Aberrant Otx2 Expression Enhances Migration and Induces Ectopic Proliferation of Hindbrain Neuronal Progenitor Cells

Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation

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

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 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

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