Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of asense/ASCL1

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Version of Record published: March 5, 2020 (This version)
Accepted: February 19, 2020
Accepted Manuscript published: February 19, 2020 (Go to version)
Received: November 6, 2019

1. Of interest
A gene regulatory network for neural induction

Katherine E Trevers, Hui-Chun Lu ... Claudio D Stern

Research Article Updated Mar 24, 2023
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Abstract

Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. Here we show that high levels of the Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, we could block tumour formation completely by re-expressing Asense (homologue of human ASCL1), which we show is a direct target of Tailless. Our results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells.

Introduction

The underlying mechanisms of glioblastoma initiation and growth have proved challenging to elucidate. This is due, in part, to the extensive molecular heterogeneity of glioblastoma, both between patients and within individual tumours. As such, there are many potential routes to tumourigenesis and the cell fate changes that contribute to glioblastoma initiation and progression remain to be fully elucidated. Cell fates can be altered in many different ways during tumourigenesis, depending upon the combination of genetic mutations present and the tumour cell of origin.

Mouse models have revealed many of the different cell types that can give rise to glioblastoma. In the central nervous system (CNS), tumours have been induced experimentally from differentiated glial cells, glial precursors and neural stem/progenitor cells (Alcantara Llaguno et al., 2015; Alcantara Llaguno et al., 2009; Bachoo et al., 2002; Chow et al., 2011; Friedmann-Morvinski et al., 2012; Holland et al., 2000; Lindberg et al., 2009; Marumoto et al., 2009). A recent study revealed that astrocyte-like neural stem cells (NSCs) in the SVZ of glioblastoma patients harbour driver mutations that are found in the patient’s tumour, suggesting that astrocyte-like NSCs are cells of origin of glioblastoma in humans (Lee et al., 2018). Neural lineages become more resistant to glioblastoma transformation as differentiation progresses, supporting stem cells or early progenitor cells as a common source of glioblastoma (Alcantara Llaguno et al., 2019). However, it is difficult to state unequivocally which cell type gives rise to tumours in mouse models of glioblastoma due in part to the lack of specific markers and driver lines. For example, both stem and progenitor cells express Nestin (Chen et al., 2009) and GFAP labels both stem cells and astrocytes (Doetsch et al., 1999). Furthermore, the mechanism through which cells within NSC lineages change identity during tumourigenesis and contribute to tumour aggressiveness remains unclear.

The Drosophila CNS has proved extremely valuable for understanding the fundamental principles of cancer (Deng, 2019; Villegas, 2019). The availability of an unparalleled Drosophila genetic toolkit and extensive knowledge of neural cell fate transitions has enabled diverse aspects of tumourigenesis to be investigated. One Drosophila model of glioblastoma is based on co-activation of EGFR and PI3K in glial cells (Chen and Read, 2019; Chen et al., 2019; Chen et al., 2018; Read et al., 2009; Read et al., 2013; Witte et al., 2009). This model recapitulates some of the features of glioblastoma, however, co-activation of EGFR and PI3K does not transform NSCs or their progeny. As a result the model does not address the contribution of neural lineages to glioblastoma (Read et al., 2009).

High levels of the orphan nuclear receptor TLX (also known as NR2E1, Nuclear Receptor Subfamily 2 Group E Member 1) have been observed in glioblastoma and been shown to correlate with poor patient prognosis (Park et al., 2010; Zou et al., 2012). TLX is expressed in adult NSCs, where it is required for neurogenesis in both the subventricular zone (SVZ) and the subgranular zone (SGZ) (Liu et al., 2008; Liu et al., 2010; Shi et al., 2004; Zhang et al., 2008; Zou et al., 2012). TLX is also expressed in glioblastoma stem cells (Zhu et al., 2014) and upregulation of TLX promotes gliomagenesis in the mouse SVZ (Liu et al., 2010). These results indicate that TLX is an important stem cell regulator both in normal and tumourigenic conditions. However, it is not known how abnormally high TLX levels affect the identity of cells in NSC lineages nor has the cell type vulnerable to TLX overexpression been identified.

In Drosophila, different NSC lineages exhibit distinct vulnerabilities to tumour-inducing mutations (Hakes and Brand, 2019). The majority of lineages arise from Type I NSCs (Figure 1A) that divide asymmetrically to self-renew and generate ganglion mother cells (GMCs), which then undergo terminal division (Figure 1B; Harding and White, 2018; Ramon-Cañellas et al., 2019). A much smaller number of Type II NSCs, by contrast, generate intermediate neural progenitors (INPs) (Figure 1B’; Bello et al., 2008; Boone and Doe, 2008; Bowman et al., 2008) that are themselves able to self-renew and produce GMCs. These transit amplifying Type II lineages more closely resemble neural lineages in the vertebrate CNS and provide an opportunity to investigate whether conserved mechanisms regulate how NSCs and their progeny respond to tumourigenic insults.

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Tll is expressed in *Drosophila* Type II NSCs.

(A) Schematic showing the position of the eight Type II NSCs (red) in each brain lobe. The majority of stem cells in the *Drosophila* brain are Type I NSCs (green). The optic lobes, which generate the adult visual processing centre, are shown in grey. (**B–B’**) Schematics showing the expression of cell fate markers in (B) Type I and (B’) Type II lineages. NSC: neural stem cell; imm INP: immature intermediate neural progenitor; mat INP: mature intermediate neural progenitor; GMC: ganglion mother cell. (C) RNA FISH shows *tll* mRNA (green) expression in Type II NSCs (solid outline) but not in their lineages (dotted outline). Type II lineages were identified by *pntP1*-GAL4 > *mCD8-GFP* expression in the central brain at wandering third instar larval stage. (**D–D’**) Immunostaining for Tll (green) shows strong expression in Type II NSCs (Dpn⁺ (red), solid outline) and weak expression in Dpn^- immature INPs (immINPs, arrow heads). Mature INPs (small Dpn⁺ cells in the lineage) do not express Tll. Type II lineages were identified by *pntP1*-GAL4 > *mCD8-GFP* expression in the central brain at wandering third instar larval stage. (E) Amino acid conservation between human TLX and *Drosophila* Tll. (F) Schematic showing that TLX (green) is expressed in NSCs and intermediate progenitors (IPs) in SVZ of the adult mouse brain (Li et al., 2012; Obernier et al., 2011). (G) Schematic showing *tll* mRNA (red) and Tll protein (green) expression in *Drosophila* Type II NSC lineages. Single section confocal images. Scale bars represent 10 µm.

Here we show that the Drosophila TLX homologue, Tailless (Tll), is required to direct the identity of Type II NSCs during development. We found that high levels of Tll are sufficient to initiate tumours from differentiating Type II NSC lineages by directing a cell fate change from INP to NSC. To identify downstream effectors of Tll action, we mapped the genome-wide targets of Tll and identified the proneural gene asense as a direct target of Tll repression, both during development and in tumourigenesis. Strikingly, we were able to rescue Tll tumours completely, and restore normal neurogenesis, by re-expressing asense. Our results demonstrate a reciprocal relationship between Tll and Asense expression and we hypothesized that this relationship might hold true in glioblastoma. We found that expression of TLX and ASCL1 (human counterparts of Tll and Asense) are also mutually exclusive in glioblastoma, suggesting a potentially conserved route to tumourigenesis.

Results

Tailless is necessary for Type II NSC identity and lineage progression

To understand the role Tailless (Tll) plays in the development of Type II NSC lineages, we first assessed its expression pattern. We found that Tll was expressed in Type II NSCs throughout larval development (Figure 1—figure supplement 1A–C). We detected tll mRNA in Type II NSCs but not in their progeny (INPs) (Figure 1C), while Tll protein was present in NSCs and at low levels in newly-born INPs (Figure 1D–D’). Tll shares a high degree of homology with human TLX (Figure 1E; Jackson et al., 1998): their DNA binding domains are 81% identical (94% similarity) and their ligand binding domains are 40% identical (77% similarity). In addition, TLX and Tll bind to the same consensus DNA sequence (Yu et al., 1994) and recruit conserved cofactors, such as Atrophin, via their ligand binding domains (Wang et al., 2006; Zhi et al., 2015). TLX is expressed in the neurogenic regions of the adult mouse brain (Liu et al., 2008; Niu et al., 2011; Shi et al., 2004; Zhang et al., 2008). In the SVZ, TLX is detected in NSCs and their progeny, intermediate progenitor cells (Figure 1F; Li et al., 2012; Obernier et al., 2011), which is very similar to the expression pattern of Tll in Drosophila Type II lineages (Figure 1G).

The enrichment of Tll expression in Type II NSCs suggested a role for Tll in regulating Type II NSC identity or proliferation. We knocked down Tll in larval NSCs using wor-GAL4 using two independent RNAi constructs that target different regions of the tll coding sequence (Figure 2—figure supplement 1A). We scored expression of Deadpan (Dpn), a Hes family bHLH-O transcription factor that is expressed in all NSCs (Bier et al., 1992), and Asense (Ase), a proneural bHLH factor expressed in Type I but not Type II NSCs (Bowman et al., 2008). Expressing either tll RNAi construct resulted in the absence of all Type II NSC in all brains assessed (i.e. all NSCs expressed Dpn and Ase) (Figure 2—figure supplement 1B–C’). We also generated tll null MARCM (Mosaic Analysis with a Repressible Cell Marker) clones (Lee and Luo, 1999). We found that Type II lineages were often labelled in wild type clones (Figure 2—figure supplement 1D–D’), demonstrating that MARCM clones could encompass Type II NSCs. However, we were unable to recover tll null Type II NSC clones, despite mutant clones being visible in other NSC lineages (Figure 2—figure supplement 1E). This suggested that tll null Type II NSCs underwent a cell fate transition that resulted in the loss of Type II markers. In support of this, quantification of the number of Type II lineages in brains with tll null clones revealed a reduction in the number of Type II lineages (Figure 2—figure supplement 1F). Furthermore, the number of absent Type II lineages in brains with tll null clones was comparable to the number of Type II lineages encompassed in MARCM clones in control brains (Figure 2—figure supplement 1F).

Next, we knocked down Tll expression specifically in Type II lineages by driving tll RNAi (tll-miRNA[s], which effectively knocked down Tll protein (Figure 2—figure supplement 1G)) with pntP1-GAL4 (Zhu et al., 2011) in combination with a ‘FLP-out’ GAL4 cassette to immortalise GAL4 expression (Figure 2—figure supplement 2A) and followed alterations in cell fate. While Dpn expression was unaffected, tll knockdown resulted in derepression of Ase in all Type II NSCs (Figure 2—figure supplement 2B), suggesting a switch in mode of neurogenesis from Type II to Type I.

To test whether Type II NSCs were transformed into Type I NSCs, we assessed lineage composition and gene expression. Type I NSCs express Dpn and Ase and segregate cortically localised Prospero (Pros) to their daughter cells (GMCs). In GMCs, Ase is expressed and Pros, a pro-differentiation transcription factor, translocates to the nucleus. In contrast, Type II NSCs express Dpn but not Ase or Pros. Type II NSCs give rise to INPs, which express Dpn, Ase and cortical Pros. INPs then generate GMCs that are Ase⁺ Pros⁺. In addition, the Ets transcription factor PointedP1 (PntP1) is expressed in Type II NSCs and immature INPs but not in Type I lineages (Zhu et al., 2011). Type II lineages can also be labelled by expression driven by a regulatory fragment of the FezF transcription factor earmuff (erm), which is expressed from INPs onwards (Pfeiffer et al., 2008; Weng et al., 2010) but not in Type I lineages.

Strikingly, upon tll knockdown, no INPs (small Dpn⁺ Ase⁺) could be found in Type II lineages and instead GMCs (Ase⁺ Pros⁺) were positioned adjacent to the NSCs (compare Figure 2A–A’’ to Figure 2B-B’’). In 6 out of 10 tll knockdown brains, at least one Type II NSC expressed Pros that localised in a crescent at the cell cortex, indicating asymmetric segregation of Pros to daughter cells (Figure 2B’’). Asymmetric segregation of Pros is a feature characteristic of Type I NSCs and INPs that is never observed in Type II NSCs (Bayraktar et al., 2010; Bello et al., 2008). Furthermore, expression of Pnt-GFP (Boisclair Lachance et al., 2014) in Type II NSCs (Figure 2—figure supplement 2C–C’) and the Type II lineage marker, erm-CD4-tdTomato (Han et al., 2011), was lost in the absence of tll (Figure 2—figure supplement 2D). We conclude that, in the absence of tll, Type II NSC lineages are transformed into Type I lineages which exhibit lower neurogenic capacity due to the lack of INPs (Figure 2C–C’ and Figure 2—figure supplement 2E–E’).

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Tll is required for Type II NSC fate and lineage progression.

(**A–A’’**) Control Type II NSCs (Dpn⁺ (red) and Ase^- (green)) generate INPs (arrowheads, Dpn⁺, Ase⁺ and Pros^- (blue)). Dotted lines outline three Type II lineages. Red asterisks (*) indicate Type II NSCs. n = 10 brains, dissected at the end of second larval instar stage. (**B–B’’**) Upon *tll* knockdown using *pntP1* >*act*-GAL4 to drive UAS-*tll*-miRNA[s], Type II NSCs express Ase and generate GMCs directly (arrowheads, Ase⁺ and Pros⁺) and exhibit Pros crescents (red arrowhead). Dotted lines outline three Type II lineages identified by *pntP1* >*act*-GAL4 driving UAS-*GFP*. Red asterisks (*) indicate Type II NSCs. n = 10 brains, dissected at the end of second larval instar stage. (**C–C’**) Schematic summarising the *tll* loss of function (LOF) phenotype in Type II NSCs. Single section confocal images. Scale bars represent 10 µm.

Tailless tumours can arise from Type II INPs and Type I NSCs

We have shown that Type II NSCs are lost when Tll is downregulated. As a corollary, we hypothesised that ectopic expression of Tll might result in excess Type II NSCs. To test this, we drove Tll expression in INPs and their progeny with erm-GAL4 (Pfeiffer et al., 2008; Weng et al., 2010) in combination with a ‘FLP-out’ GAL4 cassette to immortalise GAL4 expression (Ito et al., 1997; Figure 3A and Figure 3—figure supplement 1A–B). In control brain lobes there are only eight Type II NSCs. Tll misexpression in INPs resulted in a dramatic increase in the number of Type II NSCs, from 8 to 109 ± 12.12 per brain lobe (Figure 3B–B’ and quantified in Figure 3C). We also observed a strong reduction in the number of differentiating progeny in Type II lineages (Figure 3—figure supplement 1C–D’). We tested whether Tll expression in more differentiated neural precursors (GMCs) or post-mitotic neurons would also generate excess NSCs but, interestingly, we found that both cell types were resistant to ectopic Tll expression: we observed neither additional NSCs nor neuronal dedifferentiation (Figure 3D–E’). Therefore, ectopic Tll expression in Type II lineages promotes NSC fate at the expense of self-renewing cells, specifically INPs (Figure 3—figure supplement 1E–E’).

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Tll overexpression in INPs generates ectopic NSCs.

(A) Schematic showing the expression of *erm*-GAL4, which begins to be expressed in Type II lineages during the final stages of INP maturation. (**B–B’**) In Control (zoom), solid white outlines indicate Type II NSCs and *erm* >*act*-GAL4 is expressed in their lineages (dotted white lines). Tll OE in INPs with *erm* >*act*-GAL4 resulted in a large expansion of Type II NSCs (Dpn⁺ (red) and Ase^- (green)) in Type II lineages. Arrowheads in Tll OE – INP (zoom) highlight ectopic Type II NSCs. Zoom panels are magnifications of boxed regions in Control and Tll overexpression (OE) – INP. n = 10 brain lobes for Control and Tll. UAS-*tll* expression was restricted to larval stages with *tub*-GAL80^ts and brains were dissected at wandering third instar larval stage. (C) Quantification of the total number of Type II NSCs (Dpn⁺ Ase^-) in Control or Tll OE *erm* >*act*-GAL4 brains. Kolmogorov-Smirnov test ***, p<0.001 (p=0.000091). (**D–D’**) Expressing Tll in GMCs (using GMR71C09-GAL4 > mCD8-GFP (green)) does not result in ectopic NSCs (*i.e.* no Dpn⁺ GFP⁺ cells) nor defects in differentiation, as assessed by Pros (blue) staining. n = 10 brains for Control, n = 12 brains for Tll OE. Brains were dissected at wandering third instar larval stage. (**E–E’**) Expressing Tll in neurons using OK371-GAL4 > *mCD8-GFP* (green) does not result in ectopic NSCs (*i.e.* no Dpn⁺ GFP⁺ cells). n = 4 brains for Control and Tll. Brains were dissected at wandering third instar larval stage. Single section confocal images. Scale bars represent 30 μm in (**B, B’, E, E’**) and 10 µm in (**D, D’**).

INPs closely resemble Type I NSCs, in that they divide in the same manner and express common cell fate markers (Dpn, Ase and cortical Pros; Figure 1B–B’). We tested if Type I NSCs, which are found throughout the CNS, are also vulnerable to high levels of Tll expression. When we expressed Tll throughout the CNS, we observed large tumour-like growths in the adult brain that consisted almost entirely of NSCs (Dpn⁺ cells) (compare Figure 4A–A’). At larval stages, we found that high levels of Tll resulted in Dpn⁺ NSCs in both the central brain and ventral nerve cord, indicating that ectopic Tll can induce tumours from Type I NSCs (Figure 4B–B’).

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Tll can initiate Type II NSC tumours from Type I NSCs.

(**A–A’**) Overexpression of *Drosophila* Tll in neural lineages using *wor*-GAL4 resulted in NSC tumours (Dpn⁺ (white)) in all adult brains assessed. Control adult brains did not contain any NSCs. n = 7 brains for Control and Tll OE. UAS-*tll* expression was restricted to late larval stages with *tub*-GAL80^ts and brains were dissected from newly-eclosed adult flies. Images are projections over 15 µm (Control) or 17 µm (Tll). (**B–B’**) Overexpression of Tll during larval development with *wor*-GAL4 resulted in large tumours consisting of ectopic NSCs (Dpn⁺ (red) and *wor*-GAL4 >*mCD8-GFP* (green)) in the central brain and VNC of all brains assessed. UAS-*tll* expression was restricted to larval stages with *tub*-GAL80^ts and brains were dissected at wandering third instar larval stage. (**C–C’**) NSCs in the VNC are Type I (Dpn⁺ (red) and Ase⁺ (green)) in Control brains. Tll-induced tumours (ectopic Dpn⁺ cells) derived from Type I NSCs in the VNC are negative for Ase. UAS-*tll* expression was restricted to larval stages with *tub*-GAL80^ts and brains were dissected at wandering third instar larval stage. (**D–D’**) Tll tumours in the VNC occur at the expense of differentiating progeny (Pros (blue)). UAS-*tll* expression was restricted to larval stages with *tub*-GAL80^ts and brains were dissected at wandering third instar larval stage. (E) Schematic showing the organisation of Type I NSCs (Ase⁺ (grey)) and Type II NSCs (Ase^- (red)) in Control brains and Tll OE brains. Note that in Control brains the VNC contains only Type I NSCs, whereas Tll OE VNCs contain many ectopic Type II NSCs. (E’) Schematic showing transformation of Type I NSC lineages in the VNC to ectopic Type II NSCs when Tll is expressed at high levels. Single section confocal images unless stated otherwise. Scale bars represent 100 µm in (A-B') and 30 µm in (C-D'). n = 10 brains for all conditions unless stated otherwise.

Expression of Tll in Type I NSCs (normally Tll^-, Ase⁺) might convert them to a Type II fate (Tll⁺, Ase^-). To determine if tumour initiation occurred via conversion of Type I to Type II NSCs, we assessed the expression of Ase in tumours in the ventral nerve cord, which normally contains only Type I NSCs. Remarkably, Tll-induced tumours consisted almost entirely of NSCs that were negative for Ase, indicating a Type II-like NSC fate (Figure 4C–C’). Consistent with a change in identity to Type II NSC, the tumour NSCs lacked Pros (Figure 4D–D’; Bayraktar et al., 2010). Furthermore, a subset of these transformed Type I NSCs generated INPs (Figure 4—figure supplement 1A–A’), indicating that Tll is sufficient to induce a switch in NSC identity. Interestingly, the absence of Pros from Tll-induced hyperplasia had been reported previously (Kurusu et al., 2009) but had not been linked to a transformation from Type I to Type II NSC fate, or the ectopic appearance of INPs in the ventral nerve cord.

The appearance of the ectopic Type II-like NSCs (Dpn⁺ Ase^-) was associated with a reduction in GMCs and neurons, as assessed by expression of Pros (Figure 4D–D’). We conclude that expressing Tll in Type I lineages not only directs a change in NSC identity but also blocks differentiation in these newly transformed lineages, resulting in large NSC tumours comprised of Type II NSCs (Figure 4E–E’).

TLX/Tailless tumour initiation occurs via the reversion of INPs to NSC fate

To determine the cell of origin of Tll-induced tumours, we used G-TRACE (GAL4 technique for real-time and clonal expression) (Evans et al., 2009) to follow cell fate transformations within the Type II lineage. G-TRACE reports both current and historic GAL4 expression and so can be used to follow cell lineages (Figure 5—figure supplement 1A). erm-GAL4 driving G-TRACE labels INPs, but not Type II NSCs, in control brains (Figure 5A). Expressing high levels of Tll in INPs resulted in supernumerary Type II NSCs, which had previously expressed erm-GAL4 but lacked current expression (Figure 5A’ and quantified in Figure 5B). This would be expected if the cells had originally been INPs (erm-GAL4 expressing) and were then transformed into Type II NSCs (erm-GAL4 negative). We conclude that Tll expression is sufficient to induce a cell fate change from INP to NSC and our results implicate INP reversion to NSCs as the mechanism of tumour initiation.

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Tll/TLX overexpression results in reversion of INPs to NSC fate.

(**A–A’’**) G-TRACE reveals current (RFP (red)) and historic (EGFP (green)) *erm*-GAL4 expression (top panels). Dpn (red) and Ase (green) were used to assess the reversion of INPs to Type II NSCs (bottom panels). (A) In Control Type II lineages, NSCs (Dpn⁺ Ase^-, solid outline) are negative for both components of G-TRACE, whereas lineages show transition from RFP to EGFP (dotted outline). Overexpression (OE) of (A’) Tll or (**A’’**) human TLX in INPs resulted in ectopic Type II NSCs (Dpn⁺ Ase^-, white outlines) that express the EGFP component of the G-TRACE only (solid outline). Dpn⁺ Ase^- NSCs with dotted white outline either express neither G-TRACE component (as in Control) or express both RFP and GFP (indicating current expression of *erm*-GAL4). n = 8 brain lobes for Control and n = 10 for Tll and human TLX. Brains were dissected at wandering third instar stage. (B) Quantification of Type II NSCs expressing G-TRACE memory only (*i.e.* Dpn⁺ Ase^- and RFP^- EGFP⁺). Kolmogorov-Smirnov test ***, p<0.001 (p=0.000103). n = 7 brain lobes for Control; n = 10 brain lobes for Tll overexpression (OE). Brains were dissected at wandering third instar larval stage. (**C–C’**) Schematic showing the expression of G-TRACE with the INP-specific *erm*-GAL4 in Control brains or with Tll/TLX OE. (D) A model for how Tll/TLX generates ectopic NSCs and, consequently, tumours from INPs. Single section confocal images. Scale bars represent 15 µm.

To investigate whether human TLX could also initiate tumours from INPs via similar regulatory pathways, we expressed TLX in combination with G-TRACE in INPs. As for Tll, we observed ectopic Type II NSCs with historic erm-GAL4 expression only (Figure 5A’’). Our results indicate that both Tll and TLX can initiate tumourigenesis from neural lineages by reverting INPs to NSCs (Figure 5C). Interestingly, neither TLX nor Tll could revert post-mitotic neurons to NSCs, demonstrating that neurons are resistant to tumour initiation (see Figure 3D and Figure 5—figure supplement 2). We conclude that INPs represent a tumour-susceptible cell type and are the tumour cells of origin for TLX- and Tll-induced tumours in Drosophila (Figure 5D).

Ase restores progenitor identity and enforces differentiation to block Tailless tumours

We have shown that Tll is both necessary and sufficient to repress Ase expression during development and in tumourigenesis. To investigate if Tll represses ase directly, we identified the genome-wide Tll binding sites in vivo using Targeted DamID (TaDa) (Southall et al., 2013). We profiled Tll binding in Type II NSCs (16 per brain; approximately 700 NSCs per replicate) (Figure 6—figure supplement 1A–B) and identified Tll-binding peaks at 2495 protein-coding genes (see ‘Tll TaDa binding targets.xlsx’ for full list of Tll targets), including ase (Figure 6—figure supplement 1C). We conclude that Tll binds ase directly and represses its expression to promote Type II NSC fate. However, the loss of Ase alone is not sufficient to induce Type II fate in Type I NSCs (Bowman et al., 2008), indicating that Tll acts on additional target genes to mediate this cell fate change and tumour initiation. One potential candidate is Pros, which we found was also bound by Tll in Type II NSCs (Figure 6—figure supplement 1D). Pros is known to negatively regulate NSC proliferation (Cabernard and Doe, 2009) and is also repressed when Tll is expressed at high levels (Figure 3—figure supplement 1C–C’ and Figure 4D–D’; Kurusu et al., 2009).

As ectopic expression of Tll results in repression of ase and tumour formation, we investigated whether reinstating ase expression might be sufficient to block Tll-induced tumourigenesis. We expressed Ase together with Tll and found, remarkably, that tumourigenesis was completely abolished in all brains analysed (n = 10) (Figure 6A–A’’). Strikingly, not only did Ase prevent the production of ectopic NSCs, it also re-established normal neurogenesis in Type I NSC lineages: the production of GMCs and neurons was restored, as revealed by expression of Pros and Elav (Figure 6A–A’’ and B–B’’). However, re-introducing Ase into Tll tumours did not repress the expression of Tll (Figure 6—figure supplement 2A–A’’) nor does the ectopic expression of Ase turn off Tll in Type II NSCs (Figure 6—figure supplement 2B–B’). Therefore, by expressing Ase we were able to reinstate the normal neurogenic programme and prevent tumour initiation by Tll (Figure 6C–C’’).

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Reinstating progenitor identity prevents the formation of Tll tumours.

(**A–A’’**) Expressing Ase in combination with Tll during larval development using *wor*-GAL4 prevents tumour formation (ectopic Dpn⁺ cells (red)) and restores neuronal differentiation (Elav (green)) in all brains assessed. n = 10 brains for all conditions. Brains were dissected at wandering third instar stage. (**B–B’’**) Ase (green) rescues Tll tumours by promoting differentiation (Pros (red)). n = 9 brains for Control; n = 10 brains for Tll overexpression (Tll OE) and Ase rescue. Brains were dissected at wandering third instar larval stage. (**C–C’’**) Schematic depicting Type NSC I lineages (C) during development, (C’) with Tll-induced tumours and (**C’’**) with Ase expression in Tll tumours. Single section confocal images. Scale bars represent 30 µm.

TLX and ASCL1 appear to be mutually exclusive in human glioblastoma

We showed that, in Drosophila, Tll represses Ase both during development and in tumourigenesis. In other words, high levels of Tll correspond to low levels of Ase.

In human glioblastoma, high TLX expression is correlated with poor patient prognosis (Park et al., 2010; Zou et al., 2012). Intriguingly, ASCL1 levels also vary between human glioblastoma samples and low levels are correlated with shorter survival time (Park et al., 2017). Increasing ASCL1 levels was shown to promote terminal differentiation and attenuate tumorigenicity. Based on our results, we would predict that glioblastoma cells with high levels of TLX would exhibit low ASCL1 expression.

To determine if TLX and ASCL1 expression are mutually exclusive in glioblastoma, we analysed a previously published single-cell RNA sequencing (scRNA seq) data set that profiled glioblastoma samples from 28 patients, including both adult and pediatric tumours (Neftel et al., 2019). Our analysis identified 7,835 single cells in this data set and, following cluster annotation based on previously known markers (Neftel et al., 2019), we found that expression of both TLX and ASCL1 was restricted to the malignant glioblastoma cells (6,766 cells) (Figure 7A–A’). However, when we compared TLX and ASCL1 expression within the malignant population, we found very few cells that expressed both transcripts (Figure 7B), suggesting that TLX and ASCL1 are indeed mutually exclusive in malignant glioblastoma cells.

Figure 7

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Single cell RNA sequencing reveals that TLX and ASCL1 appear to be mutually exclusive in human glioblastoma.

(A) Uniform Manifold Approximation and Projection (UMAP) plot of 7,835 single cells coloured by TLX expression (red). Clusters were annotated based on previously known markers. TLX expression is only detected in the malignant cells. (A’) UMAP plot coloured by ASCL1 expression (green). ASCL1 expression is only detected in the malignant cells. (B) UMAP plot coloured by expression of both TLX (red) and ASCL1 (green), which appear mutually exclusive. Yellow indicates cells that express high levels of both TLX and ASCL1. Single cell RNA sequencing data and cluster markers obtained from Neftel et al. (2019).

Discussion

Our results revealed the mechanism through which high levels of the orphan nuclear receptor Tll initiate tumours in the Drosophila CNS (Figure 8A). We showed that Tll is expressed in Type II NSCs during larval development, where it is required for Type II NSC identity and subsequent lineage progression. In the absence of Tll, the proneural transcription factor Ase is derepressed in Type II NSCs. As a consequence, transit amplifying INPs are no longer generated and the resulting NSC lineages have a lower neurogenic potential.

Figure 8

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Model – Tll reverts INPs to NSC fate to initiate tumourigenesis.

(A) Schematics depicting the promotion of Type II NSC fate by Tll (red) in development and tumourigenesis. Tll must be down regulated in Type II lineages to allow differentiation. Ase (green) expression is activated during differentiation. If Tll is high in INPs, or in Type I lineages, Type II NSC fate is maintained, or induced, and tumours form. (B) In the adult mouse SVZ, TLX expression is high in NSCs and lower in intermediate progenitors (IPs) (Li et al., 2012; Obernier et al., 2011), whereas ASCL1 is high in IPs and low in NSCs (Kim et al., 2011; Parras et al., 2004). Based on our results, we predict that high levels of TLX associated with aggressive glioblastoma revert IPs through the repression of ASCL1 to promote the generation of glioblastoma stem cells.

A recent study examined the role of Tll in embryonic brain and showed that tll mutant embryos lack Type II NSCs (Curt et al., 2019). However, it was shown many years ago that tll mutant embryos fail to generate many NCSs, not just Type II NSCs, due to lack of l’sc expression that precedes NSC delamination (Younossi-Hartenstein et al., 1997). As a result, tll null mutants are not viable and the effect of tll loss of function on Type II NSCs specifically has not been addressed.

In mice, TLX is expressed in NSCs during embryonic development and in adulthood (Li et al., 2012; Li et al., 2008; Liu et al., 2008; Shi et al., 2004). Embryonic NSCs display defects in proliferation in the absence of TLX (Li et al., 2008) and the loss of TLX in adults results in the loss of transit-amplifying intermediates and reduction in neurogenesis (Li et al., 2012; Liu et al., 2008; Niu et al., 2011; Shi et al., 2004). While these effects were previously attributed to changes in the NSC cell cycle, our results suggest a cell fate change may occur due to the loss of TLX.

High levels of TLX in human glioblastoma are correlated with tumour aggressiveness (Park et al., 2010; Zou et al., 2012). High level expression of TLX results in glioblastoma-like lesions derived from SVZ NSC lineages in mouse models of glioblastoma (Liu et al., 2010) indicating that TLX can also promote glioblastoma development. However, it was not known how high TLX leads to glioblastoma, nor had the cellular origin of TLX-induced tumours been identified. TLX and its Drosophila homologue, Tll, are highly conserved proteins (Yu et al., 1994) and we found that both genes are able to revert INPs to NSC fate as a first step in tumour initiation. Ectopic expression of Tll was also sufficient to induce the expansion of NSCs throughout the Drosophila CNS, demonstrating the widespread vulnerability of NSC and progenitor populations to ectopic Tll expression.

We found that the ectopic NSCs resulting from high Tll expression are negative for Ase. We showed that Tll binds to the ase locus, suggesting that Tll directly represses ase. The absence of Ase is a hallmark of Type II NSCs. Therefore, ectopic Tll promotes a cell fate change from INP/Type I NSC to Type II NSC and thereby initiates tumourigenesis.

The capacity of Tll to induce NSC expansion had been reported previously as part of a study showing that Tll regulates the proliferation of larval mushroom body NSCs and GMCs (Kurusu et al., 2009). The authors showed that overexpressing Tll resulted in ectopic NSCs, but they did not identify the origin of these tumours and argued against a role for Tll in Type II NSC fate (Kurusu et al., 2009). Tll-induced tumourigenesis could be blocked by ectopic expression of Pros (Kurusu et al., 2009). However, ectopic Pros results in the loss of NSCs even in wild type brains (Cabernard and Doe, 2009). In contrast, Type I NSC lineages appear normal after Ase misexpression in wild type brains (Bowman et al., 2008). Furthermore, it has been reported that high levels of the human homologue of Pros, PROX1, exacerbate glioblastoma (Elsir et al., 2010; Goudarzi et al., 2018; Roodakker et al., 2016; Xu et al., 2017), arguing against PROX1 expression as a therapeutic strategy.

We found that the tumourigenic capacity of Drosophila Tll and human TLX was highly conserved (Figure 8B). Human TLX could also induce ectopic Type II NSCs from INPs through the repression of Ase. Analysis of scRNA seq from glioblastoma revealed that TLX and ASCL1 expression is mutually exclusive. It is notable that the origin of human glioblastoma has been mapped to the SVZ (Lee et al., 2018). While TLX positive NSCs have been identified in both the SVZ and dentate gyrus, high levels of TLX giving rise to glioblastoma has only been shown robustly in the SVZ (Liu et al., 2010). Furthermore, a recent study demonstrated that low expression levels of ASCL1 correlate with glioblastoma malignancy (Park et al., 2017). Ectopic expression of ASCL1 in glioblastoma stem cells was sufficient to promote neuronal differentiation. Based on our results in Drosophila, we predict that introducing ASCL1 would override the repressive effect of TLX, induce neuronal differentiation and reduce tumour growth, thereby providing an effective treatment.

Our results indicate that INPs are the tumour initiating cells in Type II NSC lineages expressing high levels of the orphan nuclear receptor Tll and potentially implicate intermediate progenitors as one of the cells of origin in TLX⁺ glioblastomas, an aggressive and lethal brain tumour. We found that Ase is a direct target of Tll and that Ase expression not only blocks Tll-induced tumourigenesis, but also reinstates a normal neural differentiation programme.

Materials and methods

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (D. melanogaster)	w¹¹¹⁸;+;+	BDSC	RRID:BSDC_3605
Genetic reagent (D. melanogaster)	Ay-GAL4, UAS-GFP	BDSC	RRID:BDSC_4411
Genetic reagent (D. melanogaster)	Ay-GAL4, UAS-lacZ(nls)	BDSC	RRID:BDSC_4410
Genetic reagent (D. melanogaster)	btd-GAL4	(Estella et al., 2003)
Genetic reagent (D. melanogaster)	erm-GAL4	BDSC	RRID:BDSC_40731	GMR9D11-GAL4
Genetic reagent (D. melanogaster)	GMR71C09-GAL4	BDSC	RRID:BDSC_39575
Genetic reagent (D. melanogaster)	insc-GAL4	(Luo et al., 1994)		GAL4^MZ1407
Genetic reagent (D. melanogaster)	pntP1^14-94-GAL4	(Zhu et al., 2011)
Genetic reagent (D. melanogaster)	wor-GAL4	(Albertson et al., 2004)
Genetic reagent (D. melanogaster)	OK371-GAL4	BDSC	RRID:BDSC_26160	VGlut^OK371
Genetic reagent (D. melanogaster)	tub-GAL80^ts	BDSC	RRID:BDSC_7018
Genetic reagent (D. melanogaster)	UAS-ase	(Brand et al., 1993)
Genetic reagent (D. melanogaster)	UAS-FLP	BDSC	RRID:BDSC_4539
Genetic reagent (D. melanogaster)	UAS-FLP	BDSC	RRID:BDSC_4540
Genetic reagent (D. melanogaster)	UAS-lacZ	(Brand and Perrimon, 1993)
Genetic reagent (D. melanogaster)	UAS-LT3-NDam	(Southall et al., 2013)
Genetic reagent (D. melanogaster)	UAS-LT3-NDam-tll	this study		Tll-Dam fusion for Targeted DamID
Genetic reagent (D. melanogaster)	UAS-mCD8-GFP	BDSC	RRID:BDSC_5130
Genetic reagent (D. melanogaster)	UAS-mCD8-GFP	BDSC	RRID:BDSC_5137
Genetic reagent (D. melanogaster)	UAS-mCD8-mCherry	BDSC	RRID:BDSC_27391
Genetic reagent (D. melanogaster)	UAS-myr-mRFP	BDSC	RRID:BDSC_7118
Genetic reagent (D. melanogaster)	UAS-myr-mRFP	BDSC	RRID:BDSC_7119
Genetic reagent (D. melanogaster)	UAS-tll	Kyoto DGRC	109680
Genetic reagent (D. melanogaster)	UAS-tll-miRNA[s]	(Lin et al., 2009)
Genetic reagent (D. melanogaster)	UAS-tll-shRNA	VDRC	330031
Genetic reagent (D. melanogaster)	UAS-TLX	this study		Human TLX under the control of UAS
Genetic reagent (D. melanogaster)	G-TRACE	BDSC	RRID:BDSC_28280
Genetic reagent (D. melanogaster)	G-TRACE	BDSC	RRID:BDSC_28281
Genetic reagent (D. melanogaster)	erm-CD4-tdTomato	(Han et al., 2011)		R9D11-CD4-tdTomato
Genetic reagent (D. melanogaster)	erm-mCD8-GFP	(Zhu et al., 2011)		R9D11-mCD8-GFP
Genetic reagent (D. melanogaster)	erm-lacZ	(Haenfler et al., 2012)		R9D11-lacZ
Genetic reagent (D. melanogaster)	Tll-GFP	BDSC	RRID:BDSC_30874
Genetic reagent (D. melanogaster)	Pnt-GFP	BDSC	RRID:BDSC_42680
Genetic reagent (D. melanogaster)	FRT82B, tub-GAL80	BDSC	RRID:BDSC_5135
Genetic reagent (D. melanogaster)	FRT82B	BDSC	RRID:BDSC_2035
Genetic reagent (D. melanogaster)	FRT82B,tll^l49/TM6B	(Pignoni et al., 1990)
Genetic reagent (D. melanogaster)	dpn>KDRTs-stop-KDRTs>GAL4	(Yang et al., 2016)
Genetic reagent (D. melanogaster)	ase-GAL80	(Neumüller et al., 2011)
Genetic reagent (D. melanogaster)	stg14-kd	(Yang et al., 2016)
Antibody	rabbit anti-Ase (polyclonal)	(Brand et al., 1993) Gift from the Jan Lab		IF 1:2,000
Antibody	chicken anti-β-Galactosidase (polyclonal)	abcam	ab9361	IF 1:1,000
Antibody	rabbit anti-β-Galactosidase (polyclonal)	Cappel (now MP Biomedicals)	55976	IF 1:10,000
Antibody	guinea pig anti-Dpn (polyclonal)	(Caygill and Brand, 2017)		IF 1:5,000
Antibody	rat anti-Elav (monoclonal)	DSHB	7E8A10 conc.	IF 1:100
Antibody	chicken anti-GFP (polyclonal)	abcam	ab13970	IF 1:2,000
Antibody	rabbit anti-PntP1 (polyclonal)	A gift from Jim Skeath		IF 1:500
Antibody	mouse anti-Pros (monoclonal)	DSHB	MR1A	IF 1:30
Antibody	rabbit anti-Tll (polyclonal)	(Kosman et al., 1998) Asian Distribution Center for Segmentation Antibodies		IF 1:300

Fly stocks and husbandry

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Drosophila melanogaster were reared in cages at 25 °C. Embryos were collected on yeasted apple juice plates. For experiments involving GAL80^ts embryos were kept at 18 °C until hatching. After hatching, larvae were transferred to a yeasted food plate and reared to the desired stage before dissection. Please see Supplementary file 1 for experimental genotypes and the temperature at which larvae were raised for each experiment.

The following GAL4 lines were used: Ay-GAL4, UAS-GFP (BL4411), Ay-GAL4, UAS-lacZ(nls) (BL4410), btd-GAL4 (Estella et al., 2003), erm-GAL4 (Pfeiffer et al., 2008; Weng et al., 2010) (R9D11-GAL4, BL40731), GMR71C09-GAL4 (Li et al., 2014) (BL39575), insc-GAL4 (GAL4^MZ1407) (Luo et al., 1994), pntP1^14-94-GAL4 (Zhu et al., 2011), wor-GAL4 (Albertson et al., 2004), OK371-GAL4 (VGlut^OK371-GAL4) (BL26160). tub-GAL80^ts (BL7018) was used to restrict GAL4 activity to larval stages as indicated.

The following UAS-transgenes were used: UAS-ase (Brand et al., 1993), UAS-FLP (BL4539 and BL4540), UAS-lacZ (Brand and Perrimon, 1993), UAS-LT3-NDam (Southall et al., 2013) and UAS-LT3-NDam-tll (this study), UAS-mCD8-GFP (BL5130 and BL5137), UAS-mCD8-mCherry (BL27391), UAS-myr-mRFP (BL7118 and BL7119), UAS-tll (Kurusu et al., 2009) (Kyoto Stock Center 109680), UAS-tll-miRNA[s] (Lin et al., 2009), UAS-tll-shRNA (VDRC 330031), UAS-TLX (this study), G-TRACE (BL28280 and BL28281). w¹¹¹⁸ was used as a reference stock.

The following reporter lines were used: erm-CD4-tdTomato (R9D11-CD4-tdTomato) (Han et al., 2011), erm-mCD8-GFP (R9D11-mCD8-GFP) (Zhu et al., 2011), erm-lacZ (R9D11-lacZ) (Haenfler et al., 2012) and Tll-GFP (Venken et al., 2009) (BL30874). Tll-GFP is a protein fusion under the control of a 20 kb insert containing the tll coding sequence and surrounding regulatory sequences. Importantly, this construct can rescue the lethality of homozygous tll^l49 mutants (data not shown). Pnt-GFP (BL42680) is a protein fusion under the control of a 90.7 kb insert containing the pnt coding sequence and surrounding regulatory sequences. This construct can rescue the lethality of pnt amorphic heteroallelic combinations (Boisclair Lachance et al., 2014).

For MARCM clone analysis, virgin female flies carrying hsFLP¹²²; wor-GAL4, UAS-mCD8-mCherry/(CyOact-GFP); FRT82B, tub-GAL80 were crossed to male flies carrying w; erm-lacZ; FRT82B or w; erm-lacZ; FRT82B, tll^l49/TM6B. tll^l49 is strong tll point mutation that is homozygous embryonic lethal (Pignoni et al., 1990). Embryos were collected on apple juice plates at 25°C and newly hatched larvae were transferred to yeasted food plates and raised at 25°C. Clones were induced by a heat shock in a water bath (5 min 37°C, 5 min rest at room temperature, 1 hr 37°C) at 24 hr ALH and larvae were dissected 72 hr later.

Immunostaining

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Brains were dissected in PBS, fixed in 4% formaldehyde/PBS for 20 min at room temperature and washed with PBS with 0.3% TritonX-100 (PBTx). Samples were blocked with 10% normal goat serum before overnight incubation with the following antisera: rabbit anti-Ase 1:2,000 (Brand et al., 1993) (a gift from the Jan lab), chicken anti-β-Galactosidase 1:1,000 (abcam ab9361), rabbit anti-β-Galactosidase 1:10,000 (Cappel), guinea pig anti-Dpn 1:5,000 (Caygill and Brand, 2017), rat anti-Elav 1:100 (DSHB, 7E8A10 conc.), chicken anti-GFP 1:2,000 (abcam ab13970), rabbit anti-PntP1 (1:500) (a gift from the Jim Skeath), mouse anti-Pros 1:30 (DSHB, MR1A), rabbit anti-Tll 1:300 (Kosman et al., 1998). Secondary antibodies conjugated to Alexa-405, Alexa-488, Alexa-546, Alexa-568, Alexa-633 all 1:500 (Life Technologies) or DyLight-405 1:200 (Jackson Laboratories) were used. Samples were mounted in Vectashield (Vector Laboratories) for imaging.

tll RNA FISH

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A set of 38 Stellaris FISH probes was designed against the tll coding sequence and labeled with Quasar 570. Third instar larval brains were fixed in 4% formaldehyde/PBS for 45 min at room temperature and then permeabilized in 70% ethanol/PBS for 6 hr at 4 °C. Brains were washed with Wash Buffer (10% formamide, 2xSSC) for 5 min before being incubated with probes (125 nM) in hybridisation buffer (100 mg/mL dextran sulfate, 10% formamide, 2xSSC) overnight at 45 °C. Brains were washed with Wash Buffer, stained with DAPI and mounted in Vectashield (Vector Laboratories) for imaging.

Image acquisition and processing

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Fluorescent images were acquired using a Leica SP8 confocal microscope. Images were analysed using Fiji (Schindelin et al., 2012), which was also used to adjust brightness and contrast in images. Adobe Illustrator was used to compile figures.

Quantification and statistical analysis

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GraphPad Prism version 7.00 for Mac OS X (www.graphpad.com) was used for statistical analysis. No data were excluded.

Sequence alignment of human TLX and Drosophila Tll

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Sequence alignment performed using EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) and UniProt alignment (https://www.uniprot.org/align/) tools.

Generation of UAS-TLX

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The coding sequence of human TLX (Jackson et al., 1998) was amplified from cDNA prepared from H9 ESCs (a kind gift from T. Otani) using the primers fwd: 5’-AGATGAATTCATGAGCAAGCCAGCCGG-3’ and rev: 5’-ATGACTCGAGTTAGATATCACTGGATTTGTACATATCTGAAAGCAGTC-3’. The amplified product was cloned (using restriction enzymes EcoR1 and XhoI) into pUAST-attB and then integrated into attP40 by standard methods.

Generation of UAS-LT3-NDam-tll

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The coding sequence of tll was amplified from an embryonic cDNA library using the primers fwd: 5’-cagaaactcatctctgaagaggatctgcgagatctaATGCAGTCGTCGGAGG-3’ and rev: 5’ acagaagtaaggttccttcacaaagatcctctagaTCAGATCTTGCGCTGACT 3’. The amplified product was cloned via Gibson assembly into pUASTattB-LT3-NDam (Southall et al., 2013) cut with BglII and XbaI and then integrated into attP40 by standard methods.

Targeted DamID

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We used a recombinase-dependent system to restrict GAL4 expression to Type II lineages (Yang et al., 2016). dpn >KDRTs-stop-KDRTs>GAL4; ase-GAL80/CyOact-GFP; + virgins were crossed to w; UAS-LT3-NDam-tll; stg14-kd or w; UAS-LT3-NDam; stg14-kd males at 25 °C. Larvae were transferred to yeasted food plates within an hour of hatching and dissected 50 hr later. Analysis was performed using the damidseq_pipeline as described previously (Marshall et al., 2016). dpn >KDRTs-stop-KDRTs>GAL4 and stg14-kd flies were provided by T. Lee (Yang et al., 2016) and ase-GAL80 flies by J. Knoblich (Neumüller et al., 2011). DamID analysis was performed as described previously (Marshall and Brand, 2015) and the Integrative Genomics Viewer (IGV, version 2.3.68) was used to visualise binding tracks aligned to release 6 of the Drosophila genome.

RNA single cell sequencing analysis

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Single cell sequencing analysis was performed using Seurat version 3. Data was obtained from Neftel et al. (2019), which was made available through the Broad Institute Single-Cell Portal (https://portals.broadinstitute.org/ single_cell/study/SCP393/single-cell-rna-seq-of-adult-and-pediatric-glioblastoma) and the Gene Expression Omnibus (GEO: GSE131928).

Data availability

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

The following previously published data sets were used

1. Neftel C
2. Laffy J
3. Filbin MG
4. Hara T
(2019) NCBI Gene Expression Omnibus
ID GSE131928. single cell RNA-seq analysis of adult and paediatric IDH-wildtype Glioblastomas.

https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE131928

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Decision letter

Claude Desplan

Reviewing Editor; New York University, United States
Utpal Banerjee

Senior Editor; University of California, Los Angeles, United States
Yan Song

Reviewer; Peking University, China

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

This paper demonstrates very elegantly that the transcription factor Tailless that is expressed in type II neuroblasts is required to repress Asense (the ortholog of ASCL1), and that its loss leads to the switch of identity from type II to type I neuroblasts. Interestingly, the paper shows that tumorgenesis resulting from overexpression of Tll is due to the reversion of intermediate neural progenitors to dividing neuroblasts, and that this is due to the Ase down-regulation. But the most exciting point of this paper is the demonstration that these brain tumors can be avoided by restoring Ase expression, which might be an important tool to eventually address why human glioblastoma have a poor prognosis when ASCL1 is also affected.

Decision letter after peer review:

Thank you for submitting your article "Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of asense/ASCL1" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Utpal Banerjee as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Yan Song (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

In short, the three reviewers are very positive about the paper and its relevance to human cancer.

One of the reviewers mentioned that the fact that overexpression of Tll causes brain tumors was known and so was its role in Type II/Type I neuroblasts, in particular with a recent paper from the Thor lab in eLife. However, all three reviewers are conscious of the mechanistic insights that your paper provides, and, more importantly, the connection to human brain tumors and the different prognosis depending on the levels of ASCL1/Ase.

However, before proceeding with publication, the reviewers would like you to add two points, which you should be able to achieve in the coming two months:

– One is to present the DamID data in more detail, in particular genes other than Ase that might play a role in the process.

– The reviewers would also like you to explore the link between Tll and the Notch pathway since some of the phenotypes are quite similar. It should be fairly simple in your hands to look at interactions between these pathways.

Reviewer #1

The orphan nuclear receptor TLX is an important neural stem cell regulator in both normal and tumorigenic conditions. TLX is expressed in neural stem cells (NSCs) during mouse embryonic development and in adulthood and is required for neurogenesis. High levels of TLX have been detected in glioblastoma and correlate with poor patient prognosis. However, the cellular origin and the molecular mechanisms underlying high TLX-induced brain tumor formation remain unclear. In the study, the authors used Drosophila larval brain neural stem cell lineages as model system to tackle these important questions. The authors showed that fly TLX homolog, Tailless (Tll), is specifically expressed in type II NSCs. Downregulation of Tll led to upregulation of Asense (Ase; fly homolog of ASCL1) and a NSC identity switch from type II to type I. The authors further showed that high levels of Tll induced tumorigenesis by reverting intermediate neural progenitors (INPs) to a NSC state and identified INPs as the cellular origin of Tll-induced tumors. More importantly, the authors identified Ase as a key direct target of Tll. Ase is downregulated in high Tll-induced brain tumors and restoring Ase expression prevented Tll-induced tumorigenesis and reinstated normal neurogenesis. Finally, the authors suggested that such mutually exclusive relationship between Tll and Ase holds true in human glioblastoma samples via analysis of single-cell RNA-seq.

Overall, this is a very interesting study of potential therapeutic values. Most experiments are well-controlled and well performed. However, before recommending publication the following points need be addressed:

1) Through DamID-seq, the authors identified Ase as the major target of Tll in NSC lineages. Indeed, high Tll-induce tumor could be completely blocked by Ase co-expression. However, since Ase inactivation itself does not lead to tumor formation (Bowman et al., 2008), downregulation of Ase seems to be a permissive but not sufficient step in Tll-induced tumorigenesis. Therefore, other self-renewal or differentiation gene(s) is very likely to be important target gene of Tll in NSC lineages. Can the authors re-examine their DamID-seq results and find out such important target gene(s)?

2) Figure 4 showed that Tll can induced tumor from type I NSCs. However, downregulation of Ase alone might lead to a type I to type II NSC identity switch, but not tumorigenesis (Bowman et al., 2008). What is the cellular origin and molecular mechanisms underlying Tll-induced tumorigenesis in type I NSC lineages?

3) Whether Ase, in turn, inhibits Tll expression in INPs or type I NSCs?

Reviewer #2

They identify a role for tll in the developing Drosophila CNS, in Type II neuroblasts (NBs). They identify ase as a key target of tll, and find that simultaneous overexpression of ase can suppress the tll overexpression effects. They furthermore use DamID to identify ase as a putative direct target of Tll. These are very interesting findings. However, three major points temper my enthusiasm for this study.

Major points:

1) The role of tll in Drosophila NB biology is not novel. tll was previously shown to be important for brain NB generation (Younossi-Hartenstein et al., 1997) and MBNB proliferation (Kurusu et al., 2009). Even more importantly, tll was recently show to be expressed in Type II NBs in the embryo and necessary for their generation/development, and tll could act with erm to convert Type I NBs in the embryonic nerve cord to Type II NBs, as well as act in the developing wing disc to generate ectopic Type II NBs (Curt et al., 2019). Against this backdrop, the current study does take major strides in our understanding of Type II NB biology.

2) In the same vein, there is a large body of work on the NB->INP->GMC->Neuron transition in Type II NBs. This has identified key "gating" roles for Notch-Dpn-E(spl), as well as for Brm, Brat, Klu, PntP1, Btd, Erm, Mediator-Complex, Barc and Trx. However, the possible connection between tll and these other regulators is not addressed (at least 24 publications). In particular, the connection between Notch pathway and tll is certainly worthy of more scrutiny, as Notch signalling, similar to tll, acts in Type II to repress Ase and Erm expression, hence preventing NBs from prematurely becoming INPs. Moreover, there is growing connection between tll/Tlx and Notch: tll mutants show loss of expression of the proneural gene l'sc (Younossi-Hartenstein et al., 1997), which is negatively regulated by Notch; tll and Notch signalling intersect in the developing Drosophila embryonic optic placodes (Mishra et al., 2018, PLoS Genet.); the C. elegans tll orthologue nhr-67 regulates both lin-12 (Notch) and lag-2 (Delta) during uterus development (Vergehese et al., 2011, Dev. Biol.); mouse tll orthologue Nr2E1 (aka Tlx) negatively regulates the canonical Notch target gene Hes1 (Luque-Molina et al., 2019, Stem Cell Reports). Hence, a key issue to address would be the intersection between tll and Notch, and given that NHRs are able to act both as transcriptional repressors and activators, two simple models emerge: (A) Tll acts with NotchICD-Su(H)-Mam on E(spl), and the obligate E(spl) repressors then act to directly repress ase, (B) alternatively, Tll acts combinatorially with E(spl) to directly repress ase. A straightforward way to test these two models would be to analyse expression of E(spl)-reporters in Tll LOF and GOF. In addition, it would be important to analyse Tll/tll expression in Notch pathway GOF and LOF.

3) DamID: Does Tll-Dam bind to other genes in the NB-INP-GMC-neuron pathway e.g., pnt, erm, E(spl), btd, klu, pros, dpn? Importantly, the evidence for Tll binding to the ase gene, but possibly also to dpn and E(spl), has bearing on the model for tll action. In addition, the DamID results are not described in great detail.

Reviewer #3

The Hakes and Brand manuscript contains detailed insightful information on the function of Drosophila Tailless. It covers both the role of Tll in normal neurogenesis and the tumourigenic effect of Tll overexpression.

The authors show that tll is required to maintain the Type II state: strikingly, upon tll loss, Type II neural stem cells are transformed into Type I, hence significantly compromising neurogenesis due to the lack of intermediate neural progenitors. They also show tll levels are critical: TLL over expression in the larval brain causes tumours derived from both Type II and Type I lineages. In the former, tumours develop as a consequence of intermediate progenitors reverting to a neural stem cell-like state. In the later, ectopic Tll results in the loss of Ase and in some cases even in the generation of intermediate neural progenitors, both suggestive of a certain degree of transdetermination from Type I towards Type II neural stem cells.

The manuscript goes on to demonstrate that Asense is a direct target of Tll repression during normal development as well as in the tumours that develop upon Tll upregulation to the extent that forcing Ase expression suppresses Tll-induced tumours

Overexpression of the human homologue TLX, which shares a remarkable level of protein sequence identity, cofactors, and function with Drosophila Tll, has been linked to the development of malignant astrocytomas. Interestingly, the authors find that TLX and ASCL1 (human Ase) are mutually exclusive in cells from human glioblastomas, which indeed is closely reminiscent of the results reported in the manuscript regarding fly neurogenesis and brain tumour development.

The reported studies take advantage of state-of-the-art techniques to perform sophisticated cell type-specific experimental manipulations to create the mutant conditions, and to trace the cell lineages of interest. The results are very well documented, presented, and discussed. Altogether, this is a high quality manuscript that I am happy to recommend for publication.

https://doi.org/10.7554/eLife.53377.sa1

Author response

Reviewer #1

[…]

Overall, this is a very interesting study of potential therapeutic values. Most experiments are well-controlled and well performed. However, before recommending publication the following points need be addressed:

1) Through DamID-seq, the authors identified Ase as the major target of Tll in NSC lineages. Indeed, high Tll-induce tumor could be completely blocked by Ase coexpression. However, since Ase inactivation itself does not lead to tumor formation (Bowman et al., 2008), downregulation of Ase seems to be a permissive but not sufficient step in Tll-induced tumorigenesis. Therefore, other self-renewal or differentiation gene(s) is very likely to be important target gene of Tll in NSC lineages. Can the authors reexamine their DamID-seq results and find out such important target gene(s)?

We have included the full list of Tll target genes as an excel file (Figure 6—figure supplement 1—source data 1). In addition to ase, Tll has multiple targets that could contribute to the Tll phenotypes we observe (including pnt, dpn, klu, wor, cycA, cycB, cycE).

2) Figure 4 showed that Tll can induced tumor from type I NSCs. However, downregulation of Ase alone might lead to a type I to type II NSC identity switch, but not tumorigenesis (Bowman et al., 2008). What is the cellular origin and molecular mechanisms underlying Tll-induced tumorigenesis in type I NSC lineages?

We showed that Tll must be downregulated in Type II lineages to allow differentiation to proceed. When expressed in Type I NSCs, Tll induces a cell fate change from Type I to Type II NSC and Tll is maintained at high levels in these transformed lineages. Once a Type I NSC has been transformed into a Type II NSC, differentiation is blocked due to continued high levels of Tll, which results in tumourigenesis. Tumourigenesis likely occurs through the inactivation of Ase in addition to other Tll target genes (see above).

3) Whether Ase, in turn, inhibits Tll expression in INPs or type I NSCs?

We show that ectopic expression of Ase in Type II NSCs does not repress Tll nor does expressing Ase in Tll-induced tumours. We have added these data as Figure 6—figure supplement 2. Therefore, Ase does not act directly on Tll but through other downstream targets.

Reviewer #2

They identify a role for tll in the developing Drosophila CNS, in Type II neuroblasts (NBs). They identify ase as a key target of tll, and find that simultaneous overexpression of ase can suppress the tll overexpression effects. They furthermore use DamID to identify ase as a putative direct target of Tll. These are very interesting findings. However, three major points temper my enthusiasm for this study.

Major points:

1) The role of tll in Drosophila NB biology is not novel. tll was previously shown to be important for brain NB generation (Younossi-Hartenstein et al., 1997) and MBNB proliferation (Kurusu et al., 2009). Even more importantly, tll was recently show to be expressed in Type II NBs in the embryo and necessary for their generation/development, and tll could act with erm to convert Type I NBs in the embryonic nerve cord to Type II NBs, as well as act in the developing wing disc to generate ectopic Type II NBs (Curt et al., 2019). Against this backdrop, the current study does take major strides in our understanding of Type II NB biology.

tll null mutants are not viable and the effect of tll loss of function on Type II NSCs specifically has never been addressed. We have identified a previously unknown role for Tll in larval Type II NSCs. Our data show that Tll is necessary and sufficient to promote Type II NSC fate. In the absence of Tll, Type II NSCs switch to a Type I identity, whereas ectopic expression of Tll alone is sufficient to induce Type II NSC fate in Type I NSCs.

It was shown some years ago (Younossi-Hartenstein et al., 1997) that tll mutant embryos fail to generate many different types of NSC (not just Type II NSCs) due to lack of l’sc expression, which precedes NSC delamination. This would explain why Curt et al., 2019 found that tll mutant embryos lack Type II NSCs. Curt et al. promoted Type II NSC identity in the embryonic brain by co-expression of Tll and Erm, not Tll alone. Later in development, Tll is indeed required for the proliferation of larval mushroom body NSCs and GMCs (Kurusu et al., 2009), demonstrating that Tll has diverse roles in brain development.

We have added these points to our Discussion.

2) In the same vein, there is a large body of work on the NB->INP->GMC->Neuron transition in Type II NBs. This has identified key "gating" roles for Notch-Dpn-E(spl), as well as for Brm, Brat, Klu, PntP1, Btd, Erm, Mediator-Complex, Barc and Trx. However, the possible connection between tll and these other regulators is not addressed (at least 24 publications). […] Hence, a key issue to address would be the intersection between tll and Notch, and given that NHRs are able to act both as transcriptional repressors and activators, two simple models emerge: (A) Tll acts with NotchICD-Su(H)-Mam on E(spl), and the obligate E(spl) repressors then act to directly repress ase,

We show that Tll binds directly to the ase locus. Therefore, the simplest explanation is that Tll acts directly on ase. Furthermore, we found that expression of the Notch signalling reporter E(spl)-mγ-GFP (Almeida and Bray., 2005) remains unchanged in Tll LOF Type II NSCs (see Author response image 1). These data argue against the reviewer’s first model that Tll acts with NotchICD-Su(H)-Mam on E(spl). More importantly to the reviewer’s point is that the published data indicate that Notch signalling represses Ase indirectly in Type II NSCs and instead maintains Type II NSC fate through the repression of Erm (Li et al., 2016).

Author response image 1

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N signalling is maintained in Tll LOF Type II NSCs.

E(spl)-mγ-GFP, a N signalling reporter (Almeida and Bray., 2005), is expressed in Control Type II NSCs (left panel, solid white outlines) and in Tll LOF Type II NSCs (*tll*-miRNA[s], right panel, solid white outlines). Dotted outlines highlight three Type II lineages identified by *pntP1*>*act*-GAL4 driving UAS-*lacZ(nls). n* = 13 brains for Control; n = 12 brains for *tll*-miRNA[s]. Brains dissected at the end of the second instar larval stage. The control panel is a projection of two 1 μm slices; the *tll*-miRNA[s] panel is a single section confocal images. Scale bars represent 15 µm.

(B) alternatively, Tll acts combinatorially with E(spl) to directly repress ase. A straightforward way to test these two models would be to analyse expression of E(spl)-reporters in Tll LOF and GOF.

We have shown that Tll GOF results in ectopic Type II NSCs. Since it is known that Type II NSCs normally have active N signalling (Zacharioudaki et al., 2012), we would expect to see an increase in E(spl)-mγ-GFP⁺ cells in Tll GOF lineages. Indeed, we found that ectopic Type II NSCs in Tll GOF lineages express the Notch signalling reporter E(spl)-mγ-GFP (Almeida and Bray., 2005) (see Author response image 2).

Author response image 2

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N signalling is active in ectopic NSCs in Tll GOF Type II lineages.

E(spl)-mγ-GFP (green), a N signalling reporter (Almeida and Bray., 2005), is expressed in Control Type II NSCs (top panels). Type II NSCs (Dpn⁺ (red) and Ase^- (blue)) are indicated with white arrowheads and lineages are outlined in dotted white lines. Likewise, N signalling is active in the ectopic Type II NSCs in Tll GOF lineages (bottom panels). Note that N signalling is also active in Type I NSCs (Dpn⁺ and Ase⁺, see arrow in Tll OE zoom panels). Zoom panels are magnifications of the boxed regions shown. *btd-*GAL4,FRT19A/FM7act-GFP;+;*tub*-GAL80^ts virgins were crossed to *w; E(spl)-mγ-GFP*; UAS-*myr-mRFP*/TM6B (Control) or *w; E(spl)-mγ-GFP*; UAS-*tll*, UAS-*myr-mRFP*/TM6B (Tll OE) males. Dotted white lines indicate *btd*-GAL4>*myr-mRFP*. GAL4 activity was restricted to larval development and brains were dissected after 3 days at 29 °C. n = 4 for Control and n = 5 for Tll OE. Single section confocal images. Scale bars represent 15 µm.

In addition, it would be important to analyse Tll/tll expression in Notch pathway GOF and LOF.

It is known that N GOF in Type II lineages results in ectopic Type II NSCs (Bowman et al., 2008; Weng et al., 2010; Xiao et al., 2012; Song et al., 2011; Farnsworth et al., 2015) and we have shown that normal Type II NSCs express Tll. Therefore, an increase in Tll⁺ cells in N GOF Type II lineages would be consistent with previous literature and the data we have presented in this study.

There are some important differences between the Tll and N LOF phenotypes. We have shown that Type II NSCs become Type I NSCs without Tll, whereas Type II NSCs appear to undergo premature differentiation without N signalling (Li et al., 2016). Although it was reported previously that Type II NSCs switch to Type I NSCs without N signalling, Erm is precociously expressed in the NSC and maintained in the lineage, i.e. they are not Type I NSCs (Li et al., 2016). In contrast, erm expression is absent entirely from Tll LOF Type II lineages. Indeed, all Type II NSC and lineage features are absent in Tll LOF Type II NSCs (repression of Ase in NSC, plus PntP1 and erm expression). We have added the effect of Tll LOF on PntP1 expression as Figure 2—figure supplement 2C-D’.

We propose that Tll and N signalling act in parallel in Type II NSCs: Tll acts upstream to establish Type II NSC identity (including repression of ase) and that N signalling acts within Type II lineages to ensure timely differentiation of INPs (by repressing erm in Type II NSCs). However, as the reviewer states, there are many genes that regulate cell fate changes in Type II lineages. How Tll interacts with each of these genes will be an interesting topic for future study.

3) DamID: Does Tll-Dam bind to other genes in the NB-INP-GMC-neuron pathway e.g., pnt, erm, E(spl), btd, klu, pros, dpn? Importantly, the evidence for Tll binding to the ase gene, but possibly also to dpn and E(spl), has bearing on the model for tll action. In addition, the DamID results are not described in great detail.

We have included the full list of Tll target genes as an excel file (Figure 6—figure supplement 1—source data 1). In addition to ase, Tll as a transcription factor has multiple targets, including pnt, erm, E(spl), btd, klu, pros and dpn, that could contribute to the Tll phenotypes we observe.

https://doi.org/10.7554/eLife.53377.sa2

Article and author information

Author details

Anna E Hakes

The Gurdon Institute and Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

Contribution
Conceptualization, Resources, Data curation, Formal analysis, Investigation, Methodology

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-8664-1014
Andrea H Brand

The Gurdon Institute and Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom

Contribution
Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Project administration

For correspondence
a.brand@gurdon.cam.ac.uk

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-2089-6954

Funding

Wellcome (103792)

Andrea H Brand

Royal Society

Andrea H Brand

Wellcome (102454)

Anna E Hakes

Wellcome (092096)

Andrea H Brand

Cancer Research UK (C6946/A14492)

Andrea H Brand

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

Acknowledgements

We should like to thank L Jan and Y N Jan, J Knoblich, M Kurusu, C-Y. Lee, T Lee, T Otani, J Skeath, S Zhu, the Asian Distribution Centre for Segmentation Antibodies, Bloomington Drosophila Stock Centre, Developmental Studies Hybridoma Bank (DSHB), the Kyoto Stock Center (DGRC), and the ViennaDrosophilaResource Center (VDRC) for reagents. We thank D J Kunz for advice on glioblastoma single cell RNA sequencing data, LYJ Tang for performing single cell RNA sequencing analysis and R Krautz for analyzing the Tll TaDa binding data. We thank L Otsuki for helpful discussions.

This work was funded by Wellcome Trust Senior Investigator Award (103792 to AHB) and the Royal Society Darwin Trust Research Professorship (to AHB) and Wellcome Trust PhD Studentship (102454 to AEH). AHB acknowledges core funding to The Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492).

Senior Editor

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

Reviewing Editor

Claude Desplan, New York University, United States

Reviewer

Yan Song, Peking University, China

Publication history

Received: November 6, 2019
Accepted: February 19, 2020
Accepted Manuscript published: February 19, 2020 (version 1)
Version of Record published: March 5, 2020 (version 2)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.