1. Intra-lineage Fate Decisions Involve Activation of Notch Receptors Basal to the Midbody in Drosophila Sensory Organ Precursor Cells 
Mateusz Trylinski, Khalil Mazouni, François Schweisguth 
Current Biology 
Volume 27, Issue 15, Pages e3 (August 2017)
DOI: /j.cub
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2. Figure 1 Two Pools of Notch along the pIIa-pIIb Interface
(A–A”) NiGFP (green) transiently accumulated at the apical pIIa-pIIb interface at t15 in living pupae. The pIIa/pIIb nuclei (outlined in A’) were marked with iRFP670nls. A faint NiGFP signal was detected along the lateral interface at t15 (arrow, A’; the apical surface area of the pIIa and pIIb cells are indicated in A). In these and other panels, the “apical” and “basal” labels refer to the x,y sections showing the apical and “lateral” populations of Notch receptors (see E).
(B and C) Quantification of nuclear NiGFP (B) and GFP-E(spl)m8-HLH (C) levels during cytokinesis (t0; metaphase-anaphase transition). Nuclear NiGFP (n ≥ 21 for each time point) and GFP-E(spl)m8-HLH (n ≥ 18) reached a plateau at t30. Data are represented as mean ± SEM.
(D–D”) Morphology of the pIIa-pIIb contact at t30 (BazCherry, magenta; DlgGFP, green; iRFP670nls, red). Two contact regions were seen apical and basal to the midbody. Note the absence of pIIa-pIIb contact at the level of the midbody (arrow in D’).
(E) Schematic representation of the apical and lateral contact regions along the pIIa-pIIb interface.
(F and F’) Quantification of the apical (F) and lateral (F’) pools of NiGFP in wild-type (green; n ≥ 21) and numbRNAi (red; n ≥ 27) pupae. Significant levels of NiGFP were transiently detected apical to the midbody in wild-type pupae. The silencing of numb led to NiGFP accumulation specifically at the lateral interface. Data are represented as mean ± SEM.
(G–G”) NiGFP (green; iRFP670nls; marking the pIIa/pIIb nuclei, red) accumulated in dots along the lateral interface (arrow) at t15 in numbRNAi pupae.
(H–H”) The loss of Psn activity in mutant clones led to the accumulation of NiGFP (green) along the lateral pIIa-pIIb interface in living mosaic pupae (arrow; pIIa/pIIb nuclei were identified using iRFP670nls, red; mutant cells were marked by the loss of a RFP marker, white; only isolated mutant SOPs were studied).
In these and all other panels, anterior is left (i.e., pIIb, left, and pIIa, right) and scale bars are 5 μm. See also Figure S1.
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3. Figure 2 Ligand Endocytosis from the Lateral pIIa-pIIb Interface
(A–A”) Dl (anti-Dl intracellular domain, green) localized mostly below the midbody (marked with MyoII, not shown; CadGFP, magenta). High-magnification views of the pIIa-pIIb interface (arrows in A and A’; pIIa and pIIb nuclei were marked with iRFP670nls, not shown) are shown as insets.
(B–C”) DlGFP (green) was detected in fixed nota (B–B”) and in living pupae at t15 (C–C”). The pIIa and pIIb nuclei were marked with iRFP670nls (red in C–C”; not shown in B–B”). A faint DlGFP signal was detected along the pIIa-pIIb interface in living pupae. By contrast, a clear anti-GFP signal was seen basal to AJs (Cad, magenta in B–B”) and midbody (MyoII; not shown) in fixed nota.
(D–D”) DlGFP (green) specifically accumulated along the lateral interface (arrow in D’) in living neur > BrdR pupae at t15, suggesting that Dl is endocytosed in a Neur-dependent manner from the lateral pIIa-pIIb interface.
(E–E”) Distribution of NeurGFP (green) at t15 in living pupae. NeurGFP localized both apical and basal (arrow in E’) to the midbody (iRFP670nls, red).
See also Figure S2.
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4. Figure 3 NICD Originated from Recently Synthetized Receptors
(A and B) Structure (A) and properties (B) of NiGFP4Cherry5 (adapted from [38]). GFP (green) and Cherry (red) are intracellular. Upon synthesis of Notch, GFP, and Cherry are in a dark immature state. NiGFP4Cherry5 molecules are more likely to become first GFP-bright and Cherry-dark due to the faster maturation of GFP relative to Cherry, before being both GFP- and Cherry-bright. GFP-bright-only molecules were detected at the cell surface and in early endosomes, whereas Cherry-bright receptors were detected into acidic intracellular compartments (where GFP is quenched at low pH).
(C and C’) Snapshots showing the fluorescence of NiGFP4Cherry5 at t30 (GFP, green and Cherry, red). The pIIa and pIIb nuclei, marked with iRFP670nls (not shown), are outlined. Nuclear NiGFP4Cherry5 was detected in pIIa only by GFP fluorescence.
(D and E) Quantification of the GFP (D) and Cherry (E) fluorescence produced by nuclear NiGFP4Cherry5 (n = 24). NICD came from a pool of GFP-bright and Cherry-dark receptors. Data are represented as mean ± SEM.
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5. Figure 4 Tracing the Origin of NICD Using Photo-Bleaching
(A) Principle of Notch photo-tracking using photo-bleaching. A loss of nuclear fluorescence upon the photo-bleaching of a specific pool of NiGFP (lateral pool, greyed) would indicate that photo-bleached receptors contribute to NICD production.
(B–B”) Selective photo-bleaching of the pIIa-pIIb interface (shown here using CadGFP, green; nuclei marked with iRFP670nls, red) in the notum. The position of the apical interface (yellow dotted box in B) did not coincide with the lateral membrane (B’) due to a posterior tilt of the pIIa-pIIb interface (B”; white dotted lines show the angle of the pIIa-pIIb interface relative to the apical-basal axis).
(C–F) Effect of selective photo-bleaching (apical pool, C and C’; lateral pool, E and E’) on nuclear NiGFP fluorescence (D and F). The apical and lateral fluorescence profiles of NiGFP at t30 (control, green) were specifically changed upon repeated photo-bleaching. Apical photo-bleaching efficiently reduced the fluorescence of apical NiGFP (C, blue) but had no effect on basal NiGFP (C’, blue profile) and vice versa (E and E’; brown profiles). Photo-bleaching the lateral pool of NiGFP specifically reduced the level of nuclear NiGFP fluorescence in pIIa at t30 (F; n = 19). By contrast, photo-bleaching the lateral pool of NiGFP had no significant effect (D; n = 21). In (C), (C’), (E), and (E’), data are represented as mean ± SEM.
(G) Live imaging of sensory cells in pupal legs. Pupae are mounted legs up (top). Pupal legs appeared as tubular epithelia at the body surface so that pIIa-pIIb cells located laterally could be imaged on side views (bottom).
(H) Distribution of NiGFP (green) and BazCherry (magenta) along the pIIa-pIIb interface in pupal legs of living pupae. The lateral pool of NiGFP was clearly observed at t15.
(I and J) Nuclear NiGFP fluorescence at t30 following selective photo-bleaching in pupal legs. Bleaching the apical pool of NiGFP had no significant effect (I; n = 19), whereas bleaching the lateral pool specifically reduced nuclear NiGFP fluorescence in pIIa (J; n = 21).
n.s., not significant; ∗∗∗p < See also Figure S3.
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6. Figure 5 Photo-Tracking Notch Using Photo-Convertible Receptors
(A) Structure of NimMaple3 (as in Figure 3A). mMaple3 was inserted at position 2388 by CRISPR-mediated HR.
(B–C’) NimMaple3 was detected at the cell cortex prior to photo-conversion (B; green channel) and was efficiently photo-converted (C’, red channel).
(D–E”) Photo-converted NimMaple3 (red/false colors) was detected in pIIa nuclei (SensGFP, green) at t30 in pupal legs. The red fluorescence emitted by NimMaple3 was measured before (D–D”) and after photo-conversion (E–E”). Raw (D’ and E’) and processed signals (D’’ and E’’; using a threshold selecting the 5% brightest pixels of pIIa) are shown. Note the strong auto-fluorescence signal from the pre-cuticle (asterisk in D and E).
(F) Plot showing the pIIa/pIIb ratio values of nuclear photo-converted NimMaple3 for different thresholds (indicated as the percentage of brightest pixels in the pIIa nucleus). Raw data (threshold value 100; lateral photo-conversion versus control) displayed statistically significant differences. Image processing efficiently extracted the signal of nuclear photo-converted NimMaple3, as shown by the increasing difference between ratio values measured before (−405 nm) and after photo-conversion (+405 nm; n = 6).
(G–G”) Photo-converted NimMaple3 (red/false colors) was detected in pIIa nuclei (SensGFP, green) at t30 in pupal legs following repeated UV illumination of the lateral domain at t15, t20, and t25.
(H) Plot showing the pIIa/pIIb ratio values of photo-converted NimMaple3 (as in F) in control cells (no photo-conversion, black) and upon photo-conversion of the apical (red) and lateral (blue) pools. Higher levels of photo-converted NICD were detected in pIIa upon photo-conversion of the lateral versus apical pool of Notch (n = 25 for each condition).
In (F)–(H), data are represented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < See also Figure S4.
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