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Cerebellar granule cell replenishment postinjury by adaptive reprogramming of Nestin+ progenitors

Nature Neuroscience volume 20, pages 1361–1370 (2017)Cite this article

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Abstract

Regeneration of several organs involves adaptive reprogramming of progenitors, but the intrinsic capacity of the developing brain to replenish lost cells remains largely unknown. Here we found that the developing cerebellum has unappreciated progenitor plasticity, since it undergoes near full growth and functional recovery following acute depletion of granule cells, the most plentiful neuron population in the brain. We demonstrate that following postnatal ablation of granule cell progenitors, Nestin-expressing progenitors, specified during mid-embryogenesis to produce astroglia and interneurons, switch their fate and generate granule neurons in mice. Moreover, Hedgehog signaling in two Nestin-expressing progenitor populations is crucial not only for the compensatory replenishment of granule neurons but also for scaling interneuron and astrocyte numbers. Thus, we provide insights into the mechanisms underlying robustness of circuit formation in the cerebellum and speculate that adaptive reprogramming of progenitors in other brain regions plays a greater role than appreciated in developmental regeneration.

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Figure 1: Irradiation of cerebella at P1 results in a major loss of the EGL by P3, but growth largely recovers and motor behavior is intact at P30.
Figure 2: The EGL is progressively replenished after injury.
Figure 3: NEPs populate the EGL and generate granule cells and rescue growth.
Figure 4: PCL NEPs expand and migrate into the EGL after injury.
Figure 5: WM NEPs delay their expansion and production of interneurons and astroglia.
Figure 6: SHH signaling is required for NEPs to expand and populate the EGL and for substantial recovery of the cerebellum.

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Acknowledgements

We thank Z. Yang for providing the Nestin-CFP line and for discussions of unpublished data. We are grateful to B. Palikuqi (in S. Rafii's lab) for technical support with initial Nes-CFP FACS and R. Sillitoe, T. Stay and E. Lackey for their advice with performing the behavior tests. We thank K. Zaret for thoughtful comments on the manuscript, and past and present members of the Joyner laboratory for discussions. We also thank the Flow Cytometry, Bio-informatics, Center for Comparative Medicine and Pathology, and Integrated Genomics Operation (IGO) core facilities of MSKCC for outstanding technical support. We also gratefully acknowledge P. Zanzonico for his help with mouse irradiation and B. Nieman for advice; Q. Chen and the MSKCC Small-Animal Imaging Core Facility for technical services; and a Shared Resources Grant from the MSKCC Geoffrey Beene Cancer Research Center, which provided funding support for the purchase of the XRad 225Cx Microirradiator. This work was supported by grants from the Brain Tumor Center at MSKCC (to A.W.), National Brain Tumor Association (to A.L.J.) and NINDS (R01 NS092096 to A.L.J. and F32 NS086163 to A.K.L.), as well as a National Cancer Institute Cancer Center Support Grant (P30 CA008748-48).

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Authors and Affiliations

  1. Developmental Biology Program, Sloan Kettering Institute, New York, New York, USA

    Alexandre Wojcinski, Andrew K Lawton, N Sumru Bayin, Zhimin Lao, Daniel N Stephen & Alexandra L Joyner

  2. Biochemistry, Cell and Molecular Biology Program, Weill Cornell Graduate School of Medical Sciences, New York, New York, USA

    Alexandra L Joyner

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Contributions

A.W. and A.L.J. conceived the project; A.W., A.K.L., N.S.B. and A.L.J. designed the research; A.W., A.K.L., N.S.B., Z.L. and D.N.S. performed the experiments; A.W., A.K.L., N.S.B. and A.L.J. analyzed the data and all authors discussed the data; A.W. and A.L.J. wrote the manuscript with contributions from all authors.

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Integrated supplementary information

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.

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.

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. 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.

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 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.

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.

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; 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.

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.

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.

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).

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). Scale bars indicate 100βm. (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.

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.

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.

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Supplementary Figures 1–12 (PDF 4975 kb)

Life Sciences Reporting Summary (PDF 142 kb)

Supplementary Table 1

Statistical details for figure legends (XLSX 17 kb)

Supplementary Table 2

Differentially expressed genes in NEPs from P5 Non-IR et IR cerebella. (XLSX 58 kb)

P6 Non-IR cerebellum shows no obvious movement of NEPs to the EGL.

Detection of native CFP fluorescence on sagittal slices of the vermis (lobule 4/5) of P6 Non-IR Nes-CFP/+ mice showing displacement of CFP+ cells. Image stacks were acquired every 3min for 5h 45min. (MOV 15867 kb)

NEPs are highly motile after irradiation.

Detection of native CFP fluorescence on sagittal slices of the vermis (lobule 4/5) of P6 IR Nes-CFP/+ mice showing movement of CFP+ cells within the EGL and between the Purkinje Cell Layer (PCL) and EGL. Image stacks were acquired every 3min for 5h 45min. (MOV 21908 kb)

PCL NEPs migrate towards the EGL after irradiation.

Magnification of native CFP fluorescence detection on sagittal slices of the vermis (lobule 4/5) of P6 IR Nes-CFP/+ mice showing movement of CFP+ cells into the EGL. Image stacks were acquired every 3min for 5h 45min. Green and purple dots indicate cells located initially in the Purkinje Cell Layer (PCL) and Molecular Layer (ML) respectively. (AVI 10374 kb)

Adult Non-IR Nes-Smo CKO mice have normal motor behavior.

Video showing motor behavior of 5-6 week old Nes-FlpoER/+;R26FSF-GFPcre/+;Smoflox/flox (Nes-Smo CKO) Non-IR mouse administered with Tm at P0. (MP4 18828 kb)

Adult IR Nes-Smo CKO mice show impaired motor behavior.

Video showing motor behavior of 5-6 week old Nes-FlpoER/+;R26FSF-GFPcre/+;Smoflox/flox (Nes-Smo CKO) mouse administered with Tm at P0 and irradiated at P1. (MP4 17017 kb)

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Wojcinski, A., Lawton, A., Bayin, N. et al. Cerebellar granule cell replenishment postinjury by adaptive reprogramming of Nestin+ progenitors. Nat Neurosci 20, 1361–1370 (2017). https://doi.org/10.1038/nn.4621

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