Cerebellum (2010) 9:537–547 The decreased cellular metabolism in the DKO fits with the disrupted cerebellar growth exhibited in the DKO. Discussion N-myc is required for normal murine and human brain growth [16, 40]. Mutation of N-myc in humans causes FS with its very large range of birth defects in a host of tissues [41]. Interestingly the range of defects in constitutive N- myc KO mice are remarkably similar to that of FS [42]. While these findings suggest a conserved role for N-myc in both human and mouse NSC that drives brain growth, the mechanisms have remained somewhat unclear. Complicat- ing KO studies in mice has been the continued presence of c-myc, which has increased expression in the N-myc KO cerebellum and appears to compensate for the loss of N- myc [36]. Our studies here using a DKO overcome this problem, and point toward a key role for Myc in maintaining essential cerebellar progenitor cell character- istics: a specific cell cycle program, high metabolism, and inhibited differentiation (i.e., maintained pluripo- tency). In the Myc DKO mice, progenitors in the cerebellum lose all these properties. The DKO behaves akin to a developmental switch turning off, telling the cerebellum, via its progenitor cells that its developmental period is largely over as early as E17.5 and more strongly by P6. Interestingly, at least at the developmen- tal stages examined, there was no clear effect of the DKO on survival. One of the most striking features of the DKO is the changes that occur in the EGL. At P6 the EGL normal consists of CGNPs that are Math1+/NeuN−/BrdU+/p27−. An IGL is already forming that consists of cells derived from CGNPs that are almost the opposite, (e.g., Math1−/ NeuN+), that have migrated inward. In the DKO at P6, the EGL has a phenotype very similar to that of the IGL, consisting of cells that are Math1−/NeuN+/BrdU−/p27+. However, like normal CGNPs of the EGL, these DKO cells still reside in the EGL domain and form a multicell layer on the outside of the developing cerebellum. They also consist of cells with small, DAPI bright nuclei, a normal feature of CGNPs in the EGL. Therefore, our data together suggest that in the absence of c- and N-myc, the DKO CGNPs have an aberrant cell fate. Instead of migrating inward, differen- tiating, exiting the cell cycle, and remodeling their nucleus into a larger, more euchromatic structure as normally occurs, the DKO CGNPs mostly fail to migrate and undergo an abnormal kind of differentiation right within the EGL, where they also exit the cell cycle. The fact that they nonetheless retain their location, layer structure, nuclear morphology and DAPI staining suggests that at one point they were CGNPs. 545 Other evidence of abnormal cell fate in the DKO comes from the Purkinje neurons that are present in the mature cerebellum. While they are overrepresented relative to granule neurons, indicating that c- and N-myc are not absolutely required for a Purkinje cell fate, the DKO Purkinje neurons are abnormal in morphology. They lack normal arborization and appear disconnected from the cerebellar neural network. This may be a cell autonomous phenotype due to loss of Myc in Purkinje progenitors, or alternatively, maybe due to the absence of granule neurons/ migrating CGNPs that normal provide instructive signals for Purkinje development. In the N-myc single nestin-cre- driven KO cerebellum, Purkinje neurons are not overrepre- sented and despite decreased granule neurons, the Purkinje neurons still appear normal in morphology and connected- ness. Therefore, the abnormal Purkinje maturation is unique to the DKO. The effects of loss of Myc on the cell cycle of cerebellar progenitors are notable. While it is not surprising that the loss of c- and N-myc leads to the near complete absence of S phase cells, the presence of normal or near normal numbers of M phase cells is unexpected. In the previous N- myc single KOs driven by nestin-cre, decreases in both S and M phase cell were observed. The fact that in the DKO, there are almost no S phase cells but are relatively normal numbers of M phase cells suggests the DKO progenitors are blocked in M phase. This notion is supported by expression microarray experiments in which a host of M phase genes are downregulated in the DKO cerebellum [43]. Since very few DKO progenitors were in S phase, it is tempting to speculate that those few “cycling” cells are mostly stuck in M phase, and that Myc is essential for normal mitosis. Further insight into Myc regulation of cerebellar devel- opment would be gained by exploring the potential role of the third main myc gene, L-myc, which is highly expressed in the EGL. Although constitutive L-myc KO mice were reported to have no phenotype of any kind, it seems probable that the high L-myc levels in the EGL reflect a function obscured in the L-myc KO because of the continued presence of very high levels of N-myc. Because Purkinje neurons are overrepresented in the DKO, we predict that L-myc plays a role in Purkinje neuron development as well. Conclusion Overall our data indicate that N- and c-myc play particu- larly important roles in cerebellar development. The functions of c- and N-Myc proteins in the cerebellum include maintaining pluripotency, metabolism, and cell cycling of progenitors.