CXCR4 signaling stimulates β-catenin transcriptional activity in rat neural progenitors

Neuroscience Letters 398 (2006) 291–295

SDF1␣/CXCR4 signaling stimulates ␤-catenin transcriptional activity in rat neural progenitors Yongquan Luo a , Jingli Cai a , Haipeng Xue a , Mark P. Mattson a,b , Mahendra S. Rao a,b,∗ a

Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD, USA b Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA Received 1 November 2005; received in revised form 3 January 2006; accepted 6 January 2006

Abstract Stromal cell-derived factor (SDF-1), by activating its cognate receptor CXCR4, plays multiple roles in cell migration, proliferation and survival in the development of the central nervous system. Recently, we have shown that functional SDF1␣/CXCR4 signaling mediates chemotaxis through extracellular signal-regulated kinase (ERK) activation in the developing spinal cord. Here, we report that SDF1␣/CXCR4 signaling activates ␤-catenin/TCF transcriptional activity in embryonic rat spinal cord neural progenitors. Stimulation of neural progenitors with SDF1␣ resulted in cytoplasmic ␤-catenin accumulation in 30 min, and lasted for approximately 240 min, while Wnt3a, a positive control, stabilized cytoplasmic ␤-catenin in 120 min. Dose–response studies indicated that the ␤-catenin stabilization effect could be detected in cells exposed to fM concentrations of SDF1␣. This SDF1␣-induced ␤-catenin stabilization effect was inhibited by pretreatment of the cells with either pertussis toxin (PTX), an inactivator of G protein-coupled receptors, or PD98059, a MEK1 inhibitor. Concomitant with ␤-catenin accumulation in the cytoplasm, SDF1␣ enhanced nuclear translocation of ␤-catenin and its binding to nuclear transcription factor T cell-specific transcription factor/lymphoid enhancer-binding factor (TCF/LEF). Furthermore, SDF1␣ increased expression of genes such as Ccnd1, 2, 3, and c-Myc known as targets of the Wnt/␤-catenin/TCF pathway. The increased expression of Ccnd1 and c-Myc by SDF1␣ was further confirmed by immunoblot analysis. Our data suggest that SDF1␣/CXCR4 signaling may interact with the Wnt/␤-catenin/TCF pathway to regulate the development of the central nervous system. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: c-Myc; Electrophoretic mobility shift assay; Gene expression; Wnt

Stromal cell-derived factor (SDF1) is a small (8–13 kDa) secreted chemokine protein and is the only known ligand for the membrane bound G protein coupled receptor CXCR4 [19,21]. Although the SDF1/CXCR4 pathway and its functions were originally identified in the immune system, emerging data suggest that this pathway plays an important role in the developing central nervous system as well. Using mice lacking either SDF1 or CXCR4, it was demonstrated that SDF1/CXCR4 signaling is required for neural precursor migration in the cerebellum, the hippocampal dentate gyrus and the cortex [1,14,17,20,22,24], pathfinding of sensory axons expressing the neurotrophin receptor TrkA [3] and embryonic retinal ganglion cell survival [2]. In cell culture systems, SDF1 enhances Sonic hedgehog-induced proliferation of cerebellar granule cells [12]. Using rat neural tubes, we were able to show that SDF1/CXCR4 signal-

∗

Corresponding author. Tel.: +1 410 558 8204; fax: +1 410 558 8249. E-mail address: [email protected] (M.S. Rao).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.01.024

ing is functional in the developing spinal cord. Expression of SDF1␣/CXCR4 is upregulated as neural stem cells at rat E10.5 differentiate into more restricted precursors at E14.5 [15,16]. At E14.5, SDF1␣ is capable of promoting neural migration and activating the ERK pathway and Ets family of transcription factors, subsequently regulating the expression of cell survival related genes. Pretreatment of the cells with specific ERK pathway inhibitor, PD98059, completely blocks SDF1␣-induced migration, indicating that activation of the ERK pathway is involved in chemotaxis [16]. ␤-Catenin is a 94 kDa protein that functions in cell adhesion, migration and transcription through interactions with different protein complexes. ␤-Catenin-associated complexes include adhesion complex, microtubule complex, destruction complex and transcription complex (reviewed in [7]). In adhesion complexes, ␤-catenin links cadherin to ␣-catenin which binds to actin-associated cytoskeletal proteins. In the cytoplasm, ␤-catenin interacts with adenomatous polysis coli (APC) and glycogen synthase kinase 3␤ (GSK3␤) linked destruction

292

Y. Luo et al. / Neuroscience Letters 398 (2006) 291–295

complex, leading to its ubiquitination and subsequent degradation by the proteasome. Interactions between the adhesion and destruction complexes result in a relatively stable pool of ␤catenin linked with adherence junction and a small and free cytosolic pool of ␤-catenin. Signaling via Wnt stimulation can inhibit the destruction complex by inactivating GSK3␤, thus, stabilizing levels of ␤-catenin in the cytoplasm. This ␤-catenin can enter the nucleus and bind to the TCF linked transcription complex, thus regulating target gene expression and cell proliferation and differentiation. In this communication, we provide evidence that SDF1␣ is capable of regulating ␤-catenin transcriptional activity in rat neural progenitor cells. We first examined whether SDF1␣ could stabilize cytoplasmic ␤-catenin in neural progenitors. Rat E14.5 spinal cord neural progenitors were prepared as described previously [15]. Briefly, experimentally naive, pregnant Sprague–Dawley rats at day 8 of gestation (Harlan) were purchased and housed individually until day E14.5. Embryos were removed and caudal neural tubes were dissected in fresh cold PBS (Invitrogen). The acutely dissected neural tubes were incubated with 0.05% Trypsin-EDTA (Invitrogen) for 10 min at 37 ◦ C and then gently triturated with a Pasteur pipette in NEP-basal medium with bFGF (20 ng/mL, Peprotech) [10,18]. The dissociated cells were plated on dishes coated with poly-l-lysine (13.3 ␮g/mL, Sigma) and laminin (15 ␮g/mL, Invitrogen) at a density of 104 /cm2 and maintained at 37 ◦ C in a 5% CO2 atmosphere in NEP-basal medium containing bFGF (20 ng/mL) for 3 days. The cultures were remained in the same medium and stimulated by adding either SDF1␣ (10 nM, Peprotech) or Wnt3a (200 ng/mL, R&D Systems) over the time course indicated in Fig. 1A. After stimulation, the cells were washed twice with cold PBS, collected in 250 ␮L of cold PBS containing 100 ␮g/mL PMSF, and 2 ␮g/mL leupeptin, followed by sonication for 10 s on ice. The cellular extracts were then centrifuged for 30 min at 100,000 × g and the supernatant (cytosolic fraction) was collected [11]. The cytoplasmic fractions were subjected to immunoblot analysis with anti-␤-catenin (1:1000, Chemicon). As shown in Fig. 1A, SDF1␣ greatly increased accumulation of ␤-catenin within 30 min, and this effect was maintained through 240 min. Wnt3a at 200 ng/mL, a positive control, also greatly enhanced the accumulation of ␤-catenin in cytoplasm. Protein loading controls were performed using coomassie blue staining on its sister membranes and showed similar staining among all lanes (last row, Fig. 1A). We then determined dose-dependent response by incubating the cells with SDF1␣ at different concentrations from 0.00001 to 10 nM for 120 min. SDF1␣, even at 10 fM, could stabilize cytoplasmic ␤-catenin (Fig. 1B). We next determined if SDF1␣-stabilized cytoplasmic ␤-catenin could be mediated by a G protein-linked ERK pathway. The E14.5 cell cultures were pretreated with PTX, an inhibitor of G protein-coupled receptor signaling, or PD98059, a specific MEK1 inhibitor, or vehicle, and were then stimulated with SDF1␣ (10 nM) for 120 min. Preliminary tests showed that either PTX or PD98059 alone at this concentration had no toxicity since no significant differences of cell viability were observed compared to control groups. The cytoplasmic fractions were prepared and used for anti-␤-catenin immunoblot. Both PTX and PD98059 completely inhibited SDF1␣ induced accumulation of

Fig. 1. SDF1␣ stabilizes cytosolic ␤-catenin in neural progenitors through PTXsensitive G protein and ERK linked pathway. Rat E14.5 cell cultures were stimulated with the indicated compounds. The cells were collected and sonicated on ice. The soluble cytosolic fractions were obtained and used (2 ␮g protein/lane) for immunoblot analysis with anti-␤-catenin. A protein loading control was performed using coomassie blue staining of sister membranes shown on the bottom row of each panel. (A) Time-course study. SDF1␣ (10 nM) significantly increased accumulation of cytosolic ␤-catenin within 30 min of stimulation. Wnt3a (200 ng/mL) served as a positive control and stabilized cytosolic ␤catenin. (B) Dose–response. The rat E14.5 cell cultures were treated with SDF1␣ for 120 min at different concentrations. SDF1␣ stabilized cytosolic ␤-catenin in a dose-dependent manner. (C) PTX and PD98059 treatments. E14.5 cell cultures were pretreated with PTX (0.01 ␮g/mL, 17 h), PD98059 (50 ␮M, 0.5 h, both labeled with +) or vehicle (water, labeled with −). Cells were then subjected to stimulation by SDF1␣ (10 nM, +) or vehicle (−) for 120 min. PTX and PD98059 completely inhibited ␤-catenin accumulation induced by SDF1 as observed in lanes 4 and 6.

cytoplasm ␤-catenin (Fig. 1C). Thus, our results indicate that the SDF1␣-enhanced ␤-catenin accumulation is mediated by PTXsensitive G protein-coupled CXCR4/ERK pathway. Since SDF1␣ stabilized cytoplasm levels of ␤-catenin, we determined if ␤-catenin translocates into the nucleus and binds to the transcription factor TCF/LEF in response to stimulation with SDF1␣. Rat E14.5 cell cultures were stimulated with SDF1␣ (10 nM) for different time periods (Fig. 2A). Nuclear extracts were prepared using Nuclear Extraction Kit (Panomics) and

Y. Luo et al. / Neuroscience Letters 398 (2006) 291–295

293

Fig. 2. SDF1␣ enhanced ␤-catenin/TCF transcription activity and their target gene expression. (A) Nuclear translocation and electrophoretic mobility shift assay. Rat E14.5 cell cultures were stimulated by SDF1␣ (10 nM) for different lengths of time. The nuclear extracts (4 ␮g/lane) were used to perform immunoblot against ␤-catenin (top row) or electrophoretic mobility shift assay. Consistent with ␤-catenin translocation into nuclei, SDF1␣ stimulated TCF/LEF DNA binding activity at 30 min, and maintained its activity at least for 240 min, cold: unlabeled excess TCF/LEF probes added as control. (B) Wnt signaling focused array analysis. Rat E14.5 cell cultures were stimulated by SDF1␣ (10 nM) for 180 min. Total RNAs were isolated and used to prepare biotin-labeled cRNA targets (5 ␮g/array). Shown is a representative image of the focused array of two independent experiments. The arrows indicate some up-regulated target genes as compared to control group. (C) Quantification of expression of some target genes by ␤-catenin/TCF/LEF transcription complex activation. Intensity of signal of each spot was measured using ImageQuant 5.2 software (Molecular Dynamics) with a local background subtraction method. A relative intensity for each spot was obtained by dividing the subtracted intensity by the average of intensity from GAPDH spots (two spots in each array). The last column shows the fold change of relative intensity of SDF1␣ treated group compared to those in controls. TF: transcription factor. (D) Immunoblot analysis. Rat E14.5 cell cultures were stimulated by SDF1␣ (10 nM) as indicated. The whole cell lysates (20 ␮g/lane) were immunoblotted with either anti-Ccnd1(1:1000, Santa Cruz) or anti-c-Myc (1:1000, Santa Cruz). Consistent with gene expression data, SDF1␣ increased Ccnd1 and c-Myc expression at protein levels. The ␤-actin immunoblot results from reprobed membranes show similar protein loadings.

immunoblotted with anti-␤-catenin. Consistent with its stabilizing ability in cytoplasm, SDF1␣ (10 nM) increased translocation of ␤-catenin into the nucleus (Fig. 2A). We then performed electrophoretic mobility shift assays using biotin-labeled TCF/LEF specific consensus DNA probes (Panomic; see sequence in [23]). Briefly, biotin-labeled specific consensus DNA sequence was incubated with 4 ␮g of the above nuclear extract proteins for 30 min at 20 ◦ C. The biotin-TCF/LEF-bound DNA complexes were separated from free probes by running through a 6% polyacrylamide gel and were then transferred to Biodyne B® membrane. The biotin-labeled DNA complex was detected by its reaction with Streptavidin-HRP conjugate and its substrate and images were acquired using a ChemoFlureTM 8900 (Alpha

Innotech Corp.). As shown in the lower panel in Fig. 2A, SDF1␣ promoted nuclear TCF/LEF DNA-binding activity which was correlated with enhanced ␤-catenin nuclear translocation. This TCF/LEF-DNA complex band was specific since it could be competed with unlabeled excess double-strand TCF/LEF DNA oligonucleotides (Fig. 2A). Using a Wnt signaling pathway-focused microarray, we examined target gene expression in response to SDF1␣ stimulated ␤-catenin/TCF/LEF transcription complex activation. Rat E14.5 cell cultures were treated with SDF1␣ (10 nM) for 180 min. Total RNAs were isolated using RNA STAT-60 (TelTest Inc). Biotin-labeled complimentary RNAs were generated using TrueLabeling-AMPTM Linear RNA Amplification Kit

294

Y. Luo et al. / Neuroscience Letters 398 (2006) 291–295

(SuperArray Bioscience Corp.). The Wnt array filters (SuperArray Bioscience Corp.) were hybridized with these biotinlabeled targets (5 ␮g/array) at 60 ◦ C for 17 h. The filters were washed and subsequently incubated with alkaline phosphataseconjugated streptavidin and CDP-Star substrate. The Chemiluminescent images were captured using FluorChemTM 8900 (Alpha Innotech Corp.). A representative image profile of two independent experiments is presented in Fig. 2B. As expected, SDF1␣ stimulation increased expression of genes that are known targets of ␤-catenin/TCF/LEF transcription complex activation. These included genes such as Ccnd1 (cyclin D1), 2, 3 for cell division and the transcription factor c-Myc as indicated by arrows in Fig. 2B. For quantification, the spot intensity was measured and normalized to the value of the housekeeping gene GAPDH. The fold changes relative to control are summarized in Fig. 2C. To confirm these results, we chose Ccnd1 and c-Myc to perform immunoblot analysis with whole cell lysates. Rat E14.5 cultures were stimulated with SDF1␣ for a time course as indicated in Fig. 2D. Whole cell lysates were obtained using 100 ␮L of ice-cold lysis buffer containing 25 mM Hepes, pH 7.5, 300 mM NaCl, 1.5 mM MgCl2 , 0.2 mM EDTA, 0.1% Triton X 100, 20 mM ␤-glycerophosphate, 0.1 mM sodium orthovanadate, 0.5 mM DTT, 100 ␮g/mL PMSF, and 2 ␮g/mL leupeptin, followed by sonication for 10 s on ice. Protein concentration was determined with a BCA protein assay kit (Pierce). As shown in Fig. 2D, SDF1␣ treatment increased levels of Ccnd1 and c-Myc proteins. In this communication, we have demonstrated that stimulation of rat neural progenitors by SDF1␣ resulted in cytoplasmic accumulation of ␤-catenin through G protein coupled CXCR4 receptor mediated ERK activation pathway. Concomitant with its ability to stabilize ␤-catenin, SDF1␣ stimulation also enhanced nuclear translocation of ␤-catenin, binding to the transcription factor TCF/LEF and target gene expression. These results extend our previous demonstration of a functional SDF1␣/CXCR4/ERK/Ets pathway in E14.5 neural progenitors. In addition, to the best of our knowledge, this is the first demonstration of ␤-catenin/TCF/LEF activation by SDF1␣ in neural progenitor cells. Moreover, our results suggest a positive cross-talk between SDF1␣/CXCR4 signaling and ␤catenin/TCF/LEF pathway that is typically referred to as the Wnt signaling pathway. There have been several reports suggesting positive modulatory signaling interactions with ␤-catenin mediated transcription. For example, stimulation of the non-receptor tyrosine kinase v-Src activates ␤-catenin/TCF mediated transcription through, at least partially, the ERK pathway in tumor cells [6]. Additionally, M1 muscarinic receptor activation leads to inactivation of GSK3␤ via PKC in GSK3␤ transgenic mice [4]. In rat E18 hippocampus cultures, stimulation of M1 receptors by specific agonist AF267B prevents amyloid-␤-peptide (A␤) induced activation of GSK3␤, destabilization of ␤-catenin and subsequent reduction of target gene expression, thus promoting neuronal survival [4]. Stimulation with prostaglandin E2 (PGE2) in HEK-293 cells also increases the TCF transcription activity involved in the activation of PKA and inhibition of GSK3␤ activity [5]. Recently, it was shown that activation of cAMP-dependent protein kinase can phosphorylate ser675 of ␤-

catenin, resulting in the inhibition of ubiquitination of ␤-catenin, thus stabilizing cytoplasm ␤-catenin and enhancing its transcriptional activity [8]. Our demonstration of SDF1␣ induced ␤-catenin/TCF transcriptional activity provides an important link to the conserved and fundamental Wnt signaling pathway in neural development. An understanding of SDF1␣ induced ␤-catenin/TCF pathway may provide a new way to study SDF1␣ mediated functions. In neural development, SDF1/CXCR4 signaling has been shown to promote cell survival and proliferation and modulate cell migration, axon growth and guidance. SDF1␣-mediated neuronal survival has been linked to the activation of MAP kinase and cAMP-dependent protein kinase (PKA) and subsequent activation of cAMP response element binding protein (CREB), as well as inhibition of GSK3␤ activity [2]. Since both activation of PKA and inhibition of GSK3␤ activity have been demonstrated to greatly stabilize ␤-catenin through inhibition of its ubiquitination and degradation to enhance TCF transcription activity [8], it could be predicted that SDF1␣-induced PKA activation and GSK3␤ inhibition may have cross-talk with ␤-catenin mediated transcription, thus, benefiting cell survival. The interaction between ␤-catenin and adhesion and microtubule complexes may participate in SDF1␣ modulated migration and axonal guidance. Since Wnt/␤-catenin/TCF signaling plays multiple functions including mitogenic stimulation, cell fate specification, and differentiation depending on the developmental stage [9,13], interaction between SDF1 and Wnt pathways for these functions should be further studied. Acknowledgements This research was supported by the National Institute on Aging Intramural Research Program, NIH, the ALS Center at Johns Hopkins, the CNS Foundation and the NIH stem cell center. MSR acknowledges the contributions of Dr. S. Rao that made undertaking this project possible. References [1] A. Bagri, T. Gurney, X. He, Y.R. Zou, D.R. Littman, M. Tessier-Lavigne, S.J. Pleasure, The chemokine SDF1 regulates migration of dentate granule cells, Development 129 (2002) 4249–4260. [2] S.H. Chalasani, F. Baribaud, C.M. Coughlan, M.J. Sunshine, V.M. Lee, R.W. Doms, D.R. Littman, J.A. Raper, The chemokine stromal cellderived factor-1 promotes the survival of embryonic retinal ganglion cells, J. Neurosci. 23 (2003) 4601–4612. [3] S.H. Chalasani, K.A. Sabelko, M.J. Sunshine, D.R. Littman, J.A. Raper, A chemokine, SDF-1, reduces the effectiveness of multiple axonal repellents and is required for normal axon pathfinding, J. Neurosci. 23 (2003) 1360–1371. [4] G.G. Farias, J.A. Godoy, F. Hernandez, J. Avila, A. Fisher, N.C. Inestrosa, M1 muscarinic receptor activation protects neurons from betaamyloid toxicity. A role for Wnt signaling pathway, Neurobiol. Dis. 17 (2004) 337–348. [5] H. Fujino, K.A. West, J.W. Regan, Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2, J. Biol. Chem. 277 (2002) 2614–2619. [6] K. Haraguchi, A. Nishida, T. Ishidate, T. Akiyama, Activation of betacatenin-TCF-mediated transcription by non-receptor tyrosine kinase vSrc, Biochem. Biophys. Res. Commun. 313 (2004) 841–844.

Y. Luo et al. / Neuroscience Letters 398 (2006) 291–295 [7] T.J. Harris, M. Peifer, Decisions, decisions: beta-catenin chooses between adhesion and transcription, Trends Cell Biol. 15 (2005) 234–237. [8] S. Hino, C. Tanji, K.I. Nakayama, A. Kikuchi, Phosphorylation of betacatenin by cyclic AMP-dependent protein kinase stabilizes beta-catenin through inhibition of its ubiquitination, Mol. Cell. Biol. 25 (2005) 9063–9072. [9] Y. Hirabayashi, Y. Itoh, H. Tabata, K. Nakajima, T. Akiyama, N. Masuyama, Y. Gotoh, The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells, Development 131 (2004) 2791–2801. [10] A.J. Kalyani, D. Piper, T. Mujtaba, M.T. Lucero, M.S. Rao, Spinal cord neuronal precursors generate multiple neuronal phenotypes in culture, J. Neurosci. 18 (1998) 7856–7868. [11] S. Kishida, H. Yamamoto, A. Kikuchi, Wnt-3a and Dvl induce neurite retraction by activating Rho-associated kinase, Mol. Cell. Biol. 24 (2004) 4487–4501. [12] R.S. Klein, J.B. Rubin, H.D. Gibson, E.N. DeHaan, X. AlvarezHernandez, R.A. Segal, A.D. Luster, SDF-1 alpha induces chemotaxis and enhances Sonic hedgehog-induced proliferation of cerebellar granule cells, Development 128 (2001) 1971–1981. [13] C.Y. Logan, R. Nusse, The Wnt signaling pathway in development and disease, Annu. Rev. Cell Dev. Biol. 20 (2004) 781–810. [14] M. Lu, E.A. Grove, R.J. Miller, Abnormal development of the hippocampal dentate gyrus in mice lacking the CXCR4 chemokine receptor, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 7090–7095. [15] Y. Luo, J. Cai, Y. Liu, H. Xue, F.J. Chrest, R.P. Wersto, M. Rao, Microarray analysis of selected genes in neural stem and progenitor cells, J. Neurochem. 83 (2002) 1481–1497. [16] Y. Luo, J. Cai, H. Xue, T. Miura, M.S. Rao, Functional SDF1 alpha/CXCR4 signaling in the developing spinal cord, J. Neurochem. 93 (2005) 452–462.

295

[17] Q. Ma, D. Jones, P.R. Borghesani, R.A. Segal, T. Nagasawa, T. Kishimoto, R.T. Bronson, T.A Springer, Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4and SDF-1-deficient mice, Proc. Natl. Acad. Sci. U.S.A. 95 (1998) 9448–9453. [18] M. Mayer-Proschel, A.J. Kalyani, T. Mujtaba, M.S. Rao, Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells, Neuron 19 (1997) 773–785. [19] E. Oberlin, A. Amara, F. Bachelerie, C. Bessia, J.L. Virelizier, F. Arenzana-Seisdedos, O. Schwartz, J.M. Heard, I. Clark-Lewis, D.F. Legler, M. Loetscher, M. Baggiolini, B. Moser, The CXC chemokine SDF-1 is the ligand for LESTR/fusin and prevents infection by T-cellline-adapted HIV-1, Nature 382 (1996) 833–835. [20] K. Reiss, R. Mentlein, J. Sievers, D. Hartmann, Stromal cell-derived factor 1 is secreted by meningeal cells and acts as chemotactic factor on neuronal stem cells of the cerebellar external granular layer, Neuroscience 115 (2002) 295–305. [21] D. Rossi, A. Zlotnik, The biology of chemokines and their receptors, Annu. Rev. Immunol. 18 (2000) 217–242. [22] R.K. Stumm, C. Zhou, T. Ara, F. Lazarini, M. Dubois-Dalcq, T. Nagasawa, V. Hollt, S. Schulz, CXCR4 regulates interneuron migration in the developing neocortex, J. Neurosci. 23 (2003) 5123– 5130. [23] M. van de Wetering, R. Cavallo, D. Dooijes, M. van Beest, J. van Es, J. Loureiro, A. Ypma, D. Hursh, T. Jones, A. Bejsovec, M. Peifer, M. Mortin, H. Clevers, Armadillo coactivates transcription driven by the product of the Drosophila segment polarity gene dTCF, Cell 88 (1997) 789–799. [24] Y. Zhu, T. Yu, X.C. Zhang, T. Nagasawa, J.Y. Wu, Y. Rao, Role of the chemokine SDF-1 as the meningeal attractant for embryonic cerebellar neurons, Nat. Neurosci. 5 (2002) 719–720.