Bioluminescence imaging to track real-time armadillo promoter activity in live Drosophila embryos

Abstract

We established a method for bioluminescence imaging (BLI) to track real-time gene expression in live Drosophila embryos. We constructed a transgenesis vector containing multiple cloning sites and enhanced green-emitting luciferase (ELuc; Emerald Luc), a brighter and pH-insensitive luciferase for promoter analysis. To evaluate the utility of BLI using an ELuc reporter together with an optimized microscope system, we visualized the expression pattern of armadillo (arm), a member of the Wnt pathway in Drosophila, throughout embryogenesis. We generated transgenic flies carrying the arm:: ELuc fusion gene, and successfully performed BLI continuously for 22 h in the same embryos. Our study showed, for the first time, that arm::Eluc expression was dramatically increased in the anterior midgut rudiment, myoblasts of the dorsal/lateral musculature, and the posterior spiracle after stage 13, and the cephalic region at stage 17. To further demonstrate the application of our BLI system, we revealed that arm transcriptional activity in embryos was modulated inversely by treatment with ionomycin or 6-bromoindirubin-3-oxime (BIO), an inhibitor and activator of Wnt/β-catenin signaling, respectively. Therefore, our microscopic BLI system is useful for monitoring gene expression in live Drosophila embryos, and for investigating regulatory mechanisms by using chemicals and mutations that might affect expression.ᅟElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-8000-8) contains supplementary material, which is available to authorized users.

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

“…Wnt signaling in segment polarity is well studied, but many additional functions have been observed 37 . Recently 38 , reported that intestinal formation during embryo development was disrupted in embryos treated with ionomycin, an inhibitor of Wnt/β-catenin signaling. By coupling optogenetics with live imaging, we observed a similar defect in intestinal development in Arm-CRY2-mCh expressing arm XM19 mutant embryos.…”

Coupling optogenetics and light-sheet microscopy, a method to study signal transductionin vivo

“…Wnt signaling in segment polarity is well studied, but many additional functions have been observed 36 . Recently, 37 reported that intestinal formation during embryo development was disrupted in embryos treated with ionomycin, an inhibitor of Wnt/β-catenin signaling. By coupling optogenetics with live imaging, we observed a similar defect in intestinal development in Arm-CRY2-mCh expressing arm XM19 mutant embryos.…”

Bioluminescence Microscopy: Design and Applications

“…This is convenient for three-dimensional image reconstruction by light sectioning. Accordingly, we constructed transgenic Drosophila melanogaster carrying an armadillo (a member of the segment polarity gene) promoter and luciferase fusion gene [31]. Figure 10 shows three-dimensional images of armadillo promoter activity from insular larva of transgenic flies reconstructed from 11 sectionalized images (front and slant views).…”

“…In our previous studies, we optimized an optical system using a short-focal-length imaging lens for bioluminescence microscopy and performed bioluminescence imaging of single live cells expressing the luciferase gene using a conventional CCD camera [20,21]. This system is commercially available now and has been widely used for gene expression analysis in chronobiology [22][23][24][25][26][27][28], neurobiology [29,30], developmental biology [31], medical research [32][33][34][35], signal transduction analysis [36][37][38], molecular interaction [39][40][41], and radiation biology [42,43]. Accordingly, in this study, we describe the concept of bioluminescence microscopy adopting a short-focal-length imaging lens and present several representative applications, including a three-dimensional analysis, to demonstrate the advantages of the short-focal-length imaging lens system.…”