Figure 6

Increased neuronal populations due to prolonged neurogenesis at the VZ as revealed by immunofluorescence staining on cerebellar sagittal sections. (A, B) Becoming active since late gestation, the prospective white matter (PWM) (dotted area) is where interneuron progenitors delaminated from the VZ proliferate, gradually fate restricted and become postmitotic. The identity of PWM as a secondary germinal site in the developing cerebellum was exemplified by identifying the BrdU positive cells within the Pax2 positive domain. (G) Proliferation within the PWM at E16.5 was reduced in Sox9-null cerebellum when compared with control (***, p < 0.005). (C, D and H) BrdU tracing experiments from E13.5 to E18.5 revealed no difference in the number of Pax2+ interneurons born at around E13.5 between controls and mutants. (E, F and I) In contrast, BrdU tracing experiments from E16.5 to E18.5 showed significantly more interneurons were born at around E16.5, as evident by increased number of double Pax2+ BrdU+ cells localized at the proximity of VZ in Sox9 mutant cerebellum (E’ and F’, arrows) (*, p < 0.05). (J, K and L) Ngn2 expression level was more than 1.3 fold higher in E16.5 Sox9 mutant cerebellum (**, p < 0.01), while that of Ascl1 and Ngn1 was not significantly different between controls and mutants (p = 0.9072 and p = 0.3896 respectively). E’ and F’ are magnified view of the area enclosed by inset in E and F. Abbreviations: egl, external granular layer; n.s., not statistically significant; vz, ventricular zone. The error bars indicate standard deviations. Scale bar: 100 μm.