The slides were mounted using Fluoromount G mounting medium containing DAPI (Southern Biotech, Birmingham, AL, USA) and images were acquired using a confocal microscope (Zeiss, Germany). We counted all the fluorescent neurons within the region of interest (such as the hippocampus or the adjacent dorsal cortex (including the entorhinal and parietal cortex) and quantified the percentage of red (mceph/mceph), green (wild-type Kv1.1) or yellow (mceph/+) in all fluorescent (red + green + yellow) neurons or astrocytes. Statistical analyses were performed with Prism software (GraphPad Software Inc., La Jolla, CA, USA) using Student's t test INCB024360
for pair-wise comparisons. P < 0.05 was considered statistically significant. click here
The soma volume was measured using Zeiss LSM Image Browser and Imaris (Bitplane, South Windsor, CT, USA). We generated mceph-MADM6 mice to study the cell-autonomous and non-cell-autonomous mechanisms for the megencephalic phenotype (Fig. 1) (Zong et al. 2005; Tasic et al. submitted). In heterozygous mceph/+ mice with cre-recombinase active during the proliferative phase, somatic recombination may cause homozygosity of the mceph mutation distal to the MADM locus so that cells are marked with different colours according to their respective genotypes. Because homozygous mutant and homozygous wild-type cells are simultaneously generated as siblings and their progeny are labelled with different colours, the MADM mice have built-in control cells within the same brain region for phenotypic analysis (Espinosa et al. 2009; Liu et al. 2011). The mouse Kv1.1 gene is located about 8.8 centimorgan distal to the ROSA26 locus on chromosome 6 where the MADM gene cassette has been introduced (Petersson et al. 2003; Zong et al. 2005; Tasic et al. submitted). To generate homozygous mceph/mceph neurons throughout the entire nervous system Ribociclib
in the mceph/+ genetic background, we crossed mceph/+;MADM6-TG/TG (MADM6-tdTomato(loxP)GFP) mice to nestin-cre;MADM6-GT/GT mice (MADM6-GFP(loxP)tdTomato). We chose the nestin-cre recombindase to induce interchromosomal recom-bination because nestin is an intermediate filament protein in the precursor cells of the nervous system and the cre-recombinase under the nestin promoter has been used to induce loxP recombination throughout the nervous system early in development (Dubois et al. 2006). With the MADM scheme, occasionally cells in the nervous system expressing the nestin driven cre-recombinase will undergo mitotic recombination and functional GFP and tdTomato sequences are restored during this interchromosomal recombination, which are then segregated and expressed in different progeny. The result is the specific labelling of clones of cells expressing GFP (green), or tdTomato (red), or both GFP and tdTomato (yellow), or neither (colourless).