Conditional activation of Bmi1 expression regulates self-renewal, apoptosis, and differentiation of neural stem/progenitor cells in vitro and in vivo

TISSUE-SPECIFIC STEM CELLS

Conditional Activation of Bmi1 Expression Regulates Self-renewal,

Apoptosis, and Differentiation of Neural Stem/Progenitor Cells

In Vitro and In Vivo

GOKHAN YADIRGI, VERONICA LEINSTER, SERENA ACQUATI, HEETA BHAGAT, OLGA SHAKHOVA, SILVIA MARINO

Blizard Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, Queen

Mary University of London, London, United Kingdom

Key Words. Bmi1 • Neural stem cells • Self-renewal • Neuronal differentiation • Survivin • Brain tumors

ABSTRACT

The Polycomb group protein Bmi1 is a key regulator ofself-renewal of embryonic and adult central nervous sys-tem stem cells, and its overexpression has been shown to

occur in several types of brain tumors. In a Cre/LoxP-based conditional transgenic mouse model, we show thatfine-tuning of Bmi1 expression in embryonic neural stem

cell (NSC) is sufficient to increase their proliferation andself-renewal potential both in vitro and in vivo. This is

linked to downregulation of both the ink4a/ARF and thep21/Foxg1 axes. However, increased and ectopic prolifera-tion induced by overexpression of Bmi1 in progenitors

committed toward a neuronal lineage during embryonic

cortical development, triggers apoptosis through a survi-vin-mediated mechanism and leads to reduced brain size.Postnatally, however, increased self-renewal capacity of

neural stem/progenitor cells (NSPC) is independent ofFoxg1 and resistance to apoptosis is observed in neuralprogenitors derived from NSC-overexpressing Bmi1. Neo-

plastic transformation is absent in mice-overexpressingBmi1 aged up to 20 months. These studies provide strong

evidence that fine tuning of Bmi1 expression is a viabletool to increase self-renewal capacity of NSCs both in vitroand in vivo without eliciting neoplastic transformation of

these cells. STEM CELLS 2011;29:700–712

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION

Stem cells are self-renewing multipotent cells that give rise toa differentiated progeny. The balanced coordination of thesetwo stem cell fates, that is, self-renewal and differentiation, isessential for embryonic development and tissue homeostasisin the adult.

During embryonic development of the mammalian centralnervous system (CNS), proliferative radial glial cells thatdivide at the ventricular surface of the developing neocortexare considered to be neural stem cells (NSC). This is becausethey self-renew and give rise to cortical neurons, astrocytes,and oligodendrocytes. In the postnatal mammalian telencepha-lon, self-renewing and multipotent NSC exist only in specificbrain areas, namely the subventricular zone (SVZ) and thedentate gyrus of the hippocampus, where they persist through-out life [1]. The relationship between embryonic and postnatalNSC is currently ill-defined, although the latter are believedto originate from embryonic NSC [1].

Bmi1 is a member of the polycomb group (PcG) genefamily of chromatin modifiers and transcriptional repressors,

the essential role of which in development and homeostasisof the CNS was first highlighted by studies on knockoutmice. Bmi1�/� mice displayed impaired self-renewal andmaintenance of SVZ NSC leading to their postnatal depletion[2, 3] as well as impaired proliferation of postnatal granulecell progenitors leading to depletion of granule neurons andconsequently to ataxia [4, 5]. The molecular mechanisms ofBmi1 function in postnatal NSC and granule cell progenitorsare mediated in part by transcriptional repression of theink4a locus [6]. This locus encodes two cell cycle inhibitors,p16Ink4a and p19ARF [7], the activities of which increase withpostnatal age and have been linked to cellular senescence[2].

However, concomitant deletion/suppression of p16Ink4a orp19ARF or both does not completely rescue the above-men-tioned defects in self-renewal of NSC and proliferation ofgranule cell progenitors derived from Bmi1�/� mice [8, 9].Another cell cycle inhibitor, namely p21WAF1/Cip1, contributesto mediating Bmi1 function in both developmental contexts[10, 11]. Other NSC functions, such as migration, have alsobeen shown to be controlled by Bmi1 in an ink4a-independentmanner [12].

Author contributions: G.Y., V.L., and S.A.: collection and/or assembly of data, data analysis and interpretation, manuscript writing;H.B.: collection and/or assembly of data; O.S.: conception and design, collection and/or assembly of data, data analysis andinterpretation; S.M.: conception and design, data analysis and interpretation, financial support, manuscript writing. G.Y. and V.L.contributed equally to this work.

Correspondence: Silvia Marino, M.D., Blizard Institute of Cell and Molecular Science, 4 Newark Street, London E1 2AT, UnitedKingdom. Telephone: 44-207-882-2585; Fax: 44-207-882-2180; e-mail: [email protected] Received August 26, 2010; accepted forpublication January 21, 2011; first published online in STEM CELLS EXPRESS February 8, 2011. VC AlphaMed Press 1066-5099/2009/$30.00/0doi: 10.1002/stem.614

STEM CELLS 2011;29:700–712 www.StemCells.com

Elevated Bmi1 expression induces tumorigenesis in thehematopoietic system in transgenic mice [13]. Increasedexpression of BMI1 has been demonstrated in humans, notonly in hematological malignancies, such as high-grade lym-phomas [14] but also in neural tumors, for example, medullo-blastomas and neuroblastomas [4, 15]. A tight regulation ofBmi1 expression might therefore not only be crucial for main-taining a balance between self-renewal and differentiation orsenescence of NSC, but it may also play a role in CNStumorigenesis, when deregulated.

Overexpression of Bmi1 in nestinþ NSC and neural pro-genitor cells (NPC) during CNS development has been shownto have little effect in vivo albeit increasing self-renewal, pro-liferation, and neuronal differentiation of these cells in vitro.Neoplastic transformation of these cells leading to braintumors was not seen in these mice [16]. However, virus-medi-ated induction of Bmi1 overexpression in NSC/NPC duringembryonic cortical development and in the postnatal brainincreased their survival and self-renewal in vivo [17]. More-over, brain tumors were observed in a percentage of thesemice. Although He et al. showed that Bmi1 mediatedincreased self-renewal in vitro through downregulation of theink4a locus, Fasano et al. showed a new association betweenBmi1 and Foxg1 in mediating increased self-renewal in vivoin their experimental set up.

In the light of these partially contradictory findings, it iscurrently unclear (a) whether or how Bmi1 overexpressionincreases the self-renewal potential of NSC in vivo, (b)whether Bmi1 overexpression influences survival and differ-entiation of non-stem neural progenitors in vivo, (c) what isthe relative contribution of the ink4a/ARF and Foxg1/p21axes to the observed phenotypes, and (d) whether Bmi1 over-expression in NSC and cells derived thereof leads to tumorformation in the mouse.

Here, we describe a Cre/LoxP-based conditional trans-genic approach to induce gain of function of Bmi1 in embry-onic and postnatal NSC and in all their progeny in vivo.

MATERIALS AND METHODS

See Supporting Information.

RESULTS

Generation of a Mouse Line for Conditional Gain ofFunction Expression of Bmi1 in the CNS

The pccall2 construct [18] was chosen to drive conditionalexpression of Bmi1. It is composed of a cytomegalovirus(CMV) enhancer/chicken b-actin promoter followed by abgeo (LacZ/neomycin) fusion gene and three copies of theSV40 polyA signal flanked by LoxP sites. The full-length mu-rine Bmi1 cDNA was inserted after the second LoxP site, infront of an IRES-eGFP (enhanced green fluorescent protein)sequence (Supporting Information Fig.S1A). The CMVenhancer/chicken b-actin promoter drives b-galactosidaseexpression and confers neomycin resistance, however, on Cre-mediated excision of bgeo, Bmi1 and eGFP will be simulta-neously expressed [19]. The pccall2-Bmi1 vector was intro-duced into TC-1 mouse ESCs. Four ESC clones showing highLacZ expression (Supporting Information Fig. S1B) and sin-gle copy integration (Supporting Information Fig.S1C) wereselected and electroporated with a Cre recombinase expres-sion vector to test the induction of Bmi1 overexpression.

Clones IB5 and IE1 showed negative LacZ expression andBmi1 protein level comparable with a medulloblastoma sam-ple (Supporting Information Fig. S1D). They were thereforeselected for injection into blastocysts to generate chimericmice. Germline transmission and line establishment wasachieved for clone IB5, from now on referred to asSTOPFloxBmi1.

Conditional Activation of Bmi1 Expression in theDeveloping Neocortex Increases Proliferation ofNSC in the Ventricular Zone and Induces Apoptosisof Ectopically Proliferating NPCs

Initially, we analyzed the expression pattern of the newly gen-erated transgenic mouse line in vivo prior to conditional acti-vation of Bmi1 expression. X-gal staining revealed robustLacZ expression in all layers of the developing neocortex atE12.5 (Fig. 1B–1D), including NSC located in the apicalregion of the ventricular zone (VZ; Fig. 1D, arrow). We,therefore, conclude that the transgene is active in embryonicNSCs and in their progeny during embryonic forebrain devel-opment in vivo.

To assess the effect of Bmi1 upregulation in NSC andNPC, we used a Nestin-Cre transgenic line, which has beenshown to successfully recombine loxP-flanked sequences inspecific tissues and to effectively express Cre recombinase inthese cells on paternal inheritance of the transgene [20](Fig. 1A). Loss of LacZ expression was indicative of recom-bination in all cells of the VZ and in a significant proportionof cells throughout all layers of the developing neocortex(Fig. 1E and 1F). To confirm activation of the construct, GFPimmunolabeling was performed on adjacent sections. Wide-spread cytoplasmic staining was seen in double mutant micewith an expression pattern similar to that seen in the X-galstaining; importantly, expression of the construct was notedin NSC (Fig. 1I, inset). Endogenous Bmi1 expression wasseen in NSC of the apical region of the VZ and the stainingwas less intense in progenitor cells throughout the developingneocortex (Fig. 1H). Double mutant mice showed higherexpression of Bmi1 in the VZ, which was thicker and showedclustering of cells strongly expressing Bmi1 (Fig. 1J). Stron-ger expression of Bmi1 was noted also in progenitor cellslocated in the intermediate zone (IZ) of double mutant mice(Fig. 1J) as compared with control (Fig. 1H), however, thesecells seem to overexpress Bmi1 less strongly than NSC. It iscurrently unclear why the expression patterns of b-galactosi-dase, GFP, and Bmi1 differ, although it is possible that theprotein turnover varies among different proteins in differentcell types.

Next, we assessed the impact of Bmi1 overexpression onproliferation and differentiation of NSC, and on the progenitorcells derived thereof in E12.5 Nestin-Cre;STOPFloxBmi1embryos compared with controls. Proliferation was assessedby immunostaining with phospho-histone H3 (pH3). Wefound considerably more proliferating cells in the VZ (Fig.1N and 1Q as compared with Fig. 1K), which were not onlylocalized at the apical region, as in the control, but also clus-tering into small aggregates close to the basal region of theVZ and within the IZ. More prominent staining for the radialglia marker RC2 was also noted in mutant mice (Fig. 1T,1Y). While analyzing these slides, we observed, in the propi-dium iodide staining, frequent condensed nuclei in the doublemutant embryos, suggestive of apoptotic cells. To test this hy-pothesis, we performed TUNEL staining, which showed mas-sive apoptosis in the area where ectopic proliferation wasnoted, mainly in the IZ and occasionally in the outermostlayers of the differentiating neocortex (Fig. 1O and 1R

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Figure 1. Analysis of forebrain development in Nestin-Cre;STOPFloxBmi1 at E12.5. (A): Schematic representation of the conditional Bmi1transgenic construct prior and upon Cre-mediated recombination. Expression of the transgene in the developing neocortex as assessed by X-galstaining of sagittal section, overview (B) and higher magnifications (C, D) of the area boxed in (B). X-gal staining is retained only in the vesselson Nestin-Cre recombination (E, F). Widespread expression of GFP in double transgenic mice ([I, G], detail of GFP expression in ventricularzone (VZ) NSC is shown in the inset) and increased number of NSC expressing higher levels of Bmi1 most prominently in the apical VZ (J, H).Increased and ectopic proliferation of neuronal progenitor cell (NPC) in Nestin-Cre;STOPFloxBmi1 as assessed by pH3 staining (K, N, Q).Increased TUNELþ apoptotic NPC (L, O) and cCSP3þ cells (M, P) but no increase in the number of apoptotic cells in VZ NSC (R). Moreprominent RC2 staining in the VZ of double-mutant mice (T, Y). Reduced number of Tbr2þ intermediate progenitors in double-mutant mice (U,

Z, V, A0). Quantification of the findings is shown in (S). *, p < .05; **, p < .01; ***, p < .001 with Student’s t test, Error bars represents SD, n� 3. Expression of neuronal differentiation markers, DCX and bIII-tubulin (W, B0, X, C0), in NPC which escaped apoptotic death. Scale bar ¼ 2mm (B), 250 lm (C, E, H, N, L, O), 125 lm (D, F, G, I, H, J, M, P, Q, R, T–C0). Abbreviations: DCX, doublecortin; DAPI, 40,6-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; GFP, green fluorescent protein; IRES, internal ribosome entry site; LV, lateral ventricle;pA, polyA; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

compared with 1L). Interestingly, no increase in apoptosiswas noted in the VZ, implying that NSC are not shunted intoapoptosis upon Bmi1-mediated induction of proliferation(Fig. 1R). Similar findings were seen in immunostaining forcleaved Caspase3 (cCSP3), a more specific marker of apopto-sis (Fig. 1M, 1P). Immunostaining for Tbr2, a marker of dif-ferentiated preplate neurons at E12.5, showed a reduction inthe overall number of progenitor cells reaching this stage ofdifferentiation as a consequence of the cell death occurred(Fig. 1U, 1Z, 1V, 1A0). However, staining for doublecortin(DCX) and bIII-tubulin revealed that the surviving cellsachieved appropriate differentiation (Fig. 1W, 1B0, 1X, 1C0).Quantification of positive cells for markers pH3, cCSP3, andTbr2 is shown (Fig. 1S).

H&E staining of sagittal sections of the developing cortexat E16.5 suggested overall reduction of the size of the fore-brain (Fig. 2A, 2C); however, not all layers appeared equallyaffected—that is, the IZ, the subcortical plate, and the corticalplate showed the most pronounced reduction in thickness(Fig. 2B, 2D, 2E [quantification]). Remarkably, the overallthickness of the VZ/SVZ layers was not affected (Fig. 2B,2D, 2E [quantification]). X-gal staining on adjacent sectionsconfirmed widespread recombination in all cortical layers(Fig. 2A, 2C, inset). Immunostaining for Sox2, a marker ofVZ progenitors, allowed us to discriminate between SVZ andVZ and although measurements of both layers did not revealsignificant difference in their thickness, a trend toward a thin-ner SVZ and a thicker VZ was observed (Fig. 2F, 2G, 2H[quantification]). Immunostaining for pH3 and quantificationof the positive cells confirmed that also at E16.5 there was anincreased number of proliferating apical VZ progenitors (Fig.2L, 2M, 2N [quantification]), whereas the number of prolifer-ating progenitors in the SVZ and the IZ were not affected(data not shown). Increased proliferation of cells located inVZ/SVZ was confirmed also with an independent marker,BrdU (Fig. 2J, 2K, 2I [quantification]). The number of Tbr2þintermediate neuronal progenitors was unaffected at this laterdevelopmental time point and neuronal differentiation wasretained as assessed by expression of bIII-tubulin and DCX(data not shown).

In conclusion, activation of Bmi1 expression during em-bryonic forebrain development leads to increased prolifera-tion of VZ stem cells. However, increased and ectopicproliferation of migrating and differentiating neuronal pro-genitors shunts them into apoptotic death at E12.5, leadingto a reduced number of these progenitors and eventually toa reduced size of the developing neocortex in the mutantmice.

Embryonic Basic Fibroblast Growth Factors NSPC-Overexpressing Bmi1 Show Increased Self-RenewalCapacity and Increased Proliferation In Vitro

To further investigate functional effect of increased Bmi1expression in embryonic NSC and NPC, two in vitro culturesystems were used, the neurosphere assay [21] and the adher-ent NSC culture system [22, 23].

The telencephalon of Nestin-Cre;STOPFloxBmi1 and con-trol mice were isolated at E16.5, dissociated into a single-cellsuspension and plated into serum-free medium containing epi-dermal growth factor and basic fibroblast growth factors. Inthese conditions, mitogen-responsive cells [NSC and otherprogenitor cells, here referred to as neural stem/progenitorcells (NSPC)] proliferate and form three-dimensional struc-tures, named neurospheres (NS). Repeated cycles of harvest-ing and dissociation of NS followed by replating of the sin-

gle-cell suspension under the same culturing conditions wereperformed to enrich for NS-forming NSPC.

X-gal staining performed on controls (STOPFloxBmi1)and double transgenic NS confirmed expression of the trans-gene in all cells of the NS and showed a loss of expression inthe double mutant (Fig. 3A–3D). Immunohistochemical stain-ing for Bmi1 and GFP confirmed that recombination and acti-vation of the construct had occurred in the double mutant NS(Fig. 3B–3E, 3C–3F). Quantitative reverse transcription poly-merase chain reaction (qRT-PCR) and semiquantitativeassessment of protein expression by Western blot analysiswas performed on NS of Nestin-Cre;STOPFloxBmi1 and con-trols and they confirmed overexpression of Bmi1 as observedin situ (Fig. 3G, 3H).

To determine whether the in vitro self-renewal capacity ofNSPC isolated from Nestin-Cre;STOPFloxBmi1 at E16.5would be increased in vitro, we dissociated and replated terti-ary NS over nine passages as a measure of their self-renewalcapacity. A significant increase in NS frequency was consis-tently noted in the cultures overexpressing Bmi1 (Fig. 3I, p <.001). Similar results were obtained when NS were dissoci-ated, replated in serial dilutions, and the total number of NSarising after 6–7 days counted (p < .001), therefore, implyingthat the effect was not dependent on the original cell densityof the culture (Supporting Information Fig.S2A, blue andpurple bars).

Next, we analyzed whether acute upregulation of Bmi1expression would elicit a similar effect. NS isolated fromSTOPFloxBmi1 E16.5 embryos were dissociated and single-cell cultures infected with Adeno-Cre [8 multiplicity ofinfection (MOI)] or Adeno-GFP (8 MOI) viruses. A total of90%–100% of the NS originating thereof showed lack ofLacZ expression upon Adeno-Cre infection, whereas LacZexpression was retained in NS infected with Adeno-GFP(data not shown). Here, GFP positivity was seen in almost100% of the NS and confirmed the high infection rate andtherefore the efficient gene delivery of the adenoviral systemused (data not shown). To control nonspecific effects ofinfection and overexpression of any exogenous proteins, NSwere isolated from control (nontransgenic) littermates andinfected in parallel with Adeno-Cre and Adeno-GFP. Allassays were performed on these cultures under the same ex-perimental conditions as described before and no significantdifference was seen between the two viruses (data notshown). Infection with Adeno-GFP will be used throughoutthe manuscript as control for all experiments where infectionwith Adeno-Cre was performed. We showed that self-renewal of NSPC was increased upon acute induction ofBmi1 overexpression in a similar fashion as upon Nestin-Cre mediated induction during embryonic development (Sup-porting Information Fig.S2A, green and red bars).

In accordance with our in vivo observation, increased pro-liferation of NSPC on activation of Bmi1 expression was con-firmed in culture by means of EdU labeling (Supporting Infor-mation Fig. S2B; p < .01) and a colorimetric assay, alamarblue, measuring the metabolic activity of the cells (Fig. 3J;p < .05 and p < .01).

To address the question whether the observed increase inproliferation on Bmi1 overexpression was due to an effecton NSC rather than on more committed precursors, we usedan adherent culture system, in which, acutely dissociated NSare seeded at clonal concentration (10,000 cells per centime-ter square) on Matrigel-coated flasks in expansion medium.Under these conditions, cultures are composed of a homoge-neous population of NSC [23], which is characterized byuniform expression of NSC markers such as Nestin, Sox2,Musashi (Fig. 3K–3N), and lack of differentiated cells as


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assessed by immunostaining for glial fibrillary acidic protein(GFAP), O4, and bIII-tubulin (data not shown). NSC can beinduced to efficient trilineage differentiation upon switchingto a differentiation inducing medium [22, 23]. Growth

curves, obtained by plotting cell counts over five passages,revealed a significant increased proliferation in double trans-genic NSC as compared with controls (Fig. 3O; p < .001and p < .01).

Figure 2. Analysis of forebrain development in Nestin-Cre;STOPFloxBmi1 at E16.5. Reduced brain size in double mutant, H&E staining (A,

C), and complete loss of X-gal staining confirming full recombination (inset). Higher magnification showing reduced cortical thickness (B, D)

and layer measurements shows that CP, SP, and IZ are most affected (E). Sox2 immunostaining reveals no significant difference in the thicknessof the VZ (F, G, H). Increased proliferation, as assessed by 2-hour BrdU pulse and detection, is observed in VZ/SVZ ([J, K, I], details of thelabeling in VZ/SVZ is shown in the inset) and confirmed with an independent proliferation marker, pH3, in the apical VZ (L, M, N). *, p < .05;***, p < .001 with Student’s t test, Error bars represents SD, n � 3. Scale bar ¼ 2 mm (A, C); 1 mm (J, K); 250 lm in inset (A, C), (B, D, F,G); 125 lm in inset (J, K), (L, M). Abbreviations: BrdU, bromodeoxyuridine; CP, cortical plate; IZ, intermediate zone; SP, subcortical plate;VZ, ventricular zone; SVZ, subventricular zone.

704 Bmi1 Overexpression in Neural Progenitor Cells

Figure 3. Increased self-renewal and proliferation of neural stem/progenitor cells (NSPC)-overexpressing Bmi1 in vitro. Loss of b-galactosidaseactivity (A, D) and gain of GFP and Bmi1 expression in neurospheres (NS) originating from NSPC isolated from Nestin-Cre;STOPFloxBmi1 (B,

E, C, F). Increased Bmi1 expression in Nestin-Cre;STOPFloxBmi1 NS is confirmed by quantitative reverse transcription polymerase chain reac-tion (G) and Western blot analysis (H). Increased self-renewal, as assessed by counting the number of NS originating from the same number ofplated cells over several passages (I). Increased alamar blue reduction over 72 hours (J) indicates increased proliferation of NSPC-overexpressingBmi1. NSC cultured on adherent conditions (K) are homogeneously expressing Nestin (L), Musashi1 (M), and Sox2 (N). Growth curves showincreased proliferation in Bmi1-overexpressing cultures (O). Overexpression of Bmi1 did not significantly increase apoptotic death in NSC cul-tured in adherent conditions (Q). However, it significantly increased the number of differentiating progenitors undergoing apoptosis in NS origi-nating from these NSPC (P). *, p < .05; **, p < .01; ***, p < .001 with Student’s t test; Error bars represents SD, n � 3. Scale bar ¼ 125 lm.Abbreviations: DAPI, 40,6-diamidino-2-phenylindole; DIV, days in vitro; GFP, green fluorescent protein.


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In summary, in vitro assays revealed that chronic andacute enhancement of Bmi1 expression increases (a) self-renewal capacity of NSPC and (b) proliferation of NSC in asimilar fashion.

A Cell Autonomous Mechanism Causes ApoptoticDeath of Differentiating NPC-Overexpressing Bmi1

In vivo analysis of embryonic cortical development of Nestin-Cre;STOPFloxBmi1 mutants had demonstrated that NSClocated in the VZ were not shunted into apoptotic death de-spite Bmi1 induced increased proliferation (Fig. 1R). AnnexinV labeling followed by flow cytometric analysis was per-formed on NSC isolated from Nestin-Cre;STOPFloxBmi1 andcultured in adherent conditions. In accordance with the invivo observation, no significant differences were notedbetween cultures overexpressing Bmi1 and control (Fig. 3Q).

We observed a striking increase of apoptotic cells in theIZ of Nestin-Cre;STOPFloxBmi1 mutants (Fig. 1O, 1R, 1P).The IZ contains mainly progenitors committed to a neuronalfate that originated from NSC upon asymmetric cell divisionand are migrating away from the ventricle. To determinewhether cell death was due to a cell autonomous or to a non-cell autonomous mechanism, we assessed the apoptotic rate ofNPC originating from NSPC in a nonphysiological environ-ment such as a cultured NS. Annexin V staining revealed asignificantly higher percentage of apoptotic cells in Nestin-Cre;STOPFloxBmi1 NS (Fig. 3P; p < .05).

These finding suggest that increased Bmi1 expression istolerated in VZ NSC leading to an increase of the stem cellpool, both in vitro and in vivo, but its downregulation isessential for survival of committed neuronal progenitorsmigrating away for the ventricular surface. Upregulation ofBmi1 expression in these progenitors shunts them into apopto-sis through a cell autonomous mechanism.

Increased Pool of Undifferentiated NSPC andDelayed Neuronal Differentiation in NS-Overex-pressing Bmi1

CNS-derived NS are morphologically and functionally hetero-geneous, in fact, they contain not only self-renewing NSPC(�1%–10% of the total number of cells) but also committedglial and neuronal progenitors as well as differentiated cells.

To confirm that the increased number of apoptotic cellswas mainly affecting neuronal committed progenitors and toanalyze the impact of Bmi1 overexpression on glial differen-tiation, we looked at the cellular composition of NS-overex-pressing Bmi1. Immunolabeling of OCT-embedded NS forMash1, an early marker of neuronal differentiation, revealed asignificant decrease in the number of positive cells in Bmi1-overexpressing NS (Fig. 4A, 4B, 4G). Concomitant stainingfor cCSP3 confirmed increased apoptosis occurring uponBmi1 overexpression, although no increase in the number ofdouble-positive cells was noted, in accordance with progeni-tors being shunted into apoptotic death at an earlier stage,possibly at the time of commitment (Fig. 4C–4F, 4G). Expres-sion analysis of markers of glial differentiation, NG2, A2B5,GFAP, in NS either by flow cytometry (Fig. 4J, 4M [quantifi-cation]) or by immunohistochemical analysis (Fig. 4H, 4I,4K, 4L), revealed a significantly higher percentage of cellscoexpressing GFAP and NG2 (79%), GFAP and A2B5 (73%),NG2 and A2B5 (54%) in Bmi1-overexpressing NS as com-pared with those of control NS, respectively 41%, 13%, and17% (p < .001 and p < .01).

Upon plating onto laminin-coated surface and withdrawalof growth factors, NS attach to the substrate and cells startmigrating radially from the NS body toward the periphery,

while they differentiate into neurons, astrocytes, and oligoden-drocytes. As NSPC differentiate, nestin expression is downre-gulated and expression of distinct neuronal and glial markerssuch as bIII-tubulin, GFAP, and O4 is activated [24]. Differ-entiation of NS is therefore a useful assay to study differentia-tion properties of NSPC in a simplified in vitro context.

We analyzed the impact of increased Bmi1 expression onthe differentiation capacity of NSPC contained in NS derivedfrom Nestin-Cre;STOPFloxBmi1 compared with controls after5 days in vitro (DIV5). The percentage of cells expressingnestin was higher (36% vs. 13%, p < .01) in Bmi1-overex-pressing cultures (data not shown and Fig. 4V). Moreover, theexpression of Musashi, another marker of undifferentiated/uncommitted NSPC [25], was retained in a significantlyhigher number of cells (50% vs. 28%, p < .01) in the mutantcultures (Fig. 4N, 4O, 4V). Although the percentage ofGFAPþ astrocytes and O4þ oligodendrocytes was similar inboth cultures (data not shown), a significant reduction in thenumber of bIII-tubulinþ neurons was noted in Nestin-Cre;S-TOPFloxBmi1 cultures (Fig. 4T, 4U, 4V; p < .01). The per-centage of cells expressing markers of immature glial and oli-godendroglial progenitors such as A2B5 (28% vs. 10%, p <.01) and NG2 (19% vs. 10.9%, p < .01) as well as coexpress-ing GFAP and A2B5 (20% vs. 3.5%, p < .01) and GFAP andNG2 (17% vs. 10.5%, p < .001) was significantly higher incultures overexpressing Bmi1 (data not shown and Fig. 4P,4Q, 4R, 4S, 4V). Similar analysis performed at DIV9 showedhigher number of O4þ oligodendrocytes (10% vs. 6.5%, p <.01), as expected, but also a higher number of bIII-tubulinþneurons (8.5% and 5.4% respectively, p < .01; Supporting In-formation Fig. S3A, S3E and S3B, S3F, S3K).

These results suggest that increased expression of Bmi1maintains NSPC in an undifferentiated state, both in NS andupon induction of differentiation and that this does nothamper initiation of glial/oligodendroglial differentiation.Downregulation of Bmi1 expression, however, is crucial forinitiation of neuronal differentiation, an effect which can beovercome at later stages of differentiation.

Overexpression of Bmi1 in Postnatal SVZ NSCIncreases Self-Renewal Capacity In Vitro andIncreases the Number of NSC In Vivo

Next, we analyzed the effect of Bmi1 overexpression on post-natal NSPC isolated from the SVZ. We studied the expressionpattern of STOPFloxBmi1 transgenic line at two postnataltimepoints, P7 and P70, by X-gal staining. At P7, LacZexpression was seen throughout the cerebral cortex, in thehippocampus, the basal ganglia, and notably, in all cells ofthe SVZ (Supporting Information Fig.S4A). A similar expres-sion pattern was seen at P70 (Supporting InformationFig.S4B). Double labeling with X-gal and Nestin showedexpression of the transgene in the vast majority of cells popu-lating the SVZ at P7 (Fig. 5A, 5B), including scattered cellslocated within and underneath the ependymal lining. Doublelabeling of these cells for X-gal and GFAP, a marker of NSCin the postnatal and adult SVZ (Fig. 5C, 5D), confirmed thatthe transgene was indeed expressed in SVZ NSC. Similaranalysis performed on adult brains (P70) showed comparableresults (Fig. 5E, 5F), in fact, the lower cellularity of the SVZallowed us to conclude that SVZ NSC expressed LacZ(Fig. 5F, arrowhead). Moreover, NS originating from NSPCisolated from the SVZ of Bmi1STOPFlox mice at p7 and p70were strongly and homogeneously LacZ positive (SupportingInformation Fig.S4C), indicating that the construct was activein these cells.


Following this, we induced Bmi1 overexpression in cul-tured P7 SVZ NSPC by adenoviral-mediated Cre delivery, aspreviously described. Similarly to what was observed withembryonic NSPC, 90%–100% of NS originating after infec-tion of dissociated NSPC lacked LacZ expression (SupportingInformation Fig.S4E). Infection with Adeno-GFP, performedin parallel, confirmed an infection rate of 100% (SupportingInformation Fig.S4D) and retained LacZ expression on adeno-viral infection (data not shown).

As predicted, overexpression of Bmi1 in postnatal NSPCled to increased self-renewal capacity of these cells, indicatedby an increased frequency of NS arising from the same num-ber of plated cells over nine passages (Fig. 5G) and in a clon-ogenic assay (Supporting Information Fig. S4F, p < .001 and

p < .01). As for embryonic NSPC, the proliferation rate ofthe SVZ NSPC upon dissociation was significantly higher incultures overexpressing Bmi1 as compared with control GFP-infected cultures (alamar blue assay, Fig. 5H, p < .01 or p <.05). However, when the apoptotic rate was measured in NPCcontained within NS, a significant reduction in the percentageof apoptotic cells was seen in NS-overexpressing Bmi1 (Fig.5I, p < .05). Similar results were obtained at p70 (data notshown).

In vivo analysis of the SVZ of Nestin-Cre;STOPFloxBmi1mice at P7 revealed increased number of label retainingBrdUþ cells located in the subependymal area of the lateralwall of the lateral ventricle (Fig. 5J, 5K, 5L [quantification])as well as small clusters of GFAPþ and GLASTþ cells in

Figure 4. Analysis of differentiation of neural stem/progenitor cells and of the cellular composition of NS-overexpressing Bmi1. Depletion ofMash1þ cells in NS-overexpressing Bmi1 (A, B). Increased number of apoptotic cells as assessed by cleaved caspase3 staining (C, D). Quantifi-cation of the findings is shown in (G). Increased number of NG2;A2B5 (histological [H, I] and flow cytometric analysis [J]), GFAP;A2B5 (K,

L) in NS upon activation of Bmi1 expression. Quantification of the findings is shown in (M). Upon induction of differentiation, increased numberof cells expressing Musashi (N, O) and coexpressing A2B5;NG2 (P, Q) A2B5;GFAP (R, S) and reduction of the number of bIII-tubulin-positivecells (T, U) is seen at DIV5 in Bmi1-overexpressing cells. *, p < .05; **, p < .01; and ***, p < .001 with Student’s t test, Error bars representsSD, n � 4. Scale bar ¼ 125 lm (A–F, H, I, K, L), 250 lm (N–U). Abbreviations: cCASP3, cleaved caspase3; DAPI, 40,6-diamidino-2-phenylin-dole; DIV, days in vitro; GFAP, glial fibrillary acidic protein; NS, neurospheres.


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Figure 5. Overexpression of Bmi1 in postnatal neural stem/progenitor cells in vitro and in vivo. In the subventricular zone (SVZ) at P7, severalX-galþnestinþ (A, B) and X-galþ;GFAPþ (C, D) cells are identified. At P70 colocalization is seen between X-gal and GFAP in several sub-ependymal NSC ([E, F], arrowhead).Increased self-renewal (G) and increased proliferation (H) of postnatal NSC/NPC-overexpressing Bmi1. Re-silience to apoptosis is seen in differentiating progenitors contained within NS-overexpressing Bmi1 (I). *, p < .05; **, p < .01; ***, p < .001with Student’s t test. Increased number of label retaining BrdUþ cells at the lateral wall of the lateral ventricle of double transgenic P7 mice ([J,K], inset shows BrdU/DAPI). Quantification of the findings is shown in (L). **, p < .01 with ANOVA. Small clusters of GFAPþ;GLASTþNSC are identified in the lateral wall of the SVZ of the mutant mice (M, N). Reduced overall size (O, P), and weight (Q) of the adult brain ofNestinCre;STOPFloxBmi1 at 8 months. ***, p < .001 with Student’s t test. Error bars represents SD, n � 3. Scale bar ¼ 250 lm (A–D), 125lm (E, F, J, K), 62.5 lm (M, N). Abbreviations: BrdU, 5-bromo-2-deoxyuridine; DAPI, 40,6-diamidino-2-phenylindole; GFAP, glial fibrillaryacidic protein; GFP, green fluorescent protein; GLAST, L-glutamate/D-aspartate transporter.


the same areas (Fig. 5M–5N, arrowheads) which were notseen in control animals. These finding are in keeping with thein vitro data and raise the possibility that the resident NSCpool is increased in vivo also postnatally.

We show here that increased Bmi1 expression in postnatalSVZ NSC increases their in vitro proliferation and self-renewal capacity, but does not predispose NPC to apoptoticdeath. Increased NSC pool in vivo is also observedpostnatally.

Reduced Overall Brain Size in Nestin-Cre;STOPFloxBmi1 Mice But No Tumors in BothNestin-Cre;STOPFloxBmi1 and inA-Cre;STOPFloxBmi1 Mice

Adult Nestin-Cre;STOPFloxBmi1 animals consistently exhib-ited a reduced overall brain size (Fig. 5O, 5P) and brainweight (Fig. 5Q, p < .001), compared with age-matched andsex-matched controls. Histological and immunohistochemicalanalysis of their brains by H&E and immunolabeling for neu-ronal (Map2, NeuN, neurofilament, Synaptophysin), glial(GFAP, myelin basic protein), and microglial (Iba1) markersrevealed that overall architecture and organization of the brainwas normal despite its reduced size (Fig. 5O, 5P and data notshown). Gross examination and histological analysis of allbrains (n ¼ 12) excluded the occurrence of brain tumors inthese mice.

We observed a dual effect of Bmi1 overexpression on ap-optosis in NS, depending on whether the cultures wereobtained from embryonic or postnatal cells with postnatal NSshowing a lower apoptotic rate, therefore, we reasoned thatactivation of Bmi1 overexpression postnatally is more likelyto elicit tumor formation. We induced Bmi1 overexpressionin adult mice by means of intraventricular injection of Adeno-Cre in STOPFloxBmi1 mice. This is a highly efficient methodto obtain recombination of LoxP alleles in cells of the SVZ,as reported in the literature [26] and as we have seen in ourexperiments in R26R mice (Supporting Information Fig. S5A,S5B). A cohort of 15 mice was kept under observation for 20months, all brains were analyzed histologically at the termina-tion of the experiment and no CNS tumors were observed.

We conclude that reduced brain size of Nestin-Cre;STOP-FloxBmi1 is probably a consequence of the depletion of dif-ferentiating progenitors during embryonic cortical develop-ment. Bmi1 overexpression is not sufficient to induce NSC/NPC or their progeny to undergo neoplastic transformation inthe mouse.

Downregulation of Survivin Is Responsible for theIncreased Apoptosis Observed in NeuronalProgenitors-Overexpressing Bmi1 In Vitro and In Vivo

We analyzed the downstream mechanisms mediating theobserved effects of Bmi1 overexpression. First, we assessedthe expression of known Bmi1 target genes, p16ink4a, p19Arf

and p21WAF1/Cip1 on NS isolated from Nestin-Cre;STOP-FloxBmi1. We show reduced levels of all three cell cycleinhibitors in embryonic NS-overexpressing Bmi1 independentof whether Bmi1 expression was activated chronically duringembryonic development in vivo (þNCre) or acutely in vitro(þAcre, Fig. 6A). A similar reduction of the expression levelsof these proteins was observed in NS derived from postnatalNSPC, and this was independent of the timepoint analyzed(Fig. 6A). In accordance with recent data supporting a corre-lation between the expression of Bmi1 and Foxg1 [17], aforebrain specific transcription factor, we also found increasedlevels of Foxg1 in embryonic NS (Fig. 6B, (p < .01). Thiswas independent of whether Bmi1 activation occurred during

embryonic development in vivo or acutely in vitro. On thecontrary, no increased expression of Foxg1 was observed inpostnatal NS at P7 and P70 (Fig. 6B).

Although deregulation of cell cycle inhibitors is seen bothin embryonic and postnatal NS, and it is likely to be responsi-ble for the increased proliferation of NSPC-overexpressingBmi1, it is intriguing that increased apoptosis upon neuronalcommitment seems to be developmental stage-specific. Sev-eral genes are known to be involved in the control of apopto-tic death of progenitors during embryonic CNS development,among them Survivin [27]. The expression of Survivin, amember of the inhibitor of apoptosis family, has been shownto be upregulated in progenitor cells upon neuronal commit-ment (EURExpress). Moreover, conditional inactivation ofthis gene during embryonic CNS development in Nestin-expressing progenitors induces massive apoptosis of develop-ing neurons [28] with a phenotype very similar to what weobserved in our Bmi1-overexpressing double-mutant mice.Consequently, we analyzed the expression of survivin bymeans of qRT-PCR in E16.5 NS and ADH cultures. Wefound a significant decrease in the expression of survivin inNS culture but not in ADH (Fig. 6C, p < .001). Also at theprotein level, reduced number of cells expressing survivinwas counted in NS upon immunolabeling (Fig. 6D, 6E, 6F).Interestingly, no significant change in the expression of survi-vin was noted in NS cultures isolated from P7 mice (Fig. 6C).In the developing embryo at E12.5 a discrete layer of cellsexpressing survivin is seen and this coincides with the layerof Tbr2þ neuronal committed progenitor cells (Fig. 6G andEurexpress). We noticed a reduced number of cells expressingthis protein in Nestin-Cre;STOPFloxBmi1 embryos (Fig. 6H),in accordance with downregulation of survivin being an effectof Bmi1 overexpression also in vivo. Quantification of thefinding is shown in Figure 6I.

The data showed that although increased proliferation ofNSC/NPC is likely to be controlled by Bmi1 through repres-sion of its canonical cell cycle inhibitor targets, the proapop-totic role is developmental stage-specific, and it is mediatedby a novel downstream effector, survivin.

DISCUSSION

We generated a transgenic mouse model, which allowed us toactivate Bmi1 expression in a cell-specific and time-controlledfashion by means of Cre-mediated recombination of LoxPsites. With this technology, we achieved a mild enhancementof Bmi1 expression in embryonic and postnatal NSC and indifferentiating NPC, in a way similar to the physiological up-regulation of transcription factors. This makes our modelideally suited to achieve a good characterization of Bmi1influence on stem cells properties.

Overexpression of Bmi1 in embryonic NSC leads toincreased self-renewal and proliferation both in vivo and invitro. This effect is independent of whether a protracted acti-vation of Bmi1 expression was induced in vivo by means ofnestin-driven Cre expression or whether acute induction ofBmi1 expression was achieved in vitro by adenoviral-medi-ated Cre delivery. Our data support and extend a recent studywhere lentiviral-mediated overexpression of Bmi1 by intra-ventricular injection at E14 led to similar results both in vitroand in vivo, although the in vivo observation in this studywas limited to 3 days after injection [17]. In contrast, overex-pression of Bmi1 driven by the Nestin promoter did not elicita similar effect in vivo [16]. Although it is currently unclearwhy Bmi1 overexpression leads to only partially overlapping


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phenotypes in different experimental model systems, it can bespeculated that either the developmental time point at whichoverexpression is achieved or the specific subpopulation ofcells targeted or gene dosage, may all contribute to explainthese differences.

Our experimental set up allowed us to assess the effect ofBmi1 overexpression in differentiating NPC over a protractedperiod of time. We show here for the first time that embryonicneural progenitors differentiating toward a neuronal lineage arehighly sensitive to enhanced and/or ectopic expression ofBmi1. In fact, failure to downregulate Bmi1 expression shuntedthese progenitors into apoptotic death. As a consequence,reduced number of Tbr2þ intermediate neuronal progenitorswere observed in the developing neocortex of the mutant miceleading to an overall reduced brain size in adult life. The apo-ptotic rate of NPC contained in NS derived from NSPC-over-expressing Bmi1 was similarly increased and led to a signifi-

cant reduction in the number of neuronal committed Mash1þprogenitors. On the contrary, glial differentiation was favoredin Bmi1-overexpressing NS. Upon induction of differentiationin vitro, the number of bIII-tubulinþ neurons originating fromembryonic NSPC-overexpressing Bmi1 was reduced. Apoptoticdeath of NPC-overexpressing Bmi1 upon neuronal commitmentis associated with downregulation of the inhibitor of apoptosisprotein survivin, the expression of which is essential for thesurvival of developing neurons during embryonic developmentin vivo [28]. Downregulation of survivin expression was notobserved in NS originating from postnatal NSPC and the num-ber of bIII-tubulinþ neurons originating from these cells wasnot affected. NSC located in the VZ did not undergo apoptosisdespite higher levels of Bmi1 expression, a phenomenon con-firmed by the absence of apoptosis induction in NSC culturedunder adherent conditions and the lack of downregulation ofsurvivin in these cells both in vitro and in vivo.

Figure 6. Downstream effectors of Bmi1 overexpression in neural stem cells (NSC) and neural progenitor cells (NPC). Western blot analysisreveals decreased p16ink4a, p19Arf and p21 WAF1/Cip1 in NS-overexpressing Bmi1 (A). Quantitative reverse transcription polymerase chain reaction(qRT-PCR) shows increased expression of Foxg1 in neurospheres (NS) originating from E16.5 neural stem/progenitor cells (NSPC)-overexpress-ing Bmi1 (B). However, similar expression levels of Foxg1 are seen in NS-overexpressing Bmi1 and controls at both postnatal time points ana-lyzed (B). qRT-PCR show downregulation of survivin in NS-overexpressing Bmi1 but not in NSC cultured as adherent monolayer or in NSisolated postnatally (C). The percentage of cells showing nuclear positivity for survivin is also reduced in NS-overexpressing Bmi1 (D, E, F).Striking reduction of survivinþ cells in the developing neocortex of E12.5 Nestin-Cre;STOPFloxBmi1 embryos ([G, H]; [I], quantification). **, p< .01; ***, p < .001 with Student’s t test. Error bars represents SD, n � 4. Scale bar ¼ 250 lm. Abbreviation: GFP, green fluorescent protein.


Previous findings [16] showed that upregulation of Bmi1expression led to reduced expression of p16Ink4a and p19Arf ina developmental stage-independent fashion. We also observethis effect in both embryonic and postnatal NS. Importantly,downregulation of the expression of these genes was observedwhen Bmi1 activation was achieved during embryonic devel-opment by means of nestin-driven Cre expression and acutelyin vitro by adenoviral-mediated Cre delivery. Downregulationof p21WAF1/Cip1 expression was also observed at all timepointsanalyzed and both upon chronic and acute overexpression ofBmi1. Although we could confirm upregulation of Foxg1 inembryonic NS upon activation of Bmi1, as recently reportedby Fasano et al. [17], we could not detect a similar upregula-tion at both postnatal timepoints analyzed. We cannot excludethat this might reflect a dosage effect. Our single copy geneapproach allows us to obtain a tightly controlled 1.8-foldincrease of Bmi1 expression compared with a potentiallygreater upregulation obtained using a lentiviral transductionapproach as Fasano et al. did [17]. However, it is also con-ceivable that downregulation of p21WAF1/Cip1, either directlythrough Bmi1 or indirectly in the context of p19Arf downregu-lation, contributes to self-renewal/proliferation control of post-natal NSPC independently of Foxg1 upregulation.

Cells with stem cell properties, so-called brain tumor stemcells, are thought to play a crucial role in initiation and main-tenance of brain tumors (reviewed in [29]. These cells expresshigh levels of Bmi1 [30], are particularly resistant to apopto-sis [31, 32] and have recently been shown to be A2B5þ [33].Overexpression of Bmi1 in postnatal SVZ NSPC in vitro ledto increased self-renewal capacity as observed in embryonicNSPC. However, resilience to apoptosis was noted in NSoriginating from postnatal NSPC-overexpressing Bmi1. More-over, we observed increased number of A2B5þ undifferenti-ated, glial progenitor cells, in these NS. It was therefore im-portant to assess whether Bmi1 overexpression in NSC andNPC in vivo increased tumorigenicity. We show that bothembryonic and postnatal activation of Bmi1 overexpression instem cells and cells derived thereof did not lead to brain tu-mor formation in mice kept under observation for up to 20months. This is in agreement with the study of He et al.where tumors were not seen when Bmi1 overexpression wasinduced in nestinþ progenitor cells. Fasano et al., however,reported that overexpression of Bmi1 by means of intraven-

tricular lentiviral injections led to tumor formation in the firstweek after birth, although no further details were given on thetype of tumor these mice developed. More studies are clearlyneeded to elucidate these differences. It is important to men-tion that He et al. and potentially also Fasano et al. achievedhigher expression levels of Bmi1 in their systems as com-pared with our single copy gene approach.

CONCLUSION

We show that fine tuning of the expression level of the PcGgene Bmi1 is a viable tool to increase self-renewal capacityof both embryonic and postnatal NSPC in the mouse and thisapproach does not impair the long-term differentiationcapacity of postnatal NSPC. However, neuronal differentiationof embryonic NSPC is hampered by concomitant downregula-tion of the expression of survivin and induction of apoptoticcell death. Upregulation of Bmi1 expression during embryonicdevelopment and in the postnatal SVZ does not lead to neo-plastic transformation of these cells.

ACKNOWLEDGMENTS

We thank Axel Behrens and Sebastian Brandner for criticallyreading this article. We are grateful to Gary Warnes for expertsupport in FACS sorting/analysis, to the BSU staff, in particular,Anthony Price, for help in the daily care of our mouse colony,and to the BICMS Experimental Pathology Facility for process-ing and cutting paraffin blocks. This work is supported in part bygrants of Oncosuisse (OCS01636-02-2005), Cancer ResearchUK (C23985/A7802), and Medical Research Council UK(G0800020, 85704) to S.M.

DISCLOSURE OF POTENTIAL CONFLICTS OF

INTEREST

The authors indicate no potential conflicts of interest.

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Conditional activation of Bmi1 expression regulates self-renewal, apoptosis, and differentiation of neural stem/progenitor cells in vitro and in vivo

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