Origin and fate of cerebellar ectopia.

<p>(A) Rare ectopia continuous with the EGL, H & E staining. (B) Image of rare Math1⁺ ectopia continuous with the EGL in GFAP:Hi-Otx2, <i>MATH1-GFP</i> mice. (C–D, F–M) Images of GFAP:Hi-Otx2 brain sections stained with the indicated antibodies. (C–D) Sections immunostained with Ki67 and Zic1 of (C) rare ectopia continuous with the EGL and (D) representative ectopia located just deep to the IGL. (E) Localization of histologically-apparent ectopia of GFAP:Hi-Otx2 mice at P3 and P7 (distance in µm). (F, G) Expression of differentiation markers in cerebellar ectopia at (F) P7 and (G) P21. (H–K) Location of partially- to fully-differentiated neurons in (H, I) cerebellum or (J, K) brainstem as evidenced by Zic1 and Ki67 staining shown at 10× mag in (H, J) wild type mice or (I, K) GFAP:Hi-Otx2 mice. (L, M) Cleaved caspase-3 staining of ectopia in the (L) cerebellum and (M) brainstem. All images except (H–K) shown at 20× mag. CB, cerebellum; BS, brainstem. Asterisk indicates <i>p</i>≤0.05 relative to P3, student's t-test. Arrows indicate ectopia. Boxes indicate location of cropped and zoomed image.</p

Cerebellar hyperplasias in GFAP:Hi-Otx2 mice are spatially distinct from dysplasia/hyperplasia observed in <i>ND2:SmoA1</i>^+/− mice.

<p>Images of H & E stained sections (20× mag) from (A) wild type mice, (B) GFAP:Hi-Otx2 mice, and (C) <i>ND2:SmoA1^+/−</i> mice. Scale bar: 100 µm. (D) Location of ectopia at P7 in GFAP:Hi-Otx2 mice relative to location of hyperplasia/dysplasia in P7 ND2:SmoA1^+/− mice. Arrows indicate regions of hyperplasia.</p

Otx2 expression is induced in various cell types in GFAP:Hi-Otx2 mice.

<p>Sections from P7 wild type (A, C–D) or GFAP:Hi-Otx2 (B, E–J) mice were immunostained with the indicated antibodies. For (A) and (B), anterior expression threshold is designated with a red arrow. (A, B) are shown at 4× magnification (mag), all others 20× mag. sc, superior colliculus; egl, external granule layer; igl, internal granule layer. White arrows indicate overlapping expression of the indicated markers in individual cells.</p

GFAP:Hi-Otx2 mice develop focal hyperplasias in the cerebellar white matter and the brainstem.

<p>(A–F) H & E stained sections from P7 hindbrains of (A, C, E) wild type and (B, D, F) GFAP:Hi-Otx2 mice. Fields shown are as follows: (A, B) whole cerebella at 4× magnification (mag), (C, D) cerebellar white matter at 10× mag, (E, F) dorsal brainstem at 10× mag. (G) reference illustration of fields shown in C–F indicated by grey boxes. (H, I) Immunofluorescent staining for Ki67 in (H) cerebellar ectopia and (I) brainstem ectopia in GFAP:Hi-Otx2 mice (20× mag). (J) Prevalence of ectopia in GFAP:Hi-Otx2 mice over time. CB, cerebellum; BS/bs, brainstem; egl, external granule layer; igl, internal granule layer; wm, white matter; IV, fourth ventricle; mvn, medial vestibular nuclei; D, dorsal; V, ventral; A, anterior; P, posterior. Black or white arrows indicate ectopia.</p

Cerebellar and brainstem ectopia express Math1.

<p>(A, C, E) Immunofluorescent staining for Pax6 in (A) wild type cerebellum, (C) GFAP:Hi-Otx2 cerebellum, and (E) GFAP:Hi-Otx2 brainstem. (B) MATH1-GFP reporter expression in the cerebellum of <i>MATH1-GFP</i> transgenic mice. (D, F) MATH1-GFP reporter expression in the (D) cerebellum and (F) brainstem of GFAP:Hi-Otx2, <i>MATH1-GFP</i> mice. 20× mag. Arrows indicate ectopia.</p

Validation of medulloblastoma DHS sites in a cohort of OTX2-expressing and -nonexpressing cells.

<p>Nuclei of medulloblastoma cell lines were treated with increasing concentrations of DNase (indicated as Units per reaction), and then the relative proportion of DNA remaining at the indicated regions were determined by qPCR. Open and solid markers indicate OTX2-expressing and -nonexpressing cell lines, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107156#pone.0107156.s002" target="_blank">Fig. S2</a> for detailed graphs.</p

OTX2 regulates its own expression through the DHS 4 enhancer.

<p>(A) Luciferase assay for enhancer activity of DHS 4 in OTX2-expressing medulloblastoma cells treated with OTX2 siRNA. (B) Western blotting for ectopic and endogenous OTX2 following transfection of (B) OTX2-expressing or (C) OTX2-nonexpressing medulloblastoma cells with EGFP-tagged OTX2. (D) RT-qPCR demonstrating induction of the OTX2 target gene IL-6 in OTX2-nonexpressing medulloblastoma cells transfected with EGFP-tagged OTX2. (E) Chromatin immunoprecipitation of endogenous OTX2 in OTX2-expressing and -nonexpressing medulloblastoma cells. *<i>p</i><0.05 relative to scramble siRNA (A) or vector control (D), **<i>p</i><0.01 relative to scramble siRNA (A) or vector control (D), †p<0.05 relative to pGL3pro, ††p<0.01 relative to pGL3pro, Student's t-test. Error bars indicate standard deviation.</p

Functional assessment of DHS sites.

<p>Luciferase assays measuring (A) orientation-sensitive promoter activity of proximal (<2 kb from the TSS) DHS sites in D283 cells, (B) enhancer activity of all DHS sites in D283 cells, (C) promoter activity of functionally-validated proximal DHS sites [underlined in (A)] in OTX2-expressing (D283, D341) and -nonexpressing (UW228) medulloblastoma cells, (D) enhancer activity of functionally-validated DHS sites [underlined in (B)] in OTX2-expressing (D283, D341) and -nonexpressing (UW228) cells, (E) insulator activity of DHS 3 in OTX2-expressing medulloblastoma cells, and (F) enhancer activity of minimal fragments and deletion mutants of DHS 4 in OTX2-expressing medulloblastoma cells. (G) Alignment of the critical Fragment A region of DHS 4 with the OTX2 position weight matrix <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107156#pone.0107156-Matys1" target="_blank">[28]</a>. *<i>p</i><0.05 relative to empty vector, **<i>p</i><0.01 relative to empty vector, †p<0.05 relative to DHS 1.pro, ††p<0.01 relative to DHS 1.pro, Student's t-test. Error bars indicate standard deviation.</p

DHS “R” mediates retinoic acid-induced OTX2 repression.

<p>(A) DNase sensitivity of DHS “R” in three OTX2-expressing (D283, D341, D721) and two OTX2-nonexpressing (D324, UW228) medulloblastoma cell lines. (B) Luciferase assays measuring enhancer activity of DHS “R” following treatment with 2 µM all-<i>trans</i> retinoic acid (ATRA) for 24 hours. (C) OTX2 mRNA level as determined by qPCR of medulloblastoma cells treated with 2 µM all-<i>trans</i> retinoic acid (ATRA) and/or 35 µM cycloheximide (CYHEX) for 8 hours. *<i>p</i><0.05 relative to DMSO, **<i>p</i><0.01 relative to DMSO, Student's t-test. Error bars indicate standard deviation.</p

Comparison of DHS sites and DGE-seq data across species.

<p>(a) Analysis pipeline. DNase-sequences from each species were aligned to the native genome and lifted over to the human genome for analysis. Regions are filtered at various steps of the analysis to remove alignment and orthology artifacts (Materials and Materials). Correlation plots of DNase-seq signals (b) and DGE-seq signals (c) expression data show that both chromatin and expression data from human (Hu), chimpanzee (Ch), and macaque (Ma) are more highly correlated between biological replicates from the same tissue within a single species. Additionally, the same cell type from different species is more similar than different cell types from the same species.</p