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. 2021 Dec 20;56(24):3393–3404.e7. doi: 10.1016/j.devcel.2021.11.016

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© 2021 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Temporal control of gene expression in the pupal dorsal thorax (notum)

Illumination settings used for photoactivation and imaging are given in Table S1A. N, number of animals.

(A) Schematic representation of the optogenetic split Gal4-Magnets used to induce gene expression by blue light photoactivation. Gal4 DBD (aa 1–147) and p65 AD (aa 425–548) are respectively fused to nMagnet and pMagnet tandems, which heterodimerize upon blue light exposure and promote Gal4-mediated activation of gene expression.

(B) Transgenes used to ubiquitously express Gal4DBD:2xnMagHigh1 and 2xpMagHigh1:AD.

(C) Dorsal view of a 14 h after pupa formation (hAPF) Drosophila pupa (top, from Bosveld et al., 2012) and a multiposition confocal image of the notum epithelium (bottom), showing the distribution of Ecad:GFP. The black box indicates the region shown in the bottom panel. Inset: close-up on Ecad:GFP distribution in the region outlined by a white box.

(D) Time-lapse images of ubi-Gal4^MagHigh>His2B:RFP (top and bottom) and Ecad:GFP (bottom) upon photoactivation. Time (h) is set to 0 at the start of the 491-nm illumination. Red asterisks indicate sensory organ precursors, the nuclei of which are located more basally.

(E) Graph of His2B:RFP fold change (mean ± SEM, N = 5) during photoactivation. Time (h) is set to 0 at the start of the 491-nm illumination.

(F) Time-lapse images of ubi-Gal4^MagHigh>His2B:RFP (top) and act-Gal4>His2B:RFP, tub-Gal80^ts (bottom). Time (h) is set to 0 at the start of the 491-nm illumination or upon temperature shift from 18°C to 29°C.

(G) Graph of the normalized His2B:RFP intensity (mean ± SEM) for the experiments shown in (F) (red, N = 5 and purple, N = 3) and upon His2B:RFP photobleaching (FRAP) in act-Gal4>His2B:RFP pupae (green, N = 4). Red and purple curves: values are normalized to the maximal ones in each condition. Green curve: values of the bleached region are normalized to the ones of the non-bleached region. Dashed lines: t_1/2 of His2B:RFP protein accumulation. See also Figures S1F and S1G.

(H) Time-lapse images of ubi-Gal4^MagHigh>His2B:RFP following a 1 h pulsed 491-nm illumination. Images are taken in the plane of the nucleus, the AJ Ecad:3xmKate2 is not visible. Time (h) is set to 0 at the end of the 491-nm illumination.

(I) Graph of normalized His2B:RFP intensity (mean ± SEM, N = 4) for the experiment shown in (H). To ensure that His2B:RFP is accurately quantified, the measured His2B: RFP intensity is corrected by the background intensity due to Ecad:3xmKate2 signal in the nuclei plane. Values are normalized to the maximal value.

Scale bars: 50 μm (C) and 10 μm (D, F, H, and inset in C).

See also Figures S1 and S2 as well as Video S1; Table S1A.