Coupling optogenetics and light-sheet microscopy, a method to study Wnt signaling during embryogenesis

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.

Parkinson's disease, aging and adult neurogenesis: Wnt/β‐catenin signalling as the key to unlock the mystery of endogenous brain repair

“…Yang et al (2014), found that optogenetic activation of VM astrocytes can enhance the DAergic differentiation of stem cells and promote brain repair in PD models in vivo and in vitro, very likely via the mediation of WβC activation. Kaur et al (2017) addressed the potential of coupling optogenetics and light-sheet microscopy to study Wnt signalling during embryogenesis, showing that WβC-signalling is required not only for Drosophila pattern formation, but also for maintenance later in development. Additionally, Zhang, Huang, et al (2016c) Ameliorates cognitive function, enhances neurogenesis, mitigates inflammation and mitochondrial dysfunction in hippocampus in a rodent model of gulf war illeness; WβC-AC involvement to be elucidated Kodali et al (2018) Exercise, environmental enrichment…”

“…Optogenetic activation of VM astrocytes enhances DAergic differentiation of NSCs and promotes brain repair in PD rodent models; WβC-AC involvement to be elucidated Yang et al (2014) Optical depolarization of DCX-expressing neuroblasts promotes cognitive recovery and maturation of newborn neurons after traumatic brain injury via WβC-AC Zhang, Huang, et al (2016c) Coupling of optogenetics and light-sheet microscopy reveals WβC-AC during embryogenesis and post-natal development Kaur et al (2017) WβC-AC can be controlled in vivo via light responsive capsules. Ambrosone et al (2016) Optical depolarization promoted the maturation of neural stem cells via WβC-AC Xia et al (2014) (Continues)…”

“…Herein, after a description of the key Wnt‐signalling components and a synopsis of adult neurogenesis in PD, we will focus on the role of WβC‐signalling as a common final pathway in mediating NSC regulation, from development to aging and PD degeneration. We aim to survey recent literature in the field supporting the upregulation of WβC as a means to re‐activate neurogenesis and incite regeneration in the injured brain, particularly in the context of modalities through which the inherent self‐repair capacities of the aged PD brain, can be engaged (Chen et al, ; Kase et al, ; Kaur, Saunders, & Tolwinski, ; Mishra et al, ; Zeng et al, ; Zhao, et al, ; Zhang et al, a,b,c; and following sections).…”

From single cells to tissue self‐organization

“…For the moment, studying self‐organization with in toto imaging during development is performed on embryonic bodies , gastruloids , organoids or small‐scale animals. Whole animals such as Hydra or C. elegans can be imaged during their entire development, while larger organisms such as D. rerio or D. melanogaster provide fundamental insights into embryonic development. These model systems are highly informative for studying evolutionary conserved mechanisms, despite limitations in their multicellular complexity and tissue functionality.…”

Modeling the Role of Wnt Signaling in Human and Drosophila Stem Cells

“…Additionally, modern imaging methods such as light-sheet microscopy and confocal microscopy allow for precise in vivo observation. These, when combined with optogenetic approaches perturbing signal transduction pathways, could translate into an in vivo 2i system, where pathways could be turned on or off and the effects could be imaged [ 68 , 69 , 70 , 71 , 72 ]. In mice, pluripotency factors have been demonstrated to be functionally redundant; therefore, given the relative difficulty of manipulating mice genetically, Drosophila may be an effective system to dissect the individual influences of each gene more carefully as related to Wnt signaling [ 38 , 73 ].…”