The source of the Lgr5 ligand, R-Spondin1, temporally varies with CGN maturation.

<p>Cerebellar sections from <i>Lgr5^EGFP-CreERT2</i> mice at increasing developmental time points <b>a</b>) P4, <b>b</b>) P8, <b>c</b>) P14 and <b>d</b>) P28 were stained for Lgr5-EGFP, Rspo1, Calb1 and DAPI. Top row panels are representative images while boxes indicate magnified regions in bottom 4 rows. Scale bars, <i>top row</i>: 100 microns, <i>bottom 4 rows</i>: 50 microns.</p

Lgr5-positive cells in the postnatal cerebellum are lineage-restricted.

<p><b>a</b>) Schematic for lineage tracing design. Cells from <i>Lgr5^EGFP-CreERT2; R26R^tdTomato</i> mice express EGFP and CreER2 when the <i>Lgr5</i> locus is active. Addition of tamoxifen (TAM) activates CreER2, which removes the STOP element in the Rosa26-tdTomato locus permanently marking the cell and its progeny with tdTomato. Cells that maintain Lgr5 expression are double positive (yellow). <b>b</b>) Examples of lineage tracing time points. Cells that were once Lgr5+ (tdTomato+) were invariably Gabra6 positive, indicating granule neuron lineage restriction. <i>Top</i>: Lineage tracing was initiated at the beginning of Lgr5 expression in the IGL (P4) and cerebellums were analyzed at P10. <i>Bottom</i>: Lineage tracing was initiated near the end of Lgr5 expression in the IGL (P14) and analyzed at P28, when the <i>Lgr5</i> is no longer active in the IGL. Scale bar, 25 microns. <b>c</b>) Lineage tracing initiated at P14 was analyzed at extended time points, P21 and P56, demonstrating Lgr5+ cells at P14 survive and integrate into the adult IGL architecture. Scale bars, <i>top</i>: 400 microns, <i>bottom</i>: 100 microns.</p

Lgr5 is expressed in the cerebellum during postnatal development.

<p>Sagittal brain immunofluorescence sections from <i>Lgr5^EGFP-CreERT2</i> mice stained for EGFP to mark Lgr5+ cells at multiple time points. <b>a</b>) Tiled 10x magnified images of a midline sagittal section were stitched together to reveal Lgr5-EGFP expression at the P14 time point. <b>b</b>) Lgr5-EGFP expression at P4, P7, P10, P14, P21, P28 and P56 in the cerebellum demonstrating that Lgr5 expression turns on at P4, ramps up expression until its peak from P10-P14 and then expression is lost permanently over the next 7–14 days. <b>c</b>) Microarray expression data of Lgr5 generated from the Cerebellar Development Transcriptome Database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114433#pone.0114433-Sato1" target="_blank">[20]</a> taken from total cerebellum RNA at indicated time points. Data acquired from <a href="http://www.cdtdb.neuroinf.jp/CDT/ReferTemporal.do?cdIdCh=CD12762.1" target="_blank">http://www.cdtdb.neuroinf.jp/CDT/ReferTemporal.do?cdIdCh=CD12762.1</a>. <b>d</b>) Olfactory Bulb (OB) at P7 and P56 reveal Lgr5-EGFP expression throughout development and adulthood. Scale bars, 400 microns.</p

Maturing CGNs progress through a transient Lgr5 phase resulting in mature CGNs.

<p><b>a</b>) Schematic of lineage tracing time course. Tamoxifen (TAM) was injected at P4 into several large litters and brains from pairs of wildtype (WT) and <i>Lgr5^EGFP-CreERT2; R26R^tdTomato</i> mice from each litter, were analyzed at the indicated time points. Period of predicted Cre activity is indicated. <b>b</b>) Multiple sections from each brain analyzed were stained for Lgr5-EGFP to mark cells currently Lgr5+, while tdTomato expression marked previously Lgr5+ cells that had undergone recombination. Shown is an example from a P10 brain that had lineage tracing initiated at P4. Yellow dots indicate cells marked as Lgr5+/tdTomato+ by image analysis algorithm (yellow arrows indicate examples), while blue dots indicate cells marked as Lgr5-/tdTomato+ (blue arrow with red outline point out examples). Scale bars, 50 microns. <b>c</b>) Quantification from an image analysis algorithm of all sections analyzed.</p

Lgr5 CGNs are non-proliferative and post-mitotic.

<p><b>a, b</b>) Cerebellar sections from <i>Lgr5^EGFP-CreERT2</i> mice were stained for the proliferation marker Ki67 and Lgr5-EGFP at both <b>a</b>) early (P7) and <b>b</b>) late (P14) stages of Lgr5 expression. Lgr5+ cells were uniformly negative for Ki67. Scale bars 50 microns. <b>c</b>) Lineage tracing was initiated at P4 in <i>Lgr5^EGFP-CreERT2; R26R^tdTomato</i> mice and cerebella analyzed for Ki67 at P10. Many other lineage trace time points were also analyzed (data not shown). Cells post-Lgr5 expression (tdTomato+) were uniformly negative for Ki67 in every time point analyzed. Note that immature granule neurons in the EGL in A-C were Ki67+, as expected. Scale bars for C <i>top</i>: 400 microns, <i>bottom</i>: 100 microns. <b>d</b>) <i>Lgr5^EGFP-CreERT2</i> mice were crossed with <i>Catnb^flox(exon3)</i> mice to yield <i>Lgr5^EGFP-CreERT2</i>; <i>Catnb^flox(exon3)</i> mice. Upon tamoxifen administration, Cre recombinase initiates expression of a constitutively active form of β-catenin in cells with an active <i>Lgr5</i> locus. <b>e</b>) Tamoxifen was administered at P4 and cerebella were analyzed at P25, when these mice die of other complications related to transgene expression. β-catenin over-activity led to prolonged Lgr5 expression in the IGL in <i>Lgr5^EGFP-CreERT2</i>; <i>Catnb^flox(exon3</i> mice (center panel), but not wild type (right panel). The normal architecture of the cerebellum was maintained in both conditions. Scale bars 400 microns. <b>f, g</b>) Lgr5+ CGNs from P25 <i>Lgr5^EGFP-CreERT2</i>; <i>Catnb^flox(exon3)</i> mice with tamoxifen administration at P4 were <b>f</b>) positive for Gabra6 and <b>g</b>) negative for Ki67. Scale bars, <i>bottom left</i>: 200 microns, <i>other 5 panels</i>: 25 microns.</p

c-Myc induces cell cycle arrest of glioma cancer stem cells.

<p>(A, B) Early passage (0 phase; M2-G₀/G₁ phase; M3-S phase; M4-G2/M phase.</p

c-Myc modulates cell cycle regulators of glioma cancer stem cells.

<p>(A) Early passage (WAF1/CIP1, cyclin D₁ (cycD1) and cyclin D₂ (cycD2) were determined by quantitative real-time PCR 3 days after infection. (B) Protein levels of c-Myc, p53, cyclin D₁, cyclin D₂, cyclin E and p21^WAF1/CIP1 were determined by immunoblotting.</p

c-Myc is highly expressed in glioma cancer stem cells.

<p>(A) CD133− and CD133+ cells were isolated from glioma surgical biopsy specimens passaged short-term in immunocompromised mice and briefly cultured. Total RNA was isolated from both CD133− and CD133+ cells. cDNA was prepared by reverse transcription. Expression of c-Myc was then determined by quantitative real-time PCR and normalized to β-actin and HPRT1. Relative mRNA levels of c-Myc in CD133− cells were assigned a value of 1. Data are represented as mean±S.E.M in this and all subsequent graphs (#: p<0.001). (B) Total cellular lysates were resolved by SDS-PAGE. Protein levels of c-Myc and Olig2 were determined by immunoblotting. Actin was blotted as the loading control. (C) Glioma cells were isolated directly from human surgical biopsy specimens, fixed in 4% paraformaldehyde following dissociation, labeled with anti-CD133-APC and anti-c-Myc-FITC, and subjected to FACS analysis. (D) Percentage of cells expressing high levels of c-Myc within either the CD133− fraction or the CD133+ fraction was demonstrated (#: p<0.001). (E) Sections of freshly frozen human glioma surgical biopsy specimens were fixed and co-stained for c-Myc (green) and Nestin (red). Nuclei were counterstained with Hoechst 33342. Representative images (630×) were demonstrated.</p

Transferrin receptor-1 and ferritin heavy and light chains in astrocytic brain tumors: Expression and prognostic value

<div><p>Astrocytic brain tumors are the most frequent primary brain tumors. Treatment with radio- and chemotherapy has increased survival making prognostic biomarkers increasingly important. The aim of the present study was to investigate the expression and prognostic value of transferrin receptor-1 (TfR1) as well as ferritin heavy (FTH) and light (FTL) chain in astrocytic brain tumors. A cohort of 111 astrocytic brain tumors (grade II-IV) was stained immunohistochemically with antibodies against TfR1, FTH, and FTL and scored semi-quantitatively. Double-immunofluorescence stainings were established to determine the phenotype of cells expressing these markers. We found that TfR1, FTH, and FTL were expressed by tumor cells in all grades. TfR1 increased with grade (p<0.001), but was not associated with prognosis in the individual grades. FTH and FTL were expressed by tumor cells and cells with microglial/macrophage morphology. Neither FTH nor FTL increased with malignancy grade, but low FTH expression by both tumor cells (p = 0.03) and microglia/macrophages (p = 0.01) correlated with shorter survival in patients anaplastic astrocytoma. FTL-positive microglia/macrophages were frequent in glioblastomas, and high FTL levels correlated with shorter survival in the whole cohort (p = 0.01) and in patients with anaplastic astrocytoma (p = 0.02). Double-immunofluorescence showed that TfR1, FTH, and FTL were co-expressed to a limited extent with the stem cell-related marker CD133. FTH and FTL were also co-expressed by IBA-1-positive microglia/macrophages. In conclusion, TfR1 was highly expressed in glioblastomas and associated with shorter survival in the whole cohort, but not in the individual malignancy grades. Low levels of FTH-positive tumor cells and microglia/macrophages were associated with poor survival in anaplastic astrocytomas, while high amounts of FTL-positive microglia/macrophages had a negative prognostic value. The results suggest that regulation of the iron metabolism in astrocytic brain tumors is complex involving both autocrine and paracrine signaling.</p></div

c-Myc knockdown abolishes xenograft tumor formation by glioma cancer stem cells.

<p>(A) T3359 CD133+ cells were infected and selected as described. After selection, cells were injected into brains of athymic BALB/c nu/nu mice (5000 cells per mouse). Four mice were injected for each group. Mice in the control group were sacrificed upon the development of neurologic signs. All the mice bearing c-Myc knockdown glioma cells did not develop neurologic signs and were sacrificed after 100 days without evidence of tumor. Kaplan-Meier survival curves are displayed. (B) Representative photographs of hematoxylin and eosin staining of intracranial xenograft tumors (10×). (C) Xenograft tissue of the control group composed of pleomorphic cells featuring high nuclear to cytoplasmic ratios, prominent nucleoli with minimal cytoplasm, brisk mitotic activity and central geographic necrosis (asterisk, 600×). (D) The control glioma xenograft exhibited focal areas of better differentiated tumor cells with relatively more eosinophilic cytoplasm and cells with eccentric cytoplasmic profiles suggestive of a gemistocytic appearance (arrows, 400×). (E) Xenograft tumor of the control group exhibits infiltration of tumor cells into the surrounding brain tissue along the margin. The mitotically active (arrowhead) infiltrating tumor cells exhibit high nuclear to cytoplasmic ratios and elongated fibrillar cytoplasm (arrow) (600×). (F) Mouse brain injected with T3359 cells expressing c-Myc shRNA showed no evidence of tumor at the needle injection site (arrows, 200×).</p