1. Stem Cells and Regenerative Medicine
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Transient nuclear Prospero induces neural progenitor quiescence

  1. Sen-Lin Lai
  2. Chris Q Doe  Is a corresponding author
  1. Howard Hughes Medical Institute, University of Oregon, United States
  2. University of Oregon, United States
Short Report
Cite this article as: eLife 2014;3:e03363 doi: 10.7554/eLife.03363
4 figures

Figures

Figure 1 with 2 supplements
Quiescent neuroblasts have a novel transcriptional profile lacking both progenitor and differentiation markers.

(AF) Proliferating NB3-3A contains nuclear Deadpan (A), cytoplasmic Mira (B), nuclear Asense (C), nuclear Worniu (D), and nuclear CycE (E) at embryonic stages 14 and 16; quantification is shown in (F). Neuroblast lineages are marked by Eg-GFP (Eg-gal4 UAS-mCD8:GFP), and the neuroblast is identified by Dpn (shown in insets) and indicated by arrows. Anterior side is up, and lateral is at right. Scale bar: 5 μm. (GL) Proliferating NB3-3T expresses all above-mentioned neuroblast markers at stage 14 (S14). At stage 16 (S16), NB3-3T is quiescent and contains Deadpan (G) and Mira (H) but lacks Asense (I), Worniu (J) and CycE (K). Quantified in (L). Scale bar: 5 μm. (F and L) Quantification; number of neuroblasts scored shown in bar. (M) Schematic summary of neuroblast marker profiles of proliferating neuroblast, quiescent neuroblast and neuron.

https://doi.org/10.7554/eLife.03363.003
Figure 1—figure supplement 1
Larval quiescent neuroblasts are Deadpan-positive but lack the progenitor markers Miranda, Asense, Cyclin E, Worniu and the differentiation markers Prospero, Elav, and Repo.

(A) The newly hatched larval (NHL) central brain contains five persistently proliferative neuroblasts (the four mushroom body neuroblasts and the lateral antennal lobe neuroblast) and ∼100 quiescent central brain neuroblasts in each lobe. (BE) Proliferative neuroblasts in the NHL contain progenitor markers Deadpan (Dpn), Asense (Ase), and Worniu (Wor) but lack the differentiation markers Prospero (Pros), Elav, and Repo and incorporate EdU (top). Quiescent neuroblasts maintain Dpn, but lack the progenitor markers and the differentiation markers, and fail to incorporate EdU (bottom).

https://doi.org/10.7554/eLife.03363.004
Figure 1—figure supplement 2
Overexpressing Asense and Cyclin E does not change the timing of neuroblast quiescence.

No significant change in the number of proliferating NB3-3T in stage 15 embryos with overexpressed Asense (AseOXN) or Cyclin E (CycEOXN). Error bars, standard deviation; n.s., not significant. Fly genotype: AseOXN, worniu-gal4 UAS-asense; CycEOXN, worniu-gal4 UAS-cyclin E.

https://doi.org/10.7554/eLife.03363.005
Figure 2 with 1 supplement
Transient low level nuclear Prospero is tightly correlated with neuroblast entry into quiescence.

(AB) NB3-3T shows transient nuclear Prospero (Pros) during entry into quiescence at stage 15 (S15) (B). Differentiated daughter cell shows strong nuclear Prospero and is indicated by the arrow. Neuroblast lineages are identified by Eg-GFP (Eg-gal4 UAS-mCD8:GFP) (A), and the neuroblast is identified by Dpn and outlined by dashed lines. Summarized in schematic below (Prospero, green); quantified in (E). Anterior side is up, and lateral is at right. Scale bar: 5 μm. (CD) Proliferating NB3-3A does not contain nuclear Prospero. Summarized in schematic below; quantified in (E). Scale bar: 5 μm. (E) Temporal identity factors schedule the timing of neuroblast entry into quiescence and the expression of nuclear Prospero. In cas mutant (cas24), neuroblast quiescence is delayed, as is the timing of nuclear Prospero. In the deficiency allele Df(2L)ED773 which removes both nubbin/pdm2 (pdm mutant), NB3-3T precociously enters quiescence, and the timing of nuclear Prospero is advanced. (F) Schematic of the timing of neuroblast entry into quiescence and the expression of nuclear Prospero.

https://doi.org/10.7554/eLife.03363.006
Figure 2—figure supplement 1
Nuclear Prospero levels are lower in stage 15 NB3-3T than in differentiating GMCs.

(A) Confocal images of a wild type NB3-3T (blue circle, identified by Eg-GFP and Dpn) and its adjacent GMC (yellow circle, identified by Eg-GFP and its smaller size) in stage 15 embryos. Scale bar: 5 μm. (B) Quantification of nuclear Prospero intensity in unit area in a neuroblast vs an adjacent GMC in stage 15 embryos. Prospero intensity in each confocal stack was measured with the open source software FIJI to obtain gray value and the nuclear area. The total Prospero intensity was summed from the confocal stacks, and then divided by nuclear volume to obtain the Prospero intensity per unit volume. Small triangles represent the intensity quantified from each individual neuroblast, and the large triangle is the average. Small circles represent the intensity quantified from each individual GMC, and the large circle is the average.

https://doi.org/10.7554/eLife.03363.007
Prospero is required for neuroblast quiescence.

(AB) Wild type and prospero mutant (pros17) stage 16 embryos. Parental neuroblasts (NB) stain for Deadpan and mγ-LacZ, whereas ectopic ‘de-differentiated’ neuroblasts stain for Deadpan but not mγ-LacZ. Proliferating neuroblasts are marked by EdU incorporation. (A) In wild type, most thoracic neuroblasts are in quiescence and do not incorporate EdU. (B) In prospero mutants, most parental neuroblasts (mγ-LacZ+) have not entered quiescence and still incorporate EdU. Ventral side is up and anterior is at left. Dashed line, boundary of neuroblast and neuron (N) layers. Scale bars: 20 μm. (CE) NB3-3T remains proliferative in prospero mutant stage 16 embryos. (C) In wild type, NB3-3T is Mira+ mγ-LacZ+ EdU−; (D) in prospero mutants, NB3-3T is Mira+ mγ-LacZ+ EdU+. The NB3-3T lineage was identified by Eg-GFP (Eg-gal4 UAS-mCD8:GFP) and the neuroblast is indicated by arrowheads. (E) Quantification. n.s., not significant; ****p < 0.00001.

https://doi.org/10.7554/eLife.03363.008
Figure 4 with 2 supplements
Prospero is sufficient to induce neuroblast quiescence.

(AC) Transient overexpression of nuclear Prospero in neuroblasts (worniu-gal4 UAS-HA:prospero tub-gal80ts) results in neuroblast quiescence (A) or differentiation (B). Third-instar larvae were shifted from 22°C to 30°C to inactivate Gal80, which resulted in nuclear Prospero in neuroblasts. Low levels of nuclear Prospero result in neuroblast quiescence (Dpn+ Elav) (A) whereas high levels of nuclear Prospero suppress Deadpan and activate Elav to induce differentiation (Dpn Elav+) (B). Prospero levels were quantified in (C). Scale bars: 5 μm. (C) Quantification of Prospero levels in quiescent neuroblasts (Dpn+ Elav) or differentiated neuroblasts (Dpn Elav+). Differentiated neuroblasts were identified by their nuclear size larger than 7 μm in diameter. Prospero intensity was determined by totaling the gray value in nucleus of confocal stacks, followed by normalization to the total gray value of DNA marker 4ʹ,6-diamidino-2-phenylindole (DAPI). Number of neuroblasts quantified shown in bar. Error bars: standard deviation. (DF) Larval neuroblast phenotype upon transient overexpression of Prospero. (D) Neuroblasts stay Deadpan+ (Dpn+) and thus do not differentiate. (E) There is an increase of quiescent neuroblasts 12 hr after Prospero induction, as determined by the elevated percentage of EdU (D) and Wor (E) neuroblasts. The quiescent neuroblasts re-enter the cell cycle and express the progenitor marker Wor 24 hr after induction. Error bars: standard deviation. n.s., not significant; *p < 0.05; **p < 0.01; ***p < 0.0001. (G) Model. Prospero levels distinguish three progenitor fates: absent for self-renewal, low for quiescence, and high for differentiation.

https://doi.org/10.7554/eLife.03363.009
Figure 4—figure supplement 1
Low nuclear Prospero induces neuroblast quiescence, whereas high Prospero induces neuroblast differentiation.

(A) Two levels of Prospero were generated by increasing time of misexpression (4 or 12 hr) with one copy of UAS-HA:prospero transgene. Whereas 2 hr can induce neuroblast quiescence (Figure 4), 4 hr and 12 hr can robustly induce neuroblast differentiation. (B) Two levels of Prospero were generated by misexpressing one vs two copies of the UAS-HA:prospero transgene for the same time (2 hr). A significant increase of the number of neuroblasts differentiated with two copies of UAS-HA:prospero transgene.

https://doi.org/10.7554/eLife.03363.010
Figure 4—figure supplement 2
Prospero is sufficient to induce embryonic neuroblast quiescence.

(AG) Transient overexpression of nuclear Prospero in neuroblasts (worniu-gal4 UAS-HA:prospero tub-gal80ts) induces neuroblast cell cycle arrest. Embryos were shifted from 22°C to 30°C for 1 hr to inactivate Gal80, which resulted in transient nuclear Prospero in NB3-3T at stage 14 (A, wild type; B, transient nuclear Prospero). Scale bars: 2 μm. (CG) Quantification of the Eg+ NB3-3T phenotype upon transient overexpression of Prospero. (C) Some neuroblasts have detectable nuclear Prospero. (D) Most neuroblasts fail to incorporate EdU. (EG) There is a slight loss of Worniu+ (E), Asense+ (F) and CycE+ (G) neuroblasts.

https://doi.org/10.7554/eLife.03363.011

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