Abstract
The Drosophila egg chamber is a powerful system to study epithelial cell collective migration and polarized basement membrane secretion. A strength of this system is the ability to capture these dynamic processes in ex vivo organ culture using high-resolution live imaging. Ex vivo culture also allows acute pharmacological or labeling treatments, extending the versatility of the system. However, many current ex vivo egg chamber culture setups do not permit easy medium exchange, preventing researchers from following individual egg chambers through multiple treatments. Here we present a method to immobilize egg chambers in an easy-to-construct flow chamber that permits imaging of the same egg chamber through repeated solution exchanges. This will allow researchers to take greater advantage of the wide variety of available pharmacological perturbations and other treatments like dyes to study dynamic processes in the egg chamber.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Horne-Badovinac S, Bilder D (2005) Mass transit: epithelial morphogenesis in the Drosophila egg chamber. Dev Dyn 232:559–574. https://doi.org/10.1002/dvdy.20286
Haigo SL, Bilder D (2011) Global tissue revolutions in a morphogenetic movement controlling elongation. Science 331:1071–1074. https://doi.org/10.1126/science.1199424
Cetera M, Juan GRR-S, Oakes PW, Lewellyn L, Fairchild MJ, Tanentzapf G, Gardel ML, Horne-Badovinac S (2014) Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber elongation. Nat Commun 5:5511. https://doi.org/10.1038/ncomms6511
Isabella AJ, Horne-Badovinac S (2016) Rab10-mediated secretion synergizes with tissue movement to build a polarized basement membrane architecture for organ morphogenesis. Dev Cell 38:47–60. https://doi.org/10.1016/j.devcel.2016.06.009
Lerner DW, McCoy D, Isabella AJ, Mahowald AP, Gerlach GF, Chaudhry TA, Horne-Badovinac S (2013) A Rab10-dependent mechanism for polarized basement membrane secretion during organ morphogenesis. Dev Cell 24:159–168. https://doi.org/10.1016/j.devcel.2012.12.005
Lewellyn L, Cetera M, Horne-Badovinac S (2013) Misshapen decreases integrin levels to promote epithelial motility and planar polarity in Drosophila. J Cell Biol 200:721–729. https://doi.org/10.1083/jcb.201209129
Viktorinová I, Dahmann C (2013) Microtubule polarity predicts direction of egg chamber rotation in Drosophila. Curr Biol 23:1472–1477. https://doi.org/10.1016/j.cub.2013.06.014
Chen D-Y, Lipari KR, Dehghan Y, Streichan SJ, Bilder D (2016) Symmetry breaking in an edgeless epithelium by Fat2-regulated microtubule polarity. Cell Rep 15:1125–1133. https://doi.org/10.1016/j.celrep.2016.04.014
Squarr AJ, Brinkmann K, Chen B, Steinbacher T, Ebnet K, Rosen MK, Bogdan S (2016) Fat2 acts through the WAVE regulatory complex to drive collective cell migration during tissue rotation. J Cell Biol 212:591–603. https://doi.org/10.1083/jcb.201508081
Viktorinová I, Henry I, Tomancak P (2017) Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations. PLoS Genet 13:e1007107. https://doi.org/10.1371/journal.pgen.1007107
Alégot H, Pouchin P, Bardot O, Mirouse V (2018) Jak-Stat pathway induces Drosophila follicle elongation by a gradient of apical contractility. elife 7:e32943. https://doi.org/10.7554/elife.32943
Dent LG, Manning SA, Kroeger B, Williams AM, Hilmi AJS, Crea L, Kondo S, Horne-Badovinac S, Harvey KF (2019) The dPix-Git complex is essential to coordinate epithelial morphogenesis and regulate myosin during Drosophila egg chamber development. PLoS Genet 15:e1008083. https://doi.org/10.1371/journal.pgen.1008083
Stedden CG, Menegas W, Zajac AL, Williams AM, Cheng S, Özkan E, Horne-Badovinac S (2019) Planar-polarized Semaphorin-5c and Plexin A promote the collective migration of epithelial cells in Drosophila. Curr Biol 29:908–920.e6. https://doi.org/10.1016/j.cub.2019.01.049
Campos FC, Dennis C, Alégot H, Fritsch C, Isabella A, Pouchin P, Bardot O, Horne-Badovinac S, Mirouse V (2020) Oriented basement membrane fibrils provide a memory for F-actin planar polarization via the Dystrophin-Dystroglycan complex during tissue elongation. Dev 147:dev186957. https://doi.org/10.1242/dev.186957
Sherrard KM, Cetera M, Horne-Badovinac S (2021) DAAM mediates the assembly of long-lived, treadmilling stress fibers in collectively migrating epithelial cells in Drosophila. elife 10:e72881. https://doi.org/10.7554/elife.72881
Williams AM, Donoughe S, Munro E, Horne-Badovinac S (2022) Fat2 polarizes the WAVE complex in trans to align cell protrusions for collective migration.eLife 11:e78343. https://doi.org/10.7554/eLife.78343
Zajac AL, Horne-Badovinac S (2022) Kinesin-directed secretion of basement membrane proteins to a subdomain of the basolateral surface in Drosophila epithelial cells. Curr Biol 32:735–748.e10. https://doi.org/10.1016/j.cub.2021.12.025
Prasad M, Jang AC-C, Montell DJ (2007) A protocol for culturing Drosophila melanogaster egg chambers for live imaging. Protoc Exch. https://doi.org/10.1038/nprot.2007.233
Prasad M, Montell DJ (2007) Cellular and molecular mechanisms of border cell migration analyzed using time-lapse live-cell imaging. Dev Cell 12:997–1005. https://doi.org/10.1016/j.devcel.2007.03.021
Cetera M, Lewellyn L, Horne-Badovinac S (2016) Drosophila, methods and protocols. Methods Mol Biol Clifton N J 1478:215–226. https://doi.org/10.1007/978-1-4939-6371-3_12
Dixit R, Ross JL (2010) Chapter 27 – Studying plus-end tracking at single molecule resolution using TIRF microscopy. Methods Cell Biol 95:543–554. https://doi.org/10.1016/s0091-679x(10)95027-9
Chanet S, Huynh J-R (2020) Collective cell sorting requires contractile cortical waves in germline cells. Curr Biol. https://doi.org/10.1016/j.cub.2020.08.045
Wilcockson SG, Ashe HL (2019) Drosophila ovarian germline stem cell Cytocensor projections dynamically receive and attenuate BMP Signaling. Dev Cell 50:296–312.e5. https://doi.org/10.1016/j.devcel.2019.05.020
Wilcockson SG, Ashe HL (2021) Live imaging of the Drosophila ovarian germline stem cell niche. Star Protoc 2:100371. https://doi.org/10.1016/j.xpro.2021.100371
Drummond-Barbosa D, Spradling AC (2001) Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev Biol 231:265–278. https://doi.org/10.1006/dbio.2000.0135
Mazzalupo S, Cooley L (2006) Illuminating the role of caspases during Drosophila oogenesis. Cell Death Differ 13:1950–1959. https://doi.org/10.1038/sj.cdd.4401892
Pritchett TL, Tanner EA, McCall K (2009) Cracking open cell death in the Drosophila ovary. Apoptosis 14:969. https://doi.org/10.1007/s10495-009-0369-z
Acknowledgments
American Heart Association 16POST2726018, American Cancer Society 132123-PF-18-025-01-CSM, and Chicago Biomedical Consortium FP064171-01-PR postdoctoral fellowships to A.L.Z., NIH T32 HD055164 to A.M.W., and work in the Horne-Badovinac Lab is supported by NIH R01s GM126047 and GM136961 to S.H-B.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Zajac, A.L., Williams, A.M., Horne-Badovinac, S. (2023). A Low-Tech Flow Chamber for Live Imaging of Drosophila Egg Chambers During Drug Treatments. In: Giedt, M.S., Tootle, T.L. (eds) Drosophila Oogenesis. Methods in Molecular Biology, vol 2626. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2970-3_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2970-3_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2969-7
Online ISBN: 978-1-0716-2970-3
eBook Packages: Springer Protocols