Cell cycle-dependent cues regulate temporal patterning of the Drosophila central brain neural stem cells

Gonzalo N Morales Chaya
Mubarak Hussain Syed author has email address

Neural Diversity Lab, Department of Biology, University of New Mexico, Albuquerque, United States
Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon, Eugene, United States

https://doi.org/10.7554/eLife.108259.1

Open access
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Figures and data

Models for early-to-late temporal progression transition of type 2 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.

Cell cycle and cytokinesis are required for temporal gene expression progression in type 2 NSCs.
(A-C) Type 2 NSC control mCherryRNAi clones (circled) at 72h ALH show normal temporal gene expression progression. Early factor Imp is off, and late factors Syp and EcR are on at 72h ALH (D-F) Cdk1RNAi type 2 NSC clone fail to down-regulate early factor Imp, and late factors Syp and EcR are not turned on. (G-I) Similarly, cytokinesis-blocked type 2NSC 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 PavarottiRNAi 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 pavarottiRNAi (p = 0.0008); EcR: 6/6 vs 0/4 in Cdk1RNAi (p = 0.0048), 6/6 vs 0/7 in pavarottiRNAi (p = 0.0006); Syp: 5/5 vs 0/11 in Cdk1RNAi (p = 0.0002), 5/5 vs 0/8 in pavarott-iRNAi (p = 0.0008). Only one clone was found per animal. (K) Representation of experimental layout for inducing clones at 0h ALH and analysis at 72h ALH. Type 2 NSCs are identified as Asense-negative large cells expressing UAS-mcd8::GFP (green insets). Scale bars represent 20um.

Switching factor Svp expresses normally in cell-cycle arrested type II NSCs.
(A) Control type 2 NSCs marked in green show Svp-LacZ expression at 48h 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 10um.

Early Svp expression is not sufficient to drive temporal factor progression in type 2 NSCs.
(A-C) Type 2 NSC control mCherryRNAi clones (circled) induced at 42h ALH and stained at 72h 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 down-regulate 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-pavarottiRNAi type 2 NSCs (n ≥ 4 clones per geno-type). 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 20um.

Cell cycle and cytokinesis are required for temporal progression in type 1 NSCs.
(A-I). Clones were induced at 0h ALH, and for (K-S), clones were induced at 42h 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 up-regulate 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 42h 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 0h and 42h ALH–induced clones. (J) In clones induced at 0h 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 pavR-NAi 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 42h 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 Cd- k1RNAi (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 20um.

pav and Cdk1RNAi type 2 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: Cd- k1RNAi, p = 0.0003; pavRNAi, p < 0.0001. only one clone was quantified per animal. Scale bar represents 20um.

Cell-cycle and cytokinesis inhibit the early-to-late transition of temporal factors in type 2 NSCs.
(A-A’’’) Type 2 NSC control UAS-mCher- ryRNAi (circled) at 72h ALH show normal temporal gene expression progression. Early factor Imp is off, and late factors Syp and EcR are on at 72h 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, cytokine- sis-blocked type 2 NSCs using pavRNAi fail to downregulate Imp and up- regulate 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 10um.

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