Figure 1

BMP signaling declines during ependymal cell differentiation. (a) Immunohistofluorescence analysis of Id1 in the LGE of the embryonic mouse brain at E14.5 or E16.5 as well as of Id1, glial fibrillary acidic protein (GFAP), and S100 in the SVZ of the lateral ventricles in the adult mouse brain (P56–P63). The boxed regions of the upper panels are shown at higher magnification in the corresponding lower panels. Arrowheads indicate Id1⁺ cells. Scale bars, 200 μm (upper panels) or 20 μm (lower panels). Data are representative of three independent experiments. (b) Quantification by immunohistofluorescence analysis of Id1⁺ cells among Sox2⁺Ascl1^– NPCs in the LGE at E14.5 and E16.5. Data are means ± SEM (n = 3 embryos for each stage). Three sections were counted per sample. Total of 500–800 cells were counted per section. **p < 0.01 (two-tailed Student’s unpaired t test). (c) Immunoblot analysis of the VZ-SVZ of the embryonic GE and corresponding postnatal basal ganglia at the indicated embryonic, postnatal, and young adult (P30) stages with antibodies to phosphorylated (p) Smad1/5/8, to Id1, to Id4, and to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, loading control). Data are representative of three independent experiments, with each experiment analyzed several times. (d) Dissociated cells of the lateral ventricular wall of P0 mice were cultured first for 5 days in vitro (DIV) in the presence of 10% FBS (proliferation medium) and then for up to 5 DIV in the absence of serum (differentiation medium). The cells were subjected to immunocytofluorescence staining with antibodies to Foxj1 and to Id1 at the indicated times. Scale bars, 20 μm. Data are representative of three independent experiments. (e) Immunoblot analysis of cultures as in (d) with antibodies to Id1, to Id4, and to GAPDH. Data are representative of three independent experiments. (f) Id1 immunofluorescence intensity in Foxj1⁺ cells (n = 395) and Foxj1^– cells (n = 484) in cultures as in (d) analyzed at 5 DIV. Each dot represents one cell, and the red bar indicates the median. Data are from three independent experiments. ***p < 0.001 (two-tailed Student’s unpaired t test).