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. Author manuscript; available in PMC: 2020 Mar 4.
Published in final edited form as: Curr Biol. 2019 Feb 14;29(5):750–762.e3. doi: 10.1016/j.cub.2019.01.039

E93 integrates neuroblast intrinsic state with developmental time to terminate neurogenesis via autophagy

Matthew C Pahl 1, Susan E Doyle 1, Sarah E Siegrist 1,1,*
PMCID: PMC6428584  NIHMSID: NIHMS1521424  PMID: 30773368

SUMMARY

Most neurogenesis occurs during development, driven by the cell divisions of neural stem cells (NSCs). We use Drosophila to understand how neurogenesis terminates once development is complete, a process critical for neural circuit formation. We identified E93, a steroid hormone-induced transcription factor that downregulates PI3-kinase levels to activate autophagy for elimination of mushroom body (MB) neuroblasts. MB neuroblasts are a subset of Drosophila NSCs that generate neurons important for memory and learning. MB neurogenesis extends into adulthood when E93 is reduced and terminates prematurely when E93 is overexpressed. E93 is expressed in MB neuroblasts during later stages of pupal development only, which includes the time when MB neuroblasts normally terminate their divisions. Cell intrinsic Imp and Syp temporal factors regulate timing of E93 expression in MB neuroblasts, while extrinsic steroid hormone receptor (EcR) activation boosts E93 levels high for termination. Imp inhibits premature expression of E93 in a Syp-dependent manner, while Syp positively regulates E93 to promote neurogenesis termination. Imp and Syp together with E93 form a temporal cassette, which consequently links early developmental neurogenesis with termination. Altogether, E93 functions as a late-acting temporal factor integrating extrinsic hormonal cues linked to developmental timing with neuroblast intrinsic temporal cues to precisely time neurogenesis ending during development.

eTOC Blurb

Pahl et. al find that E93 is required to precisely time the end of neurogenesis during development. E93 is temporally expressed in MB neuroblasts and is regulated by extrinsic hormone cues and by neuroblast intrinsic temporal factors. E93 forms a temporal cassette with Imp and Syp which links early developmental neurogenesis with termination.

INTRODUCTION

Neurogenesis starts and stops in a spatially and temporally defined manner. Most neurogenesis occurs during development, but in some animals, new neurons are also produced throughout adulthood. Unlike developmental neurogenesis, adult neurogenesis is relatively restricted. Only certain neuron types are produced in only some brain regions [1, 2]. For example, adult rodents produce olfactory bulb neurons in the SVZ for odor detection, while primates produce hippocampal neurons important for memory and learning. However, the extent of adult neurogenesis in primates, including humans, is uncertain [3, 4]. Equally important to continuing neurogenesis is to stop it once development is complete. This is because extended or ectopic neurogenesis leads to defects in neural circuitry, which is now associated with autism, mental illness, and neurodegenerative disease [5–7].

We use Drosophila to understand how extrinsic factors, local and systemic, integrate with NSC intrinsic factors to control timing and mechanism of neurogenesis termination during development. Like mammals, neurons in the Drosophila brain are generated directly from the asymmetric divisions of NSCs, known as Type I neuroblasts in Drosophila, or indirectly from a transit amplifying daughter cell, produced by a Type II neuroblast [8–11]. In Drosophila, neurogenesis completes during development and no new neurons are produced during adulthood [12–14]. This is because all neuroblasts are eliminated by terminal differentiation or apoptosis before adulthood [12, 15–17]. Most Type I and all Type II neuroblasts stop dividing during early pupal stages, except for mushroom body neuroblasts (MB neuroblasts), a Type I subset (summarized in Figure 1A). MB neuroblasts, which reside on the dorsal brain surface superficial to the MB calyx, divide several days longer, until late pupal stages, and undergo apoptosis shortly before animals emerge from their pupal case as adults [12, 18, 19].

Figure 1: E93 is necessary and sufficient to eliminate MB neuroblasts and terminate MB neurogenesis.

Figure 1:

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(A) Schematic, timing of MB versus non-MB neuroblast (NB) elimination. (B) Top schematic, highlighting position of the MB calyx, used as a landmark in locating MB neuroblasts. Below, maximum intensity projection of the region outlined. Arrows indicate MB neuroblasts. (C) Average number of MB neuroblasts per brain hemisphere in 1-day-old adults. Numbers on bars indicate number of brain hemispheres scored for each of the indicated genotypes listed below. Error bars s.e.m. (D) Times of heat-shock treatments (arrow) for GAL4 flip out experiments with GAL4 flip out cassette below (see text). (E) Left, a MB neuroblast E93 RNAi clone in a 1-day-old adult after heat shock at 0 hrs. APF. Right, a control MB neuroblast, heat shocked at 0 hours APF, fails to express E93RNAi. Control imaged at an earlier time to identify a Dpn-positive MB neuroblast, that is normally absent in adulthood, but present when E93 is knocked down. White brackets mark neuroblasts in this and all subsequent figures. (F) Percentage of MB neuroblast E93 RNAi clones with a Dpn-positive neuroblast in one-day-old animals. Time of heat shock treatments indicated below and number of clones scored in columns. (G,H) Maximum intensity projections of adult MB neuropil. Scale bar (B) 20μm and (E) 10μm. See also Figure S1 and Table S1.

Independent of when neuroblasts terminate or whether they differentiate or undergo apoptosis, all neuroblasts undergo a period of reduced growth and proliferation prior to their disappearance, suggesting this reduced growth triggers neuroblast elimination [12, 15–17]. In Type I and Type II neuroblasts that terminate early, reductions in growth and proliferation are linked to transcriptional changes in metabolic enzymes, likely induced by systemic increases in the steroid hormone ecdysone [15]. While in MB neuroblasts, reductions in growth and proliferation are due to decreased levels of PI3-kinase activity [12]. Growth-inhibited MB neuroblasts are then primed for elimination by apoptosis [12]. However, blocking MB neuroblast apoptosis alone is not sufficient to prevent their elimination, because downregulation of PI3-kinase activates autophagy in parallel to ensure MB neuroblast removal and termination of neurogenesis. Importantly, when both autophagy and apoptosis are inhibited together, MB neuroblasts persist long term and continually generate new neurons during adulthood, some of which incorporate into existing neural structures and others which mis-project axons elsewhere [12]. Autophagy together with apoptosis could allow for faster or more efficient removal of MB neuroblasts in the brain, which in Drosophila lacks a dedicated population of phagocytic macrophage-like cells.

While PI3-kinase levels affect timing of MB neurogenesis termination, it remains unclear how PI3-kinase is regulated. PI3-kinase activity is nutrient regulated in many cell types, including MB neuroblasts [20], while in the salivary gland and fat body, increasing systemic ecdysone triggers reductions in levels of PI3-kinase activity [21–23]. Two intrinsic neuroblast temporal factors, Imp (IgF-II mRNA binding protein) and Syp (Syncrip), also regulate timing of MB neurogenesis termination [24, 25]. When Imp is knocked down, MB neurogenesis terminates prematurely and when Syp is knocked down, MB neurogenesis extends into adulthood [24, 25]. Imp and Syp, both RNA binding proteins, are expressed in opposing temporal gradients in all neuroblasts, with Imp expressed at high levels first, followed by high Syp later [24–27]. Whether PI3-kinase interacts with Imp/Syp to control neurogenesis timing is not known, however it was recently shown that ecdysone is required for the Imp to Syp temporal switch in Type II neuroblasts [26]. Therefore, an ecdysone-induced temporal switch could also regulate PI3-kinase levels in MB neuroblasts and time neurogenesis ending during development.

To determine how MB neuroblasts terminate divisions during development, we carried out a targeted RNAi screen. We identified Ecdysone-induced protein 93F (referred to as E93), a pipsqueak transcription factor family member, first characterized as an ecdysone response gene required for autophagy of larval tissues during metamorphosis [28–30]. More recently, it has been shown that E93 is also expressed in Type II neuroblasts in response to ecdysone, and in the wing, where E93 modifies chromatin accessibility at temporally regulated enhancers [26, 31]. Here, we find that E93 downregulates PI3-kinase levels in MB neuroblasts to induce autophagy for MB neuroblast elimination. In the absence of E93, PI3-kinase levels remain high, autophagy fails, and neurogenesis continues into adulthood. E93 expression correlates with timing of neurogenesis termination, both occur late, and if E93 is overexpressed constitutively, neurogenesis terminates prematurely. Imp/Syp temporal factors regulate timing of E93 expression, while EcR activation regulated by systemic hormone conditions induces E93 to high levels required for neurogenesis termination. Altered Imp/Syp temporal factor expression or reduced EcR activation cause neurogenesis to either extend into adulthood or terminate prematurely. By integrating neuroblast intrinsic temporal cues with extrinsic systemic hormonal cues, E93-dependent regulation of PI3-kinase provides a mechanism for neurogenesis termination to be synchronized with timing of animal development. This ensures that the adult mushroom body contains an appropriate number of molecularly and functionally distinct neuron types necessary for animal behavior.

RESULTS

E93 functions cell-autonomously to eliminate MB neuroblasts during pupal development

We carried out a candidate RNAi screen to identify genes required to eliminate MB neuroblasts and terminate neurogenesis during development. We screened factors known to be expressed in MB neuroblasts and factors known to initiate apoptosis and/or regulate autophagy. Candidate UAS-RNAi lines were crossed to worGAL4 to knock down gene function in neuroblasts, and brains of one-day old adults were screened for presence of persisting neuroblasts. We identified the ecdysone-induced protein 93F (Eip93F in flybase), hereafter referred to as E93. Following constitutive knock down of E93 in all neuroblasts (worGAL4,UAS-E93RNAi #HMC04773), 3.5 MB neuroblasts on average were found per brain hemisphere in adult animals (Figure 1B,C), whereas no MB neuroblasts were observed in control animals (n>50, data not shown)[12, 32]. MB neuroblasts were positively identified based on Deadpan (Dpn) and pcna:GFP (S-phase activity) reporter expression and location, dorsal brain surface superficial to the MB calyx (Figure 1B)[12, 32]. We tested a second RNAi line (#KK108140) which targets a different E93 protein coding exon and observed a similar but less penetrant phenotype (Figure 1C and S1). Next, we used OK107Gal4 to restrict E93 knockdown to MB neuroblasts and their neuron progeny. Again, MB neuroblasts were observed, whereas control animals had no MB neuroblasts (Figure 1C). We conclude that E93 is required for MB neuroblast elimination and termination of MB neurogenesis during development.

To determine whether E93 is sufficient to terminate MB neurogenesis prematurely, we over-expressed a wild type version of E93 in all neuroblasts (worGAL4,UAS-E93 WT). MB neuroblasts normally terminate divisions between 78–90 hours APF (after pupal formation), whereas other Type I and Type II neuroblasts (referred to as non-MB neuroblasts) terminate divisions much earlier (Figure 1A). Following E93 overexpression, MB neuroblasts were present in brains at 96 hours ALH, but not at 48 hours APF (n=15 brain hemispheres), indicating that E93 overexpression eliminates MB neuroblasts prematurely. Consistent with this, the adult mushroom body neuropil was dramatically reduced in E93-overexpressing animals, with the majority of late-born, FasII-positive α/β MB neuron types missing (Figure 1G,H, and data not shown). We conclude that E93 is both necessary and sufficient for termination of MB neurogenesis.

To determine when E93 is required during development, we used a heat-shock inducible GAL4 flip out cassette to control timing of E93RNAi expression and E93 inactivation (Figure 1D)[33]. Freshly hatched larvae (0 hours after larval hatching, ALH) and newly formed pupae (0 hours APF) were heat shocked to produce Flippase, which mediates excision of an FRT-flanked STOP codon (Figure 1D). After STOP excision, GAL4 is produced, driven by the actin promoter, and UAS-E93RNAi expressed in some cells, including MB neuroblasts. MB neuroblast E93RNAi expressing clones were positively identified based upon co-expression of a UAS-RFP reporter, which is weak in MB neuroblasts but strong in their neuron progeny, and Eyeless (Ey), a transcription factor that specifically marks MB neuroblasts and their neuron progeny (Figure 1E and data not shown)[20, 34–36]. Following either heat shock treatment (0 hrs. ALH or 0 hrs. APF), all MB neuroblast E93RNAi clones in adult animals had one Dpn-positive neuroblast and no Dpn-positive cells were observed outside the E93RNAi clone (Figure 1E,F). This suggests that E93 is required for MB neuroblast elimination during pupal stages and functions in a cell-autonomous manner. To further define when E93 is required, we heat shocked pupae even later, at 24 or 48 hours APF. All MB neuroblast E93RNAi clones in adult animals had one Dpn-positive neuroblast, whereas control clones had no neuroblasts (Figure 1F). We conclude that E93 is required late in development to eliminate MB neuroblasts and terminate MB neurogenesis.

MB neuroblasts express E93 during later pupal stages

Some Type I and all Type II neuroblasts express E93 during late stages of larval development [26]. At 96 hours ALH, E93 was observed in Type I and Type II neuroblasts as reported (Figure 2A, asterisks and 2B), however MB neuroblasts did not express E93 at this time (Figure 2A, arrows and 2C). E93 was detected in MB neuroblasts and their neuron progeny later, during pupal stages at 36 hours APF, but not earlier (Figure 2D,E). Once expressed, MB neuroblasts maintained relatively low levels of E93 until their elimination via apoptosis during late pupal stages (Figure 2F,G, and data not shown). The late timing of E93 expression is consistent with our heat shock experiments demonstrating that E93 is required late for MB neuroblast elimination. To ensure specificity of the E93 antibody, the GAL4 flip out cassette was used to generate MB neuroblast E93RNAi clones. Compared to control clones, E93RNAi clones had reduced E93 protein levels in both MB neuroblasts and their neuron progeny (Figure 2H,I, and quantified in 6B). We conclude that E93 is expressed in MB neuroblasts during the latter half of pupal development, which includes the time when MB neuroblasts normally terminate their divisions.

Figure 2: MB neuroblasts express E93 after non-MB neuroblasts, during later stages of pupal development.

Figure 2:

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(A) Dorsal view of a wild type larval brain hemisphere, anterior up. Below, greyscale image of same brain hemisphere, labeled with Scrib to outline NB membranes. Arrows indicate MB neuroblasts, asterisks indicate some non-MB neuroblasts. (B-G) Wild type neuroblasts from indicated time points (above) stained with markers listed within panel B. (H-I) E93 is reduced in a MB neuroblast E93RNAi clone (H) compared to a control MB neuroblast clone (I). Scale bar (A) 20μm and (B) 10μm. See also Table S1.

Figure 6: Imp, Syp, and EcR regulate E93 expression in MB neuroblasts.

Figure 6:

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(A,C) Schematic summarizing changing 20-hydroxyecdysone levels (A) and timing of Imp and Syp expression in relation to E93 (C). (B) Quantification of E93 nuclear fluorescence intensities. Numbers in columns indicate number of clones scored. (D,E,G,H,J,K,M,N,P,Q) Top, colored overlay with single channel greyscale images below of MB neuroblasts. (F,I,L,O,R) Quantification of E93 nuclear fluorescence intensities. Column numbers equal number of MB neuroblasts assayed or number of clones. (L,O,R) E93 nuclear fluorescence intensities normalized to control E93 nuclear fluorescence intensities at 48 APF (B). Error bars equal S.E.M. *p values<.001, two-tailed Student’s t-tests. Scale bar (D) 10μm. See also Figure S4 and Table S1.

E93 downregulates growth, proliferation and levels of PI3-kinase in MB neuroblasts

MB neuroblasts undergo a period of reduced growth and proliferation, due in part to reductions in levels of PI3-kinase activity prior to their elimination via apoptosis [12]. Failure to downregulate PI3-kinase activity on time could allow E93RNAi MB neuroblasts to persist into adulthood [12]. We used the mitosis specific marker phospho-Histone H3 (PHH3) to assay E93RNAi MB neuroblast proliferation and the plasma membrane markers Scribble (Scrib) or Discs-large (Dlg) to measure MB neuroblast size. Compared to control MB neuroblasts, which undergo significant decreases in size and proliferation after 72 hours APF prior to their elimination [12], E93RNAi MB neuroblasts remained large and mitotically active, even in one-day-old adults (Figure 3A,B and S2A,B). Next, we assayed subcellular localization of Foxo, a downstream effector and readout for levels of PI3-kinase activity [37–39]. When PI3-kinase is active, Foxo remains cytoplasmic, and when PI3-kinase is inactive, Foxo relocates to the nucleus to regulate gene expression. At 48 hours APF, midway through pupal development, when MB neuroblasts are still large and actively proliferating, Foxo was mostly cytoplasmic in both control and E93RNAi MB neuroblast clones (Figure 3C,D,H). But from 78–90 hours APF, after MB neuroblasts reduce their growth and proliferation [12], nuclear Foxo was increased in control but not in E93RNAi MB neuroblast clones (Figure 3E,F,H). Foxo remained mostly cytoplasmic in E93RNAi MB neuroblast clones throughout pupal development and became nuclear only in adult animals (Figure 3G,H). We also assayed total Foxo protein levels and found reductions over time in control clones, but not in E93RNAi MB neuroblast clones (Figure 3I). We conclude that E93 is required to downregulate growth, proliferation and levels of PI3-kinase activity in MB neuroblasts in a timely manner, which could allow E93RNAi MB neuroblasts to persist into adulthood.

Figure 3: Failure to downregulate PI3-kinase activity on time allows E93RNAi MB neuroblasts to persist into adulthood.

Figure 3:

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(A,J,O) Average number of MB neuroblasts per brain hemisphere over time. Column numbers indicate number of hemispheres scored. Error bars indicate standard deviation. White columns (A) within colored columns indicate average number of mitotic MB neuroblasts. (B,P) Box plots of MB neuroblast diameters. Numbers at bottom indicate number MB neuroblasts analyzed. *p values<.001, two-tailed Student’s t-tests. (C-G) Top, colored overlay with single channel greyscale image below of MB neuroblasts. Markers listed within panels, genotypes and time above. (H,I) Quantification of MB neuroblast Foxo fluorescence intensities. Column numbers equal number of MB neuroblasts assayed. (H) *p values<.001, two-tailed Student’s t-tests. (I) *p value=.0003, one-way ANOVA. (K-N) Colored overlay of MB neuroblasts. Scale bar (C,K) 10μm. See also Figure S2 and Table S1.

To test this possibility, we overexpressed the regulatory subunit of PI3-kinase, UAS-dp60, to reduce levels of PI3-kinase activity in E93RNAi MB neuroblasts [40]. Essentially no MB neuroblasts were found in brains of adults (Figure 3J). To determine whether absence of dp60,E93RNAi MB neuroblasts in adults correlates with reductions in MB neuroblast growth, we assayed MB neuroblast size at 72 hours APF when dp60,E93RNAi MB neuroblasts were still present (Figure 3O). Compared to control and E93RNAi MB neuroblasts, dp60,E93RNAi MB neuroblasts were significantly smaller, similar to dp60 MB neuroblasts which terminate prematurely (Figure 3K-P). We conclude that E93 downregulates PI3-kinase to reduce MB neuroblast growth for termination of neurogenesis.

PI3-kinase is typically regulated in a nutrient-dependent manner through binding of ligand to either InR (insulin-like tyrosine kinase receptor) or Alk (anaplastic lymphoma kinase receptor) in Drosophila [41, 42]. We co-expressed UAS-InRRNAi or UAS-AlkRNAi with UAS-E93RNAi to knock down InR or Alk with E93 in MB neuroblasts. Relatively large sized MB neuroblasts were still observed in both InRRNAi,E93RNAi and AlkRNAi,E93RNAi double knock adult animals (Figure S2C-G). We conclude that E93 downregulates PI3-kinase independent of InR and Alk, two known upstream regulators of canonical PI3-kinase signaling.

E93 is required for autophagy and functions in parallel to the pro-apoptotic regulators to terminate MB neurogenesis

Both apoptosis and autophagy are required to eliminate MB neuroblasts and terminate MB neurogenesis [12](Video S1 and Figure S3A). Only by blocking both together can MB neuroblasts persist long-term into adulthood and continually produce new neurons [12]. The pro-apoptotic genes, reaper, hid, and grim are required for MB neuroblast apoptosis, however it remains unclear what regulates MB neuroblast autophagy. E93 is reported to induce autophagy of the salivary gland and midgut [29, 30], therefore we asked whether E93 also induces MB neuroblast autophagy. We used the autophagic flux reporter, UAS-GFP-mCherry-Atg8, driven by worGAL4, to visualize autophagosome formation in control MB neuroblasts [43, 44]. UAS-GFP-mCherry-Atg8 is a fusion between pH-sensitive GFP, pH-insensitive mCherry, and Atg8, a core component of the initiating phagophore, autophagosome, and final acidic autolysosome (Figure 4M). From 48 to 72 hours APF, 90% of control MB neuroblasts have 1 or 2 autophagosomes (yellow puncta, white arrow), reflecting basal autophagy (Figure 4A,J,K and S3A). After 72 hours APF, when PI3-kinase activity declines and MB neuroblast growth and proliferation ceases, an increase in number and size of autophagosomes (yellow puncta) and autolysosomes (red puncta, white arrowhead) was observed in MB neuroblasts (Figure 4C,E,J,K and S3B). In addition, autophagic flux, the change over time of autolysosomes to total puncta number (autophagosomes plus autolysosomes), increased (Figure 4K). Both results suggest that increased autophagy and flux contribute to MB neuroblast elimination via autophagy.

Figure 4: E93 regulates autophagy in MB neuroblasts.

Figure 4:

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(A-I) Top, colored overlay of MB neuroblasts. Below, colored overlay of cropped, maximum intensity projection of MB neuroblast above, single channel greyscale images below. White arrows indicate autophagomes (mCh,GFP double positive) and arrowheads indicate autolysosomes (mCh). (A-H) Co-express UAS-GFP-mCh-Atg8, (I) UAS-GFP-Atg8. (J) Quantification of autophagosomes and autolysosomes or autophagosomes only (L) over time. Black tics represent individual MB neuroblasts. Total number of MB neuroblasts assayed at top of column, red lines denote mean. (K) Distribution of percentages of autolysosomes relative to total puncta in MB neuroblasts over time (M) Schematic of autophagy flux reporter. *p values<.001, two-tailed Student’s t-tests. Scale bar (A) 10μm. See also Figure S3, Table S1, and Video S1.

Next, we assayed autophagy in E93RNAi MB neuroblasts using the same autophagy flux reporter. From 72 to 84 hours APF, E93RNAi MB neuroblasts had significantly fewer autophagosomes (yellow puncta) and autolysosomes (red puncta) compared to controls (Figure 4B,D,F,J,K and S3B). By adulthood however, autophagosome and autolysosome number did increase in some E93RNAi MB neuroblasts, suggesting that autophagy onset is delayed, which could be due to delayed reductions in PI3-kinase levels (Figure 4G,J,K and S3B). To test this, we co-expressed UAS-dp60 and UAS-GFP-Atg8 (a standard non-flux autophagy reporter) in E93RNAi MB neuroblasts. At 72 hours APF, more autophagosomes were observed in dp60,E93RNAi MB neuroblasts compared to control or E93RNAi MB neuroblasts (Figure 4A,B,H,I,L). We conclude that E93 is required to initiate MB neuroblast autophagy via downregulation of PI3-kinase.

Next, we examined E93RNAi adult brains to determine when E93RNAi MB neuroblasts terminate divisions and whether E93-induced autophagy is required for MB neuroblast elimination. We examined brains of three-day old E93RNAi adults and found no MB neuroblasts present (Figure 5A,D, n=20 brain hemispheres), which suggests presence of a back-up pathway for MB neuroblast removal [12]. This compensatory pathway is likely apoptosis. Therefore, we co-expressed UAS-miRHG, a synthetic microRNA that inhibits the pro-apoptotic genes reaper, hid, and grim, in E93RNAi MB neuroblasts [12]. In E93RNAi,miRHG adults, MB neuroblasts were found in brains of one-week and even two-week old adults, whereas no MB neuroblasts were found in miRHG adults at either of these times (Figure 5B-D)[12]. We conclude that E93 is required for autophagy and functions in parallel to the pro-apoptotic RHG pathway to eliminate MB neuroblasts and terminate MB neurogenesis.

Figure 5: MB neuroblasts persist long-term in the absence of E93 and inhibition of apoptosis.

Figure 5:

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(A-C) Colored overlay of MB neuroblasts. (D) Average number of MB neuroblasts per brain hemisphere. Column numbers indicate number of hemispheres scored. Error bars indicate S.E.M. Scale bar 10μm. See also Table S1 and Video S1.

Extrinsic EcR and intrinsic Imp/Syp temporal factors regulate E93 expression in MB neuroblasts

The steroid hormone ecdysone regulates E93 expression in the salivary gland, midgut, and in Type II neuroblasts [26, 45]. Ecdysone is released in defined pulses from the prothoracic gland into the circulating hemolymph and converted to active 20-hydroxyecdysone in peripheral tissues [46]. As a systemic factor, 20-hydroxyecdysone triggers major developmental transitions, including larval molting and pupation (Figure 6A, schematic)[47, 48]. Because 20-hydroxyecdysone levels change over time, ecdysone signaling could provide an extrinsic timer for triggering termination of MB neurogenesis through regulation of E93 (Figure 6A, schematic). To test this, we used the GAL4 flip out cassette to generate MB neuroblast clones expressing UAS-EcRRNAi to knock down EcR, the ecdysone receptor. Unfortunately, significant animal lethality resulted, which precluded further analysis. Therefore, we knocked down EcR in a neuroblast specific manner using worGAL4. At 48 hours APF, EcR expression was not detected in EcRRNAi MB neuroblasts and E93 protein levels were reduced by half compared to controls (Figure 6D,E,F and S4A-C). Consistent with moderate E93 reduction, some persisting EcRRNAi MB neuroblasts were found in adult brains (Figure S4E,F). We conclude that E93 is regulated by ecdysone signaling in MB neuroblasts, however other factors are likely to contribute.

Because E93 is expressed in MB neuroblasts during late stages only, we asked whether the intrinsic temporal factors, Imp and Syp, regulate E93 expression in MB neuroblasts. Imp and Syp are RNA binding proteins that mutually repress each other and distinguish “young” (Imp positive, Syp negative) larval MB neuroblasts from “older” (Imp negative, Syp positive) pupal MB neuroblasts (Figure 6C, schematic)[24]. We used the GAL4 flip out cassette and heat shocked animals at 0 hours ALH to generate MB neuroblast clones expressing either UAS-ImpRNAi or UAS-SypRNAi. At 72 hours ALH, control clones have no E93 as expected, however, E93 was present in ImpRNAi MB neuroblast clones (Figure 6J,K,L). This suggests that Imp inhibits premature E93 expression. Conversely, at 48 hours APF, in SypRNAi MB neuroblast clones, E93 was not detected (Figure 6G,H,I). Consistent with strong E93 reduction, we found SypRNAi MB neuroblasts in brains of adult animals (Figure S4D,F)[24,25]. We conclude that the temporal factors, Imp and Syp regulate E93 expression in MB neuroblasts: Imp inhibits premature E93, while Syp promotes late E93.

Next, we asked whether premature E93 in ImpRNAi MB neuroblasts is EcR dependent. Ecdysone triggers the Imp to Syp temporal transition in Type II neuroblasts and ImpRNAi MB neuroblasts express Syp prematurely [24,26]. We heat shocked animals at 0 hours ALH to generate MB neuroblast clones expressing both UAS-ImpRNAi and UAS-EcRRNAi. At 72 hours ALH, control clones had no E93 as expected, however E93 was still present in ImpRNAi,EcRRNAi MB neuroblast clones (Figure 6M,N,O). This suggests that Imp functions independent of EcR to inhibit E93 expression. Next, we asked whether premature E93 in ImpRNAi MB neuroblasts is Syp dependent. We heat shocked animals at 0 hours ALH to generate MB neuroblast clones expressing both UAS-ImpRNAi and UAS-SypRNAi. At 72 ALH, E93 was not detected in control or ImpRNAi,SypRNAi MB neuroblast clones (Figure 6P,Q,R). We conclude that Imp/Syp regulate E93 independent of EcR in MB neuroblasts during larval stages.

E93 is a late-acting temporal factor that subdivides the Syp temporal window into Imp/Syp and Syp/E93

We asked whether E93 functions with Imp/Syp to control neurogenesis timing during development. Imp and E93 were knocked down in MB neuroblasts. At 48 hours APF, all MB neuroblasts were present in control and E93RNAi animals, but no MB neuroblasts were observed in ImpRNAi or ImpRNAi,E93RNAi double knock down animals (Figure 7A). Therefore, although E93 is expressed prematurely in the absence of Imp, and is necessary and sufficient to eliminate MB neuroblasts, E93 is not required for the premature elimination of ImpRNAi MB neuroblasts. Next, Imp and Syp were knocked down in MB neuroblasts. MB neuroblasts were observed in ImpRNAi,SypRNAi double knock-down animals at 48 hours APF and in adults, as reported previously (Figure 7A)[24, 25]. Therefore, unlike E93, Syp is required for premature elimination of ImpRNAi MB neuroblasts. We conclude that Syp, but not E93, inhibits Imp-dependent developmental neurogenesis.

Figure 7: E93 terminates growth of SypRNAi MB neuroblasts.

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(A) Average number of MB neuroblasts per brain hemisphere. Column numbers equal number of brain hemispheres scored. (B) Percentage of MB neuroblast clones with a MB neuroblast. Column numbers indicate number of clones scored and times below indicate time of heat shock treatment. (C) Box plots of MB neuroblast diameters. Numbers at bottom indicate number MB neuroblasts analyzed. (D,E) Colored overlay of MB neuroblast with greyscale image below. (F) Model summary, see text for details. *p values<.001, two-tailed Student’s t-tests. See also Table S1.

To determine whether Syp has a late function independent of Imp, we used the heat shock GAL4 flip out cassette to control timing of Syp inactivation. All SypRNAi MB neuroblast clones had one Dpn-positive neuroblast in adult animals (Figure 7B,E). Therefore, Syp is required late and acts independently of Imp to terminate neurogenesis. Because E93 is also required late and absent in SypRNAi MB neuroblasts, we asked whether ectopic E93 could terminate SypRNAi MB neuroblast divisions. MB neuroblasts clones expressing both UAS-E93 WT and UAS-SypRNAi were generated at 48 hours APF. Only 37% of SypRNAi MB neuroblasts clones with ectopic E93 (n=43 clones) had a Dpn-positive MB neuroblast (Figure 7B,D,E). Therefore, E93 is sufficient to terminate extended SypRNAi MB neuroblast divisions. SypRNAi MB neuroblasts with ectopic E93 were also significantly smaller than SypRNAi MB neuroblasts, consistent with the notion that E93 downregulates PI3-kinase and MB neuroblast growth for termination (Figure 7C-E). We conclude that Imp, Syp, and E93 function together forming a temporal cassette that determines the timeframe of when neurogenesis occurs during development. Imp promotes neurogenesis, Syp represses Imp and promotes E93, and E93 terminates neurogenesis, thus, linking early developmental neurogenesis with termination.

DISCUSSION

Lineage-specific intrinsic and extrinsic factors both determine timing of neurogenesis and the mechanism by which neurogenesis starts and stops during development [49–51]. In Drosophila, during early larval stages, non-MB neuroblasts exit quiescence and enter/exit cell cycle in a nutrient-dependent and PI3-kinase-dependent manner [20, 52, 53]. In contrast, MB neuroblasts divide continuously, even in the absence of dietary amino acids, which requires expression of the lineage-specific Eyeless transcription factor, a Pax-6 orthologue [20, 54, 55]. Non-MB neuroblasts then stop divisions during early pupal stages, while MB neuroblast divisions continue several days longer. In MB neuroblasts, low levels of PI3-kinase pathway activity trigger MB neuroblasts to stop dividing [12], while non-MB neuroblasts are reported to stop independent of PI3-kinase [15]. Here, we report that E93, which we identified from a RNAi screen, functions in a cell-autonomous manner to downregulate PI3-kinase activity in MB neuroblasts, which leads to increased autophagy and termination of MB neurogenesis. MB neurogenesis extends into adulthood when E93 is knocked down and terminates prematurely when E93 is overexpressed.

The Imp/Syp/E93 intrinsic temporal cassette

We found that E93 expression and timing of neurogenesis termination is controlled by the temporal factors, Imp and Syp (see model Figure 7F). E93 is expressed prematurely when Imp is knocked down and is not expressed late, when Syp is knocked down. Somewhat surprising, premature elimination of ImpRNAi MB neuroblasts can be rescued by knocking down Syp simultaneously, but not E93. Therefore, although Syp and E93 are both expressed late, they are functionally distinct. This is further supported when comparing extended neurogenesis phenotypes: SypRNAi MB neuroblasts remain large and divide continuously, even in two-week-old adults (Pahl, et. al. unpublished), versus E93RNAi MB neuroblasts, which remain large and divide into the first day of adulthood only. Therefore, Syp, but not E93, inhibits Imp and early developmental neurogenesis. Syp also positively regulates E93 for termination. Syp is an RNA binding protein and could regulate E93 transcript stability. Two of the three E93 transcripts annotated in flybase (Figure S1) and identified on Northern blot contain long 3’UTRs with predicted secondary structure [28]. Determining which E93 transcripts are expressed in MB neuroblasts and whether Syp directly regulates E93 at a post-transcriptional level will be important future work.

The Imp/Syp/E93 temporal cassette is also present in non-MB neuroblasts [24–27]. However, E93 is not required for termination of non-MB neurogenesis, suggesting that E93 is a lineage-specific termination factor (Pahl. et al, unpublished). But, if all neuroblasts express E93, how could E93 function in a lineage-specific manner to terminate neurogenesis? One possibility could be the lineage-specific expression of other factors, which control timing of neurogenesis. For reasons not yet known, Imp expression is protracted in MB neuroblasts, compared to non-MB neuroblasts [24]. Protracted Imp results in delayed expression of Syp and E93 in MB neuroblasts, compared to non-MB neuroblasts. This means that MB neuroblasts express E93 during later pupal stages, when systemic nutrients may be limited and hormone conditions different, compared to non-MB neuroblasts which express E93 earlier. Declining systemic nutrients or hormones could provide a co-factor that enables E93 to induce autophagy for neuroblast elimination. Alternatively, lineage-specific expression of other factors could control mechanism of neurogenesis termination. Non-MB neuroblasts are reported to terminally differentiate, triggered by a burst of nuclear Prospero received by neuroblasts after a final symmetric cell division [16]. In this case, E93 is not required for termination because non-MB neuroblasts differentiate. In contrast MB neuroblasts undergo apoptosis, coincident with increased autophagy due to reductions in levels of PI3-kinase activity [12]. Increased autophagy could sensitize neuroblasts to apoptosis, by degrading some factor that promotes survival. This is the case in Drosophila nurse cells, in which dBruce, an inhibitor of apoptosis, becomes localized with Atg8 positive puncta and is degraded in the lysosome during autophagy [56].

Steroid hormone receptor activation and E93-dependent regulation of PI3-kinase

Both autophagy and apoptosis together are required for MB neuroblast elimination, and we found that E93 downregulates PI3-kinase activity to activate autophagy in MB neuroblasts (see model Figure 7F). While E93 is required for MB neuroblast autophagy, it is expressed well before the time when MB neuroblasts are eliminated. E93 is a transcription factor that regulates expression of thousands of genes, including some required for autophagy and PI3-kinase pathway activity [31, 57]. Identifying E93 target genes required for MB neurogenesis termination will be important future work. Other tissues, including the salivary gland and midgut, also require E93 for autophagy, and similar to MB neuroblasts, levels of PI3-kinase activity are reduced [21, 29, 30, 45]. In the salivary gland and midgut, levels of the steroid hormone ecdysone regulate E93 expression [21, 29, 30, 45]. This is likely the case for MB neuroblasts as well, since E93 levels are reduced in the absence of EcR. It is intriguing to note that 20-hydroxyecdysone levels spike midway through pupal development and correlate with timing of E93 expression in MB neuroblasts (see model Figure 7F). In the future, it will be important to determine whether MB neuroblasts respond to this or to a different ecdysone pulse.

Terminating neurogenesis in mammals

Determining how neurogenesis terminates in mammals is more difficult than Drosophila due to increased neural complexity, NSC heterogeneity, and lack of identified molecular markers. In rodents, NSCs begin “disappearing” during late development, continuing into early postnatal stages, where NSC loss correlates with increased numbers of astrocytes and ependymal cells [58, 59]. This is consistent with NSCs being eliminated by terminal differentiation, however, some could also undergo apoptosis/autophagy and significant caspase-dependent cell death is observed in regions where NSCs normally reside [60–62]. It has also been reported that cultured hippocampal NSCs undergo autophagy and then death when insulin is withdrawn, and that NSC loss can be reduced when Atg7 is knocked down [63, 64]. Imp, Syp, and E93 are all evolutionarily conserved, suggesting that the mechanism regulating neurogenesis termination could also be conserved. In fact, NSCs are also prematurely depleted in IMP1 knockdown mice [65]. However, at the moment, roles for Syp and E93 in mammalian NSC biology have not yet been explored. Future work will be needed to determine the mechanism regulating termination of neurogenesis in mammals.

STAR METHODS

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sarah E. Siegrist (ses4gr@virginia.edu).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Fly husbandry and genetics:

Animals were maintained on a standard Bloomington fly food diet, at 25°C on a 12 hour light/dark cycle. For all expe riments, embryos were collected for 0–4 or 0–6 hours intervals and aged for 18–22 hours. Sixty freshly hatched larvae were then picked and transferred to a new vial containing Bloomington fly food. For larval staging, animals were aged from larval hatching. For pupal staging, animals were aged from white prepupae. For adults, animals were aged from the time of eclosion. Genotypes used are provided in TABLE S1.

Heat Shock experiments:

Animals were shifted to 37°C for 20–30 minutes at the indicated times to generate GAL4 flip out clones. Following heat shock, animals were returned to 25°C until the desired time.

METHOD DETAILS

Immunofluorescence and confocal imaging:

Larval, pupal or adult brains were dissected in Schneider’s insect media and fixed in a solution of 4% paraformaldehyde in PEM buffer for 20 minutes (larval brains) or 30 minutes (pupal and adult brains) as described previously [12, 20, 32]. In brief, after fixation, tissues were washed in PBT (1X PBS and .1%Triton-X) and blocked overnight at 4°C in PBT with 10% goat serum. Primary and secondary antibody incubations were also performed overnight in PBT with 10% goat serum at 4°C. Images were acquired using an upright Leica SP8 confocal microscope with a 63× 1.4NA oil immersion objective and analyzed using Imaris and ImageJ software. Figures were assembled using Adobe Photoshop and Illustrator software. The primary and secondary antibodies used are provided in table below. MB neuroblasts were positively identified based on a number of criteria, including location (dorsal surface superficial to the MB calyx), axon projections from progeny (through the calyx into the mushroom body peduncle), and expression of transcription factors including Dpn, Ey, and Tll. For MB neuroblast size measurements, average neuroblast diameter was calculated by measuring the length of two perpendicular diameters taken at the cell’s widest point.

Live imaging of whole brain explants:

Pupal brains were dissected at 84 hours APF and transferred to a glass bottom culture dish (MatTek P35G-1.0–14-C) in D22 insect medium (pH 6.95) supplemented with 10%FBS and .2mg/ml insulin. Brains imaged on an inverted Zeiss700 LSM equipped with a 63× 1.4NA oil immersion objective. Z-stacks were acquired every 2 mins [32].

QUANTIFICATION AND STATISTICAL ANALYSIS

Quantification of fluorescence intensities and statistical analysis:

Cytoplasmic and nuclear Foxo and E93 levels were quantified as described previously [12, 20, 32]. In brief, MB neuroblasts membranes labeled with Scrib and nuclei labeled with Dpn were manually traced and the average Foxo (or E93) fluorescence intensity measured in either the whole cell or nucleus alone using Image J software. Background measurements were acquired from regions devoid of Foxo (or E93) expressing cells in same focal plane. In Figure 6, we report normalized average fluorescence intensity across genotypes. For box plots, the boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles, respectively. Data is presented in the text as ± standard error of the mean, unless noted and experimental data sets were tested for statistical significance using two-tailed Student’s t-tests and one-way anova.

Supplementary Material

1
2. VIDEO S1. A MB neuroblast undergoing apoptosis. Related to Figure 4 and 5.

Time-lapse movie of a pupal MB neuroblast undergoing apoptosis in a brain explant at 84 hours APF. Still images shown in Figure S3A. Maximum intensity projection of Z stack imaged every 2 mins. Time stamp is hours:mins.

Download video file (324KB, mov)

Highlights.

E93 is required to terminate MB neurogenesis in Drosophila.

E93 downregulates PI3-kinase levels to activate MB neuroblast autophagy.

E93 is a late-acting temporal factor, regulated by intrinsic Imp and Syp.

Extrinsic steroid hormone signaling boosts E93 levels for neurogenesis termination.

ACKNOWLEDGEMENTS

We thank Claude Desplan, Chris Doe, Robert Tjian, Uwe Walldorf, and the DSHB for providing antibodies used in this study. We thank the Bloomington Stock Center, Harvard TRiP, Vienna Stock center, and Zurich FlyORF for providing transgenic flies. We especially thank Chris Doe, Karsten Siller, and Conor Sipe for providing comments on the manuscript. This work was funded by NIH/NICHD (R00-HD067293) and by NIH/NIGMS(R01-GM120421).

Footnotes

DECLARATION OF INTERESTS

The authors declare no competing interests.

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Associated Data

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

Supplementary Materials

1
NIHMS1521424-supplement-1.pdf (2.5MB, pdf)
2. VIDEO S1. A MB neuroblast undergoing apoptosis. Related to Figure 4 and 5.

Time-lapse movie of a pupal MB neuroblast undergoing apoptosis in a brain explant at 84 hours APF. Still images shown in Figure S3A. Maximum intensity projection of Z stack imaged every 2 mins. Time stamp is hours:mins.

Download video file (324KB, mov)

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