Pöschl et al., Suppl. Figure 1 CN Ki67 H&E Math1-cre::SmoM2 Fl/+ Math1-cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ P0 P2 P7 EGL ML PL IGL Math1-cre a b EGL ML PL.

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Pöschl et al., Suppl. Figure 1 CN Ki67 H&E Math1-cre::SmoM2 Fl/+ Math1-cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ P0 P2 P7 EGL ML PL IGL Math1-cre a b EGL ML PL IGL EGL IGL P16 Math1-cre::SmoM2 Fl/+ Math1-cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ Math1-cre Suppl. Fig. 1: Reduced growth of Shh-medulloblastoma after activation of the Wnt-pathway (a) H&E stains of sagittal cerebellar sections. Activation of the Shh- pathway in Math1-positive cerebellar GNPs resulted in a thickened EGL at P0 and the formation of medulloblastoma that were readily detectable at P2 (Math1- cre::SmoM2 Fl/+ genotype). Co-activation of the Wnt/β-catenin-signaling-pathway (Math1-cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ mice) notably reduced growth of medulloblastoma and led to a decreased cerebellar size when compared to Math1-cre controls. Adequate cerebellar layering was not seen (see inset with higher magnification). (b) H&E stains of axial brain stem sections. Insets show immunohistochemistry using antibodies against Ki67. Shh-pathway-activation in cochlear GNPs resulted in medulloblastoma of the brainstem (Math1-cre::SmoM2 Fl/+ genotype, for anatomical orien- tation see Math1-cre control). Wnt/β-catenin-activation decreased growth of these tumors, too (Math1-cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ mice). Dotted lines indicate anatomical regions of the CN or medulloblastoma arising herein. EGL: external granule cell layer, ML: molecular layer, PL: Purkinje cell layer, IGL: internal granule cell layer, CN: cochlear nucleus.

Pöschl et al., Suppl. Figure 2 Wild type Floxed Recombined Wild type Exon 2 Exon 4Exon 3 Wild type: Floxed: Exon 2 Exon 4 loxP Recombined: Exon 2 Exon 4loxP A A A C C C B B b Tumor DNA Tail DNA H2OH2O Marker P8 P16 P  -catenin positive tumor cell nuclei [%] Relative fraction of BrdUrd + cells among GFP + granule cells [%] n. s. Math1-cre:: SmoM2 Fl/+ Math1-cre:: SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ IRES-GFP Cre-IRES-GFP * ** 425 bp 540 bp 370 bp 715 bp Ca. 1 kb Exon 3 a d c Suppl. Fig. 2: Incomplete recombination of the Ctnnb1 allele. (a) The proportion of tumor cells with nuclear β-catenin-positivity decreased over time (P8 vs. P16: p=0.005, P8 vs. P32: p=0.001). (b) Scheme showing detectable PCR-fragments depending on allelic sequence. A, B and C delineate primers that were used for the analyses shown in (c). (c) The gel electrophoresis after PCR demonstrates incomplete recombination of the floxed Ctnnb1(ex3) allele in tumor tissue of Math1- cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ mice. (d) Primary cell cultures of Math1-cre::SmoM2 Fl/+ and Math1- cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ tumors were transduced with Cre-IRES-GFP virus to allow recombination of yet unrecombined alleles. While Math1-cre::SmoM2 Fl/+ tumor cells displayed no difference in proliferation, Math1-cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ tumor cells showed a significant decrease of proliferation 24 h after transduction (p= Values were normalized to the mean of control treated cells of each genotype, respectively (value was set 1). n. s.: not significant, one asterisk: p<0.05, two asterisks p<0.01.

Pöschl et al., Suppl. Figure 3 hGFAP-cre hGFAP-cre::SmoM2 Fl/+ Ctnnb1(ex3) Fl/+ H&E β-cateninKi67 a b c H&Eβ-cateninKi67 H&Eβ-cateninKi67 Suppl. Fig. 3: Impaired proliferation in hGFAP-positive precursors with Shh after the additional activation of Wnt/β-catenin-signaling. (a, b, c) Sagittal sections of hGFAP-cre (a), hGFAP-cre::SmoM2 Fl/+ (b) and hGFAP-cre::Ctnnb1(ex3) Fl/+ SmoM2 Fl/+ (c) cerebella at P0. Higher magnifications of the external granule cell layer (EGL) of respective genotypes show immunohistochemistry using antibodies against β-catenin and Ki67. Shh-pathway-activation in hGFAP-positive precursor cells resulted in the formation of medulloblastoma that arose from the EGL and displayed a high proliferation rate (b). Constitutive co-activation Wnt/β-catenin-signaling in hGFAP- cre::Ctnnb1(ex3) Fl/+ SmoM2 Fl/+ led to a dramatically thinned EGL showing decreased proliferation, even compared to the control (a). Adequate cerebellar layering was not seen (c versus a). Nuclear positivity for β-catenin was detectable within the EGL (white arrow in the inset of c).