A Overlay GAD67 Calbindin DAPI Cerebellum (2010) 9:537–547 543 Adult P6 Control DKO Control DKO B C DAPI Math1 NeuN NeuN E17.5 Control DKO Fig. 4. Alterations in cell fate in the DKO. Control and DKO sagittal cerebellar sections stained for DAPI (DNA; blue) or the indicated marker: a Adult. GAD67 (cytoplasm of inhibitory neurons) and Calbindin (Purkinje neurons, cytoplasm, and nucleus), b NeuN at P6, and c Math1 at E17.5 development is impaired. The DKO cerebellum has a severely attenuated, Math1+ EGL at E17 (Fig. 4b). By P6, the DKO EGL is NeuN Positive and as Early as E17.5 is Mostly Math1 Negative To explore the events earlier in cerebellar development that resulted in the severe DKO phenotype at maturity, we examined the EGL at P6 (Fig. 4b). During this stage of cerebellar development, the EGL normally consists of Math1 positive, NeuN negative CGNPs. Normally, many of the CGNPs have also begun to migrate inward to form a Math1 negative, NeuN-positive developing IGL. The findings from our staining for Math1 and NeuN in control (doubly homozygously floxed, cre negative) sections were consistent with this pattern (Fig. 5b; not shown). In striking contrast, in the DKO we observed a phenotype where the putative EGL has taken on an IGL-like fate, staining negative for Math1 and positive for NeuN (arrows in DKO panels in Fig. 5b; not shown). While the DKO has an appropriately internally localized IGL consisting of strongly NeuN-positive cells, it is highly disorganized and rostrally no longer forms an actual layer. Furthermore, the DKO cells located where the EGL should normally be are moderately positive for NeuN as well. Therefore, Myc deficiency alters CGNP fate, leading to a loss of CGNP identity, likely explaining the residual EGL apparent at P21 in the DKO (Figs. 3 and 4a). To our knowledge this is the first demonstration of an IGL-like phenotype in the EGL of a KO mouse, suggesting Myc is essential for maintaining CGNP identity and blocking differentiation. As early as E17.5, the EGL in the DKO is nearly entirely lacking Math1 positive cells, consistent with the notion of a loss progenitor cell identity (Fig. 4c).