Endogenous Visualization and Manipulation of Genes in the Gap Gene Regulatory Network in Developing D. melanogaster

Abstract

The gap gene network is critical for anterior-posterior patterning of the developing Drosophila melanogaster embryo. Gap genes are well characterized and provide an excellent platform to investigate endogenous transcription in real-time. While the synthetic reporter systems are valuable for real-time visualization of gap gene transcriptional activity, they fail to capture endogenous transcription, obscuring context dependent factors that affect transcription, including potential network effects. Here, we utilize a novel CRISPR based methodology that allows us to both introduce a tagging system to visualize endogenous gap gene transcription in real-time, and also manipulate regulatory regions of genes in the network. To gain insight into the effects of multiple enhancers on endogenous gene expression, we utilize this methodology to visualize the phenotypic effects of deletions of the proximal and distal enhancers that govern kni expression on early embryonic development and adult viability. We find that the distal enhancer appears to be more strongly implicated in establishing the posterior domain of kni expression and wildtype development than previously thought. Most importantly, we the proximal and distal enhancers do not behave as discrete, modular units but rather interact synergistically to establish proper kni expression. We also addressed whether differences in expression of X-linked genes are occuring during early development by comparing endogenous transcriptional activity of gt in male and female embryos. We observe differential expression between sexes occuring in the posterior domain of gt expression between nuclear cycles 13 to 14. Finally, we conclude with a first demonstration of the visualization of simultaneous endogenous transcriptional activity of three gap genes: Kr, kni and gt. Together our results open new avenues for future study of the dynamics of regulatory interactions between multiple genes in a wild-type background. We broadly demonstrate the capability of our methodology to investigate aspects of endogenous transcription that can be widely applied to other metazoan systems.