DOI: 10.6078/d1xx33
title: Kinetic sculpting of the seven stripes of the Drosophila even-skipped gene

description: We used live imaging to visualize the transcriptional dynamics of the
 Drosophila melanogaster even-skipped gene at single-cell and high temporal
 resolution as its seven stripe expression pattern forms, and developed
 tools to characterize and visualize how transcriptional bursting varies
 over time and space. We find that despite being created by the independent
 activity of five enhancers, even-skipped stripes are sculpted by the same
 kinetic phenomena: a coupled increase of burst frequency and amplitude. By
 tracking the position and activity of individual nuclei, we show that
 stripe movement is driven by the exchange of bursting nuclei from the
 posterior to anterior stripe flanks. Our work provides a conceptual,
 theoretical and computational framework for dissecting pattern formation
 in space and time, and reveals how the coordinated transcriptional
 activity of individual nuclei shape complex developmental patterns.
description: Generation of MS2 tagged eve BAC We used
 bacterial recombineering (Warming et al. 2005) to modify a bacterial
 artificial chromosome (BAC) (Venken et al. 2006) containing the D.
 melanogaster eve gene and all of its enhancers and regulatory elements
 (BAC CH322-103K22) (Venken et al. 2009). We  replaced the coding region
 with an array of 24 MS2 stem loops fused to the D. melanogaster yellow
 gene (Figure 1B; (J. P. Bothma et al. 2014) as described below. We
 inserted our eve::MS2::yellow BAC-based construct in the D. melanogaster
 genome at chromosome 3L through ΦC31 integrase-mediated recombination (see
 Generation of fly lines), and generated a viable homozygous fly line (w-;
 +; eve::MS2::yellow) as detailed below.  Reporter
 design In principle the length of the reporter should
 not limit our ability to estimate burst parameters. However, in practice a
 reporter construct that is too short will have insufficient signal.
 Further, one that is too long will increase the dwell time of each RNA
 polymerase molecule on the gene and, as a result, our cpHMM inference will
 require too many computational resources. Our choice of reporter construct
 structure strikes a balance between these two limitations and is ideally
 suited for inferring bursting parameters in the time range where eve
 resides, as well as for boosting the signal-to-noise ratio. See  Lammers
 et al. (2020) for a more detailed discussion of reporter length-related
 tradeoffs.  Specifics of recombineering 
 We modified a CHORI BAC CH322-103K22 derived from (Venken et al.
 2009), which contained the entire eve locus and a GFP reporter instead of
 the eve coding sequence (CH322-103K22-GFP). We replaced the GFP reporter
 with MS2::yellow (6665 bp) through a two step, scarless, galK
 cassette-mediated bacterial recombineering (Warming et al. 2005). Briefly,
 we transformed our starting CH322-103K22-GFP BAC into E.coli
 recombineering strain SW102. We then electroporated the strain with a galK
 cassette flanked by 50bp-long DNA homology arms homologous to the
 MS2::yellow (6665 bp) reporter. Upon electroporation, we selected
 transformants on M63 minimal media plates with galactose as a single
 carbon source. We achieved a correct replacement of GFP sequence by galK
 cassette in the BAC context (CH322-103K22-galK), validated by observing
 the digestion patterns produced by ApaLI restriction enzyme.
 We next purified the CH322-103K22-galK BAC and transformed it
 into fresh E. coli SW102 cells. We electroporated these cells with the
 purified MS2::yellow insert and used M63 minimal media plates with
 2-deoxy-galactose to select against bacteria with a functional galK gene.
 We used colony PCR to screen for colonies with a correct MS2::yellow
 insertion (CH322-103K22-MS2) replacing the galK cassette. We validated
 this insertion by observing ApaLI, XhoI, SmaI, and EcoRI restriction
 digestion patterns and through PCR and Sanger sequencing of the insertion
 junctions. We transformed our CH322-103K22-MS2 BAC in E.coli EPI300 cells
 to induce high copy numbers and purified it with a Qiagen plasmid Midiprep
 kit. Generation of fly lines We sent
 a sample of our purified CH322-103K22-MS2 BAC to Rainbow Transgenic Flies,
 Inc. for injection in D. melanogaster embryos bearing a ΦC31 AttP
 insertion site in chromosome 3L (Bloomington stock #24871; landing site
 VK00033; cytological location 65B2). We received the flies that resulted
 from that injection and used a balancer fly line (w- ; + ; +/TM3sb) to
 obtain a viable MS2 homozygous line (w- ; + ; MS2::yellow). We used
 line  (yw; His::RFP; MCP::GFP) as the maternal source of Histone-RFP and
 MCP-GFP (Garcia et al. 2013). Embryo Collection and
 Mounting Embryo collection and mounting was done as
 specified in (Garcia and Gregor 2018). In short, we set fly crosses
 between ~30 males (w-; +; eve::MS2::yellow) and ~80 females (yw; His::RFP;
 MCP::GFP) in a plastic cage capped with a grape juice agar plate. We
 collected embryos from cages two to ten days old by adding a fresh plate
 for 30 minutes and aging for 60 minutes to target embryos 90 min or
 younger. Embryos were mounted on a gas-permeable Lumox
 Film (Sarstedt - Catalog # 94.6077.317) embedded on a microscope slide
 hollowed on the center. Then, we coated the hydrophobic side of the Lumox
 film with heptane glue and let it dry. The film allows oxygenation of
 embryos during the 2-3h long imaging sessions while heptane immobilizes
 them.  We soaked an agar plate with Halocarbon 27 oil,
 picked embryos with forceps, and laid them down on a 3 x 3 cm piece of
 paper tissue. We dechorionated embryos by adding 2 drops of bleach diluted
 in water (5.25%) on the paper tissue and incubating for 1.5 minute. We
 removed bleach with a clean tissue and rinsed with ~4 drops of distilled
 water. We then placed the tissue paper with dechorionated embryos in
 water, and picked buoyant embryos with a brush. We
 lined ~30 apparently healthy embryos on the Lumox film slide and added 2-3
 drops of Halocarbon 27 oil to avoid desiccation, and covered the embryos
 with a cover slip (Corning® Cover Glass, No.1, 18 x 18mm) for live
 imaging. Imaging and Optimization of Data
 Collection Movies of embryonic development were
 recorded on a Zeiss-800 confocal laser scanning microscope in two
 channels, (EGFP: 488 nm; TagRFP: 561 nm). We imaged embryos on a wide
 field of view, along their anterior-posterior axis, of 1024 x 256 pixels
 (202.8µm x 50.7µm), encompassing 3-5 stripes per movie. We tuned laser
 power, scanning parameters, master gain, pinhole size and laser power to
 optimize signal to noise ratio without significant photobleaching and
 phototoxicity.  For imaging, the following microscope
 settings were used: 63x oil-objective, scan mode ‘frame’, pixel size of
 0.2µm, 16 bits per pixel, bidirectional scanning at a speed of 7, line
 step of 1, laser scanner dwelling per pixel of 1.03µs, laser scanner
 averaging of 2, averaging method Mean, averaging mode Line, 488 nm laser
 power of 30µW (EGFP), 561 nm laser power of 7.5µW (TagRFP) (both powers
 were measured with a 10x air-objective), Master Gain in EGFP detector of
 550V, Master Gain in TagRFP detector of 650V, Digital Offset in both
 detectors of 0, Digital Gain in both detectors of 1.0, and a pinhole size
 of 1 airy unit under the imaging conditions mentioned above (44µm,
 0.7µm/section), laser filters EGFP:SP545 and TagRFP:LBF640. This resulted
 in an imaging time of 633 ms per frame and a full Z-stack of 21 frames in
 intervals of 0.5µm every 16.8s. Following (J. P. Bothma et al. 2014, 2015,
 2018; Lammers et al. 2020), the imaging conditions were determined not to
 affect normal development as reported by the timing of the nuclear cycles
 in early development. We stopped imaging after 50 min into nuclear cycle
 14, and took mid-sagittal and surface pictures of the whole embryo for
 localization of the recorded field of view along the embryo’s AP
 axis. Image processing We used a
 Matlab computational pipeline based on (Garcia et al. 2013; Lammers et al.
 2020) to segment and extract numeric data from our raw movies. Briefly,
 this software segments and processes the images from the two channels
 (channel 1: MCP::GFP, channel 2: Histone::RFP) on which we collected our
 data. For segmentation of channel 1, we used Fiji-Weka Segmentation 3D
 software; this machine-learning-based method relies on the manual
 segmentation of a variety of MCP::GFP labeled transcriptional foci in a
 given 21 frame Z-stack from a single dataset (EVE_D11) to produce a model
 for the segmentation of all datasets recorded under the same imaging
 conditions. Next, we segmented and tracked the Histone::RFP labeled nuclei
 on channel 2. Subsequently, we assigned MCP::GFP labeled transcriptional
 foci to their corresponding Histone::RFP labeled nuclei. Since we
 collected whole embryo pictures of each of our datasets, we were able to
 match and locate the recorded fields of view to their right position along
 the body of their corresponding embryos. Finally, we extracted position
 and fluorescence values over time of all transcriptional foci to generate
 data structures ready to use in further analyses.