Genesis of cerebellar interneurons and the prevention of neural DNA damage require XRCC1. Youngsoo Lee, Sachin Katyal, Yang Li, Sherif F. El-Khamisy, Helen R. Russell, Keith W. Caldecott and Peter J. McKinnon. SUPPLEMENTARY MATERIALS: 12 FIGURES AND 1 TABLE.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 2 Supplementary Figure 1. Xrcc1 targeting and generation of Xrcc1 Meox2-cre animals. (a) A detailed schematic diagram of the gene construct used to target Xrcc1. Exons 4 to 10 of the Xrcc1 gene are deleted after Cre excision to generate an out-of-frame mutation, encoding only a small amino-terminal portion of Xrcc1. The region of Xrcc1 deletion corresponds to domains that interact with important DNA repair factors including DNA Polβ, LIG3, and poly(adp-ribose)polymerase (PARP). Exons are indicated as E, and representative restriction enzymes are listed. Red boxes indicate PCR primers for genotyping (see main experimental procedures section for sequence). (b) Southern blot analysis of genomic DNA from Xrcc1 Nes-cre indicating genomic fragments marked as blue in (a). The numbers indicate sizes corresponding to wildtype (5.8kb), the floxed gene (3.4kb) and deleted gene (4kb) after XbaI and NheI digestion of genomic DNA. (c) Xrcc1 LoxP/LoxP ;Meox2-Cre embryos at E9.5 and E11.5 show developmental malformation. (d) Xrcc1 mutants show high levels of apoptosis (arrows). Hematoxylin/eosin (H/E) staining (x2.5) and ssdna immunoreactivity to detect apoptosis (x40) at E9.5 and E11.5. In contrast, Xrcc1 Ctrl (Xrcc1 LoxP/+ ;Nes-cre or Xrcc1 LoxP/LoxP ) showed normal morphology and levels of apoptosis.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 3 Supplementary Figure 2. DNA repair in Xrcc1-deficient primary granule neurons and cortical astrocytes. (a) Xrcc1-null cerebellar granule neurons are deficient in the repair of DNA damage induced by the alkylating agent methyl methanesulfonate (MMS). Cells were treated with increasing concentrations of MMS for 10 minutes followed by analysis using the alkali comet assay (ACA). Comet data was compiled from 100 individual neurons and is plotted as the mean comet tail moment. (b) Xrcc1-null cortical astrocytes have defective repair of DNA damage. Quiescent astrocytes were subjected to ACA analysis after treatment with DNA damaging agents (treated at 37 o C unless indicated): 14µM camptothecin (CPT) for 60 minutes (red); 0.20mg/ml MMS for 10 minutes (green); 150µM hydrogen peroxide (H 2 O 2 ) for 5 minutes on ice (dark blue) followed by recovery at 37 o C (R30, blue; R60, light blue). Comet data were compiled from 50 individual cells and are plotted as the mean comet tail moment. (c) Quiescent confluent astrocytes were triple-stained with GFAP, PCNA and DAPI; a lack of PCNA positive cells confirms quiescence and PCNA immunostaining of cycling astrocytes (inset panel) serves as a positive control for proliferation in this cell type.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 4 Supplementary Figure 3. DNA damage is present in the Xrcc1 Nes-Cre brain. (a) Abundant DNA damage during early neural development in Xrcc1 Nes-cre embryos is shown by γh2ax immunostaining and is associated mainly with proliferating regions; γh2ax foci are almost absent in similar regions of control embryos. The developing forebrain of Xrcc1 Nes-cre embryos at E13.5 and E18.5 is shown; higher magnification illustrates individual γh2ax foci. (b) Co-localization of γh2ax and 53BP1 foci in cells of the adult Xrcc1 Nes-cre brain are shown for the cerebellum, cerebral cortex (CTX), dentate gyrus (DG) and CA1 of the hippocampus, and cerebellum. In contrast, no γh2ax foci are apparent in control brains, and only pan-nuclear staining of 53BP1 occurs, typical of unstressed nuclei. PC are Purkinje cells.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 5 Supplementary Figure 4. Xrcc1 loss does not markedly affect the spinal cord, dorsal root ganglia or the trigeminal ganglia. Analysis of other Xrcc1 Nes-cre brain regions including the spinal cord (a) and peripheral nervous system regions including the trigeminal ganglia (TGG; b) and the dorsal root ganglia (DRG) were indistinguishable from controls, as judged by immunohistochemistry. The spinal cord was analyzed using 4-week-old animals, while analysis of the TGG and DRG were from E13.5 or E18.5 tissue as indicated. Differentiation markers were Tuj1 (Tubulin βiii) and superoxide dismutase 2 (SOD2), proliferation marker was Ki67 and apoptotic markers were TUNEL and activated Caspase 3 (Casp3).
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 6 Supplementary Figure 5. Analysis of interneuron networks throughout the Xrcc1 Nes-cre brain. Normal distribution of somatostatin (a), calretinin (b), parvalbumin (c) and neuropeptide Y (NPY; d) immunopositive interneurons were found in the mature Xrcc1 Nes-cre brain (arrows). No differences were found in the distribution or numbers of immunopositive cells in 2 month old brains of all animals examined, indicating that loss of interneurons in the Xrcc1 Nes-Cre brains is restricted to the cerebellum. Arrows indicate immunopositive interneurons. Scale bar is 200µm.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 7 Supplementary Figure 6. Visualization of neuronal morphology using Golgi staining. Axons in control and mutant brains were visualized using Golgi-Cox impregnation (using FD Rapid GolgiStain kit, FD NeuroTechnologies, according to the manufacturer s directions). Staining in the cerebral cortex and the cerebellar molecular layer and deep nucleus is shown. Dendritic arborization was comparable between Xrcc1 Ctrl and Xrcc1 Nes-Cre brains. Insets show examples of Purkinje cells. Although few interneurons in the molecular layer of the Xrcc1 Nes-Cre cerebellum were present, they appeared relatively normal (red arrows). Photomicrographs are at x40 magnification.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 8 Supplementary Figure 7. Proliferation is normal in brain regions outside of the cerebellum. Except for the cerebellum, normal indices of proliferation were found throughout the Xrcc1 Nes-cre brain. (a) PCNA immunopositive cells in the neocortical subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus at different time points (P0-P7) during postnatal development are similar between Xrcc1 Ctrl and Xrcc1 Nes-cre tissue (mag. x40). (b) Proliferation in the external granule cell layer (EGL) was similar between Xrcc1 Nes-Cre and Xrcc1 Ctrl tissue at different time points (P0-P7) during postnatal development (mag. x40). (c) A significant reduction of PCNA immunoreactivity was found in the white matter of the developing Xrcc1 Nes-Cre cerebellum (arrows) between postnatal days 0 to 7 (mag. x40). Scale bars are 200µm.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 9 Supplementary Figure 8. Normal embryonic Xrcc1 Nes-Cre cerebellar development. Normal cerebellar development was found in the Xrcc1 Nes-Cre embryos during embryogenesis. Representative photomicrographs are shown of the developing cerebellum at E18.5 from Xrcc1 Ctrl and Xrcc1 Nes-Cre embryos. Nissl and Ki67 staining to identify proliferating cells and Tuj1 staining to identify differentiating neurons revealed no apparent differences between Xrcc1 Nes-cre and controls.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 10 Supplementary Figure 9. Comparative analysis of progenitor cells in the ventricular zone of Xrcc1 Nes-cre embryos. Analyses of neural progenitors, assessed by Sox2 immunostaining, throughout proliferative ventricular zone regions at E13.5 or E18.5 in Xrcc1 Nes-cre embryos were similar to those found in control littermates. An increase in TUNEL positive cells was observed in the Xrcc1 Nes-cre embryonic forebrain (arrows). Sox2 (rabbit, 1;1000, Millipore) was used without antigen retrieval. (Mag. x40).
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 11 Supplementary Figure 10. Pax2-expressing interneurons are lost in the developing Xrcc1 Nes-cre cerebellum. Compared to Xrcc1 Ctrl cerebellum, the analysis of late embryonic and early postnatal development in the Xrcc1 Nes-cre cerebellum shows a profound reduction in numbers of Pax2-positive interneurons (arrows) occurs from birth (P0). Mag. x20.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 12 Supplementary Figure 11. Analysis of GABAergic networks throughout the Xrcc1 Nes-cre nervous system. Distribution of GABAergic cells as determined by GAD67 immunoreactivity was normal in most of the adult Xrcc1 Nes-cre brain at 2 month of age, including the retina, the cerebral cortex and the hippocampus. However, loss of GAD67 immunoreactivity was noted in the molecular layer of the Xrcc1 Nes-cre cerebellum (white arrows). Inactivation of p53 restored the GABAergic network in the molecular layer of the Xrcc1-deficient cerebellum. All photomicrographs are at mag. x40.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 13 Supplementary Figure 12. Xrcc1 Nes-cre ;p53 -/- mice develop medulloblastoma. (a) Hematoxylin and eosin staining (H&E) indicates the typical morphology of classic medulloblastoma, while immunostaining shows the characteristic neuronal identity of the tumor together with the presence of DNA damage, proliferation and apoptosis. Tumors were immunopositive for typical markers of medulloblastoma such as synaptophysin, but not for calbindin or the GABA neurotransmitter marker GAD67. The dashed lines demarcate normal tissue from medulloblastoma. (b) While p53 loss slightly extended survival of Xrcc1 Nes-cre mice, all Xrcc1 Nes-cre ;p53 -/- animals examined developed medulloblastoma. Kaplan-Meier curves are those from Figure 1 with additional survival data for Xrcc1 Nes-cre ;p53 -/-, Xrcc1 Nes-cre ;p53 +/- and p53 -/- animals. (c) Medulloblastomas (arrows) arise from cerebellar granule neuron precursors as shown by intercrossing Xrcc1 Nes-cre ;p53 -/- mice with Math1-GFP mice to generate Xrcc1 Nes-cre ;p53 -/- ;Math1-GFP animals and tumors that express GFP.
Lee et al, Genesis of cerebellar interneurons SUPPLEMENTARY DATA 14