Notch signaling regulates timing of CB NB elimination and neurogenesis termination.

(A) Schematic showing developmental timeframe of CB NB elimination with timeline below used for developmental staging. (B-C, G-I) Maximum intensity projections of single brain hemispheres from indicated genotypes, times, with markers listed in bottom left. Asterisks indicate the four MB NBs and arrowheads indicate some of the ectopically proliferating CB NBs (non-MB NBs). One ectopic CB NB (white box) shown at higher magnification in bottom right. (D,J) Quantification of CB NB number (excluding MB NBs) per brain hemisphere at indicated times and genotypes. Each data point represents one brain hemisphere, mean ± SEM, ***p-value≤0.001 (unpaired two-tailed Student’s t-test). (E,K) Quantification of average CB NB diameter, used as a proxy for NB size, at indicated times and genotypes. Each data point equals one CB NB (n≥4 animals per genotype), mean±SEM. (F) Single optical section of a brain hemisphere from indicated genotype at 48 hours APF with markers listed in panels with high magnification panel to right of ectopic CB NB in white box. Distribution of Notch55e11 MARCM CB NB clones containing Dpn positive NBs. Scale bar equals 20 μms (panels) or 10 μms (insets) in this and all subsequent figures. Panel genotypes listed in Supplemental Table 1.

Delta expressed in CB NBs, GMCs, and cortex glia regulates CB NB Notch activity.

(A-F) Single optical section of a brain hemisphere from the indicated genotypes at wandering L3 stages. Higher magnification image of the CB NB highlighted by the white box is shown to the right of the colored overlays. Top panels are higher magnification colored overlay with single channel grayscale images below. White brackets indicate the CB NB and yellow brackets indicate newborn GMC progeny. (G-L) Single optical section of a brain hemisphere from the indicated genotypes at 72 hours ALH. Higher magnification image of the CB NB highlighted by the white box is shown to the right of the colored overlays. Scale bar equals 20 μms (panels) and 10 μms (insets). CB: central brain; OL: optic lobe. Panel genotypes listed in Supplemental Table 1.

Delta is required to eliminate CB NBs and terminate neurogenesis.

(A,B,E) Maximum intensity projections of single brain hemispheres from indicated genotypes. Asterisks indicate the MB NBs and the white arrowheads indicate some of the ectopically proliferating CB NBs. Inset shows a higher magnification of the ectopically proliferating CB NB highlighted by the white box. (C,F,H,J) Quantification CB NB number (excluding the MB NBs). Each data point represents one brain hemisphere. Control data in (C) is the same as Figure 1D. Mean±SEM.***P≤0.001 (unpaired two-tailed Student’s t-test). (D,G,I) Quantification of CB NB size (excluding the MB NBs) in the indicated genotypes and developmental times. Each data point represents one neuroblast (n≥4 animals per genotype). Control data in (D) is the same as Figure 1E. Mean±SEM.***P≤0.001, *P≤0.033 (Kruskal-Wallis test). Scale bar equals 20 μms (panels) and 10 μms (insets). Panel genotypes listed in Supplemental Table 1.

Notch is required early to eliminate CB NBs.

(A,B) Top, schematic depicting experimental setup to temporally control knockdown of Notch signaling. Bottom, maximum intensity projections of single brain hemispheres from indicated genotypes at 48 hours APF relative to 25⁰C. Asterisks indicate the MB NBs and the white arrowheads indicate some of the ectopically proliferating CB NBs. Inset shows a higher magnification of an ectopically proliferating CB NB highlighted by the white box. (C) Quantification of CB NB number (excluding the MB NBs) in the indicated genotypes. Each data point represents one brain hemisphere. Mean±SEM. .***P≤0.001 (Welch’s t test). Scale bar equals 20 μms (panels) and 10 μms (insets). Panel genotypes listed in Supplemental Table 1.

Notch signaling refines temporal factor expression boundaries.

(A) Schematic of temporal factor expression in CB NBs during larval and pupal development. Top timeline (days) refers to developmental timing with two timelines below used for developmental staging. Larva hatch 22 hours after egg lay. (B,I) Quantification of the percentage of CB NBs (excluding the MB NBs) in the indicated genotypes and developmental times expressing the indicated temporal factors. Mean±SEM. n≥3 animals, ***P≤0.001, **P≤0.002, *P≤0.033 (Two-way ANOVA). (C-H) Single optical section of a brain hemisphere from the indicated genotypes and developmental times. Higher magnification image of the CB NB highlighted by the white box is shown below the colored overlays. White brackets indicate the CB NB. (J) Single optical section of a CB NB, colored overlay with grayscale image below. White brackets indicate the CB NB with quantification of normalized nuclear E93 intensities. Column numbers indicate the number of CB NB clones (excluding the MB NBs) scored. Mean±SEM.***P≤0.001 (Mann-Whitney test). Scale bar equals 20 μms (panels) and 10 μms in single CB NB panels. CB: central brain; OL: optic lobe. Panel genotypes listed in Supplemental Table 1.

CB NBs ectopically persist due to prolonged early factor Imp expression and reduced late factor E93 expression.

(A,B) Single optical sections of CB NBs from indicated genotypes and developmental times. Single channel grayscale images with colored overlay to the right. White brackets indicate CB NBs. Right, percentage of CB NBs (excluding the MB NBs) in the indicated genotypes and developmental times expressing the indicated temporal factors. Column number (A) indicates number of clones scored. Mean±SEM. n≥3 animals (B). (C-F) Maximum intensity projections of single brain hemispheres from indicated genotypes in one-day old adults. White arrowheads indicate some ectopically proliferating CB NBs (MB NBs are absent). Inset shows a higher magnification of an ectopically proliferating CB NB highlighted by the white box. (G) Quantification of CB NB number (excluding MB NBs) in the indicated genotypes. Each data point represents one brain hemisphere. Mean±SEM. ***P≤0.001, *P≤0.033 (Kruskal-Wallis ANOVA). (H) Quantification of CB NB size (excluding the MB NBs) in the indicated genotypes. Each data point represents one CB NB (n≥4 animals per genotype). Mean±SEM. ***P≤0.001, *P≤0.033 (Kruskal-Wallis ANOVA). Scale bar equals 20 μms (panels) and 10 μms (insets). Panel genotypes listed in Supplemental Table 1.

Delta is expressed at higher levels early and is positively regulated by the early temporal factor Imp.

(A-B) Heat map showing the list of genes that are differentially expressed in the AL NBs from 24 to 84 hours ALH. In (B) genes are color coded to show if they are also targets of either Imp (yellow) or Syp (purple) or both (red asterisk). Variance-stabilizing transformation (VST) normalized counts were used to plot the heat maps. (C) Venn diagram showing the number of target genes analyzed that are common between Notch, Imp and Syp. (D-G) Single optical section of a brain hemisphere from the indicated genotypes at 24 hours ALH expressing Delta-GFP. Higher magnification image of the CB NB highlighted by the white box is shown to the right of the single channel grayscale images. Top panels are higher magnification colored overlay with single channel grayscale images below. White brackets indicate the NB. Scale bar equals 10 μms. Panel genotypes listed in Supplemental Table 1. (H) Model of Delta-dependent Notch activation in regulation of CB NB temporal patterning.

Drosophila genotypes per figure panel

Key Resources

Temporal control of Notch activity in CB NBs. Related to Figure 4.

(A-B) Cartoon above depicts temperature shift regime. Below, single optical sections of brain hemispheres from indicated genotypes and developmental times, with markers listed in panels. Higher magnification image of the CB NB highlighted by the white box is shown to the right of the colored overlays. White arrow heads indicate some of the ectopically persisting CB NBs. Scale bar equals 20 μms (panels) and 10 μms (inset). Panel genotypes listed in Supplemental Table 1.

Imp expression is prolonged and Syp expression delayed in animals with reduced Notch pathway activity. Related to Figure 5.

(A-C) Single optical section of a brain hemisphere from the indicated genotypes at 72 hours ALH, with markers listed to left. White arrowheads indicate CB NBs with ectopic expression of either Imp-GFP or no Syp expression. (D) Quantification of the percentage of CB NBs (excluding the MB NBs) in the indicated genotypes at 72 hours ALH expressing the indicated temporal factors. Mean±SEM. n≥3 animals, ***P≤0.001 (Two-way ANOVA). (E) Quantification of the percentage of PH3 positive CB NBs (excluding the MB NBs) in the indicated genotypes at WL3 (wandering L3 stage). (F) Image of a mitotic non-MB NB at 48 hours ALH in Dl RNAi expressing animals. (G) Maximum intensity projection of a single brain hemisphere from indicated genotype and time with markers listed in bottom left. Asterisks indicate the MB NBs and arrowheads indicate some of the ectopically proliferating CB NBs (non-MB NBs). One ectopic CB NB (white box) shown at higher magnification in bottom right. Scale bar (A,G) equals 20 μms and inset (G) equals 10 μms. Panel genotypes listed in Supplemental Table 1.

Defects in temporal patterning account for defects in timing of CB NB elimination and neurogenesis termination when Notch activity is reduced. Related to Figure 6.

(A) Maximum intensity projection of single brain hemisphere from indicated genotype at indicated time. (B) Quantification of CB NB number (excluding the MB NBs) in the indicated genotypes. Each data point represents one brain hemisphere. Mean±SEM. ***P≤0.001, **P≤0.002, (Kruskal-Wallis ANOVA). Scale bar equals 20 μms. Panel genotypes listed in Supplemental Table 1.