Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling

Abstract

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine filamentous actin (F-actin) structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of myocardin-related transcription factor, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G protein-coupled receptor Smog, G protein α_q subunit, Rho1 guanosine triphosphatase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand folded gastrulation to regulate Gα_q-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gα_q-Rho1 signaling axis derived from astrocytes, an NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

Fig. 1.. F-actin structures and dynamics during qNSC reactivation.

(A) Schematic diagram of qNSC reactivation in the Drosophila larva brain. (B) Super-resolution imaging of F-actin structure achieved by ExM-SIM microscopy in Drosophila larval brain at 6-hour ALH. GFP-Moe (white) marks F-actin. Blue patches, neuropil region. (C) High magnification of qNSC from (B). Yellow arrows, F-filaments; red arrows, F-actin patches. (D and E) Orthogonal view (xz axis, D; yz axis, E) of qNSC in (C). Insert of (D) shows sections used in (D) (xz axis in yellow) and (E) (yz axis in red). (F) 3D reconstruction of (D). (G) Live imaging stills of F-actin dynamics (GFP-Moe, black) in the qNSC at 6-hour ALH. Time, mm:ss. F-actin patches, red arrows; black arrows, retrograde flow of F-actin patches in the protrusion. (H) Stills of live imaging of F-actin dynamics (GFP-Moe) in (G) in thermal theme. H, high in thermal scale; L, low in the thermal scale; white arrows, F-actin patches; open arrow, protrusion initiation segment (PIS) region. (I) Time-lapse images of GFP-utABD (black) driven by grh-GAL4 were taken before and after laser ablation. Dashed square, ablated area; red arrows, soma F-actin patches; brackets, the position of primary protrusion of qNSCs. (J) Quantification graph of soma F-actin patches of qNSCs before and after laser ablation. Before laser ablation: 16.0 ± 2.8, n = 14; after laser ablation: 7.2 ± 1.7, n = 14. Student’s t test is used for statistics. ****P < 0.0001. The means of analyzed phenotypes were shown above each column. (K) Live imaging of GFP-utABD (black, F-actin) driven by grh-GAL4. Time, hh:mm. Red arrows, F-actin patches; black arrows, apical F-actin; white arrows, absence of F-actin; bracket, primary protrusion. Dashed lines indicate the boundary of neuropil. Scale bars, 10 μm (B and K), 5 μm (C, G, and I), and 2 μm in (H).

Fig. 2.. The actin polymerization factor Dia/Formin is required for qNSC reactivation and brain development.

(A) Larval NSCs were labeled with EdU and Dpn at 24-hour ALH. Yellow arrows and dashed circles point to EdU-negative NSCs. White arrows, EdU-positive NSCs. White arrows, NSCs with cytokinesis defects (large multinucleated cells). (B) The quantification graph of EdU-negative NSCs in (A). Control (yw): 17.9 ± 4.4, n = 10; dia¹: 42.7 ± 10.9, n = 11; dia^1/Df: 47.9 ± 18.0, n = 12; dia⁵: 59.9 ± 13.1, n = 11; dia^5/Df: 69.5 ± 21.6, n = 15. (C) NSCs were stained for Dpn and Miranda (Mira). White arrows point to primary protrusion of qNSCs. (D) Quantification graph of the percentage of NSCs carrying primary protrusion. Control (yw): 5.8 ± 2.4, n = 10; dia¹: 15.1 ± 3.7, n = 8; dia⁵: 16.3 ± 2.8, n = 11. (E) The quantification graph of brain volume. Control (yw): 28.9 ± 10.2, n = 13; dia¹: 8.2 ± 2.9, n = 13; dia⁵: 10.8 ± 4.0, n = 12. (F) The size of larval brains (DNA, gray). (G) Top and middle rows: Proliferating NSCs (Dpn, cyan; EdU, red) in control (β-gal^RNAi) or dia-KD larval brains (driven by grh-GAL4). Yellow arrows, EdU-negative NSCs; white open arrows, EdU⁺ proliferative NSCs. Bottom row: NSCs labeled with Dpn/Mira. White solid arrows, primary protrusion of qNSCs. (H) The quantification graph of EdU-negative NSCs. Control (β-gal^RNAi): 8.0 ± 4.7, n = 16; dia^RNAi-1: 20.1 ± 3.3, n = 12; dia^RNAi-2: 24.7 ± 6.7, n = 13. (I) Quantification graph of the percentage of NSCs retaining primary protrusion. Control (β-gal^RNAi): 5.5 ± 2.1, n = 14; dia^RNAi-1: 13.4 ± 3.3, n = 11; dia^RNAi-2: 15.5 ± 3.4, n = 13. One-way analysis of variance (ANOVA) is used for statistics. ****P < 0.0001; ***P < 0.001; **P < 0.01. The means of analyzed phenotypes were shown above each column. Scale bars, 50 μm (F), 10 μm [A and G (top row)], and 5 μm [C and G (bottom row)].

Fig. 3.. Gα_q-Rho1 signaling promotes qNSC reactivation.

(A and B) Larval NSCs were labeled with EdU and Dpn at 24-hour ALH. Yellow arrows and dashed circles, EdU-negative NSCs; white arrows, EdU-positive NSCs. Bottom row: NSCs were labeled with Dpn and Mira. White arrows, protrusion of NSCs; white arrows, NSCs with cytokinesis defects. (C) Quantification graph of EdU-negative qNSCs in (A) and (B). Control (yw): 14.9 ± 7.1, n = 17; Gα_q^221C: 36.9 ± 8.8, n = 15; Control (β-gal^RNAi): 8.9 ± 4.6, n = 23; rho1^RNAi-1: 28.7 ± 9.3, n = 11; rho1^RNAi-2: 32.5 ± 7.7, n = 14; Rho1^N19: 25.8 ± 9.2, n = 15. (D) Quantification graph of NSCs retaining protrusion in (A) and (B). Control (yw): 6.3 ± 1.3, n = 21; Gα_q^221C: 17.9 ± 2.7, n = 18; Control (β-gal^RNAi): 5.9 ± 2.1, n = 20; rho1^RNAi-1: 17.5 ± 3.4, n = 11; rho1^RNAi-2: 16.1 ± 3.0, n = 10; Rho1^N19: 14.6 ± 2.4, n = 10. (E) Quantification graph of EdU-positive NSCs at 6-hour ALH. Control (β-galRNAi): 20.5 ± 3.6, n = 14; Gα_q^Q203L: 33.8 ± 5.3, n = 18; Rho1^GFP: 27.0 ± 5.8, n = 11. (F) Proliferating NSCs (EdU, red; Dpn, cyan) in control (β-gal^RNAi), Gα_q^Q203L, and GFP-Rho1 larval brains driven by grh-GAL4 at 6-hour ALH. White arrows, EdU⁺ NSCs. (G) qNSCs (Dpn, cyan) in control (β-gal^RNAi) and Gα_q-KD larval brains under grh-GAL4 at 6-hour ALH. F-actin (rhodamine phalloidin) marks protrusions. DiaRBD-GFP (green) marks active Rho1. (H) Quantification graph of DiaRBD-GFP levels in the protrusion in (G). Control (β-gal^RNAi): 0.68 ± 0.22, n = 23; Gα_q^RNAi-1: 0.31 ± 0.21, n = 13. (I) Proliferating NSCs (EdU, red; Dpn, cyan) in larval brains at 24-hour ALH. UAS-GFP, a negative control for suppression effect. Yellow arrows and dashed circles, EdU-negative NSCs. (J) Quantification graph of EdU-negative NSCs in (I). Control (β-gal^RNAi),GFP: 7.9 ± 4.0, n = 15; Gα_q^RNAi-1, GFP: 20.5 ± 7.4, n = 16; Gα_q^RNAi-1, Rho1^GFP: 8.5 ± 3.3, n = 16; Rho1^GFP: 10.0 ± 4.1, n = 10. One-way ANOVA (C, D, E, and J) and unpaired Student’s t test (H) are used for statistics. ***P < 0.001; **P < 0.01. The means of analyzed phenotypes were showed above each column. Scale bars, 10 μm [A, B (top row), F, and I), 5 μm [A, B (bottom row), and G (bottom row)]. ns, no significance.

Fig. 4.. Gα_q-Rho1-Dia signaling promotes NSC reactivation via actin cytoskeleton.

(A) Dia protein (red) in qNSCs (Dpn, blue; mCD8-GFP, green) in control (β-gal^RNAi), Gα_q-KD, and rho1-KD larval brains under the control of grh-GAL4 driver at 6-hour ALH. Yellow arrows, neck region of qNSC; brackets, primary protrusion. (B) Quantification graph of Dia protein levels along the soma and protrusion of qNSC in various RNAi transgenes driven by grh-GAL4 driver. (C) F-actin patches (GFP-utABD, black) in control (β-gal^RNAi), Gα_q-KD, rho1-KD, and dia-KD qNSCs under the control of grh-GAL4 driver at 6-hour ALH. Solid lines outline the soma of NSCs. Dashed lines indicate the boundary of neuropil. Brackets, primary protrusion. (D) Quantification graph of F-actin patches in the soma of qNSCs in various genotypes of (C). Control (β-gal^RNAi): 16.4 ± 3.4, n = 11; dia^RNAi-2: 8.2 ± 1.2, n = 11; rho1^RNAi-2: 8.0 ± 1.8, n = 16; Gα_q^RNAi-2: 7.9 ± 1.9, n = 13. (E) F-actin (rhodamine phalloidin, red and black) in qNSCs (Dpn, blue; mCD8-GFP, green) under grh-GAL4 driver at 6-hour ALH. Red arrows, F-actin patches. Yellow lines mark intact qNSCs. (F) Quantification graph of F-actin patches in the soma of qNSCs in various genotypes of (G). Control (β-gal^RNAi),GFP: 6.6 ± 2.4, n = 29; dia^RNAi-1, GFP: 4.6 ± 1.8, n = 29; Gα_q^RNAi-1, GFP: 3.5 ± 1.6, n = 25; Gα_q^RNAi-1, Dia^EGFP: 7.0 ± 22, n = 34. (G) Quantification graph of EdU-negative qNSCs in various genotypes of (F). Control (β-gal^RNAi),GFP: 8.3 ± 3.8, n = 10; Gα_q^RNAi-1, GFP: 18.6 ± 7.5, n = 18; Gα_q^RNAi-1, Dia^EGFP: 7.6 ± 3.1, n = 19; rho1^RNAi-1,GFP: 20.8 ± 7.3, n = 18; rho1^RNAi-1, Dia^EGFP: 9.7 ± 2.8, n = 10; Dia^EGFP: 8.8 ± 4.5, n = 13. (H) Proliferating NSCs (EdU, red; Dpn, cyan) in larval brains of various transgenes driven by grh-GAL4 at 24-hour ALH in (G). Yellow arrows and dashed circles point to EdU-negative NSCs. One-way ANOVA is used for statistics. ****P < 0.0001; ***P < 0.001; **P < 0.01. The means of analyzed phenotypes were shown above each column. Scale bars, 10 μm (H) and 5 μm (A, C, and E).

Fig. 5.. Dia promotes qNSC reactivation and brain development via transcription factor Mrtf.

(A and B) Larval NSCs were labeled with EdU and Dpn at 24-hour ALH. Yellow arrows and dashed circles, EdU-negative NSCs. (C) Quantification graph of EdU-negative NSCs in (A) and (B). Control(yw): 17.9 ± 5.2, n = 11; mrtf^D7: 51.4 ± 10.7, n = 14; mrtf^{D7/Df(3L)BSC412}: 44.2 ± 9.2, n = 10; Control (β-gal^RNAi): 9.7 ± 4.1, n = 15; mrtf^RNAi-1: 20.2 ± 8.7, n = 13; mrtf^RNAi-2: 29.0 ± 9.0, n = 11. (D) NSCs were labeled with Mrtf, Dpn, and Mira. White squares, the region for high magnification. Solid lines, NSCs. Dashed circles, nucleus. (E) Quantification graph of nuclear Mrtf expression (top) and ratio of nuclear Mrtf to cytoplasmic Mrtf (bottom). Top graph: Control (yw): 0.66 ± 0.2, n = 16; dia⁵: 0.44 ± 0.14, n = 15; mrtf^D7: 0.46 ± 0.14, n = 16. Bottom graph: Control (yw): 0.71 ± 0.06, n = 16; dia⁵: 0.59 ± 0.07, n = 15; mrtf^D7: 0.56 ± 0.06, n = 19. (F) Larval NSCs were labeled with EdU and Dpn at 24-hour ALH. Yellow arrows and dashed circles, EdU-negative NSCs. (G) Quantification graph of EdU-negative NSCs in (E). Control (yw): 14.6 ± 5.0, n = 11; dia^1/5, grh>GFP: 42.7 ± 9.6, n = 13; dia^1/5, grh>Dia^EGFP: 17.7 ± 6.1, n = 10; dia^1/5, grh>Mrtf: 18.5 ± 6.4, n = 10; grh>Mrtf: 7.8 3.0, n = 10. (H) The size of larval brains (DNA, gray) at 96-hour ALH. Dashed circles, single brain lobe. (I) Quantification graph of brain size from Control (yw): 10.1 ± 2.3, n = 11; dia^1/5, grh>GFP: 7.1 ± 1.6, n = 10; dia^1/5, grh>Dia^EGFP: 10.6 ± 2.1, n = 11; dia^1/5, grh>Mrtf: 10.1 ± 1.7, n = 11. (J) Quantification graph of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in 24-hour ALH brains from control (yw) and mrtf^Δ7. After normalization against yw control (with SD): mrtf, 0.52 ± 0.08–fold; actin5C, 0.55 ± 0.03–fold; Hsp23, 1.00 ± 0.11–fold. One-way ANOVA is used for statistics. ***P < 0.001. The means of analyzed phenotypes were shown above each column. ***P < 0.001; **P < 0.01. The means of analyzed phenotypes were shown above each column. Scale bars, 50 μm (H), 10 μm (A, B, D, and F), and 5 μm (D) (for images with high magnification).

Fig. 6.. GPCR Smog promotes qNSC reactivation via Gα_q-Dia signaling.

(A) Proliferating NSCs (EdU, red; Dpn, cyan) in control (β-gal^RNAi) and smog-KD larval brains under the control of grh-GAL4 driver at 24-hour ALH. Yellow arrows and dashed circles point to EdU-negative NSCs. (B) Quantification graph of EdU-negative NSCs of A at 24-hour ALH. Control (β-gal^RNAi): 7.9 ± 3.8, n = 14; smog^RNAi-1: 20.5 ± 8.1, n = 13; smog^RNAi-2: 25.1 ± 8.7, n = 11. (C) Quantification graph of EdU-negative NSCs in control (yw)– and smog-mutant flies in (D). Control (yw): 16.7 ± 4.9, n = 14; smog^KO: 41.6 ± 10.0, n = 11; smog^{KO/Df(2L)Exel9062}: 33.9 ± 7.6, n = 12. (D) Proliferating NSCs (EdU, red; Dpn, cyan) in larval brains at 24-hour ALH. (E) Smog::GFP localization (green) in the qNSCs [Dpn, blue; red fluorescent protein (RFP); red] in the larval brains at 6-hour ALH. Dashed lines, intact qNSCs; brackets, primary protrusions. (F) Quantification graph of Smog::GFP levels in the soma and protrusion of qNSCs. Soma: 0.19 ± 0.06, n = 10; protrusion: 0.36 ± 0.09, n = 10. (G) Dia protein (red) in control (β-gal^RNAi) and smog-KD qNSCs (Dpn, blue; mCD8-GFP, green) under the control of grh-GAL4 driver at 6-hour ALH. (H) Quantification graph of Dia protein levels along the soma and protrusion in control (β-gal^RNAi) and smog-KD qNSCs at 6-hour ALH in (G). (I) Quantification graph of EdU-negative NSCs in J. Control (β-gal^RNAi), GFP: 7.5 ± 3.7, n = 23; smog^RNAi-1, GFP: 28.5 ± 9.2, n = 13; smog^RNAi-1, Dia^EGFP: 13.8 ± 8.3, n = 13; smog^RNAi-2, GFP: 20.6 ± 5.1, n = 12; smog^RNAi-2, Gα_q^WT: 8.3 ± 3.3, n = 13 Gα_q^WT: 8.4 ± 2.8, n = 11. (J) Proliferating NSCs (EdU, red; Dpn, cyan) in larval brains at 24-hour ALH. Yellow arrows and dashed circles, EdU-negative NSCs. One-way ANOVA (B, C, and L) and unpaired Student’s t test (F and H) were used for statistics. ****P < 0.0001; *P < 0.05. The means of analyzed phenotypes were shown above each column. Scale bars, 10 μm (A, D, and J) and 5 μm (E and G).

Fig. 7.. Astrocytes, a new NSC niche, secrete Fog via dynamin to promote qNSC reactivation.

(A and B) fog mRNA expression in larval brains from the dataset of scRNA-seq (refer to Materials and Methods). (C) Astrocyte (Repo and alrm>GFP) in larval brain at 24-hour ALH. (D and E) Larval NSCs (EdU and Dpn) at 24-hour ALH. Yellow arrows and dashed circles, EdU-negative NSCs. (F) Quantification graph of EdU-negative NSCs in (C) and (D). repo>control: 7.7 ± 3.8, n = 17; repo>fog^RNAi-1: 19.3 ± 5.8, n = 15; repo>fog^RNAi-2: 24.3 ± 8.4, n = 10; alrm>control: 8.1 ± 4.8, n = 10; alrm>fog^RNAi-1: 28.5 ± 9.1, n = 14; alrm>fog^RNAi-2: 29.6 ± 9.6, n = 15. (G) Quantification graph of Fog levels in astrocytes: control, 0.88 ± 0.29, n = 15; alrm>fog^RNAi-1, 0.43 ± 0.15, n = 14. (H) Fog levels in central brain region: control, 3.57 ± 0.83, n = 5; alrm>fog^RNAi-1, 1.39 ± 0.58, n = 5. (I) Fog levels in neuropil region: control, 4.27 ± 1.05, n = 5; alrm>fog^RNAi-1, 4.73 ± 1.87, n = 5. (J) Larval brains were labeled with Fog, Pros, and GFP at 24-hour ALH. White squares, images with higher magnification on the right; white lines, brain lobe outlines; yellow lines, neuropil outlines; asterisks, central brain regions; dashed outlines, astrocytes. (K) Larval brains were labeled with Fog and Pros at 24-hour ALH. (L) Quantification graph of Fog levels in astrocytes in control: 0.64 ± 0.20, n = 43; alrm>Shi^K44A: 0.48 ± 0.16, n = 22. (M) Larval NSCs (EdU and Dpn) at 24-hour ALH. (N) Quantification graph of EdU-negative NSCs in (M). alrm>control: 8.3 ± 4, n = 12; alrm>Shi^K44A: 22.7 ± 5.5, n = 10. Yellow arrows and dashed circles, EdU-negative NSCs. One-way ANOVA (F) and two-tailed unpaired Student’s t test (G, H, I, L, and N) are used for statistics. ****P < 0.0001; **P < 0.01. The means of analyzed phenotypes were shown above each column. Scale bars, 10 μm (D, E, J, K, and M) and 5 μm (J) (for images with high magnification).

Fig. 8.. Niche Fog promotes qNSC reactivation via GPCR Smog-Gα_q-Dia pathway in NSCs.

(A) qNSCs at 6-hour ALH under the control of alrm-GAL4 driver were stained for Dia, Dpn, and F-actin. White arrows, neck region of qNSC; brackets, primary protrusion marked by F-actin. (B) Quantification graph of Dia levels along the soma and protrusion in qNSCs at 6-hour ALH in control (β-gal^RNAi) and fog-KD in astrocyte-like glia. (C) Proliferating NSCs (EdU, red; Dpn, cyan) in various genotypes at 24-hour ALH. (D) Quantification graph of EdU-negative NSCs under the control of grh-GAL4 and alrm-GAL4 in various genotypes in (C). Control: 8.3 ± 2.8, n = 10; GFP, fog^RNAi-1: 22.7 ± 8.9, n = 17; Dia^EGFP, fog^RNAi-1: 11.7 ± 2.8, n = 15; Dia^EGFP: 9.5 ± 3.3, n = 10. (E) Proliferating NSCs (EdU, red; Dpn, cyan) at 24-hour ALH. (F) Quantification graph of EdU-negative NSCs under the control of grh-GAL4 and alrm-GAL4 in various genotypes in (E). Control: 6.9 ± 2.1, n = 18; GFP, fog^RNAi-2: 23.8 ± 8.1, n = 15; Gα_q^WT, fog^RNAi-2: 9.3 ± 3.6, n = 16; Rho1^GFP, fog^RNAi-2: 7.3 ± 3.3, n = 15. Gα_q^WT: 8.7 ± 4.4, n = 15; Rho1^GFP: 8.6 ± 4.3, n = 13. (G) Proliferating NSCs (EdU, red; Dpn, cyan) in various genotypes at 24-hour ALH. (H) Quantification graph of EdU-negative NSCs under the control of alrm-GAL4 driver in various genotypes in (G). Control: 9.2 ± 2.2, n = 8; Gα_q^WT, fog^RNAi-2: 18.8 ± 6.8, n = 12; Rho1^GFP, fog^RNAi-2: 19.7 ± 8.7, n = 11; Dia^EGFP, fog^RNAi-1: 21.9 ± 9.3, n = 10; fog^RNAi-1: 21.6 ± 11.2, n = 11. One-way ANOVA was used for statistics. ****P < 0.0001; ***P < 0.001; **P < 0.01. The means of analyzed phenotypes were showed above each column. Scale bars, 10 μm (C, E, and G) and 5 μm in (A).

Fig. 9.. A working model.

(A) Fog secreted from astrocytes reactivates qNSCs for asymmetric cell division of NSC to give rise to new neurons. F-actin forms filaments and patches in qNSCs. (B) In the primary protrusion of qNSC, GPCR receptor Smog activated by Fog ligand promotes Gα_q-Rho1-Dia signaling in the protrusion, resulting in the retrograde flow of F-actin patches. In the mutants, active Rho1 and Dia cannot transport to the primary protrusion, resulting the reduction of retrograde flow of F-actin patches, leading to the defect of F-actin dynamics in the soma [please see (C)]. (C) In the soma, F-actin patches from primary protrusion promote robust F-actin polymerization and dynamics to consume G-actin, the monomer of actin. Mrtf can translocate to nucleus and promotes actin transcription to feedback to F-actin dynamics and the other unknown target genes that are required for cell proliferation. In the mutants, F-actin amount is reduced, probably because of the defect of retrograde flow of F-actin patches in the primary protrusion; therefore, more G-actin monomers may bind to Mrtf and inhibit Mrtf from nuclear translocation.