Fig. 4. The postnatal development of granule cells is altered in treated MBP-TK mice. Granule cells were analyzed in sagittal cerebellar cryosections from (A,C) wild-type and (B,D) MBP-TK 6-day-old treated mice. (A,B) In situ hybridization using a PAX6 antisense riboprobe to label granule cells in the EGL, and (C,D) immunofluorescence using an anti-phosphorylated histone 3 (PH3) antibody to reveal proliferating cells in this layer. Arrowheads delimit the thickness of the EGL in wild-type (A,C) and MBP-TK (B,D) cerebella. The arrowhead external to the cerebellum in D also indicates the reduced thickness of the EGL in the region in which the fissura intercruralis should have formed. Squares (A,B) indicate the regions that have been analyzed at higher magnification (C-F). (E,F) TUNEL fluorescent labeling of apoptotic nuclei in the EGL of treated 6-day-old (E) wild-type and (F) MBP-TK mice. Nuclei were counterstained with DAPI. (G,H) Dark-field micrographs of in situ hybridization experiments using a RU49 antisense riboprobe in cerebellar sections from 3-week-old (G) wild-type and (H) MBP-TK treated (1-20d) animals. (I-L) Double immunostaining using anti-PAX6 (I) and anti-HSV1-TK (J) revealed, as expected, a complete absence of TK-positive granule cell precursors. Nuclei were counterstained with DAPI. Scale bars: 100 µm (A,B), 35 µm (C,D), 35 µm (E,F), 100 µm (G,H), 15 µm (I-L).