1. Anterior-Posterior Gradient in Neural Stem and Daughter Cell Proliferation Governed by Spatial and Temporal Hox Control 
Ignacio Monedero Cobeta, Behzad Yaghmaeian Salmani, Stefan Thor 
Current Biology 
Volume 27, Issue 8, Pages (April 2017)
DOI: /j.cub
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2. Current Biology 2017 27, 1161-1172DOI: (10.1016/j.cub.2017.03.023)
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3. Figure 1
A-P Gradient of Cell Numbers, Type I > 0 Daughter Proliferation Switch, and NB Exit in the NB5-6 and NB3-5 Lineages
(A) During Drosophila embryonic development, ∼800 NBs form in the ventral neurogenic regions and divide to generate the VNC. NB3-5 and NB5-6 are generated in all 13 VNC segments (T1–T3 and A1–A10). VNC NBs show different daughter proliferation modes, denoted type I and type 0 [6] (GMC, ganglion mother cell; N/G, neuron/glia).
(B and C) NB5-6 lineage is marked by lbe(K)-GFP (B) and NB3-5 lineage by ham-Gal4/UAS-GFP (C) at St16 and St16+200 min, respectively, at which stages both lineages have stopped dividing.
(D and E) Cell numbers in NB5-6 (D) and NB3-5 (E) lineages, T1 to A10 (D, St16; E, St min; SD).
(F) NB3-5 exits quiescence during larval stages and expresses ham-Gal4 (NB detected by Dpn).
(G) Cell divisions of NB3-5 and its daughter cells (St12, St15, St16+100 min, and St16+200 min; segments T2, A8, and A9; dashed circles indicate dividing cells). Percentages indicate PH3 in NBs or daughter cells (n ≥ 10 lineages).
(H and I) Lineage trees of NB5-6 (H) and NB3-5 (I) in different segments of the VNC (PCD, programmed cell death; Q, quiescence; PCD in NB5-6T after cell-cycle exit was described previously) [7, 8].
See also Figure S1 and Tables S1 and S2.
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4. Figure 2
A-P Gradient of Cell Numbers, Dividing NBs, and Type I > 0 Daughter Proliferation Switches and Programmed Cell Death in the VNC
(A) Cell numbers/segment (T1–T3, A1–A7, and A8–A10) at St16 in control (black) and PCD mutants (ED225; gray). Significant differences were found between thoracic (T1–T3), anterior abdominal (A1–A7), and posterior abdominal (A8–10) regions in both control and ED225 background (Mann-Whitney U test; SD; n ≥ 10 embryos per genotype; black asterisks, control versus ED225; blue asterisks, ED225 at different segments; red asterisks, control at different segments).
(B) Cell numbers per segment (T1–A10) at St16 of control (black bars) and PCD mutants (ED225; gray bars) (Student’s t test; SD; n ≥ 10 embryos per genotype).
(C) Percentage of cells/segment in ED225 mutants compared to control. PCD removes an increasing percentage of cells in more posterior segments.
(D–E’) Mitotic NBs (D and E) and daughters (D’ and E’), marked by Pros, PH3, and Dpn, in St13 control embryo (NBs are Dpn+, Pros-asymmetric-cytoplasmic, and PH3+; daughter cells are Dpn-negative, Pros-nuclear, and PH3+; scale bar, 5 μm).
(F and G) Quantification of dividing NBs (F) and daughters (G) in control and ED225 mutants at St13 (Student’s t test; SD; n ≥ 10 embryos per genotype).
(H and I) Quantification of dividing NBs and daughters from St10–St16 in thoracic and abdominal segments (T2–T3, solid lines; A6–A10, dashed lines) of controls (H) and ED225 mutants (I). The significance represents differences between dividing NBs and daughters, comparing T2–T3 with A6–A10, at the stage indicated (Student’s t test and Mann-Whitney U test, as appropriate based on normality Shapiro-Wilk test; SD; n ≥ 10 embryos per genotype and stage).
See also Figure S2 and Tables S3 and S4.
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5. Figure 3 Temporal and Segmental BX-C Hox Expression in NBs
(A) Ubx, Abd-A, and Abd-B expression in the VNC of control embryos, segments T1–A10, at St11 (top) and St13 (bottom); scale bar, 10 μm. For each Hox protein, St11 and St13 VNCs were scanned on the same slide, using identical confocal microscopy settings; images are projections of the entire thickness of the VNC. At St11, expression of Ubx and Abd-A is weak, whereas Abd-B is more clearly expressed in segments A8–A10. At St13, expression is evident of all three Hox proteins and shows the anticipated segmental A-P extension.
(B and C) Cartoon of Hox protein expression at St11 (B) and St13 (C).
(D–F) Quantification of the protein expression levels specifically in NBs, in segments T1–A10, between St11 and St13, plotted for each segment of the VNC. Ubx (D), Abd-A (E), and Abd-B (F) are shown. Red lines represent intensity mode of the slide considered as background threshold (3.5 in D; 0.5 in E; 0.4 in F).
(G) Magnification of St13 hemisegments showing Ubx, Abd-A, and Abd-B expression in A1, A5, and A9–10, respectively (dashed lines indicate NBs; scale bar, 10 μm).
See also Figure S3.
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6. Figure 4 BX-C Hox Genes Control Daughter Proliferation Posteriorly
(A–F) Increase of mitotic daughters (white dashed circles) and mitotic NBs (yellow dashed circles) when compared to control (A, C, and E) in Ubx (B), abd-A (D), and Abd-B (F) mutants (scale bar, 10 μm).
(G–I) Quantification of dividing daughters and NBs, at St13, in control and BX-C (Student’s t test; Mann-Whitney U-test; SD; n ≥ 10 embryos per genotype).
(J) Analysis of the A9 NB3-5 lineage in control and Abd-B mutants at St16 reveals aberrant daughter and NB proliferation (Chi-square test: p ≥ for NBs and p ≥ for daughters; n ≥ 22 lineages per genotype; dashed yellow circle depicts the NB; dashed white circle depicts mitotic daughter cells; scale bar, 5 μm).
See also Figure S4.
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7. Figure 5
BX-C Hox Misexpression Suppresses Daughter and NB Proliferation Anteriorly
(A–C’) Mitotic daughters (white dashed circles) and mitotic NBs (yellow dashed circles) in control (A), pros>BX-C misexpression (B), and pros>BX-C; ED225 (C). PH3+ signal is shown for the respective genotypes control (A’), pros>BX-C misexpression (B’), and pros>BX-C; ED225 (C’).
(D and E) Quantification of dividing daughters and NBs in single and triple misexpression of Ubx, abd-A, and Abd-B using pros-Gal4 driver in control and ED225 background (Student’s t test; SD; n ≥ 10 embryos per genotype).
(F–I) Cell numbers in NB5-6T lineage in control (F) and BX-C misexpression (G) and in NB3-5T lineage in control (H) and BX-C misexpression (I).
(J–M) Quantification of cell number in NB5-6T (J) and NB3-5T (K) (Student's test; SD; n ≥ 40) and frequency of dividing NBs and daughters in NB5-6T (L) and NB3-5T (M) lineages (Chi-square test; n ≥ 60 lineages) in NB5-6 and NB3-5 lineages in pros>BX-C misexpression.
See also Figure S5.
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8. Figure 6 BX-C Hox Genes Regulate E2F1, CycE, Stg, and Dap
(A–I’) Cell-cycle protein expression in NBs (white dashed circles) of Dap in control (A, A’, D, and D’), Ubx mutant (B and B’), pros>Ubx (C and C’), abd-A mutant (E and E’), and pros>abd-A (F and F’) and for CycE in control (G and G’), Abd-B mutant (H and H’), and pros>Abd-B (I and I’) at St13.
(J–O) Quantification of expression levels in NBs (mitotic and non-mitotic) of Dap, CycE, E2F1, and Stg in Ubx mutant (J), pros>Ubx (K), abd-A mutant (L), pros>abd-A (M), Abd-B mutant (O), and pros>Abd-B (P) (Student’s t test; SD; n ≥ 10 embryos per genotype).
(P) Proportion of Dap-positive NBs at St13 in BX-C mutants and misexpression (Chi-square test; n ≥ 10 embryos per genotype).
(Q) Cartoon summarizing the regulation of Dap, CycE, E2F1, and Stg by BX-C (activation depicted by arrows; inhibition by prune end lines), concluded from mutant and misexpression phenotypes. Dashed lines denote regulation observed only in mutant or misexpression background, whereas solid lines refer to effects observed in both.
See also Figure S6.
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9. Figure 7
Model of Hox-Mediated Proliferation Control along the A-P Axis in the VNC
In the early VNC, BX-C and Antp Hox expression is absent (or low) in NBs (this study) [6, 7]. Subsequently, Hox expression commences in NBs, which triggers the type I > 0 daughter proliferation switch and ultimately NB cell-cycle exit. The overlapping gradients of Hox expression result in earlier type I > 0 proliferation switches and NB exit in more posterior segments, therefore generating fewer cells/lineage in posterior segments and developing a wedge-like profile in the VNC. PCD (crosses) plays an important role in removing both NBs and postmitotic cells, but the A-P gradient in proliferation and lineage size is evident even in PCD mutants.
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