586 research outputs found
Nos2 Inactivation Promotes the Development of Medulloblastoma in Ptch1+/β Mice by Deregulation of Gap43βDependent Granule Cell Precursor Migration
Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1+/β mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1+/β mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1+/β Nos2β/β mice compared to Ptch1+/β Nos2+/+ mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1+/+ Nos2β/β mice but not from Ptch1+/β Nos2β/β mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1+/+ Nos2β/β mice but increased in Ptch1+/β Nos2β/β mice relative to Ptch1+/β Nos2+/+ mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1+/β mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression
Quantifying Earth system interactions for sustainable food production via expert elicitation
Several safe boundaries of critical Earth system processes have already been crossed due to human perturbations; not accounting for their interactions may further narrow the safe operating space for humanity. Using expert knowledge elicitation, we explored interactions among seven variables representing Earth system processes relevant to food production, identifying many interactions little explored in Earth system literature. We found that green water and land system change affect other Earth system processes strongly, while land, freshwater and ocean components of biosphere integrity are the most impacted by other Earth system processes, most notably blue water and biogeochemical flows. We also mapped a complex network of mechanisms mediating these interactions and created a future research prioritization scheme based on interaction strengths and existing knowledge gaps. Our study improves the understanding of Earth system interactions, with sustainability implications including improved Earth system modelling and more explicit biophysical limits for future food production
Estradiol protects dermal hyaluronan/versican matrix during photo aging by release of EGF from keratinocytes
Estradiol protects dermal hyaluronan/versican matrix during photo aging by release of EGF from keratinocytes
Characteristics and functional implication of Gap43 expression in cell culture.
<p>(AβB) 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 (*pβ=β0.0234). (B) In D458 cells, <i>Gap43</i> expression is significantly decreased upon L-NAME treatment after 72 hours (***p<0.0001). (CβD) 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 (*pβ=β0.013, **pβ=β0.007). Sh39, sh42: anti-<i>Gap43</i> target shRNA, shGFP: control shRNA against GFP, shNT: non-target control shRNA.</p
<i>Nos2</i> deficiency increases the incidence of MB in <i>Ptch1</i><sup>+/β</sup> mice.
<p>(A) Kaplan-Meier analysis of MB incidence in 215 <i>Ptch1</i><sup>+/β </sup><i>Nos2</i><sup>+/+</sup> mice (orange line) versus 221 <i>Ptch1</i><sup>+/β </sup><i>Nos2</i><sup>β/β</sup> mice (red line). <i>Ptch1</i><sup>+/β </sup><i>Nos2</i><sup>β/β</sup> mice demonstrated an approximately two-fold increase in MB incidence (pβ=β0.0007, Logrank test). None of 315 wild-type (green line) and 412 <i>Ptch1</i><sup>+/+</sup><i>Nos2</i><sup>β/β</sup>mice (blue line) (control littermates) developed any MB. Vertical ticks represent censored mice. (BβE) Histological features of MBs from <i>Ptch1</i><sup>+/β </sup><i>Nos2</i><sup>+/+</sup> and <i>Ptch1</i><sup>+/β </sup><i>Nos2</i><sup>β/β</sup> mice. MBs in both genotypes (BβC, <i>Ptch1</i><sup>+/β </sup><i>Nos2</i><b><sup>+/+</sup></b>; DβE, <i>Ptch1</i><sup>+/β </sup><i>Nos2</i><sup>β/β</sup>) 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 barβ=β500 Β΅m), and cellular morphology (C, E, scale barβ=β50 Β΅m).</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<sup>+/β</sup> Nos2<sup>+/+</sup></i>, <i>Nos2</i>-deficient: <i>Ptch1<sup>+/+</sup> Nos2<sup>β/β</sup></i> and <i>Ptch1<sup>+/β</sup> Nos2<sup>β/β</sup></i>. (C) <i>Gap43</i> was differentially expressed between <i>Ptch1<sup>+/β</sup> Nos2<sup>+/+</sup></i> and <i>Ptch1<sup>+/β</sup> Nos2<sup>β/β</sup></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 (nβ=β7 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<sup>+/β</sup> Nos2<sup>+/+</sup></i> cerebella, in particular at the outer ML, scale barβ=β50 Β΅M. (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
Microarray-based gene expression profiling.
<p>(A) Hierarchical cluster analysis of normal cerebellar tissue and MB samples showed a clear separation of <i>Ptch1<sup>+/+</sup> Nos2<sup>β/β</sup></i> P9 cerebella. (B) Heat map of proliferation-associated genes downregulated in P9 cerebella of <i>Ptch1<sup>+/+</sup> Nos2<sup>β/β</sup></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<sup>+/β</sup> Nos2<sup>β/β</sup></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
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