All animal studies were carried out in outbred SW mice, under an approved IACUC animal protocol according to the institutional guidelines at New York University School of Medicine and Memorial Sloan-Kettering Cancer Center. In almost all SW mice analyzed, the most anterior lobules I and II were fused, and lobules IV and V were partially separated by a fissure. Sublobules VIa and b, as well as IXa, b, and c, were always present. The day that a plug was detected was designated as E0.5. The day of birth was designated as P0. Adults were designated as P28 or older. For cell shape analysis transgenic CAG::GPI-eGFP
mice were used [29
) reporter mice were genotyped as described [35
mice were genotyped as described [34
]. For genotyping the En2
mutant allele, the following primers were used p32 (5'-TCGGGGGAAGAAGTGTCCAATGTCC-3'), neoTGAp2 (5'-ATCGCCTTCTTGACGAGTTCTTCTGAG-3') and En2p31 (5'-GGGCCTGTACAACCATTCCACCACG-3') [49
]. The p31 and p32 primers amplify the wild-type band.
Double hemizygous males (Math1-CreER; Tau-loxP-STOP-loxP-myrGFP-IRES-nLacZ) were bred with SW females (five to six weeks old; Taconic, Hudson, NY, USA) to generate double hemizygous embryos. Tamoxifen (T-5648, Sigma, St. Louis, MO, USA) was dissolved in corn oil (Sigma C-8267) at a final concentration of 20 mg/ml. The females were given tamoxifen via gavage with animal feeding needles (Fine Science Tools, Corston, UK) at noon on E15.5 (4 mg per 40 g of body weight). Double hemizygous males (R26-CreER; R26-loxP-STOP-loxP-eYFP) were bred with SW females (five to six weeks old; Taconic) to generate double hemizygous embryos. The females were given tamoxifen at noon on E12.5 (1–2 mg per 40 g of body weight) and analyzed at E16.5/17.5. Dissected brains were immersion fixed for 20 minutes in 4% paraformaldehyde (PFA) at 4°C, cryoprotected in 15% and 30% sucrose, embedded in OCT (Tissue-Tek, Sakura Finetechnical, Japan) and sectioned at 10 μm.
Histology and immunofluorescent immunohistochemistry
For histology and immunofluorescent immunohistochemistry of mice older than P8, brains were dissected after intracardiac perfusion with 4% PFA and then immersion fixed in 4% PFA overnight at 4°C. Tissue was then processed for paraffin embedding and sectioned at 7 μm. For consistency, sections analyzed from the vermis were limited to the most medial 100–200 μm.
Immunohistochemistry using indirect immunofluorescence was performed using standard staining procedures with the following antibodies: rabbit anti-BLBP (1:2,000; Milipore, Billerica, MA, USA), mouse anti-BrdU (1:500; Becton Dickinson, Franklin Lakes, NJ, USA), mouse anti-Calbindin (1:4000; Swant, Bellizona, Switzerland), mouse anti-NeuN (1:1,000; Chemicon, Temecula, CA, USA), rat anti-GFP (1:5,000; Nacalai Technique, Kyoto, Japan), rabbit anti-GFP (1:5,000; Invitrogen Corporation, Carlsbad, CA, USA), rabbit anti-PH3 (1:500; Cell Signaling Technology, Beverly, MA, USA), anti-βgal (1:500; Biogenesis, Raleigh, NC, USA), rabbit anti-Pax6 (1:300; Chemicon), mouse anti-GFAP (1:500; Chemicon), TRITC-Phalloidin (1:2,000; Sigma), goat anti-Sema6a (1:200; R&D Systems, Minneapolis, MN, USA). Sections were mounted in Vecta Shield with DAPI (Vector Laboratories, Burlingame, CA, USA) and examined with a fluorescent microscope (DM6000, Leica, Nussloch, Germany; Axio Observer, Zeiss, Germany). Fluorescent images for Figure were captured in 1.5 μm optical sections using Zeiss Observer with Apotome setting and processed using Adobe Photoshop. Orthogonal analysis was performed to confirm co-expression of two markers.
The Cb tracings of midline sagittal sections of the vermis were done at E16.5, E17.5, E18.5, P3, P5, P7, P10 and P21 by photographing hematoxylin and eosin sections using a dissection microscope (MZ16FA, Leica) at 1× magnification, followed by careful outline of the outer most surface using Adobe Illustrator (CS2) and then overlaying the outlines on top of each other.
Bromodeoxyuridine staining and quantification of cell proliferation
To assay proliferation, pregnant females were injected intraperitoneally with 100 μg BrdU/g body weight 20 minutes before they were sacrificed. To quantify the percentage of BrdU-positive cells at E17.5 and E18.5, the percentage of BrdU-positive cells was calculated by counting the total number of cells in the EGL (DAPI positive cells) of 40–50 sections (7 μm thick) per embryo from the most medial 100–200 μm of 3 embryos at each stage. To determine the distribution of pH3 positive cells in the EGL, the number of pH3 positive cells at the base of each fissure versus the side and crown of the folia was counted at E16.5, E17.5 and E18.5. The base of the fissure was considered to be the most invaginated part of the fissure. The percentage of pH3-positive cells was then calculated from the number of pH3-positive cells per total number of granule cell precursors in each region. The total number of gcps in each region was calculated by counting the number of nuclei based on DAPI staining. The crown of folia had 100–200 nuclei at E16.5, 120–276 nuclei at E17.5, and 100–280 nuclei at E18.5. The base of fissure had 80–90 nuclei at E16.5, 100–144 nuclei at E17.5, and 100–150 nuclei at E18.5. The quantification of pH3 positive cells was done for 45 to 60 of the most medial consecutive sections (7 μm thick) of three brains for each embryonic stage (E16.5-E18.5). For En2 mutants at P0 and P1 the quantification of pH3 positive cells was done for the 25–30 most medial consecutive sections (7 μm thick) of three brains for each stage. The quantification of BrdU and βgal double positive cells was done for seven to eight of the most medial sagittal consecutive frozen sections (12 μm thick) of three brains at E18.5. Measurements of cells from multiple embryos were pooled into datasets. We defined the area of the base of the fissure in which positive cells were counted to be the most invaginated part of the fissure and an underlying fan shaped area that is 10 μm deep under the EGL (see Fig. ). We defined the area of the crown of the fissure in which positive cells were counted to be the same length of EGL as in the base of the fissure but at the top of the adjacent lobule and a 10 μm deep rectangle below this (yellow inset in Figure ).
We used ImageJ software from National Institutes of Health (NIH) to trace cell outlines and measure cell perimeters [64
]. Cells were chosen for quantification only if their outline and morphology was clearly visible. Circularity was calculated by ImageJ as a normalized ratio of area (A) to perimeter (P), with a ratio of 1 representing a circle (circularity = 4πA/P2
). By performing these calculations, circularity index distinguishes cells with round morphologies from those with more elongated morphologies.
To quantify the shape of gcps found in the bottom of the fissure and the crown of the lobe, high magnification images were taken of mid-sagittal Cb sections of CAG::GPI-eGFP transgenic mice at E16.5, E17.5 and P0. For quantification, we used 40–50 sections (7–10 μm thick) total from the most medial 300 μm of 3 embryos at each stage. Based on anti-Pax6 and anti-GFP double immunostaining, we determined that the five cell thick layer at the crown of the folia, and seven cell thick layer at the base of the fissures is the EGL and confined our measurements to this layer. Measurements of cells from multiple embryos were pooled into datasets. Sample sizes were as follow: smooth Cb at E16.5 from 3 embryos, 60 cells distributed along the AP axis of the Cb and 45 cells found in the area where EGL accumulated; the base of fissures at E17.5, 63 cells from 3 embryos; the crown of the lobes at E17.5, 70 cells from 3 embryos; the base of fissures at P0, 116 cells from 3 embryos; the crown of the lobes at P0, 125 cells from 3 embryos. For En2 mutant animals we calculated the circularity index for the outermost five cell thick layer of the cerebellar cortex. The sample sizes were as follows: both secondary and prepyramidal fissure at P0, 20–30 cells for 3 embryos; at P1, 30–35 cells from 3 embryos. Circularity index values were compared using unpaired t-tests.
E17.5 embryos were perfused with 4% PFA followed by an immersion fixation in 3% paraformaldehyde, 1% glutaraldehyde, 4% sucrose, 0.1% CaCl2 and 2.5% DMSO in 0.1 M sodium cacodylate buffer (pH 7.4). The Cb was removed and again immersion-fixed at 4°C in 4% PFA for 2 hours and post fixed with 1% osmium tetroxide at room temperature for 1.5 hours, then processed in a standard manner and embedded in Embed 812 (EMS, Hatfield, PA, USA). Semi-thin sections were cut at 1 μm and stained with 1% toluidine blue to evaluate preservation quality. Ultrathin sections (60 nm) were cut using a Leica ultracut UCT, put on formover coated copper grids and stained with uranyl acetate and lead citrate by standard methods. Stained grids were examined under a Philips CM-12 electron microscope, and photographed with a Gatan 1 k × 1 k digital camera.