Dynamic regulation of anterior-posterior patterning genes in livingDrosophilaembryos

Abstract

SummaryExpression of the gap and pair-rule genes plays an essential role in body segmentation during Drosophila embryogenesis [1–5]. However, it remains unclear how precise expression patterns of these key developmental genes arise from stochastic transcriptional activation at the single cell level. Here, I employed genome editing and live imaging approaches to comprehensively visualize regulation of the gap and pair-rule genes at the endogenous loci. Quantitative image analysis revealed that the total duration of active transcription (transcription period) is a major determinant of spatial patterning of gene expression in early embryos. The length of transcription period is regulated by the continuity of bursting activities in individual nuclei, with core expression domain producing more bursts than boundary region. Each gene exhibits distinct rate of nascent RNA production during transcriptional bursting, which contributes to gene-to-gene variability in the total output. I also provide evidence for “enhancer competition”, wherein a distal weak enhancer interferes with transcriptional activation by a strong proximal enhancer to downregulate the length of transcription period without changing the transcription rate. Analysis of endogenous hunchback (hb) locus revealed that the removal of distal shadow enhancer induces strong ectopic transcriptional activation, which suppresses refinement of broad expression domain into narrower stripe pattern at the anterior part of embryos. This study provides key insights into the link between transcriptional bursting, enhancer-promoter interaction and spatiotemporal patterning of gene expression during animal development.

CRISPR-Cas9 strategies to insert MS2 stem-loops into endogenous loci in Drosophila embryos

“…In short, the one-step CRISPR strategy uses a single cut site to introduce MS2 stem-loop sequences directly adjacent to the cleavage site into the endogenous locus together with a selectable marker that is subsequently removed ( Figure 3 A) ( Fukaya, 2020 ; Gratz et al., 2015 ; Lim et al., 2018b , 2018a ).…”

Live imaging and quantitation of nascent transcription using the MS2/MCP system in the Drosophila embryo

“…In Drosophila this technique has been used to investigate how target genes respond to different signals and transcription factor inputs ( Berrocal et al., 2020 ; Bothma et al., 2014 ; Falo-Sanjuan et al., 2019 ; Garcia et al., 2013 ; Hoppe et al., 2020 ; Lammers et al., 2020 ; Lucas et al., 2013 , 2018 ; Yamada et al., 2019 ), the function of enhancers and their contribution to transcriptional output ( Bothma et al., 2015 ; Fukaya et al., 2016 ; Koromila and Stathopoulos, 2019 ; Scholes et al., 2019 ), transcriptional memory ( Ferraro et al., 2016 ) and transvection ( Lim et al., 2018a ). The MS2 imaging system uses arrays of the MS2 bacteriophage derived stem-loop sequences that are inserted into non-coding regions of either endogenous genes ( Fukaya, 2020 ; Hoppe et al., 2020 ; Lim et al., 2018b , 2018a ) or transgenes ( Falo-Sanjuan et al., 2019 ; Fukaya et al., 2017 ; Garcia et al., 2013 ; Lucas et al., 2013 ). Once transcribed by RNA Polymerase II (Pol II), the RNA MS2 stem-loop is recognized by its cognate coat protein MCP, which binds the loop structure as a dimer ( Wu et al., 2012 ).…”

“… Optional: In order to reduce the inherent background of unbound MCP-GFP, the overall concentration can be reduced by using females that are heterozygous for His-RFP; MCP-GFP. The amount of maternally deposited His-RFP and MCP-GFP protein will be reduced but still sufficient for imaging of transcription ( Fukaya, 2020 ; Hoppe et al., 2020 ). This approach was used to acquire the example dataset supplied with this protocol.…”