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Hoechst 33258 (Molecular Probes) was used to visualize nuclear staining. All sections were examined with a Leica DMLB epi-fluorescence microscope fitted with a SPOT camera in the CWRU Genetics Imaging Facility (supported by NIH-NCRR, RR-021228). Pregnant mice were injected intraperitoneally with BrdU (Sigma) at 100?��g/g body weight 1?h prior to dissection. The percentage of BrdU+, Ki67+, pHH3+, Caspase3+ cells was calculated by dividing the number of the positive cells by the total number of nuclei (Hoechst) inside RP or other specified region. To calculate Epigenetics Compound Library clinical trial the fraction of S-phase cells within cell cycle, we divided the number of BrdU+ cells by the total number of Ki67+ cells and converted the number to a percentage. For each time point, we used 2 mid-sagittal sections from each embryo and 4�C6 embryos from at least two different litters for analysis. As the number of Caspase3+ cells is low in both WT and Gli2 mutants, we counted 3�C4 sections from each embryo Evodiamine (total 1�C5 Caspase3+ cells per embryo, with n?=?3 for Gli2 mutants and n?=?6 for WT). The number in charts was displayed as mean?��?S.E.M. Student's t-test was used to calculate the P value and to determine whether the results were significantly different from each other (** indicates P?BMS-777607 manufacturer We then calculated the percentage of EGFP+ cells within RP or in different regions of RP, and normalized the percentage against the baseline in that embryo. Results from three embryos were then used to determine whether there was a significant difference between the contribution of WT and Gli2 mutant cells. Previous studies reported a variable loss of the pituitary (3/6) in Gli2 mutants when examined at E12.5 ( Park et al., 2000). To determine how Gli2 mediates Shh signaling to influence pituitary development, we examined patterning of the pituitary gland and the production of different pituitary cell types in wild type (WT) and mutant embryos.