tubulin (see inset),*, p o0.05;**, p o 0.01;***, p o0.001. Abbreviations: 4OHT, tamoxifen-treated cultures; βIIItub, β-tubulin isotype III; β-act, β-actin; CTRL, control cultures; DCX, doublecortin; Dkk1, neural stem/progenitor cells derived from neonatal Dkk1 mice; dnTCF4, neural stem/progenitor cells derived from neonatal dnTCF4 mice; Ex3, neural stem/progenitor cells derived from neonatal Ex3 mice; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; n, number of cells or number of regions of interest.

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Table 1 Membrane properties of differentiated neonatal progenitor cells displaying a passive current pattern. 4OHT, 4-hydroxytamoxifen; Cm, membrane capacitance; CTRL, control; Dkk1, neural stem/progenitor cells derived from the frontal lobe of neonatal Dkk1 mice; dnTCF4, neural stem/progenitor cells derived from the frontal lobe of neonatal dnTCF4 mice; Ex3, neural stem/progenitor cells derived from the frontal lobe of neonatal Ex3 mice; IR, input resistance; KDR, delayed outwardly rectifying K þ current; KIR, inwardly rectifying K þ current; KIR/Cm, KDR/Cm, current densities; n, number of cells; Vm, membrane potential. Values in bold indicate significant differences between CTRL and 4OHT-treated cultures. dnTCF4

Dkk1

Ex3

Properties

CTRL

4OHT

CTRL

4OHT

CTRL

4OHT

Vm [mV] IR [MΩ] Cm [pF] KIR [pA] KIR/Cm [pA/pF] KDR [pA] KDR/Cm [pA/pF]

 83.37 0.6 73.6 7 3.1 34.4 7 2.2 74.17 7.6 2.3 7 0.2 127.0 7 17.1 3.8 7 0.5

 86.2 7 0.4** 73.2 7 2.3 31.3 7 1.6 77.2 75.4 3.97 0.3** 119.4 7 11.4 4.3 70.4

 85.2 70.4 71.2 7 2.3 36.7 7 2.0 58.0 7 4.6 2.3 7 0.2 82.3 7 7.5 3.2 7 0.3

 87.0 7 0.3* 71.6 71.8 28.27 1.2* 56.3 7 3.3 2.7 7 0.2 78.6 7 8.1 3.9 7 0.5

 85.7 70.3 69.8 7 3.2 35.472.4 71.4 7 6.1 2.77 0.2 116.4 7 12.2 4.17 0.4

 87.0 70.4 83.8 75.5 32.17 2.2 86.7 76.5 4.27 0.3** 156.5 7 18.6 5.3 70.6

* **

p o0.05. po 0.01.

Table 2 Membrane properties of differentiated neonatal progenitor cells displaying a complex current pattern. 4OHT, 4-hydroxytamoxifen; Cm, membrane capacitance; CTRL, control; Dkk1, neural stem/progenitor cells derived from the frontal lobe of neonatal Dkk1 mice; dnTCF4, neural stem/progenitor cells derived from the frontal lobe of neonatal dnTCF4 mice; Ex3, neural stem/progenitor cells derived from the frontal lobe of neonatal Ex3 mice; IR, input resistance; KA, fast activating and inactivating outwardly rectifying K þ current; KDR, delayed outwardly rectifying K þ current; KIR, inwardly rectifying K þ current; KIR/Cm, KDR/Cm, KA/Cm, current densities; n, number of cells; Vm, membrane potential. Values in bold indicate significant differences between CTRL and 4OHT-treated cultures. dnTCF4

Dkk1

Ex3

Properties

CTRL

4OHT

CTRL

4OHT

CTRL

4OHT

Vm [mV] IR [MΩ] Cm [pF] KIR [pA] KIR/Cm [pA/pF] KDR [pA] KDR/Cm [pA/pF] KA [pA] KA/Cm [pA/pF]

 83.6 71.3 202.9 7 18.0 21.97 1.7 81.2 7 6.6 4.27 0.3 514.9 7 44.2 33.774.3 290.37 35.5 23.2 7 4.0

 84.17 0.7 214.2 7 11.4 13.77 0.7*** 87.4 7 4.7 7.4 7 0.4*** 463.0 7 29.4 46.9 7 3.4 205.0 712.4* 23.1 71.6

 85.2 7 0.9 185.67 15.2 22.4 71.2 83.07 6.6 4.0 70.3 659.8 759.3 38.0 7 4.4 369.4 7 62.3 28.5 7 6.1

 86.3 7 0.7 135.878.4*** 16.7 7 0.8** 106.77 4.9* 7.87 0.5*** 434.67 38.7* 38.3 74.2 278.4 7 22.3 22.0 72.0

 86.2 7 1.0 231.4 7 27.9 17.4 7 1.8 74.2 78.2 4.37 0.4 618.5 7 61.2 43.3 74.4 229.8 724.0 20.5 73.5

 87.5 7 0.7 164.37 15.2 23.5 7 1.8 141.2 7 11.2*** 7.4 70.6** 605.8 7 40.9 36.0 7 3.5 275.6 738.3 13.4 71.6

* **

p o0.05. po 0.01. p o 0.001.

***

Table 3 Membrane properties of differentiated neonatal progenitor cells displaying an outwardly rectifying current pattern. 4OHT, 4-hydroxytamoxifen; Cm, membrane capacitance; CTRL, control; Dkk1, neural stem/progenitor cells derived from the frontal lobe of neonatal Dkk1 mice; dnTCF4, neural stem/progenitor cells derived from the frontal lobe of neonatal dnTCF4 mice; Ex3, neural stem/progenitor cells derived from the frontal lobe of neonatal Ex3 mice; IR, input resistance; KA, fast activating and inactivating outwardly rectifying K þ current; KDR, delayed outwardly rectifying K þ current; KDR/Cm, KA/Cm, current densities; n, number of cells; Vm, membrane potential. Values in bold indicate significant differences between CTRL and 4OHT-treated cultures. dnTCF4 Properties Vm [mV] IR [MΩ] Cm [pF] KDR [pA] KDR/Cm [pA/pF] KA [pA] KA/Cm [pA/pF]

CTRL  69.17 1.3 1856.0 778.0 10.67 0.4 691.0 723.2 72.1 72.2 634.5 727.5 68.77 3.1

Dkk1 4OHT  70.77 1.7 1817.7 7 98.8 7.5 7 0.3*** 637.3 7 26.0 101.0 75.1*** 479.2 7 27.7** 78.9 7 5.1

Ex3

CTRL

4OHT

 63.67 1.7 1797.7 7 95.6 11.37 0.5 830.17 34.6 87.47 3.8 795.17 38.5 92.7 7 5.1

**

 74.97 2.9 1514.3 7181.8 7.77 0.4** 881.17 79.0 132.3 714.3** 524.0749.5** 79.0 7 9.7

CTRL

4OHT

 67.07 2.1 1924.7 7 82.1 10.3 70.4 652.7 7 25.3 71.2 73.1 675.97 37.6 80.37 5.2

 78.7 7 1.6*** 1434.3 7115.9** 9.9 70.5 695.6 742.1 75.9 73.6 441.87 37.1*** 55.9 7 5.0*

*

p o0.05. po 0.01. *** p o 0.001. **

pathway via production of a stable form of β-catenin protein. The manipulation of the Wnt pathway at distinct points of the signaling relay gave us a deeper insight into the possible molecular mechanisms underlying NS/PCs differentiation in the neonatal mouse brain. In contrast to our study, other authors opted for different approaches. For example, Hirsch et al. (2007) added recombinant Wnt3a to the differentiation media, Prajerova et al.

(2010) used NS/PCs transduced with Wnt7a, and Meyers et al. (2012) employed pharmacological and heat-shock-inducible genetic manipulation of the Wnt signaling pathway. However, none of the studies listed above combined all the approaches and methods we utilized in our study. We carried out immunofluorescent staining, as well as functional studies (patch-clamp technique, Ca2 þ imaging) on NS/PCs derived from transgenic

J. Kriska et al. / Brain Research 1651 (2016) 73–87

81

Fig. 6. Voltage-dependent Na þ channels in cells with an outwardly rectifying current profile. (A) A typical current pattern of a cell expressing outwardly rectifying K þ currents, along with inwardly rectifying Na þ currents. (B) Inhibiting Wnt signaling (dnTCF4, Dkk1) causes a decrease in the incidence of cells expressing Na þ channels. In contrast, activating the pathway (Ex3) leads to an increase in the incidence, as well as in the current densities of voltage-gated Na þ channels. The number of identified cells showing Na þ currents was as follows (in brackets): dnTCF4-CTRL (4), dnTCF4-4OHT (0), Dkk1-CTRL (4), Dkk1-4OHT (0), Ex3-CTRL (1), and Ex3-4OHT (3). (C) Some of the cells were capable of generating action potentials; the trace represents such action potentials. Abbreviations: 4OHT, tamoxifen-treated cultures; CTRL, control cultures; Dkk1, neural stem/progenitor cells derived from neonatal Dkk1 mice; dnTCF4, neural stem/progenitor cells derived from neonatal dnTCF4 mice; Ex3, neural stem/progenitor cells derived from neonatal Ex3 mice; n, number of cells; Na/Cm, current densities of Na þ currents.

Fig. 7. Glutamate- and ATP-evoked Ca2 þ responses in DCX/MAP2-positive cells. (A) Suppression of Wnt signaling at the membrane level (Dkk1) significantly lowers Ca2 þ responses (% of responding cells and their average amplitude) to 100 mM glutamate application, and increases the response rate to 50 mM ATP application in DCX/MAP2-positive cells. (B) Activation of the pathway (Ex3) results in lower average amplitudes of ATP-responding cells. The number of cells used in this analysis was as follows (in brackets): Dkk1-CTRL (9), Dkk1-4OHT (6), Ex3-CTRL (7), and Ex3-4OHT (7), */#, p o0.05; ***/###, p o 0.001. Abbreviations: 4OHT, tamoxifentreated cultures; ATP, 50 mM adenosine 5′-triphosphate; CTRL, control cultures; Dkk1, neural stem/progenitor cells derived from neonatal Dkk1 mice; Ex3, neural stem/progenitor cells derived from neonatal Ex3 mice; GLU, 100 mM L-glutamic acid (glutamate).

mouse strains, which allowed us to either activate or inhibit the Wnt signaling pathway at different subcellular levels, and so we performed a comprehensive analysis of effects caused by the changes in Wnt signaling. It is worth mentioning that we obtained similar results as the above-mentioned authors. As expected, inhibition of the pathway in the nucleus using dnTCF4 mice did not

affect the levels of β-catenin expression, which accords well with the fact that the alteration in the pathway occurs downstream of the β-catenin destruction complex. This was not the case in Dkk1 and Ex3 mice, where the alterations occurred at the membrane and the protein itself respectively. Using qRT-PCR, we assessed the expression of putative Wnt signaling target genes, namely Axin 2, Sp5 and Troy. These genes are correlated positively with β-catenin signaling as shown previously (Buttitta et al., 2003; Fujimura et al., 2007; Jho et al., 2002). Wnt signaling led to a markedly higher expression of all analyzed genes, which goes along with the findings of Fafilek et al. (2013), Fujimura et al. (2007) and Hirsch et al. (2007). They found that after Wnt stimulation, stem cells express higher levels of inhibitory components of the Wnt pathway. Examples for such components are the aforementioned genes – Axin 2, Sp5 and Troy. Surprisingly, the expression of Axin 2 was not altered after Wnt signaling inhibition. This inconsistency might be due to overall low expression of Axin 2 (Ct33 values  32) in our dnTCF4 and Dkk1 cultures, which might imply that in these two models, the Wnt signaling pathway is prevented from activation, rather than being inhibited after its activation, while Wnt signaling is still able to affect the cell phenotype. On the other hand, after Wnt signaling activation, the expression of Axin 2 was much higher (Ct values  27). Thus the protein was able to act as a negative feedback loop that controls Wnt signaling activity. Additionally, we analyzed gene transcription using qRT-PCR, and found that Wnt4, Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt9a and Wnt10b, ligands, typical of both canonical and non-canonical pathways, were present in all control cultures. This correlates, to some extent, with Hirsch et al. (2007); they found Wnt3, Wnt4, Wnt5a, Wnt7a, Wnt7b and Wnt11 genes to be expressed in NS/PCs. For example, non-canonical Wnt5a enhances neurogenesis, and improves functional integration of newly derived neurons in vivo (Parish et al., 2008). Similar functions are also exercised by Wnt7a, since this Wnt ligand stimulates NS/PCs proliferation, and promotes neuronal differentiation and maturation (Qu et al., 2013). Interestingly, over-expression of Wnt7b impairs neuronal differentiation of neural progenitors, and thus antagonizes the effect of Wnt5a and Wnt7a ligands (Papachristou et al., 2014); however, other functions may also be performed by these ligands according to the developmental stages of experimental animals, as presented 33

Ct, threshold cycle.

82

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by Hirabayashi et al. (2004) and reviewed by Inestrosa and VarelaNallar (2015). Moreover, the function of Wnt ligands could be quite intriguing, as the very same ligand is able to activate both the βcatenin-dependent and non-canonical pathways (Nalesso et al., 2011). 3.2. Wnt signaling promotes differentiation of neonatal neural stem/ progenitor cells towards neuron-like cells Next, we showed that hyperactivated Wnt signaling increases the incidence of cells positive for neuronal marker β III tubulin, and decreases the incidence of GFAP-positive cells, though not significantly. Nevertheless, we suggest that there is a tendency towards neurogenesis after Wnt signaling activation, since Hirsch et al. (2007) demonstrated similar effects of Wnt signaling on the expression of β III tubulin, although the number of GFAP-positive cells remained unchanged. This discrepancy might be explained by differences in Wnt pathway activation between our and their study. We took advantage of employing transgenic mouse models, and activated the pathway in NS/PCs using the “general” intracellular signal mediator (β-catenin) in the cytoplasm, whereas Hirsch and colleagues applied Wnt3 ligand only. Furthermore, we observed that Wnt signaling causes a higher incidence of DCX/ MAP2-positive cells that displayed branched processes, indicating a more advanced developmental stage. These branched and overlayed processes could be responsible for the fact that we were not able to identify significant changes in the levels of DCX and MAP2. The observation is in concordance with previously published data from the in vitro experiments of Kunke et al. (2009). These authors transduced mouse NS/PCs with constructs producing Wnt7a or Dkk1, and showed that the molecules influenced cell fate during differentiation. Expression of Wnt7a (Wnt signaling activation) led to an increase in the incidence of MAP2-positive cells, while the numbers of GFAP-positive cells decreased. In contrast, Dkk1 (Wnt signaling inhibition) significantly increased gliogenesis at the expense of neurogenesis. In this study, the authors incubated single cells, derived from trypsinized neurospheres, for only four days. In the present study, we employed the same approach; however, we analyzed differentiated cells after eight days of in vitro differentiation. Therefore, our results complement those of Kunke et al. (2009) and show that, after Wnt signaling inhibition/activation, differentiated cells preserve similar characteristics for a longer period of time. Another study showed that Wnt signaling inhibition, using retroviral-mediated RNA interference to knockdown Frizzled-1 receptor, though in the adult hippocampus, resulted in a decrease in the percentage of cells expressing DCX and concomitantly, in increased differentiation into astrocytes (Mardones et al., 2016). In summary, from our results and previously published studies it is clear that Wnt signaling plays a crucial role in the control of neuronal progenitor differentiation. 3.3. Wnt signaling has an impact on the electrophysiological properties of differentiated neonatal neural stem/progenitor cells Previously, it has been shown that Wnt signaling activation affects the electrophysiological properties of differentiated neural precursors (Prajerova et al., 2010). Activated Wnt signaling caused a higher incidence of cells expressing outwardly rectifying K þ currents, and tetrodotoxin (TTX34)-sensitive inwardly rectifying Na þ currents, and generating action potentials. This neuron-like current pattern also prevailed in our experiments. In our work, we further disclosed that MAP2-positive cells displayed large KA and

KDR current amplitudes, which agrees with previous studies in neonatal NS/PCs transduced with Wnt7a (Prajerova et al., 2010). Concurrently, in differentiated NS/PCs upon Wnt signaling inhibition, we found that the response of MAP2/DCX-positive cells to glutamate application decreased. These data, based on calcium imaging, support the presence of the glutamate receptors on neural progenitors. As suggested by Deisseroth et al. (2004), the glutamate receptors mediate excitation-induced neurogenesis, by inhibiting expression of the glial transcription factors Hes1, and inhibitor of DNA binding 2 (Id235), and by promoting expression of the pro-neuronal transcription factor neuronal differentiation (NeuroD36). Using retrovirus-mediated gene knock-out in mice, Tashiro et al. (2006) showed that survival of maturating neurons is competitively regulated by their own NMDA receptors, during a short period after neuronal birth. Following this, we found that inhibition of the Wnt pathway led to increased numbers of cells responding to the ATP application, but nevertheless, elevated Wnt/ β-catenin signaling resulted in a lower average response amplitude. This is in contrast to observations that ATP up-regulates neuronal markers; neuron-specific class III β-tubulin (Tuj137), neuronal nuclei (NeuN38) and β-catenin expression, and thus promotes neuronal differentiation of stem cells (Tu et al., 2014). The discrepancy might be related to the usage of different cells; whereas we used neural stem cells, Tu et al. (2014) employed mesenchymal stem cells in their study. In summary, the Wnt signaling pathway regulates neurogenesis, as it drives neonatal NS/PCs differentiation into the DCX/ MAP2-positive cells, expressing outwardly rectifying K þ currents, and inwardly rectifying Na þ currents. Importantly, our results indicate that manipulating Wnt/β-catenin signaling in NS/PCs may provide novel approaches for the treatment of neurological disorders. For instance, studies of progenitor cells in the olfactory epithelium (globose basal cells) performed in neonatal and adult mice suggested a key role of Wnt signaling in progenitor cells proliferation and epithelial lesion neuroregeneration (Chen et al., 2014). Moreover, a connection between Alzheimer's disease and Wnt signaling pathway impairments has been suggested (Inestrosa et al., 2002; Inestrosa and Varela-Nallar, 2015).

4. Conclusion In this study, we employed transgenic mice that served as a suitable animal model for manipulating canonical Wnt signaling. Furthermore, we demonstrated that Wnt signaling in cell cultures derived from neonatal mice increased the number of DCX/MAP2/β III tubulin-positive cells, yet on the other hand, decreased the counts of GFAP-positive cells. Hyperactive Wnt/β-catenin signaling also increased the incidence of cells displaying an outwardly rectifying K þ current profile, together with inwardly rectifying Na þ currents. Our data indicate that the Wnt signaling pathway in the newborn mouse brain regulates the NS/PCs differentiation towards cells with features of neuroblasts or immature neurons. The possibility of regulating neurogenesis via Wnt pathway manipulation might be useful in nervous tissue regeneration in the diseased CNS. 35 36 37

34

TTX, tetrodotoxin.

38

Id2, inhibitor of DNA binding 2. NeuroD, neuronal differentiation. Tuj1, neuron-specific class III β-tubulin. NeuN, neuronal nuclei.

J. Kriska et al. / Brain Research 1651 (2016) 73–87

5. Experimental procedure 5.1. Transgenic animals All procedures involving the use of laboratory mice were performed in accordance with the European Communities Council Directive 24 November 1986 (86/609/EEC) and animal care guidelines approved by the Institute of Experimental Medicine, Academy of Sciences of the Czech Republic (Animal Care Committee on April 7, 2011; approval number 018/2011). Neonatal mice (P0-2) were used in this study. We used the following mouse strains: Rosa26-Dkk1 mice (Wu et al., 2008), the mice produce – upon Cre-mediated excision of a transcriptional blocker – the extracellular Wnt pathway inhibitor Dkk1, from the ubiquitous Rosa26 locus (Fig. 1A); Catnblox(ex3) mice harboring the floxed allele of the Ctnnb1 gene (the gene encodes β-catenin; Harada et al., 1999), the allele allows conditional stabilization of β-catenin (Fig. 1B); Rosa26-tdTomato-EGFP39/dnTCF4 mice (Janeckova et al., 2016), the mice produce from the Rosa26 locus, the dnTCF4, which functions as a nuclear Wnt pathway inhibitor (similarly to the Rosa26-Dkk1 strain, dnTCF4 expression is triggered upon Cremediated excision of a transcriptional blocker, located upstream of dnTCF4 cDNA; Fig. 1C). The mice were crossbred with general Cre deletor Rosa26-CreERT240 animals (Ventura et al., 2007; Fig. 1D). According to the resulting genotype, the mice enabled inhibition of Wnt signaling at the nuclear (genotype: Rosa26dnTCF4/CreERT2; further termed dnTCF4 mice/cells), or membrane (genotype: Rosa26Dkk1/CreERT2; further termed Dkk1 mice/cells) level. Furthermore, Ctnnb1del(ex3)/ þ Rosa26 þ /CreERT2 mice (further termed Ex3 mice/cells), upon Cre-mediated excision of exon 3 of the Ctnnb1 gene, produce a stable β-catenin variant that (hyper)activated canonical Wnt signaling. The Wnt signaling manipulation (i.e., the CreERT2 activation) was achieved by addition of 4OHT (1 μM; Sigma-Aldrich, St. Louis, MO, USA), dissolved in ethanol, into the differentiation medium. Cells treated with vehicle (ethanol) only were used as controls. 5.2. Cell culture Primary cultures were prepared from NS/PCs, isolated from neonatal mouse brains. After decapitation, the brains were quickly dissected out and the frontal lobe of the brain was isolated. Using a 1-ml-pipette, the tissue was mechanically dissociated in a 2-mlEppendorf tube with 1 ml of proliferation medium containing Neurobasal-A medium (Life Technologies, Waltham, MA, USA), supplemented with B27 (2%; Life Technologies, Waltham, MA, USA), L-glutamine (2 mM; Sigma-Aldrich, St. Louis, MO, USA), primocin (100 mg/ml; Invivogen, Toulouse, France), fibroblast growth factor-basic (bFGF41; 10 ng/ml) and epidermal growth factor (EGF42; 20 ng/ml; both were purchased from PeproTech, Rocky Hill, NJ, USA). The cells were subsequently filtered through a 70 mm cell strainer, into a 100 mm-diameter Petri dish containing 9 ml of proliferation medium. The cells were cultured as neurospheres, at 37 °C and 5% CO2. After 7 days of in vitro proliferation, the neurospheres were collected and transferred into a 12 ml Falcon tube, and centrifuged at 1020  g for 3 min. The supernatant was discarded, and 1 ml of trypsin (Sigma-Aldrich, St. Louis, MO, USA) was added to the pelleted neurospheres. After 3 min, 1 ml of trypsin inhibitor (Sigma-Aldrich, St. Louis, MO, USA) was added to the dissociated cells, to block the trypsinization. 39

EGFP, enhanced green fluorescent protein. CreERT2, Cre recombinase fused with a modified form of the estrogen receptor. 41 bFGF, basic fibroblast growth factor. 42 EGF, epidermal growth factor.

83

Subsequently, 100 ml of the cell suspension was used to count cells in the hemocytometer. The rest of the cell suspension was centrifuged at 1020  g for 3 min. The cells were plated on coverslips coated with poly-L-lysine (PLL43; Sigma-Aldrich, St. Louis, MO, USA), at the cell density 6  104 cells/cm2, treated with differentiation medium (the same composition as the proliferation medium, but without EGF, and with 20 ng/ml bFGF), and maintained at 37 °C and 5% CO2, with medium exchange on every third day. After 8–9 days of in vitro differentiation, the cells were used for electrophysiological measurements, immunocytochemistry, Western blot analysis, and qRT-PCR. To estimate the impact of Wnt signaling inhibition/activation during differentiation, 4OHT- and ethanol-treated (controls) cultures were compared. 5.3. Patch-clamp recording Cell membrane currents were recorded 8–9 days after the onset of differentiation, using the patch-clamp technique in the wholecell configuration. Recording pipettes with a tip resistance of 8– 12 MΩ were made from borosilicate capillaries (Sutter Instruments, Novato, CA, USA), using a P-97 Brown-Flaming micropipette puller (Sutter Instruments, Novato, CA, USA). Recording pipettes were filled with intracellular solution containing (in mM): 130 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 10 HEPES (pH 7.2). To visualize the recorded cells, the intracellular solution contained Alexa Fluor hydrazide 488 (A488; Molecular Probes, Carlsbad, CA, USA). The labeled cells were used for further post-recording immunocytochemical identification. All recordings were made in artificial cerebrospinal fluid (aCSF44), containing (in mM): 122 NaCl, 3 KCl, 1.5 CaCl2, 1.3 MgCl2, 1.25 Na2HPO4, 28 NaHCO3, and 10 D-glucose (osmolality 300 75 mmol/kg). The solution was continuously gassed with 5% CO2, to maintain a final pH of 7.4. All recordings were made on coverslips perfused with aCSF at room temperature. Electrophysiological data were measured with a 10 kHz sample frequency, using an EPC9 or EPC10 amplifier, controlled by PatchMaster software (HEKA Elektronik, Lambrecht/ Pfalz, Germany), and filtered using a Bessel filter. The coverslips with cells were transferred to the recording chamber of an upright Axioscop microscope (Zeiss, Gottingen, Germany), equipped with electronic micromanipulators (Luigs & Neumann, Ratingen, Germany) and a high-resolution AxioCam HR digital camera (Zeiss, Gottingen, Germany). The resting membrane potential was measured by switching the EPC9 or EPC10 amplifier to the current-clamp mode. Using FitMaster software (HEKA Elektronik, Lambrecht/Pfalz, Germany), IR was calculated from the current value of 40 ms, after the onset of the depolarizing 10 mV pulse from the holding potential, of  70 mV to  60 mV for 50 ms. Membrane capacitance was determined automatically from the Lock-in protocol by PatchMaster. Current patterns were obtained by hyper-, and by depolarizing the cell membrane from the holding potential of  70 mV to the values ranging from  160 to 40 mV, at 10 mV intervals. Pulse duration was 50 ms. In order to isolate the KDR current components, a voltage step from  70 to 60 mV was used to subtract the time- and voltage-independent currents, as described previously (Anděrová et al., 2006; Neprasova et al., 2007). To activate the KDR currents only, the cells were held at  50 mV, and the amplitude of the KDR currents was measured at 40 mV, at the end of the pulse. The KIR currents were determined at  160 mV, at the end of the pulse. The KA currents were isolated by subtracting the current traces, clamped at 110 mV from those clamped at  50 mV, and its amplitude was measured at the peak value.

40

43 44

PLL, poly-L-lysine. aCSF, artificial cerebrospinal fluid.

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Current densities were calculated by dividing the maximum current amplitudes by the corresponding Cm values, for each individual cell. Tetrodotoxin-sensitive Na þ currents were isolated by subtracting the current traces measured in solution containing 1 mM TTX (Alomone Labs, Jerusalem, Israel), from those measured in the absence of TTX. Sodium current amplitudes were measured at the peak value. Action potentials were obtained in the currentclamp mode. Current values ranged from 50 pA to 1 nA, at 50 pA intervals. Pulse duration was 300 ms. After recording, the coverslips were fixed in phosphate buffer (PB45; 0.2 M; pH 7.4), containing 4% paraformaldehyde for 9 min, and then transferred to phosphate-buffered saline (PBS46; 10 mM; pH 7.2), for post-recording identification using immunocytochemistry. 5.4. Immunocytochemistry Primary cultures attached to PLL-coated coverslips were fixed in 4% paraformaldehyde solution in 0.2 M PB (pH 7.4) for 9 min, and kept in 10 mM PBS at 4 °C for further processing. The coverslips were incubated in a blocking solution containing 5% Chemiblocker (Millipore, Billerica, MA, USA), and 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in 10 mM PBS, at 4 °C for 2 h. Next, they were incubated overnight at 4 °C with primary antibodies in PBS, containing 0.2% Triton X-100. After the overnight incubation, three 10-min washes with PBS were performed, followed by incubation with secondary antibodies for two hours at 4 °C (primary and secondary antibodies are listed in Table 4). For double labeling, a mouse monoclonal antibody directed against GFAP, and conjugated with Cy347 (1:800; Sigma-Aldrich, St. Louis, MO, USA) was subsequently applied and incubated overnight at 4 °C. Afterwards, the coverslips were washed in PBS three times for 10 min. To visualize cell nuclei, the coverslips were incubated with 300 nM 4′,6-diamidino-2-phenylindole (DAPI48; Molecular Probes, Carlsbad, CA, USA) in PBS for 5 min at room temperature. Finally, the coverslips were mounted using Aqua Poly/Mount (Polysciences Inc., Eppelheim, Germany). An LSM 5 DUO spectral confocal microscope (Zeiss, Gottingen, Germany), equipped with an Arg/HeNe laser was used for immunochemical analyses. Six fluorescent microscopy images were taken from every cell type treated with ethanol only or 4OHT. The fluorescent signals were analyzed using the ImageJ software (NIH, Bethesda, MD, USA), and obtained data were evaluated by Student’s t-test. 5.4.1. Immunocytochemistry and quantification of β-catenin signal intensity Immunocytochemical staining was performed as described previously (Doubravska et al., 2011), using mouse monoclonal antibody against β-catenin (BD Transduction Laboratories, San Jose, CA, USA), goat-anti-mouse secondary antibody, conjugated with Alexa 488 dye, and DAPI (Sigma-Aldrich, St. Louis, MO, USA). Twelve fluorescent microscopy images were taken from every cell type, treated with ethanol only or 4OHT. The Alexa 488 and DAPI fluorescent signals were analyzed using the ImageJ software (NIH, Bethesda, MD, USA), and the intensity of β-catenin staining was normalized to the cell number in every image. Obtained data were evaluated by Student’s t-test. 5.4.2. Quantification of GFAP, DCX and MAP2 signal intensity The fluorescent signals were analyzed using ImageJ software (NIH, Bethesda, MD, USA). Superimposed images of GFAP, DCX or 45 46 47 48

PB, phosphate buffer. PBS, phosphate-buffered saline. Cy3, Cyanine 3. DAPI, 4′,6-diamidino-2-phenylindole.

MAP2 staining were obtained by overlaying 4 individual confocal planes. The images were subsequently digitally filtered, and the red area, outlined by yellow, was used for quantification of the staining. The area (in mm2) corresponding to the immunoreactivity of the cells was calculated in random regions of interest (ROIs49; 318.2 mm  318.2 mm large inspected regions). In Fig. S3A-C, an example of such ROI that corresponds to DCX staining is shown. Six ROIs, from two independent cultures with similar confluence were used for the quantification of each immunostaining. 5.5. Calcium imaging measurements Coverslips with 8–9-D-old cell cultures were incubated for 45 min, in 0.5 ml of differentiation medium containing 4.5 mM Oregon-Green Bapta-1 (OGB-150) AM, and 0.09% Pluronic F-127 (Life Technologies, Waltham, MA, USA), at 37 °C in an incubator (100% humidity, 5% CO2). The coverslips were then transferred to the microscope superfusion chamber, and three measurements were made on each coverslip with a sufficient distance between the measurement regions. During the measurements, the microscope superfusion chamber was continually perfused with HEPESbased aCSF, containing (in mM): 135 NaCl, 2.7 KCl, 1 MgCl2, 2.5 CaCl2, 1 Na2HPO4, 10 glucose, 10 HEPES (pH 7.4, osmolality 305 mOsm/kg, equilibrated with O2), at a flow rate of 2.5 ml/min at room temperature. The solutions of ATP, L-glutamic acid (glutamate) (both from Sigma-Aldrich, St. Louis, MO, USA) and HEPESbased aCSF, in which part of NaCl was replaced for KCl to reach 50 mM concentration of K þ (H-aCSF50K þ ), were applied through a capillary (i.d. 250 mm), located 0.5 – 1 mm from the measurement region, and connected to a Perfusion Pressure Kit pressurized application system (flow rate 600 ml/min) controlled by a ValveBank II controller (AutoMate Scientific, Inc. Berkeley, CA, USA). The HEPES-based aCSF was applied before and after the applications with the same flow rate, so that the responses were not influenced by the application itself. OGB-1 fluorescence was detected with a TILL Photonics Imaging System installed on a Zeiss Axioskop 2 FS Plus microscope, equipped with a long-distance 40  lens (IR Achroplan 0.8 W, Zeiss, Gottingen, Germany). A digital camera (PCO Sensicam, Kelheim, Germany) was controlled by TILLvisION software. The excitation light (484 nm) was generated by a Polychrome V (TILL Photonics GmbH, Gräfelfing, Germany), filtered by a BP 450–490 excitation band-pass filter, reflected by a FT 510 beam splitter, and the emitted light was filtered by a LP 515 longpass filter (Filter Set 09, Zeiss, Gottingen, Germany). Images were acquired at 0.83 Hz and were analyzed offline. Fluorescence intensity (F51) was measured in the cell bodies and expressed as ΔF/ F0, where F0 is the baseline fluorescence intensity before drug application. The threshold for Ca2 þ responses was 110% of the baseline fluorescence, and the maximum intensity which occurred within 1 min from the onset of application was taken into account. After the measurements, immunocytochemical stainings for GFAP, DCX, MAP2 and PDGFαR were performed to confirm the identity of the measured cells. 5.6. Western blotting Cells were lysed in standard 1  RIPA buffer, containing 25 mM Tris–HCl, pH 8.0; 150 mM NaCl; 1% NP-40; 1% sodium deoxycholate; 0.1% SDS; 1 mM EDTA, and supplemented with inhibitors of proteases (Roche, Basel, Switzerland) and phosphatases (20 mM NaF; 1 mM Na3VO4). The total protein content in the homogenates 49 50 51

ROI, region of interest. OGB-1, Oregon-Green Bapta-1. F, fluorescence intensity.

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Table 4 Primary and secondary antibodies used for immunocytochemistry and Western blotting. ICC, immunocytochemistry; WB, Western blot; GFAP-Cy3, glial fibrillary acidic protein coupled with the Cy3 fluorescent dye; PDGFαR, platelet-derived growth factor alpha receptor; DCX, doublecortin; MAP2, microtubule associated protein 2; Non-pβcatenin, non-phosphorylated β-catenin; β III tubulin, β-tubulin isotype III; GAM/GAR 488/594/660, goat anti-mouse/rabbit IgG conjugated with Alexa Fluor 488/594/660. Method Primary antibody

Dilution Manufacturer

Secondary antibody

Dilution Manufacturer

ICC

GFAP-Cy3 PDGFαR

1:800 1:200

– GAM/GAR

– 1:200

– Molecular Probes, Carlsbad, CA, USA

DCX

1:500

Sigma-Aldrich, St. Louis, MO, USA Santa Cruz Biotechnology, Dallas, TX, USA Santa Cruz Biotechnology, Dallas, TX, USA Chemicon, Billerica, MA, USA Santa Cruz Biotechnology, Dallas, TX, USA BD Biosciences, San Jose, CA, USA

Horseradish

1:5000

Jackson ImmunoResearch Laboratories, West Grove, PA, USA

WB

MAP2

1:800

β-actin

1:1000

β-catenin

1:2000

Non-pβ-catenin

1:300

β III tubulin

1:400

488/594/660

peroxidase- conjugated secondary antibodies

Cell Signaling Technology, Danvers, MA, USA Sigma-Aldrich, St. Louis, MO, USA

was determined using the Pierce BCA™ protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins, supplemented with 100 mM dithiothreitol, were subjected to SDS PAGE (10–15%), and the proteins were then transferred onto nitrocellulose membrane (Hybond ECL 0.45 μM; Little Chalfont, Amersham, UK) and detected by specific antibodies (Table 4), combined with horseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). Peroxidase activity was detected using enhanced chemiluminescence (ECL52) detection reagents (Little Chalfont, Amersham, UK).

Table 5 Sequences of primers used for quantitative RT-PCR. Actb, β-actin; Sp5, SP5 transcription factor; Troy, Tumor necrosis factor receptor superfamily, member 19; Ubb, Ubiquitin; Wnt4 – 10b, Wingless/Integrated4 – 10b. Gene symbol

Sequence

Actb

Forward: 5′-GATCTGGCACCACACCTTCT-3′ Reverse: 5′-GGGGTGTTGAAGGTCTCAAA-3′

Axin2

Forward: 5′-TAGGCGGAATGAAGATGGAC-3′ Reverse: 5′-CTGGTCACCCAACAAGGAGT-3′

Sp5

Forward: 5′-GGACAGGAAACTGGGTCGTA-3′ Reverse: 5′-AATCGGGCCTAGCAAAAACT-3′

Troy

Forward: 5′-GCTCAGGATGCTCAAAGGAC-3′ Reverse: 5′-CCAGACACCAAGACTGCTCA-3′

Ubb

Forward: 5′-ATGTGAAGGCCAAGATCCAG-3′ Reverse: 5′-TAATAGCCACCCCTCAGACG-3′

5.7. RNA isolation and qRT-PCR Purification of RNA was performed using RNA Blue (Top-Bio, Prague, Czech Republic), according to the manufacturers’ protocol. Reverse transcription and qRT-PCR were performed following previously described protocols (Lukas et al., 2009), using LightCyclers 480 SYBR Green I Master (Roche Diagnostics, Indianapolis, IN, USA). The primers for qRT-PCR are listed in Table 5.

Wnt4

Forward: 5′-AACGGAACCTTGAGGTGATG-3′ Reverse: 5′-TCACAGCCACACTTCTCCAG-3′

Wnt5a

Forward: 5′-AGGAGTTCGTGGACGCTAGA-3′ Reverse: 5′-ACTTCTCCTTGAGGGCATCG-3′

5.8. Data analysis and statistics

Wnt5b

Forward: 5′-CGCTTTGGAAGATGTTGGTC-3′ Reverse: 5′-ACATCTCCGGTCTCTGCACT-3′

Four (Dkk1 mice) or six (dnTCF4 and Ex3 mice) founder mice were used to derive NS/PCs for the experiments, unless otherwise stated. Data are presented as means7S.E.M.53 (standard error of the mean) for n cells, unless otherwise stated. Student's unpaired t-test was used to determine significant differences between the experimental groups. Values of *po0.05 were considered significant, **po0.01 very significant and ***po0.001 extremely significant.

Wnt7a

Forward: 5′-GCCTGGACGAGTGTCAGTTT-3′ Reverse: 5′-TGGTACTGGCCTTGCTTCTC-3′

Wnt7b

Forward: 5′-AGTGCCAGCACCAGTTCC-3′ Reverse: 5′-CCTTCCGCCTGGTTGTAGTA-3′

Wnt9a

Forward: 5′-TCGTGGGTGTGAAGGTGATA-3′ Reverse: 5′-TGGCTTCATTGGTAGTGCTG-3′

Wnt10b

Forward: 5′-CTTCGACATGCTGGAGGAG-3′ Reverse: 5′-CCCAGCTGTCGCTTACTCAG-3′

Acknowledgements The authors would like to thank Helena Pavlikova and Marketa Hemerova for their technical assistance. Our thanks also go to Frances Zatrepalkova for her helpful comments and suggestions. This study was supported by the grants GACR P304/12/G069, from the Czech Science Foundation, and GAUK 26214 from the Grant Agency of Charles University in Prague. This publication is a result of the "Advanced Bioimaging of Living Tissues" project, reg. n. CZ.2.16/3.1.00/21527, which was financed from the budget of the European Regional Development Fund and public budgets of the 52 53

ECL, enhanced chemiluminescence. S.E.M., standard error of the mean.

Czech Republic through the Operational Programme Prague – Competitiveness. The aforementioned funding sources had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit this article for publication.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.brainres.2016.09. 026.

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