Imaging Cytonemes in Drosophila Embryos
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Imaging Cytonemes in Drosophila Embryos
Methods Mol Biol
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Conserved morphogenetic signaling proteins disperse across tissues to generate signal and signaling gradients, which in turn are considered to assign positional coordinates to the recipient cells. Recent imaging studies in Drosophila model have provided evidence for a "direct-delivery" mechanism of signal dispersion that is mediated by specialized actin-rich signaling filopodia, named cytonemes. Cytonemes establish contact between the signal-producing and target cells to directly exchange and transport the morphogenetic proteins. Although an increasing amount of evidence supports the critical role of these specialized signaling structures, imaging these highly dynamic 200 nm-thin structures in the complex three-dimensional contour of living tissues is challenging. Here, we describe the imaging methods that we optimized for studying cytonemes in Drosophila embryos.
Breathless; Cell–cell signaling; Cytonemes; Drosophila; Embryo; Engrailed; Filopodia; Morphogen; Trachea.
Drosophila ASP cytonemes. ( a) A drawing depicting the third instar larval wing disc and air sac primordium (ASP). The ASP cells project cytonemes (green) to contact the bnl (blue) and dpp sources (pink) in the disc for receiving signals. ( b) Live image of CD8:GFP marked cytonemes emanating from the growing tip region of an ASP; the image in ( b) corresponds to an area represented by the dashed square in ( a)
Embryo collection apparatus. (
a) Embryo collection cage. ( b) Grape juice agar plate fits in the plastic cap of the embryo collection cage. ( c) Cell strainer used for embryo dechorionization
Cartoon illustrating the steps for embryo imaging preparation. (
a) Live embryo imaging preparation (description in Subheading 3.4): 1. use a paintbrush to transfer embryos to the center of the cover glass; eliminate excess liquid; 2. add a drop of Halocarbon oil 27 on top of the embryos; 3. gently spread embryos in the oil and make a single layer of embryos; 4. place some wet tissue in the dish surrounding the glass bottom microwell. ( b) Slide preparation for fixed embryos (description in Subheading 3.5): (1) Leave about 100 μL of PBST in Eppendorf tube with embryos; cut off the tip of a p200 pipette tip, pipette embryos onto a microscope slide; remove the extra PBST liquid from the slide. (2) Add two drops of the VECTASHIELD on top of the embryos. (3) Use a needle to gently spread the embryos in a single layer, gently lower the coverslip (arrow) onto the embryos in VECTASHIELD without including an air bubble
Examples of cytonemes from fixed and live embryonic trachea. (
a) Confocal images of a fixed embryo at stage 14 showing dorsal tracheal branches (green), each growing toward a dorsal bnl source (red); genotype: btl-Gal4,UAS-CD8:GFP/+; bnl-LexA,lexO-CherryCAAX/+. ( b) A stage 14 embryo showing cytonemes from the dorsal (1) and ventral (2) tracheal branches without marking the bnl-source; genotype: btl-Gal4; UAS-CD8: GFP. Top panel, a z-projection image of 34 optical sections across 51 μm depth of a whole embryo, imaged in fixed condition with a 40× objective in Leica SP5X. Bottom panels, images of cytonemes from a dorsal (1) and a ventral (2) branch of a live stage 14 embryo; these cytonemes project toward the bnl-source ( see panel a); imaging condition: 40× objective with 2× zoom factor, focusing a growing tip of a branch. (c) Schematic drawing of a stage 14 trachea metamere (green) and the bnl-sources (red), arrows point to the dorsal (1) and the ventral (2) branches as shown in ( b)
Example of cytonemes from live embryonic epidermal cells that express
engrailed ( en-Gal4). ( a) Schematic drawing of a stage 12–13 embryo with engrailed-expressing stripes. ( b) CD4:IFP2 expressing cells in the engrailed stripes project short cytonemes; 40× magnification with a 2× zoom of ROI function; the region shown in ( b) corresponds to the dashed square in ( a); scale bar, 10 μm
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Research Support, N.I.H., Extramural
Drosophila Proteins / metabolism*
Drosophila melanogaster / embryology*
Drosophila melanogaster / metabolism
Drosophila melanogaster / ultrastructure*
Embryo, Nonmammalian / metabolism
Embryo, Nonmammalian / ultrastructure*
Image Processing, Computer-Assisted / methods*
Microscopy, Confocal / methods*
Pseudopodia / ultrastructure*
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