Novel Fluorescent and Photoconvertible Fusions Reveal Dorsal Activator Dynamics

Abstract

Over the last two decades, new in vivo and in cellulo imaging technologies have uncovered the inherently dynamic nature of transcriptional regulation in embryonic development and, in particular, in the fruit fly D. melanogaster. These technologies have made it possible to characterize the subnuclear and single-molecule dynamics of transcription factors. However, a lack of appropriate fluorescent protein fusions has, until now, limited these studies to only a few of the dozens of important transcription factors in the fruit fly gene regulatory network dictating early development. Here, we report the creation of four new fluorescent protein fusions to Dorsal, a member of the NF-{kappa}B/Rel family that initiates dorsal-ventral patterning. We generated and characterized two bright fluorescent protein fusions for Dorsal, meGFP and mNeonGreen, and two photoconvertible fluorescent protein fusions, mEos4a and Dendra2. We show that removal of the DsRed2 cassette commonly used to mark the CRISPR integration restores endogenous Dorsal mRNA and protein levels and enables the fusion allele to rescue a dorsal null allele, meeting the gold standard for endogenous function of a tagged protein in a fruit fly. We then demonstrate that our bright fluorescent protein fusions can be used to dissect the spatiotemporal dynamics of stable Dorsal clusters that traverse the nucleoplasm and uncovered that these clusters preferentially interact with active sites of Dorsal-modulated transcription. We further demonstrate that our photoconvertible fluorescent protein fusions make it possible to detect individual molecules of Dorsal in the nuclei of developing embryos. These new fluorescent protein fusions constitute a valuable resource for the community to elucidate the role of Dorsal activator dynamics in dictating fruit fly early embryonic development.