CANCER Copyright © 2021 Notch1 switches progenitor …

 

 

C A N C E R

Notch1 switches progenitor competence in
inducing medulloblastoma
Claudio Ballabio1†, Matteo Gianesello1†, Chiara Lago1, Konstantin Okonechnikov2,3,
Marica Anderle1, Giuseppe Aiello1, Francesco Antonica1, Tingting Zhang4, Francesca Gianno5,6,
Felice Giangaspero5,6, Bassem A. Hassan4, Stefan M. Pfister2,3,7, Luca Tiberi1*

The identity of the cell of origin is a key determinant of cancer subtype, progression, and prognosis. Group 3
medulloblastoma (MB) is a malignant childhood brain cancer with poor prognosis and few candidates as putative
cell of origin. We overexpressed the group 3 MB genetic drivers MYC and Gfi1 in different candidate cells of origin
+
cells are competent to initiate group 3 MB, and we
in the postnatal mouse cerebellum. We found that S100b
+
+
cells. We found
cells have higher levels of Notch1 pathway activity compared to Math1
observed that S100b
+
+
cells was sufficient to induce group 3 MB upon MYC/Gfi1
and Sox2
that additional activation of Notch1 in Math1
expression. Together, our data suggest that the Notch1 pathway plays a critical role in group 3 MB initiation.

Copyright © 2021
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
under a Creative
Commons Attribution
NonCommercial
License 4.0 (CC BY-NC).

INTRODUCTION
Defining the cancer cell of origin can be critical for understanding
the first steps of cancer development and finding the signals re-
quired for transformation (1, 2). The identity of certain classes of
tumors seems to be more strongly related to the cell of origin than
to the oncogenic insult that induces malignant transformation. For
example, in brain tumors, concurrent inactivation of Trp53, Nf1,
and Pten in neural progenitor cells or oligodendrocyte progenitors
triggers the development of different subtypes of glioblastoma with
distinct gene expression profiles (3, 4). BCR-ABL (breakpoint clus-
ter region-Abelson kinase fusion gene) also provides an interesting
example of an oncogene that produces different tumors depending
on the cell in which it is expressed (5). These studies suggest that the
transcriptional context of the cell of origin can determine the iden-
tity of the tumor. By contrast, in some cases, certain driver muta-
tions rather than the cell of origin mainly define the tumor profile.
Activation of Hedgehog signaling in neural stem cells, granule neural
precursors, or postmitotic granule neurons leads to development of
aggressive medulloblastomas (MBs) with similar molecular profiles
(6–8). Nevertheless, it remains partially unclear what the specific
determinants of the cell of origin required for tumor initiation are,
and whether these specific features could be activated by an oncogenic
insult only. For instance, few progenitors in the mouse brain are able
to generate group 3 MB, although several genetic mutations that able
to generate this kind of tumors have been identified (9–12).

Group 3 is the most aggressive subtype of MB, mainly affecting
children younger than 10 years of age. Recently, single-cell RNA
sequencing (scRNA-seq) studies allowed the comparison of human
cerebellar tumors with transcriptional clusters in the developing

1Armenise-Harvard Laboratory of Brain Cancer, Department CIBIO, University of
Trento, Via Sommarive 9, 38123 Trento, Italy. 2Hopp Children’s Cancer Center
Heidelberg (KiTZ), Heidelberg, Germany. 3Division of Pediatric Neurooncology,
German Cancer Research Center and German Cancer Consortium, Heidelberg,
Germany. 4Paris Brain Institute-Institut du Cerveau, Sorbonne Université, Inserm,
CNRS, Hôpital Pitié-Salpêtrière, 8, Paris, France. 5Dept. of Radiologic, Oncologic and
Anatomo Pathological Sciences, University Sapienza of Rome, Rome, Italy. 6IRCCS
Neuromed, Pozzilli, Isernia, Italy. 7Department of Pediatric Hematology and Oncology,
Heidelberg University Hospital, Heidelberg, Germany.
*Corresponding author. Email: luca.tiberi@unitn.it
†These authors contributed equally to this work.

murine cerebellum. Analysis of human group 3  MB revealed the
presence of different transcriptional clusters within the tumor, re-
sembling granule neuron precursors (GNPs), unipolar brush cells
(UBCs), Purkinje cells, and GABAergic interneuron lineages during
normal development (13, 14). This suggested that early uncommitted
human cerebellar stem cells might represent a putative group 3 MB
cell of origin (14).

The human cerebellum contains 80% of all brain neurons and
has a 750-fold larger surface area, increased neuronal numbers,
altered neuronal subtype ratios, and increased folia complexity
compared to the mouse cerebellum (15, 16). Human cerebellar de-
velopment begins 30 days after conception and is thought to be
completed by the age of 2 years (15). Human cerebellar volume in-
creases fivefold between 22 postconception weeks (PCW) and birth,
and it becomes highly foliated during the third trimester [24 to 40
gestational weeks (GW)] (15). Granule cell progenitor proliferation
peaks during this period, and it is accompanied by increased exter-
nal granule layer (EGL) thickness. In mice, cerebellar growth and
foliation are driven by granule cell progenitor proliferation between
postnatal day 1 (P1) and P14, with deficient proliferation causing
EGL thinning (17). Hence, the postnatal development of the mouse
cerebellum resembles key aspects of human embryonic develop-
ment. For this reason, the cell of origin of group 3 MB should also
be studied during postnatal mouse cerebellar development. MB
mouse models have been developed by postnatally deregulating
Myc and Trp53 ex vivo in Math1+ GNPs or in CD133+ stem cells
(11, 12, 18). It has also been demonstrated that CD133+ cerebellar
stem cells and GNPs are able to give rise to group 3 MB upon ex vivo
enforced expression of Myc and Gfi1 or Myc and Gfi1b (10, 19, 20).
Furthermore, we have recently demonstrated that MYC and Gfi1
induce group 3 MB when co-overexpressed in human brain organoids
(9). Moreover, Sox2+ astrocyte progenitors (upon Myc over-
expression ex vivo) have been proposed to give rise to group 3 MB
in the postnatal developing cerebellum (21). Notably, whether MYC
and Gfi1 induce group 3 MB in specific progenitor populations has
never been tested in vivo, during embryonic or postnatal cerebellum
development. In addition, the molecular features that render a
cerebellar progenitor competent for group 3 MB development are
still unknown.

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Ballabio et al., Sci. Adv. 2021; 7 : eabd2781 23 June 2021SCIENCE ADVANCES | RESEARCH ARTICLE RESULTS
S100b+ cells are competent to induce group 3 MB
To modify mouse cerebellar cells directly in vivo, we stably trans-
fected P0 mouse cerebella exploiting the piggyBac transposon system.
We were able to target different cell populations in the developing
cerebellum, such as Pax6+, Sox2+, Sox9+, and Calbindin+ cells (fig.
S1, A to D) (9). To investigate which postnatal progenitors/cells are
competent to potentially give rise to group 3 MB, we used mice ex-
pressing creER recombinase in GNPs (Math1-creER, 24 mice) (22),
in glial cells progenitors (Sox2-creER, 7 mice) (23), Purkinje cells,
GABAergic interneurons, and glial cells (Ascl1-creER, 24 mice)
(Fig. 1A and fig. S1E) (24). We crossed these mice with R26-loxP-
STOP-loxP-Myc (R26-LSL-Myc) transgenic mice conditionally ex-
pressing MYC under the control of the creER recombinase, and we
stably transfected the cerebella at P0 with piggyBac vectors express-
ing Gfi1 (pPB-CAG-Gfi1). In parallel, we transfected the cerebella of
mice bearing creER recombinase with piggyBac vectors expressing
both MYC and Gfi1 under the control of a loxP-STOP-loxP cassette
(pPB-LSL-MYC and pPB-LSL-Gfi1) at P0 (Fig. 1A). Subsequently,
we induced the creER- dependent removal of the LSL cassette by in-
jecting tamoxifen (TMX) at different time points (table S1; we con-
sidered only mice with transfected cells). To further identify which
progenitors are competent to give rise to group 3 MB, we also trans-
fected pPB-LSL-MYC and pPB-LSL-Gfi1 vectors together with plas-
mids expressing the cre recombinase under the control of Tbr2
(UBCs), Sox2, Math1, or S100b (astrocytic and extra-astrocytic ex-
pression in human brain) (25) regulatory elements (Fig. 1B and table
S1). Notably, we obtained tumors only when MYC and Gfi1 ex-
pression was driven by S100b promoter (3 of 16, from two dif-
ferent litters; Fig.  1C). These tumors showed few S100b+ cells
(Fig. 1D), consist ently with the observation that the tumor pheno-
type not always resembles its cell of origin (26,  27). In addition,
these tumors were pH3+, showed few glial fibrillary acidic pro-
tein–positive (GFAP+) cells, and were also positive for the group
3–specific marker natriuretic peptide receptor 3 (NPR3) (Fig. 1E
and fig. S1, F and G). Sox2-creER;R26-LSL-Myc mice developed
choroid plexus carcinoma (CPC) 1 month after TMX injection
(6 of 6; Fig. 1F) as already published (28, 29). We did not detect
MB formation with any of the other promoters, but we observed
Venus+ cells 75 days after injection (Fig. 1, G and H, and fig. S1,
H and I). We checked the expression of MYC and Gfi1 by immu-
nofluorescence (fig. S1, J to L). We were able to detect the expres-
sion of MYC in the CPC developed by Sox2-creER;R26-LSL-Myc
transgenic mice injected with TMX at P2 (fig. S1J). The expres-
sion of both Gfi1 and MYC was also observed by immunofluores-
cence in P7 CD1 mouse cerebella transfected at P0 with pMath1-cre +
pPB-LSL-MYC + pPB-LSL- Gfi1 (fig. S1, K and L).

Because the S100b promoter was able to drive cre expression in
cells competent to develop group 3 MB, we investigated the identity
of these cells. As shown in fig. S2A, S100b+ cells are present in the
mouse cerebellum at P0. In addition, we could detect Venus+/
S100b+ cells 4 days after transfection with pS100b-cre + pPB-LSL-
Venus, thus confirming that our promoter recapitulates endogenous
S100b expression (Fig. 2, A and B). Furthermore, S100b promoter
drives Venus expression not only in the ventricular zone (VZ) but
also in the white matter (WM), internal granule layer (IGL), molecu-
lar layer (ML), and EGL, and most of those cells are positive for
Sox2 and GFAP (glial cells; Fig. 2, C and D). Because the Sox2 pro-
moter is not able to render postnatal cells competent for group 3 MB

development, we investigated the presence of S100b+ (Venus+) and
Sox2− cells (Fig. 2, D and E, and fig. S2, B and C). We observed that
34.8  ±  7.7% (mean  ±  SEM, n  =  7 brains) of S100b-cre+ cells are
Sox2− 4 days after transfection, with a small subset being Sox2−/
Nestin+ (Fig. 2D). We then characterized which cells are produced
by S100b-cre+ cells by performing lineage tracing experiments.
Thirty days after transfection, we observed ependymal cells (Fig. 2F),
glial cells (fig. S2D), oligodendrocytes (Fig. 2G), Bergmann glia
(Fig.  2H), and S100b+ cells (Fig.  2I) that derive from S100b-cre+
cells transfected at P0. Hence, the population of S100b-cre+ cells at
P0 is able to generate several cerebellar populations, notably dif-
ferent types of glial cells. To study the first step of S100b cell trans-
formation, we performed lineage tracing of S100b-cre+ cells that
start to stably express MYC and Gfi1 starting from P0. As shown in
Fig. 2 (J to M), after 10 days, we observed small clusters of Venus+
cells, having a homogeneous round morphology that is different
from cells not expressing MYC and Gfi1 (Fig. 2E and fig. S2, B and
C). Furthermore, these cell clusters are mainly GFAP− and S100b−
(Fig. 2, J and M), with few Sox2+ and Sox9+ cells (Fig. 2, K and L).
We did not detect any cluster formation with other promoters driving
cre expression (Fig. 1, G and H, and fig. S1, H and I). Because
S100b+ cells are able to generate group 3 MB postnatally, we tested
whether S100b+ cells might be competent to give rise to MB also
during embryonic development. We first confirmed the embry-
onic expression of S100b in the cerebellar VZ (fig. S2E) (30), and
then we electroporated in utero pPB-LSL-MYC + pPB-LSL-Gfi1 to-
gether with pS100b-cre at embryonic day 15.5 (E15.5) (fig. S2F).
We observed formation of tumors in 3 of 12 electroporated mice
(fig. S2G) that were positive for the group 3–specific marker NPR3
(fig. S2H).

Human S100B+ cells are competent to induce group 3 MB
To test whether S100B+ cells are also present during human cerebellar
development, we performed histological analysis of S100B in human
tissues. As shown in Fig. 3 (A and B), S100B+ cells are present in the
human cerebellum at 22 and 39 GW in EGL, IGL, and ML. We
could also detect a few S100B+ cells in human group 3 MB samples
(fig. S3A). On the basis of these results, we tested whether human
S100B+ cells are competent to generate MB in human cerebellar or-
ganoids. We have recently shown that MYC and Gfi1 overexpression in
human cerebellar organoids induces group 3 MB with a methylation
profile similar to human patients (9). Therefore, we electroporated
human induced pluripotent stem cell (iPSC)–derived cerebellar or-
ganoids (9, 31) with pPB-LSL-Venus and pS100b-cre or pSox2-cre
at day 35 to target S100B+ and SOX2+ cells when these cerebellar
progenitors are present (fig. S3, B to D) (9, 31). Unfortunately, we
were unable to specifically target GNPs in human cerebellar or-
ganoids, as reliable human ATOH1 regulatory sequences are lacking.
Next, we also electroporated pPB-LSL-MYC + pPB-LSL-Gfi1
together with pS100b-cre or pSox2-cre. As shown in Fig. 3 (C and D),
MYC and Gfi1 expression in S100B-cre+ cells induced formation
of clusters of cells in 12.5% of electroporated organoids (n = 40), as
we previously observed (9). MYC and Gfi1 overexpression in
SOX2-cre+ cells did not induce cell cluster formation (0 of 40)
(Fig. 3, C and D). Furthermore, we observed clusters of proliferating
cell nuclear antigen–positive (PCNA+) cells using the S100B pro-
moter, suggesting that MYC and Gfi1 overexpression has an
oncogenic potential in S100B-cre+ cells also in human cerebellar
organoids (Fig. 3E).

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Ballabio et al., Sci. Adv. 2021; 7 : eabd2781 23 June 2021SCIENCE ADVANCES | RESEARCH ARTICLE t

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Fig. 1. Overexpression of MYC and Gfi1 in postnatal S100b+ cerebellar cells induces MB. (A and B) Schematic representation of in vivo transfection experiments to
overexpress MYC and Gfi1 in different postnatal cerebellar cell populations. (C) DAPI (4′,6-diamidino-2-phenylindole) staining and Venus immunofluorescence of sagittal
brain section of CD1 mouse 2 months after transfection with pPBase + pS100b-cre + pPB-LSL-MYC + pPB-LSL-Gfi1 + pPB-LSL-Venus at P0. (D and E) Confocal images of
Venus and S100b (D) and Venus and GFAP (E) immunofluorescence of tumors in CD1 mice 2 months after transfection with pPBase + pS100b-cre + pPB-LSL-Myc + pPB-
LSL-Gfi1 + pPB-LSL-Venus at P0. The white squares in (D) and (E) mark the regions shown at higher magnification. (F) DAPI staining and pH3 immunofluorescence of
sagittal brain section of Sox2-creER;R26-LSL-Myc mouse 1 month after TMX injection at P2. The white square in (F) marks the region shown in (F′). (G and H) DAPI staining
and Venus immunofluorescence of sagittal brain sections of Math1-creER;R26-LSL-Myc mouse (G) or Ascl1-creER;R26-LSL-Myc mouse (H) 2.5 months after transfection
with pPBase + pPB-Gfi1 + pPB-Venus at P0 and TMX injection at P2 (G) or P0 (H). Arrows point to Venus+ cells. Scale bars, 1 mm (C and F) and 100 m (D, E, G, and H). IGL,
internal granule layer; EGL, external granule layer.

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Ballabio et al., Sci. Adv. 2021; 7 : eabd2781 23 June 2021SCIENCE ADVANCES | RESEARCH ARTICLE DAPI/Venus

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Fig. 2. Lineage tracing of S100b-cre+ cells in postnatal CD1 mouse cerebellum. (A to E) Lineage tracing in P4 cerebellum transfected with pPBase + pS100b-cre + pPB-
LSL-Venus at P0. (A) Immunofluorescence of Venus and S100b. (A and A′) Higher magnifications in (A′) and (A′′). The arrow marks a S100b+/Venus+ cell. (B) Quantification
of S100b+/Venus+ cells within Venus+ cells. (C) Quantification of Venus+ cells within different regions. VZ, ventricular zone; white matter, WM; ML. molecular layer.
(D) Quantification of Venus+/GFAP+, Venus+/Sox2+, Venus+/Nestin+, Venus+/Sox2+/Nestin+, Venus+/Sox2−/Nestin+, and Venus+/Sox2−/Nestin− cells within Venus+ cells.
(E) Immunofluorescence of Venus, Sox2, and Nestin. The white square marks the region shown at higher magnification. The arrow marks a Venus+/Sox2+ cell; the arrow-
head marks a Venus+/Sox2− cell. (F to I) Immunofluorescence of Venus with GFAP (F), Olig2 (G), Sox2 (H), and S100b (I) in P30 cerebellum transfected with pPBase +
pS100b-cre + pPB-LSL-Venus at P0. (F to I and F′ to I′) Higher magnifications in (F′ ) to (I′) and (F″) to (I″). Arrows mark double-positive cells. (J to M) Immunofluorescence
of Venus and GFAP (J), Sox2 (K), Sox9 (L), and S100b (M) in P10 cerebellum transfected with pPBase + pS100b-cre + pPB-LSL-MYC + pPB-LSL-Gfi1 + pPB-LSL-Venus at P0.
(J to M) Higher magnifications in (J′) to (M′). Confocal images scale bars, 100 m (A and E to M) and 50 m in (F′ to I′). Data in (B) to (D) presented as means + SEM.

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Ballabio et al., Sci. Adv. 2021; 7 : eabd2781 23 June 2021SCIENCE ADVANCES | RESEARCH ARTICLE DAPI/S100B

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Fig. 3. Human S100B-cre+ cells are responsive to MYC and Gfi1 overexpression. (A) Confocal images of S100B immunofluorescence of human fetal cerebellum at 22 GW.
(B) Confocal images of S100B immunofluorescence of human fetal cerebellum at 39 GW. The white squares in (A) and (B) mark the regions shown at higher magnification
in (A′) and (B′), respectively. (C) Brightfield and fluorescence images of cerebellar organoids at different time points electroporated at day 35 with pPBase + pSox2-
cre + pPB-LSL-Gfi1 + pPB-LSL-MYC + pPB-LSL-Venus, or pPBase + pS100b-cre + pPB-LSL-Gfi1 + pPB-LSL-MYC + pPB-LSL-Venus. (D) Brightfield and fluorescence images of
cerebellar organoids at 74 days, electroporated at day 35 with pPBase + pSox2-cre + pPB-LSL-Gfi1 + pPB-LSL-MYC + pPB-LSL-Venus or pPBase + pS100b-cre + pPB-LSL-
Gfi1 + pPB-LSL-MYC + pPB-LSL-Venus. The white squares mark the regions shown at higher magnification. (E) Confocal images of Venus and PCNA immunofluorescence
of cerebellar organoids at day 74, electroporated at day 35 with pPBase + pS100b-cre + pPB-LSL-Gfi1 + pPB-LSL-MYC + pPB-LSL-Venus. Scale bars, 100 m (A to E).

Notch1 activation makes Math1+ progenitors competent
to induce group 3 MB
On the basis of our data, we speculated that S100b+ cells should
have specific features/pathways that are present to a lesser extent in
postnatal Math1+, Ascl1+, and Sox2+ cells. It has been found that
Notch signaling regulates fate decisions of early mouse cerebellar
progenitors (32, 33). In particular, Sox2+ progenitors at E9.5 have
high Notch1 pathway levels and are competent to give rise to Ascl1+

and Math1+ progenitors (33). We tried to target these Sox2+ early
progenitors by conditionally overexpressing Myc at E9.5 and at E13.5
(Sox2-creER;R26-LSL-Myc mouse), but we were not able to obtain
live pups, possibly due to a widespread Sox2 expression during mouse
development (n  =  2 pregnant females administered with TMX at
E9.5; n = 2 pregnant females administered with TMX at E13.5). Notably,
these early Sox2+ progenitors do not generate the Sox2+ cells residing
in the postnatal cerebellum (Sox2-creER;R26-LSL-tdTomato, TMX

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Ballabio et al., Sci. Adv. 2021; 7 : eabd2781 23 June 2021SCIENCE ADVANCES | RESEARCH ARTICLE at E10.5; fig. S4A). Furthermore, embryonic Math1+ progenitors
express low levels of Notch1 and Hes1/Hes5 target genes, and
Notch1 pathway activation is able to repress Math1 expression, thus
changing the fate of these progenitors (33). The Notch signaling
pathway plays a critical role in central nervous system development,
stem cell maintenance, and differentiation of cerebellar GNPs and
modulates epithelial-to-mesenchymal transition; and its role in
SHH MB is still controversial (34–37). Mutations in NOTCH sig-
naling genes have been described in group 3 MB (38), with especially
elevated expression of NOTCH1 in spinal metastases (39).

On the basis of these data, we tested whether the Notch1 pathway
plays a role in determining the competence of the different cerebel-
lar progenitors to generate group 3 MB. Using available scRNA-seq
data (40) from postnatal (P0 and P4) mouse cerebella, we verified the
stable cell types assignment for the different cerebellar cell popula-
tions (Fig. 4A and fig. S4B). Further analyses on the scRNA-seq data
indicated that S100b is expressed in clusters assigned to the astro-
cytic lineage (which includes glial progenitors, oligodendrocytes, glia,
and Bergmann glia; clusters  and ) and ciliated cells (cluster )
(Fig. 4, A to D) (40) and is mostly mutually exclusive with the ex-
pression of Math1 (Fig. 4, E to G), especially in the granule neurons
lineage. Therefore, we asked whether the expression of Notch path-
way genes correlates with any cell lineage also in the postnatal cere-
bellar scRNA-seq data, as in the embryonic mouse cerebellum (33).
As shown in Fig. 4 (H to P) and fig. S4 (C and D), we found that the
S100b+ cell clusters show the highest expression levels of the Notch1
receptor and of Hes1/Hes5 target genes. On the other hand, postnatal
Math1+ cells display lower levels of Notch pathway activation, similar
to the Math1+ progenitors in the embryonic mouse cerebellum (33).
To confirm these data, we tested Notch activity using a well-
known reporter for Notch signaling. We used a plasmid that drives
the expression of destabilized enhanced green fluorescent protein
(d2EGFP) under the control of the Hes5 promoter (41). The pro-
moter activity is dependent on Notch pathway activation and is re-
stricted to neural stem cells in the mouse brain (41, 42). As shown
in fig. S4 (E to G), we observed more d2EGFP expression in S100b+
cells compared to Math1+ progenitors, confirming that the Notch
pathway is more active in S100b+ cells.

Notably, we overexpressed the Notch1 constitutively active form
(N1ICD) (42,  43) with MYC in postnatal mouse cerebellum and,
together, they are sufficient to induce MB (fig. S5A). N1ICD and
Gfi1 co-overexpression was not sufficient to generate tumor, high-
lighting the Notch and MYC relation in tumor formation (fig. S5A).
To test whether the Notch pathway is also required for the forma-
tion of group 3 MB, we used the dominant negative forms of the
Notch pathway components Maml1 (DN-Maml1) and Rbpj (DN-Rbpj)
(44, 45). As shown in fig. S5B, we did not observe tumor formation
upon overexpression in S100b+ cells of DN-Maml1 or DN-Rbpj
together with MYC/Gfi1, suggesting that the Notch pathway activa-
tion is required for group 3 MB formation. On the other hand, we
tested whether increasing Notch pathway activity could make Math1+
cells competent to induce group 3 MB. To do so, we overexpressed
N1ICD in Math1+ cells, which alone did not induce MB formation
(Fig. 5A). Next, we co-overexpressed N1ICD with MYC and Gfi1 to induce
group 3 MB in Math1+ progenitors. As shown in Fig. 5 (B and C),
stable N1ICD overexpression, together with MYC/Gfi1, in Math1+
progenitors induces tumor formation in 13 of 14 mice (Fig.  5A).
These tumors are GFAP−, pH3+ (fig. S5, C and D), and are compa-
rable to human group 3  MB and to previously published group

3 MB mouse models (MYC/Gfi1, MYC/Otx2, and MYC over-
expression in Sox2+ progenitors; Fig. 5, D and E) (9, 21). We com-
pared the transcriptional profiles of tumors induced by N1ICD +
MYC/Gfi1  in Math1+ cells and tumors induced by MYC/Gfi1  in
S100b+ cells with human MB samples classified according to the
consensus subtypes (46). The unsupervised clustering confirmed
similarity of our models to group 3 MB and also most substantial
matches to groups 3 and 3 (fig. S5E). This is consistent with the
observation of GFI1 activation to be restricted to group 3 samples
and MYC amplification in group 3. Nevertheless, MYC expression
levels in group 3 are comparable to those in group 3 (fig. S5F).
We also checked the levels of expression of the key components of
the NOTCH signaling pathway among the molecular subtypes of
group 3/4 MB. We observed only a slight enrichment of NOTCH
pathway genes expression in group 3, while group 3 demonstrated
an opposite effect (fig. S5G). This suggests that NOTCH pathway
might play a pivotal role in the initial steps of tumor formation. To
study the effects of N1ICD on Math1+ progenitors, we checked the
transfected cerebella 14 days after injection: we found that N1ICD +
MYC/Gfi1-transfected cells already formed small clusters of Ki67+
cells in the EGL, unlike granule neuron progenitors transfected
with N1ICD alone (fig. S5, H to K).

To confirm that N1ICD is required for the first steps of MB for-
mation, we transfected Math1+ cells with MYC/Gfi1 and a non-
integrating plasmid encoding for N1ICD, allowing the transient
expression of the transgene. As shown in Fig. 5F, transient expres-
sion of N1ICD was sufficient to allow tumor formation driven by
MYC/Gfi1 overexpression in 7 of 28 mice (Fig. 5, A and B). These
tumors are GFAP− and pH3+ (fig. S6, A and B), resembling human
group 3 MB and other published group 3 MB mouse models. Nota-
bly, the activity of N1ICD is sufficient during early phases of tumor
formation, while being not essential for its progression. Expression
of V5-tagged N1ICD is detectable in tumors driven by MYC/
Gfi1 + N1ICD stable, whereas it is not maintained in tumors gener-
ated by transient N1ICD expression (Fig. 5G). To confirm that the
activation of the Notch pathway is not required for the tumor pro-
gression, we checked the expression levels of known Notch pathway
genes in our tumors (fig. S6C). We did not detect any up-regulation
of Notch pathway genes (Hes1, Hes5, Jag1, and Notch1) in tumors
driven by MYC/Gfi1 (under the control of S100b promoter), sug-
gesting that fully grown tumors do not rely on the Notch pathway
for their sustenance. Notch pathway genes, such as Hes5 and Jag1,
were overexpressed only in tumors stably expressing N1ICD to-
gether with MYC/Gfi1 in Math1+ cells. Last, we tested the effect of
Notch1 activation in Sox2+ progenitors as well. Notably, MYC/
Gfi1 + N1ICD overexpression in Sox2+ cells induces MB in 9 of 11 mice
(fig. S6D). Overall, our data suggest that Notch pathway plays a critical
role in group 3 MB initiation and formation in various cerebellar
progenitors, such as S100b+, Math1+, and Sox2+ cells.

DISCUSSION
Despite large effort in understanding the cell of origin of different
tumors, the relationship of the contribution of the cell of origin ver-
sus the relevant driver mutations is still elusive. Current opinion in
the field is that tumor identity can be defined by the cell of origin
and/or genetic mutation (1, 2). In the first scenario, the cells of origin
have specific features, such as the epigenetic state, that make them
competent in inducing cancer and may also direct cell tumor-initiating

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