Aging Neural Progenitors Lose Competence to Respond to Mitogenic Notch Signaling

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.

Figure 1. Old INPs lose competence to respond to Notch

(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-Notch^intra 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-Notch^intra) standardized expression of UAS-Notch^intra. 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-Notch^intra to standardize UAS-Notch^intra 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.

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

(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-eyeless^RNAi 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-eyeless^RNAi, UAS-Notch^intra) 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.

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

(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-Notch^intra). (C–C′) When Eyeless knockdown is coupled with constitutive Notch signaling in old INPs (R16B06-gal4, UAS-eyeless^RNAi, UAS-Notch^intra), 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.

Figure 4. Notch^intra in old INPs lacking Eyeless generates ectopic INPs and GMCs

(A–B) Constitutive Notch signaling in Eyeless-negative old INPs (OK107-gal4, UAS-eyeless^RNAi, UAS-Notch^intra) 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.

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

(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.

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

(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.

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

(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-N^intra) 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.