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Fig. 3. Loss of Shh signaling leads to prolonged cell cycle length and reduced
cell cycle exit of the neural progenitor/stem cells. (A-C')
After 1-hour pulse labeling of BrdU, immunostaining of parasagittal sections
was performed with anti-Ki67 (green) and anti-BrdU (red) antibodies at E13.5
(A-C) and E15.5 (A'-C'). (G) Cell cycle length was
estimated as percentage of Ki67 and BrdU double-positive cells in all
Ki67-positive cells. Smaller percentage represents longer cell cycle. At
E13.5, both Shh-CKO and Smo-CKO embryos showed significantly
prolonged cycle length (26.5±0.005% and 23.0±0.004%,
respectively, n=3) compared with wild-type embryos
(33.0±0.034%, n=3) (A-C,G). At E15.5, cell cycle length of
Shh-CKO was not significantly prolonged (19.6±0.005%,
n=3) whereas that of Smo-CKO was significantly longer
(15.4±0.003%, n=3) than that of wild type (21.5±0.027%,
n=3) (A'-C',G). (D-F') To estimate cell
cycle exit, sections were stained with anti-Ki67 and anti-BrdU antibodies 24
hours after BrdU pulse (E13.5, D-F; E15.5, D'-F'). (H) Cell
cycle exit was determined as a ratio of the cells that exited the cell cycle
(red, BrdU+/Ki67-, no longer dividing) to all cells labeled with BrdU
(red+yellow) after 24 hours labeling. In Shh-CKO embryos, the ratio
was significantly decreased at E13.5 (0.131±0.008; wild type,
0.198±0.005; n=3), but not different at E15.5
(0.230±0.021, n=3; wild type, 0.205±0.016,
n=3). In Smo-CKO embryos, the ratio was significantly
decreased compared with wild type at both E13.5 and E15.5 (0.168±0.002
and 0.069±0.013, respectively; n=3).
*P<0.01. Scale bar: 50 µm.
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