Curated Optogenetic Publication Database

Abstract: YAP, an effector of the Hippo signalling pathway, promotes organ growth and regeneration. Prolonged YAP activation results in uncontrolled proliferation and cancer. Therefore, exogenous regulation of YAP activity has potential translational applications. We present a versatile optogenetic construct (optoYAP) for manipulating YAP localisation, and consequently its activity and function. We attach a LOV2 domain that photocages a nuclear localisation signal (NLS) to the N-terminus of YAP. In 488 nm light, the LOV2 domain unfolds, exposing the NLS, which shuttles optoYAP into the nucleus. Nuclear import of optoYAP is reversible and tuneable by light intensity. In cell culture, activated optoYAP promotes YAP target gene expression and cell proliferation. Similarly, optofYap can be used in zebrafish embryos to modulate target genes. We demonstrate that optoYAP can override a cell's response to substrate stiffness to generate anchorage-independent growth. OptoYAP is functional in both cell culture and in vivo, providing a powerful tool to address basic research questions and therapeutic applications in regeneration and disease.

Abstract: With the rise of new tools, from controlled genetic manipulations and optogenetics to improved microscopy, it is now possible to make clear, quantitative and reproducible measurements of biological processes. The humble fruit fly Drosophila melanogaster, with its ease of genetic manipulation combined with excellent imaging accessibility, has become a major model system for performing quantitative in vivo measurements. Such measurements are driving a new wave of interest from physicists and engineers, who are developing a range of testable dynamic models of active systems to understand fundamental biological processes. The reproducibility of the early Drosophila embryo has been crucial for understanding how biological systems are robust to unavoidable noise during development. Insights from quantitative in vivo experiments in the Drosophila embryo are having an impact on our understanding of critical biological processes, such as how cells make decisions and how complex tissue shape emerges. Here, to highlight the power of using Drosophila embryogenesis for quantitative biology, I focus on three main areas: (1) formation and robustness of morphogen gradients; (2) how gene regulatory networks ensure precise boundary formation; and (3) how mechanical interactions drive packing and tissue folding. I further discuss how such data has driven advances in modelling.

Abstract: Development of the Drosophila embryonic mesoderm is controlled through both internal and external inputs to the mesoderm. One such factor is Heartless (Htl), a Fibroblast Growth Factor Receptor (FGFR) expressed in the mesoderm. Htl is involved in shaping the mesoderm at both early and later stages during embryogenesis. How Htl expression levels and timing of signaling affect mesoderm morphogenesis after spreading remains elusive. We have developed an optogenetic tool (Opto-htl) to control the activation of Htl signaling with precise spatiotemporal resolution in vivo. We find that the embryo is most sensitive to Htl over-activation within a developmental window of ~4 hours ranging from late stage 10 until early stage 13, which corresponds to early stages of heart morphogenesis. Opto-htl restores heart cells in htl mutants upon light activation, independent of its role in early mesoderm shaping events. We also successfully generated spatially distinct regions of Htl activity in the mesoderm using light-sheet microscopy. The developing tissue was unable to correct for the ensuing asymmetries in cell fate. Overall, Opto-htl is a powerful tool for studying spatiotemporal regulation of Htl 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.

Abstract: Morphogen gradients provide essential spatial information during development. Not only the local concentration but also duration of morphogen exposure is critical for correct cell fate decisions. Yet, how and when cells temporally integrate signals from a morphogen remains unclear. Here, we use optogenetic manipulation to switch off Bicoid-dependent transcription in the early Drosophila embryo with high temporal resolution, allowing time-specific and reversible manipulation of morphogen signalling. We find that Bicoid transcriptional activity is dispensable for embryonic viability in the first hour after fertilization, but persistently required throughout the rest of the blastoderm stage. Short interruptions of Bicoid activity alter the most anterior cell fate decisions, while prolonged inactivation expands patterning defects from anterior to posterior. Such anterior susceptibility correlates with high reliance of anterior gap gene expression on Bicoid. Therefore, cell fates exposed to higher Bicoid concentration require input for longer duration, demonstrating a previously unknown aspect of Bicoid decoding.