Pöschl et al., Suppl. Figure 1

CNKi67 Ki67 Ki67

H&E

Math1-cre::SmoM2Fl/+ Math1-cre::SmoM2Fl/+ Ctnnb1(ex3)Fl/+

P0

P2

P7

EGL

ML

PL

IGL

Math1-crea

b

EGL

ML

PL

IGL

EGL

IGL

P16

Math1-cre::SmoM2Fl/+ Math1-cre::SmoM2Fl/+ 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::SmoM2Fl/+ genotype). Co-activation of the Wnt/β-catenin-signaling-pathway (Math1-cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ mice) notably reduced growth of medulloblas-toma 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 medul-loblastoma of the brainstem (Math1-cre::SmoM2Fl/+ genotype, for anatomical orien-tation see Math1-cre control). Wnt/β-catenin-activation decreased growth of these tumors, too (Math1-cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ mice). Dotted lines indicate anato-mical 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 4loxP loxP

Recombined:

Exon 2 Exon 4loxP

A

A

A C

C

CB

B

b

Tumor DNA

Tail D

NA

H 2O

Marker

P8 P16P32

5

10

15

20

-ca

teni

n po

sitiv

etu

mor

cel

l nuc

lei [

%]

Rel

ativ

e fr

actio

n o

f B

rdU

rd+

cel

ls a

mon

g G

FP+

gra

nule

cel

ls [

%]

n. s.

Math1-cre::

SmoM2Fl/+

Math1-cre::

SmoM2Fl/+

Ctnnb1(ex3)F

l/+

0.5

1

2

4

8

16

TissueSpheres

Axin2

expres

sion

IRES-GFPCre-IRES-GFP

*

1

0.5

**

**

425 bp

540 bp

370 bp

715 bp

Ca. 1 kb

Exon 3

a

d

c

0

1.5

0

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::SmoM2Fl/+Ctnnb1(ex3)Fl/+ mice. (d) Primary cell cultures of Math1-cre::SmoM2Fl/+ and Math1-cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ tumors were transduced with Cre-IRES-GFP virus to allow recombination of yet unrecombined alleles. While Math1-cre::SmoM2Fl/+ tumor cells displayed no difference in proliferation, Math1-cre::SmoM2Fl/+Ctnnb1(ex3)Fl/+ tumor cells showed a significant decrease of proliferation 24 h after transduction (p=0.023. 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::SmoM2Fl/+hGFAP-cre::SmoM2Fl/+

Ctnnb1(ex3)Fl/+

H&E H&E H&E

H&E β-catenin Ki67

a b c

H&E β-catenin Ki67 H&E β-catenin Ki67

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::SmoM2Fl/+ (b) and hGFAP-cre::Ctnnb1(ex3)Fl/+SmoM2Fl/+ (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/+SmoM2Fl/+ 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).