Fig. 3. The three-dimensional structure of the neurospheres compared with the developing neuroepithelium. (A) Expression of ß1 on 14 µm cryostat sections of neurospheres grown in EGF from P0 rat pups. Note that high-ß1-expressing cells are present at the edge of the spheres. (B) Co-labelling of high-ß1-expressing cells (green) with anti-nestin antibodies (red) is seen at the edge of the neurosphere. (C) The cells in the centre of the neurosphere, shown by DAPI staining (blue), do not stain for nestin (red). Expression of GFAP (D, red), nestin/GFAP (E, green and red, respectively) and ß3 tubulin (F, red) in neurospheres grown from P0 rat pups shows that differentiated cells are present mostly in the center. Nuclei are counterstained with DAPI (blue). Proliferation in the developing VZ and in the neurospheres is shown in panels G-K. In the E15.5 developing rat brain, cells divide in the VZ, as shown by the expression of phosphorylated histone H3 (P-H3, red; H,I), whereas ß3 tubulin⁺ cells (green; H,G) do not express P-H3. (G,I) Higher magnification views of the ß3 tubulin⁺ region (G) and the phosphorylated histone H3+ region (I) seen in H, as indicated by the boxes. Likewise in the neurospheres BrdU incorporation (green nuclei, J) occurs at the periphery only, and not in the postmitotic ß3 tubulin⁺ cells in the centre (red cells, K). Note that the yellow (non nuclear) staining seen in the centre of the sphere is due to background. (L) The cells at the edge of the sphere express the EGF receptor. In K and L nuclei are counterstained with DAPI (blue). Scale bar: 15 µm in B; 20 µm in A; 25 µm in C-G,L,I; 30 µm in J,K; 40 µm in H.