Gene Expression Differences between Prom1⁺ and Prom1⁻ Endothelium (downregulated).

<p>Functionally relevant genes downregulated in prom1⁺ endothelial cells compared to prom1⁻ endothelial cells.</p

Isolated Prom1⁺ embryonic lateral ventricle and SVZ cells in culture give rise to neurospheres expressing neural cell markers but not vascular markers.

<p>Z-stack confocal images of cultured neurospheres generated from Prom1⁺ cells of E12.5 forebrain of prominin-1^lacZ/+ mice in serum free media and in the presence of bFGF revealed co-immunostaining for ß-galactosidase (green) and both Sox2 (A) and Musashi (B) stem cell markers. Z-stack confocal images of secondary differentiated neurospheres induced by removing bFGF and plating on polyL-ornithine and fibronectin coated dishes from Prom1⁺ cells of E16.5–E17.5 forebrain of prominin-1^lacZ/+ mice. Prom1⁺ cells (ß-galactosidase (green) in culture give rise to 3 different neural lineages (C) Olig 2 oligodendrocytes, (D) GFAP astrocytes and (E) Tuj1 neuron-specific progenitor neuronal marker.</p

Prom1⁺ cells are present throughout the brain of an eight-week old <i>Prom1^lacZ/+</i> mouse model.

<p>(A) X-gal staining of Prom1 cells in sagittal section of the brain. (B–F, left panel) Low-power and respective magnified images (C-G, right panel) of anteroposterior coronal sections showing the distribution of ß-galactosidase activity in the hippocampus (B, C), occipital cortex (D, E) and cerebellum (F, G). Abbreviations: CPu, caudate/putamen; Ent, enthorinal cortex; fi, fimbria; GrDG, granular layer dentate gyrus; GrL, granular layer cerebellum; hipp, hippocampus; LV, lateral ventricle; MolDG, molecular layer dentate gyrus; MoL, molecular layer cerebellum; OB, olfactory bulb; OC, occipital cortex; PcL, Purkinje cell layer; RMS, rostral migratory stream; Tc, temporal cortex; Th, thalamus; WM, white matter; 3V, third ventricle.</p

Prom1⁺ neural cells are distributed in the hippocampus and cerebellum of <i>Prom1^lacZ/+</i> in an eight-week old mouse.

<p>(A) Z-stack confocal image showing coimmunostaining for ß-galactosidase (green) and GFAP (red) astrocytic cells in the hippocampus and (B) calbindin (red) Purkinje cells in the cerebellum. (C) Electron micrograph shows electron-dense X-gal crystals (arrow) localized in the cytoplasm of Purkinje cells. Abbreviations: N, nucleus; PC, Purkinje cell.</p

Prom1 is expressed by endothelial and tumor cells and both Prom1⁺ and Prom1⁻ cells contribute to glioma growth.

<p>H&E sections of brain tumor of 2 to 4 month old mouse generated by crossing <i>Prom1^lacZ/+</i> and Ntv-a;<i>Ink4a-Arf^−/−</i> mice infected with RCAS-PDGF. Tumors (A) were characterized by pseudopalisading necrosis (C) and microvascular proliferation (E), characteristic of glioblastoma. X-gal staining revealed ß-galactosidase activity throughout the tumor (B), at the periphery of pseudopalisading necrosis (D) and within microvascular proliferation“glomeruloid tuft” (F). (G) Z-stack and confocal optical sections revealed that some tumor cells coimmunostained for ß-galactosidase (green) and GFAP (red) and had astrocytic morphology (inset). (H) Other cells within the “glomeruloid tuft” coimmunostained for ß-galactosidase (green) and CD31 endothelial cells (red). (I) Kaplan-Meier analysis comparing survival of adult wild-type mice implanted with tumor cells dissociated from Prom1⁺ (N = 8) and Prom1⁻ neurospheres (N = 8) from PDGF-induced tumors in Ntv-a;<i>Ink4a-Arf^−/− Prom1^lacZ/+</i> mice. (J) Survival curve of adult (8 weeks) wild-type mice implanted with Prom1⁺ (N = 5) and Prom1⁻ (N = 8) PDGF-induced tumors immediately after their cells were dissociated.</p

Tumors with endothelial cells expressing Prom1⁺ are larger and exhibit dynamic microvascular endothelial proliferation compared to tumors containing Prom1⁻ endothelium.

<p>(A) Stereotactic co-implantation into the adult mouse brain of cells dissociated from PDGF-induced tumorspheres in combination with sorted VEGFR2-GFP Prom1⁺ endothelial cells (N = 12) form tumors at four weeks after implantation with maximal cross-sectional area that is significantly larger than those produced by co-implantation with VEGFR2-GFP Prom1⁻ endothelium (N = 10). (B) Immunohistochemical detection of CD31 endothelial cells counterstained with hematoxylin confirmed paucity of CD31 endothelial cells in tumors implanted with VEGFR2-GFP Prom1⁻ endothelial cells, but (C) marked immunoreactivity in tumors implanted with VEGFR2-GFP Prom1⁺ endothelium. (D) Confocal images showing multiple areas of coimmunostaining for VEGFR2-GFP Prom1⁺ (green) and CD31 endothelial cells (red) (Inset, higher magnification). (E) Confocal images showing rare areas of coimmunostaining for VEGFR2-GFP Prom1⁻ (green) and CD31 (red) endothelial cells. Error bars depict the s.e.m. Scale bar, 50 µm (B, C) or 100 µm (D, E). *P<0.03.</p

Endothelial cells are among the Prom1⁺ cell population in the brain.

<p>(A) Z-stack confocal image showing co-immunostaining for ß-galactosidase (green) and CD31 endothelial cells (red). (B) Flow cytometry confirms the presence of CD31 endothelial cells among Prom1⁺ cells in the brain of a six-week wild-type mouse. (C) Likewise, Prom1⁺ cells are identified among VEGFR2 expressing cells in a reporter mouse in which VEGFR2 promoter drives GFP. CD31 endothelial cells (red) appeared either isolated or (D) wrapped around astrocytes (blue) in the VZ and SVZ areas. (E) Electron micrograph section of <i>Prom1^lacZ/+</i> mouse brain corresponding to areas previously identified by X-gal staining showing rod-shaped electron-dense structures in the endothelial cell cytoplasm of a representative capillary vessel. Arrows, X-gal crystals; E, endothelial cell; L, lumen; P, pericyte; Pt, protrusion.</p

Gene Expression Differences between Prom1⁺ and Prom1⁻ Endothelium (upregulated).

<p>Functionally relevant genes upregulated in prom1⁺ endothelial cells compared to prom1⁻ endothelial cells.</p