Article Figures & Data
Figures
- Fig. 1.
Effects of different growth factor conditions on mouse E14 striatal cells. A–D, Photographs of neurospheres present in cultures grown for 7 d without EGF and FGF-2 (A) or in the presence of EGF (B), FGF-2 (C), or EGF plus FGF-2 (D). E, Number of viable cells in cultures of striatal cells grown as neurospheres for 7 d in the presence of the indicated growth factors. Data represent the means of seven independent experiments. F–H, Rate of BrdU incorporation in striatal cells grown in vitro for 1 (F), 3 (G), and 6 (H) d in the presence of the indicated growth factors. Data represent the means of four different experiments.E, #Significantly different from EGF and FGF (p < 0.001). G, *Significantly different from EGF and control (p < 0.001).
- Fig. 2.
FGF-2-induced CREB phosphorylation in mouse E14 striatal cells cultured in vitro for 24 hr.A, Examples of phospho-CREB immunostaining (right panels) and corresponding phase-contrast pictures (left panels) of unstimulated cells (unst.) or after stimulation with the indicated growth factors. Arrowsindicate phospho-CREB-immunopositive cells. Scale bar, 10 μm.B, Quantitative analysis of CREB phosphorylation in unstimulated cells (U) and in cells stimulated with EGF (E), FGF-2 (F), or a combination of EGF and FGF-2 (E/F). Data represent the means of five independent experiments. For each condition >5000 cells were counted. *Significantly different from unstimulated (p < 0.001).
- Fig. 3.
FGF-2-induced CREB phosphorylation in precursor cells and neurons present in mouse E14 striatal cells culturedin vitro for 24 hr. Examples of double immunostaining after stimulation of the cells with the indicated growth factors are shown. Phospho-CREB and nestin immunoreactivity (left panels) and phospho-CREB and TuJ1 immunoreactivity (right panels) are shown. Phospho-CREB immunoreactivity is viewed through rhodamine filters. Nestin and TuJ1 immunoreactivity is viewed through fluorescein filters. Arrows indicate double-immunopositive cells; arrowheads indicate cells immunopositive only for phospho-CREB; unst., unstimulated cells. Scale bar, 15 μm.
- Fig. 4.
Characterization of FGF-2-responsive mouse E14 striatal cells cultured in vitro for 24 hr. Quantitative analysis of phospho-CREB and nestin (A) or phospho-CREB and TuJ1 (B) double-immunopositive cells in unstimulated cultures (U) or in cultures stimulated with EGF (E), FGF-2 (F), or with a combination of both EGF and FGF-2 (E/F). Data represent the means of four independent experiments. For each condition a total of 3000 cells were counted. *Significantly different from U(p < 0.001).
- Fig. 5.
EGF- and FGF-2-induced CREB phosphorylation in precursor cells and neurons present in mouse E14 striatal cultures grown for 6 d in the presence of EGF plus FGF-2. A, B, Left panels, Examples of phospho-CREB immunostainings in unstimulated (unst.) cells and after stimulation with the indicated growth factors. Right panels, Nestin (A) and TuJ1 (B) immunoreactivity within the same visual fields. Arrowsindicate double-immunopositive cells. Scale bar, 15 μm.
- Fig. 6.
Characterization of growth factor-responsive cells in cultures of striatal precursor cells grown for 6 d in the presence of EGF (6 DIV EGF) and FGF-2 (6 DIV FGF-2) or in the presence of both (6 DIV EGF+FGF-2). Quantitative analysis of the double-immunopositive cells (A, phospho-CREB and nestin; B, phospho-CREB and TuJ1) in unstimulated cultures (U) and in cultures stimulated with EGF (E), FGF-2 (F), or a combination of EGF and FGF-2 (E/F). Data represent the means of three independent experiments. For each condition >1000 cells were counted. A, *Significantly different from U (p < 0.01);#Significantly different from E andF (p < 0.05) and fromU (p < 0.001).B, *Significantly different from unst. (p < 0.001). The differences in the numbers of TuJ1 and phospho-CREB double-immunopositive cells inU and E are not significant.
- Fig. 7.
BrdU and phopho-CREB double immunostaining. Examples of phospho-CREB immunostainings in unstimulated cultures (unst.) and after stimulation with the indicated growth factors (top panels) and BrdU-immunopositive precursors within the same visual fields (bottom panels).Arrows indicate double-immunopositive cells. Scale bar, 5 μm.
- Fig. 8.
Characterization of growth factor-responsive cells in cultures of striatal precursor cells primed for 24 hr with EGF (A, C) or FGF-2 (B, D). Graphical representation of the percentage of double-immunopositive phospho-CREB and nestin (A, B) and phospho-CREB and TuJ1, (C, D) cells in unstimulated cultures (U) and in cultures stimulated with EGF (E), FGF-2 (F), or a combination of EGF and FGF-2 (E/F). Data represent the means of three independent experiments. For each condition a total of 600 cells were counted. A, B, *Significantly different from U(p < 0.01). C, D, *Significantly different from U(p < 0.001).
- Fig. 9.
Model illustrating the generation of neural precursors responding to both EGF and FGF-2 during in vitro development. Neural precursors responding to FGF-2 (Fres) after 1 d in vitro(DIV) acquire EGF responsiveness later duringin vitro development, giving rise to precursors responding to both growth factors (E/Fres). The effects of the different growth factor conditions on this transition are illustrated: FGF-2, but not EGF, promotes the change in growth factor responsiveness; EGF plus FGF-2 has a synergistic effect on the growth of E/Fres cells. The numbers of E/Fres cells indicate their approximate growth rate under the different culture conditions.
Tables
EGF FGF-2 EGF + FGF-2 Small 85.0 ± 17.0 110.4 ± 17.8 162.1 ± 33.6 Medium 11.2 ± 3.1 41.2 ± 9.3 44.5 ± 6.0 Large 2.1 ± 0.9 4.2 ± 1.0 12.4 ± 2.3 Total no. 98.6 ± 18.9 155.8 ± 0.8 219.0 ± 38.9 Cells were grown for 6 d in vitro in the presence of the indicated growth factors. Values represent means ± SEM of counts obtained from eight different experiments.
- Table 2.
Effects of growth factor conditions on expression of differentiation markers in E14 striatal cells
nestin TuJ1 GFAP Gal-C 1 DIV (5) 46.9 ± 4.7 45.2 ± 2.9 <5 <5 6 DIV EGF (4) 64.5 ± 3.2 2.9 ± 1.3 0 <1 6 DIV FGF-2 (4) 47.6 ± 3.3 15.6 ± 3.8 0 <1 6 DIV EGF + FGF-2(4) 73.9 ± 5.8 4.3 ± 0.8 0 <1 6 DIV EGF + differentiation (4) ND 36.7 ± 6.69 62.4 ± 3.8 7.3 ± 0.2 6 DIV FGF-2 + differentiation (4) ND 39.8 ± 5.0 64.5 ± 7.4 8.4 ± 0.1 6 DIV EGF + FGF-2 + differentiation (4) ND 39.1 ± 5.0 65.6 ± 10.1 8.3 ± 0.35 Immunocytochemical detection of differentiation markers in cells after 1 d in vitro (1 DIV) in the absence of EGF and FGF-2 or after culturing for 6 d (6 DIV) in the presence of the indicated growth factors. For the growth factor-treated cultures the cellular phenotype was also assessed after differentiation in vitro (+ differentiation). Values represent means of percent total cell ± SEM; the number of experiments is indicated in parentheses. ND, Not detected.
- Table 3.
Effects of 24 h growth factor starvation on proliferation of mouse E14 striatal cells
Growth factors at DIV 0 Growth factors at DIV 1 cell no. cluster no. cell no. cluster no. EGF 42.5 ± 15.1 34.0 ± 3.5 51.2 ± 20.4 32.0 ± 5.8 FGF-2 99.7 ± 23.5 81.5 ± 23.6 113.7 ± 36.9 100.0 ± 34.2 EGF + FGF-2 512.2 ± 136.58 146.2 ± 68.1 557.5 ± 129.6 183.0 ± 76.1 Striatal cells were plated onto a poly-l-lysine–laminin substrate. The indicated growth factors were added either at the time of plating (DIV 0) or 24 h after plating (DIV 1). Values represent means ± SEM of total counts obtained from two different experiments.
