Cell cycle-dependent cues regulate temporal patterning of the Drosophila central brain neural stem cells
- Neural Diversity Lab, Department of Biology, University of New Mexico, United States
- Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, United States
Figures
Figure 1
Models for early-to-late temporal progression transition of type 2 neural stem cells (NSCs).
(A) Depicts timing of NSC temporal factor expression in type 2 NSCs during Drosophila larval development. (B) Wild-type NSCs undergo normal early-to-late temporal factor progression. (C) Cell cycle progression as a regulator to allow timely expression of factors in NSCs. (D) Cytokinesis-dependent regulation of temporal gene expression.
Figure 2 with 2 supplements
Cell cycle and cytokinesis are required for temporal gene expression progression in type 2 neural stem cells (NSCs).
(A–C) Type 2 NSC control mCherryRNAi clones (circled) at 72 hr after larval hatching (ALH) show normal temporal gene expression progression. Early factor Imp is off, and late factors Syp and EcR are on at 72 hr ALH (D–F) Cdk1RNAi type 2 NSC clone fails to downregulate early factor Imp, and late factors Syp and EcR are not turned on. (G–I) Similarly, cytokinesis-blocked type 2 NSC clones using pavRNAi fail to downregulate Imp and upregulate Syp and EcR. (J) Quantification of early (Imp) and late (EcR, Syp) temporal factor expression in mCherryRNAi, Cdk1RNAi, and pavRNAi type 2 NSCs (n≥4 clones per genotype). Statistical analysis by Fisher’s exact test: Imp: 0/5 vs 11/11 clones in Cdk1RNAi (p=0.0002), 0/5 vs 8/8 in pavRNAi (p=0.0008); EcR: 6/6 vs 0/4 in Cdk1RNAi (p=0.0048), 6/6 vs 0/7 in pavRNAi (p=0.0006); Syp: 5/5 vs 0/11 in Cdk1RNAi (p=0.0002), 5/5 vs 0/8 in pavRNAi (p=0.0008). Only one clone was found per animal. (K) Representation of experimental layout for inducing clones at 0 hr ALH and analysis at 72 hr ALH. Type 2 NSCs are identified as Asense-negative large cells expressing UAS-mcd8::GFP (green insets). Scale bars represent 20 µm.
-
Figure 2—source data 1
Number of T2 NSC clones expressing temporal factors on 0h ALH heat-shocked animals.
- https://cdn.elifesciences.org/articles/108259/elife-108259-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
pav and Cdk1RNAi type 2 neural stem cell (NSC) clones are larger in volume.
Confocal scans with their respective Imaris reconstruction are shown on the right for mCherryRNAi (A–A’), Cdk1RNAi (B–B’), and pavRNAi (C–C’) clones. (D) Quantification of log10-transformed clone volumes (μm3); n=5 clones per condition, plotted as mean ± SEM. Overall treatment effect by Welch’s ANOVA, p<0.0001. Post hoc Dunnett’s T3 vs mCherryRNAi: Cdk1RNAi, p=0.0003; pavRNAi, p<0.0001. Only one clone was quantified per animal. Type 2 (E–G) and type 1 (E’–G’) clones show negative and positive nuclear Asense (Ase) staining, respectively, in (E, E’) mCherryRNAi, (F, F’) Cdk1RNAi, and (G, G’) pavRNAi. Scale bar represents 20 µm.
-
Figure 2—figure supplement 1—source data 1
Raw clone volume values per genotype.
- https://cdn.elifesciences.org/articles/108259/elife-108259-fig2-figsupp1-data1-v1.xlsx
Figure 2—figure supplement 2
Cell cycle and cytokinesis inhibit the early-to-late transition of temporal factors in type 2 neural stem cells (NSCs).
(A–A’’’) Type 2 NSC control UAS-mCherryRNAi (circled) at 72 hr after larval hatching (ALH) shows normal temporal gene expression progression. Early factor Imp is off, and late factors Syp and EcR are on at 72 hr ALH (B–B’’’’). Cdk1RNAi type 2 NSCs fail to downregulate early factor Imp, and late factors Syp and EcR are not turned on. (C–C’’’) Similarly, cytokinesis-blocked type 2 NSCs using pavRNAi fail to downregulate Imp and upregulate Syp and EcR. (D) Quantifications of early and late temporal factor expression in control, cell cycle, and cytokinesis-blocked type 2 NSCs; n=6 for each genotype. Type 2 NSCs are identified as large cells expressing UAS-mcd8::GFP. Scale bars represent 10 µm.
Figure 3
Switching factor Svp is expressed normally in cell cycle-arrested type 2 neural stem cells (NSCs).
(A) Control type 2 NSCs marked in green show Svp-LacZ expression at 48 hr after larval hatching (ALH). (B) Cell cycle-arrested (Cdk1RNAi) type 2 NSCs express Svp-LacZ similar to the control. (C) Quantification of LacZ expression in control and cell cycle-blocked type 2 NSCs, n=6 for each genotype. Unpaired t-test, p-value = 0.9168. Scale bars represent 10 µm.
-
Figure 3—source data 1
Raw values showing percent of T2 NSC expressing svp-LacZ per brain.
- https://cdn.elifesciences.org/articles/108259/elife-108259-fig3-data1-v1.xlsx
Figure 4
Early Svp expression is not sufficient to drive temporal factor progression in type 2 neural stem cells (NSCs).
(A–C) Type 2 NSC control mCherryRNAi clones (circled) induced at 42 hr after larval hatching (ALH) and stained at 72 hr ALH show normal progression of temporal factors. Early factor Imp is off, and late factors Syp and EcR are on. (D–F) Cdk1RNAi clones show consistent failure to downregulate early factor Imp and activate Syp and EcR. (G–I) Likewise, pavRNAi clones fail to express EcR and Syp and consistently express early factor Imp. (J) Quantification of early (Imp) and late (EcR, Syp) temporal factor expression in control (UAS-mCherryRNAi), UAS-Cdk1RNAi, and UAS-pavRNAi type 2 NSCs (n≥4 clones per genotype). Statistical analysis by Fisher’s exact test: Imp: Cdk1RNAi vs control p=0.0002, pavRNAi vs control p=0.0079; EcR: Cdk1RNAi vs control p=0.0079, pavRNAi vs control p=0.0079; Syp: Cdk1RNAi vs control p=0.0002, pavRNAi vs control p=0.0079. Only one clone was found per animal. (K) Representation of experimental layout. Clones are identified as Asense-negative large cells expressing UAS-mcd8::GFP (green insets). Scale bars represent 20 µm.
-
Figure 4—source data 1
Number of T2 NSC clones expressing temporal factors on 42h ALH heat-shocked animals.
- https://cdn.elifesciences.org/articles/108259/elife-108259-fig4-data1-v1.xlsx
Figure 5
Cell cycle and cytokinesis are required for temporal progression in type 1 neural stem cells (NSCs).
(A–I). Clones were induced at 0 hr after larval hatching (ALH), and for (K–S), clones were induced at 42 hr ALH. (A–C) Type 1 NSC control RNAi clones show normal expression of temporal factor progression. (D–F) Cell cycle-blocked type 1 NSC clones using Cdk1RNAi show failure to upregulate EcR and Syp, and consistent expression of early factor Imp. (G–I) pavRNAi type I NSC clones fail to downregulate early factor Imp, and late factors Syp and EcR fail to express. (K–M) Control type 1 NSC clones induced at 42 hr ALH show normal expression of early and late factors, while Cdk1RNAi clones (N–P) and pavRNAi clones (Q–S) fail to downregulate Imp and fail to activate late factors Syp and EcR. (J, T) Quantification of early and late factor expression of 0 hr and 42 hr ALH–induced clones. (J) In clones induced at 0 hr ALH, Imp was expressed in 10/10 Cdk1RNAi clones (2/13 control, p=0.000067) and 12/12 pavRNAi clones (2/13 control, p=0.000020). EcR was not detected in Cdk1RNAi (0/11) or pavRNAi clones (0/11), while it was expressed in 12/12 control clones (p=0.000001 for both). Syp was absent in Cdk1RNAi (0/10) and pavRNAi clones (0/12), while present in 11/13 control clones (p=0.000067 and p=0.000020, respectively). (T) In clones induced at 42 hr ALH, Imp expression persisted in 10/10 Cdk1RNAi (0/10 control, p=0.000011) and 13/13 pavRNAi clones (0/10 control, p=0.000001). EcR expression was not detected in Cdk1RNAi (0/8) or pavRNAi clones (0/13), compared to 10/10 control (p=0.000023 and p=0.000001). Syp expression was not detected in Cdk1RNAi (0/10) or pavRNAi clones (0/13) but was regular in 10/10 control (p=0.000011 and p=0.000001). Only one clone was found per animal. For each genotype, n≥8. Type 1 clones are identified as Asense-positive, large cells expressing mcd8::GFP (green insets). Scale bars represent 20 µm.
-
Figure 5—source data 1
Number of T1 NSC clones expressing temporal factors on 0h and 42h ALH heat-shocked animals.
- https://cdn.elifesciences.org/articles/108259/elife-108259-fig5-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-Deadpan (Rat, Monoclonal) | Abcam | Cat# ab195173; RRID:AB_2687586 | 1:300 |
| Antibody | Anti-GFP (Chicken, Polyclonal) | Aves Lab | Cat# GFP-1010; RRID:AB_2307313 | 1:1500 |
| Antibody | Anti-Asense (Rabbit, Clonality unknown) | Chen-Yu Lee | 1:1000 | |
| Antibody | Anti-IMP (Rat, Clonality unknown) | Claude Desplan | 1:200 | |
| Antibody | Anti β -GAL (Rabbit, Clonality unknown) | MP Biomedicals | Cat# 559762; RRID:AB_2335286 | 1:1000 |
| Antibody | Anti-EcR-B1 (Mouse, Monoclonal) | Carl Thummel | 1:2000 | |
| Antibody | Anti-Syncrip (Guinea pig, Polyclonal) | Ilan Davis | 1:2000 | |
| Chemical compound | 16% Paraformaldehyde | Electron Microscopy Sciences | Cat# 15710 | |
| Chemical compound | Triton X-100 | Sigma-Aldrich | Cat# T8787 | |
| Chemical compound | Apple Juice | Martinelli & Co | ||
| Chemical compound | Schneider’s Insect medium | Sigma-Aldrich | Cat# S0146 | |
| Chemical compound | Agar | Sigma-Aldrich | Cat# A1296 | |
| Chemical compound | DPX mounting medium | Sigma-Aldrich | Cat# 06522 | |
| Chemical compound | Sucrose | Research Products International | Cat# 57-50-1 | |
| Chemical compound | Xylene | Fisher Scientific | Cat# 1330-20-7, 100-41-4 | |
| Genetic reagent (D. melanogaster) | hsFlp,UAS-mCD8GFP; Act-FRTstopFRT-GAL4 | Syed Lab | ||
| Genetic reagent (D. melanogaster) | UAS-pavRNAi | BDSC | BDSC_43963; RRID:BDSC_43963 | |
| Genetic reagent (D. melanogaster) | UAS-Cdk1RNAi | BDSC | BDSC_36117; RRID:BDSC_36117 | |
| Genetic reagent (D. melanogaster) | UAS-mCherryRNAi | BDSC | BDSC_35785; RRID:BDSC_35785 | |
| Genetic reagent (D. melanogaster) | Wor-GAL4, Ase-GAL80; 20xUASmcd8GFP | Syed Lab | ||
| Genetic reagent (D. melanogaster) | Wor-GAL4, Ase-GAL80; tub-GAL80ts | Syed Lab | ||
| Genetic reagent (D. melanogaster) | UAS-mcd8GFP (II) | BDSC | BDSC_5137; RRID:BDSC_5137 | |
| Genetic reagent (D. melanogaster) | 20xUAS-mcd8GFP (III) | BDSC | BDSC_32194; RRID:BDSC_32194 | |
| Genetic reagent (D. melanogaster) | svp-LacZ | BDSC | BDSC_26669; RRID:BDSC_26669 | |
| Software, algorthim | ImageJ | Fiji | RRID:SCR_002285 | Version: 2.9.0/1.53t |
| Software, algorthim | Adobe Illustrator | Adobe Systems | RRID:SCR_010279 | Version 24.005.20307 |
| Software, algorthim | GraphPad Prism 9 | GraphPad Software | RRID:SCR_002798; https://www.graphpad.com/ | |
| Software, algorthim | Imaris v9.9 and above | Oxford Instruments | RRID:SCR_007370; https://www.oxinst.com/news/imaris-launches-version-9.9-with-machine-learning-and-open-source-connections |
Additional files
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Cell cycle-dependent cues regulate temporal patterning of the Drosophila central brain neural stem cells
eLife 14:RP108259.
https://doi.org/10.7554/eLife.108259.3
