PhotoGal4: A Versatile Light-Dependent Switch for Spatiotemporal Control of Gene Expression in Drosophila Explants

Abstract

Summary We present here PhotoGal4, a phytochrome B-based optogenetic switch for fine-tuned spatiotemporal control of gene expression in Drosophila explants. This switch integrates the light-dependent interaction between phytochrome B and PIF6 from plants with regulatory elements from the yeast Gal4/UAS system. We found that PhotoGal4 efficiently activates and deactivates gene expression upon red- or far-red-light irradiation, respectively. In addition, this optogenetic tool reacts to different illumination conditions, allowing for fine modulation of the light-dependent response. Importantly, by simply focusing a laser beam, PhotoGal4 induces intricate patterns of expression in a customized manner. For instance, we successfully sketched personalized patterns of GFP fluorescence such as emoji-like shapes or letterform logos in Drosophila explants, which illustrates the exquisite precision and versatility of this tool. Hence, we anticipate that PhotoGal4 will expand the powerful Drosophila toolbox and will provide a new avenue to investigate intricate and complex problems in biomedical research.

Rapid and robust optogenetic control of gene expression in Drosophila

“…Accordingly, there have been several attempts to develop optogenetic binary systems (de Mena et al, 2018). Some success has been reported with optogenetic Gal4 approaches in cell culture, zebrafish embryos, Drosophila, and mice (Liu et al, 2012;Wang et al, 2012;Pathak et al, 2017;de Mena and Rincon-Limas, 2020;Mruk et al, 2020;Yamada et al, 2020). These advances are significant, but they come with some limitations: (i) the long light exposure (in the order of hours) necessary to induce phenotypes or reporter expression (de Mena and Rincon-Limas, 2020; Mruk et al, 2020), likely incompatible with live imaging or the study of rapid biological processes; (ii) leakiness in the dark (Mruk et al, 2020;Pathak et al, 2017;Yamada et al, 2020); (iii) the lack of a fully transgenic optogenetic Gal4/UAS system (Liu et al, 2012;Pathak et al, 2017;Yamada et al, 2020); or (iv) the requirement of an exogenous chromophore, which is only readily administered to tissue explants (de Mena and Rincon-Limas, 2020).…”

Spatial and temporal control of expression with light-gated LOV-LexA

“…Other light-gated expression systems have been developed in Drosophila, including the cryptochrome split-LexA and Photo-Gal4 [59,60]. We expressed the cryptochrome split-LexA with the same driver used to test LOV-LexA, LC10s-SS2 [20], and found that cryptochrome split-LexA system is leaky in flies raised at 18ºC and kept in the dark (data not shown).…”

“…The spatial resolution provided by Photo-GAL4 remains unmatched by LOV-LexA in vivo, due to the scattering that occurs once light traverses the cuticle in pupal and adult flies. Given that Photo-GAL4 relies on PhyB and requires addition of the chromophore PCB, normally absent in animal cells, it is currently limited to ex vivo studies [59]. The chromophore providing LOV with light sensitivity is flavin mononucleotide that exists in animal cells, making the LOV-LexA system solely dependent on delivery of light.…”

“…In the Photo-GAL4 expression system, the PhyB and PIF were added to each split-GAL4 half instead of the zinc finger, turning the reconstitution of a complete GAL4 dependent on red light [59]. To function however, Photo-GAL4 requires addition of the PCB, a chromophore that is absent in animal cells but essential for photosensitivity of PhyB [58], limiting Photo-GAL4 to use in ex vivo experiments [59]. The cryptochrome split-LexA system similarly draws on insertion of cryptochrome 2 (CRY2) and its binding partner, the cryptochrome interacting protein (CIB), into the LexA DNA binding domain and the transactivator domain, to gate reformation of the full LexA-transactivator with blue light [60,61].…”

“…Phytochromes (Phy) are sensitive to red and far-red light when bound to the phycocyanobilin (PCB) chromophore, and translocate to the nucleus in presence of light to bind the phytochrome interacting factor (PIF) [58]. In the Photo-GAL4 expression system, the PhyB and PIF were added to each split-GAL4 half instead of the zinc finger, turning the reconstitution of a complete GAL4 dependent on red light [59]. To function however, Photo-GAL4 requires addition of the PCB, a chromophore that is absent in animal cells but essential for photosensitivity of PhyB [58], limiting Photo-GAL4 to use in ex vivo experiments [59].…”