Joerg D Hoeck

Anett Jandke

Sophia M Blake

Emma Nye

Bradley Spencer-Dene

Sebastian Brandner

Axel Behrens
email: axel.behrens@cancer.org.uk

Fbw7 controls neural stem cell differentiation and progenitor apoptosis via Notch and c-Jun

Keywords: Fbw7, c-Jun, Notch, ubiquitylation, E3 ligase, neural stem cell

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. However, the mechanisms underlying the decision of a stem cell to either self-renew or differentiate are incompletely understood. We demonstrate here that Fbw7 (F-box and WD repeat domain containing 7), the substrate recognition component of an SCF (complex of SKP1, CUL1 and F-box protein)-type E3 ubiquitin ligase, is a key regulator of murine NSC/NPC viability and differentiation. The absence of Fbw7 caused severely impaired stem cell differentiation and increased progenitor cell death. Fbw7 deficiency resulted in accumulation of the SCF(Fbw7) substrates active Notch1 and N-terminally phosphorylated c-Jun. Genetic and pharmacological rescue experiments identified c-Jun as a key substrate of Fbw7 in controlling progenitor cell viability, whereas inhibition of Notch signalling alleviated the block in stem cell differentiation. Thus Fbw7 controls neurogenesis by antagonising Notch and JNK/c-Jun signalling.

INTRODUCTION

Neural stem cells (NSCs) produce the differentiated cell types of the nervous system, namely neurons and glia. The first NSCs to appear during embryogenesis are neuroepithelial cells.

Subsequently, other stem cell populations arise, i.e. radial glia cells (RGCs) at later stages of embryonic development and stem cells with astroglial characteristics in the adult [START_REF] Merkle | Neural stem cells in mammalian development[END_REF][START_REF] Gotz | Radial glial cells defined and major intermediates between embryonic stem cells and CNS neurons[END_REF] . NSCs have been suggested as a potential therapeutic tool for neurodegenerative diseases and neural injuries. However, a pre-requisite to manipulate stem cell behaviour is to better understand the mechanisms that govern NSC homeostasis and differentiation. Fbw7 (also known as Fbxw7, hCdc4, hSel-10) is a member of the F-box family of proteins, which function as substrate recognition adaptors for SCF (complex of SKP1, CUL1 and Fbox protein)-type ubiquitin ligases [START_REF] Ho | F-box proteins: the key to protein degradation[END_REF] . SCF(Fbw7) targets several important oncoproteins, including c-Myc, c-Jun, CyclinE1 and Notch, for ubiquitin-dependent proteolysis and Fbw7 mutations have been detected in a variety of human cancers, indicating that Fbw7 is a tumour suppressor [START_REF] Welcker | FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation[END_REF][START_REF] Tan | The Fbxw7/hCdc4 tumor suppressor in human cancer[END_REF][START_REF] Fuchs | Tumor suppressor activities of the Fbw7 E3 ubiquitin ligase receptor[END_REF] . Several Fbw7 substrates have been implicated in the control of neural differentiation and viability. The AP-1 transcription factor c-Jun is an important regulator of neuronal viability, especially when phosphorylated by the JNK group of MAP kinases [START_REF] Behrens | Amino-terminal phosphorylation of c-Jun regulates stressinduced apoptosis and cellular proliferation[END_REF][START_REF] Besirli | The limited role of NH2-terminal c-Jun phosphorylation in neuronal apoptosis: identification of the nuclear pore complex as a potential target of the JNK pathway[END_REF][START_REF] Raivich | The AP-1 transcription factor c-Jun is required for efficient axonal regeneration[END_REF] . The Notch signalling pathway controls neural differentiation and is known to maintain NSC character and to inhibit neurogenesis. The Notch-Hes pathway is necessary for self-renewing cell division and thus, maintenance of the neural precursor population [START_REF] Yoon | Mind bomb 1-expressing intermediate progenitors generate notch signaling to maintain radial glial cells[END_REF][START_REF] Mizutani | Differential Notch signalling distinguishes neural stem cells from intermediate progenitors[END_REF][START_REF] Corbin | Regulation of neural progenitor cell development in the nervous system[END_REF][START_REF] Yoon | Notch signaling in the mammalian central nervous system: insights from mouse mutants[END_REF] . However, the significance of the regulation of c-Jun, Notch and other substrates by SCF(Fbw7) during neurogenesis has remained unclear to date. In this study we identify the E3 ubiquitin ligase Fbw7 as a key regulator of neural stem cell differentiation and progenitor viability. We demonstrate that Fbw7 acts as a molecular switch that antagonises Notch activity and JNK/c-Jun signalling to enable neural stem cell differentiation and progenitor survival.

RESULTS

Conditional inactivation of Fbw7 in the nervous system

To investigate the significance of Fbw7 expression and function during neural development, we generated conditional knock-out mice in which exon 5 of fbw7 was flanked by loxP sites (Supplementary Fig. 1a), since mice lacking Fbw7 die during early embryonic development due to placental and vascular defects [START_REF] Tetzlaff | Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein[END_REF][START_REF] Tsunematsu | Mouse Fbw7/Sel-10/Cdc4 is required for notch degradation during vascular development[END_REF] . Deletion of exon 5 removes most of the F-box, an essential domain of Fbw7, and in addition disrupts the fbw7 open reading frame preventing production of detectable Fbw7 protein (Jandke et al. in preparation, and below).

Floxed fbw7 (fbw7 f/f ) mice were crossed to Nestin-Cre transgenic mice previously shown to provide efficient NSC-specific Cre activity [START_REF] Raivich | The AP-1 transcription factor c-Jun is required for efficient axonal regeneration[END_REF] , with no recombination detectable in neural crestderived cells [START_REF] Kramer | Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb[END_REF] . In situ hybridisation revealed loss of fbw7 expression in fbw7 f/f ; Nestin-Cre + mutant mice (fbw7 ΔN mice) confirming efficient Cre-mediated deletion (Supplementary Fig. 1b). fbw7 ΔN mice were not obtained at weaning age and died perinatally (data not shown).

Fbw7 controls cell number and differentiation in the brain

While the overall structure of fbw7 ΔN mutant brains was normal, histological analysis of E18.5 mutant embryos revealed severe defects in neurogenesis. The size and thickness of brain regions such as the cortex was unaffected by the absence of Fbw7 (Supplementary Fig. 2), whereas the cellularity in several regions of the developing fbw7 ΔN brain was significantly reduced. In the E18.5 midbrain tectum and forebrain cortex, cell numbers were decreased by around 30% in areas where committed progenitors and differentiated cells reside, namely the tectal mantle layer (ML) as well as the cortical subventricular zone (SVZ), intermediate zone (IZ) and cortical plate (CP) (Fig. 1a-d). Similarly, the cellularity in fbw7 ΔN embryos was decreased in other brain regions such as the cerebellar anlage and the thalamus (Supplementary Fig. 3). In contrast, the cellularity in compartments harbouring immature cells was either unaffected, as in the cortical ventricular zone (VZ), or increased, as seen in the tectal VZ (Fig. 1a-d). Whilst we could not detect a statistically significant difference in the number of proliferating cells (Fig. 1e,g), the number of apoptotic cells marked by active Caspase 3 was significantly increased in the tectal ML at E16.5 (Fig. 1f,g).

To better understand the role of Fbw7 during brain development, we performed in situ hybridisation for fbw7 and immunohistochemistry (IHC) for markers characterising neural stem cells, progenitors and differentiated cells. fbw7 mRNA was highly expressed at E14.5 in the cortical VZ and SVZ, indicating a role for Fbw7 in stem cells and progenitors (Fig. 2a).

Expression of the neural stem cell and progenitor marker Nestin was already slightly increased in the E14.5 cortex of fbw7 ΔN mice. At E16.5 and E18.5, this difference in Nestin expression was more pronounced (Fig. 2b-d). The overall number of proliferating cells in the SVZ was not affected at various stages of development (Supplementary Fig. 4).

Immunoreactivity for the radial glia markers glutamate aspartate transporter (GLAST) and

Brain lipid binding protein (BLBP) was augmented (Fig. 2c,e,f). Indeed, quantification revealed a significant increase in BLBP-positive cells in the SVZ of the fbw7 ΔN E18.5 cortex (Fig. 2l). In contrast, the abundance of intermediate progenitor cells marked by Tbr2 was diminished in the mutant cortex (Fig. 2g,l). Besides the decrease in Tbr2-positive intermediate progenitors, the proportion of Doublecortin-positive neuronal progenitors was reduced in the mutant SVZ throughout embryonic brain development (Supplementary Fig. 5).

Consequently the number of cells expressing markers for neurons, NeuN, Tbr1, Ctip2 and Brn2, was decreased to varying extent (Fig. 2h-l). Since there is a substantial reduction in total cell number in areas of differentiated cells (Fig. 1a,c) and a reduced percentage of neurons, the absolute number of neurons is reduced by more than 50% in the fbw7 ΔN cortex.

In contrast to neurogenesis, absence of fbw7 did not have a significant effect on gliogenesis. GFAP-positive cells were hardly detectable in the IZ and CP of both the control and the mutant cortex at E18.5 and there was no significant difference in the number of cells expressing the astroglia marker S100 (Supplementary Fig. 6a-c). There was also no change in the percentage of cells expressing the oligodendroglia marker NG2 (Supplementary Fig. 6d,e). Similar to the cortex, an accumulation of cells expressing markers of immature cells and a reduction in NeuN-positive cells was also seen in the fbw7 ΔN tectum (Supplementary Fig. 7). It is likely that the severe reduction of neurons in fbw7 ΔN brains contributes to the perinatal lethality.

Fbw7 controls neurosphere cell survival and differentiation

To further investigate the function of Fbw7 in NSCs/NPCs, we prepared neurosphere cultures from E14.5 embryos. Neurospheres consist predominantly of neural progenitors and to a small extent of stem cells and differentiated cells [START_REF] Reynolds | Neural stem cells and neurospheres--re-evaluating the relationship[END_REF] . Neurospheres derived from fbw7 ΔN embryos were on average smaller in size and the number of neurospheres was significantly decreased (Fig. 3a-c). The loss of Fbw7 function had no significant effect on NSC/NPC proliferation analysed by staining for pH3 and CFSE dilution (Supplementary Fig. 8). Whilst the large majority of cells in both control and fbw7 ΔN neurospheres expressed Nestin, the percentage of cells positive for Musashi-1 (Msi-1), which marks more immature cells, was greatly increased in fbw7 ΔN neurospheres (Fig. 3d,e). Similar to our observations in the cortex, the number of neurosphere cells expressing glial markers was not significantly altered in the absence of Fbw7 (Supplementary Fig. 6f-i). Moreover, TUNEL assay revealed substantially increased cell death in fbw7 ΔN neurospheres (Fig. 3f,g). Thus it appears that a decrease in cell differentiation, accompanied by excessive progenitor cell death, reduces the efficiency of fbw7 ΔN neurosphere formation. This predicts that the rescue of cell death would improve neurosphere formation, an experiment we describe below.

We next investigated the differentiation potential of Fbw7-deficient NSCs/NPCs. Cultured NSCs/NPCs from control mice readily formed a monolayer of cells after induction of differentiation, but fbw7 ΔN mutant NSCs/NPCs often retained the ball-shaped neurosphere morphology (Supplementary Fig. 9), indicative of a problem to undergo the differentiation programme. 5 days after the onset of differentiation, the percentage of Nestin-positive NSCs/NPCs was low in control cultures. In contrast, a higher number of fbw7 ΔN neural cells retained Nestin expression (Fig. 4a,d). To further characterise the Nestin-positive cells that accumulated in fbw7 ΔN differentiation cultures, we investigated the expression of the classical RGC marker RC2. The majority of Nestin-positive cells were also RC2-positive (Fig. 4a,d), and the absence of Fbw7 also led to increased numbers of cells expressing two further RGC markers, BLBP and Vimentin (Supplementary Fig. 10a), indicating that RGCs represent the main immature cell population accumulating in fbw7 ΔN differentiation cultures. Additionally, CD133-positive neuroepithelial stem cells were very rare in control cultures 5 days after the onset of differentiation, whereas in fbw7-deficient cultures CD133-positive cells were readily detectable (Supplementary Fig. 10b). A similar increase in Nestin-positive cells was also observed when adherent NSC cultures were induced to differentiate (Supplementary Fig. 11a-d). The accumulation of immature neural cells in fbw7 ΔN neurosphere cultures was also observed after prolonged time (11 days) under differentiation conditions, indicating that Fbw7 inactivation does not only delay, but genuinely blocks efficient neural differentiation (Supplementary Fig. 12). Conversely, the percentage of differentiated cells expressing the neuronal marker Map2 was reduced (Fig. 4b,d) whereas the frequency of astrocytes expressing Connexin43 and oligodendrocytes marked by O4 were not significantly altered in mutant differentiation cultures (Fig. 4b-d). Triple IF staining revealed that the vast majority of both control and fbw7 ΔN neurospheres gave rise to differentiated cells of all 3 lineages (Supplementary Fig. 13). Taken together, the absence of Fbw7 results in decreased neurogenesis and an accumulation of cells expressing radial glia markers in cultured neurospheres.

Fbw7 antagonises c-Jun to regulate neural cell viability

The fbw7 locus encodes 3 different Fbw7 isoforms which are generated by alternative splicing of the first exon [START_REF] Welcker | FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation[END_REF] . Whereas the α and β fbw7 splice variants were expressed in neurospheres, the γ isoform was undetectable. fbw7 α and β were also present in total adult brain RNA.

Notably, other organs showed different fbw7 splicing patterns (Supplementary Fig. 14).

Fbw7 protein was detected in control neurosphere extracts but was absent in fbw7 ΔN neurospheres (Fig. 5a). To understand the mechanism of Fbw7 function, we investigated the expression levels of known substrates of SCF(Fbw7). Whilst phosphorylated c-Myc and phosphorylated CyclinE protein levels were not substantially altered, there was a significant increase in the levels of active Notch1 and phosphorylated c-Jun (Fig. 5a and Supplementary Fig. 11e). Phosphorylated c-Jun is an important regulator of neuronal viability [START_REF] Behrens | Amino-terminal phosphorylation of c-Jun regulates stressinduced apoptosis and cellular proliferation[END_REF][START_REF] Besirli | The limited role of NH2-terminal c-Jun phosphorylation in neuronal apoptosis: identification of the nuclear pore complex as a potential target of the JNK pathway[END_REF][START_REF] Raivich | The AP-1 transcription factor c-Jun is required for efficient axonal regeneration[END_REF] , and we have previously demonstrated that depletion of Fbw7 leads to cell death in PC12 neuronal-like cells due to increased levels of phosphorylated c-Jun [START_REF] Nateri | The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling[END_REF] . Thus we investigated whether increased c-Jun function contributed to cell death in the absence of Fbw7 during neurogenesis. As in neurosphere cultures, the expression level of phosphorylated c-Jun was also greatly increased in the brains of E18.5 fbw7 ΔN mutant embryos (Fig. 5b). To assess the functional importance of c-Jun downstream of Fbw7, one allele of the c-jun gene was inactivated in a fbw7 ΔN -mutant background (fbw7 f/f ; c-jun f/+ ; Nestin-Cre + or fbw7 ΔN ; c-jun ΔN/+ mutant mice). Heterozygous deletion markedly reduced the levels of phosphorylated c-Jun in the brain of Fbw7-deficient embryos (Fig. 5b). Genetic reduction of c-Jun function significantly rescued the defect in cellularity in the mantle layer of the midbrain tectum, but the number of stem cells in the tectal ventricular zone was still elevated in fbw7 ΔN ; c-jun ΔN/+ embryos (Fig. 5c and Supplementary Fig. 16). Likewise, neurosphere formation in vitro was substantially increased in fbw7 ΔN ; c-jun ΔN/+ cells compared to fbw7 ΔN single mutants, which was accompanied by a considerable reduction in TUNEL-positive apoptotic cells in fbw7 ΔN ; c-jun ΔN/+ neurospheres (Fig. 5d,e), indicating that c-Jun-mediated cell death is causally involved in defective fbw7 ΔN neurosphere formation.

An important mechanism of c-Jun-mediated control of neuronal viability is the transcriptional regulation of pro-apoptotic members of the Bcl2 family of proteins [START_REF] Bossy-Wetzel | Induction of apoptosis by the transcription factor c-Jun[END_REF][START_REF] Whitfield | Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF][START_REF] Ma | dp5/HRK is a c-Jun target gene and required for apoptosis induced by potassium deprivation in cerebellar granule neurons[END_REF] . mRNA levels of cjun, which is autoregulated by the c-Jun protein itself [START_REF] Angel | The jun proto-oncogene is positively autoregulated by its product, Jun/AP1[END_REF] , were increased in the absence of Fbw7. Likewise, the expression of the pro-apoptotic genes bad and bim were augmented to varying extents. The expression of the anti-apoptotic bcl2 gene was unaffected (Fig. 5f). As cells with the highest levels of phosphorylated c-Jun are expected to undergo apoptosis in neurosphere cultures, it is plausible that this analysis underestimates the effect of Fbw7 inactivation on c-Jun-mediated gene activation. Heterozygosity for c-jun reduced the expression of the pro-apoptotic c-Jun target genes (Fig. 5f). In summary, negative regulation of c-Jun activity by Fbw7 is essential to prevent excessive cell death during neurogenesis.

Thus downregulation of c-Jun in the fbw7 ΔN background rescued neurosphere formation and the number of apoptotic neurosphere cells in vitro and the reduction in cell number in vivo.

Fbw7 antagonises Notch to allow NSC differentiation

Heterozygosity for c-jun did not rescue the differentiation defect and the increased number of cells with radial glia identity in differentiating fbw7 ΔN neurosphere cultures, arguing that c-Jun protein levels are not critical for Fbw7-regulated neural differentiation (Supplementary Fig. 17). In contrast, deregulated Notch signalling was a possible candidate for the differentiation defects observed in fbw7 ΔN mutants in vivo and in vitro. Overexpression of the activating intracellular fragment of Notch1, the NICD1, has been shown to be sufficient to promote radial glia identity during embryogenesis while abrogation of Notch signalling leads to depletion of RGCs [START_REF] Yoon | Mind bomb 1-expressing intermediate progenitors generate notch signaling to maintain radial glial cells[END_REF][START_REF] Gaiano | Radial glial identity is promoted by Notch1 signaling in the murine forebrain[END_REF] . However, the molecular mechanism that antagonises Notch activity to allow neural differentiation to occur is not known.

The increased protein expression of NICD1 seen by immunoblotting was confirmed by IHC (Fig. 6a), indicating that Fbw7 regulates NICD1 levels both in vitro and in vivo. Our observation that BLBP levels were higher in fbw7 ΔN mutant embryos and neurosphere cultures (Fig. 2c,f,l and Supplementary Figs. 7 and10a) was a first indication that increased Notch immunoreactivity translated into increased Notch function, since BLBP was reported to be a direct target of the Notch signalling pathway [START_REF] Anthony | Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells[END_REF] .

To address the functional significance of increased Notch activity in Fbw7-regulated neural differentiation, we performed genetic and pharmacological rescue experiments. One allele of the Notch1 gene [START_REF] Radtke | Deficient T cell fate specification in mice with an induced inactivation of Notch1[END_REF] was inactivated in a fbw7 ΔN -mutant background (fbw7 f/f ; Notch1 Δ/+ ; Nestin-Cre + or fbw7 ΔN ; Notch1 Δ/+ mutant mice). In addition, we made use of the γ-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-Alanyl)]-S-phenylglycine t-butyl ester (DAPT), which prevents the release of the activating intracellular fragment of Notch proteins [START_REF] Selkoe | Notch and Presenilin: regulated intramembrane proteolysis links development and degeneration[END_REF] . The deletion of one Notch1 allele in the fbw7 ΔN background reduced the levels of NICD1 (Fig. 6a). Notch1 heterozygosity attenuated the accumulation of BLBP-positive cells and Nestin immunoreactivity and partially rescued the number of NeuN-positive neurons in the fbw7 ΔNmutant cortex (Fig. 6b-d and Supplementary Fig. 18a,b). mRNA levels of the classical Notch target genes hes5 and hey1 were augmented in fbw7 ΔN neurosphere cultures, whereas hes1 levels were not significantly altered (Fig. 6e and Supplementary Figs. 11f and19a).

Heterozygosity for Notch1 as well as DAPT treatment resulted in a significant reduction in the expression of hes5 and hey1 (Fig. 6e and Supplementary Fig. 19a).

Consequently, we next determined whether aberrant Notch activity contributes to the differentiation defects in fbw7 ΔN neurosphere cultures. Both Notch1 heterozygosity or DAPT treatment resulted in a substantial reduction in the percentage of Nestin-positive cells in fbw7 ΔN mutant differentiation cultures and the number of Map2-positive neurons was significantly increased in fbw7 ΔN ; Notch1 Δ/+ in comparison to fbw7 ΔN cultures (Fig. 6f,g and Supplementary Figs. 18c,d and19b,c). This suggests that Fbw7 is essential to antagonise Notch signalling to allow neural differentiation to occur.

DISCUSSION

This study identifies Fbw7 as a key regulator of neural progenitor viability and neural stem cell differentiation. We detected increased immunoreactivity for Nestin, GLAST and BLBP in the fbw7-mutant brain, suggesting that Fbw7 deficiency increases the number of immature cells. It is worth noting that GLAST and BLBP are also expressed in some astrocytes postnatally [START_REF] Gotz | Radial glial cells defined and major intermediates between embryonic stem cells and CNS neurons[END_REF][START_REF] Anthony | Radial glia serve as neuronal progenitors in all regions of the central nervous system[END_REF] . Thus it was possible that the supernumerary GLAST-positive and BLBPpositive cells in the fbw7 ΔN cortex were generated due to an increase in astrogliogenesis.

However, expression of the astrocyte markers GFAP, S100 and Connexin43 was unaltered in fbw7-mutant cells (Fig. 4b,d and Supplementary Fig. f,g). The increased percentage of cells positive for RC2 and other RGC markers in differentiating neurospheres (Fig. 4a,d and Supplementary Fig. 10a) together with the known function of Notch signalling to promote RGC fate [START_REF] Gaiano | Radial glial identity is promoted by Notch1 signaling in the murine forebrain[END_REF][START_REF] Patten | Sequential signaling through Notch1 and erbB receptors mediates radial glia differentiation[END_REF] is in agreement with a role for Fbw7 in RGC development.

While we have obtained no evidence for a defect in gliogenesis, Fbw7 deficiency leads to decreased numbers of neurons (Fig. 2h-l). The loss of Fbw7 led to a reduction of Tbr2positive intermediate progenitors (Fig. 2g,l). There was also a significant decrease in Doublecortin-positive neuroblasts in the mutant SVZ throughout embryonic brain development, indicative of a defect in neurogenesis (Supplementary Fig. 5). It is conceivable that Fbw7 deficiency inhibits neural stem cell differentiation at the expense of neuronal progenitors, a phenotype consistent with a gain of Notch function [START_REF] Gaiano | Radial glial identity is promoted by Notch1 signaling in the murine forebrain[END_REF] . Furthermore, Fbw7 deficiency increases progenitor cell death, which leads to a further reduction in neuronal number. However, a defect in neuronal maturation, and thus marker expression, may also contribute to the fbw7 ΔN phenotype. SCF(Fbw7) has several substrates with diverse biological activities. Downregulation of the Fbw7-substrate c-Jun in the fbw7 ΔN background rescued the increased number of apoptotic neurosphere cells and the reduced number of cells in areas of differentiated cells in the brain.

Regulation of c-Jun by Fbw7 is essential to control neural cell viability. Paradoxically, JNK activity is very high in neurons [START_REF] Nateri | The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling[END_REF][START_REF] Coffey | c-Jun N-terminal protein kinase (JNK) 2/3 is specifically activated by stress, mediating c-Jun activation, in the presence of constitutive JNK1 activity in cerebellar neurons[END_REF] , and a role for JNK signalling in neuronal migration and maintenance of cytoskeletal integrity of neuronal cells has been described [START_REF] Wang | Targeted deletion of the mitogen-activated protein kinase kinase 4 gene in the nervous system causes severe brain developmental defects and premature death[END_REF][START_REF] Chang | JNK1 Is Required for Maintenance of Neuronal Microtubules and Controls Phosphorylation of Microtubule-Associated Proteins[END_REF] . Therefore SCF(Fbw7) may function to inactivate apoptotic c-Jun-dependent JNK signalling, allowing neural cells to tolerate potentially neurotoxic JNK activity. c-Jun is not involved in the regulation of neural differentiation by Fbw7. This process is controlled by Fbw7 through the regulation of Notch activity. Downregulation of the Fbw7substrate Notch in the fbw7 ΔN background alleviated the block in cell differentiation and rescued neuronal numbers. While it is known that Notch activity needs to be downregulated for neuronal differentiation to occur [START_REF] Corbin | Regulation of neural progenitor cell development in the nervous system[END_REF] , the mechanism that controls this transition has remained enigmatic. This work shows for the first time that Fbw7 is an important molecular switch that is required for attenuation of Notch activity during neurogenesis. It is noteworthy that SCF(Fbw7) can target various members of the Notch family of proteins [START_REF] Tetzlaff | Defective cardiovascular development and elevated cyclin E and Notch proteins in mice lacking the Fbw7 F-box protein[END_REF] , making it wellsuited as a general antagonist of Notch function. Decreased Notch pathway activity in mice lacking Notch1, rbp-j (cbf1) or the Notch ligand dll1 as well as in hes1 single and hes1/hes5 double mutants results in precocious neuronal differentiation [START_REF] Corbin | Regulation of neural progenitor cell development in the nervous system[END_REF][START_REF] Yoon | Notch signaling in the mammalian central nervous system: insights from mouse mutants[END_REF] . hes5 and hes1 are well known targets of the Notch signalling pathway [START_REF] Ohtsuka | Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation[END_REF] , and are crucial mediators of Notch function in the brain. However, in many instances hes1 and hes5 are differentially regulated. Notch-1 or RBP-J-mutant embryos show reduced expression of hes5, but hes1 expression was normal [START_REF] Yoon | Notch signaling in the mammalian central nervous system: insights from mouse mutants[END_REF][START_REF] De La Pompa | Conservation of the Notch signalling pathway in mammalian neurogenesis[END_REF] . Similarly, γ-secretase inhibition of Notch activity in neural progenitors was reported to have a much stronger effect on hes5 compared to hes1 [START_REF] Nelson | Transient inactivation of Notch signaling synchronizes differentiation of neural progenitor cells[END_REF] . In our mouse model fbw7 deletion induces hes5 mRNA increase while having a mild effect on hes1, suggesting that fbw7 affects Notch1 with an overall balance towards hes5. This is in line with previous observations in the mouse retina, in which glial cell specification is modulated by hes5 in a hes1-independent manner [START_REF] Hojo | Glial cell fate specification modulated by the bHLH gene Hes5 in mouse retina[END_REF] .

Fbw7 appears to use distinct substrates to regulate different biological processes, however the molecular mechanism of how this occurs remains unknown. For instance, c-Myc protein accumulates in fbw7-deficient hematopoietic stem cells [START_REF] Thompson | Control of hematopoietic stem cell quiescence by the E3 ubiquitin ligase Fbw7[END_REF] . In contrast, Notch1 and c-Jun are increased upon fbw7 deletion in mouse embryonic fibroblasts (MEFs), but no effect is found on CyclinE or c-Myc in these cells [START_REF] Mao | Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene[END_REF] . In the developing brain, the main SCF(Fbw7) substrates affected are Notch1 and c-Jun, while c-Myc and CyclinE protein levels are not greatly altered. One potential explanation could be the relative abundance of Fbw7 isoforms. The Fbw7γ isoform has been shown to mediate degradation of c-Myc and CyclinE [START_REF] Welcker | A nucleolar isoform of the Fbw7 ubiquitin ligase regulates c-Myc and cell size[END_REF][START_REF] Sangfelt | Both SCF(Cdc4alpha) and SCF(Cdc4gamma) are required for cyclin E turnover in cell lines that do not overexpress cyclin E[END_REF][START_REF] Van Drogen | Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms[END_REF] . The absence of Fbw7γ in neural stem cells and progenitors (Supplementary Fig. 14) could therefore impede efficient degradation of CyclinE or c-Myc. Thus differential expression of Fbw7 isoforms may contribute to cell-type specific substrate degradation.

The function of Fbw7 in stem cells is highly dependent on the organ system. Whereas we demonstrate here that Fbw7 is required for neural differentiation, inactivation of Fbw7 in hematopoietic stem cells (HSCs) causes defective maintenance of quiescence and premature depletion of HSCs [START_REF] Thompson | Control of hematopoietic stem cell quiescence by the E3 ubiquitin ligase Fbw7[END_REF][START_REF] Matsuoka | Fbxw7 acts as a critical fail-safe against premature loss of hematopoietic stem cells and development of T-ALL[END_REF] . Thus the lack of Fbw7 leads to opposite functional consequences in HSCs and NSCs. It is plausible that cell-type specific differential regulation of Fbw7 and its substrates is one mechanism that could explain the distinct roles of Fbw7 in different types of stem cells. (d) Quantification of BLBP-positive cells in the IZ and the CP of the fbw7 f/f , fbw7 ΔN and fbw7 ΔN ; Notch1 Δ/+ E18.5 cortex. n = 3. (e) qRT-PCR analysis of hes5, hey1 and hes1 transcripts in fbw7 f/f , fbw7 f/f ; Notch1 Δ/+ , fbw7 ΔN and fbw7 ΔN ; Notch1 Δ/+ neurospheres. The data are normalised to gapdh and represented as fold change over RNA levels in fbw7 f/f neurospheres, which is set to 1. (f) Immunocytochemistry for Nestin (green) on fbw7 f/f , fbw7 f/f ; Notch1 Δ/+ , fbw7 ΔN and fbw7 ΔN ; Notch1 Δ/+ neurosphere cultures after 5 days under differentiation. (a) qRT-PCR analysis of hes5, hey1 and hes1 transcripts in fbw7 f/f and fbw7 ΔN

MATERIALS AND METHODS

Mouse Lines

The fbw7 (floxed exon 5) transgenic construct was electroporated into embryonic stem cells, stable transfectants were selected and mice generated according to standard protocols 7 .

Details will be described in Jandke et al., in preparation. c-jun f/f , Notch1 f/f and Nestin-Cre mice have been described [START_REF] Raivich | The AP-1 transcription factor c-Jun is required for efficient axonal regeneration[END_REF][START_REF] Radtke | Deficient T cell fate specification in mice with an induced inactivation of Notch1[END_REF] . All experiments involving mice were approved by the London Research Institute (LRI) Animal Ethics Committee following Home Office guidelines.

Cell culture

Neural cells were isolated from E14.5 fore-and midbrains of fbw7 f/f , fbw7 f/f ; Notch1 Δ/+ , fbw7 ΔN , fbw7 ΔN ; c-Jun ΔN/+ and fbw7 ΔN ; Notch1 Δ/+ mouse embryos. Cells were cultured under self-renewal conditions in Neurobasal Medium (Invitrogen) supplemented with 1% Penicillin/Streptomycin (10000 U/ml; Invitrogen), 1% L-glutamine (200 mM; Invitrogen), 2% B27 supplement (Invitrogen) and human epidermal growth factor (EGF; 20 ng/ml; PeproTech) and fibroblast growth factor (FGF-basic; 20 ng/ml; PeproTech). All experiments were performed using secondary and tertiary neurospheres.

Adherent NSC cultures were derived as previously described [START_REF] Pollard | Adherent neural stem (NS) cells from fetal and adult forebrain[END_REF] with minor modifications. Briefly, cells were cultured in Neurobasal Medium (Invitrogen) supplemented with 1% Penicillin/Streptomycin (10000 U/ml; Invitrogen), 1% L-glutamine (200 mM; Invitrogen), 2% B27 supplement (Invitrogen), 1% N-2 supplement (Invitrogen), 20 ng/ml EGF (PeproTech), 20 ng/ml FGF-basic (PeproTech) and 1 μg/ml laminin (Sigma).

For differentiation, growth factors were withdrawn from the medium and 10% NeuroCult ® Differentiation Supplement (StemCell Technologies) was added. Under differentiation conditions, 5 x 10 5 cells were plated on poly-L-ornithine (0.01% solution; Sigma; diluted 1:10 in 150 mM disodium tetraborate; Sigma) coated cover slips.

Single cell suspensions were made from neurospheres using AccuMax (PAA Laboratories).

To assess neurosphere formation, cells were plated in limiting dilutions from 500 to 4 cells in 96-well-plates. Neurospheres were counted after 2 weeks in culture.

To examine cell division rates, neurosphere-derived cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) using the CellTrace ™ CFSE Cell Proliferation Kit (Invitrogen) according to the manufacturer's instructions. FACSmeasurements for CFSE intensity were performed every 48 h after CFSE-staining for one week using an LSR II Flow Cytometer (BD). By means of DAPI-staining, viable cells (DAPInegative) were selected. Analysis was carried out using CellQuest ™ Pro (BD) and FlowJo (Tree Star) software.

For the inhibition of Notch signalling, neurospheres were treated with 1 μΜ N-[N- (3,5-Difluorophenacetyl-L-Alanyl)]-S-phenylglycine t-butyl ester (DAPT; Sigma) for 72 h.

Immunohistochemistry and immunocytochemistry

Mouse brains were dissected from E10.5, E14.5, E16.5 and E18.5 fbw7 f/f , fbw7 ΔN , fbw7 ΔN ; c-Jun ΔN/+ and fbw7 ΔN ; Notch1 Δ/+ mouse embryos. Brains were fixed in 10% neutral buffered formalin overnight, transferred to 70% ethanol, processed and embedded into paraffin.

Neurospheres were fixed in 10% neutral buffered formalin overnight and processed using the Shandon Cytoblock ® Cell Preparation System (Thermo Fisher Scientific). Sections were cut (for brain sections: sagittally) at 4 μm for Haematoxylin & Eosin (H&E) staining, 3,3'-Diaminobenzidine (DAB) staining, TUNEL (TdT-mediated dUTP-biotin nick end labeling) and fluorescent immunohistochemistry. To define comparable sections, brains were cut alongside the medial-lateral axis with every 10 th section stained with H&E. For cell counting, all cells in the tectal ML and VZ as well as in the CP, IZ, SVZ and VZ of the cortex were counted in an area of the same width. Layer boundaries were determined by cell morphology and layer-specific marker expression.

Figure 1

 1 Figure 1 Fbw7 controls cell number and survival in the brain. (a) Haematoxylin and eosin (H&E) staining of the E18.5 forebrain from fbw7 f/f and fbw7 ΔN mouse embryos. Rectangles

Figure 2

 2 Figure 2 Fbw7 controls stem cell differentiation and neurogenesis in the brain. (a) fbw7 (exon

Figure 3

 3 Figure 3 Absence of Fbw7 leads to retention of markers for immature cells and induces apoptosis in vitro. (a) Phase contrast pictures of fbw7 f/f and fbw7 ΔN neurosphere cultures under

Figure 6

 6 Figure 6 Fbw7 controls stem cell differentiation by antagonising Notch. (a)

  neurospheres treated with DMSO (control) or 1 μM DAPT. The data are normalised to gapdh and represented as fold change over RNA levels in fbw7 f/f + DMSO neurospheres, which is set to 1. (b) Immunocytochemistry for Nestin (green) on fbw7 f/f and fbw7 ΔN neurosphere cultures after 5 days under differentiation conditions treated with DMSO (control) or 1 μM DAPT. White squares mark areas shown in high magnification in panels on the right. DNA (blue) was counterstained with Hoechst 33342. Scale bars: 100 μm. (c) Histogram showing the percentage of Nestin-positive cells in fbw7 f/f and fbw7 ΔN neurosphere cultures after 5 days under differentiation conditions treated with DMSO (control) or 1 μM DAPT. n = 5. Error bars represent the s.e.m. Statistical significance: n.s.: not significant; **: p ≤ 0.01; ***: p ≤ 0.001 (unpaired t test). J.D.H. designed and conducted the majority of the experiments, did the data analyses and cowrote the manuscript. A.J. generated the fbw7 f/f mice and performed the radioactive ISH. S.M.B. performed the experiments on adherent NSC cultures. E.N. did the IHC stainings. B.S.-D. performed the non-radioactive ISH. S.B. gave advice on neurosphere sectioning. A.B. supervised the project, directed the experiments and wrote the manuscript.

ACKNOWLEDGEMENTS

We are grateful to the Animal Unit, Equipment Park, FACS Lab and the Experimental Pathology Lab at the London Research Institute for technical help and advice on histology.

We are grateful to F. Radke for floxed Notch1 mice. We thank R. Sancho for the data on fbw7 isoform expression in various tissues. We thank C. R. Da Costa, S. Marino and A. Martin-Villalba for critical reading of the manuscript. S.M.B. is supported by a Federation of the Societies of Biochemistry and Molecular Biology (FEBS) Long Term Fellowship. Part of this work is supported by an MRC project grant (85704) to A.B. The London Research Institute is funded by Cancer Research UK.

neurosphere sections. DNA (blue) was counterstained with DAPI. Scale bars: 100 μm. (e) Quantification of Musashi1-positive cells (Msi1 + ) per neurosphere in fbw7 f/f and fbw7 ΔN neurosphere cultures; n = 3. (f) DAB staining for TUNEL (TdT-mediated dUTP-biotin nick end labeling) positive cells on fbw7 f/f and fbw7 ΔN

In situ hybridisation

Radioactive in situ hybridisation (ISH) was performed by the CRUK in situ hybridisation service at the LRI. The in situ probe corresponded to exon 2-5 of the murine fbw7 cDNA. Non-radioactive ISH was performed as previously described [START_REF] Behrens | Impaired intervertebral disc formation in the absence of Jun[END_REF] with minor modifications. For fbw7 ISH, DIG-labelled 800 bp PCR product (exons 6 to 10) was used.

Western Blot Analysis

Neurospheres were suspended in cell lysis buffer (Cell Signaling) supplemented with protease inhibitor cocktail (Sigma). Western blot analysis was performed as previously described [START_REF] Nateri | Interaction of phosphorylated c-Jun with TCF4 regulates intestinal cancer development[END_REF] .

For immunoblotting, antibodies against activated Notch1 (Abcam), c-Jun (Santa Cruz Biotechnology), serine 73 phosphorylated c-Jun (Cell Signaling), Fbw7 (LRI/CRUK; Abcam), threonine 58/serine 62 phosphorylated c-Myc (Cell Signaling), threonine 395 phosphorylated cyclinE (Santa Cruz Biotechnology) and β-Actin (Sigma) were used.