A chromatin remodelling SWI/SNF subunit, Snr1, regulates neural stem cell determination and differentiation

Abstract

Coordinated spatio-temporal regulation of the determination and differentiation of neural stem cells is essential for brain development. Failure to integrate multiple factors leads to defective brain structures or tumour formation. Previous studies suggest changes of chromatin state are needed to direct neural stem cell differentiation, but the mechanisms are unclear. Analysis of Snr1, the Drosophila orthologue of SMARCB1, an ATP-dependent chromatin remodelling protein, identified a key role in regulating the transition of neuroepithelial cells into neural stem cells and subsequent differentiation of neural stem cells into the cells needed to build the brain. Loss of Snr1 in neuroepithelial cells leads to premature neural stem cell formation. Additionally, loss of Snr1 in neural stem cells results in inappropriate perdurance of neural stem cells into adulthood. Snr1 reduction in neuroepithelial or neural stem cells leads to the differential expression of target genes. We find that Snr1 is associated with the actively transcribed chromatin region of these target genes. Thus, Snr1 likely regulates the chromatin state in neuroepithelial cells and maintains chromatin state in neural stem cells for proper brain development.

Keywords: Drosophila; Differentiation; Neural stem cell; Neuroblast; Neuroepithelial cells; Optic lobe; SMARCB1; SWI/SNF complex; Snr1.

Fig. 1.

Snr1 is expressed in neuroepithelial and neuroblast cells in the optic lobe, and is required for optic lobe development. (A) Diagram of the Drosophila third instar larval brain. Optic lobe neuroepithelial cells (NE) represented in blue and medulla neuroblasts (NBs) in green. CB, central brain; OL, optic lobe; VNC, ventral nerve cord. Gray square indicates the plane of cross-section shown in B. Created with BioRender.com. (B) Diagram of a cross-section of the optic lobe showing the transition zone (TZ), ganglion mother cells (GMCs) and neurons located in the medulla. Created with BioRender.com. (C,C′) Neuroblasts expressing GFP (green). (D,D′) GFP (green) and Snr1^RNAi are expressed in all neuroblasts. The OL is outlined in yellow in C,D. Scale bars: 100 µm in C-D′. (E-E″) Larval optic lobe imaged in cross-section showing the transition zone (bracket) from neuroepithelial cells to neuroblasts. The transition zone is identified by a change in cell shape, as marked by DE-cadherin (DE-cad). Scale bar: 20 µm. (F-F″) Wild type w¹¹¹⁸ brain. (G-G″) Snr1 knocked down in neuroepithelial cells (c855a) by Snr1^RNAi. (H-H″) Snr1 knocked down in neuroblasts (insc) by Snr1^RNAi. (F-H″) Neuroepithelial cells marked by PatJ (green), neuroblasts marked by Miranda (Mira) (red). In F″, G″ and H″, DAPI is in blue and Discs Large (Dlg) is in white. Scale bars: 50 µm. (I) Neuroepithelial volume in control (66,759±15,201 µm³) and c855a>Snr1^RNAi (19,138±8075 µm³) brains. P=1e-5, n=10. (J) Optic lobe neuroblast numbers in control (416±53) and insc>Snr1^RNAi (358±58) brains. P=0.03, n=10. (K) Central brain neuroblasts in control (85±4) and insc>Snr1^RNAi (91±5) brains. P=0.07, n=10. *P<0.05, ***P<0.001; ns, not significant.

Fig. 2.

Snr1 is required for the transition from neuroepithelial cells to neuroblasts. (A) Timeline of larval development showing timing of clonal induction and dissections after larval hatching (ALH). (B-C′) Snr1 mutant clones in the optic lobe have altered location and morphology. (B,B′) Control MARCM clone at surface of brain imaged in cross-section. (C,C′) Snr1^R3 MARCM clone located deeper inside the brain showing ectopic expression of Deadpan (Dpn; arrowheads). Cell junctions are marked by DE-cad, neuroblasts are marked by Dpn, MARCM clones are marked by GFP and GMCs are marked by Pros. (D-E″) Snr1 mutant clones deep in the medulla express markers for neuroepithelial cells and neuroblasts. (D-D″) Control optic lobe MARCM clone marked by GFP. Clone is outlined by a white dashed line. (E-E″) Snr1^R3 optic lobe MARCM clone marked by GFP. Clone is outlined by a white dashed line. Neuroepithelial cells marked by PatJ (red) and neuroblasts marked by Dpn (white). (F-F′) Snr1^R3 clone cells on the surface of the brain are smaller (arrowheads) and express neuroepithelial (NE) cell marker. Neuroepithelial cell junctions are marked by PatJ (magenta) and MARCM clones are marked by GFP (green). (G-H‴) MARCM clones imaged in cross-section at mid third instar stage. Brackets indicate transition zone. Arrows indicate ectopic Dpn⁺ cells. Scale bars: 20 µm.

Fig. 3.

Snr1 mutant cells maintain expression of neuroblast marker and mis-express neuronal marker. (A) Twin spot clone labelled with RFP, DNA marked with DAPI and neuroblasts marked with Deadpan (Dpn). Snr1^R3 clones are negative for RFP (outlined with a yellow dashed line) and the corresponding wild-type clones are RFP positive (outlined with a white dashed line). In merged images, RFP is in red and Dpn is in white. (B) Expression of HA-Snr1 partially rescued clone morphology and ectopic Dpn expression in Snr1^R3 clones. MARCM clones express GFP (green). In the merged image, Dpn is shown in white, HA in red and Dlg in blue. (C) Snr1^R3 MARCM clone with disrupted Elav expression (magenta). Scale bars: 20 µm in A-C. (D) Volume of control (9894±4011 µm³) and Snr1^R3 (3119±1093 µm³) clones. P=1e-3, n=9. (E) Number of Dpn⁺ cells per volume of control (1.45e-3±3.72e-4) and Snr1^R3 (2.38e-3±5.98e-4) clones. P=8e-4, n=9. (F) Control FLP-out clone labelled with EdU. (G) FLP-out clone expressing Snr1^RNAi labelled with EdU. (F,G) Mitotic cells labelled with phospho-histone H3 (pH3). Clones are outlined with white dashed lines. Scale bars: 20 µm in F,G. (H) Percentage of EdU⁺ cells in control (29±6) and Snr1^RNAi (18±7) clones. P=1e-3, n=10. **P<0.01, ***P<0.001.

Fig. 4.

Cells expressing the neuroblast marker Deadpan are present in Snr1^R3 clones in the adult optic lobe. Clones generated at 31 h after larval hatching (ALH). (A-E) Control MARCM clones marked with GFP. (A′-E′) Higher magnification images of region outlined in E. (F-J) Snr1^R3 MARCM clone marked with GFP. (F′-J′) Higher magnification images of region outlined in J. In the merge, DAPI is in blue, GFP is in green, Dpn is in white and Snr1 is in red. Scale bars: 50 µm in E,J; 20 µm in E′,J′.

Fig. 5.

Notch signalling is reduced in Snr1 knockdown cells. (A) Combined UMAP of optic lobe neuroepithelial cells and neuroblasts from control brains and brains with knockdown of Snr1 in neuroepithelial cells. (B) UMAP showing distribution of optic lobe cells from the control brains (CTRL, red) and the Snr1 knockdown brains (KD, blue). (C,D) UMAPs showing expression of Notch targets E(spl)m 4-BFM and E(spl)mγ-HLH in neuroepithelial cells and neuroblasts represented in A and B. (E) Average expression of E(spl) genes in control and the Snr1 knockdown in neuroepithelial cells.

Fig. 6.

E(spl)mγ expression is reduced in neuroepithelial and neuroblasts in Snr1 knockdown cells. (A-A″) E(spl)mγ-GFP expression in the optic lobe medulla at the mid 3rd instar stage. E(spl)mγ-GFP is expressed in neuroepithelial cells (NE, outlined with solid white lines). Expression is reduced in the transition zone (TZ) and re-expressed in neuroblasts (NB, outlined with yellow dotted lines). (B-B″) Snr1^RNAi clone (outlined with a yellow dashed line) has reduced E(spl)mγ-GFP and Dpn expression. (C-C″) E(spl)mγ-GFP expression at the mid 3rd instar stage in cross-section. (D-D″) The Snr1^RNAi clone prematurely extruded from neuroepithelium. (C,C′,D,D′) The transition zone is marked by the bracket. (E-E″) E(spl)mγ-GFP expression in late 3rd instar brain. (F-F″) E(spl)mγ-GFP expression in Snr1 knockdown clone (outlined with a yellow dashed line). Merged images show GFP in green, Dpn in white, DAPI in blue and the clone in red. Scale bars: 20 µm.

Fig. 7.

Expression of Broad (br) and Eip93F is reduced in Snr1 knockdown cells. (A-D) Violin plots showing expression of Snr1 (log2FC=-1.17, P=6e-11), br (log2FC=-1.94, P=1e-8), Eip93F (log2FC=-2.34, P=2e-11) and inscuteable (insc) (P=0.10) in neuroblasts from control brains and from brains expressing Snr1^RNAi in neuroblasts. ***P<0.001; ns, not significant. (E) FLP-out control clones. (E′) Higher magnification images of the region outlined in E. Clone is outlined by a yellow dashed line. (F) FLP-out clones expressing Snr1^RNAi. (F′) Higher magnification image of the region outlined in F. Clone is outlined by a yellow dashed line. In merged images in E-F′, Snr1 is shown in green, Broad-Core (Br-C) is in white, RFP is in red and DAPI is in blue. (G) FLP-out control clones. (G′) Higher magnification image of region outlined in G. Clone is outlined by a yellow dashed line. (H) FLP-out clones expressing Snr1^RNAi. (H′) Higher magnification image of region outlined in H. Clone is outlined by a yellow dashed line. In merged images in G-H′, Snr1 is shown in green, Eip93F (E93) is in white, RFP is in red and DAPI is in blue. Scale bars: 50 µm in E,F,G,H; 20 µm in E′,F′,G′,H′.

Fig. 8.

Chromatin profiling reveals Snr1 occupancy at genes involved in brain development. (A) Proportion of total protein-coding genes bound by Snr1. (B) Over-represented gene ontology terms associated with genes bound by Snr1. (C) Venn diagram comparing genes significantly downregulated in neuroblasts identified by scRNA-Seq with genes found in regions of chromatin bound by Snr1. Differentially expressed genes were identified from brains with Snr1 knockdown in neuroepithelial cells (NE knockdown) or Snr1 knockdown in neuroblasts (NB knockdown). (D) Chromatin profiling plots showing replicate experiments. Representative isoforms of broad (br) and Eip93F are shown. (E) Chromatin profiles of Enhancer of split complex genes.