544 A Control DKO DAPI pH3 pH3 DAPI BrdU BrdU B C Cerebellum (2010) 9:537–547 Control DKO p27 Control DKO Nucleolin Fig. 5. Myc DKO decreases progenitor cell cycling at P6. a Control and DKO E17.5 cerebella were stained with the indicated markers. Green is phosphoH3; pH 3, a mitotic marker. Red is BrdU, incorporated in S phase cells. Blue is DAPI. Images in a were taken at 20× and scale bars=100 μM. b Staining for p27KIP1(green). c- and N-Myc are essential for CGNP cell cycling and metabolism To examine cell cycle characteristics of control and Myc DKO cerebellar precursors, we stained E17 sections with anti-phosphoH3 antibody, a marker of mitosis, as well as anti-BrdU antibody following BrdU labeling (Fig. 5a). DKO cerebellar exhibited no clear change in mitotic labeling cells in the rhombic lip, neuro- epithelium and EGL. In contrast, all germinal regions of the DKO showed a near complete absence of S phase cells as determined by BrdU staining, while control neuroepithe- lium, rhombic lip, and EGL exhibited a very high proportion of S phase cells following a 2 h label, reflective of the established very high proliferative rate in cerebellar precursors at this stage. The absence of a clear change in M phase cells together with a complete loss of S phase cells suggests the few DKO cells that are cycling having difficulty transiting M phase and there may be an M phase Yellow and white arrows indicate neuroepithelium and EGL, respec- tively. Bars=200 μM. c Control and DKO E17.5 sagittal cerebellar sections were stained with nucleolin (green), a marker of cell metbolism. Images were taken at 40× and scale bars=100 μM block. To analyze the cell cycle further at P6, p27KIP1 staining was conducted (Fig. 5b). The control P6 cerebel- lum has a handful of weakly p27-positive cells including a few in the EGL, but none in the neuroepithelium. The DKO cerebellum shows a striking induction of p27 throughout both the EGL (white arrow) and the neuroepithelium (yellow arrow). These data indicate that almost all DKO CGNPs and NSC are strongly p27 positive, consistent with the cell cycle exit suggested by the BrdU staining. Myc has been previously linked to maintenance of cell metabolism, leading us to examine whether the DKO affects cerebellar precursor metabolism. To study potential metabolic differences, we stained with antibody against the established metabolic marker, nucleolin (Fig. 5c). The DKO cells in the EGL and in the interior of the cerebellum exhibited a pronounced decrease in nucleolin staining, reflecting im- paired protein metabolism in the absence of c- and N-myc.