EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells

Neuroscience Letters 385 (2005) 204–209

EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells Mark Katakowski a,b , Zhenggang Zhang a , Ana C. deCarvalho a , Michael Chopp a,b,∗ a

Department of Neurology, Henry Ford Health Sciences Center, 2799 West Grand Boulevard, Detroit, MI 48202, USA b Department of Physics, Oakland University, Rochester MI, 48309, USA Received 18 February 2005; received in revised form 16 May 2005; accepted 19 May 2005

Abstract The subventricular germinal zone (SVZ) retains an active population of stem cells and neural precursor cells throughout adulthood. EphrinB signaling mediates angiogenesis and vasculogenesis in the developing and adult brain. Recent studies indicate that molecules involved in angiogenesis often influence neurogenesis as well. However, little work has been done considering a role for EphB2/EphrinB in adult neural precursor cells. We therefore examined whether the EphB2 receptor tyrosine kinase could directly effect proliferation of SVZ neural precursors and/or direct the cell fate of SVZ cells in vitro. Here, we found that clustered EphB2 increased bromodeoxyuridine (BrdU) incorporation and proliferation of SVZ neurosphere cultures. Immunostaining and RT-PCR analysis for ␤-tubulin III (Tuj1) and GFAP indicated 4-day treatment with EphB2 promoted a neuronal phenotype, suggesting that the EphB2 receptor might also direct SVZ cell fate. EphB2 transiently downregulated SVZ cell mRNA of Notch1 and Zic1, genes that regulate neurogenesis and neuronal differentiation. Notch1 has been implicated in apoptosis of neural precursors, however, a cell viability assay revealed no statistical difference between EphB2-treated and control cultures. When SVZ neurospheres were cultured upon Matrigel, EphB2 attenuated radial migration of SVZ cells in vitro. These results demonstrate that EphB2/EphrinB signaling directly induces SVZ proliferation, decreases migration, and promotes a neuronal fate of SVZ neural precursors independent of cell survival. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Subventricular zone; EphB2; Ephrin; Neurogenesis; Notch1; Neural precursors

The subventricular zone (SVZ) consists of a population of cells that lines the subependyma of the lateral ventricles. This region contains both stem cells and neural precursors that maintain their multi-potentiality throughout adult life [2]. Brain trauma, epilepsy, and ischemia activate SVZ proliferation and may result in the migration of SVZ neuroblasts to the site of injury [7,24]. Previously, we found that ischemic stroke induces SVZ neurogenesis and stimulates the migration of SVZ neural precursors [13,25]. Recent work to uncover the molecular signals that regulate these SVZ transformations suggest that molecules that play a role in angiogenesis such as VEGF, Notch and Slit, may also be involved in SVZ neurogenesis and migration as well [6,12,17]. EphB2 is a membrane-bound receptor tyrosine kinase that binds to the membrane-bound EphrinB1–3 ligands lead∗

Corresponding author. Tel.: +1 313 916 2227; fax: +1 313 916 1318. E-mail address: [email protected] (M. Chopp).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2005.05.060

ing to bidirectional signaling between adjacent cells [18]. The EphB2/EphrinB2 receptor–ligand interaction directs endothelial precursor cell fate in vivo, and promotes the proliferation and migration of endothelial cells in vitro [1,14]. In a prior study, Conover et al. [8] reported that intraventricular injection of the ectodomain of EphB2 over 3.5 days into adult mouse resulted in increased proliferation of SVZ astrocytes lining the lateral ventricle and disruption of SVZ neuroblast migration in the rostral migratory stream. These data suggest that EphB2/EphrinB signaling may mediate SVZ modifications within the adult brain. However, EphB2 can bind to brain endothelial cells in addition to cells of the SVZ and possibly indirectly affect SVZ populations. Therefore, to clarify a direct influence of EphB2 upon adult SVZ proliferation, migration and cell fate, we chose to investigate the effects of EphB2 upon SVZ cultures in vitro. The adult SVZ consists of three primary cell types, Type-A Tuj1+ proliferative neuroblasts, Type-B GFAP+

M. Katakowski et al. / Neuroscience Letters 385 (2005) 204–209

self-renewing neural stem cells, and Type-C Nestin+/GFAP− transit-amplifying secondary precursors [2]. When isolated, dissociated and cultured in the presence of epidermal growth factor (EGF), these cells can be expanded as neurospheres composed of cells that retain characteristics of SVZ precursor cells for several passages [10]. Ephrin-B1 is not expressed in the normal adult SVZ, whereas the ligands EphrinB2/3 have been localized to SVZ astrocytes (Type-B cells) and the EphB2 receptor is expressed by both Type-B and Type-C cells [8]. Thus, addition of EphB2 to SVZ cultures could potentially activate EphrinB2/3 ligand signaling in SVZ astrocytes, or competitively antagonize EphB2 activity in SVZ astrocytes and Type-C secondary precursors. Here, we show that clustered EphB2-Fc increases SVZ cell proliferation, promotes a neuronal phenotype, and alters Notch1 and Zic1 gene expression in vitro. EphB2 significantly decreases, but does not inhibit SVZ cell migration in vitro. Assessment of cell viability indicated that the effects of EphB2-Fc upon SVZ precursor cells were independent of cell survival. SVZ neurosphere culture: SVZ cells were dissociated from male adult C57 mice (2–3 months) subventricular zone tissue and cultured, as previously reported [23]. The generated neurospheres were passed by mechanical dissociation and reseeded as single cells at a density of 20 cells per microliter. All cells used in experiments were passages 2–5. For all experiments, SVZ cells were used 7–10 days after the preceding passage. EphB2 clustering and treatment: Soluble EphB2 is not sufficient to activate membrane-bound Ephrin ligands unless clustered [19]. In preliminary experiments, we found that unclustered 2 ug/ml EphB2 had no effect upon SVZ cell proliferation as measured by MTT or in vitro SVZ cell migration in Matrigel (results not shown). Recombinant mouse EphB2 fused to the Fc fragment of human IgG (R&D Systems, MN) was added to serum-free media containing 200 ug/ml goat anti-human-Fc monoclonal antibody (Jackson ImmunoResearch, PA) at a concentration of 20 ug/ml and incubated for 1.5 h at room temperature. In all experiments, EphB2-Fc was added to cultures at 2 ug/ml. For controls, serum-free media containing anti-human-Fc antibody only was added. Statistical analysis: Student’s t-test was employed to compare the means of control and experimental groups using Slide Write Plus software (Encinitas, CA). Samples were considered independent (unpaired) for determining whether to reject the null hypotheses. A P-value < 0.05 was considered statistically significant. All experiments were performed at least twice. Proliferation assay: One thousand cells were added to each well of a u-shaped bottom 96 well plate (n = 12 wells per experimental group). Cells were treated with clustered EphB2-Fc or anti-Fc for 4 days in 300 ul medium. At 4 days, 5 mg/ml MTT in PBS was added at 0.5 mg/ml and cells were incubated for 4 h. Plates were centrifuged and 280 ul of medium was removed. SDS (10%, 180 ul) was added to each well, and the plate was incubated at 37 ◦ C overnight.

205

Absorbance was read at 540 nm, and groups were compared using Student’s t-test. BrdU incorporation: Prior to EphB2-Fc treatment, SVZ neurospheres were passaged into 10 ml of medium containing 4 × 105 cells. BrdU was added to control or EphB2-Fctreated cultures at 10 ␮M for 24 h. SVZ cells were dissociated and BrdU incorporation was measured with a BrdU Flow Kit (BD Biosciences, CA) as instructed, and quantified on a FACSCalibur flow cytometer (BD Biosciences, CA). Live/dead cell viability assay: Cells were dissociated and plated onto glass coverslips precoated with 2 ug/cm2 laminin (Sigma, MO) in 24-well culture plates. After 4 h, clustered EphB2-Fc or control antibody was added to SVZ cultures. Cell viability was assessed with a Live/Dead Viability/Cytotoxicity Kit (Molecular Probes, OR). Cells were imaged and counted using Metamorph software (Universal Imaging, PA) at 20× objective (12 wells per group with 3 images per well). Average live cell numbers per well were determined and groups were compared using Student’s t-test. Real-time RT-PCR: Quantitative PCR was performed using SYBR Green real-time PCR system. Total RNA was extracted using an Absolutely RNA Miniprep kit (Stratagene, CA). cDNA was prepared from total RNA using oligo(dT), dNTP mix, First-Strand Buffer, DTT, RNaseOUT, and Superscipt III (Invitrogen, CA). Real-time RT-PCR was performed on an ABI 7000 PCR instrument (Applied Biosystems, CA). Primers employed were: ␤-actin; 5 -cca tca tga agt gtg acg ttg-3 (fwd), 5 -caa tga tct tga tct tca tgg tg-3 (rev). Notch1; 5 -gta ctg tac aga gga tgt gga cga-3 (fwd), 5 -cca ttg aca cac aca cag ttg tag-3 (rev), Zic-1; 5 -aaa gaa tcc tga aac tcg aga cac-3 (fwd), 5 -ttt tac tca gtc ctg gat tta ccg-3 (rev), ␤tubulin III; 5 -ctc cca ggt taa agt cct tca gta-3 (fwd), 5 -gca aca taa ata cag agg tgg cta-3 (rev), and GFAP; 5 -acc att cct gta cag act ttc tcc-3 (fwd), 5 -agt ctt tac cac gat gtt cct ctt-3 (rev). Each sample was tested in triplicate and relative gene expression was determined using the 2−∆∆CT method [15]. Real-time RT-PCR was performed for three separate experiments, yielding similar results. RT-PCR: The RT-PCR reaction system contained Taq Master, Taq Buffer with Mg+ , and Taq DNA polymerase (Eppendorf, NY). Amplification was carried out in a Master gradient thermal cycler (Eppendorf, NY) with 30 cycles of denaturation. Electrophoresis was performed in a 2% agarose gel and imaged using an Electrophoresis Systems transiluminator (Fisher Scientific, PA) and Alpha Imager software (Alpha Innotech, CA). Migration assay: 96 well plates were coated with 30 ul Matrigel per well (BD Biosciences, CA). Individual neurospheres (80–110 um diameter) were placed in each well and cultured in 250 ul of medium. Neurospheres were treated after 3 h in culture. At 4 days, migration was quantified as the mean difference between the leading edge of radially migrating cells and the original neurosphere diameter (n = 15 per group). Mean migration between groups was compared using Student’s t-test.

206

M. Katakowski et al. / Neuroscience Letters 385 (2005) 204–209

Fig. 1. SVZ cell proliferation. (A) Treatment of SVZ cells with EphB2-Fc increased metabolism of MTT at 4 days. Error bars are S.D. ( P < 0.01). (B and C) Flow cytometry analysis indicates treatment of SVZ cells with EphB2-Fc increased BrdU incorporation over 24 h. (B) Histogram overlay of positive gated events for Control (solid black) and EphB2-Fc-treated (gray outline) cells. Events under M1 represent cells positive for BrdU as determined by SVZ cell controls processed but lacking BrdU exposure. (C) Graph of BrdU positive gated events for all flow cytometry experiments, indicating EphB2-Fc treatment increases SVZ cell BrdU incorporation. Error bars are S.D. ( P < 0.05).

Immunohistochemistry: SVZ cells were passaged in flasks containing clustered EphB2-Fc or anti-human-Fc. At 4 days, neurospheres were dissociated and cells were plated at 5000 cells/cm2 on glass coverslips precoated with 2 ug/cm2 laminin. After 14 h, cells were fixed in 4% paraformaldehyde for 15 min, 100% methanol for 10 min at −20 ◦ C, then blocked in 1% BSA containing 0.01% Triton X-100 for 1 h at room temperature. Cells were incubated with primary antibodies against ␤-tubulin III (Tuj1) (Covance, NJ) or GFAP (Sigma, MO) at 4 ◦ C overnight, washed in PBS and treated with Cy3 conjugated secondary antibody (Jackson ImmunoResearch, PA) at 4 ◦ C for 48 h, then stained with DAPI (Vector Laboratories, CA). Cells were imaged at 10× objective in 8 wells per group, with three fields of view per well. Average immuno-positive cell numbers for each well were determined, and experimental groups were compared using Student’s t-test. DAPI+/Tuj1− versus DAPI+/Tuj1+ or DAPI+/GFAP− versus DAPI+/GFAP+ ratios were determined with Metamorph imaging software. Immunohistochemistry was performed three times, yielding similar results. To determine if the EphB2 receptor could directly stimulate expansion of SVZ cell cultures, we employed the MTT assay to evaluate cell proliferation. Optical absorbance

indicated that 2 ug/ml EphB2-Fc significantly (P < 0.01) increased MTT cleavage indicating a greater number of SVZ cells in EphB2-Fc-treated cultures compared to control (Fig. 1A). In a preliminary experiment, we tested a dose range of 0.5–4.0 ug/ml EphB2-Fc in the MTT assay. Although all EphB2-Fc doses increased MTT cleavage compared to control, the 2 ug/ml concentration was determined to be the most significant. The MTT assay does not distinguish between proliferation and cell viability. Therefore, we also measured BrdU incorporation as a direct measure of DNA replication. As determined by flow cytometry, treatment of SVZ cells with EphB2-Fc increased BrdU incorporation as compared to control when exposed to BrdU for 24 h. Of 7000 gated events per group, 47.1 ± 7.0% were positive for anti-BrdU fluorescein in the EphB2-Fc-treated cells compared to 69.3 ± 10.1% positive gated events in control (Fig. 1B–D). These data strongly suggest that the EphB2 receptor directly acts as a mitogenic agonist for SVZ cells. As SVZ neurospheres consist of multipotent precursors, we determined whether EphB2-Fc could induce cell differentiation. Immunostaining for the ␤-tubulin III or GFAP revealed that EphB2-Fc treatment resulted in a significant (P < 0.05) increase in the percentage of Tuj1+ neuroblasts in SVZ cell populations compared to control (Fig. 2A, B and

Fig. 2. Immunocytochemistry for Tuj1+ neuroblasts or GFAP+ SVZ astrocytes. EphB2-Fc increases Tuj1+ neuroblasts and decreases GFAP+ astrocytes after 96 h in vitro. (A and B) Cy3 (red) indicates Tuj1+ neuroblasts, and DAPI (shown green for contrast) labels all cell nuclei in control (A), and EphB2-Fc-treated cells (B), respectively. (C and D) Cy3 (red) indicates GFAP+ astrocytes, and DAPI (green) labels all cell nuclei in control (C), and EphB2-Fc-treated cells (D), respectively. Blue arrows indicate immuno-postive cells whereas yellow arrows indicate nuclei staining only. Bars in B and D are 35 ␮m. (E) Graph of Tuj1+/DAPI+ or GFAP+/DAPI+ cells counted in control and EphB2-Fc-treated cultures. Error bars are S.D. ( P < 0.05).

M. Katakowski et al. / Neuroscience Letters 385 (2005) 204–209

207

Fig. 3. RT-PCR analysis. (A) RT-PCR indicates that 96 h treatment with EphB2-Fc increases mRNA for ␤-tubulin III and slightly decreases GFAP mRNA in SVZ cell cultures. (B) Quantitative real-time RT-PCR verifies RT-PCR results, indicating that ␤-tubulin III and GFAP mRNA levels were 135 ± 5 and 91 ± 5% of control following 96 h EphB2-Fc treatment. (C) RT-PCR shows 24 h treatment with EphB2-Fc decreases Notch1 and Zic1 mRNA in SVZ cell cultures. (D) Quantitative real-time RT-PCR showing Notch1 and Zic1 mRNA levels at 1–4 days, indicating transient down-regulation of Notch1 and Zic1 following EphB2 treatment. Graphs in indicate mRNA levels normalized relative to control. Error bars are S.D. ( P < 0.05, P < 0.01).

E). Coincidently, the fraction of SVZ cells positive for GFAP was decreased by EphB2 (Fig. 2C–E). In addition to protein expression, we measured ␤-tubulin III and GFAP mRNA levels in EphB2-Fc-treated SVZ cells. RT-PCR indicated an increase in ␤-tubulin III mRNA while GFAP mRNA decreased compared to control (Fig. 3A). Real-time RT-PCR verified that 4-day EphB2-Fc treatment increased ␤-tubulin III mRNA to 135% of control while GFAP mRNA decreased slightly to 91% of control (Fig. 3B). These results further demonstrate that EphB2 promotes a neuronal phenotype in SVZ cells. Notch1 and Zic1 are genes implicated in regulation of neurogenesis and neuronal differentiation of neural precursor cells [4]. SVZ cell Notch1 and Zic1 mRNA was measured by semi-quantitative and real-time RT-PCR. RT-PCR revealed that EphB2 decreased Notch1 expression at 24 and 48 h, yet Notch1 mRNA then recovered, becoming slightly overexpressed at 3 and 4 days (Fig. 3C and D). EphB2-Fc likewise decreased Zic1 mRNA level at 24 h, but Zic1 expression also recovered and was slightly over-expressed compared to control at days 2–4 (Fig. 3C and D). These data suggest that EphB2-Fc may induce SVZ neuronal differentiation of SVZ neural precursors via transient down-regulation of Notch1 and/or Zic1. Decreased Notch1 activity has been reported to increase neural precursor apoptosis [16]. Either a loss of astrocytes or increase of neuroblasts could lead to an EphB2 promoted neuronal phenotype independent of differentiation. Thus, we determined whether EphB2 affected SVZ cell viability. At 24 and 96 h, no difference was observed between clustered EphB2-Fc-treated or anti-human-Fc-treated control cultures (Fig. 4). Since EphB2 did not enhance or reduce SVZ cell viability, the resulting increase in Tuj1+ neuroblasts seen in EphB2-Fc-treated cultures can most reasonably be explained via enhanced neuronal differentiation. To determine whether EphB2 could directly alter SVZ cell motility, we treated SVZ neurospheres with EphB2-Fc in an in vitro migration assay. SVZ neurospheres cultured upon Matrigel migrate outwards radially over the course of a few days. We found that EphB2-Fc slightly, but significantly (P < 0.05) attenuated the migration of SVZ precursor cells in vitro (Fig. 5). This suggests that the EphB2 disruption of

Fig. 4. Cell viability assay. (A) and (B) indicate live (green) and dead (red) cells in control and EphB2-Fc-treated cultures, respectively, at 24 h. Bar in (B) is 25 ␮m. (C) Graph showing the percentage of viable cells of total cells counted in SVZ cultures at 24 and 96 h. EphB2-Fc treatment had no significant effect upon SVZ cell viability at both time points (P > 0.05). Error bars are S.D.

Fig. 5. SVZ Matrigel migration assay. SVZ neurospheres cultured on Matrigel 4 days revealed that EphB2-Fc (B) decreased the rate of SVZ cells radially migrating out of neurospheres compared to control (A). Arrows indicate leading edge of cell migration. Bar in B is 800 ␮m. (C) Graph of average radial migration distance at 4 days. Error bars are S.D. ( P < 0.05).

208

M. Katakowski et al. / Neuroscience Letters 385 (2005) 204–209

SVZ precursor migration in vivo may be due in part to direct EphB2/EphrinB signaling within the cells. Brain injury, disease, and a variety of molecules can lead to SVZ neurogenesis and altered SVZ cell migration [12,24]. Recent studies suggest that molecules involved in angiogenesis, endothelial precursor migration and differentiation may mediate neurogenesis, neural precursor migration and neuronal differentiation as well [6,12,17]. This apparent trend of shared signaling amongst endothelial and neural precursor cells, lead us to examine a function for EphB2 in adult SVZ cells. In the present study, we found that direct exposure of clustered EphB2-Fc increased proliferation and neuronal differentiation of SVZ precursor cells in vitro. Treatment of SVZ cells with clustered EphB2-Fc increased the ratio of Tuj1-positive SVZ neuroblasts to GFAP-positive SVZ astrocytes. As cell viability was not altered by EphB2, it is likely that the increase in neuroblasts was due to differentiation rather than selective apoptosis. EphB2-Fc treatment transiently decreased SVZ Notch1 and Zic1 mRNA, yet prolonged exposure resulted in recovery and slightly increased Notch1 and Zic1 gene expression. Active Notch1 maintains neurogenesis or promotes a glial cell fate, and loss of Notch1 induces neuronal differentiation [6,11]. Zic1 is thought to be an upstream regulator of Notch1 and Zic proteins can inhibit neuronal differentiation via activation of Notch signals [3,4]. These findings suggest that EphB2 is capable of inducing neuronal differentiation of SVZ precursor cells, possibly via regulation of Notch1 and/or Zic1. Migration of SVZ cells out of neurospheres cultured on Matrigel was decreased by EphB2-Fc treatment. These results are consistent with the previous finding that intraventricularly infused EphB2 disrupted SVZ neuroblast migration through the rostral migratory stream [8]. In response to spinal cord lesion, EphB2 activated EphrinB2 on astrocytes helps to restrict migration of meningeal fibroblasts [5]. Similar activity in SVZ astrocytes might restrict cell migration out of SVZ neurospheres. Nonetheless, the mechanisms underlying EphB2 reduction of SVZ cell migration are likely more complex, and are beyond the scope of this investigation. However, our results indicate that EphB2 disruption of SVZ precursor cell migration is due in part to EphB2/EphrinB signaling within the SVZ cells themselves. In contrast to our finding that EphB2-Fc increases neurogenesis, as previously mentioned, an earlier study reported that intraventricular infusion of the EphB2 ectodomain into the adult rat leads to an increase in SVZ astrocytes and a reduction in SVZ neuroblasts [8]. Ligands for EphB2 are abundant in brain, and in addition to SVZ cells, the ligand EphrinB2 is expressed in neurons and brain vasculature [18]. Consequently, global effects may result from ventricular infusion of EphB2 that are dissimilar to the effects of direct EphB2 signaling in SVZ precursor cells in vitro. The regulation of SVZ Notch1 by EphB2 is particularly interesting. To our knowledge, there has been no report of regulation of Notch1 by EphB2/EphrinB activity. Whether

this is a direct effect of EphB2 or due to an indirect signaling pathway, requires further investigation. However, it is worth noting that mutant mice lacking the Notch1 ligand Delta1 show decreased expression of EphB2 and EphrinB2 in neural crest cells and somites, respectively, suggesting an underlying connection [9]. Prior studies localizing Notch1 and EphrinB within SVZ cell populations provide basis for cautious speculation. In the embryonic forebrain, Notch1 activity is localized to the radial glial-like astrocytic precursors of the ventricular zone, whereas Notch1 activity was not detected in neuronal precursors [22]. Likewise, EphrinB2 is found in SVZ astrocyte precursors, but not SVZ neuroblasts [8]. A similar coincidence of Notch1 and EphrinB2 expression is found in blood vessels where the proteins are expressed in arteries and absent in veins [21]. Notch1/EphrinB2 colocalization in the SVZ suggests that EphB2 activation of EphrinB2 could lead to modified Notch1 gene expression within SVZ astrocytes by a yet unknown mechanism. Regulation of Notch1 may then direct neurogenesis of SVZ astrocytes. Another possibility is that Notch1 indicates alterations in mitotic symmetry of proliferating SVZ precursor cells, which could promote a neuronal phenotype as well [20,26]. This is speculation however, and more work is required to verify if these are sound hypotheses. In the present study, we demonstrate that the EphrinB receptor EphB2 induces neurogenesis, neuronal differentiation and decreases migration of adult SVZ precursor cells in vitro. Our data indicate that EphB2, an abundant receptor tyrosine kinase in the brain, is a potent mediator of SVZ neurogenesis and thus may be relevant to SVZ transformations in the adult brain.

Acknowledgment This work was supported by NINDS grants PO1NS42345, RO1HL64766 and RO1NS43324.

References [1] R.H. Adams, G.A. Wilkinson, C. Weiss, F. Diella, N.W. Gale, U. Deutsch, W. Risau, R. Klein, Roles of EphrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/venous domains, vascular morphogenesis, and sprouting angiogenesis, Genes Dev. 13 (1999) 295–306. [2] A. Alvarez-Buylla, J.M. Garcia-Verdugo, Neurogenesis in adult subventricular zone, J. Neurosci. 22 (2002) 629–634. [3] J. Aruga, The role of Zic genes in neural development, Mol. Cell. Neurosci. 26 (2004) 205–221. [4] J. Aruga, T. Tohmonda, S. Homma, K. Mikoshiba, Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation, Dev. Biol. 244 (2002) 329–341. [5] L.Q. Bundesen, T.A. Scheel, B.S. Bregman, L.F. Kromer, Ephrin-B2 and EphB2 regulation of astrocyte–meningeal fibroblast interactions in response to spinal cord lesions in adult rats, J. Neurosci. 23 (2003) 7789–7800. [6] C.B. Chambers, Y. Peng, H. Nguyen, N. Gaiano, G. Fishell, J.S. Nye, Spatiotemporal selectivity of response to Notch1 signals in

M. Katakowski et al. / Neuroscience Letters 385 (2005) 204–209

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

mammalian forebrain precursors, Development 128 (2001) 689– 702. X.H. Chen, A. Iwata, M. Nonaka, K.D. Browne, D.H. Smith, Neurogenesis and glial proliferation persist for at least one year in the subventricular zone following brain trauma in rats, J. Neurotrauma 20 (2003) 623–631. J.C. Conover, F. Doetsch, J.M. Garcia-Verdugo, N.W. Gale, G.D. Yancopoulos, A. Alvarez-Buylla, Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone, Nat. Neurosci. 3 (2000) 1091–1097. M.E. De Bellard, W. Ching, A. Gossler, M. Bronner-Fraser, Disruption of segmental neural crest migration and ephrin expression in delta-1 null mice, Dev. Biol. 249 (2002) 121–130. F. Doetsch, L. Petreanu, I. Caille, J.M. Garcia-Verdugo, A. AlvarezBuylla, EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells, Neuron 36 (2002) 1021– 1034. C.H. Faux, A.M. Turnley, R. Epa, R. Cappai, P.F. Bartlett, Interactions between fibroblast growth factors and Notch regulate neuronal differentiation, J. Neurosci. 21 (2001) 5587–5596. K. Jin, Y. Zhu, Y. Sun, X.O. Mao, L. Xie, D.A. Greenberg, Vascular endothelial growth factor (VEGF) stimulates neurogenesis in vitro and in vivo, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 11946–11950. M. Katakowski, Z.G. Zhang, J. Chen, R. Zhang, Y. Wang, H. Jiang, L.J. Zhang, A.M. Robin, Y. Li, M. Choo, Phosphoinositide 3-kinase promotes adult subventricular neuroblast migration after stroke, J. Neurosci. Res. 74 (2003) 494–501. I. Kim, Y.S. Ryu, H.J. Kwak, S.Y. Ahn, J.L. Oh, G.D. Yancopoulos, N.W. Gale, G.Y. Koh, EphB ligand, ephrinB2, suppresses the VEGFand angiopoietin-induced Ras/mitogen-activated protein kinase pathway in venous endothelial cells, FASEB J. 16 (2002) 1126–1128. K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆CT method, Methods 25 (2001) 402–408. S. Lutolf, F. Radtke, M. Aguet, U. Suter, V. Taylor, Notch1 is required for neuronal and glial differentiation in the cerebellum, Development 129 (2002) 373–385.

209

[17] K.T. Nguyen-Ba-Charvet, N. Picard-Riera, M. Tessier-Lavigne, A. Baron-Van Evercooren, C. Sotelo, A. Chedotal, Multiple roles for Slits in the control of cell migration in the rostral migratory stream, J. Neurosci. 24 (2004) 1497–1506. [18] A. Palmer, R. Klein, Multiple roles of ephrins in morphogenesis, neuronal networking and brain function, Genes Dev. 17 (2003) 1429–1450. [19] D. Samuel, N.W. Gale, T.H. Aldrich, P.C. Maisonpierre, V. Lhotak, T. Pawson, M. Goldfarb, G.D. Yancopolous, Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity, Science 266 (1994) 816–819. [20] Q. Shen, W. Zhong, Y.N. Jan, S. Temple, Asymmetric Numb distribution is critical for asymmetric cell division of cerebral cortical stem cells and neuroblasts, Development 129 (2002) 4843–4853. [21] D.C. Sullivan, R. Bicknell, New molecular pathways in angiogenesis, Br. J. Cancer 89 (2003) 228–231. [22] A. Tokunaga, J. Kohyama, T. Yoshida, K. Nakao, K. Sawamoto, H. Okano, Mapping spatio-temporal activation of Notch signaling during neurogenesis and gliogenesis in the developing mouse brain, J. Neurochem. 90 (2004) 142–154. [23] L. Wang, Z.G. Zhang, R. Zhang, M.S. Hafner, H.K. Wong, Z.X. Jiao, M. Chopp, Erythropoietin up-regulates SOCS2 in neuronal progenitor cells derived from SVZ of adult rat, Neuroreport 15 (2004) 1225–1229. [24] R. Zhang, Z.G. Zhang, L. Wang, Y. Wang, A. Gousev, L. Zhang, K.L. Ho, C. Morshead, M. M.Chopp, Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats, J. Cereb. Blood Flow Metab. 24 (2004) 441–448. [25] R. Zhang, Z.G. Zhang, L. Zhang, M. Chopp, Proliferation and differentiation of progenitor cells in the cortex and the subventricular zone in the adult rat after focal cerebral ischemia, Neuroscience 105 (2001) 33–41. [26] R. Zhang, Z.G. Zhang, C. Zhang, L. Zhang, A.M. Robin, Y. Wang, M. Lu, M. Chopp, Stroke transiently increases subventricular zone cell division from asymmetric to symmetric and increases neuronal differentiation in the adult rat, J. Neurosci. 24 (2004) 5810–5815.