Microarray-based CGH analysis of MB samples.

<p>(A–B) Alteration frequency plots of <i>Ptch1^+/− Nos2^−/−</i> and <i>Ptch1^+/− Nos2^+/+</i> tumors show a common trisomy of chromosome 6 and deleted regions around the <i>Ptch1</i> locus (chromosome 13), green: gains, red: losses. Arrows denote frequent aberrations, black: common, white: differential. Color shading indicates percentage of available signals for a respective oligonucelotide (% of samples), light: high percentage, dark: low percentage. Concurrent gains and losses of X and Y chromosomes, or <i>vice versa</i>, reflect sample against reference hybridizations of different gender. The most consistent difference between MBs of both genotypes affected a small region on chromosome 14 harboring the <i>Entpd4</i> gene. (C) QRT-PCR of <i>Entpd4</i> showed no significant difference in an expanded set of tumor samples. Relative expression values are normalized to housekeeping genes. Error bars reflect SEM (standard error of the mean).</p

Characteristics and functional implication of Gap43 expression in cell culture.

<p>(A–B) Gene expression of <i>Gap43</i> was reduced upon inhibition of NO synthases. Expression values were obtained from qRT-PCR measurements and indicate linear expression values normalized to a pool of housekeeping genes. Error bars reflect SEM of three replicates. (A) In c17.2 cells, <i>Gap43</i> expression is significantly decreased upon L-NAME treatment after 120 hours (*p = 0.0234). (B) In D458 cells, <i>Gap43</i> expression is significantly decreased upon L-NAME treatment after 72 hours (***p<0.0001). (C–D) Functional analyses were performed after knockdown of <i>Gap43</i> in neuronal progenitor cells (c17.2). (C) <i>Gap43</i> and <i>Ptch1</i> showed inverse gene expression behavior measured by qRT-PCR and normalized to non-target control. (D) Cells exhibited reduced migration upon decrease of Gap43 protein levels. The percentage of migrated cells was normalized to non-target control. Significant decrease of migration in knockdown samples is indicated by asterisks (*p = 0.013, **p = 0.007). Sh39, sh42: anti-<i>Gap43</i> target shRNA, shGFP: control shRNA against GFP, shNT: non-target control shRNA.</p

Microarray-based gene expression profiling.

<p>(A) Hierarchical cluster analysis of normal cerebellar tissue and MB samples showed a clear separation of <i>Ptch1^+/+ Nos2^−/−</i> P9 cerebella. (B) Heat map of proliferation-associated genes downregulated in P9 cerebella of <i>Ptch1^+/+ Nos2^−/−</i> versus wild-type mice. Depicted samples include postnatal cerebellar tissue samples of each genotype and all MB cases. Values are normalized to gene-wise average for better visualization. (C) Gene expression of <i>Ptch1</i> was increased in P9 cerebellar tissue samples of <i>Nos2</i>-deficient mice. <i>Ptch2</i> was increased over <i>Ptch1</i> expression only in <i>Ptch1^+/− Nos2^−/−</i> P9 cerebella and MBs. Values are indicated as log2 ratios of sample against Universal Reference RNA (Stratagene). Error bars reflect SEM. AU: approximately unbiased p-values by multiple bootstrap resampling [%], BP: boostrap probability p-values by normal bootstrap resampling [%]. MB: medulloblastoma, CB: cerebellum, P9: postnatal day 9, 6W: 6 weeks after birth, 1Y: 1 year after birth.</p

<i>Nos2</i> deficiency increases the incidence of MB in <i>Ptch1</i>^+/− mice.

<p>(A) Kaplan-Meier analysis of MB incidence in 215 <i>Ptch1</i>^+/−<i>Nos2</i>^+/+ mice (orange line) versus 221 <i>Ptch1</i>^+/−<i>Nos2</i>^−/− mice (red line). <i>Ptch1</i>^+/−<i>Nos2</i>^−/− mice demonstrated an approximately two-fold increase in MB incidence (p = 0.0007, Logrank test). None of 315 wild-type (green line) and 412 <i>Ptch1</i>^+/+<i>Nos2</i>^−/−mice (blue line) (control littermates) developed any MB. Vertical ticks represent censored mice. (B–E) Histological features of MBs from <i>Ptch1</i>^+/−<i>Nos2</i>^+/+ and <i>Ptch1</i>^+/−<i>Nos2</i>^−/− mice. MBs in both genotypes (B–C, <i>Ptch1</i>^+/−<i>Nos2</i><b>^+/+</b>; D–E, <i>Ptch1</i>^+/−<i>Nos2</i>^−/−) were densely cellular primitive neuroectodermal tumors of the cerebellum corresponding histologically to the classic subtype of human MB. Hematoxylin eosin-stained sections showed no obvious differences between the genotypes concerning gross growth pattern, with well demarcated growth in the cerebellar cortex (B, D, scale bar = 500 µm), and cellular morphology (C, E, scale bar = 50 µm).</p

Identification of <i>Nos2</i>-regulated candidate genes.

<p>(A) The overlap between three group comparisons of expression profiles revealed <i>Gap43</i> and <i>Stmn1</i> as commonly deregulated by <i>Nos2</i> inactivation according to the microarray data. (B) <i>Gap43</i> gene expression was <i>Nos2</i>-dependent during different developmental stages of cerebellar development. <i>Nos2</i>-sufficient: wild-type and <i>Ptch1^+/− Nos2^+/+</i>, <i>Nos2</i>-deficient: <i>Ptch1^+/+ Nos2^−/−</i> and <i>Ptch1^+/− Nos2^−/−</i>. (C) <i>Gap43</i> was differentially expressed between <i>Ptch1^+/− Nos2^+/+</i> and <i>Ptch1^+/− Nos2^−/−</i> MB samples. Values in (B) and (C) were obtained from the microarray data and indicate log2 ratios of sample against Universal Reference RNA (Stratagene). (D) Differential <i>Gap43</i> gene expression was confirmed by qRT-PCR in an expanded tumor sample set (n = 7 per genotype). Linear expression values are normalized to housekeeping genes. Significant expression differences between groups are indicated by asterisks (**p<0.01). Error bars reflect SEM. (E) Immunofluorescent co-staining of Gap43 (green) and Ki-67 (red) on FFPE sections from P9 cerebella. Blue: DAPI-stained nuclei. Overview sections (upper left corner) were acquired by wide-field microscopy and detail sections represent confocal laser scanning microscopy images. Intense Gap43 signal was observed in wild-type and <i>Ptch1^+/− Nos2^+/+</i> cerebella, in particular at the outer ML, scale bar = 50 µM. (F) Quantification of Gap43 staining showing intensity histograms of the EGL and ML area (upper panel), as well as intensity distribution of the different genotypes with subtracted wild-type signal (lower panel). Black arrow indicates a common peak of nucleus background signal. EGL: external granule layer, ML: molecular layer, and IGL: internal granule layer.</p

Proliferation and accumulation of GCPs in postnatal cerebellum.

<p>(A) Immunofluorescent co-staining of NeuN (green) and Ki-67 (red) on FFPE sections from P9 cerebella showed an increased accumulation of proliferating GCP in the EGL of <i>Ptch1^+/− Nos2^−/−</i> mice. Overview sections were acquired by wide-field microscopy and detail images by confocal laser scanning microscopy. Blue: DAPI-stained nuclei. White arrows denote proliferating granule cells in the IGL. (B) High magnification images of the EGL and ML displayed altered morphologies especially in <i>Ptch1^+/− Nos2^−/−</i> and <i>Ptch1^+/+ Nos2^−/−</i> mice. (C) Cell counts from immunofluorescence images. Numbers of proliferating (Ki-67+) and non-proliferating (Ki67−) cells normalized to the length of the EGL edge show significant enrichment of dividing cells in <i>Ptch1^+/− Nos2^−/−</i> mice and correspondingly low numbers for <i>Ptch1^+/+ Nos2^−/−</i> mice. Ratios of dividing to non-dividing cells (Ki-67+, Ki-67− NeuN+) are indicated for each genotype. Significant differences are indicated by asterisks (*p<0.05). Scale bars = 50 µM. EGL: external granule layer, ML: molecular layer, IGL: internal granule layer.</p