Kaur, P., Saunders, T.E. & Tolwinski, N.S. Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis. Sci Rep 7, 16636 (2017). https://doi.org/10.1038/s41598-017-16879-0
Abstract:
Optogenetics allows precise, fast and reversible intervention in biological processes. Light-sheet microscopy allows observation of the full course of Drosophila embryonic development from egg to larva. Bringing the two approaches together allows unparalleled precision into the temporal regulation of signaling pathways and cellular processes in vivo. To develop this method, we investigated the regulation of canonical Wnt signaling during anterior-posterior patterning of the Drosophila embryonic epidermis. Cryptochrome 2 (CRY2) from Arabidopsis Thaliana was fused to mCherry fluorescent protein and Drosophila β–catenin to form an easy to visualize optogenetic switch. Blue light illumination caused oligomerization of the fusion protein and inhibited downstream Wnt signaling in vitro and in vivo. Temporal inactivation of β–catenin confirmed that Wnt signaling is required not only for Drosophila pattern formation, but also for maintenance later in development. We anticipate that this method will be easily extendable to other developmental signaling pathways and many other experimental systems.
License type:
http://creativecommons.org/licenses/by/4.0/
Funding Info:
This work was supported by an Academic Research Fund (AcRF) grant (MOE2014-T2–2–039) of the Ministry of Education, Singapore and Yale-NUS grant IG16-BS002 to NST. TES was supported by a National Research Foundation NRF Fellowship (NRF2012NRF-NRFF001-094).