Therefore , in our model the decrease ofCxcl3andEfna4and the increase ofCxcl12after ablation ofTis21inPtch1heterozygous mice would synergize in impairing the migration of GCPs from the EGL. Shh-dependent deregulation of the clathrin-mediated chemotaxis operating in the primary cilium through the Cxcl3-Cxcr2 axis; (ii) a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype, i. e., the neural cell type involved in group 3 MB; (iii) the identification of a subset of putative drug targets for MB, involved, among the others, in the regulation of Hippo signaling and centrosome assembly. Finally, our findings define also the role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms, influencing the fate of the GCPs. Keywords: medulloblastoma model, cerebellar precursor cell, drug target, Sonic Hedgehog, primary cilium, neural migration, retina, chemokines == Introduction == About 30% of medulloblastomas (MBs), the pediatric tumor of the cerebellum, originates from the granule neuron precursor cells (GCPs) located in the external granular layer (EGL), at the surface of the developing cerebellum, in consequence of hyperactivation of the Sonic Hedgehog (Shh) pathway (Kadin et al., 1970; Schller et al., 2008; Yang et al., 2008; Gibson et al., 2010; Northcott et al., 2012). Other MB subtypes may originate N6-Cyclohexyladenosine from neural precursors of the cerebellar embryonic anlage, different from GCPs and dependent on Wnt signaling, or from GCPs with activation of different pathways (group 3), or also from neural precursors of unknown origin (group 4; Northcott et al., 2012). GCPs intensely proliferate postnatally in the EGL, before exiting the cell cycle and migrating inward to form the mature internal granular layer (IGL; Hatten, 1999). GCPs in the EGL are forced to divide by the proliferative molecule Shh, secreted by Purkinje neurons (Dahmane and Ruiz i Altaba, 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999). It is believed that the prolonged mitotic activity of the GCPs, consequent to hyperactivation of the Shh pathway, makes them potential targets of transforming insults (Wang and Zoghbi, 2001). We have previously shown that mice lacking one allele ofPtch1, which develop MB with low frequency as result of the N6-Cyclohexyladenosine activation of the Shh pathway (Hahn et al., 1998), when crossed with mice knockout for the MB suppressorTis21develop MB with very high frequency (Farioli-Vecchioli et al., 2012a, b). We identified as responsible for this effect a defect of migration of the GCPs that, remaining for N6-Cyclohexyladenosine N6-Cyclohexyladenosine a longer period in the EGL under the proliferative influence of Shh, developed tumor more frequently. Whole-genome analyses of expression and function indicated that the key molecule responsible for the lack of migration of GCPs is the chemokine Cxcl3 (Farioli-Vecchioli et al., 2012a). Together withCxcl3, we identified other 187 gene sequences, 163 of which have a functional product, whose expression in double mutantPtch1heterozygous/Tis21knockout mice was modified, relative toPtch1heterozygous mice inTis21wild-type background (single mutants; Farioli-Vecchioli et al., 2012a). The set of genes whose expression significantly differs in the comparisonPtch1+//Tis21wild-type vs . Ptch1+//Tis21KOwill be hereafter defined as Set A (Figure1). == Figure 1 . == A Venn diagram showing four genotype pairwise comparisons and the intersection of their differentially expressed gene/sequences set AD. Set A corresponds to the pairwise comparisonPtch1+//Tis21KOvs. Ptch1+//Tis21+/+; Set B refers toPtch1+//Tis21+/+vs. wild type; Set C concernsPtch1+//Tis21KOvs. Ptch1+/+/Tis21KO; Set N6-Cyclohexyladenosine D represents the double-knockout contribution in background wild type. Here, we aimed to expand the functional investigation of the previous whole-genome analysis of gene expression alterations occurring at the onset of tumorigenesis in the GCPs, in order to further examine the set of genes whose expression is modified inPtch1heterozygous/Tis21knockout double mutant mice relative toPtch1heterozygous/Tis21wild-type mice (Set A). Given thatTis21mutation has a strong tumorigenic effect inPtch1heterozygous background, with a high increase of MB frequency, we assumed that the transcriptional changes occurring in the Set A of 163 genes afterTis21ablation inPtch1background were at the origin of the increased tumorigenicity observed. These genes, referred to asTis21-dependentgiven that their expression is by definition modified by the ablation ofTis21inPtch1heterozygous backgroundwill be divided in up-regulated and down-regulated, relative toPtch1heterozygous/Tis21wild-type mice. It is worth noting that among the genes in Set A whose expression is down-regulated abound those with tumor-inhibitory activity (e. g., Pag1, PadI4, Lats2, andCxcl3), while among the up-regulated genes are present tumor facilitators (e. g., Rab18, Dek). Phenotypically, our genomic data LRP11 antibody refer to GCPs at a very early pre-neoplastic stage, having been isolated from 7 day-old mice, i. e., when the neoplastic lesions have not yet emerged. The data analyzed interestingly lead to: (i) a link between the Shh signaling and the impairment of the GCPs migration, through a Shh-dependent deregulation of the receptor-mediated endocytosis pathway; (ii) a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype/toward the neural cell type.