Development of Threshold sensitive NF-kB reporters for Live Imaging of NF-kB transcription in Drosophila melanogaster

Abstract

In Drosophila, NF-ΚB family of transcriptional regulators mediate early dorsoventral axis patterning and host defences through the Toll and Immunodeficient (IMD) pathways. The three major NF-ΚB molecules in Drosophila include Dorsal, Dorsal-like Immune factor (Dif) and Relish (REL). During dorsoventral (D/V) patterning in the early embryo, asymmetric Toll signalling sets up a nucleocytoplasmic Dorsal gradient in the ventral and lateral regions. This along with the Dpp/Sog gradient sets up the D/V axis through threshold dependent gene expression leading to the specification of Ectoderm, Neuroectoderm amd Mesoderm. In the larval and adult stages, the Toll/Dorsal-Dif and IMD/REL signal transduction pathways upregulate transcription of defense genes in response to infection and are also involved in blood cell proliferation. Investigators have routinely used antibodies against Dorsal/Dif/REL and also antibodies or in-situ probes to monitor activation of the NF-Κ B targets in Development and host defense. In the last decade, a large number of fluorescence based methods have been developed to monitor the Dorsal gradient and also to measure NF-ΚB activation in the fat body and lymph gland. A very interesting problem in Development is the formation of the Dorsal nucleoctoplasmic gradient in the blastoderm and the effect of this gradient on cell-fate specification. The most recent efforts include use of Dorsal-fusions with fluorescent reporters such as eGFP to measure the spatiotemporal expression of the Dorsal gradient. In this study, in order to avoid the problems inherent in a Dorsal-fluoroscent protein fusion, we attempt to develop an alternative method to visualize the Dorsal gradient without disturbing Dorsal. Our method relies on using promoter elements, both native and artificial, that will respond to different concentration of nuclear Dorsal (or Dif). We hypothesize, that by using promoters of different sensitivity, and by fusing these promoters to a variety of bright colored fluoresent reporters (eGFP, mCherry, Venus, Cerulean), we will be able to visualize increasing amounts of Dorsal in the nucleus by different colors. This concept, when applied to a fly embryo will help us visualize, live, the Drosophila gradient in multiple overlapping colors in the early embryo and will also allow us to visualize different strengths of Toll/Dorsal-Dif activation in the fat body and lymph gland.