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The Journal of Neuroscience, July 23, 2003, 23(16):6638-6650
Previous Article | Next Article
A New Role for the Cell Adhesion Molecule L1 in Neural Precursor Cell Proliferation, Differentiation, and Transmitter-Specific Subtype Generation
Marcel Dihné,1 Christian Bernreuther,1 Mirjam Sibbe,1 Werner Paulus,2 andMelitta Schachner1 1Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, D-20251 Hamburg, Germany, and 2Institut für Neuropathologie, Universität Münster, D-48129 Münster, Germany
Abstract
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Abstract
Introduction
Adhesion molecules play important roles in the development and regeneration of the CNS and PNS. We found that the immunoglobulin superfamily recognition molecule L1 influences proliferation and differentiation of neural precursor cells. Substrate-coated L1 reduced proliferation of precursor cells in a dose-dependent manner and increased neuronal and decreased astrocytic differentiation when compared with poly-L-lysine or laminin substrates. Enhancement of neuronal differentiation was more effective if L1 was offered via the cell surface of transfected fibroblasts compared with substrate-coated purified L1. Furthermore, L1 decreased cholinergic-subtype differentiation and accelerated GABAergic differentiation of precursor cell-derived neurons in comparison with poly-L-lysine or laminin. Generation of dopaminergic neurons was not influenced by L1. Experiments with precursor cells generated from L1-deficient mice indicate that L1 acts via heterophilic interaction on proliferation and differentiation of L1-negative precursor cells and via a homophilic or L1 coreceptor-mediated interaction on maturation of precursor cell-derived L1-positive neurons. Clonal analysis revealed that L1 equally inhibits proliferation of monopotential, bipotential, and multipotential precursor cells, but selectively enhances neuronal differentiation of multipotential and bipotential neuron-astrocyte precursors. Our observations support a new role for L1 or L1 ligands in neural precursor cell proliferation and differentiation.
Key words: acetylcholine; adrenalin; cell adhesion molecules; differentiation; dopamine; GABA; L1; neural stem cells; proliferation
Introduction
Neural precursor cells have been the focus of increased attention over recent years because of their potential in cell replacement and gene therapy in the adult brain of vertebrates (Brüstle et al., 1997, 1999
Materials and Methods
Neural precursor cells. All experiments were approved by the University and State of Hamburg Animal Care Committees. Fourteen-day-old embryos from wild-type C57BL/6J mice or C57BL/6J mice ubiquitously expressing enhanced green fluorescent protein (GFP) under the control of the chicken actin promotor were used (Okabe et al., 1997Generation of L1-deficient neural precursor cells. For generation of L1-deficient precursor cells, the C57BL/6J L1/tTA knock-in mutant was used. In this mutant, expression of L1 was abolished by insertion of a tetracycline-controlled transactivator (tTA) (Gossen and Bujard, 1992
Purification of L1 from mouse brain homogenate. The cell adhesion molecule L1 was immunoaffinity purified from brain homogenate of adult C57BL/6J mice as described by Rathjen and Schachner (1984
Substrate-coating of L1, laminin, and poly-L-lysine. For coating of poly-L-lysine (PLL), coverslips were washed overnight in 100% ethanol. After they were washed five times in distilled water, coverslips were incubated overnight at 4°C in 0.01% PLL (Sigma-Aldrich, Deisenhofen, Germany). After coating, coverslips were washed three times in distilled water and air-dried. For coating of laminin (Sigma) or L1, PLL-precoated coverslips were treated overnight at 4°C with 100 µl of coating solution containing laminin or L1 in HBSS (PAA Laboratories GmbH, Linz, Austria) at the concentrations indicated. Before stem cells were seeded, coated coverslips were washed two times in PBS.
Production of L1-expressing fibroblasts. To produce L1-expressing fibroblasts, we used the MEF/3T3 Tet-Off Cell Line (Clontech, Palo Alto, CA), which contains the regulator plasmid of the Tet-Off-System coding for the tTA and for neomycin resistance. For L1 expression, the pBI-2 expression plasmid carrying a bidirectional tTA-responsive promotor that drives both the luciferase marker gene and the gene of interest was used (Baron et al., 1995
Measurement of luciferase activity. After lysis buffer (25 mM Tris, pH 7.8, 2 mM dithiothreitol, 2 mM diaminocyclohexanetetra-acetic acid, 10% glycerol, 1% Triton X-100) was added, cells were frozen at -20°C for 5 min. After thawing, cells were centrifuged for 15 sec at 12,000 x g. Ten microliters of supernatant were mixed with 250 µl of assay solution containing 25 mM glycerol and 15 mM MgSO4. Immediately after adding 0.125 mM D-luciferin (Sigma), which is converted by luciferase to the fluorescent end product oxyluciferin, flashes of light generated by this reaction were measured with a luminometer (Lumat LB 9501, Berthold, Bad Wildbad, Germany).
Immunoblotting. Nontransfected, parental (L1-), or L1-transfected (L1+) fibroblasts were lysed with buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P40, 1 mM Na2P2O7, 1mM NaF, 1 mM EDTA, 2 mM NaVO4, and 2% Complete Protease inhibitor (Roche Biochemicals, Mannheim, Germany). Total protein was measured using the BCA Protein Assay Kit (Pierce, Rockford, IL). Proteins were separated by SDS-PAGE (8% gels) by loading 3 µg of protein in each lane and blotted onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). After the membranes were blocked with 3% milk powder, L1 was detected after incubation with a polyclonal rabbit L1 antibody (1:4000) (kindly provided by Melanie Richter, Zentrum für Molekulare Neurobiologie Hamburg, Hamburg, Germany) at 4°C overnight and with appropriate horseradish peroxidase-conjugated secondary antibody using an ECL detection kit (Amersham Biosciences, Braunschweig, Germany).
Generation of a fibroblast monolayer. Parental and L1-transfected murine embryonic fibroblasts were expanded in DMEM high glucose (Biochrom) containing 10% fetal bovine serum (TET system approved, Clontech) and 200 mM glutamine (PAA). For collection of adherent cells, they were trypsinized with 0.05% trypsin/EDTA (PAA Laboratories GmbH, Austria). Fibroblasts were seeded onto 15 mm glass coverslips placed into 12-well plates at a density of 100,000 cells/ml (1 ml per well). At confluency after 6 d, fibroblasts were treated with 10 µg/ml mitomycin-C (Roth, Karlsruhe, Germany) for 3 hr at 37°C to inhibit proliferation.
Attachment, proliferation, and differentiation of neural precursor cells. Attachment of dissociated precursors was assessed by cell counting 12 or 24 hr after plating on coverslips coated with different concentrations of substrates. For assessment of overall proliferation of neural precursor cells and for determination of the proportion of proliferating neuronal precursor cells, bromodeoxyuridine (BrdU) (10 µmol; Sigma) was administered in EGF-FGF-containing culture medium 8 hr before cells were fixed with 4% paraformaldehyde. To detect neuronal precursor cells, double-immunolabeling for BrdU and protein gene product 9.5 (pGp 9.5) as a marker for young neurons was used. Antibodies to pGp 9.5 are from rabbit and label the same immature neurons as
-tubulin antibodies (this was confirmed by double immunocytochemistry for
For immunocytochemistry with cell type-specific markers, cells were washed in PBS and treated with 0.1% bovine serum albumin (Sigma) in PBS for 40 min. Primary antibodies, used at 4°C overnight, were monoclonal mouse antibodies to
Assessment of cell death. To determine cell death by the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) method, the In Situ Cell Death Detection Kit (Roche Biochemicals) was used according to the manufacturer's instructions.
Clonal analysis. To achieve clonal growth from single neurosphere-derived precursor cells, GFP+ precursors were plated at low density (1 cell/6 mm2) into 35 mmø cell culture dishes coated with poly-L-lysine (0.01%), laminin (20 µg/ml), or L1 (6 µg/ml). For complete dissociation, neurospheres were treated with undiluted Accutase (PAA) for 10 min at 37°C before triturating. Two hours after seeding, only well dissociated single GFP+ cells were marked by scratching the bottom of the culture dishes. Because we could not achieve clonal growth at cell concentrations under 90 cells/6 mm2, we established a mixed cell culture system with GFP+ precursors at 1 cell/6 mm2 and "supporter" GFP- precursors at 125 cells cells/6 mm2. Clonal analysis was performed only with GFP+ clones. This technique provides a cell culture system in which single precursor cell clones can be observed within their normal environment. Plating single cells at low density without "supporter" cells could lead to a selective survival of only certain precursors that do not reflect the diversity of precursor cells. Preliminary experiments demonstrated that 3 d of expansion time in the presence of EGF and FGF were optimal to obtain clearly isolated GFP+ clones situated within a population of GFP- cells. Only GFP+ clones separated by a distance of at least 500 µm were considered for clonal analysis to rule out the possibility of inter-clonal migration of cells. Precursor cells were allowed to differentiate without growth factors for 10 d after the 3 d expansion period. This procedure reduced the proportion of nestin+ immature precursor cells to
5-10% in comparison with a differentiation period of only 5 d when
Cell counting and statistics. To measure the total populations of precursor cells, GFP+ cells or DAPI+ nuclei (e.g., in the case of GFP- precursor cells from L1-deficient mice) were counted. Control experiments were performed to check that all GFP+ cells were also DAPI+ and vice versa. Experiments were repeated with independent cultures at least three times in duplicate. For clonal analysis, four independent experiments were performed. On every coverslip, at least 200 GFP+ or DAPI+ cells were counted. Percentages of double-labeled cells (cell type-specific marker-GFP+ or -DAPI+ cells) were counted and averaged. SEM was calculated. Student's t test was used for statistical evaluation.
Results
To verify whether the observation that L1 is expressed only on postmitotic neurons also applies for neurons derived from neural precursor cells, L1 immunocytochemistry was performed. Neurospheres and dissociated precursor cells under the influence of growth factors (EGF and FGF) were L1 negative. One day after initiating differentiation by growth factor withdrawal, some cells maintained on a PLL substrate were first detected to express L1 (Fig. 1A-C). Double immunocytochemistry with cell type-specific markers showed that L1 was expressed only by precursor-derived
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Figure 1. L1 expression on postmitotic neurons generated from neural precursor cells. One day after starting precursor cell differentiation, some GFP+ cells (A) are also L1+ (B) and
L1 inhibits neural precursor cell proliferation
To determine precursor cell proliferation, nestin+ cultures were maintained 8 hr in BrdU, after growing for 6 d in the presence of growth factors. Percentages of BrdU+ precursor cells were measured on substrate-coated L1 (1-15 µg/ml) and, for control, on laminin (5-50 µg/ml) or PLL. Offering L1 as a substrate was preferred over administration of L1 in solution because L1 as a substrate is more efficient than soluble L1 in enhancing neurite outgrowth or neuronal survival (Chen et al., 1999
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Figure 2. Substrate-coated L1 inhibits precursor cell proliferation. To measure the influence of different substrates on proliferation, dissociated precursor cells were plated on PLL, L1, or laminin substrates. Six days after plating, an 8 hr BrdU pulse was administered, and the percentage of BrdU+ cells was determined. Photomicrographs show more GFP+ precursor cells on PLL (A) than on L1 (B) substrate. BrdU+ cells are shown in corresponding figures of identical fields (A', B'). C, Percentages of all cells having incorporated BrdU when grown on substrates consisting of different concentrations of L1 or heat-inactivated L1 are shown in relation to BrdU incorporation on PLL substrate, which was set to 100%. D, Percentage of BrdU-labeled precursor cells on laminin substrate at different concentrations is shown in relation to BrdU incorporation on PLL substrate, which was set to 100%. E, Time course of BrdU incorporation of precursor cells growing on the indicated substrates is shown. Decreasing amounts of BrdU+ cells were detected at time points that precursor cells reached 80-90% confluency. Scale bar, 50 µm. *p < 0.05 versus the corresponding time point on PLL substrate.
To investigate whether proliferation rates depended on the day after plating and thus on different cell densities, dissociated precursor cells were maintained for 8 hr in BrdU at different times after plating on L1 (6 µg/ml), laminin (20 µg/ml), or PLL substrate in the presence of growth factors (Fig. 2E). Until the eighth day after cell plating, BrdU incorporation always tended to be higher, although not significantly, on the laminin substrate (59-62% BrdU+ cells of all cells) than on the PLL substrate (51-56% BrdU+ cells of all cells). From the eighth day onward, after plating cells on PLL or laminin substrate, precursor cells decreased their proliferation, possibly because of the fact that they had reached 80-90% confluency. During the first 12 d after plating, precursor cells on L1 substrate proliferated significantly less when compared with PLL substrate, with only 30-35% of all cells incorporating BrdU. Probably because of the lower proliferation rate, precursor cells maintained on L1 substrate took a longer time to become confluent, and thus a decrease in proliferation was only seen 14 d after plating. When L1 was administered in solution at a concentration of 10 µg/ml, a similar decrease in proliferation was seen as for substrate-coated L1 (data not shown).
L1 enhances neuronal differentiation of precursor cells
To investigate whether L1 may affect differentiation of precursor cells, cultures growing on different substrates were monitored with cell type-specific markers by immunocytochemistry. Neurons were identified by the marker
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Figure 3. Substrate-coated L1 enhances neuronal yield after precursor cell differentiation. Three days after plating, in the presence of growth factors, most of the precursor cells remain nestin+, independent of the substrate. A, A', GFP+ and nestin+ cells in the same microscopic field. At this time point,
When precursor cells were maintained on substrate-coated L1 (6 µg/ml) for 10 d (first5dinthe presence of growth factors and then for another 5 d in their absence), the percentage of
Neural precursor cells attach equally well to different substrates
Because differences in proliferation and differentiation of precursor cells may be caused by differences in cell density that in turn may depend on differences in neural precursor cell attachment to different substrates, we investigated the efficiency of attachment of precursor cells after plating onto L1 substrate in comparison with PLL in the presence of growth factors. To allow complete attachment of cells and to control for the impact of different substrates on proliferation, two time points after seeding the cells were chosen: 12 and 24 hr. Substrate-coated L1 did not lead to a significant alteration of precursor cell attachment at coating concentrations from 3 to 15µg/ml when compared with PLL (Table 1).
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Table 1. Attachment of precursor cells to substrate-coated L1 in relation to attachment to the PLL substrate
L1 does not influence cell death
To investigate whether L1 may influence the extent of precursor cell death, the percentage of TUNEL+ cells in the total cell population was determined 5 and 10 d after the cells were seeded (first 5 d in the presence of growth factors and then for another 5 d in their absence). The number of total cells was assessed by DAPI staining, not by measuring GFP, because the green fluorescent labeling would have interfered with detection of labeled DNA by TUNEL staining. As mentioned before, total cell numbers by counting GFP+ or DAPI+ cells were not significantly different from each other. Percentages of TUNEL+ cells were not different when precursor cells were maintained on PLL, L1, or laminin at different concentrations (Fig. 4A,B). On all substrates, the percentage of TUNEL+ cells was between 5.9 and 8.5%. We also investigated whether
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Figure 4. Death of precursor cells on laminin, L1, or PLL substrate, 5 d after plating with growth factors or 10 d after plating (5 d without and 5 d with growth factors) is similar. A, TUNEL labeling of precursor cells grown on PLL, L1, or laminin substrate. B, Percentage of TUNEL+ cells of all cells as detected by DAPI staining. Scale bar, 250 µm. Values are means + SEM.
L1 expressed by fibroblasts also enhances neuronal differentiation of precursor cells
To investigate whether L1 exposed on the surface of live cells would also influence proliferation and differentiation of neural precursor cells, embryonic murine fibroblasts were transfected to express L1 under a bidirectional promotor that also drives the luciferase reporter gene (Baron et al., 1995
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Figure 5. Regulated L1 expression by transfected murine fibroblasts under control of a tetracycline derivative. A, As detected by immunocytochemistry, neither parental fibroblasts (L1-) nor L1-transfected fibroblasts (L1+) in the presence of doxycycline express L1. In the absence of doxycycline, L1-transfected fibroblasts were L1+. B, As detected by immunoblotting, L1 expression was seen in transfected fibroblasts in the absence of doxycycline but not in parental cells. The two L1 bands are detected at 220 and 160 kDa.
Dissociated precursor cells were grown on a confluent layer of L1+ or L1- fibroblasts in the presence of growth factors. Attachment of precursor cells to L1+ or L1- fibroblasts 12 or 24 hr after plating was similar. Ten days after plating (first5dinthe presence of growth factors and then for another 5 d in their absence), the percentage of
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Figure 6. L1 expressed by fibroblasts influences proliferation and differentiation of precursor cells. Precursor cells were plated on a monolayer of parental or L1-expressing fibroblasts. Immunofluorescence photomicrographs of
L1-induced changes in precursor cell development are caused by heterophilic interaction
Having shown that L1 influences precursor cell proliferation and differentiation, we investigated whether homophilic or heterophilic interactions with L1 are involved. Although precursor cells do not immunocytochemically express L1 (see first paragraph in Results) and only become L1+ after differentiation into
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Figure 7. Precursor cells generated from L1-deficient mice. A-C, Sphere diameter, proliferation, and differentiation of precursor cells generated from L1-deficient mice are similar when compared with precursor cells from wild-type mice. A, Diameter of neurospheres from L1-deficient or wild-type mice. B, Percentages of BrdU+ precursor cells from L1-deficient or wild-type mice cultured on PLL or L1 substrates are given for different time points after plating. C, Percentage of cell type-specific marker expression in precursor cell-derived descendents from L1-deficient or wild-type mice. *p < 0.05 versus the corresponding time point on PLL substrate.
L1 influences differentiation of
To investigate whether L1 may influence the differentiation of precursor cell-derived neurons into a particular neurotransmitter phenotype, immunocytochemistry was performed with markers for GABAergic neurons (GAD), catecholaminergic neurons (TH), and cholinergic neurons (VAChT). Double immunocytochemistry was performed with antibodies against
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Figure 8. Generation of different neuronal subtypes from neural precursor cells over a time period of 20 d. A-C, Immunocytochemistry for vesicular acetylcholine transporter (VAChT) for cholinergic neurons and glutamate dehydrogenase (GAD) for GABAergic neurons. Time course of the percentage of VAChT (D) or GAD (E) expression of all
GABAergic neurons were already detected in higher amounts 5 d after starting differentiation, with 4.3 ± 0.4 times more GABAergic neurons on L1 than on PLL substrate (Fig. 8D). Ten days after starting differentiation, the percentage of GABAergic neurons was higher than after 5 d on PLL and L1, with neurons plated on L1 showing 1.7 ± 0.5 times more GABAergic phenotypes than those on PLL substrate. Fifteen and 20 d after starting differentiation, the percentage of GABAergic neurons plated on PLL was approximately equal to that on L1 substrate.
To control for possible differences in cell densities between precursors maintained on PLL or L1 substrate, precursor cells on L1 substrate were allowed to proliferate for an additional 3 d. When equal cell densities were reached (5 d on PLL and 8 d on L1 substrate), both cultures were allowed to differentiate for 10 d without growth factors. Even with identical cell densities, differences in percentages of cholinergic neurons (15.3 ± 2.1% on PLL; 8.6 ± 1.8% on L1) and GABAergic neurons (10.6 ± 1.9% on PLL; 19.5 ± 2.4% on L1) were seen. When precursor cells were maintained at higher concentrations of substrate-coated L1 (9 and 12 µg/ml), results were similar to those obtained with coating concentrations of 6 µg/ml L1 (data not shown). Furthermore, L1 exposed on the cell surface of fibroblasts also influenced neurotransmitter-specific maturation of
To investigate whether neurotransmitter-specific differentiation of neurons was caused by homophilic or heterophilic L1 interactions, experiments were performed with precursor cells from L1-deficient mice seeded on PLL or L1 substrate. Substrate-coated L1 did not influence the neurotransmitter-specific maturation of precursor-derived neurons from L1-deficient mice (L1- GABAergic neurons-
L1- cholinergic neurons-
L1 inhibits proliferation of monopotential, bipotential, and multipotential precursor cell clones
To investigate whether the increased number of neurons after precursor cell differentiation on the L1 substrate might be caused by selectively enhanced proliferation of neuronal precursors versus an inhibited proliferation of glial precursor cells, a clonal analysis was performed. First, we compared the number of cells within different types of clones (monopotential, bipotential, and multipotential) derived from single neural precursor cells. Both monopotential neuronal clones and monopotential astrocytic clones showed a significant reduction in cell number of 46% (p < 0.01) or 38% (p < 0.05), respectively, when grown for 15 d on the L1 substrate in comparison with PLL or laminin substrates (Fig. 9A,B). This observation rules out the possibility that enhanced proliferation of monopotential neuronal but not glial precursor cells leads to the overall increased percentage of neurons in differentiated cultures. This conclusion is supported further by the observation that the number of proliferating neuronal precursors identified immunocytochemically by BrdU+-pGp+ double labeling is decreased on the L1 substrate (data not shown). Also the bipotential (Fig. 10A-C) and multipotential (Fig. 10D) clones proliferated less when grown on an L1 substrate compared with PLL or laminin substrates. To rule out possible differences in the extent of cell death when comparing different clone types and different substrates, quantification of TUNEL-positive cells within monopotential neuronal or astrocytic clones was performed. No significant differences in number of TUNEL-positive cells on different substrates were detected (data not shown).
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Figure 9. L1 inhibits proliferation of monopotential neuronal or astrocytic clones. The number of cells within individual monopotential neuronal (A) or astrocytic (B) clones was determined after clones were maintained for 15 d on PLL, laminin, or L1 substrates. Diagrams indicate the averaged number of cells per clone. Left and right photomicrographs show representative GFP-labeled neuronal (A) and astrocytic (B) clones on L1 or PLL substrates. Clone type was assessed by triple-label immunocytochemistry for
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Figure 10. L1 inhibits proliferation of bipotential (A-C) and multipotential (D) clones. The number of cells within individual clones was determined after clones were maintained for 15 d on PLL, laminin, or L1 substrates. Diagrams indicate the averaged number of cells per clone. Values are means + SEM. *p < 0.05 versus the corresponding clone type on laminin and PLL.
L1 influences selectively the neuronal differentiation of precursor cells within multipotential and bipotential neuron-astrocyte clones
To assess whether the increased number of neurons on the L1 substrate is caused by changes in the proportion of different cell types only within certain clone types, we determined the percentage of neurons, astrocytes, and oligodendrocytes within individual bipotential and multipotential clones. Indeed, L1 increased selectively the percentage of neurons in neuron-astrocyte bipotential clones (2.8-fold versus PLL substrate, p < 0.05; 2.1-fold versus laminin substrate, p < 0.05) and in multipotential clones (3.9-fold versus PLL substrate, p < 0.001; 3.2-fold versus laminin substrate, p < 0.001) (Fig. 11). The neuron-oligodendrocyte ratio in bipotential neuron-oligodendrocyte clones was not influenced by L1. Thus, when the uncloned population was assayed for neuronal differentiation (see above), it is the neuronal precursor pool derived from multipotential and bipotential neuron-astrocyte precursors that is responsible for the overall twofold (in comparison with PLL, p < 0.05) and 1.9-fold (in comparison with laminin, p < 0.05) increase in number of neurons after maintenance on the L1 substrate for 10 d.
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Figure 11. Percentages of neurons, oligodendrocytes, and astrocytes within different clone types maintained on PLL, L1, or laminin substrates. Individual precursor cell clones maintained on PLL, L1, or laminin were scored for their numbers of
L1 does not change the initial lineage potential of individual precursor cells
To investigate whether the commitment of the different monopotential, bipotential, and multipotential precursor cells is stable after maintaining single cells for 15 d on L1, laminin, or PLL substrates, quantification of different clone types on different substrates was performed. No differences were detected concerning the proportion of different clone types on different substrates (Fig. 12). Thus, L1 does not appear to influence the initial lineage-potential of the monopotential, bipotential, and multipotential clones but modulates their ensuing individual lineage decision.
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Figure 12. Percentages of different clone types on PLL, laminin, or L1 substrate. After precursor cells were maintained on PLL, laminin, or L1 substrates for 15 d, the distribution of different clone types was scored for each substrate. No significant differences were found between different substrates. Values are means + SEM. Clone types were as follows: Multi, multipotential; N+O, neuronoligodendrocyte bipotential; N+A, neuron-astrocyte bipotential; O+A, oligodendrocyte-astrocyte bipotential; A, astrocytic monopotential; N, neuronal monopotential.
Discussion
Our present study demonstrates that the cell adhesion molecule L1 modifies neural precursor cell proliferation, differentiation, and neuronal subtype-specific development, first via heterophilic mechanisms and then via a homophilic or L1 coreceptor-mediated interaction. L1 inhibits proliferation of different precursor cell types cultured in the presence of growth factors via heterophilic interactions. After precursor cell differentiation is initiated, L1 enhances neuronal differentiation and inhibits astrocytic differentiation, also via heterophilic interactions. During differentiation of precursor cells into mature neurons, L1 inhibits maturation of cholinergic neurons and accelerates maturation of GABAergic neurons. This effect depends on the presence of homophilic L1 or L1 coreceptor-dependent mechanisms. The rare event of maturation into dopaminergic neurons was not influenced by L1 under our culture conditions. In contrast to L1, the extracellular matrix molecule laminin did not influence precursor cell proliferation and differentiation significantly when compared with poly-L-lysine, which was used as reference in this study.L1 inhibits in a dose-dependent manner precursor cell proliferation in the presence of EGF and FGF. Because L1 does not change the percentage of TUNEL+ precursor cells in comparison with laminin or PLL, reduction of proliferation is not caused by apoptosis. Because the anti-proliferative effect of L1 was detected over a culture period of 2 weeks after plating, cell density does not appear to play a role. In contrast to the anti-proliferative effect of substrate-coated L1, L1+ fibroblasts tended to but did not significantly decrease proliferation of precursor cells in comparison with L1- fibroblasts. However, precursor cell proliferation was in general significantly lower when cultured on fibroblasts, independent of their L1 expression, and thus it is possible that a fibroblast-induced inhibition of precursor proliferation reduces the contribution of L1. Because the anti-proliferative effect of substrate-coated L1 is clearly dose dependent, it is also conceivable that L1 is not expressed in sufficient amounts on the surface of fibroblasts to influence precursor cell proliferation. However, because the amount of L1 expressed on fibroblasts is sufficient to enhance neurogenesis, the former explanation appears more reasonable. Although substrate-coated L1 was able to decrease precursor cell proliferation, precursors did not differentiate as long as growth factors were present. Thus, L1 can counteract the proliferative effect of growth factors but cannot initiate differentiation in the presence of growth factors.
Substrate-coated L1 led to an approximately twofold higher number of
Different mechanisms by which L1 influences the generation of
Another possibility by which the percentages of neurons and astrocytes may be influenced is to promote survival or to inhibit apoptosis of a particular subgroup of cells (Kirschenbaum and Goldman, 1995
A third possibility by which the percentages of neurons and astrocytes may be influenced on the L1 substrate is an instructive role in lineage decision. This instructive role for L1 is most likely, because L1 does not increase proliferation or reduce apoptotic cell death. This possibility is supported by the observation that enhanced neuronal differentiation selectively occurs within multipotential and neuron-astrocyte bipotential clones but not within neuron-oligodendrocyte bipotential clones.
The interrelationship between proliferation and differentiation of stem cells remains tentative so far, because a reduction in proliferation does not mean that stem cells automatically start to differentiate and lose multipotentiality (Morshead and van der Kooy, 1992
This apparent instructive role of L1 for neuronal differentiation might be an explanation for the reduced number of hippocampal neurons in L1-deficient mice (Demyanenko et al., 1999
It has been reported that the neural cell adhesion molecule (NCAM) reduces proliferation of hippocampus-derived precursor cells and enhances neuronal differentiation (Amoureux et al., 2000
L1 influences neural precursor cell proliferation and differentiation via heterophilic interactions, because our data show that precursor cells derived from L1-deficient mice also were inhibited in proliferation and enhanced in neuronal differentiation by substrate-coated or membrane-bound L1. Because L1 has been shown to bind heterophilically to recognition molecules such as F3-F11-contactin, CD24, and integrins (Brümmendorf and Rathjen, 1993
In contrast to heterophilic influences on precursor cell proliferation and differentiation, the transmitter subtype of precursor cell-derived neurons was influenced only if L1 was present both in the substrate and on immature neurons. This constellation is consistent with a possible homophilic interaction between exogenous and endogenous L1, but also with a heterophilic interaction between exogenous L1 and a so far unknown coreceptor for L1 that depends on endogenous L1 for signal transduction. Although 5 and 10 d after starting differentiation no significant differences were detected between cholinergic neurons on PLL or L1 substrate, 15 and 20 d after starting differentiation percentages of cholinergic neurons were significantly lower on L1 substrate. Furthermore, the L1 substrate accelerates maturation of GABAergic neurons, because percentages of GABAergic neurons 5 and 10 d after starting differentiation on L1 substrate were higher when compared with PLL substrate. However, 15 and 20 d after starting differentiation, no significant differences in percentages of GABAergic neurons on L1 or PLL substrates could be detected, indicating that L1 merely accelerates GABAergic differentiation. These effects were independent of the form of presentation of L1, whether substrate coated or surface membrane bound. Interestingly, we only occasionally observed dopaminergic neurons, independent of whether precursors were cultured on PLL or L1 substrate. The fact that L1 is first expressed on postmitotic neurons points to the interesting possibility that the neurotransmitter subtype decision is made, at least under the circumstances of our study, after a cell has begun to differentiate into a neuron. During CNS development, L1 thus could influence neural precursors sequentially. First, L1 could act via heterophilic action on multipotential and bipotential precursor cells and then via homophilic or L1 coreceptor-mediated mechanisms on immature neurons that start to express L1. The mechanisms by which L1 influences transmitter subtype-specific differentiation of neurons remain to be investigated.
Footnotes
Received Apr. 7, 2003; revised May. 27, 2003; accepted May. 30, 2003.This work was supported by the Deutsche Forschungsgemeinschaft (Di 881/1-1; Scha 185/32-1). We are grateful to Masaru Okabe for providing the transgenic mouse expressing EGFP, to Michael Kutsche and Peggy Putthoff for providing the L1-deficient mouse, and to Melanie Richter for generating a polyclonal L1 antibody. We are particularly grateful to Marius Ader and Udo Bartsch for helpful discussions and critical comments on this manuscript.
Correspondence should be addressed to Dr. Marcel Dihné, Zentrum für Molekulare Neurobiologie Hamburg, Martinstrasse 52, 20246 Hamburg, Germany. E-mail: marcel.dihne{at}zmnh.uni-hamburg.de.
Copyright © 2003 Society for Neuroscience 0270-6474/03/236638-13$15.00/0
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