Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation

Jelle van den Ameele; Andrea H Brand

doi:10.7554/eLife.47887

. 2019 Sep 12;8:e47887. doi: 10.7554/eLife.47887

Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation

Jelle van den Ameele ^1,², Andrea H Brand ^1,^2,^✉

Editors: Claude Desplan³, Utpal Banerjee⁴

PMCID: PMC6763261 PMID: 31513013

Abstract

Translating advances in cancer research to clinical applications requires better insight into the metabolism of normal cells and tumour cells in vivo. Much effort has focused on understanding how glycolysis and oxidative phosphorylation (OxPhos) support proliferation, while their impact on other aspects of development and tumourigenesis remain largely unexplored. We found that inhibition of OxPhos in neural stem cells (NSCs) or tumours in the Drosophila brain not only decreases proliferation, but also affects many different aspects of stem cell behaviour. In NSCs, OxPhos dysfunction leads to a protracted G₁/S-phase and results in delayed temporal patterning and reduced neuronal diversity. As a consequence, NSCs fail to undergo terminal differentiation, leading to prolonged neurogenesis into adulthood. Similarly, in brain tumours inhibition of OxPhos slows proliferation and prevents differentiation, resulting in reduced tumour heterogeneity. Thus, in vivo, highly proliferative stem cells and tumour cells require OxPhos for efficient growth and generation of diversity.

Research organism: D. melanogaster

Introduction

The observation that some cancer cells rely primarily on aerobic glycolysis for energy and biomass production (the Warburg effect) (Vander Heiden et al., 2009; Warburg, 1956) has often led to the assumption that the other main source of ATP, mitochondrial oxidative phosphorylation (OxPhos), is dispensable. However, it is becoming increasingly clear that many tumours do require mitochondrial activity for energy and biosynthesis and OxPhos is now frequently exploited as a therapeutic target in cancer (Gui et al., 2016; Molina et al., 2018; Shi et al., 2019; Weinberg and Chandel, 2015). OxPhos takes place at the inner mitochondrial membrane in five large protein complexes (Complex I-V), which together form the respiratory chain. Complexes I-IV transfer electrons from NADH to O₂ and use the released energy to translocate protons from the mitochondrial matrix into the intermembrane space. The resulting electrochemical gradient is then used by Complex V (ATP synthase) to generate ATP from ADP. Apart from the production of ATP, OxPhos is also directly involved in the generation of NAD⁺, orotate, fumarate and reactive oxygen species (ROS) and thus affects many cellular processes, such as nucleotide synthesis (Birsoy et al., 2015; Sullivan et al., 2018; Sullivan et al., 2015), signalling pathway activity (Chandel, 2014) and epigenetic modifications (Lu and Thompson, 2012). The Warburg effect has since been interpreted as a normal adaptation to the metabolic requirements of proliferation, both in cancer cells and proliferating stem cells (Vander Heiden et al., 2009). High glycolytic flux is thought to be required for a constant supply of biomass while OxPhos, apart from its role in production of ATP, primarily maintains the cellular redox balance (Birsoy et al., 2015; Sullivan et al., 2015; Titov et al., 2016).

However, metabolic flux in cancer cells can be influenced by extrinsic and intrinsic factors such as substrate availability, oncogenic mutations and the tumour’s tissue and cell type of origin (Hu et al., 2013; Mayers et al., 2016; Vander Heiden and DeBerardinis, 2017). Brain tumours in particular recapitulate many features of their tissue of origin and grow along a hierarchy reminiscent of normal brain development (Azzarelli et al., 2018; Genovese et al., 2018; Lan et al., 2017; Lee et al., 2018; Tiberi et al., 2014). An integrated understanding of the interactions between metabolism and cell identity in vivo, during both tumourigenesis and normal development, is therefore crucial to translate advances in cancer research to clinical applications.

Development of the Drosophila central nervous system (CNS) has been used extensively as a powerful reductionist model of human brain development and tumourigenesis in vivo (Brand and Livesey, 2011; Hakes and Brand, 2019; Villegas, 2019). The CNS of Drosophila develops from rapidly cycling embryonic and larval neural stem cells (NSCs) that generate a wide variety of neurons and glia. Neuronal diversity is achieved primarily by spatial and temporal patterning, which confers specific identities on NSCs and their progeny according to their location and developmental time (Miyares and Lee, 2019; Technau et al., 2006). Neural stem cells (NSCs) in Drosophila and mammals are thought to generate ATP through aerobic glycolysis rather than OxPhos, whereas their neuronal progeny switch to mitochondrial respiration upon differentiation (Agathocleous et al., 2012; Beckervordersandforth et al., 2017; Hall et al., 2012; Homem et al., 2014; Lange et al., 2016; Tennessen et al., 2014; Tennessen et al., 2011; Zheng et al., 2016). Upregulation of aerobic glycolysis, reminiscent of the Warburg effect, has also been described in a number of Drosophila tumour paradigms (Eichenlaub et al., 2018; Wang et al., 2016; Wong et al., 2019). However, the interpretation that mitochondrial respiration is dispensable for normal Drosophila NSCs (Homem et al., 2014) contrasts with the clear requirement for OxPhos to support cell cycle progression in the Drosophila eye disc (Mandal et al., 2010; Mandal et al., 2005; Owusu-Ansah et al., 2008). Here, we investigate whether, and to what extent, Drosophila NSCs and brain tumours rely on oxidative phosphorylation.

Results

OxPhos is required for brain tumour growth and heterogeneity

We first examined whether OxPhos is required in tumours generated by loss of the transcription factor, Prospero (Pros) (Caussinus and Gonzalez, 2005; Choksi et al., 2006), in which differentiating daughter-cells revert to a NSC-like fate (Choksi et al., 2006) (Figure 1h). pros tumours are invasive upon transplantation and exhibit genomic instability over time (Caussinus and Gonzalez, 2005). We used RNAi to knock down subunits of complex I (NDUFS1) or complex V (ATPsynα) in NSCs and tumour cells with a NSC-specific driver, Worniu-GAL4 (Albertson et al., 2004). The complex I RNAi line has been validated previously (Garcia et al., 2017; Hermle et al., 2017; Owusu-Ansah et al., 2013; Pletcher et al., 2019); expression of the complex V RNAi in NSCs strongly reduced the levels of ATPsynα (Figure 1—figure supplement 2a–c). We also assessed mitochondrial morphology by stimulated emission-depletion (STED) super-resolution microscopy of mitochondria-targeted GFP (Rizzuto et al., 1995). Both RNAi lines caused fragmentation of mitochondria (Figure 1—figure supplement 2d–f), a known consequence of OxPhos dysfunction in mouse and human cells (Duvezin-Caubet et al., 2006).

Figure 1. — (**a–g**) phospho Histone H3 (pH3) staining in the CNS of third instar larvae (L3) with NSC-specific expression (Wor-GAL4;Tub-GAL80^ts) of control RNAi (a), Pros-RNAi (**b,c**), aPKC-CAAX (**d,e**) or Brat-RNAi (**f, g**), either without (**b,d,f**) or with (**c,e,g**) RNAi against a complex I subunit (NDUFS1). Maximum intensity projections through the entire CNS; dashed lines outline the CNS. (h) NSC lineages before and after tumourigenic transformation. (**i,j**) Brain size (i) and mitotic index of Dpn+ tumour cells (j) from L3 larvae expressing the indicated transgenes in NSCs. Datapoints indicate individual brains from one to four biological replicates. (**k,l**) Dpn (red, **k’,l’**) and Imp (green, **k’’,l’’**) immunostaining in Pros-RNAi tumours, without (k) or with (l) a complex I RNAi. Scale bars are 100 µm (**a–g**) or 10 µm (**k,l**).

Figure 1—figure supplement 1. — (**a–g**) phospho Histone H3 (pH3) staining in the CNS of third instar larvae (L3) with NSC-specific expression (Wor-GAL4;Tub-GAL80^ts) of control RNAi (a), Pros-RNAi (**b,c**), aPKC-CAAX (**d,e**) or Brat-RNAi (**f, g**), either without (**b,d,f**) or with (**c,e,g**) RNAi against a complex I subunit (NDUFS1). Maximum intensity projections through the entire CNS; dashed lines outline the CNS. (h) NSC lineages before and after tumourigenic transformation. (**i,j**) Brain size (i) and mitotic index of Dpn+ tumour cells (j) from L3 larvae expressing the indicated transgenes in NSCs. Datapoints indicate individual brains from one to four biological replicates. (**k,l**) Dpn (red, **k’,l’**) and Imp (green, **k’’,l’’**) immunostaining in Pros-RNAi tumours, without (k) or with (l) a complex I RNAi. Scale bars are 100 µm (**a–g**) or 10 µm (**k,l**).

To our surprise, inhibition of OxPhos through knockdown of mitochondrial complex I or V in pros tumours caused a decrease in tumour growth and an overall reduction in brain size (Figure 1a–c,i). This result was comparable to the effect observed upon inhibition of glycolysis with an RNAi against aldolase (Figure 1—figure supplement 1a–c). This suggests that neither glycolysis nor OxPhos are sufficient to support brain tumour growth in vivo.

Next, we tested the requirement for OxPhos in different types of brain tumours. Constitutive activation of aPKC (aPKC-CAAX) leads to symmetric division of NSCs in the Drosophila brain (Lee et al., 2006) (Figure 1h), whereas loss of brat results in dedifferentiation of the progeny of type II NSCs (Bowman et al., 2008) (Figure 1h). In both aPKC-CAAX and brat tumours we found that knockdown of the complex I subunit, NDUFS1, strongly inhibited tumour growth and decreased overall brain size (Figure 1d–g,i). This was accompanied by a significant decrease in the mitotic index of tumourigenic NSCs (Figure 1j), consistent with mitochondrial metabolism playing a key role in regulating the proliferation rate of brain tumour cells. There was no obvious increase in apoptosis upon OxPhos inhibition in pros tumours, as assessed by TUNEL-staining (Figure 1—figure supplement 1d–f).

Growth of pros mutant tumours is sustained by a small proportion of highly proliferative stem cells that express Imp (IGF-II mRNA-binding protein) (Genovese et al., 2018; Narbonne-Reveau et al., 2016). These tumour stem cells self-renew and generate more differentiated Imp-negative tumour cells with limited self-renewal capacity. We assessed whether OxPhos inhibition promotes the differentiation of these Imp-positive stem cells towards Imp-negative tumour cells, which could result in inhibition of tumour growth (Genovese et al., 2018). However, after knockdown of complex I by targeted RNAi, most tumourigenic NSCs in pros and aPKC-CAAX tumours remained Imp-positive and differentiation into Imp-negative cells was reduced (Figure 1k,l—figure supplement 1g). Our results suggest that OxPhos inhibition does not lead to more aggressive tumours, but rather slows it down by decreasing the proliferation rate of the tumour cells.

NSC proliferation depends on OxPhos

We found that, as for tumour cells, inhibition of OxPhos in NSCs throughout development resulted in smaller brains (Figure 1i; Figure 2a–d). This could not be explained by an overall developmental delay, as larval and pupal body length was similar to controls (Figure 2—figure supplement 1). In contrast, inhibition of glycolysis by NSC-specific knockdown of phosphofructokinase (PFK), aldolase or phosphoglycerate kinase (PGK) had no effect on brain size and knockdown of pyruvate kinase (PyK) only caused a slight reduction (Figure 2d—figure supplement 2a-d and data not shown).

Figure 2—figure supplement 1. — (**a–c**) pH3 staining in the CNS of L3 larvae. Maximum intensity projections through the entire CNS; dashed lines outline the CNS. (d) Brain size from L3 larvae. (**e,f**) Mitotic index (e) and 15 min EdU incorporation (f) in NSCs expressing the indicated RNAi (Wor-GAL4;Tub-GAL80^ts). (g) Stills from time-lapse imaging of NSCs (Figure 2—video 1) in the early third instar larval VNC with NSC-specific expression of GFP or Complex I RNAi. Arrowheads indicate mitoses of selected NSCs. Datapoints indicate individual brains from four (e), one (f) and two to four (d) biological replicates. Scale bars are 5 µm (g) or 100 µm (**a–c**).

Complex I or V knockdown did not cause an increase in apoptosis in the VNC of third instar larvae (L3) (Figure 2—figure supplement 2e–k). However, mitotic index (Figure 2e) and incorporation of the S-phase marker 5-ethynyl-2’-deoxyuridine (EdU) (Figure 2f) were significantly reduced, indicating that NSCs rely on OxPhos for proliferation. Live imaging of NSCs in the ventral nerve cord (VNC) after complex I knockdown confirmed a striking increase in cell cycle time: NSC division was rarely observed in a 3 hr time window, whereas control NSCs divided between one and three times (Figure 2g; Figure 2—videos 1 and 2).

To investigate whether RNAi-mediated OxPhos inhibition affects ATP production in NSCs, we measured ATP concentration in vivo using a genetically encoded ATP FRET sensor (Tsuyama et al., 2013). ATP concentration in NSCs in the L3 VNC was similar between controls and complex V knockdown (Figure 1—figure supplement 2g–i). Acute pharmacological inhibition of glycolysis through application of 2-deoxyglucose to ex vivo cultured brains caused a drop in ATP levels in both conditions. However, this drop was significantly more rapid and severe in NSCs with prior complex V inhibition (Figure 1—figure supplement 2g–i). This suggests that mitochondrial dysfunction results in rewiring of NSC metabolism to rely more on glycolysis for ATP production.

OxPhos is required for temporal patterning of NSCs and their progeny

In order to generate the diversity of neurons and glia within the CNS, NSCs undergo temporal patterning. This allows them to generate progeny with different identities according to their developmental time (Miyares and Lee, 2019). Drosophila NSCs in the larval VNC progress from an early identity marked by cytoplasmic Imp and nuclear Chinmo, to a late identity marked by cytoplasmic Syncrip (Syp) and nuclear Broad (Liu et al., 2015; Maurange et al., 2008) (Figure 3a). We found that inhibition of OxPhos caused a defect in temporal patterning of larval NSCs. After knockdown of complex I, one third of NSCs in the VNC (32.0 ± 4.0%, mean ± s.e.m., n = 11 VNCs) failed to downregulate Imp expression (Figure 3b-e—figure supplement 1a-c) and some (9.1 ± 1.4%, n = 14 VNCs) even failed to differentiate into Syp-positive NSCs at the end of larval life (Figure 3—figure supplement 1d–g). This is reminiscent of the failure to downregulate Imp and reduced differentiation in NSC-derived tumours. Immunostaining for other temporal markers (Maurange et al., 2008; Miyares and Lee, 2019) revealed a delay in the downregulation of the early temporal factors Castor and Chinmo, a decreased peak of expression of the switching factor Sevenup, and delayed upregulation of the late temporal factor Broad (Figure 3e—figure supplement 1h). Similar results were observed after knockdown of other subunits of complex I (NDUFA10, NDUFV1) or V (ATPsynα, ATPsynγ) in the VNC (Figure 3d—figure supplement 1i–m), and after OxPhos inhibition in the central brain (CB) (Figure 3—figure supplement 1n–p). Importantly, this was accompanied by a significant reduction in the number of NSC progeny expressing Broad and lacking Chinmo, indicators of late neuronal identity (Figure 3f-h—figure supplement 2). We conclude that OxPhos is required for NSCs to progress from an early to a late temporal fate.

Figure 3. — (a) Scheme of the major temporal transitions in larval NSCs. (**b–d**) Dpn and Imp expression in the VNC of L3 larvae. Arrowheads indicate Imp-positive NSCs. (e) Percentage of Dpn-positive NSCs in the thoracic VNC that express the indicated temporal marker at different time points after larval hatching (ALH) at 25°C. (**f,g**) Dpn (NSCs), Chinmo and Broad in the VNC of L3 larvae. (h) Absolute number of cells per NSC lineage in the VNC that express Chinmo or Broad; graph indicates mean + /- s.e.m. of 6 clones. Datapoints indicate individual brains from four (d), two (e) and one (h) biological replicates. Scale bars are 10 µm.

Figure 3—figure supplement 1. — (a) Scheme of the major temporal transitions in larval NSCs. (**b–d**) Dpn and Imp expression in the VNC of L3 larvae. Arrowheads indicate Imp-positive NSCs. (e) Percentage of Dpn-positive NSCs in the thoracic VNC that express the indicated temporal marker at different time points after larval hatching (ALH) at 25°C. (**f,g**) Dpn (NSCs), Chinmo and Broad in the VNC of L3 larvae. (h) Absolute number of cells per NSC lineage in the VNC that express Chinmo or Broad; graph indicates mean + /- s.e.m. of 6 clones. Datapoints indicate individual brains from four (d), two (e) and one (h) biological replicates. Scale bars are 10 µm.

Temporal patterning of NSCs is regulated at the G₁/S transition

To test directly whether increasing cell cycle length inhibits NSC temporal progression, we slowed the cell cycle by expression of Myt1, Wee1 (Price et al., 2002) or both, which delay the G₂/M transition (Figure 4g) and strongly decrease final brain size (Figure 4—figure supplement 1a). However, this did not affect NSC temporal progression and no Imp-positive NSCs could be detected at the end of neurogenesis (Figure 4d–f,h). Next, we tested whether inhibition of the G₁/S transition affects temporal progression by expression of Dacapo (Dap; the p21/p27/p57 homologue), or an activated form of Rb (Rbf280). Strikingly, many NSCs in the VNC expressed Imp continuously (Dap: 7.8 ± 1.2%, n = 15 VNCs; Rb: 24.2 ± 3.4%, n = 10 VNCs) (Figure 4a–c,h). When Dap and Rbf280 were co-expressed, a majority of NSCs remained positive for the early NSC marker Chinmo (80.4 ± 1.1%, n = 4 VNCs) (Figure 4—figure supplement 1b,c). The block in temporal patterning correlated with the decrease in mitotic index (Figure 4h—figure supplement 1d,e). Our data suggest that temporal patterning and generation of neuronal diversity are linked to cell cycle progression and that regulation occurs at the G₁/S rather than the G₂/M transition.

Figure 4. — (**a–f**) Dpn (NSCs), pH3 (mitosis) and Imp in the VNC of L3 larvae after NSC-specific expression of the indicated transgene. Arrowheads indicate Imp-positive NSCs. (g) Scheme depicting activity of the regulators of the G₁/S and G₂/M transitions that are used for misexpression in this study, and the Fly-FUCCI transgenes. (h) Percentage of Dpn-positive NSCs that express Imp in L3 larvae. (**i–k**) L3 larvae with NSC-specific expression of the Fly-FUCCI system, together with control RNAi or complex I RNAi. Outlines indicate Dpn-positive nuclei (**i,j**). The percentage of Dpn-positive NSCs in the VNC that are positive for either GFP (G₁), RFP (S), a combination of GFP and RFP (G₂/M) or none (G₁/S transition); graphs indicate mean of 8 and 9 brains from one biological replicate (k). Datapoints indicate individual brains from two or three biological replicates (h). Scale bars are 10 µm.

Figure 4—figure supplement 1. — (**a–f**) Dpn (NSCs), pH3 (mitosis) and Imp in the VNC of L3 larvae after NSC-specific expression of the indicated transgene. Arrowheads indicate Imp-positive NSCs. (g) Scheme depicting activity of the regulators of the G₁/S and G₂/M transitions that are used for misexpression in this study, and the Fly-FUCCI transgenes. (h) Percentage of Dpn-positive NSCs that express Imp in L3 larvae. (**i–k**) L3 larvae with NSC-specific expression of the Fly-FUCCI system, together with control RNAi or complex I RNAi. Outlines indicate Dpn-positive nuclei (**i,j**). The percentage of Dpn-positive NSCs in the VNC that are positive for either GFP (G₁), RFP (S), a combination of GFP and RFP (G₂/M) or none (G₁/S transition); graphs indicate mean of 8 and 9 brains from one biological replicate (k). Datapoints indicate individual brains from two or three biological replicates (h). Scale bars are 10 µm.

There is growing evidence for cross talk between mitochondrial metabolism and cell cycle progression at the G₁/S transition (Mandal et al., 2010; Mandal et al., 2005; Mitra et al., 2009; Owusu-Ansah et al., 2008; Schieke et al., 2008). Therefore, we assessed cell cycle stage after knockdown of complex I using Fly-Fucci (Zielke et al., 2014) (Figure 4g). We found an increase in the number of cells in G₁ (26.5 ± 1.6%, n = 9 control VNCs vs. 34.9 ± 1.8%, n = 8 complex I RNAi VNCs) and at the G₁/S transition (14.5 ± 2.3%, n = 9 control VNCs vs. 24.3 ± 1.7%, n = 8 complex I RNAi VNCs) (Figure 4i–k). Our results suggest that OxPhos dysfunction causes activation of the G₁/S checkpoint and this in turn results in delayed temporal patterning of NSCs.

Activation of the G₁/S checkpoint upon downregulation of OxPhos activity has been observed in various tissues in Drosophila (DiGregorio et al., 2001; Mandal et al., 2005). In the eye disc, G₁/S delay upon complex I dysfunction was caused by increased production of ROS and JNK-pathway activity, while complex IV dysfunction decreased the ATP/AMP ratio and activated the G₁/S checkpoint through AMPK and p53 (Mandal et al., 2010; Mandal et al., 2005; Owusu-Ansah et al., 2008). Our preliminary data suggest that decreasing ROS does not rescue the proliferation or temporal patterning defects of complex I or V inhibition (data not shown) and nor does knock down of AMPK or p53 (data not shown). Moreover, clones mutant for ampk in a background where all NSCs continue to express complex I or V RNAi enhanced rather than suppressed the temporal patterning defect (Figure 4—figure supplement 2a–d). Therefore, it remains to be seen which pathway activates the G₁/S checkpoint in NSCs with mitochondrial dysfunction.

OxPhos dysfunction and prolonged G₁/S interfere with termination of proliferation

The adult CNS in Drosophila does not normally contain NSCs (Kato et al., 2009; Siegrist et al., 2010; von Trotha et al., 2009). NSCs stop dividing in the first 20–30 hr after pupariation at which time they differentiate or undergo apoptosis (Figure 3a) (Homem et al., 2014; Ito and Hotta, 1992; Maurange et al., 2008; Siegrist et al., 2010; Truman and Bate, 1988). It was previously shown that knocking down complex III or IV subunits in NSCs prevents termination of proliferation at the onset of pupal life (Homem et al., 2014). The authors suggested that pupariation is accompanied by a metabolic switch from glycolysis to OxPhos that results in NSC shrinkage and cell cycle exit. Similarly, we found that when complex I or V subunits were knocked down, NSCs, identified by Dpn-expression and continued expression of GFP from a NSC-specific GAL4-driver (Worniu-GAL4), were maintained into the adult VNC and CB (Figure 5a-c—figure supplement 1a–d). Of the 133 NSCs in the larval VNC (Birkholz et al., 2015; Lacin and Truman, 2016), an average of 30.8 ± 3.1 and 20.2 ± 3.3 persisted into adulthood when complex I or V were inhibited respectively (Figure 5c). These NSCs continued to proliferate and generate neuronal progeny (Figure 5a-c—figure supplement 1a-j). NSCs also persisted in the adult CB and VNC when the G₁/S, but not G₂/M, transition was delayed, independent of OxPhos dysfunction (Figure 5d-g—figure supplement 1k-n).

Figure 5. — (**a–g**) ElaV (neurons), GFP (NSCs, Wor-GAL4 >mCD8 GFP), Dpn (NSCs) and pH3 (mitosis) in the pharate adult CB or VNC. Maximum intensity projections through the CB or VNC; dashed lines mark the outline of the CNS. (**c,g**) Total number of GFP-expressing NSCs in the pharate adult CB or VNC. (**h,i**) Dpn (NSCs), RFP (negatively marked clones) and Imp in the pharate adult CNS. Arrowheads indicate Dpn-positive NSCs. Dashed outlines mark RFP-negative clones. (j) Percentage of all Dpn-positive NSCs in the pharate adult CNS (CB and VNC) that are part of an RFP-negative clone. (k) OxPhos inhibition prevents terminal differentiation; this is rescued by timely removal of Imp. Datapoints indicate individual brains (**c,g**) or clones (j) from one biological replicate. Scale bars are 50 µm (**a,b**), 20 µm (b’) or 10 µm (h, i).

Figure 5—figure supplement 1. — (**a–g**) ElaV (neurons), GFP (NSCs, Wor-GAL4 >mCD8 GFP), Dpn (NSCs) and pH3 (mitosis) in the pharate adult CB or VNC. Maximum intensity projections through the CB or VNC; dashed lines mark the outline of the CNS. (**c,g**) Total number of GFP-expressing NSCs in the pharate adult CB or VNC. (**h,i**) Dpn (NSCs), RFP (negatively marked clones) and Imp in the pharate adult CNS. Arrowheads indicate Dpn-positive NSCs. Dashed outlines mark RFP-negative clones. (j) Percentage of all Dpn-positive NSCs in the pharate adult CNS (CB and VNC) that are part of an RFP-negative clone. (k) OxPhos inhibition prevents terminal differentiation; this is rescued by timely removal of Imp. Datapoints indicate individual brains (**c,g**) or clones (j) from one biological replicate. Scale bars are 50 µm (**a,b**), 20 µm (b’) or 10 µm (h, i).

Timely cell cycle exit of Drosophila NSCs at the end of neurogenesis was shown to depend on normal progression through the larval temporal cascade (Maurange et al., 2008; Yang et al., 2017). We therefore asked whether the defect in termination of proliferation caused by OxPhos inhibition could be due to delayed temporal patterning during larval life, as opposed to a metabolic switch at pupariation. To test this, we restored the temporal identity in NSCs in which complex I was downregulated by removing Imp at 48 hr or 72 hr ALH. Deletion of Imp significantly decreased adult neurogenesis (Figure 5h–k), consistent with a direct relationship between temporal patterning defects and the adult persistence of NSCs upon OxPhos dysfunction. Together, our data indicate that the previously observed defect in termination of NSC proliferation is a consequence of the earlier temporal patterning defects caused by OxPhos dysfunction.

Discussion

Significant progress has been made in identifying the signalling pathways and transcription factors that regulate stem cell transitions during brain development and homeostasis (Taverna et al., 2014; Tiberi et al., 2012). In contrast, our understanding of the metabolic changes that accompany, or drive, these transitions is still limited (Knobloch and Jessberger, 2017). Here we show that the metabolic requirements of highly proliferative NSCs in the Drosophila brain, as well as the tumour cells they generate upon transformation, cannot be met by aerobic glycolysis alone. Instead, Drosophila NSCs require OxPhos for key aspects of their behaviour: proliferation, generation of diversity through temporal patterning, and termination of proliferation (Figure 6). Respiratory activity may provide an explanation for the strong increase in ROS production that has been observed in NSCs upon hypoxia (Bailey et al., 2015) and for the developmental lethality caused by CNS-specific mutation of the mitochondrial genome (Chen et al., 2015). While OxPhos dysfunction affects both normal NSCs and tumour cells in the brain, inhibition of glycolysis only affects tumour growth (Figure 1—figure supplement 1) but not normal brain development (Figure 2). This is reminiscent of the upregulation of aerobic glycolysis in Hipk, EGFR or PDGF/VEGF-induced tumours in the Drosophila wing disc (Eichenlaub et al., 2018; Wang et al., 2016; Wong et al., 2019). Future experiments will determine the origin and consequences of this tumour-specific reliance on glycolysis in the brain.

Figure 6. — We propose a model, whereby highly proliferative Drosophila NSCs also rely on OxPhos for most aspects of their behaviour. In particular, the G₁/S transition depends on OxPhos activity and perturbation of this transition, either directly, or indirectly through OxPhos inhibition, results in delayed temporal patterning. This in turn prevents NSCs from terminating proliferation at the appropriate time, causing neurogenesis to persist into the adult. A similar dependence on OxPhos can be seen in brain tumours, where both proliferation and differentiation require mitochondrial activity, presumably through a similar mechanism to that found in normal NSCs.

Our results contrast with previous findings suggesting that OxPhos is dispensable during normal NSC development and in brain tumours, and is only activated at the end of neurogenesis as part of a metabolic switch to induce termination of NSC proliferation (Homem et al., 2014). While our experiments do not directly address whether this metabolic switch takes place, the results provide an alternate interpretation. We find that sustained OxPhos activity throughout NSC development is required for normal temporal patterning. Prolonged expression of early temporal markers makes NSCs unresponsive to the developmental cues that govern cell cycle exit (Maurange et al., 2008; Yang et al., 2017) and we show that restoring temporal progression by timely depletion of the early temporal factor Imp enhances termination of proliferation in spite of continued OxPhos inhibition. Our findings thus integrate key aspects of NSC and tumour cell biology (Figure 6) : OxPhos-dependent proliferation is required for temporal patterning and differentiation at the G₁/S transition of the cell cycle. This enables NSCs to undergo normal aging and to respond to the developmental cues that instruct termination of proliferation. Interestingly, adult neurogenesis in the subventricular zone of the mammalian brain depends on p57-induced slowing of the cell cycle during embryonic development (Furutachi et al., 2015). It is not known whether p57 expression or mitochondrial dysfunction also affects the temporal identity of mammalian NSCs. Importantly, the effects we observed are specific to the G₁/S transition: activation of the G₂/M checkpoint did not affect temporal patterning or termination of proliferation. Our results therefore demonstrate that the size and composition of Drosophila NSC lineages are not strictly predetermined (Birkholz et al., 2015) but rather controlled by both intrinsic and extrinsic factors. Single-cell sequencing data indicate that metabolic differences exist between NSCs in different regions of the brain or at different developmental stages (Davie et al., 2018; Genovese et al., 2018) and it will be interesting to assess whether all NSCs are similarly affected by OxPhos dysfunction and G₁/S delay or whether specific lineages show stereotypical responses, as has been shown for entry into quiescence, where arrest in G₂ or G₀ is predetermined (Otsuki and Brand, 2018).

Our study indicates that OxPhos might constitute a targetable metabolic vulnerability of cancer. Small molecule inhibitors of OxPhos are currently being developed and tested in clinical trials to treat various forms of cancer (Gui et al., 2016; Molina et al., 2018; Shi et al., 2019; Weinberg and Chandel, 2015). However, we find that the in vivo impact of OxPhos dysfunction is much more complex than mere inhibition of proliferation. A better understanding of the interactions between metabolism, differentiation and tumour heterogeneity in vivo has the potential to uncover novel therapeutic approaches.

Materials and methods

Key resources table.

Reagent type (species)	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (D. melanogaster)	mCherry-TRIP	BDSC	RRID: BDSC_35785	Control RNAi
Genetic reagent (D. melanogaster)	w¹¹¹⁸;+;+	BDSC	RRID: BDSC_3605
Genetic reagent (D. melanogaster)	ND75-TRIP	BDSC	RRID: BDSC_33911	Complex I RNAi
Genetic reagent (D. melanogaster)	Blw-TRIP	BDSC	RRID: BDSC_28059	Complex V RNAi
Genetic reagent (D. melanogaster)	Pros-TRIP	BDSC	RRID: BDSC_42538
Genetic reagent (D. melanogaster)	Brat-TRIP	BDSC	RRID: BDSC_28590
Genetic reagent (D. melanogaster)	ND42-TRIP	BDSC	RRID: BDSC_32998
Genetic reagent (D. melanogaster)	ND51-TRIP	BDSC	RRID: BDSC_36701
Genetic reagent (D. melanogaster)	Blw-RNAi-KK	VDRC	34663
Genetic reagent (D. melanogaster)	ATPsynβ-TRIP	BDSC	RRID: BDSC_28056
Genetic reagent (D. melanogaster)	ATPsynγ-TRIP	BDSC	RRID: BDSC_28723
Genetic reagent (D. melanogaster)	ATPsynO-TRIP	BDSC	RRID: BDSC_43265
Genetic reagent (D. melanogaster)	PFK-TRIP	BDSC	RRID: BDSC_34336
Genetic reagent (D. melanogaster)	Aldolase-TRIP	BDSC	RRID: BDSC_26301
Genetic reagent (D. melanogaster)	PyK-TRIP	BDSC	RRID: BDSC_35218
Genetic reagent (D. melanogaster)	PGK-RNAi-KK	VDRC	110081
Genetic reagent (D. melanogaster)	UASp-EGFP-Myt1	BDSC	RRID: BDSC_65393
Genetic reagent (D. melanogaster)	UASt-dWee1	(Price et al., 2002) PMID: 12072468
Genetic reagent (D. melanogaster)	UASt-Dap	(Lane et al., 1996) PMID: 8980229
Genetic reagent (D. melanogaster)	UAS-Rbf-280	(Duman-Scheel et al., 2002) PMID: 12015606
Genetic reagent (D. melanogaster)	UASt-aPKC.CAAXWT	(Lee et al., 2006; Sotillos et al., 2004) PMID: 16357871, 15302858
Genetic reagent (D. melanogaster)	UAS-mito-HA-GFP,e1	BDSC	RRID: BDSC_8443
Genetic reagent (D. melanogaster)	UAS-AT1.03-NL on III	(Tsuyama et al., 2013) PMID: 23875533
Genetic reagent (D. melanogaster)	UAS-AT1.03-RK on III	(Tsuyama et al., 2013) PMID: 23875533
Genetic reagent (D. melanogaster)	UAS-GFP-E2F1.1–230, UAS-mRFP1-NLS-CycB.1–266	(Zielke et al., 2014) PMID: 24726363		Fly FUCCI
Genetic reagent (D. melanogaster)	Worniu-GAL4 on II	(Albertson et al., 2004) PMID: 15536119
Genetic reagent (D. melanogaster)	Cas::GFP FlyFos line	VDRC	318476
Genetic reagent (D. melanogaster)	Ubi-FRT-Stop-FRT-GFP	BDSC	RRID: BDSC_32251
Genetic reagent (D. melanogaster)	Imp⁸	(Munro et al., 2006) PMID: 16476777		Imp mutant
Genetic reagent (D. melanogaster)	Ampkα³	(Haack et al., 2013) PMID: 24337115		AMPK mutant
Antibody	rat anti-PH3 (monoclonal)	Abcam	ab10543 RRID: AB_2295065	IF, 1/500
Antibody	rabbit anti-PH3 (polyclonal)	Merck Millipore	06–570 RRID: AB_310177	IF, 1/500
Antibody	guinea pig anti-Dpn (polyclonal)	James Skeath		IF, 1/10,000
Antibody	rabbit anti-Imp (polyclonal)	(Geng and Macdonald, 2006) PMID: 17030623		IF, 1/600
Antibody	guinea pig anti-Syp (polyclonal)	(McDermott et al., 2012) PMID: 23213441		IF, 1/1000
Antibody	chicken anti-GFP (polyclonal)	Abcam	ab13970 RRID: AB_300798	IF, 1/2000
Antibody	rat anti-Mira (polyclonal)	Chris Doe		IF, 1/500
Antibody	rat anti-Chinmo (polyclonal)	(Wu et al., 2012) PMID: 22814608		IF, 1/500
Antibody	mouse anti-Broad (monoclonal)	DSHB	25E9.07	IF, 1/100
Antibody	rabbit anti-RFP (polyclonal)	Abcam	ab62341 RRID: AB_945213	IF, 1/500
Antibody	rat anti-ElaV (monoclonal)	DSHB	7E8A10	IF, 1/100
Antibody	mouse anti-Sevenup (polyclonal)	(Kanai et al., 2005) PMID: 15691762		IF, 1/200
Antibody	mouse anti-ATPsynα (monoclonal)	Abcam	ab14748 RRID: AB_301447	IF, 1/100
Antibody	GFP-booster Atto647N	Chromotek	gba647n RRID: AB_2629215	IF, 1/500 for STED
Commercial assay or kit	ApopTag Red In Situ Apoptosis Detection kit	Merkc Millipore	S7165
Commercial assay or kit	Click-iT EdU Alexa Fluor 647 Imaging Kit	Invitrogen	C10340
Chemical compound, drug	2-deoxyglucose	Sigma	D8375	200 mM final concentration

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Fly husbandry

Drosophila melanogaster were reared in cages at 25°C. For most experiments, embryos were collected on food plates for 3 hr and transferred to 29°C until analysis. Unless indicated otherwise, larvae were matched for developmental timing at wandering third instar (L3). For time-course experiments, embryos were collected on yeasted apple juice plates and larvae were transferred to a fresh yeasted food plate within 2 hr of hatching (designated 0 hr ALH) and grown at 25°C until the desired stage. For clonal analysis, embryos and larvae were grown at 25°C and heat-shocked when indicated for 20 min in a 37°C water bath.

Fly stocks

The following stocks were used: mCherry-TRIP (Bl#35785) was used as control RNAi and w¹¹¹⁸;+;+ as control. Unless otherwise indicated all Complex I RNAi data are from ND75-TRIP (NDUFS1; Bl#33911)(Owusu-Ansah et al., 2013) and all Complex V RNAi data from Blw-TRIP (ATPsynα; Bl#28059)(Teixeira et al., 2015). The other UAS-lines used were: Pros-TRIP (Bl#42538); Brat-TRIP (Bl#28590); ND42-TRIP (Bl#32998)(Garcia et al., 2017); ND51-TRIP (Bl#36701) (Garcia et al., 2017); Blw-RNAi-KK (VDRC#34663) (Teixeira et al., 2015); ATPsynγ-TRIP (Bl#28723) (Teixeira et al., 2015); PFK-TRIP (Bl#34336); Aldolase-TRIP (Bl#26301); PyK-TRIP (Bl#35218); PGK-RNAi-KK (VDRC#110081); UASp-EGFP-Myt1 (Bl#65393); UASt-dWee1 (Price et al., 2002); UASt-Dap (Lane et al., 1996); UAS-Rbf-280 (Duman-Scheel et al., 2002); UASt-aPKC.CAAXWT (Lee et al., 2006; Sotillos et al., 2004); UAS-mito-HA-GFP,e1 (Bl#8443); UAS-AT1.03-NL and UAS-AT1.03-RK on III (Tsuyama et al., 2013); Fly-FUCCI (Zielke et al., 2014) was UAS-GFP-E2F1.1–230, UAS-mRFP1-NLS-CycB.1–266. All RNAis against OxPhos or glycolysis components caused developmental lethality upon ubiquitous expression with Tubulin-GAL4 on III. The GAL4-driver used throughout the study was Worniu-GAL4 on II (Albertson et al., 2004), either on its own, or recombined with UAS-mCD8-GFP on II and Tub-GAL80^ts on III. Castor was visualised with Cas::GFP FlyFos line (VDRC#318476). The genotypes for mitotic recombination clones (Figure 3h; Figure 3—figure supplement 2d–f; Figure 4—figure supplement 2; Figure 5h–j) were as follows: yw,FRT19a (control), yw,Imp⁸,FRT19a (Imp-mutant; Munro et al., 2006) or yw,Ampkα³,FRT19a (AMPK-mutant; Haack et al., 2013)/yw,hsflp,ubi-RFP,FRT19a;Wor-Gal4;+ or ND75-TRIP/+ or Blw-TRIP/+. This resulted in NSC lineages which were randomly marked upon heat-shock by mitotic recombination, whereby all NSCs in the CNS continued to express the RNAi. The genotypes for flip-out clones (Figure 5—figure supplement 1e–j) were as follows: yw,hsflp/+;Wor-Gal4;Ubi-FRT-Stop-FRT-GFP (from Bl#32251) (Evans et al., 2009)/+ or ND75-TRIP or Blw-TRIP.

Immunostaining, EdU and TUNEL

Larval brains were dissected in PBS with 0.3% Triton (PBST), fixed in 4% formaldehyde/PBST for 20 min and washed three times in PBST. For EdU incorporation, freshly dissected brains were immersed in PBS containing 200 ug/ml EdU for 15 min, rinsed twice in PBS and then fixed. EdU detection was performed using a Click-iT EdU Alexa Fluor 647 Imaging Kit (Invitrogen C10340) according to the manufacturer’s instructions. For immunostaining, brains were incubated with primary antibodies in PBST overnight at 4°C, washed with PBST, incubated with Alexa Fluor-conjugated secondary antibodies (Life Technologies) or a GFP-nanobody coupled to Atto647N (Chromotek gba647n) diluted 1/500 in PBST overnight at 4°C and washed with PBST. Brains were mounted in Prolong Diamond Antifade Mountant (Invitrogen). TUNEL staining was done using the ApopTag Red In Situ Apoptosis Detection Kit (Merck Millipore S7165) according to the manufacturer’s instructions.

Antibodies

The following primary antibodies were used: rat anti-PH3 (1/500, Abcam ab10543); rabbit anti-PH3 (1/500, Merck Millipore, 06–570); guinea pig anti-Dpn (1/10,000, gift of James Skeath); rabbit anti-Imp (1/600, gift of Paul MacDonald; Geng and Macdonald, 2006); guinea pig anti-Syp (1/1000, gift of Ilan Davis; McDermott et al., 2012); chicken anti-GFP (1/2000, Abcam ab13970), rat anti-Mira (1/500, gift of Chris Doe); rat anti-Chinmo (1/500, gift of Nicholas Sokol; Wu et al., 2012); mouse anti-Broad (1/100, DSHB 25E9.07); rabbit anti-RFP (1/500, Abcam ab62341); rat anti-ElaV (1/100, DSHB 7E8A10); mouse anti-Sevenup (1/200, gift of Yasushi Hiromi; Kanai et al., 2005); mouse anti-ATPsynα (1/100, Abcam ab14748).

Imaging and image processing

Fluorescent images were acquired using a Leica SP8 confocal microscope and analysed using ImageJ. For the larval CNS, we imaged the thoracic segments of the VNC from the ventral side until the neuropil, or the ventral regions of the CB; for the adult CNS, the entire VNC or CB was imaged. All images are single sections, unless indicated otherwise. For live imaging, third instar larval brains were dissected at room temperature in Schneider’s insect medium (Sigma S0146), mounted in Schneider’s medium with 10% FBS on low 35 mm Ibitreat dishes (Ibidi 80136) and imaged on an inverted Leica SP8 confocal microscope at room temperature. Z-stacks of the ventral side of the thoracic VNC were made at the indicated intervals for 3 hr. For live in vivo ATP measurements with an ATP FRET sensor for Drosophila (Imamura et al., 2009; Tsuyama et al., 2013), confocal settings were as follows: 405 nm excitation and simultaneous detection at 445–490 nm (CFP) and 530–760 nm (FRET); 2-DG (Sigma D8375) was added to the medium to a final concentration of 200 mM. Ratios were calculated for mean FRET/CFP intensity per NSC. Stimulated emission depletion (STED) super-resolution imaging was performed on a custom STED microscope as described in Trovisco et al. (2016) with a 100x UPlanSApo 1.35 NA silicone oil immersion objective lens (Olympus, Japan) over a region of 20 μm² (1024 × 1024 pixels). Images were processed using ImageJ. Timestamps were generated with a custom-built OverTime ImageJ plugin (Richard Butler). Figures were compiled in Adobe Illustrator.

Quantifications and statistical analysis

For quantification of NSCs, Dpn- or Mira-positive NSC on the ventral side of the thoracic VNC at the indicated stage were counted. For TUNEL-staining all TUNEL-positive cells were quantified throughout the entire thickness of the thoracic VNC. To quantify adult NSCs, all GFP-positive lineages were counted throughout the entire VNC or CB; in the control CB, GFP perdures until pharate adult stage in eight mushroom body lineages, which terminate proliferation only at the end of pupal life. To quantify adult NSCs upon Imp-mutation (Figure 5j), all Dpn-positive cells were counted in the VNC or CB. Mitotic index is the number of pH3-positive cells among Dpn-positive cells. For quantification of tumour mitotic index, over 200 Dpn-positive cells were quantified in each thoracic VNC. For brain size, the area of CNS maximum projections was measured.

Graphs were generated in R or Excel. Box-and-whisker plots depict median, interquartile range (box) and 1.5IQR below and above the first and third quartiles respectively (whiskers). Bar graphs, line graphs and values in the text indicate mean ± s.e.m. Datapoints indicate the value of individual VNCs or CBs, apart from Figure 5—figure supplement 1j where datapoints depict individual clones. One biological replicate is defined as the result of one parental cross.

Statistical tests were performed in R. All datasets were first checked for normal distribution with a Shapiro-Wilk test, and then ANOVA was performed with a post-hoc Tukey test. When data were not normal distributed, Kruskal-Wallis test was performed with post-hoc Dunn test and Bonferroni adjustment for multiple comparisons. For time course experiments (Figure 3e—figure supplement 1h), the two conditions at individual time-points were compared with a two-sided Mann-Whitney U test. ATP measurements (AT1.03-NL) were normalised for each NSC to t = 0 when 2-DG was added to the medium, and to the mean values from VNCs that expressed an ATP-insensitive sensor (AT1.03-RK) and were imaged in the same experiment. Modelling of the dynamics of ATP levels was done in R, based on the assumption of exponential decay. Significance is shown compared to control samples, unless indicated otherwise, with the following symbols: *p<0.05; **p<0.01; ***p<0.001; n.s. p≥0.05.

Acknowledgements

We thank C Doe, I Davis, Y Hiromi, P MacDonald, J Skeath, N Sokol for antisera, and D St Johnston, T Uemura, the Bloomington Drosophila Stock Centre and Vienna Drosophila Resource Centre for Drosophila stocks. We thank R Butler (Gurdon Institute Imaging Facility) for the OverTime Fiji plugin, G Sirinakis for help with super-resolution imaging, R Krautz for statistical modelling and Y Bellaiche, W Staels, F Munoz-Martinez, PF Chinnery and members of the Brand lab for discussion and comments on the manuscript. This work was funded by Wellcome Trust Senior Investigator Award (103792) and Royal Society Darwin Trust Research Professorship to AHB. JvdA was supported by EMBO Long-term Fellowship (ALTF 1600_2014) and Wellcome Trust Postdoctoral Training Fellowship for Clinicians (105839). AHB acknowledges core funding to the Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492).

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Contributor Information

Andrea H Brand, Email: a.brand@gurdon.cam.ac.uk.

Claude Desplan, New York University, United States.

Utpal Banerjee, University of California, Los Angeles, United States.

Funding Information

This paper was supported by the following grants:

Wellcome Trust 103792 to Andrea H Brand.
Royal Society to Andrea H Brand.
European Molecular Biology Organization ALTF 1600_2014 to Jelle van den Ameele.
Wellcome Trust 105839 to Jelle van den Ameele.
Wellcome Trust 092096 to Andrea H Brand.
Cancer Research UK C6946/A14492 to Andrea H Brand.

Additional information

Competing interests

No competing interests declared.

Author contributions

Conceptualization, Resources, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Writing—review and editing.

Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing.

Additional files

Transparent reporting form

elife-47887-transrepform.docx^{(66.8KB, docx)}

DOI: 10.7554/eLife.47887.019

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

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eLife. doi: 10.7554/eLife.47887.021

Decision letter

Editor: Claude Desplan¹

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "OxPhos-dependent stem cell proliferation drives temporal patterning and tumour growth in the developing brain" for consideration by eLife. Your article has been reviewed by four peer reviewers, and the evaluation has been overseen by Claude Desplan as Reviewing Editor and Utpal Banerjee as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. The four reviewers agree on the significance and importance of the paper and the quality of the data presented, especially since the Warburg effect is the topic of so much discussion. It was thought that normal NSC and brat tumors do well without OxPhos while the manuscript proves that this might not be the case. The paper clearly makes this point, which is important.

However, there are several serious criticisms that need to be addressed before the paper can be considered.

- A better presentation of the problem.

- One major concern is that the paper does not really examine glycolysis; it demonstrates that knocking down two essential components of OxPhos compromises normal NSC development as well as larval brain tumor growth. Refocusing the text on OxPhos while discussing the results in the context of Homem et al. and Mandal et al. would significantly improve the manuscript. It should be noted that the manuscript neither confirms nor discards the previously claimed Glycolysis-to-OxPhos transition.

- The lack of metabolic characterization of mutants. A revised version should examine mitochondria morphology, their membrane potential, and ATP levels.

- One reviewer would also like the paper to address the possibility that these are glial effects.

- Delete the over-interpretations. In particular, the title must be changed to reflect this: "drive" is an overstatement as the manuscript shows it to be "necessary".

The reviews below are quite detailed and should help you improve the manuscript for publication.

Reviewer #1:

This manuscript by van den Ameele and Brand addresses the interesting question of metabolic reprogramming during tumor formation or stem cell behavior. Authors use Drosophila neural stem cells as a model and show that reducing OxPhos by the knock down of specific components of the respiratory chain complexes, either during uncontrolled NSC proliferation or during normal NSC life, alters their proliferation, blocks the cells in G₁/S and affects the terminal differentiation of NSCs. This finding may be of interest in the context of many studies showing that tumor cells as well as stem cells require OxPhos for their proliferative activity.

However, the conclusions of the experiments are in some instances overstated. The general context of the study, presented as a re-evaluation of the Warburg hypothesis for NSC-derived tumors, is also somehow misleading for two reasons:

1) Most of the experiments presented are made in the context of normal NSC development.

2) One cannot exclude a more trivial interpretation of the data, whereby OxPhos inhibition triggers a general cellular stress response leading to G₁/S arrest. In which case, the study fails addressing the role of OxPhos versus glycolytic pathways in the maintenance of stem cell identity or tumor formation, as stated in the title, Abstract and Discussion.

The authors may want to consider rewiring their manuscript and highlight the major strength of the paper, i.e. the demonstration that a proper sequence of cell cycle progression in the NSCs is required for their differentiation program to be completed.

1) The title is misleading. The authors do not address whether OxPhos is sufficient for NSC or tumor proliferation. Therefore, "drive" seems inappropriate. The Abstract is also misleading (see above).

2) Figure 1 lacks an important piece of information: what driver is used for the knockdown? Figure 1I lacks controls RNAi + Complex1 RNAi, which is on the next figure. Inhibition of glycolysis should be presented as a negative control.

3) A control for apoptosis should be presented in the case of tumors.

4) Figure 3: why only 10% NSCs fail to differentiate into Syp-positive, when up to 50% fail to reduce Imp expression upon CI knock-down?

5) Title of the last Results paragraph ("OxPhos dysfunction…) is confusing, as the data is: why do CI or CV silenced-NSCs continue to proliferate in adults if they arrest in G₁/S during larva? How can one know that the same cells that arrest in G1 in larva are the one that proliferate in adult? More explanation/data is needed to establish this result.

Reviewer #2:

In 2014, Homem et al., reported that in the transition from larval to pupal stages, oxygen consumption rate (OCR) increases and lactate level goes down, and documented a concomitant increase in the expression of genes encoding several enzymes that control rate-limiting steps of related metabolic pathways including the Krebs cycle. They concluded that terminal differentiation of Drosophila NSCs is brought about by induction of respiratory chain and oxidative phosphorylation (OxPhos). Moreover, because brat mutant NSCs do not terminally differentiate, Homem and colleagues hypothesised that loss of brat must render NSCs unable to respond to this developmental program.

In the submitted manuscript, van den Ameele and Brand set out to re-asses such a Warburg-like effect both in NSCs and in larval brain tumours. They show that NSCs require OxPhos activity not only for timely termination of cell proliferation, but also, critically, through NSC development to control cell cycle progression, which is essential to control temporal patterning and to ensure neuronal diversity. They also show that OxPhos activity is required to maintain a normal rate of cell proliferation and cell heterogeneity in different types of larval brain tumours. From these data they conclude that both wild-type and tumoral NSCs require OxPhos for proliferation and to generate cellular diversity.

These results challenge those from the previous article on two accounts. Firstly, they very strongly suggest that the key to timely termination of NSC proliferation is not a switch from glycolysis to OxPhos at the onset of pupal life, but substained OxPhos activity throughout NSC development. Secondly they demonstrate three different larval brain tumour types (pros, aPKC-CAAX, and, brat itself) are in fact tightly dependent upon OxPhos. Thus, in short, aerobic glycolysis is not sufficient for normal NSCs development and sustained tumour growth in the Drosophila brain.

The reported work has been carried out to high standards; experiments are well designed and controlled; and results are thoroughly documented and conveniently discussed.

These are important results. They are also timely because the Warburg effect it is only too often assumed to be a unescapable trait of human cancer (when in fact it is not) and, by extension of Drosophila tumours too.

The one key standing question that the authors have not addressed is whether or not the reported Glycolysis-to-OxPhos transition takes place. The finding that OxPhos is needed throughout NSC development casts serious doubts on the physiological need for such a switch to mostly respiratory metabolism. Moreover, the proposed switch rests on slim evidence, i.e. a 1.2 fold increase in OCR (and that is comparing entire brains, not NSCs).

Other points:

"We assessed whether OxPhos inhibition promotes the differentiation and early depletion of the Imp-positive stem cells. This would result in Imp-negative tumour cells with limited self-renewal capacity (Genovese et al., 2018)". I do not grasp the rationale behind this experiment.

The article "in vivo genetic dissection of tumour growth and the Warburg effect" (Wang et al., 2016) should be cited and discussed. It makes a strong claim on the relevance of the Warburg effect in a different type of tumour in flies.

Figure 1H, At first, I could not appreciate the blue rim on the aPKC-CAAX cell cartoon (it looked all green to me). Using better contrasted colours might help.

Reviewer #3:

This manuscript examines the requirement for OXPHOS during development of Drosophila neuronal stem cells. Using an RNAi-based approach, the authors demonstrate that disruption of either complex I or complex V slows both normal brain growth and tumor growth. Moreover, this growth phenotype stems from a decreased cell proliferation and defects in temporal patterning.

Overall, I like the experimental foundation of this paper and I think it will ultimately be quite interesting. While there are experimental shortcomings (see below), the major issue is in regard to the Introduction and Discussion, which fail to place the reader in an appropriate mindset, and as a result, fail to highlight interesting findings. Specifically, the Introduction and Discussion are underdeveloped and devoted a significant number of sentences to providing an overly simplistic discussion of aerobic glycolysis. The text in these sections leads the reader to believe that the paper is devoted to aerobic glycolysis, as evident the final sentence which states that the goal of the study is to examine the role of the Warburg effect in NSCs. Similarly, the first paragraph concludes with a sentence about aerobic glycolysis. But nowhere in the manuscript do the authors examine the role of aerobic glycolysis within NSCs. There are no assays dedicated to studying glycolytic metabolism nor do they disrupt glycolysis in these cells. In fact, they never even measure lactate production – the hallmark byproduct of aerobic glycolysis. I'm not sure why the paper is setup in this manner, other than the possibility that the authors are really annoyed with the inappropriate manner by which other labs sometimes use the term "aerobic glycolysis."

This unwarranted focus on aerobic glycolysis produces a manuscript that lacks direction. All experiments are devoted to studying OXPHOS, yet the reader is focused on glycolysis. I recommend that the authors entirely rewrite the Introduction and Discussion with a focus on the importance of OXPHOS in both cancer and normal developmental growth. OXPHOS is clearly essential in both cancer and developmental growth. In fact, the most recent in vivo 13C-glucose labeling experiments in human patients suggest that most tumors are highly dependent on OXPHOS. Here the authors describe a new system for studying the link between OXPHOS and cell proliferation! Describe why these findings regarding OXPHOS are important and forget about aerobic glycolysis – unless the authors really want to devote a significant amount to experimental effort (far beyond the scope of a simple revision) to prove their hypothesis that aerobic glycolysis (as defined by elevated glucose consumption and oxygen-independent lactate production) is not important in this context.

As a second offshoot of my concern regarding the text, the results herein are clearly related to the studies by Homem et al., 2014, and Mandal et al., 2005, yet, little effort is made to discuss the results in the context of these earlier studies. I don't think that this previous work detracts from the novelty of the study and the lack of discussion is annoying.

My other major concern is that the results are descriptive and lack some rather basic metabolic characterizations. Specifically, there is no metabolic characterization of the cells expressing the complex I and complex V RNAi constructs. Since the manuscript argues that these RNAi treatments are disrupting the cell cycle as the result of defects in OXPHOS, the resulting metabolic defects should be characterized. At a minimum, the authors should examine mitochondrial morphology and ATP levels. All of these parameters can be measured using previously published reagents.

Finally, the Results section would be enhanced with a few simple experiments that would make sense in light of previous CoVa (tenured) studies (Mandal et al., 2005), which demonstrate that disruption of Complex IV results in a G₁/S arrest that is dependent on AMPK and p53. The authors briefly elude to these similarities, but fail to address the model. A few simple genetic experiments, such as determining if RNAi-depletion of AMPK suppresses the OXPHOS-induced defects, would significantly enhance the impact of the study.

Reviewer #4:

The role of metabolism in modulating development and cancer progression is a fascinating and active area of research. As the authors argue, many studies have focused on the role of aerobic fermentation and one carbon metabolism in fueling growth, but relatively little attention has been paid to OxPhos. In this study the authors argue that oxphos is necessary for proliferation, temporal patterning and differentiation of NSCs. This is a good study that exploits the tools of Drosophila melanogaster to address an important question.

I am asking the authors to address the points below:

While the data showing that KD of OxPhos affects different aspects of NSCs are strong, the argument that OxPhos is affected is only genetic. Given the strength of the authors' claim and the fact that changing the mRNA levels of metabolic enzymes often results in compensatory or unforeseen effects, the authors should validate that their manipulations affect OxPhos using different assays. This would make the manuscript much stronger, as there would be direct, rather than genetic (inferential), data about OxPhos.

OxPhos is dispensable for neuronal function, but required in glia (Volkenhoff, 2015). Since NSC can give rise to glial precursors, and glia in turn can support NSCs proliferation and differentiation, is it possible that the phenotypes observed are due to an absence or change in glia?

Given that the brain controls growth of the organism, could changes in brain size, especially when the manipulations result in a smaller brain, be because the larvae are smaller? A way to compare brain size to body length or to another independent metric would be beneficial.

The authors use the word "cause" in the Results and Discussion section; however, their experiments show that OxPhos is necessary for proliferation, not that it is sufficient, thus there is no direct evidence that it "causes or drives" temporal patterning, proliferation, and differentiation.

There is no GAL4 control in the figures.

There is no validation of the efficacy of the RNAi transgenes by qPCR.

eLife. 2019 Sep 12;8:e47887. doi: 10.7554/eLife.47887.022

Author response

[…] There are several serious criticisms that need to be addressed before the paper can be considered.

- A better presentation of the problem.

- One major concern is that the paper does not really examine glycolysis; it demonstrates that knocking down two essential components of OxPhos compromises normal NSC development as well as larval brain tumor growth. Refocusing the text on OxPhos while discussing the results in the context of Homem et al. and Mandal et al. would significantly improve the manuscript. It should be noted that the manuscript neither confirms nor discards the previously claimed Glycolysis-to-OxPhos transition.

As suggested by the editor and reviewers 1 and 3, the text has now been refocused on the role of OxPhos in NSCs and brain tumours, and we provide more discussion of Homem et al. and Mandal et al.

- The introduction describes the role of OxPhos in proliferating cells. We introduce normal development of the Drosophila CNS and present the Drosophila literature on metabolism in tumour cells (Eichenlaub et al., 2018; Wang et al., 2016; Wong et al., 2019). We also introduce the papers from the Banerjee lab (Mandal et al., 2010, 2005; Owusu-Ansah et al., 2008).

- We performed knockdown of glycolysis enzymes in NSCs and in tumour cells and included these data in Figure 1, Figure 1—figure supplement 1, 2 and Figure 2—figure supplement 1.

- The Discussion now clearly states that based on our results we cannot confirm nor discard the presence or absence of the metabolic switch described in Homem et al. However, we also explain that our data provide an alternative interpretation of how OxPhos dysfunction affects termination of NSC proliferation.

- The lack of metabolic characterization of mutants. A revised version should examine mitochondria morphology, their membrane potential, and ATP levels.

To further validate the effect of knockdown of complex I (NDUFS1) and complex V (ATPsynα) on mitochondrial metabolism, we performed the following experiments (Figure 1—figure supplement 2):

- We assessed mitochondrial morphology using STED super-resolution microscopy (conventional confocal microscopy did not resolve individual mitochondria in an intact brain). Both complex I and complex V knockdown showed increased fragmentation of mitochondria in NSCs (Figure 1—figure supplement 2D-F).

- We measured ATP concentration in vivo using a previously validated ATP sensor (Tsuyama et al., 2013). Baseline ATP levels were not affected, but after complex V knockdown, NSCs showed a stronger drop in ATP levels upon acute pharmacological inhibition of glycolysis (Figure 1—figure supplement 2G-I), suggesting that this knockdown affects mitochondrial metabolism and results in rewiring cellular metabolism to rely more on glycolysis.

- One reviewer would also like the paper to address the possibility that these are glial effects.

We did knock down the same complex I and complex V subunits in glia using Repo-Gal4 and assessed the effects on NSCs. In addition, we temporally restricted complex I knockdown to a period in development when cortex glia have already been generated. The results are described in the response to reviewer 4.

- Delete the over-interpretations. In particular, the title must be changed to reflect this: "drive" is an overstatement as the manuscript shows it to be "necessary".

The title, Abstract and Discussion have been changed to reflect that OxPhos is necessary rather than sufficient for the phenotype. The new title is: “Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation”

The reviews below are quite detailed and should help you improve the manuscript for publication.

Reviewer #1:

[…] The conclusions of the experiments are in some instances overstated. The general context of the study, presented as a re-evaluation of the Warburg hypothesis for NSC-derived tumors, is also somehow misleading for two reasons:

1) Most of the experiments presented are made in the context of normal NSC development.

The Introduction has now been refocused:

- We introduce the importance of OxPhos in proliferating cells.

- We introduce the papers that investigate metabolism of NSCs, in vertebrates as well as in Drosophila.

- We describe normal development of the Drosophila CNS and highlight the Drosophila literature on metabolism in tumour cells (Eichenlaub et al., 2018; Wang et al., 2016; Wong et al., 2019).

- The aim of the paper, as stated at the end of the Introduction now focusses on investigating the role of OxPhos in proliferating NSCs and in tumour cells.

2) One cannot exclude a more trivial interpretation of the data, whereby OxPhos inhibition triggers a general cellular stress response leading to G₁/S arrest. In which case, the study fails addressing the role of OxPhos versus glycolytic pathways in the maintenance of stem cell identity or tumor formation, as stated in the title, Abstract and Discussion.

We have now performed knockdown of glycolysis enzymes in NSCs and in tumour cells and included these data in Figure 1, Figure 1—figure supplement 1, 2 and Figure 2—figure supplement 1. This shows that NSC-specific knockdown of PFK, aldolase and PGK does not affect brain size, but knockdown of PyK results in smaller brains. We also tested aldolase knockdown in Pros tumours and found that it, in contrast to normal NSCs, their growth is significantly decreased.

We agree that the phenotypes we observe are probably mediated through activation of cellular stress pathways, which, among others, lead to G₁/S delay, similar to what was shown in Owusu-Ansah et al., 2008 where complex I dysfunction in the Drosophila eye disc leads to ROS production and activation of the stress-response through the JNK-pathway.

An in-depth analysis of the pathways mediating this response is beyond the scope of our paper but we have now included the following results:

- Figure 1—figure supplement 2G-I: baseline ATP levels are not decreased upon chronic inhibition of complex V in NSCs. However, ATP levels drop more significantly upon acute inhibition of glycolysis when complex V is inhibited than in the control NSCs. This suggests that part of the response is an increased reliance on glycolysis.

- Figure 4—figure supplement 2: Ampk mutation does not rescue proliferation or temporal patterning, and actually makes it worse. This might suggest that AMPK plays a compensatory role in the response to OxPhos dysfunction.

The authors may want to consider rewiring their manuscript and highlight the major strength of the paper, i.e. the demonstration that a proper sequence of cell cycle progression in the NSCs is required for their differentiation program to be completed.

The text has been refocused on the role of OxPhos in NSCs and brain tumours. The Introduction now also describes normal development of the Drosophila CNS, and the role of temporal patterning in generation of neuronal diversity. In the Discussion, we provide more explanation of the differences with Homem et al., and discuss how our findings may impact on the current understanding of NSC diversity and normal temporal patterning of NSCs.

1) The title is misleading. The authors do not address whether OxPhos is sufficient for NSC or tumor proliferation. Therefore, "drive" seems inappropriate. The Abstract is also misleading (see above).

2) Figure 1 lacks an important piece of information: what driver is used for the knockdown? Figure 1I lacks controls RNAi + Complex1 RNAi, which is on the next figure. Inhibition of glycolysis should be presented as a negative control.

- The driver used for the knockdown is Worniu-Gal4. This is now mentioned in the text and figure legend.

- We performed additional experiments and included the data from the control RNAi + Complex I or V RNAi crosses in Figure 1l.

- We knocked down aldolase in Pros tumours and observed a significant decrease in tumour growth. These data are now included in Figure 1—figure supplement 1A-C.

3) A control for apoptosis should be presented in the case of tumors.

TUNEL staining in the Pros-RNAi brains is now presented in Figure 1—figure supplement 1. We observed no obvious increase in apoptosis upon knockdown of complex I or complex V.

4) Figure 3: why only 10% NSCs fail to differentiate into Syp-positive, when up to 50% fail to reduce Imp expression upon CI knock-down?

The transition from Imp- to Syp-expression is gradual (Liu et al., 2015), and many cells that fail to downregulate Imp upon OxPhos knockdown do express Syp anyway. This is illustrated in Author response image 1 (NSCs with complex I RNAi), whereby Worniu>GFP-positive NSCs that remain Imp-positive often are Syp-positive as well (double-positive NSCs are indicated by arrowheads).

5) Title of the last Results paragraph ("OxPhos dysfunction…) is confusing, as the data is: why do CI or CV silenced-NSCs continue to proliferate in adults if they arrest in G₁/S during larva? How can one know that the same cells that arrest in G1 in larva are the one that proliferate in adult? More explanation/data is needed to establish this result.

The NSCs do not “arrest” in G₁ upon expression of Dap or Rbf280. The transition is delayed rather than blocked, since some are in mitosis (pH3+) and they continue to generate neuronal (ElaV+) progeny (See for example in Figure 5—figure supplement 1l).

Adult NSCs are identified by expression of Dpn, as well as by GFP under the control of the NSC-specific Worniu-GAL4. The same GAL4-driver is used for overexpression or knockdown during development and is expressed in more than 90% of the NSCs in the VNC. Because there are no Dpn-positive cells in the adult brain that are GFP-negative (data not shown), it seems unlikely that the few NSCs which did not express to the UAS-transgene are the ones that continue to proliferate in the adult.

The title and the wording of the last Results paragraph have been changed to avoid confusion, and we explain better how adult NSCs are identified.

Reviewer #2:

[…] The one key standing question that the authors have not addressed is whether or not the reported Glycolysis-to-OxPhos transition takes place. The finding that OxPhos is needed throughout NSC development casts serious doubts on the physiological need for such a switch to mostly respiratory metabolism. Moreover, the proposed switch rests on slim evidence, i.e. a 1.2 fold increase in OCR (and that is comparing entire brains, not NSCs).

We agree with the reviewer that the existence of such a switch seems less likely in view of our results. We comment on this in the Discussion, and explain an alternative, and in our view more plausible, interpretation of the data: OxPhos-dependent proliferation is required for temporal patterning and differentiation at the G₁/S transition of the cell cycle; this enables NSCs to undergo normal aging and respond to the developmental cues that instruct termination of proliferation.

Other points:

"We assessed whether OxPhos inhibition promotes the differentiation and early depletion of the Imp-positive stem cells. This would result in Imp-negative tumour cells with limited self-renewal capacity (Genovese et al., 2018)". I do not grasp the rationale behind this experiment.

Growth of pros mutant tumours is known to be sustained by a small proportion of highly proliferative stem cells expressing Imp (Genovese et al., 2018; Narbonne-Reveau et al., 2016). These Imp-positive cells self-renew, but also generate more differentiated tumour-cells that limit tumour growth. Because OxPhos dysfunction reduces tumour growth (Figure 1A-J), we wanted to exclude the possibility that this was a result of increased differentiation towards Imp-negative cells with limited self-renewal capacity. This is now explained in the Results section.

The article "in vivo genetic dissection of tumour growth and the Warburg effect" (Wang et al., 2016) should be cited and discussed. It makes a strong claim on the relevance of the Warburg effect in a different type of tumour in flies.

The article is now presented in the Introduction.

Figure 1H, At first, I could not appreciate the blue rim on the aPKC-CAAX cell cartoon (it looked all green to me). Using better contrasted colours might help.

The colour in the figure has been changed.

Reviewer #3:

This manuscript examines the requirement for OXPHOS during development of Drosophila neuronal stem cells. Using an RNAi-based approach, the authors demonstrate that disruption of either complex I or complex V slows both normal brain growth and tumor growth. Moreover, this growth phenotype stems from a decreased cell proliferation and defects in temporal patterning.

Overall, I like the experimental foundation of this paper and I think it will ultimately be quite interesting. While there are experimental shortcomings (see below), the major issue is in regard to the Introduction and Discussion, which fail to place the reader in an appropriate mindset, and as a result, fail to highlight interesting findings. Specifically, the Introduction and Discussion are underdeveloped and devoted a significant number of sentences to providing an overly simplistic discussion of aerobic glycolysis. The text in these sections leads the reader to believe that the paper is devoted to aerobic glycolysis, as evident the final sentence which states that the goal of the study is to examine the role of the Warburg effect in NSCs. Similarly, the first paragraph concludes with a sentence about aerobic glycolysis. But nowhere in the manuscript do the authors examine the role of aerobic glycolysis within NSCs. There are no assays dedicated to studying glycolytic metabolism nor do they disrupt glycolysis in these cells. In fact, they never even measure lactate production – the hallmark byproduct of aerobic glycolysis. I'm not sure why the paper is setup in this manner, other than the possibility that the authors are really annoyed with the inappropriate manner by which other labs sometimes use the term "aerobic glycolysis."

This unwarranted focus on aerobic glycolysis produces a manuscript that lacks direction. All experiments are devoted to studying OXPHOS, yet the reader is focused on glycolysis. I recommend that the authors entirely rewrite the Introduction and Discussion with a focus on the importance of OXPHOS in both cancer and normal developmental growth. OXPHOS is clearly essential in both cancer and developmental growth. In fact, the most recent in vivo 13C-glucose labeling experiments in human patients suggest that most tumors are highly dependent on OXPHOS. Here the authors describe a new system for studying the link between OXPHOS and cell proliferation! Describe why these findings regarding OxPhos are important and forget about aerobic glycolysis – unless the authors really want to devote a significant amount to experimental effort (far beyond the scope of a simple revision) to prove their hypothesis that aerobic glycolysis (as defined by elevated glucose consumption and oxygen-independent lactate production) is not important in this context.

We have refocused the manuscript on the importance of OxPhos in proliferating NSCs.

- The Introduction explains the importance of OxPhos in proliferating cells.

- The Discussion has been expanded considerably. We provide more explanation of the discrepancies with Homem et al. We discuss how our findings may impact on the current understanding of NSC diversity and normal temporal patterning of NSCs. We also present our findings in the context of research from the Banerjee lab (Mandal et al., 2010, 2005; Owusu-Ansah et al., 2008).

- We have now performed knockdown of glycolysis enzymes in NSCs and in tumour cells and included these data in Figure 1, Figure 1—figure supplement 1, 2 and Figure 2—figure supplement 1. This shows that NSC-specific knockdown of PFK, aldolase and PGK does not affect brain size, but knockdown of PyK results in smaller brains. We also tested aldolase knockdown in Pros tumours and found that, in contrast to normal NSCs, their growth is significantly decreased.

As a second offshoot of my concern regarding the text, the results herein are clearly related to the studies by Homem et al., 2014, and Mandal et al., 2005, yet, little effort is made to discuss the results in the context of these earlier studies. I don't think that this previous work detracts from the novelty of the study and the lack of discussion is annoying.

- Mandal et al. and Homem et al. are better explained in the Introduction, and we mention both papers as a motivation to conduct our experiments.

- We highlight the differences with Homem et al. and discuss in more detail an alternative interpretation of their data based on our findings.

- Additional experiments (cf below), based on the work from Mandal et al., have now been included in the Results section (Figure 4—figure supplement 2) and are interpreted in the context of (Mandal et al., 2010, 2005; Owusu-Ansah et al., 2008).

My other major concern is that the results are descriptive and lack some rather basic metabolic characterizations. Specifically, there is no metabolic characterization of the cells expressing the complex I and complex V RNAi constructs. Since the manuscript argues that these RNAi treatments are disrupting the cell cycle as the result of defects in OXPHOS, the resulting metabolic defects should be characterized. At a minimum, the authors should examine mitochondrial morphology and ATP levels. All of these parameters can be measured using previously published reagents.

The complex I RNAi line has been used extensively and validated in several previous publications (e.g. Garcia et al., 2017; Hermle et al., 2017; Owusu-Ansah et al., 2013; Pletcher et al., 2019), which we now cite in the Results section.

To further validate the effect of knockdown of complex I (NDUFS1) and complex V (ATPsynα) on mitochondrial metabolism, we performed the following experiments (Figure 1—figure supplement 2):

Finally, the Results section would be enhanced with a few simple experiments that would make sense in light of previous CoVa (tenured) studies (Mandal et al., 2005), which demonstrate that disruption of Complex IV results in a G₁/S arrest that is dependent on AMPK and p53. The authors briefly elude to these similarities, but fail to address the model. A few simple genetic experiments, such as determining if RNAi-depletion of AMPK suppresses the OXPHOS-induced defects, would significantly enhance the impact of the study.

In view of the reviewers’ comments, we assessed both retrograde signalling pathways that were shown to cause G₁/S arrest by (Mandal et al., 2010, 2005; Owusu-Ansah et al., 2008). Our preliminary data suggest that decreasing ROS does not rescue the proliferation or temporal patterning defects of complex I or V inhibition (data not shown). We also knocked down Ampk and p53 by RNAi, and these did not rescue the effect of OxPhos knockdown (complex I RNAi) either (data not shown).

We next generated Ampk mutant clones through mitotic recombination, in a background where all NSCs continue to express the complex I or complex V RNAi. This again did not rescue proliferation or temporal patterning, and in fact enhanced rather than suppressed the phenotype. We included these experiments in the Results section (Figure 4—figure supplement 2) and interpret them in the context of (Mandal et al., 2010, 2005; Owusu-Ansah et al., 2008).

Reviewer #4:

[…] I am asking the authors to address the points below:

While the data showing that KD of OxPhos affects different aspects of NSCs are strong, the argument that OxPhos is affected is only genetic. Given the strength of the authors' claim and the fact that changing the mRNA levels of metabolic enzymes often results in compensatory or unforeseen effects, the authors should validate that their manipulations affect OxPhos using different assays. This would make the manuscript much stronger, as there would be direct, rather than genetic (inferential), data about OxPhos.

To further validate the effect of knockdown of complex I (NDUFS1) and complex V (ATPsynα) on mitochondrial metabolism, we performed the following experiments (Figure 1—figure supplement 2):

OxPhos is dispensable for neuronal function, but required in glia (Volkenhoff, 2015). Since NSC can give rise to glial precursors, and glia in turn can support NSCs proliferation and differentiation, is it possible that the phenotypes observed are due to an absence or change in glia?

All literature so far, in mammals and Drosophila (recently reviewed in Magistretti and Alleman, 2018), including the paper mentioned (Volkenhoff et al., 2015), suggest that glial metabolism is mainly glycolytic, to support the function of neurons through secretion of lactate and alanine.

Based on the reviewer’s suggestion, we knocked down the same complex I and complex V subunits in glia using Repo-Gal4. Glial knockdown resulted in a decreased mitotic index of NSCs (Author response image 2D). However this did not affect temporal patterning, as assessed by the number of Imp-positive NSCs at the end of neurogenesis (Author response image 2E).

In addition, we temporally restricted complex I knockdown, and expressed the RNAi from L1 onwards. At this stage, all cortex glia have already been generated and thus are not affected by the expression of RNAi in NSCs. This also prevented termination of proliferation as shown in Author response image 3 and indicates that the phenotype is not due to an effect on glia.

Given that the brain controls growth of the organism, could changes in brain size, especially when the manipulations result in a smaller brain, be because the larvae are smaller? A way to compare brain size to body length or to another independent metric would be beneficial.

L3 larvae were always collected at wandering stage in order to synchronize developmental timing. In addition, we quantified larval length at the stage of dissection, and pupal length as a measure for final developmental size. The results are included in Figure 2—figure supplement 2 and did not reveal significant differences between the experimental conditions.

The authors use the word "cause" in the Results and Discussion section; however, their experiments show that OxPhos is necessary for proliferation, not that it is sufficient, thus there is no direct evidence that it "causes or drives" temporal patterning, proliferation, and differentiation.

There is no GAL4 control in the figures.

As a control, we crossed the same GAL4-line to an RNAi against mCherry. This controls for the presence of GAL4, the RNAi and the genetic background.

There is no validation of the efficacy of the RNAi transgenes by qPCR.

- The complex I RNAi line has been extensively used and validated in several previous publications (e.g. Garcia et al., 2017; Hermle et al., 2017; Owusu-Ansah et al., 2013; Pletcher et al., 2019), which we now mention in the Results section.

- We now validated the complex V RNAi line (against Blw) by staining for the Blw protein (ATPsynα), which showed a clear downregulation (Figure 1—figure supplement 2A-C).

- We also performed a more functional validation of the RNAi-lines as explained above.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Transparent reporting form

elife-47887-transrepform.docx^{(66.8KB, docx)}

DOI: 10.7554/eLife.47887.019

Data Availability Statement

All data generated or analysed during this study are included in the manuscript and supporting files.

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PERMALINK

Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation

Jelle van den Ameele

Andrea H Brand

Roles

Abstract

Introduction

Results

OxPhos is required for brain tumour growth and heterogeneity

Figure 1. Brain tumours require OxPhos for growth.

Figure 1—figure supplement 1. Brain tumours require OxPhos and glycolysis for their growth.

Figure 1—figure supplement 2. OxPhos RNAi in NSCs affects mitochondrial function.

NSC proliferation depends on OxPhos

Figure 2. OxPhos inhibition decreases NSC proliferation.

Figure 2—figure supplement 1. OxPhos inhibition does not affect body size.

Figure 2—figure supplement 2. OxPhos inhibition does not increase apoptosis.

Figure 2—video 1. Mitochondrial dysfunction increases cell cycle length.

Figure 2—video 2. Mitochondrial dysfunction increases cell cycle length.

OxPhos is required for temporal patterning of NSCs and their progeny

Figure 3. OxPhos is required for temporal patterning of NSC and their progeny.

Figure 3—figure supplement 1. Mitochondrial dysfunction in NSCs delays temporal patterning.

Figure 3—figure supplement 2. Delayed temporal patterning of NSCs affects their progeny.

Temporal patterning of NSCs is regulated at the G1/S transition

Figure 4. G1/S progression drives temporal patterning.

Figure 4—figure supplement 1. G1/S and G2/M delay results in smaller brains.

Figure 4—figure supplement 2. AMPK deletion does not rescue the temporal patterning defect caused by OxPhos inhibition.

OxPhos dysfunction and prolonged G1/S interfere with termination of proliferation

Figure 5. NSCs require OxPhos for termination of proliferation.

Figure 5—figure supplement 1. Adult neurogenesis upon OxPhos knockdown and G1/S delay.

Discussion

Figure 6. Model of the role of OxPhos in Drosophila NSCs and tumour cells.

Materials and methods

Key resources table.

Fly husbandry

Fly stocks

Immunostaining, EdU and TUNEL

Antibodies

Imaging and image processing

Quantifications and statistical analysis

Acknowledgements

Funding Statement

Contributor Information

Funding Information

Additional information

Competing interests

Author contributions

Additional files

Data availability

References

Decision letter

Roles

Author response

Author response image 1.

Author response image 2.

Author response image 3.

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Temporal patterning of NSCs is regulated at the G₁/S transition

Figure 4. G₁/S progression drives temporal patterning.

Figure 4—figure supplement 1. G₁/S and G₂/M delay results in smaller brains.

OxPhos dysfunction and prolonged G₁/S interfere with termination of proliferation

Figure 5—figure supplement 1. Adult neurogenesis upon OxPhos knockdown and G₁/S delay.