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. 2022 Mar 4;13:1185. doi: 10.1038/s41467-022-28587-z

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — A Schematic depiction of htl>FRT>stop>FRT>Gal4 construct and its application to generate FLIP-out clones exclusively in AMPs (also see Supplementary Fig. 1E, F). B-B” Wing disc harboring random CD8:GFP-marked AMP clones (hs-Flp; UAS-CD8:GFP; htl>FRT>stop>FRT>Gal4) showing orthogonal and lateral polarity of AMPs and AMP cytonemes relative to disc plane; arrow and dashed arrow, proximal and distal AMPs/AMP cytonemes, respectively; arrowhead, distal small non-polar cells; *, adherent distal AMPs; phalloidin (red), actin-rich disc-AMP junction and cell-cortex (also see Supplementary Fig. 1G–H”); B’ GFP channel of B; B”’ Violin plots showing angles (Theta, θ; see Fig. 1G) between proximal and distal AMPs and their cytonemes relative to their underlying disc plane (see Supplementary Table 2 for statistical analyses). C–E Comparison of AMP cytonemes (arrows) marked by various fluorescent proteins driven by different transcription drivers, in fixed and live tissues, as indicated; arrowhead, actin-rich (phalloidin-marked) apical-junction of disc epithelium; E Graphs comparing length and numbers (count/100 μm length of AMP-disc interface) of orthogonal cytonemes (n = >125 cytonemes for each genotype/condition, imaged from >5 wing disc/genotype under fixed condition and four discs under live condition; see “Source Data” for statistics). F Actin-rich cytonemes (arrow) from wing disc cells expressing mCherryCAAX and Lifeact:GFP (fixed tissue). G–J Live dynamics of AMP cytonemes; G 3D-rendered image showing live cytonemes captured from p-to-d direction of the tissue; H dynamics of cytonemes (arrow) (2 min time-lapse, also see Supplementary Movies 1–4); I Graphs showing numbers of niche-occupying cytonemes over time within selected ROIs; three graph colors, three discs; J Graph showing the distribution of cytonemes lifetimes (n = 77 cytonemes; also see Supplementary Fig. 2D). Source data for B”‘, I, and J are provided as a “Source Data” file. C–J Genetic crosses: enhancer-Gal4/-LexA x UAS-/LexO-fluorescent protein (FP), as indicated. Scale bars: 20 μm; 5 μm (H).