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. Author manuscript; available in PMC: 2016 Dec 7.
Published in final edited form as: Curr Biol. 2015 Nov 12;25(23):3058–3068. doi: 10.1016/j.cub.2015.10.027

Aging neural progenitors lose competence to respond to mitogenic Notch signaling

Dylan R Farnsworth 1,2, Omer Ali Bayraktar 1,2,3, Chris Q Doe 1,2,3,*
PMCID: PMC4679507  NIHMSID: NIHMS731253  PMID: 26585279

Abstract

Drosophila neural stem cells (neuroblasts) are a powerful model system for investigating stem cell self-renewal, specification of temporal identity, and progressive restriction in competence. Notch signaling is a conserved cue that is an important determinant of cell fate in many contexts across animal development; for example mammalian T cell differentiation in the thymus and neuroblast specification in Drosophila are both regulated by Notch signaling. However, Notch also functions as a mitogen, and constitutive Notch signaling potentiates T cell leukemia as well as Drosophila neuroblast tumors. While the role of Notch signaling has been studied in these and other cell types, it remains unclear how stem cells and progenitors change competence to respond to Notch over time. Notch is required in type II neuroblasts for normal development of their transit amplifying progeny, intermediate neural progenitors (INPs). Here we find that aging INPs lose competence to respond to constitutively active Notch signaling. Moreover, we show that reducing the levels of the old INP temporal transcription factor Eyeless/Pax6 allows Notch signaling to promote the de-differentiation of INP progeny into ectopic INPs, thereby creating a proliferative mass of ectopic progenitors in the brain. These findings provide a new system for studying progenitor competence, and identify a novel role for the conserved transcription factor Eyeless/Pax6 in blocking Notch signaling during development.

Introduction

Development of complex structures like the human central nervous system (CNS) requires the production of a staggering diversity of cell types from a relatively small pool of progenitors. Spatial cues generate progenitor diversity, whereas subsequent temporal cues allow single progenitors to produce a series of distinct neuronal and glial cell types [1, 2]. Recently it has become clear that progenitors change competence to respond to spatial and temporal cues, potentially allowing a single cue to generate distinct outputs [2–6]. For example, mammalian cortical progenitors gradually lose competence to form early-born cell types. When developmentally advanced progenitors are transplanted into their native region in younger hosts, they fail to produce the deep layer neurons typically born in this cortical environment [7]. Similarly, Drosophila embryonic neuroblasts (NBs) are initially competent to respond to the early temporal transcription factors Hunchback or Krüppel, but later lose competence to respond to these cues [8–10]. Although there has been excellent progress on identifying spatial and temporal patterning cues, much less is known about how progenitors change competence. Do progenitors pass through discrete competence windows where distinct cells types are born in response to the same cue? What are the mechanisms that restrict competence? Are there many mechanisms, or might there be a small number of highly conserved mechanisms?

Drosophila neural progenitors are a model system to investigate how competence to respond to cell fate cues changes over time. Drosophila neuroblasts arise in the early embryo and can persist throughout larval stages. Most neuroblasts undergo a “type I” mode of division in which they divide asymmetrically to generate a series of smaller ganglion mother cells (GMCs) that each produces a pair of neurons or glia (Figure 1A). There are well-characterized spatial and temporal patterning cues that act on embryonic type I neuroblasts to generate neural diversity, as well as evidence for at least two distinct neuroblast competence windows that may produce different responses to early temporal identity factors [reviewed in [2, 6, 11–13]].

Figure 1. Old INPs lose competence to respond to Notch.

Figure 1

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(A) Eight type II NBs are found in the central brain (CB) of each larval brain lobe (OL = optic lobe, VNC = ventral nerve cord).

(A′–A″) Summary of type I and type II NB cell lineages. Type I NBs self-renew and produce GMCs which divide to make two neurons or glia. Type II NBs make INPs which transit amplify their lineage. R9D11-gal4 is expressed in young INPs and their progeny but not the parental NB, whereas OK107-gal4 is expressed in old INPs and their progeny but not other cells in the lineage.

(B–B′) Wild type third instar larvae expressing GFP in young INP lineages (R9D11-gal4 UAS-GFP) show the normal number of Dpn+ Ase− type II neuroblasts (8±0 per lobe; n=3).

(C–C′) Expression of constitutively active Notch in young INPs (R9D11-gal4 UAS-Notchintra UAS-GFP) produces ectopic Dpn+ Ase− type II neuroblasts (34±1 per lobe, n=3).

(D–D′) A permanent lineage tracing system in young INPs (UAS-Flp, UAS-FRT-Stop-FRT-actin-gal4, UAS-Notchintra) standardized expression of UAS-Notchintra. This also produced ectopic type II NBs.

(E–E′) Old INPs are labeled by OK107-gal4 driving membrane GFP, without generating ectopic type II neuroblasts (8±0 per lobe; n=3).

(F–F′) Old INPs do not generate ectopic Dpn+ NBs in response to constitutive Notch signaling (8±0; n=3).

(G–G′) Using OK107-gal4, UAS-Flp, UAS-FRT-Stop-FRT-actin-gal4, UAS-Notchintra to standardize UAS-Notchintra expression levels did not produce ectopic Dpn+ NBs (8±0 per lobe; n=3).

(H, I, J) Summary and quantification of results. Images are a single, one micron plane through a whole brain lobe. Yellow outline = INP lineages in central brain.

All panels show third instar larvae; scale bar = 10 μm.

More recently, our lab and others have identified eight larval neuroblasts per brain lobe that undergo a more complex “type II” mode of division (Figure 1A′). Type II neuroblasts generate a series of smaller intermediate neural progenitors (INPs) that act as transit amplifying cells; each INP undergoes a series molecularly asymmetric divisions to self-renew and produce about six GMCs, each of which makes a pair of neurons or glia (Figure 1A″) [14–16]. Type I and II neuroblasts can also be distinguished by molecular markers; type I neuroblasts contain the transcription factors Deadpan (Dpn), Worniu (Wor), and Asense (Ase) whereas the type II neuroblasts contain Dpn, Wor, and Pointed P1 (PntP1). Spatial and temporal patterning factors acting on larval neuroblasts have been identified [17–22], and we have recently identified three INP temporal transcription factors: Dichaete (D), Grainy head (Grh), and Eyeless (Ey) [23]. Despite this progress, currently nothing is known about how larval neuroblasts or INPs change competence to respond to cell fate or mitogenic cues.

Here we established a new system for investigating progenitor competence, INPs of the type II neuroblast lineages. In type II neuroblasts, Notch signaling is active and is required to maintain neuroblast identity and proliferation [16, 24–27]. This is a highly conserved function, as Notch signaling also promotes self-renewal and proliferation of mammalian neural progenitors and stem cells [28–32]. Drosophila type II neuroblasts divide asymmetrically to produce immature INPs that lack active Notch signaling due in part to partitioning of the Notch inhibitor Numb selectively into the newborn INP. Overexpression of the Notch intracellular domain (Notchintra) can bypass this block and induce de-differentiation of the new-born INP back into a type II neuroblast, leading to “neuroblast tumors” [16, 25, 26, 33]. Here we investigate how INPs change competence to respond to Notch signaling over time. We confirm that expression of constitutively active Notchintra in young INPs results in the formation of neuroblast tumors, but in striking contrast old INPs have no detectable response to precisely the same level of Notchintra. Thus, INP competence to respond to Notch signaling changes over time, although the mechanism preventing old INPs from responding to Notchintra remains unknown. Here, we identify a second mechanism that prevents GMCs from responding to Notch signaling: reducing the level of the old INP temporal transcription factor Eyeless/Pax6 resulted in de-differentiation of GMCs into INPs, leading to a proliferative mass of INP/GMC cell types that failed to initiate neuronal/glial differentiation. This defines a new role for the conserved Eyeless/Pax6 transcription factor in preventing progenitors from responding to Notch signaling.

Results

Old INPs lose competence to respond to Notchintra signaling

As a starting point for our studies, we confirmed previous reports showing that constitutively active Notch (Notchintra) in young INPs triggered INP de-differentiation into ectopic Dpn+ Ase− type II neuroblasts (Figure 1C–C′ and data not shown; quantified in Figure 1J) [and see Figure 6C in [25]]. Next, to determine whether old INPs remained competent to de-differentiate into type II neuroblasts in response to Notch signaling, we expressed Notchintra using OK107-gal4, which is specifically expressed in old INPs within type II lineages [23]. As expected, expression of GFP alone in old INPs did not produce any ectopic Dpn+ Ase− Type II neuroblasts (Figure 1E and data not shown; quantified in Figure 1J). Interestingly, expression of Notchintra alone in old INPs also did not generate any ectopic neuroblasts (Figure 1F; quantified in Figure 1J), in contrast to its potent induction of ectopic neuroblasts when expressed in young INPs. There are two possible interpretations of these results: (a) the OK107-gal4 line produced lower levels of Notchintra compared to R9D11-gal4, leading to insufficient Notchintra to induce neuroblast identity; or (b) old INPs have lost competence to respond to Notchintra.

To ensure equal Notchintra levels in young or old INPs, we used a “flp out” expression method [23]. We used the young INP R9D11-gal4 line or the old INP OK107-gal4 line to drive expression of UAS-Flp, which catalyzes excision of transcriptional stop sequences in the actin-FRT-stop-FRT-gal4 gene. Thus, this method results in permanent expression of actin-gal4 in either young INPs or old INPs, thereby ensuring equal levels of expression of the UAS-Notchintra gene. As expected, actin-gal4 driving UAS-Notchintra in young INPs induced a large number of ectopic Dpn+ Ase− Type II neuroblasts (Figure 1D and data not shown; quantified in Figure 1J; summarized in Figure 1H). In contrast, actin-gal4 driving UAS-Notchintra in old INPs did not generate any Dpn+ Ase− neuroblasts (Figure 1G and data not shown; quantified in Figure 1J; summarized in Figure 1I). In addition, Notchintra protein levels are indistinguishable among these genotypes (Figure S1). We conclude that old INPs have lost competence to form neuroblasts in response to Notch signaling.

Eyeless restricts the competence of old INPs, or their progeny, to respond to Notchintra signaling

We have shown that young and old INPs differ in their competence to respond to Notch signaling. What might be the cause of these differences? The recent identification of the transcription factor Eyeless expressed in old INPs provides a good candidate. We hypothesized that Eyeless may block Notch signaling in old INPs or their progeny.

We have previously shown that loss of Eyeless causes old INPs to delay the termination of their lineages by several additional divisions, but no ectopic neuroblasts or INPs are formed [23]. To test whether loss of Eyeless increased the competence of old INPs to respond to Notch signaling, we used our previously well-characterized UAS-eyelessRNAi transgene [23] to eliminate all detectable Eyeless protein concurrent with expression of UAS-Notchintra (OK107-gal4, UAS-mCD8-GFP, UAS-Notchintra, UAS-eyelessRNAi). Confirming previous findings [23], Eyeless RNAi removes all detectable Eyeless protein without generating any ectopic Dpn+ Ase− neuroblasts and very few Dpn+ Ase+ INPs (Figure 2A,B and data not shown; quantified in Figure 2D). In contrast, removing all detectable Eyeless together with expression of Notchintra led to the formation of many ectopic Dpn+ neuroblasts or INPs (Figure 2C; quantified in Figure 2D). There are several possible explanations for the observed phenotype: (a) the ectopic Dpn+ cells could arise from the OK107-gal4 expressing optic lobe or mushroom body that have migrated into medial brain regions where the type II lineages are located; (b) the ectopic Dpn+ cells could be due to Notchintra in the optic lobe or mushroom body lineages, leading to indirect effects on the type II lineages; or (c) the ectopic Dpn+ cells could be due to the action of Notchintra within the type II lineages.

Figure 2. Eyeless restricts the competence of old INPs to respond to Notch signaling.

Figure 2

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(A–C) Overexpression of Notch in Eyeless-negative old INPs generates ectopic Deadpan+ presumptive INPs. (A–A″) OK107-gal4 driving membrane GFP labels old INPs that express Eyeless and Deadpan. (B–B″) OK107-gal4 UAS-eyelessRNAi results in efficient knockdown of Ey in old INPs, but does not generate ectopic Deadpan+ NBs or INPs. (C–E) Constitutive Notch signaling in Eyeless-negative old INPs (OK107-gal4, UAS-eyelessRNAi, UAS-Notchintra) generates many ectopic Dpn+ (C) presumptive INPs expressing Grh (D–E) in the dorsomedial brain. Images are a single, one micron plane through a whole brain lobe (A–D) or zoomed in to the dorsal-anterior central brain (E).

All panels show third instar larvae; scale bar = 10 μm.

To distinguish between Notchintra acting directly or indirectly on type II lineages, we used the R16B06-gal4 line. R16B06-gal4 contains an eyeless fragment driving gal4 expression [34, 35] and can be used to target Notchintra expression specifically to old Eyeless+ INPs without additional larval brain expression in the optic lobe or mushroom body (Figure S2). Using R16B06-gal4 to drive expression of GFP alone or Notchintra alone did not produce any ectopic Dpn+ cells (Figure 3A–B; quantified in Figure 3F; summarized in Figure 3G). In contrast, using R16B06-gal4 to express UAS-GFP UAS-eyelessRNAi UAS-Notchintra together in old INPs produced many ectopic Dpn+ cells (Figure 3C–C′); quantified in Figure 3F; summarized in Figure 3G), which we provisionally assign an INP identity because most cells have the Dpn+ Ase+ molecular profile of INPs (Figure 3D–D‴). This is in contrast to the ectopic Dpn+ Ase− Type II neuroblasts formed from young INPs dedifferentiating in response to Notch (Figure 3E–E‴). We conclude that Eyeless restricts the competence of old INPs, or their progeny, to respond to Notchintra signaling.

Figure 3. Old INPs labeled by R16B06-gal4 also lose competence to respond to Notch.

Figure 3

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(A–A′) Old INPs in the central brain are labeled by R16B06-gal4 driving membrane-bound GFP.

(B–B′) Old INPs labeled by R16B06-gal4 do not produce ectopic Dpn+ cells in response to constitutive notch signaling (R16B06-gal4, UAS-Notchintra).

(C–C′) When Eyeless knockdown is coupled with constitutive Notch signaling in old INPs (R16B06-gal4, UAS-eyelessRNAi, UAS-Notchintra), many ectopic Dpn+ cells are produced.

(D–D‴) The ectopic cells produced from constitutive Notch signaling coupled with Ey knockdown in old INPs labeled by R16B06-gal4 have an INP-like identity (Dpn+ Ase+). (E–E‴) Ectopic cells produced from constitutive Notch expression in young INPs are Dpn+ but do not express Ase, indicating a Type II NB-like identity. (F,G) Summary of results.Images are a single, one micron plane through a whole brain lobe (A–C) or zoomed in to the dorsal-anterior central brain (D–E). All panels show third instar larvae; scale bar = 10 μm.

Eyeless blocks Notchintra from inducing GMC-to-INP dedifferentiation

Next, we wanted to verify the INP identity of the ectopic Dpn+ cells induced by Notchintra, and determine their developmental origin. Using the old INP lines R16B06-gal4 or OK107-gal4 to concurrently eliminate Eyeless protein and induce Notchintra, we find the vast majority of ectopic cells are Dpn+ Ase+ consistent with an INP identity (Figure 4A,B). In addition, most of the ectopic cells were also Grh+ (Figure 4C,D) consistent with the molecular profile of Eyeless-negative INPs [23]. We conclude that the majority of the ectopic cells induced by Notch in old Eyeless-negative INP lineages have the molecular characteristics of INPs.

Figure 4. Notchintra in old INPs lacking Eyeless generates ectopic INPs and GMCs.

Figure 4

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(A–B) Constitutive Notch signaling in Eyeless-negative old INPs (OK107-gal4, UAS-eyelessRNAi, UAS-Notchintra) generates many ectopic Dpn+ Grh+ cells. (C) presumptive INPs expressing Grh (D–E) in the dorsomedial brain.

Images are a single, one micron plane through a whole brain lobe (A–D) or zoomed in to the dorsal-anterior central brain (E).(C) Wild-type old INPs normally express Dpn and Asense (Ase).

(D) Overexpression of Notch in old INPs generates ectopic Dpn+ Ase+ INPs. Images are a single, one micron plane zoomed in to the dorsal-anterior central brain (D–F).

All panels show third instar larvae; scale bar = 10 μm.

The large number of ectopic INPs could form by two mechanism: via symmetric cell divisions to expand the INP pool (i.e. one INP produces two INPs following mitosis), or via a normal asymmetric cell division to generate a self-renewed INP and a GMC that subsequently de-differentiates into an INP (similar to the role of Notchintra in promoting young INP de-differentiation into a type II neuroblast). To distinguish these alternatives we assayed mitotic INPs to determine if they performed a symmetric or asymmetric cell division. Wild type INPs are phospho-histone H3 (PH3) positive during mitosis (Figure 5A‴), and divide asymmetrically to localize the Miranda scaffolding protein and Prospero transcription factor cargo to the basal cortex (Figure 5A–A″) thereby partitioning Prospero into the GMC daughter cell, where it enters the nucleus at interphase. We find that the Notch-induced ectopic INPs also undergo asymmetric cell division, forming Miranda/Prospero crescents during mitosis (Figure 5B–B″), are PH3+ and localize Prospero to the nucleus during interphase. Furthermore, Pros+ GMCs can be identified throughout the proliferative mass (Figure 5C). Interestingly, nuclear Prospero is insufficient to drive neuronal differentiation in this population (see next section). Thus, INPs undergo asymmetric division to generate INP and GMC daughter cells, although the GMC fate does not appear to be maintained. We propose that loss of Eyeless allows Notchintra to induce GMC > INP de-differentiation.

Figure 5. Asymmetrical cell division is maintained in ectopic INP-like cells.

Figure 5

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(A–A‴) Wild-type INPs expressing OK107-gal4 UAS-GFP are GFP+ (A) and divide asymmetrically with basally localized crescents of Miranda (Mira; A′) and Prospero (Pros; A″) (white arrow marks basal crescent). The GFP+ cells marked by yellow dashed lines are in interphase (Pros+, PH3−). (B–B‴) Ectopic INP-like cells also asymmetrically localize Pros and Mira and have PH3+ chromosomes. (C–C‴) Pros+, Dpn− GMC-like cells are found in the proliferating mass generated from constitutive Notch signaling in old INPs where Eyeless is knocked down. All panels show third instar larvae; scale bar = 10 μm.

Next, we determined whether the GMCs in the EyelessRNAi Notchintra expressing population always de-differentiate or whether they can sometimes produce differentiated neurons. In wild type, the pan-neuronal Elav protein is detected in all neurons but not in neuroblasts or INPs [14–16, 36], and as expected we observe Elav+ neurons within R16B06-gal4, “flp-out,” UAS-GFP permanently marked old INP lineages (Figure 6A,B; quantified in Figure 6E). In contrast, the EyelessRNAi Notchintra population contained few or no Elav+ neurons (Figure 6C,D; quantified in Figure 6E). In addition, this population never expressed markers for differentiated neurons derived from old INPs like Twin of Eyeless (Toy) or from young INPs like Brain-specific homeobox (Bsh) (data not shown). We conclude that loss of Eyeless allows Notchintra to induce GMC > INP de-differentiation which maintains INP proliferation and nearly completely blocks neuronal differentiation (summarized in Figure 6F). This highlights the loss of competence that INPs undergo as they age, and identifies a novel function for the conserved Eyeless/Pax6 transcription factor: to block Notch signaling.

Figure 6. Notch signaling induces GMC to INP de-differentiation within old INP lineages in the absence of Eyeless.

Figure 6

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(A–B) Old INPs lineages are permanently labeled by R16B06-gal4 “flp-out” driving membrane GFP. (A–A′) Wild-type, old INP lineages labeled with GFP produce differentiated neurons marked by Elav. (B–B′) High-magnification images show Dpn+ INPs and Elav+ neurons in these GFP+ lineages.

(C–C′) Eyeless knockdown and constitutive Notch signaling in old INPs produces ectopic cells at the expense of Elav+ differentiated cells. (D–D′) High magnification images show striking loss of Elav+ cells in GFP+, old INP lineages, while many ectopic cells express Dpn+.

(E) Quantification of Elav+ neurons in GFP+ old INP lineages.

(F) Model of asymmetric cell division in wild-type and ectopic INP-like cell phenotype for old INPs responding to Notch in the absence of Eyeless.

All panels show third instar larvae; scale bar = 10 μm.

Eyeless blocks Notchintra from inducing direct target gene expression

Old INP lineages are non-responsive to the potent Notchintra mitogenic signal, at least in part due to the presence of the Eyeless/Pax6 transcription factor. Where in the Notch signaling pathway does Eyeless act? We can conclude it acts after ligand binding and proteolytic cleavage of Notch, because these steps are bypassed by overexpression of Notchintra; furthermore, we’ve shown that nuclear import of Notchintra is normal (Figure S1). Furthermore, gene expression driven by a synthetic Notch response element [37] was observed when Notchintra was expressed in old INPs, indicating that the Notchintra protein is functional (Figure S3). Does Eyeless block expression of Notch direct target genes in GMCs? There are four proposed direct Notch target genes in the larval CNS: E(spl)mγ, dpn, hey, and Myc [33, 37–40]. Here we focus on Dpn and E(spl)mγ because their expression has been detected in INPs, and Myc because it is detected in neuroblasts [33]. In contrast, Hey is detected only in a subset of post-mitotic neurons [39] and is not likely to be relevant to the GMC > INP dedifferentiation step.

In wild type, Eyeless+ old INPs normally express the Notch target genes dpn, E(spl)mγ, and the NRE-GFP Notch reporter gene whereas these genes are not expressed in GMC progeny (Figure 7A–A‴, see also Figure S3). Similarly, forced expression of Notchintra in old INPs results in Notch target gene expression in INPs but not GMCs (Figure 7B–B‴, see also Figure S3B,B′; data not shown). In contrast, forced expression of Notchintra in old INPs that lack Eyeless (EyelessRNAi Notchintra) results in Dpn expression in both INPs as well as some GMCs (Figure 7C–C‴, quantified in 7D). We conclude that Eyeless functions in GMCs to prevent Notchintra from activating target gene expression.

Figure 7. Derepression of Deadpan in old INP progeny is induced by loss of Eyeless and constitutive Notch signaling.

Figure 7

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(A–A‴) Wild-type, old INPs give berth to GMC progeny that express Pros but not Dpn. (B–B‴) Constitutive Notch signaling in old INPs and their progeny (UAS-Nintra) does not induce expression of Dpn. (C–C‴) Loss of Ey function and constitutive Notch signaling in old INPs and their progeny produce many ectopic GMC-like cells which express Pros and have derepressed Dpn. (D–F) Schematic of results. (G) Quantification of cells with nuclear Pros and Dpn per brain lobe. (A–B) White arrows show Pros+, Dpn− GMCs. (C) Arrows show ectopic Pros+, Dpn+ double positive cells. All panels show third instar larvae; scale bar = 10 μm.

Discussion

Here we report three new findings. First, we show that young INPs undergo an INP > neuroblast dedifferentiation in response elevated Notch signaling, whereas old INPs are completely resistant elevated Notch signaling; thus, old INPs lose competence to generate tumors in response to Notch signaling. Second, we show Notch signaling can induce GMC > INP de-differentiation in the absence of the late INP temporal transcription factor Eyeless/Pax6. Third, we show that Eyeless/Pax6 blocks Notch signaling by preventing transcriptional activation of several direct target genes.

Why do old INP lineages lack competence to respond to potent Notchintra signaling? A simple model is old INPs may undergo chromatin remodeling to silence Notch target genes. The SWI/SNF chromatin remodeling complex helps commit INPs to a limited proliferative potential and prevent their de-differentiation into neuroblasts [41, 42]. These factors are expressed throughout the lifespan of INPs, and may directly silence Notch target genes.

We have shown that Notchintra can promote GMCs > INP dedifferentiation, but that this effect of Notchintra can be completely blocked by the conserved Eyeless/Pax6 transcription factor. How does Eyeless block Notch signaling? One model is that Eyeless recruits the SWI/SNF complex to block activation of the Notch target genes Dpn and E(spl)mγ - which are normally expressed in INPs but not GMCs [37, 38] - preventing them from becoming transcriptionally activated by Notch signaling. Supporting this notion, the Eyeless-related Pax6 protein binds the SWI/SNF-related BAF complex to regulate the expression of neurogenic transcription factors in murine adult neural progenitors [43]. In addition, a switch in BAF subunits has been shown to direct the transition from proliferation to differentiation in mammalian neural progenitors [44], raising the possibility that both Drosophila and mammals use similar pathways to regulate progenitor choice of differentiation or proliferation.

Our finding that Eyeless can block the activity of constitutively active Notchintra signaling raises several questions. First, why does Eyeless block expression of the Notch target genes dpn and E(spl)mγ in GMCs but not INPs? An attractive model is that there is a co-factor present in GMCs but not INPs (such as Prospero) that acts with Eyeless to block Notch target gene expression. Consistent with this model is the observation that reducing Prospero from GMCs results in dedifferentiation into neuroblasts that express the Notch target genes dpn, E(spl)mγ, and Myc [16, 45–47]. Second, can misexpression of Notch target genes bypass the tumor suppressor function of Eyeless? We misexpressed the Notch target genes dpn, E(spl)mγ, and Myc in old INPs, but we detected no ectopic INPs (data not shown); perhaps two or more target genes, or a currently unknown Notch target gene, are required to induce a GMC>INP dedifferentiation. Third, why doesn’t loss of Eyeless alone trigger GMC dedifferentiation? One possibility is that endogenous Notch signaling is too low to induce dedifferentiation either due to absence of a Notch pathway component or lack of access to ligand. Fourth, can misexpression of Eyeless block Notchintra-induced young INP > neuroblast dedifferentiation? We attempted to answer this question by misexpressing Notchintra and Eyeless together in young INPs (R9D11-gal4 UAS-GFP UAS-Notchintra UAS-Eyeless). Surprisingly, the young INPs had no detectable Eyeless protein (Figure S4), although they had high GFP levels and despite UAS-GFP and UAS-Eyeless being coexpressed, due to an unknown mechanism blocking Eyeless translation in young INPs. Consequently, the expected “neuroblast tumor” phenotype was observed and we could not determine the role of Ey in blocking young INP tumors. The mechanism preventing Eyeless protein expression is an interesting area for future investigation, particularly to determine if a similar mechanism is used to regulate its mammalian ortholog, Pax6.

Notch signaling is well conserved and has been shown to initiate diverse cell fate outcomes in a context dependent fashion. For example, constitutively active Notch signaling in hematopoietic stem cells (HSCs) in mouse bone marrow is sufficient to generate extra-thymic T cells [48], but the competence to respond to Notch in these cells requires functional pre-T cell receptor (TCR) signaling. Furthermore, restoration of competence to respond to Notch in TCR mutant HSCs with a TCR transgene and active Notch1 signaling potentiates these tissues to form T cell leukemia [48]. In addition, the transcription factor Ikaros has been shown to control the availability of Notch targets genes during T cell differentiation and loss of Ikaros generates T cell leukemias in mice [49]. The tumor suppressor function of Ikaros in controlling the response to Notch signaling in T cells is strikingly similar to the function of Eyeless we report here. Similar to Type II neuroblasts, T cell precursors rely on endogenous levels of Notch signaling to properly specify progeny, but are also sensitive to Notch as a mitogen, and must maintain homeostatic proliferation through the careful regulation of Notch signaling [49]. In the case of pre-T cells, it appears that competence to respond to Notch is established by TCR expression, and final T cell differentiation requires Notch signaling provided in the thymus, spatially controlling T cell development. Thus, in Drosophila as well as mammalian tissues, Notch signaling must be precisely regulated to ensure normal development. In addition, it is clear that cells also regulate their competence to respond to Notch, enabling multiple, context-dependent outcomes from a single extrinsic cue.

Eyeless and its mammalian ortholog Pax6 were initially defined as master regulators of eye development, and have since been shown to play essential roles in other cell types [50]. Eyeless was recently identified as a temporal identity factor in INPs, and is essential for proper development of the Drosophila adult central complex [23]. Pax6 expression is a reliable marker of mammalian cortical progenitors and is under both spatial and temporal control. Both Pax6/Eyeless transcription factors and Notch signaling are well conserved between Drosophila and mammals. Understanding how these factors interact to regulate progenitor competence may provide insight into mammalian neural development and tissue repair following injury or disease.

Methods

Fly genetics

Mutant larvae were generated in vial collections incubated at 28–30°C using 3–5 day old females. Larvae were collected at third instar for dissection based on a combination of timing and morphology.

Immunohistochemistry

Larval brains were fixed in 4% paraformaldehyde in PBST (phosphate-buffered saline plus 0.3% Triton-X100; Sigma Aldrich) for 25 min at room temperature. Normal goat and donkey serum (5%) in PBST was used as a pre-staining blocking solution and staining buffer. Primary antibody staining was performed overnight at 4°C. The following primary antibodies were used: chicken antibody to GFP (1:2000; Aves Laboratories, Tigard, OR), rat antibody to Dpn (1:50; Doe lab), rabbit antibody to Ase (1:2000; C.-Y. Lee lab, Univ. Michigan), guinea pig antibody to D (1:500; J. Nambu), rabbit antibody to Ey (1:3500; U. Walldorf), guinea pig antibody to Mira (1:1000; Doe lab), mouse antibody to Pros (1:1000; Doe lab), guinea pig antibody to Toy (1:500; U. Walldorf) and mouse antibody to Notchintra (1:50; Developmental Studies Hybridoma Bank, Iowa City, Iowa). Secondary antibody staining was performed at room temperature for two hours (1:500; Molecular Probes, Eugene, OR, or Jackson Immunoresearch, West Grove, PA). After staining, brains were kept at 4°C in Vectashield (Vector Laboratories, Burlingame, CA) prior to imaging.

Imaging and analysis

Images were obtained using a Zeiss LSM710 confocal microscope. Image processing and analysis was performed in FIJI [51].

Supplementary Material

supplement

Acknowledgments

We would like to thank Sen-Lin Lai, Ingo Braasch, Julia Ganz, Bruce Bowerman, and the anonymous reviewers for comments that improved this manuscript and Sen-Lin Lai for mentorship. This work was supported by an NIH Genetics training grant T32 GM007413 (DRF) and the Howard Hughes Medical Institute, where CQD is an Investigator.

Footnotes

Author Contributions

DRF did all experiments and co-wrote the manuscript; OAB participated in the characterization of R16B06-gal4 and the design of the study; CQD guided the project and co-wrote the manuscript.

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