Anchoring center formation involves changes in gcp shape. Sagittal sections of the Cb of CAG::GPI-eGFP transgenic mice to label cell membranes. (a-a2) At E16.5, anti-GFP immunostaining reveals round gcps throughout the AP axis of the EGL. (a1) Anti-Pax6 (red) and anti-GFP (green) immunostaining confirms that cells in the outermost layer are gcps. (a2) Anti-pH3 and anti-GFP immunostaining shows that round gcps are proliferating (arrowheads). White box in a indicates the region in a1, a2. (b-b2) At E17.5, anti-GFP and anti-Pax6 immunostaining reveals elongated gcps (blue dotted line) at emerging anchoring centers, in contrast to the more round gcps (red dotted line) in the adjacent areas (b2). Asterisk indicates the fissure. (c-c2) The gcp shape differences are more evident in the fissures at P0. Elongated gcps (blue dotted line) are found at the base of the fissures and appear organized; their longitudinal axes aligned with the plane of the fissure. At the crown of the folia, gcps are round and less organized (red dotted line). (d) Anti-pH3 immunostaining indicates that the elongated gcps at the base of the fissure are proliferating at P0. (e) Circularity index of the gcps (blue at the base of fissure, red at the crown of the folia). Bar height indicates the mean value of each data set, and error bars indicate standard error. An asterisk indicates statistically significant differences in ci between the base of the fissure and the crown of the folia for each data set (p < 0.0001). (f) Electron micrograph of the emerging primary anchoring center at E17.5 confirms that gcps located in the fissure (red asterisks) are elongated and organized in a plane parallel to the fissure (black asterisk). Gcps located between the fissures are more rounded (blue asterisks). Inset is a bright field image of the same area. White dotted lines indicate EGL. Bv, blood vessel; gcp, granule cell precursors. Scale bars: (a) 100 μm; (c) 50 μm; (a1, a2, b, d) 15 μm; (b1, b2, c1, c2) 10 μm.