Fig. 2
The increased apoptosis and reduced proliferation of cells in KO neural tubes and NPCs. A Increased cell apoptosis in KO neural tubes. Neural tubes at the level of the rostral hindbrain (at upper 1/3 of the body length measured from the cranial extreme; sectioned transversally) of e9.5 KO embryos with exencephaly and normal WT counterparts were determined by TUNEL. The percentage (means ± SD) of TUNEL-positive cells among total cells in randomly selected view areas in the neural folds and adjacent areas is presented in a bar graph at the right. Results were based on the counting of three different sections per fetus (KO: 3 fetuses; WT: 3 fetuses). B Characterization of NPCs. WT NPCs were stained with NPC markers Sox2 (pseudo-red) and Nestin (pseudo-green). C, D Reduced KO NPC proliferation. Different numbers of NPCs, as indicated, were cultured in the presence of a fixed concentration of EGF (20 ng/ml) (C), or a fixed number of NPCs were cultured in the presence of different concentrations of EGF as indicated (D). After 72 h, proliferation was measured by an MTS-based CellTiter96 AQueous Assay. Samples were in triplicate, and means ± SD of OD490 nm (after subtracting background absorbance based on OD490 nm of wells without cells) of a representative experiment out of four independent ones are shown. E Slower progression of KO NPCs from the G1 phase to the S phase. NPCs were blocked at the late G1 phase with aphidicolin for 8 h. The cells were then released from the blockages, and their DNA content was measured by flow cytometry at different time points as indicated. A set of representative histograms is shown at the left, with DNA content stained with PI (propidium iodine). Results from 6 independent experiments are summarized at the right, with mean ± SD of the percentage of cells in the G1, S, and G2/M phase at different time points indicated. F Augmented apoptosis of KO NPCs cultured in the presence of different concentrations of EGF. NPCs were cultured for 20 h, and their apoptosis was measured by annexin V staining followed by flow cytometry. Representative histograms are shown (left panel). G A summary of the flow cytometry data (means ± SD) of four independent experiments is presented in a bar graph (right panel). Data in A and E were analyzed with paired two-way Student’s t test. *p < 0.05; **p < 0.01. Data in C, D, and G were analyzed by two-factor with replication ANOVA, and p-values for the WT versus KO groups were indicated