Comparative characterization of the human and mouse third ventricle germinal zones

Abstract

Recent evidence indicates differences in neural stem cell biology in different brain regions. For example, we demonstrated that neurofibromatosis 1 (NF1) tumor suppressor gene inactivation leads to increased neural stem cell proliferation and gliogenesis in the optic chiasm and brainstem but not in the cerebral cortex. The differential effect of Nf1 inactivation in the optic nerve and brainstem (in which gliomas commonly form in children with NF1) versus the cortex (in which gliomas rarely develop) suggests the existence of distinct ventricular zones for gliomagenesis in children and in adults. Here, we characterized the third ventricle subventricular zone (tv-SVZ) in young and adult mouse and human brains. In children, but not adult humans, the tv-SVZ contains nestin-positive, glial fibrillary acidic protein-positive, brain fatty acid binding protein-positive, and sox2-positive cells with radial processes and prominent cilia. In contrast, the tv-SVZ in young mice contains sox2-positive progenitor cells and ciliated ependymal lining cells but lacks glial fibrillary acidic protein-positive, nestin-positive radial glia. As in the lateral ventricle SVZ, proliferation in the human and murine tv-SVZ decreases with age. The tv-SVZ in adult mice lacks the hypocellular subventricular zone observed in adult human specimens. Collectively, these data indicate the existence of a subventricular zone relevant to our understanding of glioma formation in children and will assist interpretation of genetically engineered mouse glioma models.

Figure 1

Age-dependent changes in the human third ventricle subventricular zone (tv-SVZ). (A, B) Section of the third ventricle from a child (A) reveals a single layered tall columnar epithelium with prominent cilia at the ventricular surface (inset). In an adult the ependymal layer is flattened and contains a single layer of cuboidal cells and there is marked reduction of lining cilia (B). (C, D) Acetylated tubulin immunostaining reveals abundant cilia in a pediatric (C) and few in an adult (D) tv-SVZ. Scale bars = 20 μm. (E, F) Electron microscopy demonstrates a relative abundance of cilia in a pediatric tv-SVZ (E; arrow), and the presence of lipopigment in lining ependymal cells in an adult tv-SVZ (F; arrow). A, B: Hematoxylin and eosin.

Figure 2

Radial glia in the third ventricle subventricular zone (tv-SVZ) of children. (A-D) Immunostaining for glial fibrillary acidic protein (GFAP) (A, B) and nestin (C, D) demonstrate moderate staining in the ependymal lining of tv-SVZ in a child (A, C) and an adult (B, D). Radial processes (inset, arrows) were only observed in the tv-SVZ of the child; there is a cobweb-like pattern of expression in the adult third ventricle subependymal zone (SEZ) (inset). Arrowheads in C and D indicate blood vessels as internal positive immunostaining controls. (E) Radial glia in a pediatric tv-SVZ co-label (yellow) for GFAP (green) and nestin (red). Inset shows radial processes double positive for GFAP and nestin. (F) The radial glia also express brain fatty acid binding protein (BLBP: red, DAPI: blue). BLBP expression is also seen in isolated cells within the SEZ. (G) A small number of cells within the lining ependyma and tv-SEZ are Sox2-immunoreactive (arrows). Scale bars = 20 μm.

Figure 3

Age-dependent changes in neuronal architecture in the human third ventricle subventricular zone (tv-SVZ). (A-D) Immunostaining for (A, B) polysialated neural cell adhesion molecules (PSA-NCAM) (A, B) and Tuj-1 (C, D) shows intense staining of the processes abutting the third ventricle SEZ in a child (A, C), that is absent in the adult SEZ (B, D). The hypocellular region in the adult tv-SVZ is denoted by bars. Scale bar = 20 μm.

Figure 4

Proliferation in the human third ventricle subventricular zone (tv-SVZ) decreases with age. (A) Diagram of the region containing the human third ventricle (red box). (B, C) Ki67 immunolabeling of the tv-SVZ in representative specimens from a child (B) and an adult (C). (D) Mean and SD of Ki67 labeling indices (p < 0.005). Scale bar: B, C = 20 μm.

Figure 5

Proliferating cells in the human third ventricle subventricular zone (tv-SVZ) are glial fibrillary acidic protein (GFAP)- and nestin-immunoreactive. (A) A small population of GFAP-positive cells in the third ventricle subependymal zone (SEZ) co-labels with Ki67. Inset demonstrates a representative Ki67-positive, GFAP-positive cell. (B) Similar results are shown with GFAP/Ki67 immunofluorescence double labeling. The arrow denotes a representative GFAP-positive, Ki67-positive cell. (C) A small number of nestin-positive cells in the human third ventricle SEZ co-labels with Ki67. Inset demonstrates a representative nestin-positive, Ki67-positive cell. Scale bar = 20 μm.

Figure 6

Age-dependent changes in the cellular composition of the mouse third ventricle subventricular zone (tv-SVZ). (A, B) Representative hematoxylin and eosin-stained sections of the third ventricle in young and adult mice. (C, D) Glial fibrillary acidic protein (GFAP)-positive cells are barely detected in the third ventricle in a young mouse (C) whereas an adult mouse exhibits focal GFAP immunoreactivity with diffuse expression in ependymal cells (D). (E, F) Rare nestin-positive cells in a young mouse (E); an adult moue ependymal layer contains numerous nestin-immunopositive cells (F). Arrowheads indicate blood vessels, which serve as internal positive controls. (G, H) Acetylated tubulin immunostaining illustrates age-dependent reduction in the number of cilia. The cilia appear relatively shorter than those in the human tv-SVZ.

Figure 7

Age-dependent similarities in the cellular composition of the mouse third ventricle subventricular zone (tv-SVZ). (A-D) Similar patterns of polysialated neural cell adhesion molecule (PSA-NCAM) (A, B) and Tuj-1 in the tv-SEZ (C, D) are observed in young mice (A, C) and adult mice (B, D). No hypocellular zone is detected in the adult mouse tv-SVZ. Scale bar: 20 μm.

Figure 8

Proliferation in the mouse third ventricle subventricular zone (tv-SVZ) decreases with age. (A) Diagram of the region containing the mouse third ventricle (red box). (B, C) Ki67 immunolabeling of the tv-SVZ in a young mouse (B) and an adult mouse (C). Scale bar: 20 μm. (D) Mean and SD for the Ki67 labeling indices (p < 0.005).

Figure 9

Age-dependent decreases in ventricular zone proliferation in the mouse and human third ventricle subventricular zone (tv-SVZ) and lateral ventricle SVZ (lv-SVZ). (A-D) The Ki67 labeling index is reduced in the human lv-SVZ as a function of age (p = 0.01) (A). The Ki67 labeling index is reduced in the murine lv-SVZ as a function of age (p = 0.0004) (C). Diagrams of the regions containing the human (B) and mouse (D) lv-SVZ (red boxes).