Supplementary Figure 1

Irradiation does not lead to major cell death in the SOX2+ layers.

(A- D) FIHC detection of the indicated proteins and dapi on midsagittal sections (lobule IV/V) of Non-IR (A-B)

and IR (C-D) mice at the indicated ages (n=4). External granule layer (EGL), molecular layer (ML), Purkinje

Cell Layer (PCL) and white matter (WM)+ internal granule layer (IGL) are delimited by yellow dotted lines.

White arrowheads in C indicate TUNEL+ cells present in the PCL. Black scale bar indicates 100μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 2

NEPs generate interneurons (INs) and astrocytes but not granule neurons (GCs) in the WT cerebellum.

(A-H) FIHC detection of the indicated proteins and dapi on sagittal sections of the vermis (lobule III) of Nes-

FlpoER/+; R26FSF-TDTom/+

; Atoh1-GFP/+ (Nes-TDTom; Atoh1-GFP), Nes-FlpoER/+; R26MASTR/+

(Nes-EGFP-

Cre) and Nes-CFP/+ mice at the indicated ages. (B) Nes-FlpoER given tamoxifen at P0 initially (P1) mainly

marks SOX2+ progenitors, and not Atoh1+ (GFP+) GCPs (backwards arrow in Fig. 1A), and give rise at P8 to

S100+ astrocytes (astro), Bergmann glia (Bg) and PAX2+ interneurons (INs) (arrows C, E, F), only rare

PAX6+ GCPs or GCs (backward arrows in D)(See Fig. 3 for quantifications). Higher magnification images are

shown in (’ and ’’) and white matter (WM), internal granule layer (IGL), PCL and molecular layer (ML) are

delimited by white doted lines. (G-H) FIHC detection of the indicated proteins and dapi on sagittal sections of

the vermis (lobule IV/V) of P4 Nes-CFP/+ mice. Scale bars indicate 50μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 3

Nes-CFP+ cells can self-renew and are multipotent in vitro.

(A) P4 Nes-CFP expression in SOX+ NSCs. (B) Flow cytometric analysis of P4 cerebellar Nes-CFP+ cells.

Nature Neuroscience: doi:10.1038/nn.4621

Nes-CFP+ cells represent 6.19 % of the total population. (C-F) FACS-isolated CFP+ cells form neurospheres

after 7 days, when cultured in neurosphere media containing bFGF and EGF. (C and D) Neurospheres

expresses CFP (D) and Sox2 (D’). Bright-field view in (C). (E and F) Graphs showing the number of

neurospheres formed when CF+ and CFP- cells are plated at the indicated numbers (60 cells/well: p<0.0001,

t(4)=28; 600 cells/well: p=0.0031, t(4)=6.369; 6000 cells/well: p=0.0042, t(4)=5.884) (E) and indicated serial

passages (secondary: p<0.0001, t(4)=18.30; tertiary: p=0.0408, t(4)=2.98; primary vs secondary: p=0.0155, t(4)=4.049; primary vs tertiary: p=0.2999, t(4)=1.19; secondary vs tertiary: p=09523, t(4)=0.06369) (F) (n=3

experiments). (G-J) Nes-CFP+ neurospheres were dissociated, plated as a monolayer and were grown in either

neurosphere media containing bFGF and EGF or differentiation media with 10% serum for 7 days. The levels of

CFP and SOX2 (G-G’), GFAP (H-H’), TUJ1(I-I’) and O4 (J-J’) are shown using immunofluorescent

microscopy to assess multipotency. Nes-CFP+ cells showed morphological changes and were able to

differentiate into neurons, astrocytes and oligodendrocytes when subjected to differentiation. Graphical data are

present as means ± SEM and significance was determined with the two-tailed Student’s t test, ****P<0.0001,

**P<0.01, *P<0.05. All scale bars indicate 100 μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 4

Tamoxifen administration delays the response of NEPs to injury.

(A-D) FIHC detection of the indicated proteins, EdU and dapi on sagittal sections at the indicated ages of the

vermis (lobule IV/V) of Non-IR (A, B) and IR (C, D) Nes-CFP/+mice administrated with Tm at P0. (E-J)

Graphs of the changes in density at the indicated stages of CFP+ cells per mm of PCL (E) (n=3; P4 no Tm:

p=0.017, t(4)=3.917; P4 Tm: p=0.127, t(4)=1.924; P6 no Tm: p=0.027, t(4)=3.418; P6 Tm: p=0.068, t(4)=2.48;

P4 Non-IR Tm vs no Tm: p=0.634, t(4)=0.514; P6 Non-IR Tm vs no Tm: p=0.033, t(4)=3.171; P4 IR Tm vs no

Tm: p=0.02, t(4)=3.738; P6 IR Tm vs no Tm: p=0.044, t(4)=2.898) and per mm2 of IGL+WM (H) (n=3; P4 no

Nature Neuroscience: doi:10.1038/nn.4621

Tm: p=0.737, t(4)=0.36; P4 Tm: p=0.219, t(4)=1.46; P6 no Tm: p=0.179, t(4)=1.63; P6 Tm: p=0.027, t(4)=3.42;

P4 Non-IR Tm vs no Tm: p=0.596, t(4)=0.56; P6 Non-IR Tm vs no Tm: p=0.0044, t(4)=5.79; P4 IR Tm vs no

Tm: p=0.428, t(4)=0.88; P6 IR Tm vs no Tm: p=0.01, t(4)=4.53), the proportion of proliferating (Ki67+) CFP+

cells in the PCL (F) (n=3; P4 no Tm: p=0.0011, t(4)=8.5; P4 Tm: p=0.764, t(4)=0.322; P6 no Tm: p=0.03,

t(4)=3.25; P6 Tm: p=0.541, t(4)=0.67; P4 Non-IR Tm vs no Tm: p=0.589, t(4)=0.586; P6 Non-IR Tm vs no

Tm: p=0.36, t(4)=1.03; P4 IR Tm vs no Tm: p=0.096, t(4)=2.16; P6 IR Tm vs no Tm: p=0.01, t(4)=4.57) and in

the WM+IGL (I) (n=3; P4 no Tm: p=0.054, t(4)=2.7; P4 Tm: p=0.0042, t(4)=5.86; P6 no Tm: p=0.506,

t(4)=0.73; P6 Tm: p=0.557, t(4)=0.64; P4 Non-IR Tm vs no Tm: p=0.097, t(4)=2.16; P6 Non-IR Tm vs no Tm:

p=0.11, t(4)=2.05; P4 IR Tm vs no Tm: p=0.943, t(4)=0.075; P6 IR Tm vs no Tm: p=0.012, t(4)=4.4), the

proliferation index in PCL (% [Ki67+ GFP+ EdU+] cells of all [GFP+Ki67+] cells) (G) (n=3; P4 no Tm:

p=0.717, t(4)=0.389; P4 Tm: p=0.678, t(4)=0.678; P6 no Tm: p=0.457, t(4)=0.457; P6 Tm: p=0.36, t(4)=0.363;

P4 Non-IR Tm vs no Tm: p=0.263, t(4)=1.3; P6 Non-IR Tm vs no Tm: p=0.108, t(4)=2.06; P4 IR Tm vs no

Tm: p=0.759, t(4)=0.33; P6 IR Tm vs no Tm: p=0.348, t(4)=1.06) and in the WM+IGL (J) (n=3; P4 no Tm:

p=0.127, t(4)=1.92; P4 Tm: p=0.0088, t(4)=4.78; P6 no Tm: p=0.18, t(4)=1.62; P6 Tm: p=0.109, t(4)=2.06; P4

Non-IR Tm vs no Tm: p=0.822, t(4)=0.24; P6 Non-IR Tm vs no Tm: p=0.535, t(4)=0.68; P4 IR Tm vs no Tm:

p=0.0429, t(4)=2.93; P6 IR Tm vs no Tm: p=0.384, t(4)=0.98). All of the analyses were performed on 3 midline

sections per brain and for lobule IV/V. All graphical data are present as means ± SEM and the significance was

determined with using two-tailed Student’s t test, ***P<0.001, **P<0.01, *P<0.05. Scale bars indicate 100 μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 5

Completion of cerebellum development is delayed after injury.

(A-B) H&E staining of sagittal sections of the vermis of P16 Nes-FlpoER/+; R26FSF-TDtom/+

(Nes-TDTom) mice

given Tm at P0 with or without irradiation. (‘ and “) are high magnifications of the yellow dotted boxed areas

indicated in A and B. Yellow arrowheads indicate the thicker EGL in the IR condition than control. Scale bars

indicate 500 μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 6

Diphtheria toxin-induced GCP cell death triggers the replenishment of the EGL by NEPs.

(A) Schematic representation of the experimental design. (B-C) FIHC detection of Diphtheria toxin receptor

(DTR) and Ki67 proteins and dapi on sagittal sections of the vermis (lobule IV/V) (B) and the para-vermis (C)

of P1 Atoh1-tTA/+;TRE-Cre/+;R26LSL-DTR/+

(Atoh-DTR) mice not injected with DT. Yellow dotted circles in C

and C’ indicate the position of the cerebellar nuclei (CN). (D-E) FIHC detection of PAX6 protein, TUNEL and

dapi on sagittal sections of the vermis (lobule IV/V) of P2 Atoh1-tTA/+;R26LSL-DTR/+

(Control) and Atoh-DTR

mice (DT at P1). Note the increase of TUNEL staining in the EGL of Atoh-DTR mice compared to controls (no

DT). (F-I) H&E and FIHC detection of the indicated proteins and dapi on sagittal sections of the vermis of P4

Control; Nes-CFP/+ (F-G) and Atoh-DTR; Nes-CFP/+ (H-I) mice (DT at P1). G and I are from lobule IV/V.

Yellow dotted lines in G’ and I’ indicate the EGL. Red arrowhead in I’ indicates the presence of CFP+ cells in

the EGL. (J-K) FIHC detection of the indicated proteins and dapi on sagittal sections of the vermis (fissure

between lobule IV/V and VI) of P6 Control; Nes-FlpoER/+; R26FSF-TDTom/+

(Nes-TDTom) (J) and Atoh-DTR;

Nature Neuroscience: doi:10.1038/nn.4621

Nes-FlpoER/+; R26FSF-TDTom/+

(K) mice (Tm at P0 and DT at P1). Yellow dotted lines in J’ and K’ indicate the

EGL. Red arrowhead in K’ indicates the presence of TDTom+ cells in the EGL. Black and white scale bars

indicate 500 and 100 μm, respectively.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 7

NEPs progressively extinguished Nestin expression in the EGL

(A-C) FIHC detection of CFP on sagittal sections of the vermis (lobule IV/V) of IR Nes-CFP/+ mice at the

indicated ages. The EGL is delimitated by yellow doted lines. Red arrowheads indicate the progressive decrease

of CFP staining in the EGL. Scale bar indicates 100μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 8

NEPs are highly motile after EGL depletion.

(A-H) Detection of native CFP fluorescence on sagittal slices of the vermis (lobule IV/V) of P6 Non-IR (A-D)

and IR (E-H) Nes-CFP/+mice showing tracks of CFP+ cells in each layer during the 5h45min of imaging. The

length of the tracks represents the distance travelled by the cells. The color code for the tracks is as indicated.

Black scale bar indicates 100μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 9

Irradiation changes the gene expression profile of NEPs.

(A) Heat map showing differential gene expression between Non-IR and IR P5 NEPs after irradiation of the CB

at P1. Color code is as indicated. (B) Gene Ontology categories of the genes significantly changed by ≥1.5 fold

in P5 IR NEPs compared to Non-IR. Gene Ontology was generated using DAVID Bioinformatics Resources

6.7. (C-F) In situ hybridization of Gli1 on P5 (C, E) and P8 (D, F) midsagittal cerebellar sections (lobule IV/V).

Yellow arrowheads indicate the PCL expression is higher after IR. Yellow asterisks indicate the PCL expression

is similar in both Non-IR and IR CB at P8. (G) Q-rtPCR analysis of the indicated genes in Nes-CFP+ NEPs

isolated from P4 (Gli1: p=0.0533, t(4)=2.7; Ptch1: p=0.0632, t(4)=2.551; Ptch2: p=0.0526, t(4)=2.727) and P6

(Gli1: p=0.1654, t(4)=1.695; Ptch1: p=0.1943, t(4)=1.6; Ptch2: p=0.1557, t(4)=1.746) Non-IR and IR cerebella

(n=3 FACS experiments/genotype).

Nature Neuroscience: doi:10.1038/nn.4621

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 10

SHH signaling regulates the expansion of NEPs and the fate of NEP-derived cells.

(A-F) FIHC detection of the indicated proteins and dapi on midsagittal sections (lobule VIII) of P8 control Nes-

FlpoER/+; R26MASTR/+

(Nes-GFP, n=3, A and D) and experimental Nes-FlpoER/+; R26MASTR/+

; Smolox/lox

(Nes-

Smo CKO, n=3, B and E) oe Nes-FlpoER/+; R26MASTR/SmoM2-YPF

(Nes-SmoM2, n=6, C and F) mice (Tm at P0).

(G-J) Graphs showing changes in the number of GFP+ cells in each layer (as

indicated in A) of P8 mice that also expressed SOX2 only (EGL: Nes-GFP vs Nes-Smo CKO: p=N/A; Nes-

GFP vs Nes-SmoM2: p= 0.185, t(7)=1.47; Nes-Smo CKO vs Nes-SmoM2: p=0.185, t(7)=1.47; ML: Nes-GFP vs

Nes-Smo CKO: p=0.238, t(4)=1.387; Nes-GFP vs Nes-SmoM2: p= 0.203, t(7)=1.403; Nes-Smo CKO vs Nes-

SmoM2: p=0.051, t(7)=2.38; PCL: Nes-GFP vs Nes-Smo CKO: p=0.031, t(4)=3.25; Nes-GFP vs Nes-SmoM2:

p=0.779, t(7)=0.29; Nes-Smo CKO vs Nes-SmoM2: p=0.053, t(5.24)=2.49; IGL: Nes-GFP vs Nes-Smo CKO:

p=0.0036, t(4)=6.13; Nes-GFP vs Nes-SmoM2: p= 0.0017, t(7)=4.92; Nes-Smo CKO vs Nes-SmoM2: p=0.0294,

t(7)=2.73; WM: Nes-GFP vs Nes-Smo CKO: p=0.353, t(4)=1.05; Nes-GFP vs Nes-SmoM2: p= 0.54, t(7)=0.64;

Nes-Smo CKO vs Nes-SmoM2: p=0.158, t(7)=1.58) (G), PAX2 only (EGL: Nes-GFP vs Nes-Smo CKO:

p=0.597, t(4)=0.57; Nes-GFP vs Nes-SmoM2: p= 0.051, t(2.13)=3.99; Nes-Smo CKO vs Nes-SmoM2: p=0.0001,

t(7)=7.64; ML: Nes-GFP vs Nes-Smo CKO: p=0.142, t(4)=1.83; Nes-GFP vs Nes-SmoM2: p= 0.0155,

t(7)=3.18; Nes-Smo CKO vs Nes-SmoM2: p=0.124, t(7)=1.75; PCL: Nes-GFP vs Nes-Smo CKO: p=0.525,

t(4)=0.695; Nes-GFP vs Nes-SmoM2: p=0.966, t(7)=0.044; Nes-Smo CKO vs Nes-SmoM2: p=0.667, t(7)=0.44;

IGL: Nes-GFP vs Nes-Smo CKO: p=0.0299, t(4)=3.3; Nes-GFP vs Nes-SmoM2: p= 0.86, t(7)=0.184; Nes-Smo

CKO vs Nes-SmoM2: p=0.0127, t(5.46)=3.64; WM: Nes-GFP vs Nes-Smo CKO: p=0.0014, t(4)=7.88; Nes-

GFP vs Nes-SmoM2: p= 0.982, t(7)=0.023; Nes-Smo CKO vs Nes-SmoM2: p=0.0034, t(7)=4.34) (H), S100β

only (EGL: Nes-GFP vs Nes-Smo CKO: p=0.0963, t(4)=2.165; Nes-GFP vs Nes-SmoM2: p= 0.389, t(7)=0.918;

Nes-Smo CKO vs Nes-SmoM2: p=0.901, t(7)=0.129; ML: Nes-GFP vs Nes-Smo CKO: p=0.686, t(4)=0.435;

Nes-GFP vs Nes-SmoM2: p=0.406, t(7)=0.884; Nes-Smo CKO vs Nes-SmoM2: p=0.243, t(7)=1.27; PCL: Nes-

GFP vs Nes-Smo CKO: p=0.51, t(4)=0.722; Nes-GFP vs Nes-SmoM2: p=0.018, t(7)=3.07; Nes-Smo CKO vs

Nes-SmoM2: p=0.032, t(7)=2.68; IGL: Nes-GFP vs Nes-Smo CKO: p=0.379, t(4)=0.99; Nes-GFP vs Nes-

SmoM2: p= 0.99, t(7)=0.012; Nes-Smo CKO vs Nes-SmoM2: p=0.571, t(7)=0.594; WM: Nes-GFP vs Nes-Smo

CKO: p=0.01, t(3.04)=4.524; Nes-GFP vs Nes-SmoM2: p= 0.725, t(7)=0.366; Nes-Smo CKO vs Nes-SmoM2:

p=0.345, t(5.426)=1.458) (I) and both SOX2 and S100β (EGL: Nes-GFP vs Nes-Smo CKO: p>0.9999, t(4)=0;

Nes-GFP vs Nes-SmoM2: p= 0.078, t(7)=2.06; Nes-Smo CKO vs Nes-SmoM2: p=0.078, t(7)=2.06; ML: Nes-

GFP vs Nes-Smo CKO: p=0.493, t(4)=0.754; Nes-GFP vs Nes-SmoM2: p= 0.082, t(7)=2.028; Nes-Smo CKO vs

Nes-SmoM2: p=0.06, t(7)=2.238; PCL: Nes-GFP vs Nes-Smo CKO: p=0.018, t(3.475)=3.87; Nes-GFP vs Nes-

SmoM2: p=0.0039, t(7)=4.23; Nes-Smo CKO vs Nes-SmoM2: p=0.0005, t(7)=5.99; IGL: Nes-GFP vs Nes-Smo

CKO: p=0.064, t(4)=2.546; Nes-GFP vs Nes-SmoM2: p= 0.61, t(7)=0.532; Nes-Smo CKO vs Nes-SmoM2:

p=0.0029, t(7)=4.483; WM: Nes-GFP vs Nes-Smo CKO: p=0.086, t(4)=2.269; Nes-GFP vs Nes-SmoM2: p=

0.174, t(7)=1.514; Nes-Smo CKO vs Nes-SmoM2: p=0.01, t(7)=3.42) (J). All of the analyses were performed on

3 midline sections per brain. All graphical data are presented as means ± SEM and the significance was

determined with two-tailed Student’s t test, ***P<0.001, **P<0.01, *P<0.05. Scale bars indicate 100 μm. (K-

P) H&E and FIHC detection of the indicated proteins and dapi on sagittal sections of the vermis of P8 (A-C)

and P12 (D-F) Nes-FlpoER/+; R26MASTR/SmoM2-YPF

(Nes-SmoM2) mice given Tm at P0. (B-C and E-F) are high

magnifications of the yellow dotted boxed areas indicated in A and D. Yellow dotted lines indicate the EGL (E)

and molecular layer (ML). Black and white scale bars indicate 500 and 50 μm, respectively.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 11

Loss of Smo specifically in NEPs results in a lack of expansion and migrate to the EGL after injury.

(A-F) FIHC detection of GFP on cerebellar midsagittal sections at P12 (Lobule IV/V) of control Nes-GFP

Non-IR (solid black) and IR (dashed black) and mutant Nes-Smo CKO Non-IR (solid red) and IR (dashed red)

mice given Tm at P0. Yellow dotted lines outline the different layers. Scale bars indicate 100μm.

Nature Neuroscience: doi:10.1038/nn.4621

Supplementary Figure 12

Model of cellular responses during regeneration of the developing cerebellum.

Depletion of the External Granule Layer (EGL) during the first days of postnatal cerebellar development results

in up-regulation of Hedgehog (HH)-signaling in the Purkinje Cell Layer (PCL), which leads to the expansion

and migration of Nestin-Expressing Progenitors (NEPs) in the PCL that normally produce Astrocytes (Astro)

and Bergmann Glia (Bg). Once in the EGL, NEP-derived cells progressively lose their Neural Stem Cell (NSC)

markers (SOX2 and Nestin), they initiate expression of Granule Cell (GC) lineage-specific genes (Pax6 and

Atoh1) and expand to replenish the EGL. Concomitantly, down-regulation of HH-signaling in White Matter

(WM) NEPs induces a transient reduction of production of interneurons and astrocytes by the WM bipotent

progenitors. Thus, injury of the EGL stimulates a cell-cell communication system that coordinates the responses

of the different NEP populations during recovery, leading to a reset of the postnatal developmental clock of the

cerebellum to re-establish the correct proportions of cerebellar cell types and ensure normal cerebellar circuit

formation.

Nature Neuroscience: doi:10.1038/nn.4621