Control DKO 542 A Overlay NeuN GFAP DAPI B Overlay Control CNPase TUJ DAPI Cerebellum (2010) 9:537–547 DKO Fig. 3. Loss of c- and N-myc severely disrupts cerebellar develop- ment. Control and DKO Sagittal cerebellar sections from mature animals stained for DAPI (DNA; blue) or the indicated marker: a NeuN (predominantly granule neurons) and GFAP (astroglia), b CNPase (oligodendrocytes), and TUJ (BTUBIII; predominantly morphology. Purkinje neurons, which normally are present at an enormous underrepresentation compared to granule neurons, are also strikingly overrepresented in the DKO (Fig. 3a versus b, BTUJ versus NeuN staining). This is confirmed by Calbindin staining where a significant fraction of total cells in the DKO cerebellum at P21 appear to be Purkinje neurons (Fig. 4a). Evidence of Arrested and Impaired Development in the Mature DKO Cerebellum While GAD67+ inhibitory neurons appear to be present in the DKO, their location, staining, and morphology are all very abnormal (Fig. 4a), suggesting a failure to mature Purkinje neurons). Blue arrows in (a, b) points toward DAPI stained residual, abnormal EGL. Images were taken at 20× and scale bars= 100 μM. Note that the control images are from the tip of just one folium, whereas the DKO images are nearly the entire cerebellum normally. Similarly, although c- and N-myc are not strictly required for Purkinje neuron generation, nonetheless the DKO Purkinje neurons exhibited evidence of impaired maturity. The Purkinje neurons present are scattered and are quite abnormal in morphology (Fig. 3, BTUJ and Fig. 4a, Calbindin staining). While staining positive for both TUJ and Calbindin, the staining of DKO Purkinje neurons is abnormally intense. The Purkinje neurons appear to be disconnected and do not form a visible neural network. There is an absence of the normal beautiful arborization, very evident in the control (Electronic supplementary material, Fig. S2). It is unclear if these Myc-deficient Purkinje neurons possess more than rudimentary axons and dendrites, suggesting that although they are present their