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.
Highlights
Dissections of signaling pathways and their downstream processes have centered on in vitro cell culture models and in vivo model organism studies using small molecule, knockdown and overexpression approaches
Blue light can induce oligomerization of A. thaliana Cryptochrome 2 (CRY2) to form ‘photobodies’ in plant and mammalian cells[6,16]. This leads to sequestration and functional inactivation of the protein. We tested this mechanism by fusing the photolyase homology region (PHR) of CRY2 and mCherry to the C terminus of Drosophila β–catenin or Arm protein (Arm-CRY2-mCh)
Various optogenetic systems have been described to date such as the Cryptochrome 2 (CRY2), Phytochrome B (PHYB), LOV and Dronpa systems
Summary
Dissections of signaling pathways and their downstream processes have centered on in vitro cell culture models and in vivo model organism studies using small molecule, knockdown and overexpression approaches. The recent discovery of optogenetic tools has provided a new and effective toolbox for spatial and temporal regulation of proteins in various in vitro and in vivo systems[2,3,4,5]. As β-catenin protein levels increase, it enters the nucleus where it regulates transcription of target genes by associating with the transcription factor TCF13–15 This basic signaling mechanism is widely conserved, and critical in embryogenesis, but whether β-catenin signaling is needed in a constant widespread manner, versus in a temporally and/or spatially restricted manner was not known, because the tools needed to address this question were lacking. We describe our method for applying CRY2 protein fusions to investigate the spatial and temporal regulation of β-catenin protein in the developing embryo
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