Heat-mediated manipulation of gene expression by IR-LEGO in the developing genitalia in Drosophila

Abstract

Manipulating gene expression in a tissue-specific and temporally controlled manner is essential for understanding the function of the focal genes. Still, in many cases, the limited availability of specific promotors to drive ectopic manipulation remains a restricting factor in developing organs, even in Drosophila. Developing external genitalia is one such organ with a complex anatomical structure shaped by a joint regulatory network of many transcription factors. To overcome the restriction, we employed the infrared laser-evoked gene operator system (IR-LEGO), in which infrared laser (1,480 nm) irradiation induces gene expression under the control of a heat shock promoter. Pupal genital structures were irradiated at approximately 24 or 48 hours after puparium formation. We tested a range of laser power and depth to the target structure by a reporter assay using green fluorescent protein, which was induced under the control of the heat shock protein 70 promoter (hs-GAL4). In previous studies, the IR-LEGO has been used as a tool to induce ectopic transgene expression. In this study, we attempted to knock down genes such as yellow (y) and odd-paired (opa) ectopically by RNAi using the GAL4/UAS system. The results demonstrated that this technique has a high potential in manipulating transcript abundance levels in small groups of cells in specific genital structures to unravel novel functions of genes involved in the morphogenesis of species-specific and rapidly evolving anatomical structures. Article summaryManipulating gene expression is essential for understanding the function of the genes at specific regions of the body. In particular, knowing how different genes contribute to shaping complex anatomical structures, such as insect external genitalia, is an intriguing question. While manipulating gene expression precisely at a specific time and space remains challenging, we show that we can knock down a gene in a specific group of cells in the developing genitalia of Drosophila pupae, using infrared laser irradiation. This is a new approach to investigating how activity of genes shapes the rapidly evolving male genitalia in insects.