Upregulation of E2F1 in cerebellar neuroprogenitor cellsand cell cycle arrest during postnatal brain development

Daniela E. Suzuki & Carolina B. Ariza &

Marimélia A. Porcionatto & Oswaldo Keith Okamoto

Received: 11 January 2011 /Accepted: 11 May 2011 /Published online: 26 May 2011 / Editor: T. Okamoto# The Society for In Vitro Biology 2011

Abstract In the developing cerebellum, proliferation ofgranular neuroprogenitor (GNP) cells lasts until the earlypostnatal stages when terminal maturation of the cerebellarcortex occurs. GNPs are considered cell targets forneoplastic transformation, and disturbances in cerebellarGNP cell proliferation may contribute to the developmentof pediatric medulloblastoma. At the molecular level,proliferation of GNPs is regulated through an orchestratedaction of the SHH, NOTCH, and WNT pathways, but theunderlying mechanisms still need to be dissected. Here, wereport that expression of the E2F1 transcription factor in ratGNPs is inversely correlated with cell proliferation rateduring postnatal development, as opposed to its traditionalSHH-dependent induction of cell cycle. Proliferation ofGNPs peaked at postnatal day 3 (P3), with a subsequentcontinuing decrease in proliferation rates occurring until

P12. Such gradual decline in proliferating neuroprogenitorsparalleled the extent of cerebellum maturation confirmed byhistological analysis with cresyl violet staining and temporalexpression profiling of SHH, NOTCH2, and WNT4 genes. Atime course analysis of E2F1 expression in GNPs revealedsignificantly increased levels at P12, correlating withdecreased cell proliferation. Expression of the cell cycleinhibitor p18Ink4c, a target of E2F1, was also significantlyhigher at P12. Conversely, increased E2F1 expression didnot correlate with either SMAC/DIABLO and BCL2 expres-sion profiles or apoptosis of cerebellar cells. Altogether,these results suggest that E2F1 may also be involved in theinhibition of GNP proliferation during rat postnataldevelopment despite its conventional mitogenic effects.

Keywords Gene expression . Neuroprogenitors . Cellularproliferation . Central nervous system . Development .

Transcription factor . E2F1

Introduction

During mammalian development, maturation of the cerebel-lum is concluded in the early postnatal period. Throughoutthis process, major changes at the molecular and cellularlevels occur in a time-specific manner, involving theactivation of neural stem cells, proliferation of neuralprogenitors, differentiation into specialized cells, migration,and integration into functional neural circuits (Chizhkov andMillen 2003). In its mature configuration, the cerebellumcontains nearly half the amount of total neurons in thecentral nervous system (CNS) despite representing only 10%of the total brain volume (Wang and Zoghbi 2001).

Regulation of cerebellar neurogenesis involves modulationof different biochemical pathways such as those signaled by

Electronic supplementary material The online version of this article(doi:10.1007/s11626-011-9426-3) contains supplementary material,which is available to authorized users.

D. E. Suzuki :O. K. OkamotoDisciplina de Neurologia Experimental, Departamento deNeurologia/Neurocirurgia, UNIFESP,São Paulo, Brazil

C. B. Ariza :M. A. PorcionattoDisciplina de Biologia Molecular,Departamento de Bioquímica, UNIFESP,São Paulo, Brazil

O. K. Okamoto (*)Centro de Estudos do Genoma Humano, Departamento deGenética e Biologia Evolutiva, Instituto de Biociências,Universidade de São Paulo,Rua do Matão, 277,CEP 05508-090, São Paulo, São Paulo, Brazile-mail: keith.okamoto@usp.br

In Vitro Cell.Dev.Biol.—Animal (2011) 47:492–499DOI 10.1007/s11626-011-9426-3

SHH, WNT, and NOTCH proteins, which act in a concertedfashion (Clark et al. 2007). Some of these pathwayscontrol the G1-S phase transition of the cell cycle andinteract with pathways that modulate growth (MEK/ERK),survival (PI3K/AKT), and death of eukaryotic cells. However,the molecular mechanisms controlling the proliferation ofneural progenitors along with its lineage commitment anddifferentiation are not completely elucidated.

Genetic or epigenetic alterations affecting the expressionof critical genes in neurogenesis may contribute toneoplastic transformation. Indeed, mutations or ectopicexpression of genes belonging to NOTCH, WNT, andSHH pathways are frequently observed in human tumors,and their involvement in the development of gliomas iswell known (Dyer 2004; Pomeroy 2004; Clark et al. 2007).

The downstream targets of some of these pathwaysinclude genes encoding E2F transcription factors involvedin the regulation of cell cycle progression, cell differentiation,and apoptosis (Müller et al. 2001; DeGregori 2002;Polager et al. 2002). The mammalian family of E2Ftranscription factors comprised activators (E2F1–E2F3)and repressors (E2F4–E2F8) of gene expression, with dualroles in tumor promotion and suppression (Chen et al.2009). In humans, aberrant expression of the E2F2 membercorrelates with malignancy of astrocytomas and is involved inthe transformation of astrocytes (Okamoto et al. 2007).

Under physiological conditions, expression of E2Fmembers is tissue-specific and is implicated in manyaspects of the CNS development, but the biological roleof each member remains unclear. Recent studies inanimal models suggest that E2F1 is involved in theregulation of granule cell number within the postnatalcerebellum. However, E2F1 deficiency in transgenicmice has been correlated with both loss of granuleneurons (Cooper-Kuhn et al. 2002) and increasedproliferation of cerebellar neurons (Wang et al. 2007).Since cerebellar granular neuroprogenitors (GNPs) aretargets for neoplastic transformation, the better understandingof E2F transcription factors in GCP cell proliferation controlis of great interest.

In this study, a time course analysis of E2F1 expressionin rat cerebellar progenitor cells was performed throughoutthe postnatal development. Our findings revealed anupregulation of E2F1 which correlated with the decreasedproliferative activity of GNP cells, suggesting a tumorsuppression activity rather than a pro-oncogenic role duringnormal postnatal maturation of rat cerebellum.

Material and Methods

Isolation of cerebellar progenitor cells. Wistar rats werehoused under standard controlled conditions (7:00A.M./7:00P.M.

light/dark cycle, 20–22°C, 45–55% humidity) with foodand water ad libitum. Pregnant rats were isolated insingle cages once pregnancy became evident. Day ofbirth was considered P0. All efforts were made tominimize animal suffering following the proposal of theInternational Ethical Guideline for Biomedical Research(CIOMS/OMS, 1985). The study was approved by theEthics Committee for Animal Research of the FederalUniversity of São Paulo, Brazil (CEP 2054/07). Ratpups were anesthetized and euthanized by decapitationat different postnatal days (P1, P3, P6, P9, and P12).Brains were assessed and the cerebellum was identifiedand dissected based on its position and morphology (i.e., thedorsal portion of the metencephalon, superficial to thepons, and bordered posteriorly by the lumen of thefourth ventricle and anteriorly by the mesencephalon;Klein et al. 2005).

Neuroprogenitor cells were isolated from neonatal ratcerebellum as previously described (Klein et al. 2001).Briefly, the cerebella were incubated with 0.1% Trypsin inHanks solution (15 mM HEPES, 36 mM glucose, EBSS)containing 125 U/mL DNase (Sigma-Aldrich Corporation,St. Louis, MO) for 20 min. The reaction was blocked by theaddition of Dulbecco’s modified Eagle’s medium lowglucose (DMEM-LG) containing 20% of fetal bovineserum (FBS). Tissue was minced to obtain single cellsuspension and then centrifuged, washed, and plated inplastic culture plates with DMEM/F12 (1:1) culturemedium (Invitrogen, San Diego, CA) containing 24 mMKCl, 15 mM HEPES, 36 mM glucose, 20% FBS, and10% penicillin–streptomycin (ICN Biomedicals Inc,Irvine, CA) for 30 min. Under these conditions, glialcells attached to the culture plate while neuroprogenitorsremained in suspension. Cells in suspension wereharvested by gentle centrifugation at 300×g for 10 min,suspended in culture medium, and kept at 37°C under 5%CO2 for further analysis. Cell cultures were evaluated on adaily basis by phase contrast microscopy.

Neuroprogenitor cell proliferation assay. Neuroprogenitorsisolated from the cerebella were suspended in DMEM/F12(1:1) with 10% FBS and transferred to poly-D-lysine-coatedplates at an initial cell density of 1×106 cells/mL. After24 h at 37°C under 5% CO2, cells were incubated with[3H]-thymidine (0.5 μCi/mL) for an additional 24-h period.The radioactivity incorporated by cells was measured usingUltima-GoldTM (Packard Instruments Company Inc.,Downers Grove, IL) in a Liquid Scintillation Analyzer(TRI-CARB 2100TR, Packard Instruments Company Inc.,Downers Grove, IL). Assays were conducted in triplicate.

Quantification of gene expression by real-time RT-PCR. Total RNA was extracted from cerebellar neuroproge-

UPREGULATION OF E2F1 IN CEREBELLAR NP CELLS AND CELL CYCLE ARREST 493

nitor cells using the RNeasy® Mini Kit (cat. no. 74104;Qiagen Inc., Valencia, CA) according to the manufacturer’sinstructions. RNA integrity and quantity was evaluated bymeasuring absorbance at 260 and 280 nm (NANO DROP,Qiagen Inc.). A260/A280 ratios in the range of 1.8–2.0 wereconsidered satisfactory for purity standards. Denaturingagarose gel electrophoresis was used to assess the quality ofRNA samples. One microgram of total RNA was used tosynthesize cDNA by extension with oligo-dT primers and200 U of Superscript II Reverse Transcriptase (Invitrogen LifeTechnologies). Quantitative reverse transcription polymerasechain reaction was performed in a MX 3005P thermal cycler(Stratagene, La Jolla, CA) by the SYBR Green approach(Platinum SYBR Green qPCR SuperMix-UDG, InvitrogenLife Technologies). Amplification specificity was assessedby dissociation curve analysis. Normalization of quantitativedata was based on the expression of the housekeeping genehypoxanthine guanine phosphoribosyl transferase (HPRT).Quantification was based on linear regression analysis fromstandard curves determined for each gene studied, withamplification efficiencies ranging from 90% to 100%. Allsamples were analyzed simultaneously in a single PCR runand under identical conditions. Amplification reactions,containing 2 μL of 1:10 diluted cDNA and 10 pmol of eachprimer, were conducted in duplicate. Basic amplificationcycle suggested by the manufacturer was used, with anannealing temperature of 60°C. Amplicons were generatedusing the following primer pairs: HPRT: Forward (F) 5′-CTCATG GAC TGATTATGG ACA GGA C-3′ and reverse (R)5′-GCA GGT CAG CAA AGA ACT TAT AGC C-3′;p18INK4c: (F) 5′-ACC GAA CTG GTT TTG CTG TC-3′ and(R) 5′-GGG CAG GTT CCC TTC ATTAT-3′; E2F1: (F) 5′-TGA AGG TGC AGA AAC GAC GCA T-3′ and (R) 5′-AAA GCA GTT GCA GCT GAG TGG T-3′; NESTIN: (F)5′-AGC AAC TGG CAC ACC TCA AG-3′ and (R) 5′-GGTGTC TGC AAG AGA AAG TTC-3′; NOTCH2: (F) 5′-AGGCCC CTT GCC CTC TAT GT-3′ and (R) 5′-TGATAG GTTGGG ACA ATG GTC TG-3′; SHH: (F) 5′-GCT GGA TTCGAC TGG GTC TA-3′ and (R) 5′-GAA GGT GAG GAAGTC GCT GT-3′; WNT4: (F) 5′-CAC CAT GCA CCT CTCCCA GC-3′ and (R) 5′-ATG GAG CCG ATC CGG TCCAG-3′; BCL2: (F) 5′-CGA CTT TGC AGA GAT GTC CA-3′ and (R) 5′-CAT CCA CAG AGC GAT GTT GT-3′;SMAC/DIABLO: (F) 5′-CTC GGA GCG TAA CCT TTCTG-3′ and (R) 5′-TCC TCA TCA GTG CTT CGT TG-3′.

Analysis of apoptotic cells. Rat pups were euthanized atdifferent postnatal ages (P1, P3, P6, P9, and P12) andperfused with 4% paraformaldehyde (PFA; in PBS 0.1 M,pH 7.4) solution. Brains were dissected, fixed in 4% PFAfor 14–16 h at room temperature, cryoprotected with 30%sucrose (in PBS 0.1 M, pH 7.4), and frozen into coldisopentane (Sigma-Aldrich Corporation). The cerebella

were then cryosectioned in 20-μm-thick slices, which wereused for in situ apoptosis detection by the terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP Nickand labeling (TUNEL) assay using the ApopTag in situApoptosis Kit Detection (CHEMICON International,Temecula, CA) and following the manufacturer’s proto-col. Brain sections were counterstained with 100 ng/mLDAPI (Molecular Probes, Eugene, OR) and mounted onmicroscope slides using Fluormount G. Fluorescentimages were visualized on microscopes (Nikon EclipseE600 and Carl Zeiss confocal Axiovert 100M) andcaptured by a digital camera Evolution MP Color (MediaCybernetics, Silver Spring, MD) with either QCapturePro(QImaging, Surrey, BC, Canada) or LSM Image Browser(Carl Zeiss, Jena, Germany) softwares.

Statistical analyses. All experiments used five animals perpostnatal age group (n=5 per experimental group), andthree independent experiments were carried out. Data wereanalyzed by ANOVA with Tukey as post hoc test.Significance was established at the p≤0.05 level. Resultsare expressed as mean±SD.

Results

Time course analysis of cerebellar neuroprogenitor cellproliferation during postnatal development. Neuroprogeni-tor cells could be successfully isolated from rat cerebellumat different postnatal ages, although with lower efficiencyat P9 and P12. Neuroprogenitors from P1 and P3cerebella formed neurospheres within 72 h in culture,characterized by a core of cells under clonal expansionsurrounded by more differentiated cells, some of whichdisplaying developing neurites. An example of such cellagglomerates upon culture of P1 neuroprogenitors isshown in Fig. 1. Neurospheres were rarely observed invitro in P6, P9, and P12 cell preparations.

Proliferative activity could be detected ex vivo inpopulations of neuroprogenitor cells obtained at allpostnatal ages tested (P1–P12). Cell proliferation wasbasal at P1 and attained maximum activity at P3,gradually decreasing thereafter until reaching baseline atP12. [3H]-thymidine incorporation was significantlyhigher in P3 neuroprogenitors compared with those ofcells from all other postnatal ages (Fig. 1B).

These data are consistent with a decline in prolifera-tion of granular progenitors as they become moredifferentiated along with the postnatal development ofthe cerebellum. Progression of cerebellar neurogenesiscould be attested by histological analysis (ElectronicSupplementary Materials (ESM) Fig. 1) and by time

494 SUZUKI ET AL.

course expression analysis of genes belonging to signalingpathways regulating neurogenesis. The SHH pathway isknown to be involved in the maintenance and proliferationof cerebellar neuroprogenitors. As shown in Fig. 1,expression of SHH was highest at P1 and significantlylower at P3 and P6. No SHH expression could be detectedfrom P9 to P12. These data were in agreement with the

expression pattern found for NESTIN, a marker of neuralstem cells and neuroprogenitors. NESTIN expression wasmaximum at P1, displaying a sharp decrease from P3 toP12. Accordingly, expression of WNT4 and NOTCH2genes regulating neuronal differentiation peaked later onat P9, displaying lower levels at P12, but still significantlyhigher than the expression levels detected at P1 and P3.

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Figure 1. Proliferation of neuroprogenitor cells isolated fromneonatal rat cerebellum. A Phase contrast microscopy showingneurospheres derived from P1 neuroprogenitors after 3 days in vitro(magnification, ×300; scale bar, 200 μm). B Proliferation rates ofcerebellar neuroprogenitors isolated at different postnatal ages. Cell

proliferation was estimated by incorporation of [3H]-thymidine(0.5 μCi/mL). C Expression of NESTIN, SHH, WNT4, and NOTCH2in cerebellar progenitor cells during postnatal development. Geneexpression was measured by real-time PCR using HPRT as referencegene. Statistical significance: *p<0.05; **p<0.01; ***p<0.005.

UPREGULATION OF E2F1 IN CEREBELLAR NP CELLS AND CELL CYCLE ARREST 495

E2F1 expression in cerebellar neuroprogenitors correlateswith decreased cell proliferation rate at P12. Despite itsconventional positive effect on cell proliferation, expressionof E2F1 was basal from P1 to P9, increasing significantlyonly at P12 (p<0.01, Fig. 2A). Consistent with aninhibitory effect on cell proliferation, expression of acyclin-dependent kinase inhibitor p18Ink4c was also higherat P12 compared with all other ages (p<0.01, Fig. 2B). Inaddition to an inhibitory effect on cell cycle progression,E2F1 transcription factor is also able to induce apoptosis. Inorder to verify this possibility, expression of genes encodingpro-apoptotic and anti-apoptotic proteins was evaluated inneuroprogenitor cells during the course of postnatal cerebellardevelopment. Expression of SMAC/DIABLO encoding a pro-apoptotic mitochondrial protein was significantly higher atP9 when compared with all other postnatal ages, decreasingsomewhat at P12. However, a similar expression pattern wasobserved for the anti-apoptotic gene BCL2 encodinganother mitochondrial protein. BCL2 expression wassignificantly higher at P9 and P12 when compared withP1, P3, and P6 (Fig. 2C, D). Accordingly, when evaluated

in situ, no discrepancies in apoptotic cells were foundregarding postnatal age or specific cerebellar regions(ESM Fig. 2). A few cerebellar neurons positively stainedby the TUNEL assay and counterstained with DAPI werefound in both EGL and IGL from P1 to P12.

Discussion

During postnatal cerebellar maturation, a decline in prolif-erative capacity is expected to occur as neuroprogenitorcells differentiate to constitute the cerebellar cortex. In thisstudy, the proliferative activity of rat cerebellar neuro-progenitors was found to be maximum at P3, reaching basallevels at P12. A similar peak of precursor cell proliferationhas been reported in mice, although occurring at later stagesin postnatal development, varying from P4 to P8 (Hattenet al. 1997). The basal levels of cell proliferation found atP12 were in accordance with a higher degree of celldifferentiation at this age. In addition to the histological

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Figure 2. Expression of E2F1, p18 Ink4c, SMAC/DIABLO, and BCL2 genes in cerebellar progenitor cells during postnatal development. Geneexpression was measured by real-time PCR using HPRT as reference gene. Statistical significance: *p<0.05; **p<0.01; ***p<0.005.

496 SUZUKI ET AL.

evidences, the process of cell differentiation was sup-ported by the temporal expression pattern of NESTIN andother specific genes in granular progenitors. SHHexpression was maximum at P1, decreasing significantlyafterwards until becoming undetectable as of P9. Indeed,the SHH signaling pathway is known to stimulateproliferation and inhibit the differentiation of neuroproge-nitor cells (Wechsler-Reya and Scott 1999; Dyer 2004;Lewis et al. 2004). Additionally, genes mediating neuronaldifferentiation such as WNT4 and NOTCH2 were foundmore highly expressed in neuroprogenitors isolated at P9and P12, when cerebellar foliation was well advanced.

According to Solecki et al. (2001), NOTCH and SHHsignaling pathways can stimulate the proliferation ofcerebellar granule neuron precursors by co-activatingHES1 transcription factor. In this study, although simulta-neous expression of SHH and NOTCH2 was detected ingranular progenitor cells at early postnatal ages, theirrespective expression kinetics were found to be different.No expression of SHH was found at P9 and P12, whenNOTCH2 expression attained highest levels, suggestingadditional roles for NOTCH2 during postnatal developmentof the rat cerebellum.

A somewhat unexpected expression profile was foundfor E2F1 which displayed a peak of expression in granularprogenitor cells at P12, when cell proliferation reachedbaseline levels. E2F transcription factors are usually knownby their positive regulation of the cell cycle progression.A SHH-driven mitogenic effect of E2F1 has beenrecently reported in mouse cerebellar GNPs and medul-loblastoma cells (Bhatia et al. 2011). However, some

studies attribute to the E2F1 member an inhibitory effecton cell proliferation (Morris et al. 2008). Consistent withthis notion, temporal expression profile of the cell cycleinhibitor p18Ink4c was similar to that of E2F1, withmaximum levels in granule progenitors occurring at P12.Also known as CDKN2C (cyclin-dependent kinase inhibitor2C), p18Ink4c inhibits CDK4 and CDK6, thus suppressingcell cycle (Zindy et al. 1997). According to Blais et al.(2002), the p18Ink4c gene promoter contains bindingelements for E2F, and expression of p18Ink4c has beenshown to be directly regulated by E2F1. Moreover, E2F1has been recently reported to be dispensable for progenitorcell division and to partner with Rb to facilitate the cellcycle exit in differentiating cells (Chong et al. 2009).Therefore, it seems reasonable to assume that a similarmechanism of cell cycle control could occur in neuro-progenitor cells during cerebellar maturation.

E2F1 has also been implicated in the regulation ofapoptosis (Xie et al. 2006; Morris et al. 2008). E2F1 iscapable of inducing apoptosis through the p53 pathway(Wu and Levine 1994; Phillips and Vousden 2001). Xie etal. (2006) reported that the E2F1 is also able to induce theexpression of SMAC/DIABLO, directly modulating theapoptotic mitochondrial pathway. In this study, however,the expression of both SMAC/DIABLO and BCL2 wereequivalent and significantly increased at P9 and P12,indicating no predisposition toward pro-apoptotic condi-tions. The results obtained by the TUNEL techniquerevealed only a small amount of apoptotic cells in thecerebellum, detected in both EGL and IGL. No obviouscorrelation with the degree of cerebellar maturation was

NOTCH2 WNT4

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Figure 3. Temporal expressionpattern correlations among SHH,NOTCH2, WNT4, E2F1, andp18Ink4c genes in neuroprogeni-tor cells during postnataldevelopment of rat cerebellum.A proposed model for E2F1regulation of neuroprogenitorcell proliferation is depicted inthe scheme, involving p18Ink4c

and interaction with the WNTpathway.

UPREGULATION OF E2F1 IN CEREBELLAR NP CELLS AND CELL CYCLE ARREST 497

noticed. In addition, maximum SMAC/DIABLO expressionoccurred long before the peak of E2F1 expression,suggesting alternative mechanisms of SMAC/DIABLOregulation.

Another interesting temporal gene expression correlationwas found between E2F1 and WNT4. A recent studyreported that expression of E2F1 can be regulated by thecanonical WNT pathway, an effect mediated by TCF andLEF-1 transcription factors (Zhou et al. 2008; Abramovaet al. 2010). On the other hand, Hughes and Brady (2005)reported that the E2F1 positively regulates the expressionof tumor suppressor gene Axin2, which is an inhibitor ofWNT. Although direct evidences of such molecularinteractions have not been generated in this study, thetemporal gene expression correlations detected duringcerebellar postnatal development support a regulatoryfeedback loop with E2F1 as a central player modulatingthe WNT pathway in cerebellar neuroprogenitor cells(Fig. 3).

Altogether, these findings strengthen a potential role ofE2F1 in inhibiting the proliferation of GCP at the end of thepostnatal development of rat cerebellum. Such regulationmediated by E2F1 should be important for appropriateneurogenesis to occur, given that abnormalities in thisprocess are related to the formation of different tumors(Johnson and DeGregori 2006). In the CNS, there areexperimental evidences supporting the involvement of E2Fgenes in the development of medulloblastoma (Olson et al.2007), most of them focusing on a pro-oncogenic activity.Considering an additional potential tumor suppressor roleof E2F1, our results suggest that an aberrant expression ofE2F1 leading to a loss of function could also affect theproliferation of GNPs and contribute to pathologicalcerebellar development.

Acknowledgments This work was supported by grants fromFAPESP, CNPq, and INCT-CETGEN. Daniela Emi Suzuki andCarolina Batista Ariza were recipients of CAPES and FAPESPfellowships, respectively. Oswaldo K. Okamoto and Marimélia A.Porcionatto are recipients of research fellowships from CNPq.

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