Apoptosis after 2 Gy ionising radiation (IR) only arises in the 2 subdomains containing progenitor cells.

<p>(A) Scheme showing the lineage of neural stem cells (NSCs) within the subventricular zone (SVZ). Glial Fibrillary Acidic Protein (GFAP)⁺ neural stem cells (NSCs; light blue) are predominantly quiescent. 5%–10% are activated, however, and can produce mammalian Achaete-scute homologue 1 (Mash1)⁺ transit amplifying progenitors (TAPs; green), which in turn give rise to doublecortin⁺ (Dcx⁺) neuroblasts (NBs; red). 50% of NBs are proliferative. NSCs, TAPs, and NBs are also called type B, C, and A cells, respectively. (B) Graphical illustration showing the subdomain structure around the lateral ventricle. The lineage of cells arising from these subdomains is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s001" target="_blank">S1A Fig</a>. The colour code (black, ventral; blue, medial; orange, dorsal; yellow, dorsolateral) will be used throughout. (C) Quantification of GFAP⁺, Mash1⁺, Dcx⁺, and Ki67⁺ cells within the 4 subdomains of untreated control mice. In this and all subsequent figures, the percentages are normalised to the number of 4′,6-diamidino-2-phenylindole (DAPI) positive cells. (D) Images showing GFAP⁺ cells (magenta), Ki67⁺ cells (red), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)⁺ cells (green), and DAPI staining (grey) within the 4 subdomains. Separate colour channels are given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s001" target="_blank">S1E Fig</a>. Apoptosis is only observed in the dorsolateral and ventral subdomains. (E) Quantification of TUNEL⁺ cells in the entire lateral ventricle (LV) in control and irradiated mice at 6 h post 2 Gy. (F) Quantification of TUNEL⁺ cells within each subdomain at 6 h post 2 Gy. Experiments were carried out on 3-month-old mice and represent the mean ± SEM of <i>n</i> ≥ 3 mice for each condition. Scale bars, 25 μm. Student <i>t</i> test, *** <i>P</i> < 0.001. Underlying data can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s007" target="_blank">S1 Data</a> file.</p

Arrest of proliferation and progenitor marker loss are additional DNA damage responses to 2 Gy ionising radiation (IR).

<p>(A) Apoptosis ceases by 48 h post 2 Gy. Apoptosis in the lateral ventricle (LV) was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) staining at 6 h and 48 h post 2 Gy and quantified as the total number of TUNEL⁺ cells per LV. (B-C) Proliferation (Ki67) and the neurobloast (NB) marker (doublecortin [Dcx]) are diminished by 48 h post 2 Gy. The quantification of Ki67⁺ and Dcx⁺ cells in the ventral (panel B) and dorsolateral (panel C) subdomains at 6 h and 48 h post 2 Gy. Grey columns, Ki67; red columns, Dcx. (D) A schematic showing the protocol for bromodeoxyuridine (BrdU)/ethynyl deoxyuridine (EdU) labelling of replicating cells post 2 Gy. (E) The assessment of percentage of BrdU⁺ or EdU⁺ cells in the ventral and dorsolateral subdomains in unirradiated control mice or at 6 h following 2 Gy. (F) The representative images from the experiment shown in panel D-E. (G-I) Dose-response analysis of apoptosis and Ki67⁺ cells, respectively. Adult mice were exposed to the indicated dose of IR and quantified for apoptosis (TUNEL) in the LV (panel G) or Ki67⁺ cells in the ventral or dorsolateral subdomains (panel H) at 6 h post IR. Panel I shows representative images. For panels G and H, black and yellow columns show analysis of the ventral and dorsolateral subdomains, respectively. Experiments were carried out on 3-month-old mice and results represent the mean ± SEM of <i>n</i> ≥ 3 mice for each condition. Scale bars, 25 μm. Student <i>t</i> test, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, ns = not significant. Underlying data can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s007" target="_blank">S1 Data</a> file.</p

DNA damage responses in the neural stem cells (NSCs).

<p>(A-B) Activated Glial Fibrillary Acidic Protein (GFAP)⁺ stem cells do not undergo apoptosis. Panel A shows the protocol for quantifying apoptosis in activated stem cells. In brief, 3-month-old adult mice are exposed to 2 Gy, and, 7 days later, given a second exposure to 2 Gy, then the level of GFAP⁺ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)⁺ cells out of the total GFAP⁺ cells were quantified 6 h later in the ventral (black) or dorsolateral (yellow) subdomains. The level of activated NSCs (GFAP⁺Ki67⁺) was about 4-fold higher at 7 days after the first exposure (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.g005" target="_blank">Fig 5C</a>), but the level of apoptosis after the second exposure was similar to that observed after a single exposure to 2 Gy. (C-D) Neural stem and progenitor cells show similar levels of double-strand break (DSB) induction and repair. Three-month-old adult mice were exposed to 100 mGy or 2 Gy, and p53-binding protein 1 (53BP1) foci (a DSB marker) were quantified 0.5 h and 6 h later in doublecortin (Dcx⁺) and GFAP⁺ cells. Panel D shows representative images. We have previously observed one-third the level of 53BP1 foci in our tissue analysis compared with that found using cultured fibroblasts [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.ref024" target="_blank">24</a>]. The numbers observed here are consistent with this (approximately 25 foci are observed at 0.5 h post 2 Gy in cultured fibroblasts). The reduced efficiency of DSB detection in vivo could be a consequence of a failure to detect all DSBs throughout the depth of the cell in a tissue section. (E) Stem cells do not show senescence at 7 days post 2 Gy. Senescence was assessed using β-galactosidase (SA-β-Gal) and nuclear fast red (NFR) as counterstain in individual subdomains of the lateral ventricle (LV). SA-β-Gal⁺ cells were observed endogenously in the kidney of aged (21-month-old) mice. For quantification, <i>n</i> = 2 mice for each condition were analysed. Experiments in A-D were carried out on 3-month-old mice, and results represent the mean ± SEM of <i>n</i> ≥ 3 mice for each condition. Student <i>t</i> test, ns = not significant. Underlying data can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s007" target="_blank">S1 Data</a> file.</p

Fifty percent of the progenitor cells undergo apoptosis after 2 Gy regardless of proliferative status.

<p>(A) The percentage of Ki67⁺ cells of each cell type analysed (Glial Fibrillary Acidic Protein [GFAP]⁺, mammalian Achaete-scute homologue 1 [Mash1]⁺ and doublecortin⁺ [Dcx⁺]) in the ventral and dorsolateral subdomains of the subventricular zone (SVZ). Only low numbers of Ki67⁺ cells were detected in the medial and dorsal subdomains (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.g001" target="_blank">Fig 1C</a>). (B) The percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)⁺ cells of each cell type analysed at 6 h post 2 Gy. (C) A scheme showing the protocol to assess percentage of replicating cells undergoing apoptosis using bromodeoxyuridine (BrdU) incorporation to assess replication. Mice were given 2 injections of BrdU at 4 h and 1 h prior to exposure to 2 Gy IR and assessed for apoptosis (cleaved caspase-3 [Casp3⁺]) and/or BrdU incorporation 6 h later. (D) The quantification of BrdU⁺Casp3⁺ cells out of the total BrdU⁺ cells. Assessment of Casp3⁺BrdU⁺ cells out of the total Casp3⁺ cells is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s002" target="_blank">S2B Fig</a>. (E) The representative images of labelled cells for the experiment shown in panels C-D. Black and yellow columns show analysis of the ventral and dorsolateral subdomains, respectively. Experiments were carried out on 3-month-old mice and represent the mean ± SEM of <i>n</i> ≥ 3 mice for each condition. Scale bars, 25 μm. Student <i>t</i> test, ns = not significant. Underlying data can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s007" target="_blank">S1 Data</a> file.</p

DNA damage-induced neuronal differentiation.

<p>(A-B) The quantification of the percentage of neuron-specific class III β-tubulin(Tuj1)⁺ and microtubule-associated protein 2 (MAP2)⁺ cells in the ventral and dorsolateral subdomains of control and irradiated mice at 48 h post 2 Gy. (C) The representative images for the experiment shown in panel A, showing loss of Tuj1⁺ cells at 48 h following IR. (D) A schematic showing the protocol for bromodeoxyuridine (BrdU) labelling. (E) Percentages of BrdU⁺ cells in the ventral and dorsolateral subdomains in unirradiated control mice and at 48 h following 2 Gy. (F) Percentages of Dcx⁺BrdU⁺, Tuj1⁺BrdU⁺, and MAP2⁺BrdU⁺ cells out of the total BrdU⁺ cells in the ventral and dorsolateral subdomains in unirradiated control mice and at 48 h following 2 Gy. For this analysis, >50 BrdU⁺ cells were scored per mouse from 3 mice per treatment condition. For the irradiated group, this required cell scoring on at least 10 tissue sections per mouse. (G) The representative images for the experiment shown in panel F. Insets magnify cells indicated in the dashed box showing the presence of double-labelled BrdU⁺MAP2⁺ cells at 48 h after 2 Gy. Experiments were carried out on 3-month-old mice, and results represent the mean ± SEM of <i>n</i> ≥ 3 mice for each condition. Scale bars, 25 μm. Student <i>t</i> test, * <i>P</i> < 0.05, ns = not significant. Underlying data can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s007" target="_blank">S1 Data</a> file.</p

Neonatal mice do not efficiently arrest proliferation after 2 Gy.

<p>(A-C) Neonatal (P5) mice show enhanced proliferative cells and reduced Glial Fibrillary Acidic Protein (GFAP)⁺ cells in each subdomain relative to adult mice. The percentage of Ki67⁺ cells (panel A) and GFAP⁺ cells (panel B) in each lateral ventricle subdomain were quantified in P5 and adult mice. Panel C shows representative images of control mice at P5. (D) The level of apoptosis per lateral ventricle (LV) is enhanced in P5 compared to adult mice. (E) The level of apoptosis per cell is similar in the ventral and dorsolateral subdomains in P5 and adult mice. Apoptosis is assessed as the percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL)⁺ cells out of the total 4′,6-diamidino-2-phenylindole (DAPI)⁺ cells in each subdomain. (F) Representative images of P5 mice from the experiment shown in panel E. (G) Proliferation is not efficiently arrested in P5 mice post 2 Gy. The percentage of Ki67⁺ cells were quantified in each subdomain at P5, P7, and P9 mice either with or without exposure to 2 Gy on P5 as indicated. P5, P7, and P9 respectively correspond to 6 h, 48 h, and 4 days post irradiation at P5. In panel G, Student <i>t</i> tests were performed relative to the control group at P5. (H) The level of GFAP⁺Ki67⁺ cells out of the total GFAP⁺ cells were assessed at the specified times post 2 Gy in each subdomain as indicated. The results and Student <i>t</i> tests are expressed relative to the level in untreated mice of the same age. Experiments were carried out on P5, P7, and P9 mice, and results represent the mean ± SEM of <i>n</i> ≥ 3 mice for each condition. cc, corpus callosum. Str, striatum. Scale bars, 25 μm. Student <i>t</i> test, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, ns = not significant. Panels C and F were originated from 4-channel images. Underlying data can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s007" target="_blank">S1 Data</a> file.</p

Temporal analysis of the response to 2 Gy within distinct subdomains of the lateral ventricle.

<p>(A-D) Adult mice were exposed to 2 Gy and the percentage of Ki67⁺ (panel A), Glial Fibrillary Acidic Protein (GFAP)⁺ (panel B), GFAP⁺Ki67⁺ (panel C), and doublecortin (Dcx⁺) (panel D) cells assessed in the ventral or dorsolateral subdomains as indicated. (E-F) The quantification (panel E) of the percentage of GFAP⁺Ki67⁺ cells in all subdomains and representative images (panel F) at 7 days after 2 Gy. (G-H) The quantification (panel G) of the percentage of Dcx⁺Ki67⁺ cells amongst the Dcx⁺ cells in the ventral and dorsolateral subdomains of unirradiated control mice and at 7 and 14 days post 2 Gy ionising radiation (IR) and representative images (panel H). Experiments were carried out on 3-month-old mice and results represent the mean ± SEM of <i>n</i> ≥ 3 mice for each condition. Scale bars, 25 μm. Student <i>t</i> test was performed relative to the control group, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001, ns = not significant. Underlying data can be found in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001264#pbio.2001264.s007" target="_blank">S1 Data</a> file.</p

ORC1 Meier-Gorlin syndrome and IFT43 Sensenbrenner syndrome fibroblasts exhibit impaired chondroinduction.

<p>a–b) Phase contrast images (40×) of control (C) hTERT, ORC1-deficient MGS (ORC1-P4hTert) and <i>IFT43</i>-mutated Sensenbrenner (IFT43) patient derived fibroblasts at 0 hr and 24 hr following addition to aggrecan coated plates. Size distribution of aggregates from control (C), ORC1 and IFT43 fibroblasts following 24 hr micromass culture in aggrecan coated plates (n = 350 aggregates scored per line). Larger aggregate size was a feature of control fibroblasts following chondroinduction compared to ORC1 and IFT43 cells. c–d) Semi-quantitative RT-PCR amplification of <i>VEGFA</i> isoform a (upper band) and isoform c (lower band) either uninduced (Und) or during chondroinduction. Both isoforms were induced in control fibroblasts (C) upon chondroinduction. Whilst IFT43 cells exhibited higher endogenous levels of <i>VEGFA</i> isoform c, it was not maintained upon chondroinduction. Isoform a also was not induced after chrondroinduction. Similar findings were observed for ORC1 cells, although the high endogenous level of isoform c reduced less dramatically than that in IFT43 cells but did not increase in as in control cells. <i>ELP4</i> was used as an amplification control. Panel (d) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in three independent experiments. e) Type I collagen represents a negative marker for chondroinduction as its levels decrease as differentiated chondrocytes secrete a specific extracellular matrix. Consistent with this, <i>COL1A1</i> levels, as monitored by quantitative RT-PCR were found to decrease in control fibroblasts (C) during chondroinduction. Interestingly, both ORC1 and IFT43 defective patient derived cells exhibited similar levels of endogenous <i>COL1A1</i> compared to control but by 48 h, the levels had less dramatically diminished compared to control cells. The results represent the mean of three experiments. f–g) Analysis of a control hTERT cell line treated with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for <i>ORC1, 4, 6, CDC6</i> or <i>CDT1</i>. Cells were uninduced (Und) or induced on a chondrogenic matrix then assayed for <i>VEGFA</i> expression as detailed in (c–d). Panel (g) shows the combined quantification for isoforms a and c from the above panel. Similar findings have been observed in two independent experiments.</p

Deficiency in origin licensing proteins dramatically impairs cilia formation.

<p>a–b) Control (C) or ORC1-deficient cells were induced to enter G0 by serum starvation for 24 or 48 hr and processed to identify cilia using anti-acetylated tubulin and anti-γ-tubulin antibodies to mark the entire cilia or the basal body, respectively. Lower panel shows that in ORC1-hTERT fibroblasts immunostaining with α-acetylated tubulin reveals extended perinuclear microtubular arrays around the centrosome in distinction to the ordered alignment in control cells and as reported for other cilia defective cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360-Mill1" target="_blank">[56]</a>. c) Control (C) or ORC1-deficient hTERT cells were monitored for long term cilia formation as above after the indicated numbers of days of serum depletion. d) Origin licensing proteins were knocked down with siRNA in control hTERT cells, serum starved for 24 hrs then analysed for cilia formation as above. Although a marked defect is observed in cilia formation up to 48 h post serum starvation, cilia can form in around 50% of the cells when examined 4–5 days post serum starvation. e) ORC1-P4 hTERT cells were transfected with empty plasmid or plasmid expressing GFP-tagged <i>ORC1</i> cDNA and positive cells detected with anti-GFP antibodies. The percent of GFP⁺ cells, representing those that have been successfully transfected, with cilia was assessed as in panel (d). <i>ORC1</i> cDNA expression resulted in rescue of the defect in cilia formation. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen.1003360.s002" target="_blank">Figure S2a</a> shows cilia formation in a gfp⁺ versus gfp⁻ cells.</p

Deficiency in origin licensing proteins impairs cilia function in response to platelet-derived growth factor (PDGF).

<p>a–b) Fibroblasts were induced to enter G0 phase following serum depletion for 48 hr. PDGF-AA or –BB and BrdU was then added and the percentage of S phase cells, monitored as BrdU⁺ cells, was estimated by immunofluorescence 11 (a) and 24 hr (b) later. The receptor for PDGF-AA is located in cilia whilst the –BB receptor is on the cell membrane. c) Analysis as in a) following the indicated siRNA. d) Cellular localisation of PDGFR-α or β. Anti-PDGFR-α or –β antibodies were used to examine the localisation of the two PDGF isoforms in control (C) or ORC1-P4 hTERT cells. PDGFR-α localised to the cilia, identified using anti-acetylated tubulin in control and ORC1-P4 cells although fewer cilia formed in the latter cells. PDGFR-β showed pan cellular localisation but did not co-localise with the cilium. e) Cells were induced to enter G0 phase following serum depletion for 48 hr. Serum was then re-added and the fraction of BrdU⁺ S phase cells monitored at the indicated times. The top panel shows the results with a control (C) primary fibroblasts, 48BR, primary fibroblasts from Sensenbrenner syndrome patients (<i>IFT43</i>-mutated and <i>WDR35</i>-mutated), PCNT defective fibroblasts and an ORC1 deficient line MGS cells. Both Sensenbrenner syndrome lines and PCNT cells showed a delayed S phase entry, similar to ORC1 defective MGS, compared to the control primary line. f) Analysis of a control hTERT immortalised cell line either without knockdown (C), treatment with control siRNA oligonucleotides (siC) or with oligonucleotides specific (si) for <i>ORC1, ORC4, ORC6, CDC6</i> or <i>CDT1</i>. Knockdown efficiency was assessed and was similar to that observed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003360#pgen-1003360-g002" target="_blank">Figure 2</a>. Note that the control hTERT immortalised line (C) enters S phase more rapidly that the primary fibroblasts line making it difficult to allow a direct comparison between the S phase entry kinetic defects observed in the Sensenbrenner syndrome primary lines in (e).</p