Cell cycle heterogeneity directs the timing of neural stem cell activation from quiescence

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Science  06 Apr 2018:
Vol. 360, Issue 6384, pp. 99-102
DOI: 10.1126/science.aan8795

Staging quiescent cells

Tissue-specific stem cells either divide or wait in a quiescent state until needed by the body. Quiescent stem cells have been thought to reside in the G0 stage before activating to reenter the cell cycle. However, Otsuki and Brand now show that most quiescent stem cells in the Drosophila brain are arrested in G2. Cells in the two phases display differences; for example, G2 stem cells awaken more quickly than G0 stem cells, with the conserved pseudokinase Tribbles playing a regulatory role. Elucidating the different pathways and mechanisms underlying quiescence could help to inform regenerative drug design.

Science, this issue p. 99


Quiescent stem cells in adult tissues can be activated for homeostasis or repair. Neural stem cells (NSCs) in Drosophila are reactivated from quiescence in response to nutrition by the insulin signaling pathway. It is widely accepted that quiescent stem cells are arrested in G0. In this study, however, we demonstrate that quiescent NSCs (qNSCs) are arrested in either G2 or G0. G2-G0 heterogeneity directs NSC behavior: G2 qNSCs reactivate before G0 qNSCs. In addition, we show that the evolutionarily conserved pseudokinase Tribbles (Trbl) induces G2 NSCs to enter quiescence by promoting degradation of Cdc25String and that it subsequently maintains quiescence by inhibiting Akt activation. Insulin signaling overrides repression of Akt and silences trbl transcription, allowing NSCs to exit quiescence. Our results have implications for identifying and manipulating quiescent stem cells for regenerative purposes.

Neural stem cells (NSCs) in Drosophila, like those in mammals, proliferate during embryogenesis, become quiescent in the late embryo, and then proliferate again (reactivate) postembryonically to produce neurons and glia (Fig. 1A and fig. S1A) (1, 2). A nutritional stimulus induces reactivation (3); specifically, dietary amino acids induce glial cells in the blood-brain barrier to secrete Drosophila insulin-like peptides (dILPs) (4, 5). dILPs activate the insulin signaling pathway in neighboring quiescent NSCs (qNSCs), prompting the NSCs to exit quiescence (4, 6).

Fig. 1 qNSCs are arrested in G0 or G2.

(A) qNSCs are smaller than proliferating NSCs and extend a primary process, which is retracted upon activation from quiescence. Proliferating NSCs in the embryo do not exhibit a primary process before entering quiescence. The cell cycle status of qNSCs is not known (as indicated by a question mark). (B) Among qNSCs (green), 73% ± 0.79% of cells expressed CycA (red). n = 10 tVNCs, ~150 NSCs each. (C) Cell cycle phase assessment with FUCCI and pH3. (D) Percentages of NSCs (outlined in bottom panels) in each cell cycle phase during quiescence. Colors correspond to phases as shown in (C). n = 5 tVNCs, ~150 NSCs each. ?, undetermined. (E) DAPI (4′,6-diamidino-2-phenylindole) intensities of CycA+ and CycA qNSC nuclei were significantly different (P = 2.20 × 10−16, Kolmogorov-Smirnov test). n = 10 tVNCs, ~75 NSCs each. Arbitrary units (A.U.) were defined such that 1 A.U. equals the mean DAPI intensity of the CycA population. Error bars indicate SEM. (F) Features of G2 and G0 qNSCs. Images are single-section confocal images, unless indicated otherwise, and anterior is up in this and all subsequent figures.

Quiescent stem cells are widely accepted to be arrested in G0, a poorly understood state characterized by a 2n DNA content and a lack of expression of cell cycle progression factors (7). We assessed whether Drosophila qNSCs are arrested in G0. As expected, we did not detect the M phase marker phospho–histone H3 (pH3) in qNSCs (fig. S1B). Previous studies demonstrated that qNSCs do not express the G1 marker cyclin E or incorporate the S phase marker 5-bromo-2′-deoxyuridine (BrdU) or 5-ethynyl-2′-deoxyuridine (EdU) (1, 3, 6, 8). However, we found that 73% of qNSCs expressed the G2 markers cyclin A (CycA) and cyclin B (CycB) (Fig. 1B and fig. S1C). This finding suggests that most qNSCs are arrested in G2 and that qNSCs are arrested heterogeneously in the cell cycle.

We verified that ~75% of qNSCs were arrested in G2 by comparing the fluorescent ubiquitination-based cell cycle indicator (FUCCI)–pH3 profiles of qNSCs and proliferating NSCs (Fig. 1, C and D, and fig. S1D) (9, 10). CycA-positive (CycA+) qNSCs had twice the DNA content of CycA-negative (CycA) qNSCs (Fig. 1E) and larger nuclei [30.5 ± 0.66 μm3 versus 18.1 ± 0.32 μm3; n = 10 thoracic ventral nerve cords (tVNCs), with ~75 NSCs each] than CycA qNSCs. Thus, qNSCs exhibited two types of stem cell quiescence: The majority were arrested in G2, and a minority were arrested in G0 (Fig. 1F). G2 quiescence has not been reported previously for stem cells in mammals or Drosophila.

The choice of G2 or G0 arrest could be stochastic or preprogrammed. We found seven G0 qNSCs in the first thoracic hemisegment, T1, and eight G0 qNSCs each in T2 and T3. A consistent subset of qNSCs were always arrested in G0, namely, NB2-2, NB2-4, NB2-5, NB3-4, NB5-3, and NB7-4 (cells are named according to their spatial origin in the neuroectoderm) (Fig. 2, A and B; fig. S2; and table S1). Of these qNSCs, NB2-4 disappears from T1 during embryogenesis (11, 12), explaining why fewer qNSCs are arrested in G0 in T1 than in T2 and T3. NB5-4 and NB5-7 were arrested in G2 in 50% of hemisegments but were not always arrested in the same cell cycle phase on either side of the midline (fig. S2F). We conclude that, with the exception of NB5-4 and NB5-7, the choice of G2 or G0 quiescence is entirely invariant.

Fig. 2 G2 qNSCs reactivate before G0 qNSCs.

(A) Maximum-intensity projection of a tVNC hemisegment, stained for G2 (yellow) and G0 (green; circled) qNSCs. Dashed lines indicate hemisegment boundaries. (B) Positions and identities of G0 qNSCs (green) within a hemisegment. The dashed line represents the midline. MN, median neuroblast. [Schematic modified from (12)] (C) Quantification of Wor+ G0 or G2 qNSCs. n = 10 tVNCs, ~150 NSCs each, per time point. ***P < 1.39 × 10−5, two-tailed paired t tests. Red lines indicate medians. (D) NB3-4 (Eg+; arrowheads) remains small and Wor negative at 20 hours ALH, while neighboring G2 qNSCs have reactivated.

Is G2-G0 heterogeneity in qNSCs significant? We assessed the reactivation of G2 and G0 qNSCs by tracking the expression of the reactivation marker worniu (wor) (fig. S3). More than 86% of G2 qNSCs reactivated by 20 hours after larval hatching (ALH), compared with 20% of G0 qNSCs (n = 10 tVNCs, ~150 NSCs each) (Fig. 2C). For example, NB3-4, a G0 qNSC, reactivated in fewer than 7% of hemisegments (n = 10 tVNCs, six hemisegments each) (Fig. 2D). All NSCs reactivated by 48 hours ALH (fig. S3C). Thus, G2 qNSCs are faster-reactivating stem cells than G0 qNSCs.

We next profiled gene expression in qNSCs using targeted DamID (TaDa) (13), identifying 1656 genes. Corresponding Gene Ontology (GO) terms included “nervous system development” (35 genes; corrected P value, 2.70 × 10−6) and “neuroblast [NSC] development” (10 genes; corrected P value, 8.40 × 10−4) (tables S2 and S3). To identify quiescence-specific genes, we eliminated genes common to quiescent and proliferating NSCs (13), such as deadpan (dpn) (fig. S4, A and B). tribbles (trbl) is one of the most significantly expressed protein-encoding genes specific to qNSCs (fig. S4C). trbl encodes an evolutionarily conserved pseudokinase with three human homologs that have been implicated in insulin and mitogen-activated protein kinase signaling [reviewed in (14)]. We confirmed that trbl labels quiescent but not proliferating NSCs in vivo (Fig. 3A and fig. S4, D to F). To date, no other gene that labels qNSCs specifically has been identified.

Fig. 3 trbl regulates G2 qNSCs.

(A) trbl reporter expression (green) in NSCs (red) before, during, and after quiescence. Emb, embryonic stage. (B) Quantification of proliferating NSCs in control (CTRL) tVNCs (n = 10 tVNCs) versus trblEP3519 mutant tVNCs (n = 8 tVNCs), with ~120 NSCs per tVNC. ***P = 7.06 × 10−14, Student’s t test. (C) Trbl protein expression (green) in G2 (CycA+) or G0 (CycA) qNSCs (red). Trbl GFSTF, Trbl tagged with EGFP-FlAsH-StrepII-TEV-3×Flag. (D) Quantification of qNSCs in tVNCs with GFP-Trbl expression driven by grh-GAL4 (grh-GAL4>GFP-Trbl tVNCs). “−” and “+” denote GFP-Trbl and GFP-Trbl+ NSCs, respectively. n = 9 tVNCs, ~150 NSCs each. ***P = 3.90 × 10−4, Wilcoxon signed-rank test. Red lines in (B) and (D) indicate medians. (E) In grh-GAL4>GFP-Trbl brains, GFP-Trbl+ NSCs (indicated by green outlines) do not incorporate EdU, but control NSCs (indicated by yellow arrowheads) do.

trbl is necessary for quiescence entry, as NSCs continued to divide during late embryogenesis in trbl hypomorphic mutants or when trbl was knocked down specifically in NSCs (Fig. 3B and fig. S5, A to D). trbl regulates quiescence entry specifically, without affecting division mode or cell viability (fig. S5, E and F). The ectopically dividing NSCs in the trblEP3519 mutant were G2, not G0, qNSCs (fig. S5G). G2 but not G0 qNSCs also became significantly smaller in trblEP3519 mutants (fig. S5, H to J). As embryonic NSCs do not regrow between cell divisions (15), the size reduction is consistent with excessive divisions. Consistent with a function in G2 quiescence, Trbl was expressed primarily in G2 qNSCs (Fig. 3C and fig. S5K).

Trbl is also required to maintain quiescence. RNA interference–mediated knockdown of trbl in qNSCs caused NSCs to leave quiescence and divide (fig. S5, L and M). We generated transgenic flies carrying upstream activation sequence–green fluorescent protein (GFP)–Trbl and drove expression with grainyhead (grh)GAL4 (4) to assess whether Trbl is sufficient to maintain G2 quiescence. grh-GAL4 expression is initiated at quiescence entry and occurs in ~67% of NSCs, allowing comparison between neighboring GFP-Trbl–expressing and nonexpressing NSCs. Almost all (91.8 ± 0.88%; n = 10 tVNCs, ~120 NSCs each) GFP-Trbl–expressing NSCs remained in G2 quiescence and expressed CycA (Fig. 3, D and E, and fig. S6). GFP-Trbl–expressing NSCs retained the primary process that is extended specifically by quiescent NSCs (Fig. 1A), unlike control NSCs, which had begun to divide (fig. S6, B and C) (1, 4, 16). Thus, Trbl is sufficient to maintain G2 quiescence.

In the embryonic mesoderm, trbl induces G2 arrest by promoting Cdc25String protein degradation (1719). We found that Cdc25String protein was reduced in NSCs at quiescence entry but that cdc25string mRNA was maintained (Fig. 4A). Therefore, Cdc25String is regulated posttranscriptionally at quiescence entry. Significantly more NSCs were positive for Cdc25String protein in trblEP3519 mutants than in controls (Fig. 4B and fig. S7, A and B). This increase in Cdc25String is sufficient to explain the excessive NSC proliferation in trbl mutants (fig. S7, C and D). Thus, Trbl initiates quiescence entry by promoting Cdc25String protein degradation during late embryogenesis.

Fig. 4 Trbl induces and maintains quiescence through different mechanisms.

(A) (Top) Maximum-intensity projections showing Cdc25String protein (prot.) (green) expression in NSCs (red) before and during quiescence (percentages are means ± SEM). n = 10 tVNCs, ~130 NSCs each, per time point. (Bottom) In situ hybridization against cdc25string mRNA at the same stages. (B) Percentages of Cdc25String protein+ NSCs in control (trblEP3519 heterozygote) tVNCs versus mutant tVNCs. n = 10 tVNCs, ~110 NSCs each, per genotype. ***P = 9.08 × 10−5, Kolmogorov-Smirnov test. (C and D) Quantification of qNSCs in epistasis experiments between GFP-Trbl and AktACT (C) or PI3KACT (D). n > 10 tVNCs, ~80 NSCs each, per condition. ***P < 3.39 × 10−9; ns, not significant (P > 0.05), one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. In (D), there is no significant difference between GFP-Trbl alone and GFP-Trbl plus PI3KACT. Red lines in (B) to (D) indicate medians. (E) Three-step model for Trbl activity.

Trbl also maintains NSC quiescence postembryonically; however, it must act through another mechanism, as Cdc25String is no longer expressed in postembryonic qNSCs (fig. S8A). Trbl is known to inhibit insulin signaling by binding Akt and preventing its phosphorylation (fig. S8B) (20). Consistent with this, Trbl-expressing NSCs had less phosphorylated translation initiation factor 4E–binding protein (p4E-BP) than control NSCs (fig. S8C). If Trbl inhibits Akt to maintain quiescence, constitutively active Akt (myr-Akt; hereafter AktACT) (21) should counteract Trbl-induced quiescence. AktACT fully rescued NSC reactivation (Fig. 4C and fig. S8D). In contrast, as Trbl is thought to act downstream of phosphatidylinositol 3-kinase (PI3K) (20), constitutively active PI3K (dp110CAAX; hereafter PI3KACT) (22) should not rescue reactivation, which it did not (Fig. 4D and fig. S8D). Thus, Trbl maintains quiescence by blocking activation of Akt. This role is specific to postembryonic NSCs, as embryonic NSCs do not depend on insulin signaling to proliferate (fig. S8E).

trbl expression must be repressed to allow NSC reactivation. We found that insulin signaling is necessary and sufficient to repress trbl transcription. NSCs misexpressing phosphatase and tensin homolog (PTEN), an insulin pathway inhibitor, failed to down-regulate trbl transcription (fig. S8F). In contrast, activating the insulin pathway by expressing AktACT in NSCs was sufficient to switch off trbl transcription (fig. S8G).

We have discovered the mechanisms by which Drosophila NSCs enter, remain in, and exit quiescence in response to nutrition (Fig. 4E). First, Trbl pseudokinase promotes degradation of Cdc25String protein to induce quiescence; second, it blocks insulin signaling by inhibiting Akt in the same NSCs to maintain quiescence; and third, it is overridden by nutrition-dependent secretion of dILPs from blood-brain barrier glia, which activate insulin signaling in qNSCs, repress trbl expression, and enable reactivation.

We found that qNSCs are preprogrammed for arrest in G2 or G0, contrary to accepted doctrine. G2 qNSCs are the first to reactivate and generate neurons; this is followed by reactivation of G0 qNSCs. This pattern may ensure that neurons form the correct circuits in the appropriate order during brain development. G2 arrest also enables high-fidelity homologous recombination–mediated repair in response to DNA damage, preserving genomic integrity during quiescence. Quiescent stem cells in mammals may also arrest in G2, with implications for isolating and manipulating quiescent stem cells for therapeutic purposes.

Supplementary Materials

Materials and Methods

Figs. S1 to S8

Tables S1 to S3

References (2341)

References and Notes

Acknowledgments: We thank P. Callaerts, F. Díaz-Benjumea, J. Dods, C. Q. Doe, B. Edgar, E. Higginbotham, Y. Kimata, J. Ng, J. Urban, U. Walldorf, E. Wieschaus, Bloomington Drosophila Stock Centre, and the Developmental Studies Hybridoma Bank (DSHB) for generously providing reagents; T. Southall and O. J. Marshall for updating the TaDa microarray data to Release 6 of the Drosophila genome and for gene expression analysis; and F. Doetsch, A. C. Delgado, F. J. Livesey, D. St. Johnston, and the Brand laboratory members for discussion. Funding: This work was funded by the Royal Society Darwin Trust Research Professorship, Wellcome Trust Senior Investigator award 103792, and Wellcome Trust Programme grant 092545 to A.H.B. and by Wellcome Trust Ph.D. Studentship stipend 097423 to L.O. A.H.B acknowledges core funding to the Gurdon Institute from the Wellcome Trust (grant 092096) and Cancer Research UK (CRUK) (grant C6946/A14492). Author contributions: L.O. and A.H.B. designed the experiments, analyzed the data, and wrote the manuscript. L.O. performed the experiments. Competing interests: The authors declare no conflict of interest. Data and materials accessibility: Microarray data have been deposited with the Gene Expression Omnibus under accession number GSE81745.
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