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First published online September 27, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 173 -181
doi:10.1634/stemcells.2007-0293; www.StemCells.com
© 2008 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

c-Myb Is Required for Neural Progenitor Cell Proliferation and Maintenance of the Neural Stem Cell Niche in Adult Brain

Jordane Malaterrea, Theo Mantamadiotisa,b, Sebastian Dworkina, Sally Lightowlera, Qing Yangc, Mark I. Ransomed, Ann M. Turnleyd, Nancy R. Nicholse, Nikla R. Emambokusf, Jon Framptonf, Robert G. Ramsaya,b

aDifferentiation and Transcription Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia;
bDepartment of Pharmaceutical Biology, Monash University, Parkville, Victoria, Australia;
cHoward Florey Institute, Parkville, Victoria, Australia;
dCentre for Neuroscience, University of Melbourne, Parkville, Victoria, Australia;
eDepartment of Physiology, Monash University, Clayton, Victoria, Australia;
fInstitute for Biomedical Research, Birmingham University, Birmingham, United Kingdom

Key Words. Neural stem cells • c-Myb • Ependymal cells • Transcription factor • Mice

Correspondence: Correspondence: Theo Mantamadiotis, Ph.D., Department of Pharmaceutical Biology, Victorian College of Pharmacy, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia. Telephone: 61-3-9903-9574; Fax: 61-3-9903-9638; e-mail: Theo.Mantamadiotis{at}vcp.monash.edu.au

Received on April 22, 2007; accepted for publication on September 18, 2007.

First published online in STEM CELLS EXPRESS  September 27, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Ongoing production of neurons in adult brain is restricted to specialized neurogenic niches. Deregulated expression of genes controlling homeostasis of neural progenitor cell division and/or their microenvironment underpins a spectrum of brain pathologies. Using conditional gene deletion, we show that the proto-oncogene c-myb regulates neural progenitor cell proliferation and maintains ependymal cell integrity in mice. These two cellular compartments constitute the neurogenic niche in the adult brain. Brains devoid of c-Myb showed enlarged ventricular spaces, ependymal cell abnormalities, and reduced neurogenesis. Neural progenitor cells lacking c-Myb showed a reduced intrinsic proliferative capacity and reduction of Sox-2 and Pax-6 expression. These data point to an important role for c-Myb in the neurogenic niche of the adult brain.

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


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
In mammals, the generation of new nerve cells, referred to as neurogenesis, is extensive early in development but becomes largely restricted to two discrete areas in adult brain: the subgranular zone of dentate gyrus [1] and the subventricular zone (SVZ) [2]. The SVZ neurogenic niche is composed of a heterogeneous population of cells [3, 4], with two cell compartments distinguished on the basis of their proliferative potential. The nonproliferative compartment is composed of ependymal cells, which are essential for maintaining the neurogenic niche by providing necessary factors, many of which are yet to be identified. By producing soluble molecules such as noggin, a bone morphogenetic protein antagonist, ependymal cells can direct neural stem/progenitor cell (NSPC) fate toward a neurogenic lineage [5]. The proliferative compartment is composed of stem cells and transit-amplifying progenitor daughter cells; these cells give rise to neuroblasts, which eventually migrate tangentially via the rostral migratory stream (RMS) to reach the olfactory bulb (OB), where they functionally differentiate and integrate existing networks [6].

Hydrocephalus in humans is associated with defects in cerebrospinal fluid (CSF) circulation and/or production, resulting in enlargement of brain ventricular spaces and massive loss of brain parenchyma [7]. Recent studies have shown a causal link between ciliated ependymal cell dysfunction and hydrocephalus [8]. Ependymal cells constitute the specialized epithelium lining the ventricular spaces and play a necessary role in production, transport, and filtration of CSF molecules [9, 10]. Although neurogenic abnormalities and ependymal cell function defects are often associated with hydrocephalus [11, 12], the genetic alterations underlying these defects are poorly understood [7].

The c-myb gene encodes a proto-oncogene transcription factor, highly expressed in proliferating cells of both the developing hematopoietic system and in human leukemias [13], maintaining cells in an undifferentiated state by repressing terminal differentiation and driving proliferation [14, 15]. In addition to its role in the hematopoietic system, we have previously shown that c-myb is required for normal colon development [16] and that it is highly expressed in numerous colon cancer cell lines [15, 17]. Our recent data extended these findings to show that c-Myb specifically regulates progenitor cell homeostasis in the colon crypt [18] and skin growth [19], implying that c-Myb may have a more general role in modulating progenitor cells in many more tissues than previously thought. However, less is known about the role of c-myb in the proliferative compartments of other tissues. Although c-myb mRNA expression has been reported previously in mouse and rat brain [20, 21], its function in normal brain development and cellular proliferation remains unexplored. Previous work in our laboratory has shown that c-myb mRNA levels in the brain decrease throughout the course of murine embryogenesis [20] but persist in neurogenic foci in the adult brain, implying that c-Myb may play a role in neurogenic zones where stem/progenitor cells reside.

Mice devoid of c-Myb display a block in definitive hematopoiesis resulting in early embryonic lethality [22, 23] and severe defects in colon architecture [16]. Here, we show that c-Myb is expressed in NSPCs in the neurogenic regions of the adult mouse brain. To study the role of c-Myb in NSPC function, we used the Cre/loxP recombination system to specifically disrupt c-Myb expression in NSPCs. Although mice with brain-specific loss of c-myb (c-mybnesCre mutant mice) were viable, the loss of c-Myb specifically in NSPCs and their progeny resulted in a distinct phenotype, including enlarged ventricular spaces and olfactory bulb hypertrophy. One-fifth of the mutants exhibited severe hydrocephalus, associated with massive loss of brain parenchyma. At the cellular level, we found ependymal cell layer alteration, with a decrease in both the length and number of cilia and impaired neurogenesis. Analysis of proliferation dynamics both in vivo and in neurosphere cultures shows that loss of c-Myb causes specific cell-cycle defects. Altogether, our data suggest c-myb has an important role in supporting neurogenesis in the adult brain.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mice
To generate the nervous system-specific c-myb mutant mice, we used mice in which c-myb exons 3–6 were flanked with loxP (F) sites [23]. Mice harboring the c-mybF allele were crossed with transgenic mice possessing the Cre recombinase gene under the control of the nestin promoter and enhancer [24, 25]. Control mice used were c-mybF/F or littermate c-mybNesCre mice. Severely hydrocephalic mutants were excluded from all cell counting and neurogenesis analysis.

Histology
Mice were perfused with cold 4% paraformaldehyde (PFA). Depending on the age of the mice, either brains were dissected (adult) or whole heads (embryonic day [E] 18.5) were postfixed overnight in 4% PFA at 4°C prior to embedding in paraffin wax. Paraffin blocks were sectioned on a microtome at a thickness of 6 µm; sections were cleared of paraffin and rehydrated through an ethanol series.

5-Bromo-2'-deoxyuridine Labeling and Detection
For the 5-bromo-2'-deoxyuridine (BrdU) pulse experiments, mice were injected intraperitoneally with 100 mg/kg BrdU and sacrificed 2.5 hours later. Following blocking of endogenous peroxidase activity in 3% hydrogen peroxide for 10 minutes, sections were rinsed in phosphate-buffered saline (PBS) and treated with 2 N HCl in PBS with 0.1% Triton X-100 for 30 minutes. After rinses, sections were processed for staining with mouse monoclonal anti-BrdU (1:500; Dako, Glostrup, Denmark, http://www.dako.com) and the ImmPress Kit (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). Staining was visualized with diaminobenzidine (DAB; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com) and H2O2. For the 2-week BrdU pulse experiments, mice received BrdU in their drinking water ad libitum at 1 mg/ml. BrdU antibodies (1:100) were detected using fluorescent antibodies Alexa Fluor 594 goat anti-mouse IgG (1:400; Molecular Probes).

Immunohistochemistry
For c-Myb monoclonal antibody 1.1 [26], S-100 (rabbit anti-S-100, 1:2,000; Dako), and Polaris (rabbit sera anti-Polaris, 1:500; kindly provided by Dr B. Yoder [27]) staining, sections were subjected to pressure antigen retrieval in EDTA 1 mM pH 8 buffer. Primary antibodies were detected using the ImmPress Kit (Vector Laboratories) kit, and staining was visualized with DAB and H2O2.

Double-Labeling Experiments
For BrdU/glial fibrillary acidic protein (GFAP) or doublecortin (DCX) labeling, following antigen retrieval, adjacent sections were incubated overnight at 4°C in goat anti-doublecortin antibody 1:100 in PBS (DCX [C18]; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) or in rabbit anti-GFAP antibody 1:200 in PBS (DakoCytomation). BrdU labeling was performed as previously described. For c-Myb and GFAP, DCX, or S-100 double labeling, high-pH antigen retrieval solution (DakoCytomation) was used for c-Myb detection. For phosphohistone-H3 (PH3) and proliferating cell nuclear antigen (PCNA) double labeling, coronal sections were stained after citric acid antigen retrieval for PCNA and PH3 cell cycle marker, respectively, using a mouse anti-PCNA antibody (1:100; BD Biosciences, San Diego, http://www.bdbiosciences.com) and a rabbit anti-PH3 antibody (1:250; Millipore, Billerica, MA, http://www.millipore.com). Secondary detection was performed using species-appropriate fluorescent antibodies: Alexa Fluor 488 goat anti-rabbit IgG (for S-100, GFAP, and PH3), Alexa Fluor 488 donkey anti-goat IgG (for DCX), and Alexa Fluor 594 goat anti-mouse IgG (for PCNA, BrdU, and Myb 1.1) (1:800; Molecular Probes, Eugene, OR, http://probes.invitrogen.com). Sections were then mounted in a fluorescent mounting medium (DakoCytomation).

Electron Microscopy
For scanning electron microscopy, tissues were immersion-fixed in 2% glutaraldehyde, 2.5% paraformaldehyde in 0.08 M Sorensen's PBS buffer for 2 hours at room temperature. Tissues were washed in 0.08 M Sorensen's PBS three times for 5 minutes per wash, postfixed in 2% OsO4 in PBS for 1 hours, and then washed in distilled water three times for 5 minutes each and dehydrated through a graded series of ethanol, 50%, 70%, 90%, 95%, and twice at 100% for 20 minutes each before "critical point drying." Finally tissues were sputter-coated with gold, mounted onto sample stubs, and viewed in the scanning electron microscope. For transmission electron microscopy (TEM), tissue was cut into 1-mm square blocks and fixed with 2.5% PFA, 2.5% glutaraldehyde in 0.8 M Sorensen's buffer. After rinses in 0.8 M Sorensen's buffer, pH 7.4, secondary fixation was performed in 2% OsO4 in 0.1 M Sorensen's buffer, pH 7.4. Samples were gradually dehydrated and infiltrated with 1:1 Spurr's resin and 100% acetone overnight, followed by 1:1 Spurr's resin and 100% ethanol for 2 hours.

Terminal Deoxynucleotidyl Transferase dUTP Nick-End Labeling
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed as described previously [25]. Paraffin sections of embryos were cleared of paraffin and rehydrated through an ethanol series. Following a water rinse, sections were incubated for 10 minutes in 20 µg/ml proteinase K and for 10 minutes in 3% H2O2 at room temperature. Staining was performed using the In Situ Cell Detection Kit (Roche, Basel, Switzerland, http://www.roche-applied-science.com) according to the manufacturer's instructions.

Cell Counts
BrdU+ cells were counted in the left and right SVZ (2.5-hour or 2-week pulse) from a series of five coronal sections (0.98 mm from bregma, according to Paxinos stereotaxic coordinates), every fifth section per animal (n = 3). Cells were counted on a Zeiss microscope with Magnifier digital camera (Carl Zeiss, Jena, Germany, http://www.zeiss.com). To quantify DCX-labeled cell numbers, cells were scored on five equivalent coronal sections (every third section) throughout the left and right RMS (from bregma 1.98) per animal (n = 3). Result from left and right section counts were averaged, and results were expressed as average number of positive cells per hemisphere.

Double labeling of cells for specific marker (PCNA/PH3, DCX/BrdU, or GFAP/BrdU), were analyzed through the z-plane in 0.3-µm optical sections using a confocal microscope (FV1000; Bio-Rad, Hercules, CA, http://www.bio-rad.com). When necessary, sections were rotated in the z-plane. Three equivalent planes in each of three sections (granular and periglomerular layers of the OB) of each animal (n = 3) were scanned at a magnification of x100.

Neurosphere Cultures
NSPCs were derived from adult mutant mice and control littermates and maintained as neurospheres as previously described [28]. The neurosphere cultures were passaged every 7 days for 10 passages, and the cumulative number of cells generated over this time was calculated. For differentiation studies, dissociated neurospheres were plated on poly-DL-ornithine/laminin-coated eight-well chamber slides (Falcon) at 1 x 105 cells per well and differentiated for 3 days in mitogen-free medium in the presence of 1% fetal calf serum. Following fixation in 4% PFA, neurons were detected by immunostaining with mouse anti-βIII-tubulin (1:2,000; Promega, Madison, WI, http://www.promega.com) and sheep anti-mouse-Cy3 (1:1,000; Jackson Immunoresearch), whereas astrocytes were detected using rabbit anti-GFAP (1:500; Dako) and goat anti-rabbit-Cy2 (1:1,000; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com). For BrdU proliferation assays, 104 NSPCs per cm2 were plated onto poly-L-ornithine/laminin-coated slides. After 4 days, cultures were BrdU pulsed for 1 hour prior to 4% PFA fixation and BrdU detection. For self-renewal assays and neurosphere diameter analysis, neurospheres were dissociated and replated at 104 cells per milliliter to minimize cell aggregation [2, 29]. The number of regenerated neurospheres was counted after 4 days, and diameters of at least 15 randomly chosen neurospheres were measured after 8 days. Neurospheres were used between passage 1 and 5 for proliferation, self-renewal, and quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) determination of c-myb, sox-2, and pax-6 mRNA.

RT-PCR Analysis
RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's instructions. Genomic DNA was removed from RNA by addition of RNase-free DNase 1 (1 U/µl; Promega). First-strand cDNA was synthesized with Superscript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. All real-time RT-PCRs were conducted using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). Eight microliters of cDNA (1:10 dilution) was combined with 10 µl of SYBR Green PCR Master Mix (Applied Biosystems) and 200 nM sense and antisense oligonucleotides (Geneworks, Adelaide, South Australia, Australia, http://www.geneworks.com.au) and amplified using temperatures of 50°C for 2 minutes and 95°C for 10 minutes. These initial steps were followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Expression of all genes was normalized to gapdh or β2-microglobulin housekeeping genes to determine relative levels of mRNA transcripts.

Statistical Analysis
For both in vivo and in vitro experiments, statistical analyses were conducted using the StatView (SAS Institute, Cary, NC, http://www.sas.com) computer package. Significance was determined using the two-tailed Student t test or the Mann-Whitney U test.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
c-Myb Is Expressed in the SVZ Neurogenic Niche
To extend our previous analysis of c-myb expression in the developing and adult mouse brain, we performed in situ hybridization. During embryogenesis, we found that c-myb mRNA is highly expressed in the neuroepithelium at embryonic day (E) 12.5 (supplemental online Fig. 1A). In the adult mouse brain, c-myb mRNA and protein (Fig. 1B, 1C, 1E, 1H, 1K; supplemental online Fig. 1F) are strongly expressed in neurogenic zones (SVZ) compared with non-neurogenic areas (cortex; Fig. 1A, 1B). These observations are consistent with the view that c-myb is highly expressed in immature, proliferating cell compartments. To investigate which cell types in the SVZ expressed c-Myb, we performed dual immunolabeling for c-Myb with DCX, GFAP, and S-100 or Polaris to identify migrating neuroblasts, astrocytes, and ependymal cells, respectively. Our results show that c-Myb is expressed within virtually all cells of the neurogenic niche: DCX+ neuroblasts, S-100+ and polaris+ ependymal cells, GFAP+ and S-100+ SVZ astrocytes. Some c-Myb+ cells that do not stain for DCX or GFAP may represent transit-amplifying progenitors (Fig. 1D–1L; supplemental online Fig. 2).


Figure 1
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  		Figure 1. Expression of c-Myb in adult brain. (A): Diagram showing a coronal view of a mouse brain through the SVZ. The CX and OB are also marked. (B): Relative levels of c-myb mRNA were determined by quantitative reverse transcription polymerase chain reaction in control SVZ and CX, showing the highest c-myb levels in the SVZ. (C): In situ analysis of c-myb on adult mouse brain showed extensive mRNA distribution in the SVZ. (D–L): Double labeling with anti-c-Myb and anti-GFAP (D–F), anti-doublecortin (G–I), or S-100 antibodies (J–L) on adult mouse SVZ sections. c-Myb protein was expressed in SVZ astrocytes expressing GFAP (white arrowheads [F]) or S-100 (white arrows [L]), DCX+ neuroblast (white arrowhead [I]), and SVZ S-100+ ependymal cells (white arrowhead [L]). Abbreviations: CX, cortex; GFAP, glial fibrillary acidic protein; OB, olfactory bulb; SVZ, subventricular zone; V, ventricle.

 
Loss of c-Myb in Brain Results in Enlarged Ventricles
To further explore c-Myb function in brain, we crossed mice harboring loxP-modified, "floxed" alleles of c-myb [23] and a nestin promoter/enhancer driven Cre-recombinase (Cre) transgene to generate mice devoid of c-Myb in neural stem cells and all cells derived from these (supplemental online Fig. 1B). This specific nestin-Cre transgenic line has been widely used to achieve robust nervous system-specific recombination of many floxed genes [24, 25], and it has been previously reported that the action of nestin-driven Cre expression results in efficient gene deletion of loxP-flanked sequences within the creb1 gene by E12.5 and essentially complete deletion in postmitotic neurons by E16 [25]. We show here that c-myb mRNA is lost in brains harboring floxed c-myb alleles and the nestin-Cre transgene (supplemental online Fig. 1C). Immunohistochemical detection of c-Myb showed that loss of c-Myb protein expression is extensive in the developing brain by E18.5 (supplemental online Fig. 1E) and in the adult SVZ (supplemental online Fig. 1G). c-mybnesCre mutant mice developed normally in utero, thus bypassing the embryonic lethality evident in c-myb–/– mice [22]. Although brain structure was relatively normal at birth, the OBs were significantly smaller in c-mybnesCre brains (Fig. 3D). By 2 months of age, c-mybnesCre mutant mouse brains (100% penetrance) exhibited a fourfold (p < .005) enlargement of lateral ventricles (1.35 ± 0.511 mm2; n = 4), compared with controls (0.33 ± 0.18 mm2; n = 4) as shown by histology (Fig. 2A, 2B) and sequential MRI scanning (supplemental online Fig. 3A, 3B). Further morphometric analysis of other brain regions, such as the striatum and thalamus, indicated that there were no significant changes in these regions. However, 19% (9 of 47) of the mutants exhibited obvious hydrocephalus, with ventricle enlargement associated with cortical thickening and massive loss of striata, characteristic of hydrocephalus (Fig. 2C).


Figure 2
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  		Figure 2. The ependymal cell layer is disrupted in c-mybnesCre mutant mice. Images of H&E-stained control (A), c-mybnesCre mutant (B), and c-mybnesCre mutant with hydrocephalus (C) mouse brains. Scale bar = 1 mm. S-100 (D, E) and Polaris (F, G) immunolabeling revealed ependymal cell layer disorganization, including decreased length and number of cilia in c-mybnesCre mutant (black arrow [E]) compared with control (black arrow [D]) or clumped cilia (black arrows [G]) by comparison with control (F). Scale bars = 10 µm (D, E) and 20 µm (F, G). Scanning electron microscopy performed on control (H), c-mybnesCre mutant (I), and c-mybnesCre mutant with hydrocephalus (J). Scale bars = 5 µm (H–J). Transmission electron micrograph performed on ultrathin section in control (K), c-mybnesCre mutant mice (L), and c-mybnesCre mutant mice with hydrocephalus (M). Magnification is x4,000, showing normal axoneme structure in c-mybnesCre mutants compared with control.

 


Figure 3
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  		Figure 3. Neurogenic defects in c-mybnesCre M brains. The number of BrdU+ cells following 2 weeks of labeling was quantified by counting cells in the SVZ of C (A) and M (B) mice. Scale bars = 100 µm. Results displayed in (C) show significantly fewer (*, p < .05) positive cells in c-mybnesCre M mice compared with C. (D–H): Whole mouse brains from adult M and C mice were dissected to show the relatively smaller olfactory bulbs (black arrows and square brackets) in c-mybnesCre M mice compared with C (D). The OB to total brain weight ratio from c-mybnesCre M mice was significantly reduced (F). The RMS showed a trend toward decreased size in c-mybnesCre M (n = 3) (G), and DCX+ cell number was significantly reduced (n = 3) (E, H). Data shown are mean ± SEM; n = 3; *, p < .05. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; C, control; DCX, doublecortin; M, mutant; OB, olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular zone.

 
To investigate whether hydrocephalus in our model was due to disruption of CSF circulation, we examined coronal sections throughout the brain, paying special attention to the aqueduct of Sylvius (supplemental online Fig. 3C, 3D), the canal that connects the third and fourth ventricles, and the choroid plexus (supplemental online Fig. 3E, 3F), which generates CSF. No differences were observed in these structures between control and mutant mice, indicating that mechanical disruption of CSF flow was unlikely to be an underlying cause of the enlarged ventricles.

Ependymal Cells Are Disorganized and Display Shorter and Fewer Cilia in c-mybnesCre Mutant Mice
Ependymal cells are a specialized epithelial cell type lining the ventricles of the brain and are formed late during embryogenesis (E14–E16) from radial glia in the ventricular zone [30]. Ependymal cell differentiation and apical surface cilia formation occurs during the first postnatal week [30]. To identify these cells, we used immunohistochemistry (IHC) for the secretatory protein S-100. We found that the ependymal cells in c-mybnesCre mutant mice were highly disorganized, including disruption in nuclear alignment (Fig. 2D, 2E). Apical S-100 labeling was also disrupted in c-mybnesCre mutant mice with a more diffuse pattern, suggesting defective secretatory function. To investigate these cells further, we examined the expression of Polaris, a protein component of the intraflagellar transport system necessary for cilia formation, by IHC. Homozygous hypomorphic mutation of the polaris gene results in cilia loss and hydrocephalus [31]. Polaris staining of ependymal cells evidenced shorter and fewer cilia, often clumped and aggregated in c-mybnesCre mutant mouse ependymal cells (Fig. 2F, 2G). The distribution of the Polaris protein also differed in the brains of c-mybnesCre mutant mice. Control mice showed characteristic deposition of Polaris at the apical surface of ependymal cells, as well as within the cilia proper, whereas c-mybnesCre mutant mice showed distribution throughout the cytoplasm.

To investigate whether the structure of cilia was also disrupted, we performed scanning electron microscopy of the ventricular surfaces (Fig. 2H–2J). Densely populated, long cilia were seen in control samples (Fig. 2H), whereas fewer cilia were obvious in mybnesCre samples (Fig. 2I), and only a few long cilia were seen in hydrocephalus samples (Fig. 2J). Motile cilia are composed of a cylindrical arrangement of nine doublet microtubules and a central pair of singlet microtubules forming the axoneme. Transmission electron microscopy analysis on ultrathin brain sections through subventricular sections showed that the axoneme of mutant cilia showed no defects in c-mybnesCre mutant mouse ependymal cells (Fig. 2K–2M). Although motile cilia are required for CSF circulation, loss of cilia is also associated with ventricular enlargement in both humans and mice [8]. These data suggest that the ependymal cell defects observed in the c-mybnesCre mutant brains may in part underpin the enlarged ventricular spaces observed in mutants.

Reduced Cell Birth and Proliferation Alters Early Neurogenesis in c-mybnesCre Mutant Mice
Ependymal cells regulate CSF flow [9, 10] and thus the movement of soluble growth/survival factors and the establishment of chemotactic gradients in the brain [32]. Consequently, disruption of the ependymal cell layer may alter the function of the adjacent SVZ cells in the neurogenic niche. Therefore, we hypothesized that this observed ependymal cell layer defect had an impact upon neurogenesis in vivo.

To measure the extent to which new cells were generated and the level of proliferation, we used a cumulative BrdU labeling protocol. BrdU is a thymidine analog that is incorporated in the DNA during the S-phase of cell cycle. Control and c-mybnesCre mutant mice received the BrdU in drinking water for 2 weeks. This protocol allows identification of fast-proliferating cells and their progeny, as well as slowly dividing cells. We found significantly fewer BrdU+ cells in the SVZ of c-mybnesCre mutant mice, suggesting that c-Myb loss compromises the proliferative capacity of NSPCs (Fig. 3A–3C). TUNEL of brain sections showed that c-Myb loss did not result in increased cell death compared with control brains, implying that the reduced cell birth in mutants could not be attributed to cell loss by increased apoptosis (supplemental online Fig. 3G, 3H). In addition, we analyzed the mRNA levels of a recognized c-Myb target gene, the cell survival factor bcl-2 [33], by quantitative reverse transcription (qRT)-PCR and found no change in expression (data not shown).

Newly generated neuroblasts from the rodent SVZ migrate predominantly through the RMS and integrate within the OBs. Since c-mybnesCre mutant mice exhibited significantly smaller (size and weight) OBs (Fig. 3D, 3F), we investigated whether this was due to reduced production of neuroblasts migrating from the SVZ. To measure neuronal birth, we used DCX as an early marker of neurogenesis to identify migrating neuroblasts en route to the OB [34]. We found that the number of DCX+ cells was significantly reduced as quantified on coronal sections throughout the RMS, indicating a potential defect in the generation of neuroblasts from the SVZ of c-mybnesCre mutant mice. The relative cross-sectional area of the RMS was also used as a surrogate measure of migrating neuroblasts (DCX+ cells) and was reduced in c-mybnesCre mutant mice compared with controls, but this difference was not significant, although fewer DCX+ cells were observed (Fig. 3E, 3G, 3H). As c-Myb regulates the differentiation of hematopoietic progenitor cells [23], we tested whether the differentiation potential was affected in cells that arise from the SVZ neurogenic zone. In the mouse, newly generated cells in the SVZ reach the OB 5–7 days later to integrate within the granular and periglomerular layers [35]. We performed double-labeling experiments for BrdU and DCX or GFAP following 2 weeks of BrdU treatment. Analysis of double-stained cells revealed no difference in the percentage of cells coexpressing BrdU and either DCX or GFAP in the granular and glomerular layer of the OB, suggesting that loss of c-Myb does not affect cell fate (supplemental online Fig. 4E, 4F).

Cell Cycle Progression Is Delayed in the Brains of c-mybnesCre Mutant Mice
To further investigate the observed reduction in proliferative capacity of cells in the c-mybnesCre mutant SVZ, we hypothesized that NSPCs from c-mybnesCre mice may have perturbations in the cell cycle, consistent with this role attributed to c-Myb in other cell types [36–38]. To measure cycling cells actively engaged in S-phase, mice were injected with the exogenous S-phase marker BrdU and sacrificed after a 2.5-hour exposure. c-mybnesCre mutant brains showed significantly fewer BrdU-labeled cells in the SVZ, suggesting that there were fewer proliferating NSPCs in S-phase (Fig. 4A–4C). By using double labeling with PCNA and the G2/M marker PH3, we were able to measure the fraction of cycling cells (PCNA+) in the mitotic phase (PH3+) (Fig. 4D–4I). The data reveal that c-mybnesCre brains exhibited significantly fewer PH3+/PCNA+ cells compared with control (Fig. 4J). Taken together, these data lead us to conclude that c-Myb is required to maintain normal progression through the G1/S and G2/M transitions of the cell cycle (Fig. 4K).


Figure 4
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  		Figure 4. c-mybnesCre M have proliferation defects in the SVZ. (A, B): Coronal sections through the SVZ were labeled for BrdU following a 2.5-hour BrdU pulse, showing fewer S-phase-labeled cells (black arrows) in the SVZ of c-mybnesCre M mice (B) as compared with C (A). (C): Quantitation of BrdU+ cells showed significant reduction in c-mybnesCre M compared with C (n = 3). Data shown are mean ± SEM; *, p < .05. Scale bars = 20 µm. (D–I): Coronal sections from c-mybnesCre M (G–I) and C (D–F) mice were stained with antibodies against PCNA (red) and PH3 (green), and double-labeled cells were analyzed by confocal microscopy. The percentage of PCNA+ cells engaged in mitosis PH3+ was greatly reduced in c-mybnesCre M as compared with C (J). Results are expressed as mean percentage ± SEM of PCNA+ cells expressing PH3 (n = 3; *, p < .05) compared with C. Scale bar = 20 µm (A–I). (K): Schematic depicting the cell cycle in adult mouse neural progenitor cells (from [56]) indicating the phases where BrdU and PH3 label cells. c-Myb loss impacts upon at least two transition points of the cell cycle, G1/S and G2/M. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; C, control; M, mutant; PCNA, proliferating cell nuclear antigen; PH3, phosphohistone-H3; SVZ, subventricular zone.

 
Reduced Neural Progenitor Cell Proliferation Is Independent of Cellular Niche
Our findings show that neurogenesis is defective in c-mybnesCre mutant brains because of delayed progression of NSPCs through the cell cycle. To determine whether these effects extended beyond the defects in the ependymal layer of c-mybnesCre mutant mice, we used the neurosphere assay system to measure cell-intrinsic effects of c-Myb loss on NSPC proliferation.

When we compared the relative c-myb mRNA expression in control adult SVZ-derived neurospheres grown in the presence of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) (proliferating NSPCs) or cells withdrawn from bFGF and EGF in the presence of serum (differentiated neurons and glia), we found that c-myb mRNA was five times lower in the differentiated cells (supplemental online Fig. 4A), as was the marker of immature neural cells, nestin. This shows that c-Myb is highly expressed in NSPCs, but expression declines in differentiated postmitotic cells.

To gauge the proliferative potential of mutant and control NSPCs, cells were grown over 10 passages, reseeding dissociated neurospheres after each passage at the same density. In growth factor conditions that allow the propagation of neurospheres, where 80%–90% of cells are stem and progenitor cells [39], neurospheres derived from c-mybnesCre mutant mice showed significantly reduced growth potential over time, where mybnesCre cells reached only 60% total cells compared with wild-type cells over 10 weeks. Furthermore, the slope of the line corresponding to growth rate was significantly different from that of control (p < .0001), according to linear regression analysis (Fig. 5B).


Figure 5
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  		Figure 5. Neural progenitor cells lacking c-Myb exhibit reduced proliferation but no change in differentiation. (A): To confirm c-myb loss in neurospheres, we performed reverse transcription polymerase chain reaction and showed that c-Myb is absent in c-MybnesCre neurospheres (M), but not in C. The expression of the transcription factor creb1 and housekeeping gene gapdh were unchanged. (B): Neural progenitor cell (NPC) growth was reduced in c-mybnesCre as measured by cumulative cell number in neurosphere cultures over 10 passages. To test whether the proliferations/slopes of the growth curve of M and C cultures were significantly different, data were extrapolated using linear regression analysis. The curve slopes were significantly different (p < .0001) according to this analysis, demonstrating a clear proliferation difference. (C): The number of neurospheres was unchanged in c-mybnesCre M neurosphere culture; however, c-mybnesCre M neurospheres were smaller in diameter compared with C (average neurosphere diameter ± SEM; *, p < .05; n = 3 per genotype) (D). (E–K): For BrdU proliferation assays, NPCs were grown as adherent cell colonies in the presence of mitogens over 4 days and were exposed to 1-hour BrdU incubation. C (F–H) and M (I–K) cultures were labeled for BrdU (green) and counterstained with the nuclear marker PI (red). The percentage of BrdU-labeled cells was quantified in a total of 20 colonies per slide (performed in duplicate [E]). Results are expressed as mean ± SEM; **, p < .01; n = 3 per genotype. Scale bars = 20 µm. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; C, control; M, mutant; PI, propidium iodide.

 
To further characterize whether the defect in c-mybnesCre NSPC propagation was due to a cell-intrinsic deficiency in self-renewal, we examined the potential of c-mybnesCre NSPCs to form neurospheres following dissociation of neurospheres into single cells (self-renewal; Fig. 5C). We found no difference in the number of neurospheres generated from c-mybnesCre NSPCs, implying that c-Myb-deficient NSPCs retained their self-renewal capacity. This indicated that a defect in proliferation was responsible for the decreased cell number in c-mybnesCre-derived neurospheres. This correlates with a reduced diameter (size) of c-Myb mutant neurospheres compared with control (Fig. 5D; supplemental online Fig. 5A). Similarly, when single cells were seeded at clonal dilution (10 µl at 1 cell per microliter) in Terasaki wells, we found that mutant cells generated the same number of neurospheres after 6 days but at a slower rate (supplemental online Fig. 5B–5G). To investigate cellular proliferation independent of the SVZ niche, we performed proliferation analyses by exposing dissociated neurospheres (grown as monolayers) to BrdU for 1 hour (Fig. 5F–5K). As was seen in the in vivo analysis, cultured NSPCs derived from c-mybnesCre mutant mice exhibited significantly fewer BrdU+ cells than controls (Fig. 5E), indicating that the proliferation defects seen in the SVZ of c-mybnesCre mutant mice are likely to results from both cell-intrinsic and niche-dependent mechanisms following c-Myb loss.

To establish whether NSPCs derived from c-mybnesCre mutant mice retained multipotency in vitro, we induced neurosphere differentiation and analyzed cell differentiation into neurons and glia. In accordance with our in vivo data, c-Myb-deficient cells retained the potential to generate both neurons and astrocytes (supplemental online Fig. 4B). The percentage of these cells was not different compared with control cultures (supplemental online Fig. 4C–4D), indicating that loss of c-Myb does not specifically affect neuronal or glial differentiation.

Loss of c-Myb Decreases pax-6 and sox-2 mRNA Expression in Neurospheres
To identify genes involved in c-Myb-mediated regulation of NSPC proliferation, we hypothesized that the expression of pax-6, a c-Myb target gene identified during quail development [40] and involved in neural development and proliferation [41] may be affected by c-myb loss in brain. Pax-6 heterozygous mutant mice display phenotypic parallels to c-mybnesCre mutant mice seen in our study [42]. Sequence alignments of the pax-6 promoters of human and mouse revealed the existence of conserved c-Myb DNA-binding consensus sequences (supplemental online Fig. 6). Further analysis of the Pax gene family promoters showed that pax-6 promoters (P0 and P1) exhibit higher frequency of potential DNA-binding sites for c-Myb compared with other member of the pax family of genes (supplemental online Fig. 7). Analysis of pax-6 mRNA expression in mutant and control neurospheres by qRT-PCR showed that pax-6 mRNA expression was significantly reduced in c-mybnesCre mutant neurospheres (Fig. 6B), suggesting that c-Myb may regulate pax-6 expression in NSPCs. We also investigated the expression of the Pax-6 transcription partner Sox-2 [43] and identified conserved potential DNA-binding sites for c-Myb in the sox-2 promoter region in both human and mouse (supplemental online Fig. 8). Indeed, sox-2 deletion results in defective neurogenesis associated with ependymal cell layer disruption [44], which is also observed in c-mybnesCre mutant mice. We asked whether c-Myb loss induced a change in the sox-2 expression level in mutant neurospheres and found using qRT-PCR analysis showed that c-Myb loss induced a change in sox-2 expression in mutant neurospheres (Fig. 6A), similar to pax-6 mRNA, indicating that c-Myb may directly or indirectly regulate pax-6 and sox-2 expression.


Figure 6
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  		Figure 6. The role of c-Myb in the maintenance of neurogenesis and cell cycle progression in the stem cell niche. Both sox-2 (A) and pax-6 mRNA (B) expression is decreased in c-mybnesCre M compared with C neurospheres as measured by quantitative reverse transcription polymerase chain reaction. Results are expressed as mean relative expression to β2-microglobulin; n ≥ 3; *, p < .05 by the Student t test. (C): Proposed model showing the roles of c-Myb in the neurogenic niche: The different cell types that constitute the neural stem cell niche are depicted (modified from [57]): (a) GFAP+ S-100– stem cells give rise to DCX– GFAP– transit-amplifying cells, which generate DCX+ GFAP– neuroblasts; (b) DCX+ cells are ensheathed by the cellular processes of S-100+ GFAP+ non-neurogenic astrocytes; and (c) S-100+ ependymal cells promote neurogenesis [5] and are essential for cerebrospinal fluid microcirculation. Thus, c-Myb is expressed in cells constituting the neural stem/progenitor cell niche, and our data support the view that in the absence of c-Myb, the neurogenic niche is functionally impaired. Abbreviations: C, control; DCX, doublecortin; GFAP, glial fibrillary acidic protein; M, mutant.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In this study, we showed that c-Myb, a hematopoietic transcription factor and proto-oncogene, has an important role in neural progenitor cell proliferation and maintenance of the neurogenic niche; this is the first study addressing c-Myb function in neural cells. By in situ hybridization and IHC we showed that c-Myb is expressed in neurogenic regions of the adult mouse brain and that c-Myb expression declines when NSPCs differentiate.

Using a brain-specific c-myb knockout mouse model (c-mybnesCre mutant mice), we showed that c-Myb has a role in postnatal brain development and homeostasis. No obvious phenotype could be seen at the histological level when analyzing c-Myb mutant embryos. However, the consequences of c-Myb loss after birth were apparent when ependymal cell differentiation occurs and presumably ependymal cell function becomes important. c-Myb loss caused ependymal cell layer alteration and was associated with enlarged lateral ventricles or hydrocephalus and reduced NSPC proliferation, suggesting that the effect on neurogenesis in vivo results from the synergistic effects of both extrinsic (ependymal cell defect) and intrinsic mechanisms. It is notable that NSPCs in adult brain are closely associated with ependymal cells, which together constitute the neurogenic niche. Alteration of the stem cell niche results in NSPC dysfunction [45, 46]. Ependymal cells play a key role in the local mixing of CSF molecules and mediate much of the CSF flow throughout the brain. Thus, CSF is thought to influence neurogenesis, although the specific factors in CSF that do this are presently unknown [32]. Choroid plexus cells are specialized ependymal cells that have also been shown to influence NSPCs, emphasizing the functional importance of ependymal cells in supporting the neurogenic niche in adult brain [47] [48]. Alterations to molecules associated with ependymal cilia function may alter NSPC activity. For example ASPM (abnormal spindle-like microcephaly associated) [12] has been proposed to regulate cerebral cortical size as well as neurogenesis. Neurogenic transcription factors also appear to be involved in ependymal cell homeostasis. For example, sox-2-deficient mice display reduced neurogenesis and ependymal cell disruption [44], comparable with the observed defects we report here in c-Myb-deficient mice. We found that sox-2 expression was decreased in c-Myb-deficient neurospheres compared with controls, suggesting that c-Myb regulates sox-2 expression. Inhibition of sox-2 in mouse embryonic brain leads to reduced S-phase entry and cell cycle exit [49]. Similarly, our in vivo and in vitro experiments revealed that fewer c-mybnesCre mutant NSPCs incorporated BrdU and show reduced progress into mitosis. This is consistent with the role of c-Myb in G1/S-phase transition in mouse T and B cells [36, 38], and progression through the G2/M phase in Drosophila is dependent upon the single ortholog D-Myb [37].

Concomitantly, we found that c-Myb loss reduced pax-6 expression in neurospheres. In vivo, Pax-6 is expressed in the adult SVZ neurogenic zone [50, 51] and is a regulator of NSPC proliferation [41, 52]. We thus hypothesize that together with its interacting partner Sox-2, Pax-6 may be part of the c-Myb signaling pathway, which regulates NSPC proliferation (Fig. 6C). Moreover, we found the following: (a) Similar to Sox-2 [44], c-Myb is expressed in the ependyma, and c-Myb loss resulted in abnormal ependymal cell morphogenesis, particularly in the ciliation of these cells, an important characteristic of ependymal cell function. We further propose that c-Myb and Sox-2 are required for ependymal cell differentiation and thus for maintenance of the neurogenic niche and CSF circulation. (b) c-Myb is also expressed in SVZ cells, similar to Sox-2 [44, 53] and Pax-6 [54, 55]. c-Myb may regulate both Sox-2 and Pax-6 gene expression to maintain cell cycle progression and neuroblast generation. This is similar to the hematopoietic stem cell niche, where c-Myb regulates c-Kit in progenitor cells on the one hand, while also regulating stem cell factor (c-Kit ligand) expression in fetal liver stromal cells [14].

In rodents, reduced neurogenesis results in smaller OBs, as many of the newborn cells in the SVZ migrate via the RMS to the OB [2]. Consistent with this, the OBs of c-mybnesCre mutant mice were smaller than those in controls. Similarly to the neurogenic defects seen in our c-mybnesCre mutant model system, spontaneous mutation of pax-6 leads to reduced OB size in heterozygous mice [42]. We speculate that brain-specific deletion of pax-6 might mimic the cell proliferation defects seen in our c-mybnesCre model.

Maintenance of the NSPC niche in the mammalian adult brain is dependent on the balance of NSPC proliferation and ependymal cell integrity, both of which are critical for maintenance of overall normal brain cellularity. As c-Myb has a role in both NSPC proliferation and ependymal cell integrity and since brain-specific c-Myb loss in mice results in hydrocephaly, perturbation of c-Myb function might contribute to the development of enlarged ventricular spaces and perhaps to development of hydrocephalus.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Prof. Larry J. Marsh (University of California, Irvine, Irvine, CA) and members of the Differentiation and Transcription laboratory for helpful discussions. We acknowledge Sarah Ellis, Stephen Asquith, and Julie Crewe for TEM work. We also thank Dr. Maree Overall and Tina Isaakidis for critical reading of the manuscript. J.M. was supported by the Fondation pour la Recherche Medicale. J.F. is supported by the Wellcome Trust. A.M.T. and R.G.R. are supported by the National Health and Medical Research Council of Australia. T.M. thanks Prof. Günther Schütz and colleagues for support in initiating this project. J.M. and T.M. contributed equally to this work. N.R.E. is currently affiliated with Cell Press, Boston, MA. T.M., S.D., and R.G.R. are honorary members of the Department of Pathology, University of Melbourne, Victoria, Western Australia.


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Top
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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