Title: Elevated FOXG1 supports exit from quiescence in neural stem cells through FoxO6

The molecular mechanisms controlling the balance of quiescence and proliferation in adult neural stem cells (NSCs) are often deregulated in brain cancers such as glioblastoma (GBM). Previously, we reported that FOXG1, a forebrain-restricted neurodevelopmental transcription factor, is frequently upregulated in glioblastoma stem cells (GSCs) and limits the effects of cytostatic pathways, in part by repression of the tumour suppressor Foxo3 . Here, we show that increased FOXG1 upregulates FoxO6, a more recently discovered FoxO family member with potential oncogenic functions. Although genetic ablation of FoxO6 in proliferating NSCs has no effect on the cell cycle or entry into quiescence, we find that FoxO6 -null NSCs can no longer efficiently exit quiescence following FOXG1 elevation. Increased FoxO6 results in the formation of large acidic vacuoles, reminiscent of Pak1-regulated macropinocytosis. Consistently, Pak1 expression is upregulated by FOXG1 overexpression and downregulated upon FoxO6 loss in proliferative NSCs. These data suggest a pro-oncogenic role for FoxO6 in controlling the exit from quiescence in NSCs, and shed light on the functions of this underexplored FoxO family member. is a downstream target activated FOXG1. FOXG1-V5 overexpression were plated at a density of 10 cells/mm 2 (10,000 cells per well of a 6 well plate), in NSC media in the absence of EGF/FGF-2 and supplemented with BMP4 (10 ng/ml) (Bulstrode et al , 2017). After 24 h, media was replaced fully with NSC media containing EGF/FGF-2 with or without Dox (1000 ng/ml). Media was then replaced every 3-4 days. Following 10-12 days, plates were fixed using 4% PFA for 10 min at room temperature. Colonies were stained using methylene blue for 30 min and imaged on a Celigo™ Image Cytometer (Nexcelom Bioscience). Colonies were counted manually using the Cell Counter plugin on FIJI, or the % pixel area of the well covered by colonies quantified using FIJI Image thresholding and Particle Analysis functions. Three technical replicates were averaged to give the mean number of colonies per biological replicate.


Introduction
Stem cell fate is orchestrated by gene regulatory networks of 'lineage-specific' master regulatory transcription factors (Graf & Enver, 2009). Just as tissues rely on these factors for proper development, cancers can subvert developmental networks to impose a stem cell-like state that underpins tumour growth (Roy & Hebrok, 2015;Huilgol et al, 2019). Glioblastoma multiforme (GBM), the most common and aggressive primary adult brain cancer, is driven by glioblastoma stem cells (GSCs) that display neural stem cell (NSC) characteristics (Singh et al, 2003;Pollard et al, 2009;Richards et al, 2021). GSCs frequently overexpress key neurodevelopmental transcription factors to drive their self-renewal and restrict differentiation (Engström et al, 2012;Carén et al, 2015;Suva et al, 2014;Singh et al, 2017). One such factor is the Forkhead box transcription factor, FOXG1. FOXG1 has important roles in telencephalon development and in vitro reprogramming (Lujan et al, 2012;Xuan et al, 1995;Bulstrode et al, 2017). It is one of the most consistently overexpressed genes across GBM molecular subtypes, and high levels are associated with adverse outcomes (Engström et al, 2012;Robertson et al, 2015;Wang et al, 2018;Verginelli et al, 2013). Understanding the molecular mechanisms through which FOXG1 operates in NSCs and GSCs is therefore of great interest.
Both GSCs and genetically normal NSCs are known to be heterogeneous with regards to cell cycle status, with cells spanning a continuum from dormant to quiescent and proliferative states (non-cycling, slowcycling and fast cycling, respectively) (Codega et al, 2014;Dulken et al, 2017;Marqués-Torrejón et al, NSCs expanded in culture have overlapping gene regulatory networks with GBMs and provide a genetically tractable experimental in vitro model that has been useful in delineating the pathways controlling GSC quiescence (Ying et al, 2003;Sun et al, 2008;Conti et al, 2005;Pollard et al, 2009;Bulstrode et al, 2017;Carén et al, 2015;Bressan et al, 2021;Marqués-Torrejón et al, 2021). Bone-morphogenetic protein 4 (BMP4) induces quiescence of NSCs in vitro and in vivo (Bond et al, 2012;Martynoga et al, 2013;Mira et al, 2007;Sun et al, 2011;Marqués-Torrejón et al, 2021), while the mitogens EGF and FGF-2 stimulate proliferation. Previously, we demonstrated that overexpression of the GBM-associated master regulators FOXG1 and SOX2 drives quiescent mouse NSCs into a proliferative radial glia-like state (Bulstrode et al, 2017) and induces transcriptional changes at many key cell cycle and epigenetic regulators. In particular, FoxO3, which induces quiescence and prevents premature NSC differentiation, is directly repressed by FOXG1 (Renault et al, 2009;Bulstrode et al, 2017). FOXG1 is therefore an important regulator of quiescence in NSCs and GSCs. Determining the genes and pathways operating downstream of elevated FOXG1 will therefore help our understanding of normal NSC development, adult NSC homeostasis and GBM biology.
The FoxO family are key downstream effectors of PI3K-Akt signalling, controlling genes governing diverse cellular processes including proliferation, metabolism, differentiation, and apoptosis. While FoxO factors can have context-dependent roles in supporting cellular resilience, they are most well-known for their tumour suppressive functions in tissue homeostasis, ageing and cancer (Hornsveld et al, 2018;Dansen & Burgering, 2008). FoxO1/3/4 are broadly expressed during development and adulthood and, while discrete roles have been identified, they appear to regulate common target genes in vitro with likely significant redundancies (Paik et al, 2007).
FoxO6 is the most recently identified FoxO member; it was initially reported to be expressed mainly within the CNS of adult mammals (Hoekman et al, 2006), but may also have roles in other tissues such as liver and muscle (Kim et al, 2011). Compared to FoxO1/3/4, it has several unique molecular characteristics: FoxO6 has a low sequence homology (~30%) to other FoxO factors, lacks one of three consensus PKB phosphorylation sites and the presence of a nuclear export signal is debated (Kim et al, 2013). Unlike other FoxO members, FoxO6 does not undergo complete nucleo-cytoplasmic shuttling in response to PI3K-Aktmediated phosphorylation (van der Heide et al, 2005;Jacobs et al, 2003). These features suggest a distinct cellular function. Indeed, in several cancers FOXO6 is elevated and has oncogenic roles, triggering increased proliferation and progression (Qinyu et al, 2013;Rothenberg et al, 2015;Wang et al, 2017;Lallemand et al, 2018).
Here, we demonstrate that FoxO6 is transcriptionally activated downstream of elevated FOXG1 in both mouse NSCs and GSCs, and is necessary for FOXG1-driven exit from quiescence. Following forced expression of FoxO6, we observed a stimulation of macropinocytosis, a cellular process involved in nutrient uptake that requires Pak1-regulated actin cytoskeleton remodelling. Gain-and loss-of-function mechanistic studies demonstrate Pak1 is upregulated by FOXG1 overexpression and downregulated upon FoxO6 loss in proliferative NSCs. Altogether, these data suggest a functional pro-oncogenic role for FoxO6 in the regulatory transitions, such as cell shape and metabolic changes, that must be initiated as cells exit from quiescence into proliferation.
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FOXG1 transcriptionally activates FoxO6 in mouse NSCs and GSCs
Previously we found that overexpression of the master regulators FOXG1 and SOX2 supports cell cycle reentry of quiescent mouse NSCs. ChIP-seq and RNA-seq data identified FoxO6 as a strong candidate FOXG1/SOX2-regulated target gene (Bulstrode et al, 2017). Here, we hypothesise that, in contrast to FoxO3, FoxO6 may have unique roles in supporting proliferation. To investigate the effect of elevated FOXG1 on FoxO6 expression -thereby mimicking the increased levels seen in GBMs -we used clonal adult mouse NSC lines harbouring a Doxycycline (Dox)-inducible FOXG1-V5 construct, as reported previously (Bulstrode et al, 2017).
Elevated FOXG1-V5 was found to significantly increase FoxO6 expression in proliferating NSCs ( Figure   1A). After 24 h, FoxO6 levels increased by ~17-fold and ~4-fold in two independent clonal NSC lines, termed 'F6' and 'F11-19', respectively. To circumvent a lack of FoxO6-specific antibodies, an HA tag was inserted using CRISPR/Cas9-mediated homologous recombination at the 3' end of FoxO6 in F6 cells ( Figure 1B). In agreement with mRNA upregulation, we observed a clear induction of FoxO6 protein in response to elevated FOXG1 (+Dox) (Figures 1C and D). These data indicate that FoxO6 mRNA and protein expression are activated downstream of FOXG1 in normal NSCs.
We next explored FoxO6 levels in a mouse GBM model cell line (IENS), which expresses FOXG1 at higher levels than mouse NSCs (Bulstrode et al, 2017). We tested if Foxg1 ablation affected FoxO6 expression.
Elevated Foxg1 is therefore necessary for FoxO6 expression in GSCs and is sufficient to induce increased FoxO6 expression in NSCs.
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FOXG1 induction of FoxO6 occurs early during the exit of NSCs from quiescence
We next assessed FoxO6 levels during the early phases of NSC exit from quiescence following FOXG1 overexpression. We used quiescence conditions previously shown to induce cell cycle exit, downregulation of NSC markers, and upregulation of quiescent marker expression, namely: treatment with BMP4 at low density for 24 h (Figure 2A) (Bulstrode et al, 2017). Following exchange of BMP4 for culture media with EGF and FGF-2, FOXG1 induction (+Dox) drives cells to re-enter cell cycle and form NSC-like colonies ( Figure 2B-F). As expected, we find that Dox addition induced a 235-fold upregulation in FOXG1 expression by 24h compared to the non-BMP treated control (EGF/FGF-2) ( Figure 2G). FoxO6 was dramatically upregulated at these early timepoints, prior to any visible proliferative response, with a ~6.5fold upregulation by 24h compared to the non-BMP4 treated control, ~16-fold higher than without Dox ( Figure 2H). Increased FoxO6 expression is therefore an early part of the response to FOXG1 in the transition from quiescence to proliferation, consistent with it being an important functional downstream effector.

FOXG1-induced reactivation of quiescent NSCs is significantly impaired upon FoxO6 loss
To assess whether FoxO6 was required for FOXG1-induced quiescence exit, CRISPR/Cas9 was used to generate clonal adult mouse NSC lines with bi-allelic deletion of the first FoxO6 coding exon ( Figure S1A-B). qRT-PCR analysis confirmed loss of FoxO6, with a >98% decrease in expression following CRISPR treatment compared to untargeted parental cells ( Figure 3A).
FoxO6 -/clonal cells displayed a bipolar phase-bright morphology characteristic of proliferative NSCs ( Figure 3B). Although confluence analysis suggested a marginally reduced growth compared to parental cells, EdU incorporation did not indicate any significant changes in proliferation ( Figure 3C-D).
Furthermore, FoxO6 -/cells were found to form typical NSC colonies in EGF/FGF-2 ( Figure 3E). This . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 indicated that FoxO6 is not necessary for NSC proliferation or colony formation under optimal selfrenewing conditions. Following BMP4 treatment at low density for 24hr, both parental and FoxO6 -/-NSCs displayed a characteristic change to a stellate astrocytic morphology ( Figure 3F). ICC analysis confirmed a decrease in NESTIN and increase in GFAP expression in both parental and FoxO6 -/-NSCs after 3 days of BMP4 treatment ( Figure 3G). qRT-PCR analyses showed upregulation of astrocytic/quiescence markers (Gfap, Aqp4, Id1, Cd9) and downregulation of NSC and cell cycle markers (Nestin,Olig2,Egfr,cMyc,Plk1,Cdk4) in parental and FoxO6 -/cells ( Figure 3H). Cell cycle exit following BMP4 treatment in both parental and FoxO6 -/cells was confirmed by loss of EdU incorporation ( Figure 3I). Proliferation and quiescence entry analyses in additional FoxO6 -/clonal lines showed consistent results, with only one out of three KO lines displaying altered proliferation ( Figure S1C-E). These results suggest that NSC identity is not lost following FoxO6 disruption, and that FoxO6 is not required for cytostatic BMP response and entry into the quiescent state.
We next tested whether FoxO6 was essential for exit from quiescence. Parental and FoxO6 -/-NSCs were transfected with the Dox-inducible FOXG1-V5 overexpression construct using the PiggyBac transposase system. Quiescence exit was assessed following BMP4 treatment and return of cells to EGF/FGF-2 for 10 days, with or without FOXG1-V5 induction (-/+ Dox), as previously mentioned. Similar levels of transgene induction were achieved in both parental and FoxO6 -/populations with inducible FOXG1, as assessed by FOXG1-V5 qRT-PCR and ICC ( Figure 4A-C). Strikingly, FoxO6 -/cells showed dramatically reduced capacity to reform proliferative NSC colonies ( Figure 4D-F) and, unlike the parental cells, did not highly upregulate the NSC marker Nestin or proliferative marker Ki67 upon FOXG1 induction ( Figure S2A).
FoxO6 -/-NSCs showed striking morphological changes from the typical 'fried-egg', stellate astrocytic morphology, to an elongated spindle shape that was distinct from the typical bipolar phase-bright morphology of parental cells with FOXG1 induction ( Figure S2B). After an extended period (25 days) only . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10.1101/2022.06.30.498283 doi: bioRxiv preprint minor evidence of colony formation was seen in the FoxO6 -/population ( Figure S2C). Together these data suggest FoxO6 is a key downstream effector of elevated FOXG1, required for efficient transition from quiescence to proliferation. Without FoxO6, quiescent cells fail to undergo the shape changes and cell cycle re-entry typical of quiescence exit in NSCs.

Elevated FoxO6 induces the formation of large acidic vacuoles by macropinocytosis
To explore the specific pathways through which FoxO6 might operate to stimulate quiescence exit, we next tested the effects of forced FoxO6 expression. We established adult mouse NSCs with Dox-inducible We used these transfected cells to explore cellular responses to elevated FoxO6 by microscopy. Strikingly, prominent vacuolisation was observed upon FoxO6 overexpression across multiple clonal lines ( Figure 5A-B). Live cell imaging across a time-course revealed vacuole formation occurred within 10-11 hours of Dox addition, coincident with mCherry expression ( Figure 5D). Neither treatment of untransfected adult mouse NSCs with Dox nor overexpression of an alternative transcription factor (using the same plasmid constructs with only the gene of interest substituted) resulted in vacuole formation, indicating that the phenotype was specific to FoxO6 overexpression and not a result of Dox treatment, the HA tag or mCherry overexpression ( Figure S3A-B).
We next characterised the resulting vacuoles using various imaging methods. We first ruled out these structures being lipid droplets or enlarged lysosomes, which have been implicated in quiescence regulation (Ramosaj et al, 2021;Leeman et al, 2018). Staining with the neutral lipid dye BODIPY did not reveal colocalisation with the vacuoles ( Figure S3C). In contrast, following LysoView™ incubation, we observed . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10.1101/2022.06.30.498283 doi: bioRxiv preprint strong fluorescent signal co-localised with the vacuoles indicating their acidification ( Figure 5D).
Interestingly, not all structures showed equal LysoView™ accumulation, suggesting they were at different stages of acidification and maturation, and therefore not simply enlarged lysosomes. Consistently, ICC for the lysosomal membrane marker, LAMP1, and the early endosomal marker, EEA1, did not reveal uniform co-localisation with the vacuoles ( Figure 5E) and Western blot analysis showed no clear increase in EEA1 nor LAMP1 expression upon FoxO6-HA induction ( Figure S3E).
Uptake of a fluorescent EGF ligand revealed much smaller puncta, indicating the vacuoles were not formed by receptor-mediated endocytosis ( Figure S3D). Instead, the vacuole size (as large as 6 microns in diameter ( Figure 5F)) was strongly suggestive of non-selective macropinocytosis -an actin-driven process by which extracellular contents are engulfed and processed along the endosomal pathway (Swanson & Watts, 1995;Lim & Gleeson, 2011;Kerr & Teasdale, 2009). To test this hypothesis, we incubated cells with large molecular weight 70kDa FITC-dextran, a well-established marker of macropinocytosis (Galenkamp et al, 2019;Commisso et al, 2014). In cells treated overnight with Dox, clear FITC-dextran uptake was visible within the vacuoles ( Figure 5F). Flow cytometry quantification of FITC-dextran uptake confirmed an increase in the percentage of 'Dextran high' cells following Dox addition compared to -Dox controls across three FoxO6-HA inducible cell lines ( Figure 5G and S3F).
EdU analysis suggested vacuolisation did not per se provide a growth advantage; instead, highly vacuolated cells were associated with EdU negativity following a 24h pulse ( Figure S3G-H). The vacuolated cells could be passaged and remained in culture ( Figure S3I), ruling out a novel form of cell death induced by hyperactivated macropinocytosis in cancer named methuosis (Song et al, 2021;Overmeyer et al, 2008).
Together these observations suggest macropinocytosis, or the pathways that stimulate it, may be an important feature of FoxO6 activity as cells exit quiescence. Macropinocytosis in cancer is associated with nutrient acquisition to aid proliferation (Recouvreux & Commisso, 2017;Commisso et al, 2013). The lack . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10.1101/2022.06.30.498283 doi: bioRxiv preprint of proliferative advantage conferred by FoxO6 overexpression is consistent with the need for other supporting pathways downstream of FOXG1 for quiescence exit.

Pak1 expression is upregulated upon FOXG1 elevation and downregulated upon FoxO6 loss in proliferative NSCs
The p21 (Cdc42/Rac)-activated kinase, Pak1, is a specific regulator of macropinocytosis controlling actin cytoskeleton dynamics (Dharmawardhane et al, 2000), and has been reported as a To investigate a potential involvement of Pak1, we explored if its levels were modulated by FOXG1 or FoxO6. qRT-PCR and Western blot analysis revealed an increase in Pak1 expression in proliferating NSCs (compared to the -Dox control) upon FOXG1 induction ( Figures 6A-B). Furthermore, Pak1 expression was decreased in FoxO6 -/proliferative NSCs by both qRT-PCR and Western blot analysis ( Figure 6C-E). This suggests that in proliferative culture conditions, FoxO6 is needed to sustain Pak1 expression, consistent with a potentially important role in the earliest phases of cell cycle re-entry during quiescence exit. Finally, qRT-PCR analysis of FOXG1, FoxO6 and Pak1 following FOXG1-induced quiescence exit (in F6 cells) revealed higher levels of Pak1 in +Dox compared to the -Dox control, coincident with FOXG1 inducing FoxO6 upregulation ( Figure 6F). In summary, our findings show that FOXG1-mediated induction of FoxO6 is required for efficient quiescence exit of NSCs. We speculate this might operate through modulation of a Pak1-macropinocytosis-related pathway that is initiated as cell undergo regulatory changes (such as shape and nutrient requirements) in preparation for cell cycle re-entry.
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Discussion
Understanding the molecular mechanisms governing control of NSC quiescence has important implications in GBM, a highly aggressive adult brain cancer in which quiescent NSC-like stem cells drive relapse. Our findings here extend our previous observations that high levels of FOXG1 and SOX2 drive a proliferative radial glial-like NSC phenotype, in part through repression of the tumour suppressor FoxO3 (Bulstrode et al, 2017). Here, we show that FoxO6, an underexplored FoxO member, is a downstream target activated by elevated FOXG1.
Whilst FoxO3 has been well-described as a tumour suppressor that preserves NSC quiescence (Renault et al, 2009;Liu et al, 2018), our data suggest FoxO6 has an opposite, pro-proliferative, role in FOXG1induced quiescence exit of NSCs. FoxO6 loss did not impair NSC proliferation or BMP4-induced quiescence entry. We observed NSC and cell cycle marker downregulation, astrocytic/quiescence marker upregulation, morphological changes and cell cycle exit upon BMP4 treatment, all indicating entry into a quiescent state (Codega et al, 2014;Dulken et al, 2017;Llorens-Bobadilla et al, 2015). However, FoxO6 loss was found to significantly impair FOXG1-induced exit from quiescence. Elevated FOXO6 has indeed been associated with stimulating proliferation and progression in several cancers (Qinyu et al, 2013;Rothenberg et al, 2015;Wang et al, 2017;Lallemand et al, 2018). FoxO6 has also been reported to transcriptionally control SOX2, STAT3 and Hippo signalling, all reported to control NSC and GSC self- Civiero & Greggio, 2018), this led us to investigate Pak1 levels in relation to FOXG1 and FoxO6. Our data . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10. 1101/2022 suggest in proliferating NSCs (with mitogens EGF and FGF-2), FoxO6 is required to sustain Pak1 expression and FOXG1 induction can result in even higher Pak1 levels. Whilst this is not functionally important in sustaining NSC proliferation, as shown by the lack of proliferation defects upon Foxg1 or FoxO6 deletion (Bulstrode et al, 2017), these changes in Pak1 levels may impact regulatory transitions, such as cell shape and metabolic requirements, as cells prepare to exit quiescence into the proliferative radial-glia like state. Our data lead us to speculate a working model in which FoxO6 is activated downstream of FOXG1, and in turn will trigger a Pak1-related pathway that alters actin dynamics and related cell shape/nutrient sensing pathways to facilitate exit from quiescence.
As vacuolisation was not observed upon FOXG1 overexpression ( Figure S3J), it is possible that FoxO6induced macropinocytosis represents a stalled state, with other pathways downstream of FOXG1 necessary to be activated concomitantly to ensure cell cycle re-entry, e.g., through increased pinocytic flux that cannot be assessed within our experimental timeframes. Indeed, active Pak1 has been found to modulate pinocytic cycling, enhancing both FITC-dextran uptake and efflux (Dharmawardhane et al, 2000). It is plausible that such an enhancement in pinocytic cycling may aid rewiring of the metabolome required for the transition from quiescence to proliferation (Lee et al, 2017;Wani et al, 2022;Adusumilli et al, 2021). This will require further deeper exploration in future studies. Alternatively, hyperactivation of signalling pathways upon FoxO6 overexpression may result in macropinocytosis as a metabolic stress response. Hyperactivation of Ras signalling, canonical Wnt and PI3K signalling have all been shown to play roles in inducing macropinocytosis (Overmeyer et al, 2008;Tejeda-Muñoz et al, 2019;Recouvreux & Commisso, 2017).
Interestingly, FOXG1 was recently found to synergise with Wnt signalling in driving quiescence exit in GBM (Robertson et al, submitted). The activity of FoxO factors is controlled by phosphorylation downstream of IGF/PI3K/AKT signalling (Hay, 2011;Jiramongkol & Lam, 2020). Pak1 is upregulated in various cancer types (Huynh et al, 2015), integrates various signalling pathways, such as PI3K and Ras, and has been reported to phosphorylate and inactivate FoxO1 in breast cancer (Mazumdar et al, 2003) and . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10. 1101/2022 Foxo6 in liver ageing (Kim et al, 2015). It is therefore also possible that FoxO6 elevation results in signalling activation that in turn reinforces phosphorylation and deactivation of FoxO tumour suppressors.
FoxO factors are known to modulate metabolic functions in homeostasis and cancer (Yadav et al, 2018;Kim et al, 2011;Chung et al, 2013;Paik et al, 2007). FoxO3 protects NSCs against oxidative stress and controls their glucose metabolism to ensure optimal self-renewal (Yeo et al, 2013;Renault et al, 2009), in part through Myc inhibition (Peck et al, 2013). In contrast, FoxO6 promotes gastric cancer cell proliferation through c-Myc induction (Qinyu et al, 2013), and its loss inhibits colorectal cancer cell proliferation, invasion and glycolysis, with decreased PI3K/Akt/mTOR pathway activation (Li et al, 2019). In GBM, mTORC2 signalling controls glycolytic metabolism through inhibition of FoxO1/3 and de-repression of c-Myc (Masui et al, 2013). Elevated FOXG1, itself implicated in regulating mitochondrial functions (Pancrazi et al, 2015), may therefore alter FoxO3 and FoxO6 expression to result in deregulated energetics that drive a proliferative state and/or oppose quiescence. Macropinocytosis in cancer has been reported to aid nutrient uptake (Recouvreux & Commisso, 2017;Commisso et al, 2013); the role of FoxO6 in linking GSC state transitions with metabolism will therefore be an interesting avenue for further exploration.
With respect to normal NSCs, FoxO6's roles in the developing and adult brain are less well-defined than FoxO3 (Sun et al, 2018;Salih et al, 2012). The changing spatial pattern of FoxO6 expression during mouse brain development suggests different functions at distinct stages; yet, the NSC number at E18 is unchanged in FoxO6 null mice (Hoekman et al, 2006;Paap et al, 2016). Cortical FoxO6 levels decrease significantly after birth, with adulthood expression regulating synapse formation in the hippocampal CA1/3 regions, as well as cerebellar neuronal polarity (Salih et al, 2012;De La Torre-Ubieta et al, 2010). Like Foxg1, FoxO6's homeostatic roles may therefore be subtle in adulthood, and mostly involved in neural plasticity (Yu et al, 2019). This is in keeping with our finding that basal FoxO6 levels are low in adult NSCs and not required for sustained proliferation, but are important for cell state transitions. If the FoxO6 levels activated by elevated FOXG1 represent an acquired dependency of GBM, there may be a therapeutic window to . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10.1101/2022.06.30.498283 doi: bioRxiv preprint target this pathway. However, given the poorly understood roles of FoxO6, further work is needed to determine its specific value as a therapeutic target. Regardless, the balance between these three FOX family members -Foxg1, FoxO3 and FoxO6 -has been revealed by our studies, and others, to be a key signalling node in the context of GBM quiescence control and warrants further investigation. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. Selection for FOXG1-V5 expression cassette integration was then commenced by supplementing NSC media with Dox and blasticidin (5 µg/ml). All mock transfected control cells died within seven days of selection. The surviving transfected population were then expanded in NSC media and Dox-inducible FOXG1-V5 expression was confirmed by ICC and qRT-PCR. Cells were reselected for stable transgene expression between independent experiments. The resulting population were expanded for 3-4 days in NSC media prior to functional assays, during which time existing FOXG1-V5 protein was degraded. Stable transgene integration using the PiggyBac system was also used to derive ANS4 cells with Dox-inducible FoxO6-HA-IRES-mCherry overexpression. The TetOn FoxO6-HA-IRES-mCherry vector was derived using the extensible mammalian modular assembly toolkit (EMMA) system (Martella et al, 2017). All . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022.  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10.1101/2022.06.30.498283 doi: bioRxiv preprint

PCR-based genotyping of genetically engineered cell lines
Genomic DNA (gDNA) isolation from bulk transfected cells and clonal cell lines was performed using the DNeasy Blood and Tissue kit (Qiagen), according to the manufacturer's protocol. DNA concentrations were quantified using a NanoDrop TM spectrophotometer. All primers were designed using Primer3 software (http://primer3.ut.ee). To identify NHEJ-based indel formation, the region flanking the gRNA target site was amplified using gene-specific primers. In case of Foxg1 deletion from IENS-GFP, primers were designed flanking the 5' and 3' gRNA targeting sites. For validation of FoxO6 gene deletion primers were designed as described in (Bressan et al, 2017) (PCR1, 2 and 3). For validation of HA tag knock-in at the FoxO6 locus, primers were designed flanking the tag, outside of the 77-bp 5' and 3' homology arms. PCR products were analysed using 1-2.5% agarose gels with EtBr and GeneRuler TM 1kB plus DNA ladder (Thermo Scientific). Gels were imaged on a UV gel reader or Bio-Rad ChemiDoc TM Imager. PerkinElmer's Operetta High-content imaging system and Columbus software. The following primary . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022.

Cell proliferation assays
Confluence analysis and growth curves were determined using the IncuCyte™ live cell imaging system (Essen Bioscience). Cells were plated at ~25 cells/mm 2 in NSC media (EGF/FGF-2) in triplicate wells and imaged periodically until confluence was reached. For analysis of proliferation rates, cells were incubated in NSC media (EGF/FGF-2), supplemented with 10 µM EdU for 24 h. Cells were then fixed in 4% PFA for 10 min at room temperature and stained with the Click-iT EdU Alexa Fluor 647 assay kit (Life Technologies) according to manufacturer's instructions. Imaging was performed using the Nikon TiE microscope and NIS software. For each condition, triplicate wells were analysed (4x4 10x stitched images per well). The total cell number was determined by DAPI staining. Quantification was performed using the Image thresholding and Particle Analysis functions on FIJI software.

Colony formation assays
Colony formation in NSC media (EGF/FGF-2) was assessed by plating cells at a density of 1 cell/mm 2 (1000 cells per well of a 6 well plate, with 6 replicate wells). Media was changed every 3-4 days. Following 10 days, plates were fixed using 4% PFA for 10 min at room temperature. Colonies were stained using methylene blue for 30 min. Plates were washed gently with deionised water and allowed to dry. Plates were then imaged on a Celigo™ Image Cytometer (Nexcelom Bioscience). Colonies were counted manually using the Cell Counter plugin on FIJI, or the % pixel area of the well covered by colonies was quantified using FIJI Image thresholding and Particle Analysis functions.
For assessment of colony formation following BMP4 treatment, cells with Dox-inducible FOXG1-V5 overexpression were plated at a density of 10 cells/mm 2 (10,000 cells per well of a 6 well plate), in NSC media in the absence of EGF/FGF-2 and supplemented with BMP4 (10 ng/ml) (Bulstrode et al, 2017). After 24 h, media was replaced fully with NSC media containing EGF/FGF-2 with or without Dox (1000 ng/ml).
Media was then replaced every 3-4 days. Following 10-12 days, plates were fixed using 4% PFA for 10 min at room temperature. Colonies were stained using methylene blue for 30 min and imaged on a Celigo™ Image Cytometer (Nexcelom Bioscience). Colonies were counted manually using the Cell Counter plugin on FIJI, or the % pixel area of the well covered by colonies quantified using FIJI Image thresholding and Particle Analysis functions. Three technical replicates were averaged to give the mean number of colonies per biological replicate.
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Imaging analysis of vacuoles
Live imaging following FoxO6-HA induction was performed using the Nikon TiE microscope. Imaging began 4 hours after Dox addition and images were obtained every 10 minutes for ~18 hr. For lysosome assessment, 1000x LysoView488™ stock solution (70057 Biotium) was diluted to 1x in NSC media. Cells were incubated in media containing 1x LysoView488™ for 30 min at 37°C prior to imaging. For lipid droplet assessment, BODIPY 493/503 (Invitrogen D3922, 5 mg/ml) was used. For analysis of Dextran uptake, 70000 MW FITC-Dextran (Invitrogen, 070621, 20 mg/ml) was diluted 1:20 to 1 mg/ml in NSC media and added to cells overnight coincident with Dox addition if appropriate. Dextran uptake was visualised in the green channel by imaging and flow cytometry. EGF uptake was visualised by incubating vacuolated cells with media containing 100 ng/ml of EGFR ligand conjugated a fluorophore (EGF-647, E35351, Thermo Fisher Scientific) for 1 hr prior to washing and imaging.

Statistical analyses
Statistical analyses were performed in GraphPad Prism 7. Biological replicates were considered as different passage numbers of same cell line plated in independent experiments. Mean and SEM or SD, and n numbers, are shown in the figure legends. Due to small sample sizes, tests for normality and distribution were of limited value. However, this was not considered to be an impediment to parametric analysis with small n numbers (de Winter, 2013). Statistical tests used are indicated in the figure legends. For qRT-PCR data, statistics calculated from ddCt values. Where two-tailed one-sample t-tests are used, this is based on the null hypothesis that log2(FC)/-ddCt equals zero (i.e. equal to the calibrator sample). Paired Students ttests were used where samples (e.g. wild-type and FoxO6 KO cells) must be matched due to variation between biological replicates (e.g. growth analysis, colony assays following BMP4 treatment). Where significant, p values are indicated in Figures as asterisks, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
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Figure 1 | Elevated FOXG1 transcriptionally activates FoxO6 in mouse NSCs and GSCs
(A) qRT-PCR analysis of FOXG1 transgene and endogenous FoxO6 expression in two independent adult mouse NSC lines ('F6' and 'F11-19') with Dox-inducible FOXG1-V5 expression grown in NSC media with or without Dox for 24 h. Expression shown relative to -Dox (in which log2(FC) = 0). Mean +/-SEM. n=2/3 independent experiments, respectively. Each data point shows the mean of one experiment performed in technical duplicates. Two-tailed one sample t-test. * P ≤ 0.05.
(B) Schematic of HDR-mediated knock-in of an HA epitope tag at the 3' end of last FoxO6 coding exon in F6 cells. PCR genotyping of the bulk transfected F6 cell population revealed a 196 bp product, indicating the presence of cells with insertion of the HA tag at the 3' end of FoxO6.
(D) Western immunoblot analysis of FOXG1-V5 and FoxO6-HA protein expression in tagged F6 NSCs following Dox addition for 4 days in EGF/FGF-2. GAPDH was used as a loading control. (G) Western immunoblot confirming loss of Foxg1 protein in the two independent IENS-GFP knock-out clonal lines (KO 58 and KO 59). GAPDH was used as a loading control.
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Figure 2
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10.1101/2022.06.30.498283 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022  (I) EdU incorporation after treatment with BMP4 or EGF/FGF-2 for 24h, followed by a 24h EdU pulse in EGF/FGF-2. Mean +/-SEM. n=2 independent experiments. Each data point shows the mean one experiment, performed in technical triplicates.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022.  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022.  (C) Live imaging following Dox addition to clonal NSCs with Dox-inducible FoxO6-HA-IRES-mCherry expression (C71). Dox was added 4 hours prior to imaging. Images were obtained every 10 minutes for ~18 hours. Scale bar 100 um or 25 um.
(F) Live imaging of 70 kDa FITC-dextran uptake and mCherry expression following incubation with Dox overnight in FoxO6-inducible cell line (C59). Scale bar 25 um.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022.  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022.  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10. 1101/2022 Figure S1 | FoxO6 is not essential for NSC proliferation or response to BMP4, related to Figure 3. (A) Schematic of FoxO6 locus following CRISPR/Cas9-mediated knockout strategy. Yellow triangles show the sgRNA target sites, resulting in a 178 bp deletion in allele 1 in FoxO6 KO clone 53. Exon 1 of allele 2 is replaced by an EF1a-puromycin cassette.
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Figure S2
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. (B) Representative brightfield images following fixation of colony assay plate and staining with methylene blue.
-/--/-. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. ; https://doi.org/10.1101/2022.06.30.498283 doi: bioRxiv preprint

Figure S3
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 1, 2022. (D) EGF-647 uptake after a pulse of 1 hr shows puncta representative of receptor-mediated endocytosis (C71 incubated overnight with Dox prior to EGF-647 pulse). Scale bar 25 um.
(F) Phase contrast images show vacuole formation in FoxO6-HA inducible cell lines following Dox+Dextran overnight incubation, prior to flow cytometry analysis. Scale bar 100 um.
(I) Phase-contrast images of Dox treated cells in culture (c71). Vacuolated cells remain after 5 days in Dox and following Dox removal.
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