DOI: 10.5061/dryad.tmpg4f56p
title: Data from: Drosophila medulla neuroblast termination via apoptosis, differentiation and gliogenic switch is scheduled by the depletion of the neuroepithelial stem cell pool

description: The brain is consisted of diverse neurons arising from a limited number of
 neural stem cells. Drosophila neural stem cells called
 neuroblasts (NBs) produces specific neural lineages of various lineage
 sizes depending on their location in the brain. In
 the Drosophila visual processing centre - the optic
 lobes (OLs), medulla NBs derived from the neuroepithelium (NE) give rise
 to neurons and glia cells of the medulla cortex. The timing and the
 mechanisms responsible for the cessation of medulla NBs are so far not
 known. In this study, we show that the termination of medulla NBs during
 early pupal development is determined by the exhaustion of the NE stem
 cell pool. Hence, altering NE-NB transition during larval neurogenesis
 disrupts the timely termination of medulla NBs. Medulla NBs terminate
 neurogenesis via a combination of apoptosis, terminal symmetric division
 via Prospero, and a switch to gliogenesis via Glial Cell Missing (Gcm),
 however, these processes occur independently of each other. We also show
 that temporal progression of the medulla NBs is mostly not required for
 their termination. As the Drosophila OL shares a similar mode of
 division with mammalian neurogenesis, understanding when and how these
 progenitors cease proliferation during development can have important
 implications for mammalian brain size determination and regulation of its
 overall function.
description: <strong>Fly stocks and husbandry </strong>
 The fly strains used were detailed in the extended Materials and
 Methods in the paper. Fly stocks were reared on standard media at
 25<sup>o</sup>C. For larval dissection, brains were dissected
 at wandering L3 stages (approximately 120h ALH). For pupal dissection,
 white pupae were selected and allowed to age to the desired
 stages.  Knockdown and overexpression experiments using
 <em>eyR16F10-GAL4</em> were moved to
 29<sup>o</sup>C after overnight embryo collection until
 dissection. Experiments using
 <em>tub-GAL80<sup>ts</sup>;eyR16F10-GAL4,
 </em>embryos were reared at 25<sup>o</sup>C for one day,
 larvae were then moved to repressive 18<sup>o</sup>C for 5
 days to inhibit GAL4 activity, and transferred to permissive
 29<sup>o</sup>C to induce GAL4-mediated transgene expression
 for 2 days prior to dissection. Knockdown and overexpression flip-out
 clones were induced at 24h ALH at 37<sup>o</sup>C for 10 min
 and then allowed to develop at 29<sup>o</sup>C until
 dissection except for <em>pros RNAi</em> clones that were let
 developed at 25<sup>o</sup>C to allow a mild knockdown of
 <em>pros</em>. <em>FRT42D</em> and
 <em>FRT42D;UAS-tll</em> MARCM clones were induced at 24h ALH
 (Figure 2A-D) or 48h ALH (Figure 5-figure supplement 2A-B) at
 37<sup>o</sup>C for 15 min. <em>FRT82B</em> and
 <em>FRT82B,pros<sup>17</sup></em> MARCM clones
 were induced at 24h ALH at 37<sup>o</sup>C for 8
 min. <strong>Immunostaining
 </strong> Larval and pupal brains were dissected
 in phosphate buffered saline (PBS), fixed in 4% formaldehyde for 20 min
 and rinsed three time in 0.3% or 0.5% PBST (PBS + 0.5% Triton),
 respectively at room temperature. For immunostaining, brains were
 incubated in primary antibodies overnight at 4<sup>o</sup>C,
 followed by two washes in 0.3-0.5% PBST and then an overnight secondary
 antibody incubation at 4<sup>o</sup>C or 3h at room
 temperature. Brains were afterwards rinsed two times in 0.3-0.5% PBST and
 incubated in 50% glycerol in PBS for 20 min. Samples were mounted in 80%
 glycerol in PBS, on glass slides and sealed with coverslips (22x22 mm,
 No.1.5, Knittel) for image acquisition. The primary antibodies used were:
 rat anti-Dpn (1:200, Abcam 195172), chick anti-GFP (1:1000, Abcam 13970),
 mouse anti-EcR<sup>common</sup> (1:50, DSHB Ag10.2), rabbit
 anti-Dcp-1 (1:100, Cell Signalling 95785), rat anti-Mira (1:100, Abcam
 197788), rat anti-Pros (a gift from Fumio Matsuzaki), rabbit
 anti-ß-Galactosidase (1:200, a gift from Helena Richardson), rabbit
 anti-Ase (a gift from Lily Jan and Yuh Nung Jan), mouse anti-Repo (1:50,
 DSHB 8D12), rabbit anti-PatJ (1:500, a gift from Helena Richardson),
 anti-Ey (1:50, DSHB), guinea pig anti-Slp (1:200, a gift from Kuniaki
 Saito), rabbit anti-Tll (1:200, a gift from Kuniaki Saito). Secondary
 donkey antibodies conjugated to Alexa 555 and Alexa 647, and goat
 antibodies conjugated to Alexa 405, 488, 555, and 647 (Molecular Probes)
 were used at 1:500. <strong>Image acquisition and
 processing</strong> Images were acquired using
 Olympus FV3000 confocal microscope with 40x (NA 0.95, UPLSAPO) and 60x (NA
 1.30, UPLSAPO) objectives. Δz = 1.5 μm. Images were processed using Fiji
 (https://imagej.net/Fiji).  Quantification was
 performed in Fiji or via 3D reconstruction in Imaris (Bitplane). To
 quantify NB numbers (Figure 1C, 4E, Figure 1-figure supplement 1N, 2E,
 Figure 3-figure supplement 1G), the numbers of Dpn<sup>+</sup>
 cells in the OPC were measured. To quantify the nuclear sizes of the
 medulla NBs (Figure 1E), nuclear diameters identified by Dpn were
 measured. To quantify NB and NE volumes in the OPC (Figure 2A), the
 Dpn<sup>+ </sup>and PatJ<sup>+</sup> volumes were
 measured for the NBs and the NE, respectively. To quantify the ratio of
 NBs (Figure 2J, 2M, 3P, 4I, 5I, 5P, Figure 1-figure supplement 1E, Figure
 5-figure supplement 2I) or glia (Figure 5D) in clones,
 GFP<sup>+</sup> clones were first detected and
 GFP<sup>+</sup> volumes were measured. The
 GFP<sup>+</sup> areas were then utilised to make a mask for
 Dpn<sup>+</sup> or Repo<sup>+</sup> cells within
 the clones, which was in turn used to measure NB or glial cell numbers,
 respectively. The NB or glial ratio in clones is calculated as the ratio
 of the number of GFP<sup>+</sup>Dpn<sup>+</sup> or
 GFP<sup>+</sup>Repo<sup>+</sup> cells over the
 GFP<sup>+</sup> volume. To quantify the distributions of
 apoptotic NBs expressing Ey, Slp, and Tll (hereby referred as tTF) (Figure
 3K), Dpn::GFP<sup>+</sup>Dcp-1<sup>+</sup> cells
 were first identified and used as a mask for cells that are positive for
 tTFs. The ratio of each tTF is calculated as the ratio of the number of
 GFP<sup>+</sup>Dcp-1<sup>+</sup>tTF<sup>+
 </sup>cells over the number of
 GFP<sup>+</sup>Dcp-1<sup>+</sup> cells. To
 quantify the number of glia in the medulla (Figure 5-figure supplement
 2H), the total numbers of Repo<sup>+</sup> cells in the deep
 section of the medulla were measured. Images were assembled in Affinity
 Publisher 2. Schematics were created in Affinity Design 2. Scale bars = 10
 μm or 50 μm per indicated in figures.
 <strong>Statistical analyses </strong>
 The biological replicates in our experiments are represented by a
 minimum of three animals per genotype. The n number reflects brain lobe
 numbers unless otherwise specified. Statistical analyses were performed,
 and graphs were plotted in GraphPad Prism 9. In graphs, data is
 represented as mean ± standard error of the mean (SEM). For comparisons
 between two conditions, P-values were calculated by non-parametric
 Mann-Whitney tests when data if not normally distributed. For comparisons
 between more than two experimental conditions, P-values were calculated by
 ordinary one-way ANOVA tests for normally distributed data or data with
 small sampling size, i.e. n&lt;5 (Figure 1C, 2E). For non-normally
 distributed data, Kruskal-Wallis tests were employed. Holm-Šídák's or
 Dunn’s tests were used to correct for multiple comparisons following
 one-way ANOVA and Kruskal-Wallis tests, respectively. For the comparisons
 of apoptotic NBs expressing different temporal factors at 12-16h APF
 (Figure 3K), ordinary two-way ANOVA test was used, followed by
 Šídák's test for multiple comparisons. <em>(*) p&lt;0.05
 (**) p&lt; 0.01, (***) p&lt; 0.001, (****) p &lt; 0.0001, (ns)
 p&gt;0.0. </em>If quantifications were not performed, the number
 of samples in which specific phenotypes were observed, is mentioned in the
 text.
description: Source data associated with the paper "*Drosophila* medulla
 neuroblast termination via apoptosis, differentiation and gliogenic switch
 is scheduled by the depletion of the neuroepithelial stem cell pool"
 by Nguyen and Cheng, 2014. ### Description of the data and file structure
 ##### General information Source Data for representative images are in
 either TIF or PNG formats. Source images are structured according to the
 figures in the original manuscript (see "Source image file
 structure" for detailed information). Source Data for statistical
 analysis and data visualization. One GraphPad project XML file (GraphPad
 Prism software package) is structured according to the figures in the
 original manuscript. Abbreviations: NB: neuroblast APF: after pupal
 formation ALH: after larval hatching NE: neuroepithelial tTF: temporal
 transcription factor Slp: sloppy-paired mChe: mCherry med12: mediator 12
 Ey: eyeless Red: RedStinger EcR: ecdysone receptor DN: dominant negative
 Dpn: Deadpan n: notch l'sc: lethal of scute Atg1: autophagy-related 1
 pros: prospero Repo: reveresed polarity gcm: glial cell missing Tll:
 tailless Area of investigations: the medulla of the *Drosophila* optic
 lobe ##### File structure of source data and statistical analysis Figure
 1: Full quantifications.pzfx-file; data tables 1C, 1E 1C-Medulla NB
 number: No. Dpn+ cells 1E-w1118 NB nuclear size: Nuclear diameter [µm]
 Figure 1-figure supplement 1: Full quantifications.pzfx-file; data tables
 1-S1E, 1-S1K, 1-S1N 1-S1E-hsFLP&gt;mCheRi/med12Ri/ND75/dp110caax: No. Dpn+
 cells/V.GFP+ (x10^-3) 1-S1K-ey,Red: ey+Dpn+/Dpn+
 1-S1N-ey&gt;mCheRi/med12Ri: No. Dpn+ cells Figure 1-figure supplement 2:
 Full quantifications.pzfx-file; data tables 1-S2E
 1-S2E-ey&gt;mCheRi/EcRDN/Ri: No. Dpn+ cells Figure 2: Full
 quantifications.pzfx-file; data tables 2E, 2J, 2M 2E-FRT42D 12-30h APF
 (2E-NE and 2E-NB): Volumes of the NE or the NBs in the medulla [µm^3]
 2J-hsFLP&gt;mCheRi/nRi: No. Dpn+ cells/V.GFP+ (x10^-3)
 2M-hsFLP&gt;lacZ/l'sc: No. Dpn+ cells/V.GFP+ (x10^-3) Figure 3: Full
 quantifications.pzfx-file; data tables 3K, 3P 3K-Apoptosis-tTFs 12-16h
 APF: Ratio of Dpn+Dcp+1 cells 3P-hsFLP&gt;mCheRi/p35: No. Dpn+
 cells/V.GFP+ (x10^-3) Figure 3-figure supplement 1: Full
 quantifications.pzfx-file; data tables 3-S1G
 3-S1G-ey&gt;mCheRi/p35/Atg1Ri: No. Dpn+ cells Figure 4: Full
 quantifications.pzfx-file; data tables 4E, 4I 4E-ey&gt;lacZ/pros: No. Dpn+
 cells 4I-hsFLP&gt;mCheRu/prosRi: No. Dpn+ cells/V.GFP+ (x10^-3) Figure 5:
 Full quantifications.pzfx-file; data tables 5D, 5I, 5P
 5D-hsFLP&gt;mCheRu/prosRi: No.Repo+ cells/V.GFP+ (x10^-3)
 5I-hsFLP&gt;lacZ/gcm: No. Dpn+ cells/V.GFP+ (x10^-3)
 5P-hsFLP&gt;mCheRi/gcmRi: No. Dpn+ cells/V.GFP+ (x10^-3) Figure 5-figure
 supplement 2: Full quantifications.pzfx-file; data tables 5-S2H, 5-S2I
 5-S2H-hsFLP&gt;mCheRi/tllRi L3 Repo: No.Repo+ cells
 5-S2I-hsFLP&gt;mCheRi/tllRi 12h APF Dpn: No. Dpn+ cells/V.GFP+ (x10^-3)