A zebrafish live imaging model reveals differential responses of microglia towards glioblastoma cells in vivo

Glioblastoma multiforme is the most common and deadliest form of brain cancer. Glioblastomas are infiltrated by a high number of microglia, which promote tumor growth and surrounding tissue invasion. However, it is unclear how microglia and glioma cells physically interact and if there are differences, depending on glioma cell type. Hence, we have developed a novel live imaging assay to study microglia–glioma interactions in vivo in the zebrafish brain. We transplanted well-established human glioblastoma cell lines, U87 and U251, into transgenic zebrafish lines with labelled macrophages/microglia. Our confocal live imaging results show distinct interactions between microglia and U87, as well as U251 glioblastoma cells that differ in number and nature. Importantly these interactions do not appear to be antitumoral as zebrafish microglia do not engulf and phagocytose the human glioblastoma cells. Finally, xenotransplants into the irf8(−/−) zebrafish mutant that lacks microglia, as well as pharmacological inhibition of the CSF-1 receptor (CSF-1R) on microglia, confirm a prominent role for zebrafish microglia in promoting human glioblastoma cell growth. This new model will be an important tool for drug screening and the development of future immunotherapeutics targeting microglia within glioma

PCNA directs type 2 RNase H activity on DNA replication and repair substrates

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi–Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids

Pleiotropic Effects of Sox2 during the Development of the Zebrafish Epithalamus

The zebrafish epithalamus is part of the diencephalon and encompasses three major components: the pineal, the parapineal and the habenular nuclei. Using sox2 knockdown, we show here that this key transcriptional regulator has pleiotropic effects during the development of these structures. Sox2 negatively regulates pineal neurogenesis. Also, Sox2 is identified as the unknown factor responsible for pineal photoreceptor prepatterning and performs this function independently of the BMP signaling. The correct levels of sox2 are critical for the functionally important asymmetrical positioning of the parapineal organ and for the migration of parapineal cells as a coherent structure. Deviations from this strict control result in defects associated with abnormal habenular laterality, which we have documented and quantified in sox2 morphants

Det er ikkje nok å berre løfta ein - alle må løftast i Yrkesfaglærarløftet

Figure S1. Western blots showing that the reduction in RAD51 levels after cisplatin treatment in melanoma cells is not seen in other cell types. Blots support the RAD51 quantification shown in Fig. 2 of the main text. Figure S2. Images showing that RAD51 repair foci are not induced by short term cisplatin treatment of melanoma cells. Figure S3. The cisplatin-induced reduction in RAD51 levels in melanoma cells is not affected by proteasome or lysosome inhibitor treatment. Figure S4. Melanoma cells show only a weak cisplatin-induced G2 arrest. Figure S5. Specificity of antibodies to translesion synthesis DNA Polymerases. Figure S6. Western blots showing increased levels of DNA Polymerase zeta in cisplatin-treated melanoma cells. Blots support the DNA Pol ζ and Pol η expression data shown in Fig. 7 of the main text. (PDF 2393 kb

The parapineal and habenular defects are coupled in <i>sox2</i> morphants.

<p>(<b>A–C</b>) In control embryos, the left-sided parapineal projects towards the left habenula. As a result, the left habenula has denser neuropils than the right, as judged by phalloidin staining. (<b>D–F</b>) In <i>sox2</i> morphants with left-sided parapineal projections, the left habenula is larger than the right. (<b>G–I</b>) Morphants with right-sided parapineal organs display reverse habenular asymmetries, whereas (<b>J–L</b>) morphants with bilateral parapineal projections have symmetric habenulae. (<b>M</b>) The average volume of the left (blue bars) and right (red bars) habenular neuropils, as judged by the volume of phalloidin-positive areas within the habenulae. y-axis show volume in µm³. (<b>N</b>) Average asymmetry index in control (purple bar) and <i>sox2</i> morphants (orange bars). 3D reconstructions of confocal images at 4 dpf, arrows show parapineal projections and blue lines surround the habenular neuropils, error bars represent ± standard error, (<b>M</b>) * = p-value <0.05 and ** = p-value <0.001 (Wilcoxon test).</p

Suboptimal doses of <i>sox2</i> morpholinos result in increased number of PhRs and abnormal positioning of the parapineal organ.

a<p>Average number of PhRs (± standard error, number of embryos analyzed).</p>b<p>Percentage of embryos with left, right or bilateral parapineal projections (number of embryos in each category).</p

Sox2 controls PhR cell fate independently of BMP.

<p>(<b>A</b>) The transgenic line <i>Tg(aanat2:GFP)</i> marks the PhRs. (<b>B</b>) The transgenic line <i>Tg(BRE:GFP)</i> is as a BMP signaling reporter. (<b>C</b>) In <i>sox2</i> morphant embryos, the number of PhRs is increased at 24 hpf, when compared to controls (<b>A</b>). (<b>D</b>) The number of BMP-responsive cells at 24 hpf is unaffected when <i>sox2</i> is knocked down. (<b>E–F</b>) The number of PhRs (<b>E</b>) and BMP-responsive cells (<b>F</b>) is similar in control embryos at 28 hpf. (<b>G–H</b>) The number of PhRs (<b>G</b>) is higher than the number of BMP-responsive cells (<b>H</b>) at 28 hpf, in <i>sox2</i> morphants. (<b>I</b>) The average number of PhRs (<i>Tg(aanat2:GFP)</i>-positive cells) and BMP-responsive cells (<i>Tg(BRE:GFP)</i>-positive cells) in control and <i>sox2</i> morphant embryos, at 24 and 28 hpf. Confocal maximum projections, scale bars = 25 µm, error bars represent ± standard error, ** = p-value <0.001 (MWU test). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s008" target="_blank">Figure S8</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s014" target="_blank">Movie S3</a></b>.</p

A model for pineal cell-fate determination.

<p>Cell-fate determination has two phases: a prepatterning phase, followed by a determination phase. Sox2 (Sox2 levels or Sox2 along with a partner protein (X)) is important during the prepatterning phase, where is inhibits cells from adopting a PhR fate. In contrast, the Notch and BMP pathways are important during the determination fate, where BMP induces the PhR fate and Notch inhibits the PN fate. A yet-to-be-identified modulator (Y) is responsible for inducing the PN fate.</p

Sox2 inhibits the PhR cell fate.

<p>(<b>A–C</b>) <i>Tg(aanat2:GFP)</i> drives GFP expression in the pineal PhRs. (<b>B</b>) Isl1 labels the pineal cells. (<b>D–F</b>) Knockdown of <i>sox2</i> results in increased number of PhRs. (<b>G</b>) The average number of PhRs in control (purple bar) and <i>sox2</i> morphants (orange bar). (<b>H–J</b>) <i>Tg(elavl3:GFP)</i> drives GFP expression specifically in the PNs. (<b>K–M</b>) The knockdown of <i>sox2</i> does not affect the number of PNs. (<b>N</b>) The average number of PNs in controls (purple bar) and <i>sox2</i> morphants (orange bar) does not significantly differ. Confocal maximum projections of 28 hpf embryos, scale bars = 25 µm, error bars represent ± standard error, ** = p-value <0.001 (MWU test). See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s004" target="_blank">Figure S4</a></b>.</p

Knockdown of <i>sox2</i> results in abnormal parapineal development and disruption of the habenular asymmetries.

<p>(<b>A</b>) <i>gfi1ab</i> is expressed in parapineal cells on the left side of the brain in control embryos. (<b>B–D</b>) <i>sox2</i> morphants are categorized into three groups according to <i>gfi1ab</i> expression: left-sided expression (<b>B</b>), right-sided (<b>C</b>) and embryos with scattered <i>gfi1ab</i> cells (<b>D</b>). (<b>E</b>) <i>kctd12.1</i> is asymmetrically expressed in the habenulae, with a broader expression domain in the left than the right habenula. (<b>F–H</b>) In <i>sox2</i> morphants <i>kctd12.1</i> expression can be: asymmetric with more on the left side similar to control embryos (<b>F</b>), asymmetric with more on the right side (<b>G</b>) or symmetric (<b>H</b>). (<b>I</b>) A table showing the percentage of embryos with normal, reversed or bilateral parapineal organs, using different staining methods. Scale bars = 25 µm. See also <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s009" target="_blank">Figure S9</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s010" target="_blank">S10</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087546#pone.0087546.s015" target="_blank">Movie S4</a>.</b></p