2 MAPK pathway

Molecular and Cellular Neuroscience 46 (2011) 296–307

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Molecular and Cellular Neuroscience j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m c n e

CHL1 negatively regulates the proliferation and neuronal differentiation of neural progenitor cells through activation of the ERK1/2 MAPK pathway Xin Huang a, Ling-ling Zhu a,b,⁎, Tong Zhao a, Li-ying Wu a, Kui-wu Wu a, Melitta Schachner c, Zhi-Cheng Xiao b,d,e,⁎, Ming Fan a,⁎ a

Department of Brain Protection and Plasticity, Institute of Basic Medical Sciences, No. 27 Taiping Rd, Beijing 100850, China Institute of Molecular and Cell Biology, Singapore Department of Cell Biology and Neuroscience, Rutgers University in the State of New Jersey, 604 Allison Road, Nelson Laboratory D251, Piscataway, NJ 08854, USA d Institute of Molecular and Clinical Medicine, Kunming Medical College, China e R&D China, GlaxoSmithKline, Shanghai, China b c

a r t i c l e

i n f o

Article history: Received 18 May 2010 Revised 26 September 2010 Accepted 29 September 2010 Available online 8 October 2010 Keywords: CHL1 Neural progenitor cells Proliferation Differentiation ERK1/2 MAPK

a b s t r a c t Neural recognition molecules of the immunoglobulin superfamily play important roles in the development and regeneration of nervous system. Close Homologue of L1 (CHL1) is a member of the L1 family of recognition molecules which are expressed during neuronal development, suggesting a potential role in neural progenitor cells (NPCs). Here, we investigated the role of CHL1 in the proliferation and differentiation of NPCs both in vivo and in vitro, and the possible mechanism involved. The number of BrdU-positive cells in the subventricular zone (SVZ) significantly increased in CHL1−/− mice compared with CHL1+/+ mice. Moreover, there were more Tuj1-positive cells in the cortical plate region in CHL1−/− mice than in CHL1+/+ controls. To further examine the function of CHL1 in the proliferation and differentiation of NPCs, NPCs from CHL1−/− mice versus littermate wild-type mice were isolated and cultured in vitro. NPCs derived from CHL1−/− mice showed increased proliferation and self-renewal ability compared with CHL1+/+ mice. In the course of differentiation, CHL1 deficiency enhanced neuronal differentiation in the absence of growth factors. Furthermore, CHL1 deficiency on the proliferation of NPCs is accompanied by means of enhanced activation of ERK1/2 mitogen-activated protein kinase (MAPK) and the inhibitor of ERK1/2 MAPK eliminates the effect of CHL1 deficiency on the proliferation of NPCs. Our results first describe the negative modulation of the proliferation and neuronal differentiation of NPCs by CHL1/ERK1/2 MAPK signaling. © 2010 Elsevier Inc. All rights reserved.

Introduction During embryonic development, NPCs are self-renewing and give rise to neurons, astrocytes and oligodendrocytes in the CNS. In the developing cerebral cortex, these proliferative neural stem cells differentiate into progenitor cells and migrate from the ventricular zone (VZ) to superficial layers of the cortical plate (CP) along the fibers of radial glial cells (Temple, 2001). The proliferation, differentiation and migration are fundamental processes in the formation of the cerebral cortex, and investigation of the ability of NPCs to self-renew and differentiate is important for understanding both normal brain development and the potential for cell replacement and gene therapy

⁎ Corresponding authors. L. Zhu is to be contacted at the Department of Brain Protection and Plasticity, Institute of Basic Medical Sciences,@ No. 27 Taiping Road, Beijing 100850, China. Fax: + 86 10 68213039. M. Fan, fax: + 86 10 68213039. Z.-C. Xiao, GlaxoSmithKline, Building 3, 898 Halei Road, Zhangjiang Hi-tech Park, Pudong, Shanghai 201203, China. Fax: + 86 21 61590700. E-mail addresses: [email protected] (L. Zhu), [email protected] (Z.-C. Xiao), [email protected] (M. Fan). 1044-7431/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2010.09.013

(Gage, 2002; Temple, 2001). The extracellular environment (or “niche”) can nurture stem cells and enable them to maintain tissue homeostasis after lesions (Wurmser et al., 2004). Within niches, neural recognition molecules expressed at the cell surface and in the extracellular matrix provide cells with information regarding their cellular environment and activate intracellular signaling pathways so that cells carry out appropriate cellular functions during development and in the adult nervous system (Moore and Lemischka, 2006). The elucidation of signaling pathways and molecules important for maintenance and differentiation of neural stem cells is necessary in order to evaluate the ability of such cells to serve as potential cellular therapeutics (Gage, 1998). Recent studies have identified various intrinsic and extrinsic cellular mechanisms that regulate the balance of self-renewal and differentiation both in vitro and in vivo (Kriegstein et al., 2006). The L1 family of recognition molecules regulates NPC proliferation, differentiation and neuronal subtype-specific development via heterophilic and homophilic mechanisms and by receptor-mediated cell interactions (Dihne et al., 2003). Close Homologue of L1 (CHL1) is a member of the L1 family, the expression of which appears to be restricted to the nervous system. CHL1

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has been shown to play important roles in neuronal growth, axon guidance, regeneration and synaptic plasticity both in vitro and in vivo (Holm et al., 1996; Hillenbrand et al., 1999). The important role of CHL1 in regeneration after trauma is increasingly recognized. Upregulation of expression of CHL1 by reactive astrocytes in the glial scar reduces axonal regeneration and inhibits functional recovery after spinal cord injury (Jakovcevski et al., 2007). In course of differentiation of NPCs, CHL1 expression is down-regulated (Gurok et al., 2004). CHL1 is also expressed in migrating neuronal precursors and participates in cortical differentiation and formation (Demyanenko et al., 2004). The expression pattern of CHL1 suggests that it may be important in regulating the functions of NPCs. However, the functional role of CHL1 in influencing proliferation and differentiation of NPCs remains unclear. To further understand the role of CHL1, we examined the proliferation and differentiation of NPCs using CHL1-deficient (CHL1−/−) mice and wild-type (CHL1+/+) littermates. Several mitogenic signaling pathways that participate in the regulating NPC proliferation have been described (Reinhard and Frank-D, 2003). One particular pathway is known as ERK1/2/MAPK cascades which are involved in the proliferation of stem cells. During the proliferation of NPCs, ERK1/2/MAPK was enhanced in a dose- and timedependent manner by activation of known FGF receptor-dependent signaling pathways (Barnabe-Heider et al., 2003; Osamu et al., 2008). The Ras/ERK/MAPK cascade was required for cell cycle progression of NPCs from G1 to S-phase and inhibited astroglial cell differentiation by repressing the JAK/STAT pathway (Meloche and Pouyssegur, 2007). CHL1 expression was enhanced in a dose- and time-dependent manner by activation of FGF receptor-dependent signaling pathways (Jakovcevski et al., 2007). The possible mechanism of CHL1 between ERK1/2 MAPK is unclear. Here, we demonstrate increased self-renewal and neuronal differentiation of NPCs in CHL1−/− mice, indicating that CHL1 has a negative impact on the proliferation and neuronal differentiation of NPCs both in vivo and in vitro. Activation of ERK1/2 MAPK was enhanced in CHL1−/− NPCs. These findings suggest that CHL1 plays a critical role in regulating the proliferation and neuronal differentiation of NPCs through the activation of ERK1/2 MAPK signaling. Results CHL1 deficiency increases proliferation and neurogenesis in cortex at E14.5 To assess a potential role of CHL1 in proliferation and neuronal differentiation of NPCs during development, we investigated CHL1 expression during cortical development. Immunofluorescence staining of coronal sections from embryonic mice showed that CHL1 was extensively expressed in the cerebral cortex at E14.5, consistent with a previous report (Demyanenko et al., 2004). CHL1 expression was also observed in the ventricular/subventricular zone (VZ/SVZ) although its expression in these regions was weaker than in the cortical plate. Double immunofluorescence staining of CHL1 and the NPC marker nestin showed that CHL1 expression in the VZ/SVZ at E14.5 colocalized with cytoplasm and cell membranes of nestinpositive cells (Fig. 1A). These observations indicate that CHL1 may play a role in cortical progenitor cells at embryonic stage. To evaluate the effect of CHL1 deficiency on proliferation of NPCs in VZ/SVZ, where the proliferating cells give rise to cortical neurons, BrdU was injected into pregnant CHL1+/− mice at E14.5. Embryos were dissected 1 h after injection, brains were paraffin-sectioned for BrdUstaining using standard methods (Ciccolini and Svendsen, 1998), and the numbers of BrdU-positive cells were counted. Fig. 1B shows that, BrdU-positive cells were observed mainly in the VZ/SVZ. In CHL1−/− mice, the BrdU-labeled cells were distributed in a broad band, while in CHL1+/+ littermates, BrdU-positive cells were confined to a narrow region. There was a significant increase (about 20%) in the total number of BrdU-positive cells at the VZ/SVZ in CHL1−/− mice compared with CHL1+/+ littermates (Fig. 1C).

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Apoptosis is critical for the regulation of total cell number in the developing brain (Depaepe et al., 2005). To determine whether the enhancement of BrdU-labeled cells in CHL1−/− mice was associated with increased progenitor cell death, we examined apoptosis in the developing cortex of CHL1−/− and CHL1+/+ mice. TUNEL assays were performed on sections of E12.5, E14.5 and E16.5 embryos. At E14.5, apoptotic cells were detected infrequently in the cortex (Fig. 1D), and the number of TUNEL-positive cells was not different between CHL1−/− and CHL1+/+ littermates. However, at E16.5, the number of TUNEL-positive cells was decreased in the cortex of CHL1−/− animals compared with their CHL1+/+ littermates (Fig. 1E). This result indicates that CHL1 deficiency does not result in extensive cell death. During development, neurons are produced in the VZ/SVZ and migrate to the cortical plate in an orderly, spatially organized manner (Kriegstein et al., 2006). In order to analyze the role of CHL1 deficiency in cortical neurogenesis and to determine whether the number of NPCs in CHL1−/− mice increased as a result of precocious differentiation, we examined early neurogenesis in these mice and in their littermate controls. We used Tuj1 immunoreactivity as a marker of neuronal differentiation. Tuj1-positive immunofluorescence in the cortical layers of CHL1−/− mice at E14.5 was present in a thicker layer than in CHL1+/+ mice (Fig. 1F). There were more Tuj1positive cells in the cortical plate in CHL1−/− mice than in CHL1+/+ littermates (Fig. 1G). These results demonstrate that loss of the CHL1 function results in increased proliferation of NPCs and increased numbers of neurons in the cortical plate at E14.5, and these suggest that CHL1 is required for the development of NPCs, especially for the proliferation and neurogenesis of cortical progenitor cells. CHL1 is expressed in NPCs derived from embryonic cortex To further explore the role of CHL1 in neurogenesis, NPCs were isolated from the cortex of E14.5 embryos and the expression of the CHL1 protein was examined during proliferation of NPCs in vitro. Double immunofluorescence staining for CHL1 and the neural progenitor marker nestin showed that the majority of NPCs were nestin-immunoreactive and that all nestin-immunoreactive cells expressed CHL1 (Fig. 2A). As shown by Western blotting, CHL1 was stably expressed in proliferating progenitor cells in vitro, with 185 kDa and 165 kDa immunoreactive bands present on days 1, 3 and 5 (Fig. 2B). CHL1 deficiency increases the proliferation of NPCs in vitro To determine the effect of CHL1 deficiency on the proliferation of NPCs in vitro, NPCs were isolated from the cortices of CHL1−/− mice and CHL1+/+ littermates at E14.5. Cells were plated in 24-well plates at a density of 5 × 104 cells/mL in a DMEM/F12 medium containing EGF and FGF-2. NPCs formed multipotent neurospheres after 5 days in culture; the second- to fourth-formed neurospheres were dissociated into single cells and used in neurosphere formation assays. The total number of neurospheres in each well was counted. We found that the number of neurospheres obtained in neurosphere formation assays was increased by almost 2-fold in CHL1−/− cells compared with CHL1+/+ controls (Figs. 3A and B), indicating that CHL1 deficiency leads to an increase in the formation of neurospheres. Capacity for self-renewal is one of the key characteristics of stem/ progenitor cells in vitro (Matthias et al., 2006). To determine whether CHL1 deficiency promotes NPC proliferation, clone formation and BrdU incorporation were measured; clone formation was taken as a measure of self-renewal capacity. Single cells from primary cultured neurospheres were planted on 96-well plates at low density (5 × 103 cells/mL). After 7 days in culture, the secondary neurospheres were formed and were counted. The number of neurospheres was increased by 1.5-fold in CHL1−/− cells compared with controls

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Fig. 2. Expression of CHL1 in NPCs derived from embryonic cortex at E14.5. (A) Double immunofluorescence staining for CHL1 (red) and nestin (green) in NPCs from E14.5 cortex. Nuclei were counterstained with DAPI (blue) (a–d). Control staining in NPCs without CHL1 antibody (red) (e–h). Scale bars, 20 μm. (B) Western blot analysis of CHL1 protein expression in NPCs. CHL1 is highly expressed in proliferating progenitor cells in vitro, with 185 kDa and 165 kDa bands recognized on the 1st, 3rd, and 5th days of proliferation in culture.

(Fig. 3C). This result indicates that CHL1−/− NPCs undergo greater numbers of self-renewing divisions in vitro than CHL1+/+ controls. After addition of BrdU to the culture medium at a final concentration of 10 μM for 24 h, the BrdU-positive cells were measured by BrdU immunofluorescence. The number of BrdU-positive cells in the cultures from CHL1−/− mice was higher than in cultures from CHL1+/+ controls (Fig. 3D); the percentage of BrdU-positive cells in these cultures was increased about 1.5-fold compared to CHL1+/+ controls (Fig. 3E), suggesting that more CHL1−/− NPCs went into DNA synthesis phase of cell cycle. Taken together, these results demonstrate that CHL1 deficiency increases the proliferation of NPCs in vitro. These results led us to question whether addition of CHL1 could inhibit the proliferation of NPCs. To test this, CHL1 proteins were coated onto coverslips, and cell proliferation on these substrates was assayed. NPCs from CHL1−/− and CHL1+/+ were plated separately

onto coverslips coated with CHL1 (5 μg/mL) or BSA (5 μg/mL) and PLL as controls. BrdU was added to the culture medium 24 h later, and the percentage of BrdU-positive cells was counted (Fig. 3G). CHL1+/+ NPCs cultured on CHL1-coated coverslips resulted in significantly decreased proliferation (17.3 ± 3.7%) compared with those on BSAcoated substrates (20.4 ± 4.7%) (Fig. 3H). The percentage of BrdUpositive cells for CHL1−/− NPCs were also decreased after growth on CHL1-coated coverslips as compared to those on BSA-coated coverslip controls (Fig. 3H). These results suggest that CHL1 deficiency increases the proliferation of NPCs in vitro, and substrate with CHL1 decreases the proliferation of neural progenitors in vitro. To further test the involvement of loss of CHL1 function on NPCs, the dissociated NPCs from wild-type mice were cultured and treated with CHL1 antibody to perturb CHL1 function. BrdU-positive cells increased in CHL1 antibody-treated groups (21.24 ± 1.02%) compared

Fig. 1. Enhanced proliferation and neuronal differentiation in CHL1−/− mice in vivo. (A) Immunofluorescence staining for CHL1 (red) in SVZ at E14.5. CHL1-positive cells are shown in red (a) and Nestin-positive cells in green (b); the two markers colocalized in the VZ/SVZ (c). Scale bars, 25 μm. (B) Representative photomicrographs of BrdU-labeled cells in the coronal cortex of CHL1−/− (b) and CHL1+/+ (a) mice at E14.5; (c and d) are magnifications of subpanels (a and b). Scale bars, 100 μm. (C) Statistical analysis of the number of BrdU-labeled cells in E14.5 CHL1−/− mice and a littermate control. The number of BrdU-labeled cells in CHL1−/− VZ/SVZ at E14.5 was higher than in the control. (D) Representative photographs of TUNEL-positive cells in brain section. TUNEL-positive cells (green) were observed in coronal brain sections from CHL1+/+ (a, c, e) and CHL1−/− (b, d, f) mice at E12.5, E14.5 and E16.5. Nuclei are stained with DAPI (blue). Scale bars, 100 μm. (E) Statistical analysis of the total number of TUNEL-positive cells in cortex. The percentage of TUNEL-positive cells was not obviously different between control and CHL1−/− mice at 12.5 and 14.5, while it was markedly decreased in cortex at E16.5 in CHL1−/− compared with CHL1+/+ mice. (F) Representative photomicrographs of Tuj1-positive cells in cortex from CHL1−/− (b and d) and CHL1+/+ (a and c) at E14.5. Subpanels (c and d) are magnifications of (a and b). Tuj1-positive cortical layers (red) in CHL1−/− mice are thicker than in CHL1+/+ mice. (G) The qualification of the Tuj1+ cells in cortex at E14.5. The areas of Tuj1+ cells were obviously increased in cortex at E14.5 in CHL1−/− mice compared with CHL1+/+. Scale bars, 100 μm. MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone; SVZ, subventricular zone.

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Fig. 3. CHL1 deficiency increases NPC proliferation and self-renewal in vitro. (A) Representative images of neurosphere formation by cells from CHL1+/+ mice (a) and CHL1−/− littermate (b). (B) Quantification of neurosphere number in CHL1−/− NPCs compared with CHL1+/+ controls. The number of neurospheres formed by cells from CHL1−/− mice after a 5 day culture was greater than in controls. (C) Analysis of the clonal formation of NPCs. The number of secondary formed neurospheres was increased by 1.5-fold in CHL1−/− cells compared with CHL1+/+ controls. (D) Representative images of BrdU-labeled cells (red) from CHL1−/− (b) and CHL1+/+ littermate (a). (E) Quantification of BrdU-positive cells following a 24 h BrdU pulse in neurospheres. Measurements were carried out by dissociating the neurospheres into single cells and counting BrdU-positive cells as a percentage of the total. BrdU-positive cells increased in CHL1−/− NPCs compared to CHL1+/+ controls. (F) The average diameter of neurospheres was measured after 5 days of culture. The average diameter of neurospheres from CHL1−/− cells was larger than in cultures of comparably treated CHL1+/+ cells. (G) Representative images of NPCs from CHL1−/− (c, d) and CHL1+/+ littermate (a, b) plated on CHL1-coated (b, d) or BSA-coated (a, c) coverslips for BrdU-staining (red). (H) Quantification of BrdU-positive cells. For CHL1+/+ NPCs, the percentage of BrdU-positive cells on CHL1-coated coverslips decreased significantly compared with those on BSA-coated substrates. As for CHL1−/− NPCs, the percentage of BrdUpositive cells also decreased on CHL1-coated coverslips as compared with BSA-coated control. **p b 0.01 compared with BSA-coated group; #p b 0.05 compared with CHL1−/− control. Scale bars, 100 μm. (I) Representative images of BrdU-labeled cells (black) were treated with control (a) or anti-CHL1 antibodies (b). NPCs were prepared from wild-type mice at E14.5 and treated with anti-CHL1 antibody (50 μg/mL). (J) The percentage of BrdU-positive cells treated with anti-CHL1 antibody compared to control antibody was calculated. Measurements were carried out by dissociating the neurospheres into single cells and counting BrdU-positive cells as a percentage of the total. BrdU-positive cells increased in anti-CHL1 antibody-treated groups compared to controls. (K) The mean diameter of neurospheres from cells treated with anti-CHL1 antibody compared to control antibody was measured. The average diameter of neurospheres from cells treated with anti-CHL1 antibody was greater than control cells. Bar, 100 μm. *p b 0.05 compared with control.

to controls (18.39 ± 0.68%) (Figs. 3I and J). Treatment with CHL1 antibodies increased the average diameter of neurospheres (87.20 ± 2.18 μm) compared with CHL1+/+ (80.87 ± 1.65 μm) (Fig. 3K). These

data show that loss of CHL1 function enhanced self-renewal and proliferation in NPCs, suggesting that CHL1 negatively regulate the proliferation of NPCs in vitro.

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Fig. 4. Enhanced neuronal differentiation and decreased glial differentiation in CHL1−/− NPCs. (A) Representative images of differentiated NPCs from CHL1−/− (b, d, f) and CHL1+/+ controls (a, c, e). Neurons were labeled with Tuj1 (red) (a, b), astrocytes were labeled with GFAP (green) (c, d) and oligodendrocytes were labeled with CNPase (green) (e, f). Nuclei were stained by DAPI (blue). Scale bars, 50 μm. (B) Quantification of the percentage of Tuj1+, GFAP+ and CNPase+ cells. Enhanced neuronal differentiation and decreased glial differentiation in CHL1−/− NPCs compared with controls. (C) Western blot analyses of Tuj1, GFAP and CNPase expression after differentiation of NPCs. (D) The relative intensity of Tuj1, GFAP and CNPase expression compared with β-actin. Expression of Tuj1 from CHL1−/− NPCs was increased, while expression of GFAP was reduced compared with CHL1+/+ controls. **p b 0.01 compared with control.

Characteristics of the differentiation ability of CHL1−/− NPCs NPCs have the ability to differentiate into neurons, astrocytes and oligodendrocytes (Merkle and Alvarez-Buylla, 2006). CHL1 downregulation in the course of differentiation of NPCs has been reported (Gurok et al., 2004). We assessed whether CHL1 affects the differentiation potential of NPCs. NPCs from E14.5 littermates were maintained for 5 days in culture without growth factors, followed by staining with three cell-type markers: β-tubulin III (Tuj1) for neurons,

GFAP for astrocytes and CNPase for oligodendrocytes (Fig. 4A). The numbers of neurons, astrocytes and oligodendrocytes were counted. We found that CHL1-deficient NPCs exhibited changes in their potential to give rise to cells in the two lineages. The percentage of Tuj1-positive neurons differentiated from CHL1−/− NPCs (14.7 ± 1.5%) was increased compared to CHL1+/+ control (10.7 ± 0.6%), while the percentage of astrocytes was significantly reduced in CHL1−/− NPCs (Fig. 4B). The expression of cell-type-specific markers by NPCs was further studied by Western blot analysis (Fig. 4C). GFAP

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Fig. 5. Phospho-ERK1/2 level is elevated in CHL1−/− NPCs. (A) Western blot analysis was performed to detect the expression of MAPK in NPCs from CHL1−/− and CHL1+/+ controls. (B, C, D) Quantification of the relative intensity of phospho-ERK, phospho-p38, and phospho-JNK related to overall protein separately. Phospho-ERK1/2 levels in CHL1−/− NPCs were greatly elevated at each time point compared to CHL1+/+ controls (B), while there were no changes in phospho-p38 (C) or phospho-JNK (D) between CHL1−/− and CHL1+/+. (E) Western blot analysis of the expression of phospho-ERK1/2 in CHL1−/− and CHL1+/+ cortex. (F) Quantification of the relative intensity of phospho-ERK. Expression of phospho-ERK1/2 was increased in CHL1−/− compared with CHL1+/+ littermates in cortex. **p b 0.01, *p b 0.05 compared with control.

expression was reduced in CHL1−/− NPCs compared with CHL1+/+ controls. The expression of Tuj1 in CHL1−/− NPCs was increased, which was consistent with the aforementioned data (Fig. 4D). These results further demonstrate that CHL1 negatively regulates the neuronal differentiation of NPCs.

Phospho-ERK1/2 levels are elevated in CHL1−/− NPCs It has been shown that ERK1/2 signaling is activated during the proliferation of NPCs in response to growth factors and that it is involved in the survival and growth of NPCs (Barnabe-Heider et al., 2003;

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Osamu et al., 2008). To investigate whether ERK1/2 signaling contributes to CHL1 deficiency-regulated proliferation of NPCs, we examined the expression pattern and activation of MAPK during NPC proliferation. Protein was extracted from CHL1−/− progenitor cells and CHL1+/+ controls on days 1, 3, 5, and 7 of culture. Expression of ERK1/2, JNK and p38 was detected by Western blot analysis (Fig. 5A). ERK1/2 signaling was activated on days 1, 3, and 5 and decreased on day 7 during proliferation in culture of NPCs from both CHL1−/− and CHL1+/+ embryos. However, phospho-ERK1/2 levels were greatly elevated in CHL1−/− NPCs compared to CHL1+/+ controls (Fig. 5B). In contrast, phospho-JNK and phospho-p38 expression levels showed no obvious differences between CHL1−/− NPCs and CHL1+/+ controls (Figs. 5C and D). To test whether phospho-ERK1/2 was also increased in CHL1−/− tissue in vivo, we examined the expression of phospho-ERK1/2 in cortical brain tissue. We found that phospho-ERK1/2 expression was also enhanced in CHL1−/− brain tissue as compared with CHL1+/+ (Fig. 5E), with significant differences in the phospho-ERK1/2 levels (Fig. 5F). These data suggest that CHL1 deficiency may specifically enhance the activation of phospho-ERK1/2 signaling during the proliferation of NPCs both in vitro and in vivo. The ERK1/2 signaling pathway is involved in CHL1−/− regulated proliferation of NPCs To address whether ERK1/2 signaling is directly or indirectly related to the enhancement of self-renewal and proliferation in CHL1−/− NPCs, we measured the proliferation of NPCs after blockade of MAPK kinase (MEK) activity by the inhibitor PD98059, which in turn inhibits ERK1/2 phosphorylation. In our previous work, we showed that PD98059 decreased neurosphere formation in a dose-dependent

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manner and that growth of NPCs was inhibited in a time-dependent manner after treatment with 20 μM PD98059, indicating that ERK1/2 is essential for the proliferation of NPCs (Huang et al., 2008). Here, the critical participation of ERK1/2 in the regulation of CHL1−/− NPCs was further explored by analysis of neurosphere formation and cell growth after treatment with PD98059. Cell proliferation was measured using the CCK-8 assay after treatment with 20 μM and 40 μM PD98059. While 20 μM PD98059 almost completely blocked the growth of NPCs from CHL1+/+, 40 μM PD98059 only blocked one-third of the growth of NPCs from CHL1−/− (Fig. 6A). The number and diameter of neurospheres were also examined in cells treated with 20 μM PD98059 after 5 days in culture. Treatment with PD98059 markedly reduced the number of neurospheres formed by CHL1−/− progenitor cells (Fig. 6B). The average diameter of neurospheres from CHL1−/− cells (54.3 ± 13.2 μm) was also increased compared with CHL1+/+ (42.8 ± 11.3 μm) (Fig. 6C). These data show that the enhancement of self-renewal and proliferation in CHL1−/− NPCs can be blocked by PD98059. Collectively, the results of these experiments demonstrate that enhanced activation of ERK1/2 signaling is involved in CHL1−/− regulated proliferation of NPCs. Some mitogenic factors have been implicated in the processes of cortical cell proliferation and differentiation (Doherty and Walsh, 1996). These growth factors have been shown to promote neurogenesis when added to cultures of stem cells derived from different regions of the brain (Kolkova et al., 2006). We therefore examined whether the enhanced proliferation of CHL1−/− NPCs in vitro was related to EGF and/or FGF-2. Cells were cultured in the absence of EGF or FGF-2 or in the absence of both, and growth curves were determined by CCK-8 assay. An enhancement of proliferation in CHL1−/− NPCs was observed on days 1, 3, 5, and 7 when cells were cultured in the absence of EGF (Fig. 6E). There was no clear difference

Fig. 6. ERK1/2 signaling pathway is involved in CHL1−/− regulated proliferation of NPCs. (A) PD98059 inhibits the proliferation of CHL1−/− NPCs. Cell proliferation was tested by CCK-8 assay after treatment with 20 μM or 40 μM PD98059. PD98059 at 20 μM almost completely blocked the growth of NPCs from CHL1+/+, while 40 μM PD98059 only blocked one-third of the growth of cells from CHL1−/− compared with CHL1+/+ controls. (B) The number of neurospheres was examined on the 5th day after the cells were treated with 20 μM PD98059. Treatment with PD98059 markedly reduced the number of neurospheres from CHL1−/−. (C) The average diameter of neurospheres was measured on the 5th day after cells were treated with 20 μM PD98059. The average diameter of neurospheres from CHL1−/− cells was lower than in cultures of comparably treated CHL1+/+ cells. (D, E, F) Cells were cultured in the absence of EGF, FGF-2 or both and growth curves were determined by CCK-8 assay. The growth of CHL1−/− NPCs was unchanged compared to controls when cells were cultured in the presence of EGF (D). The growth of CHL1−/− NPCs was increased on the 3rd, 5th, and 7th days compared with CHL1+/+ controls when cells were cultured with FGF-2 (E). The growth of CHL1−/− NPCs was different from that of CHL1+/+ controls on the 3rd, 5th, and 7th days when cells were cultured in the absence of both EGF and FGF-2 (F). **p b 0.01 compared with control.

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between CHL1−/− NPCs and CHL1+/+ controls in the absence of FGF-2 (Fig. 6D). When cells were cultured in the absence of both growth factors, the difference between CHL1−/− progenitor cells and CHL1+/+ controls was marked. NPCs from CHL1+/+ stopped growing, while CHL1−/− derived progenitor cells continued to proliferate without EGF and FGF-2 (Fig. 6F). These data indicate that an FGF-dependent signaling pathway may be involved in the activation of ERK1/2 MAPK signaling in proliferation of CHL1−/− NPCs. Discussion The present study provides novel evidence for a role of CHL1 in the proliferation and differentiation of NPCs. Our data show that CHL1 deficiency significantly increases the self-renewal capacity of progenitor cells and their neuronal differentiation both in vivo and in vitro. Further in vitro study showed that an ERK1/2 MAPK inhibitor, PD98059, eliminated the proliferation of NPCs from CHL1−/− mice. Thus, CHL1 is an essential endogenous factor negatively regulating the proliferation and neuronal differentiation of NPCs. Role of CHL1 in the proliferation and differentiation of NPCs Neural recognition molecules play important roles in specifying cell–cell interactions during development, regeneration and plasticity of the nervous system (Kamiguchi and Lemmon, 2000). CHL1 is a member of the L1 family of neural recognition molecules, itself a subgroup of the immunoglobulin superfamily characterized by six immunoglobulin-like domains (Hortsch, 2000). Evidence for an important role of CHL1 was also obtained from analysis of CHL1deficient mice, in which displacement of pyramidal neurons in layer V of the visual cortex, misguided axonal projections and aberrant connectivity in both the olfactory bulb and the hippocampus and alterations in the organization of hippocampal mossy fibers, olfactory axon projections and stellate axon arbors in the cerebellum have been demonstrated (Nikonenko et al., 2006; Pratte et al., 2003; Maness and Schachner, 2007; Buhusi et al., 2003). CHL1 expression is distinct from other members of the L1 family. It is more broadly expressed in the cortex at embryonic day. L1 family members are primarily expressed on neurons and glia, but their expression on neural progenitor cells is not clear. Here, CHL1 was detected to express in NPCs both in vivo and in vitro. We also observed that CHL1 was expressed in the VZ/SVZ in vivo, where it colocalizes with nestin-positive cells. Furthermore, CHL1 is stably expressed in embryonic NPCs during proliferation by Western blot assay. This indicates that CHL1 was expressed by neural precursor cells. Thus, CHL1 may play a crucial role in influencing the proliferation and differentiation of NPCs. The recognition molecules L1 have been reported to regulate neural precursor cell proliferation and neuronal subtype-specific differentiation (Dihne et al., 2003). The proliferation of neural precursor cells from the lateral and medial ganglionic eminences was reduced when the cells were grown on L1-coated coverslips, while neuronal differentiation increased in the presence of growth factors. However, no differences were detected in the proliferation and differentiation of precursor cells from L1-deficient and wild-type mice when grown under the same culture conditions. In contrast to L1, here, we demonstrated that CHL1 was involved not only in regulation of proliferation but also in differentiation of NPCs. Compared with CHL1+/+ controls, the neuronal differentiation of NPCs from CHL1−/− increased, while the number of GFAP+ astrocytes was reduced, suggesting that CHL1−/− brain generates more neurons at the expense of astrocyte generation. Neural recognition molecules participate in homophilic and heterophilic interactions with neighboring cells or with the extracellular matrix through diverse mechanisms (Maness and Schachner, 2007). L1 regulate neural precursor cell proliferation and neuronal

subtype-specific differentiation via a homophilic or L1 coreceptormediated interaction (Dihne et al., 2003). CHL1 has been shown to bind heterophilically to recognition molecules such as integrins and NB-3 in different cell lines. CHL1 and β1 integrins associate directly or indirectly on the cell surface, and share signaling intermediates (Src, PI3 kinase, and ERK1/2) necessary for migration (Buhusi et al., 2003). Another recognition molecule NB-3 associates with CHL1 regulate apical dendrite growth by PTPα interacts (Ye et al., 2008). In addition, semaphorin family, neuropilin/plexin coreceptors and integrins may be involved in CHL1-dependent signaling (Barnabe-Heider and Miller, 2003). From our studies, the proliferation and differentiation on CHL1-Fc substrate were reduced in CHL1−/− NPCs compared to CHL1+/+ NPCs, while loss of CHL1 function with CHL1-antibodies enhanced the proliferation of NPCs. Our findings suggested that CHL1 may influence NPC proliferation via homophilic interactions. CHL1 may generate intracellular signals by homophilic interaction or may perturb endogenous molecular interactions and prevent normal signaling events generated by other recognition interactions. However, the heterophilic and homophilic interaction involved in the proliferation and differentiation of CHL1-deficient NPCs remains unclear, and more possible mechanisms require further study to confirm.

Possible role of ERK1/2 signaling in CHL1−/− regulated proliferation of NPCs MAPK pathway plays a key role in regulating cell proliferation and differentiation, which also is involved in neurogenesis in the determination of either cell survival or apoptosis (Pollard et al., 2008). The activation of MAPK by MEK1 is important for the proliferation of NPCs. The mechanism by which CHL1 deficiency enhanced activation of ERK1/2 MAPK is unclear, but a link between FGF-2 and enhanced CHL1 expression after optic nerve crush suggests that CHL1 deficiency may activate FGF receptor-dependent signaling pathways (Jakovcevski et al., 2007; Pratte et al., 2003). Generally, NPCs derived from E14.5 rodent can be expanded in neurospheres or two-dimensional adherent cultures by using EGF and/or FGF-2 as mitogens (Michael et al., 2009); the proliferation of neural stem cells isolated from embryonic and adult forebrain is actively regulated by both EGF and FGF-2 signaling (Ciccolini and Svendsen, 1998; Gage, 2000). CHL1 expression is enhanced by the activation of FGF receptordependent signaling pathways in cultured astrocytes, and FGF-2 enhances the expression of CHL1 when injected into spinal cord. This upregulation of CHL1 expression can be observed both in vitro and in vivo (Reynolds et al., 1992; Rolf et al., 2003; Jakovcevski et al., 2007; Pratte et al., 2003). In agreement with previous observations, our study confirmed that an FGF-dependent signaling pathway may be involved in the activation of ERK1/2 MAPK signaling in proliferation of CHL1−/− NPCs. The withdrawal of mitogenic EGF and FGF-2 led to cessation of growth of CHL1+/+ derived neural progenitor cells, while CHL1−/− derived progenitors continued to proliferate. This could be explained through the activation of ERK1/2 signaling via the FGF-2 receptor, which is critical in maintaining the proliferation of NPCs in general. In this manner, FGF-2 may increase the expression of CHL1, inhibiting the activation of ERK1/2 and in turn decreasing proliferation of CHL1+/+ NPCs. In conclusion, our in vivo and in vitro observations provide the first evidence that CHL1 is expressed in NPCs and that it acts as an internal regulator of proliferation and differentiation of NPCs through inhibition of the ERK1/2 pathway. However, the exact mechanisms by which ERK1/2 signaling regulates cell proliferation and intercellular communication with CHL1 in the NPCs remain unknown. Future studies will provide insight into these issues. Our findings may help to further the understanding of the function of CHL1 in the central nervous system, with potential applications for neural lesion treatment.

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Experimental methods Mice The generation of a CHL1-deficient (CHL1−/−) mouse in the C57BL/6 genetic background has been described previously (MontagSallaz et al., 2002). Heterozygous mice were mated to generate wildtype (CHL1+/+) and knockout (CHL1−/−) pairs, and littermates were used in all experiments. Genotyping was performed by tail DNA analysis. Mice were maintained at the animal facility with free access to water and food in accordance with institutional guidelines. The Institutional Animal Care and Use Committee (IACUC) of the Academy of Military Medical Sciences approved all experiments involving mice. Immunofluorescence Brains were isolated from embryonic day 14.5 mice (E14.5) and fixed in 4% paraformaldehyde at 4 °C overnight. Brains were cryoprotected by incubation in sucrose solutions of increasing concentration (10–30%), coronally sectioned at 8 μm on a cryostat and stored at − 20 °C until use. Sections were incubated in blocking solution containing 1% bovine serum albumin (BSA) and 10% normal goat serum in PBST (0.3% Triton X-100 in PBS) for 1 h and then incubated with anti-CHL1 antibody (1:200; R&D), rabbit anti-nestin antibody (1:500; Chemicon) or rabbit anti-β-tubulin III antibody (1:2000; Sigma) at 4 °C overnight. Sections were incubated with Alexa Fluor 488 or 594-conjugated secondary antibodies (1:500, Molecular Probes). Nuclei were counterstained with DAPI-containing mounting medium (Vector Laboratories). Fluorescence was visualized using a Zeiss LSM5 confocal laser-scanning microscope. Cultured embryonal NPCs were fixed in 4% paraformaldehyde in PBS for 30 min at room temperature (RT). Blocking was carried out for 30 min in PBST containing 3% normal goat serum (NGS). Primary and secondary antibodies were diluted in PBST containing 1% NGS. Cells were incubated with primary antibodies overnight at 4 °C and with secondary antibodies at room temperature for 1 h and then mounted with DAPI. The antibodies were used as follows: mouse anti-CHL1 (1:200; Abnova), rabbit anti-nestin (1:1000; Chemicon), mouse antiglial fibrillary acidic protein (GFAP) (1:2000; Chemicon), rabbit antiTuj1 antibody (1:2000; Sigma), and mouse anti-2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase) (1:1000; Sigma). The secondary antibodies were Alexa Fluor 488 or 594-conjugated secondary antibodies (Molecular Probes). Images were photographed using an Olympus microscope (IX71).

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labeling (TUNEL) method, the In Situ Cell Death Detection Kit (Roche Biochemicals) was used according to the manufacturer's instructions. The number of TUNEL-positive cells was counted in cortex on each section, and at least 20 sections were counted in each brain. At least 6 brain samples were analyzed. Culture of NPCs from embryos NPCs were derived from cortices at E14.5 and dissociated by 0.125% trypsinase (Zhao et al., 2008). Genotyping was performed at the same time by embryo tail DNA analysis. Cells were cultured in a DMEM/F12 medium containing 1% N2 and B27 supplement (Invitrogen), 1% penicillin and streptomycin antibiotic mixture (Hyclone), 20 ng/mL epidermal growth factor (EGF) and 20 ng/mL fibroblast growth factor-2 (FGF-2) (Invitrogen). Cells from CHL1−/− mice and CHL1+/+ littermates were plated at a density of 5 × 104 cells/mL in 6well plates for expansion. NPCs formed multipotent neurospheres after 5 days in culture, and the 2nd to 4th generated neurospheres were used in the following experiments. Neurosphere formation assay NPCs from CHL1−/− and CHL1+/+ littermates were dissociated into single cells and used for neurosphere formation assay (Raballo et al., 2000). Single cells derived from neurospheres were placed in 24-well plates at 5 × 104 cells/mL per well (Costar) for 5 days, at which time the total number of neurospheres in each well was counted. Neurospheres were counted under an optical microscope by observers who were blind to the experimental conditions. Measurement of diameter Neurospheres were photographed, and the diameter of each neurosphere was measured using IPP 6.0 software. In each group, at least 300 to 500 neurospheres were measured, and experiments were replicated at least three times. Clonal analysis To measure clonal growth from single neurosphere-derived precursor cells, neurospheres were dissociated into single cells and plated into 96-well plates at low density, approximately 5 × 103 cells/mL (Molofsky et al., 2005). Neurospheres were counted 5–10 days later to determine the number of secondary formed neurospheres.

BrdU incorporation BrdU incorporation Pregnant mice at E14.5 were injected with BrdU i.p. (50 mg/kg of body weight, Sigma) dissolved in PBS. One hour later, embryos were dissected, and their brains were fixed for paraffin sections and processed for BrdU-immunoreactive staining (Zhu et al., 2005). Sections were then fixed in 4% PFA for 15 min at 4 °C. After three washes in PBS, sections were treated for 30 min in 2 N HCl at 37 °C and for 10 min in 0.1 M sodium borate, pH 8.5. BrdU-positive cells were detected using a specific mouse anti-BrdU antibody (1:1000, Neomarker) for immunocytochemistry. Quantitation and statistical analysis were carried out as described below. The number of BrdUpositive cells was counted in at least 20 non-overlapping fields in cortex on each section, and at least 10 sections were counted in each brain. Six–eight brains were analyzed per genotype. Cell death assay Embryos of E12.5, E14.5 and E16.5 littermates were collected and brains were sectioned. For determination of cell death by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-

BrdU was added to the culture medium at a final concentration of 10 μM for 24 h, at which time BrdU-positive cells were measured by BrdU immunofluorescence. Cells were fixed on coverslips and pretreated with 2 N HCl to denature DNA, then incubated with mouse anti-BrdU antibody (1:1000, Neomarker) at 4 °C overnight. BrdU-positive cells were counted under a fluorescent microscope; at least 20 fields were randomly chosen for counting. The total number of cells in each field was determined by DAPI staining. Cell proliferation assay Cells were seeded at 1 × 104 cells per well in 96-well plates (Costar). Cell counting kit-8 (CCK-8) was added to the wells (10 μL per well) at the end of the experimental period according to the manufacturer's instructions (Dojindo Laboratories). After 4 h of incubation at 37 °C, the absorbance of each well at 450 nm was recorded on an enzyme-linked immunosorbent assay reader (BioRad, USA).

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Substrate-coating of poly-L-lysine, BSA and CHL1

Appendix A. Supplementary data

For coating of poly-L-lysine (PLL), coverslips were washed five times in distilled water and then incubated overnight at 4 °C in 0.1% PLL (Sigma). After coating, coverslips were washed three times in distilled water and air-dried. BSA (Sigma) or CHL1 (R&D system) was coated on coverslips overnight at 4 °C in PBS at the indicated concentrations as previously described (Dihne et al., 2003; Ye et al., 2008).

Supplementary data to this article can be found online at doi:10.1016/j.mcn.2010.09.013.

Perturbation of CHL1 in NPCs NPCs were prepared from wild-type mice at E14.5 and were treated with control or anti-CHL1 antibodies (50 μg/mL) (Sakurai et al., 2001). BrdU was added to the culture medium at a final concentration of 10 μM for 24 h, at which time BrdU-positive cells were measured by BrdU immunohistochemistry. The percentage of BrdU-positive cells treated with anti-CHL1 antibody compared to those of control antibody was calculated. Differentiation assay The 2nd to 4th generated neurospheres were plated on poly-Llysine coated coverslips in a DMEM/F12 medium containing 1% N2 and B27-supplement, and 1% fetal bovine serum without EGF and FGF-2. Cells were fixed with 4% paraformaldehyde in PBS after 5 day differentiation and processed for immunofluorescence as described above. Western blotting Cells were harvested at the desired time points, and total protein was extracted. Equal amounts of protein (50–100 μg) were boiled in sample buffer, separated by electrophoresis on 8–12% SDS-PAGE gels, and transferred to nitrocellulose membrane for 1–3 h. Membranes were blocked in 5% skim milk powder in TBS-T (TBS plus 0.5% Tween 20) or in 3% BSA in TBS-T for 2 h at room temperature and then incubated overnight at 4 °C with primary antibody. Primary antibodies included: Total ERK (1:1000, Cell Signaling Technology), phospho-ERK (1:1000, Cell Signaling Technology), JNK1 (F-3) (1:1000, Santa Cruz), phospho-JNK (Thr183/Tyr185) (1:1000, Santa Cruz), p38 (M0800) (1:1000, Sigma), phospho-p38 (Thr180/Tyr182) (1:1000, Sigma), and β-actin (1:1000, Santa Cruz). Membranes were treated with goat anti-mouse or -rabbit HRP-conjugated secondary antibodies (1:2000, Santa Cruz). Complexes on the membrane were visualized using an enhanced chemiluminescence (ECL) detection system (Amersham Biosciences). Statistical analysis All experiments were repeated at least three times, and the measurements were performed by the observers who were blind to the groups. Data are presented as group mean values with SEM. Asterisks identify experimental groups that were significantly different from control groups according to the Student's T test or one-way ANOVA. For all analyses, P-values b 0.05 were considered significant. Acknowledgments This work was supported by a grant from the Natural Sciences Foundation of China, No. 30670792 and 30870799; the National Basic Research Program of China, No. 2006CB504100; and the Beijing Natural Science Foundation, No. 5092023. We would like to thank Prof. Zhang Tianming for his feedback and suggestions.

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