Temporal dynamics of pair-rule stripes in livingDrosophila embryosBomyi Lima,1, Takashi Fukayab,2, Tyler Heistb, and Michael Levineb,c,1

aDepartment of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104; bLewis–Sigler Institute for IntegrativeGenomics, Princeton University, Princeton, NJ 08544; and cDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544

Contributed by Michael Levine, June 29, 2018 (sent for review June 21, 2018; reviewed by Christine Rushlow and Alexander Stark)

Traditional studies of gene regulation in the Drosophila embryocentered primarily on the analysis of fixed tissues. These methodsprovided considerable insight into the spatial control of gene ac-tivity, such as the borders of eve stripe 2, but yielded only limitedinformation about temporal dynamics. The advent of quantitativelive-imaging and genome-editing methods permits the detailedexamination of the temporal control of endogenous gene activity.Here, we present evidence that the pair-rule genes fushi tarazu(ftz) and even-skipped (eve) undergo dynamic shifts in gene ex-pression. We observe sequential anterior shifting of the stripesalong the anterior to posterior axis, with stripe 1 exhibiting move-ment before stripe 2 and the more posterior stripes. Conversely,posterior stripes shift over greater distances (two or three nuclei)than anterior stripes (one or two nuclei). Shifting of the ftz andeve stripes are slightly offset, with ftzmoving faster than eve. Thisobservation is consistent with previous genetic studies, suggestingthat eve is epistatic to ftz. The precision of pair-rule temporaldynamics might depend on enhancer–enhancer interactions withinthe eve locus, since removal of the endogenous eve stripe 1 en-hancer via CRISPR/Cas9 genome editing led to precocious and ex-panded expression of eve stripe 2. These observations raise thepossibility of an added layer of complexity in the positional infor-mation encoded by the segmentation gene regulatory network.

live imaging | Drosophila embryo | MS2-PP7 | even-skipped | fushi tarazu

The segmentation of the Drosophila embryo represents one ofthe most extensively examined gene regulatory networks in

animal development. Maternal gradients of Bicoid, Hunchback,and Caudal establish sequential, overlapping patterns of gapgene expression, which, in turn, delineate pair-rule stripes thatsubdivide the embryo into a repeating series of body seg-ments (1–3). The classical view of this system invokes thresh-old responses of pair-rule stripe enhancers to static gradientsof maternal and gap gene regulatory factors (4–6). However,quantitative imaging studies have revealed anterior shifts in gapgene expression, particularly in posterior regions of cellularizingembryos (7–9). These shifts are thought to enrich the positionalinformation encoded by this system through dynamic changes inthe distribution of regulatory gradients over time. We sought toexplore how these dynamic changes in gap gene expression wouldbe read out at the level of the pair-rule genes that they regulate.We analyzed the temporal dynamics of the pair-rule genes

fushi tarazu (ftz) and even-skipped (eve), which are responsible forspecifying the even and odd parasegments, respectively (10).MS2 and PP7 RNA stem loops were inserted into the 3′UTRs ofthe endogenous loci using CRISPR/Cas9-mediated genomeediting (11–14). Visualization of the expression patterns in livingembryos reveals highly dynamic shifts in each of the stripes, in-cluding those located in anterior regions of the embryo. Theseshifts are particularly striking for ftz, in that the initial sites of ex-pression correspond to the interstripe regions of the mature stripes.Simultaneous visualization of ftz and eve expression suggests rapidshifting of the ftz stripes, followed by a slight lag in refinement of theeve stripes. We propose that the differential rates of ftz and evetemporal dynamics contribute to the positional information enco-ded by the segmentation network. Moreover, we provide evidence

that enhancer–enhancer interactions within the eve locus may beimportant for the precision of expression.

Results and Discussionftz is regulated by four separate enhancers located upstream anddownstream of the transcription unit (15, 16). Three of the en-hancers initiate stripes 3+6, 2+7, and 1+5. There is no stripe 4enhancer, and thus its expression depends on the classical “Zebra”enhancer, which directs all seven expression stripes at later stagesfollowing initiation by the individual stripe enhancers (3, 17). Wecreated separate ftz alleles containing 24 copies of either MS2 orPP7 RNA stem loops inserted into the 3′ UTR of the endogenouslocus (Fig. 1A). Fluorescent in situ hybridization (FISH) of ftz-MS2 revealed expression patterns comparable to those seen inwild-type yw embryos (SI Appendix, Fig. S1). Moreover, homozy-gotes of these alleles were fully viable, fertile, and phenotypicallyindistinguishable from wild-type flies. This suggests that the stemloops do not significantly affect either transcription or translation.Twelve embryos expressing either ftz-MS2 or ftz-PP7 were

analyzed, and similar results were obtained for both alleles.Nuclei were visualized with His2AV-mRFP or His2AV-eBFP2,allowing us to trace transcriptional activity from individual nucleiduring nuclear cycle (nc) 14. There was a clear anterior shift inthe ftz expression pattern for all stripes during a 15–30-min in-terval of nc 14 (Fig. 1 B and C, SI Appendix, Fig. S2, and MovieS1). Strikingly, while each ftz stripe was formed at different timepoints in nc 14, the initial ftz transcripts were always first de-tected in the interstripe regions of the mature stripes (Fig. 1 Band C and Movie S1). Comparison of ftz-MS2 mRNA accumu-lation along the anteroposterior (AP) axis between early andlate nc 14 revealed clear anterior shifts in stripe positions (SIAppendix, Fig. S3). Moreover, there was an anterior-to-posterior

Significance

Classical studies of gene activity in development focused onspatial limits due to the use of fixed tissues for analysis. Theadvent of live-imaging methods provides an opportunity toexamine the temporal control of gene expression. Here, weused quantitative live-imaging methods to visualize dynamicshifts in the endogenous expression of eve and ftz pair-rulestripes in the early Drosophila embryo. We suggest thatthese temporal dynamics add to the complexity encoded by thesegmentation gene network.

Author contributions: B.L., T.F., and M.L. designed research; B.L., T.F., and T.H. performedresearch; B.L. analyzed data; and B.L., T.F., and M.L. wrote the paper.

Reviewers: C.R., New York University; and A.S., Research Institute of Molecular Pathology.

Conflict of interest statement: C.R. and B.L. are coauthors of a 2015 paper. They did notcollaborate directly on this work.

Published under the PNAS license.1To whom correspondence may be addressed. Email: bomyilim@seas.upenn.edu or msl2@princeton.edu.

2Present address: Institute for Quantitative Biosciences, The University of Tokyo, Tokyo113-0032, Japan.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1810430115/-/DCSupplemental.

Published online July 30, 2018.

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wave in the temporal shifts whereby stripe 1 shifted earlier thanstripe 2, followed by the more posterior stripes (Fig. 1C). Thesesequential shifts are evocative of the wavefront of gene expres-sion seen during somitogenesis in vertebrates (18, 19). Following

the shifts, there was a refinement in each of the stripes during thelatter periods of nc 14, such that the anterior boundary of eachstripe was fixed and the posterior nuclei gradually lost tran-scriptional activity (Fig. 1B).

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Fig. 1. Dynamics of endogenous ftz expression pattern. (A) Schematic of endogenous ftz gene locus and ftz enhancers. Here, 24 repeats of MS2 or PP7 stem loops areinserted into the 3′ UTR. (B) Snapshots from an embryo expressing ftz-MS2. Nuclei that will formmature ftz stripes are false-colored in red. Mature ftz stripes are definedas the nuclei that exhibit active transcription after 40 min into nc 14. Nuclei that show active transcription in a given frame are false-colored in green. Yellow nucleirepresent those that show active transcriptionwithin themature ftz domain. Nuclei were visualizedwith His2Av-mRFP and are shown in blue. Note that stripes are initiallyformed at one to three cells posterior to their final position. Time representsminutes after the onset of nc 14. (C) Average spatial profile of ftz transcriptional activity alongthe AP axis during nc 14. Each plot corresponds to the snapshot shown in B. The AP axis was divided into 35 bins, and an average transcriptional activity from all the nucleiwithin each bin was plotted. The error bar represents ±SEM of all the nuclei within each bin. Blue columns represent the location of mature ftz stripes 1–7.

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eve is regulated by five separate enhancers located upstreamand downstream of the transcription unit (20, 21). As for ftz, wecreated separate eve alleles tagged in the 3′ UTR with eitherMS2 or PP7 (Fig. 2A), both of which are fully viable as homo-zygotes. Furthermore, eve-MS2 expression was found to be sim-ilar to wild-type expression on FISH (SI Appendix, Fig. S1). Atotal of 17 embryos carrying the eve-MS2 or eve-PP7 allelewere imaged. Strong transcriptional activity was observed nearthe stripe 1 domain, followed by the formation of more posteriorstripes (Movie S2). Anterior shifting of each eve stripe was seen,

with the posterior stripes displaying more significant movementthan stripes 1, 2, and 3 (Fig. 2 and SI Appendix, Figs. S4 and S5).Nonetheless, there was an anterior-to-posterior wave in dy-namics, similar to that seen for ftz (Fig. 2C and SI Appendix,Fig. S4C).To determine the relative dynamics of the ftz and eve expres-

sion patterns, we examined embryos containing both ftz-PP7 andeve-MS2 alleles (Fig. 3 and Movie S3). Dual-color live imagingrevealed a slightly offset timing of expression dynamics. Forexample, eve and ftz transcripts were initially expressed in

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Fig. 2. Dynamics of endogenous eve expression pattern. (A) Schematic of endogenous eve gene locus and eve enhancers. Here, 24 repeats of MS2 or PP7 stemloops are inserted into the 3′ UTR. (B) Snapshots from an embryo expressing eve-MS2, showing stripes 1–4. Nuclei that will form mature eve stripes are false-colored in red, and nuclei with active transcription in a given frame are false-colored in green. Yellow represents actively transcribing nuclei within the mature evedomain. Nuclei were visualized with His2Av-mRFP and are shown in blue. Mature eve stripes are defined as the nuclei that exhibit active transcription after 40 mininto nc 14. Each stripe shifts from posterior to anterior, and anterior stripes shift earlier than posterior stripes. Time represents minutes after the onset of nc 14. (C)Average spatial profile of eve transcriptional activity along the AP axis during nc 14. The plot corresponds to the snapshot shown in B. Error bars represent ±SEMof all the nuclei within each of the 25 bins along the AP axis. Blue columns represent the location of mature eve stripes 1–4.

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neighboring domains. During the first 20 min of nc 14, there wasa slight overlap between eve and ftz stripes, whereby the anteriorborder of each ftz stripe was tightly juxtaposed to the posterioredge of the corresponding eve stripe (e.g., 20 min; Fig. 3). Thetwo sets of stripes did not adopt their defining complementaryand evenly spaced expression patterns until late phases of cel-lularization (e.g., 40 min; Fig. 3). Because overlap occurred onlyat the posterior boundary of eve and the anterior boundary of ftz,the simplest interpretation is that ftz stripes shift faster than eve.This idea is consistent with previous genetic studies suggesting

dominance of eve in the pair-rule gene hierarchy, with eveexerting a strong repressive effect on ftz expression (22, 23).To determine the relative rates of ftz and eve temporal dy-

namics, we conducted various quantitative analyses. We mea-sured the fraction of active nuclei for ftz and eve within themature expression limits. In general, eve nascent transcriptspersisted longer within the ftz expression domains than do ftztranscripts within the eve domains (SI Appendix, Fig. S6). Forexample, ftz transcripts were rapidly lost in the eve 2 domain aseve transcripts are being activated. On the other hand, eve

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Fig. 3. Relative dynamics of ftz and eve. (A) Snapshots from an embryo expressing ftz-PP7 and eve-MS2, showing stripes 1–4. Nuclei that exhibit active ftzand active eve transcripts in a given frame are false-colored in red and green, respectively. Nuclei are shown in yellow when both ftz and eve are expressed ina given nucleus. Nuclei were visualized with His2Av-eBFP2 and are shown in blue. Time represents the minutes after the onset of nc 14. (B) Average spatialprofile of ftz (red) and eve (green) transcriptional activity along the AP axis during nc 14. Error bars represent ±SEM of all the nuclei within each of the 25 bins alongthe AP axis. Red and green columns represent the location of mature ftz and eve stripes 1–4, respectively.

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transcripts were detected in the ftz 2 domain even after the onsetof ftz transcription (SI Appendix, Fig. S6, Top). These resultssupport the idea that ftz stripes shift faster than eve. This mightbe due to asymmetric repression of ftz by Eve (22, 23).Previous studies have documented the spatial precision of

individual pair-rule stripes, such as eve stripe 2 (24–26). Ourstudy suggests that there are similar constraints on the temporaldynamics. The anterior stripes shift first and cover a distance of justone or two nuclei, while the posterior stripes shift last and moveover greater distances of two or three nuclei. Stripe 1 displays abroad expression profile at the onset of nc 14, with expressionextending into the future limits of eve stripe 2 (Fig. 2 andMovie S2).Stripe 2 begins to form only after stripe 1 begins to refine and re-treat from the stripe 2 territory. Thus, it is possible that stripe 1exerts an “organizing” activity in the patterning of the early embryo.To explore this idea, we removed the stripe 1 enhancer, which islocated downstream of the eve transcription unit, by CRISPR/Cas9genome editing (Fig. 4A). The stripe 1 deficiency was created in thecontext of the MS2-tagged allele. Trans-heterozygotes containingthis MS2 allele along with the unmodified PP7-tagged allele werevisualized using two-color imaging with MCP-GFP and mCherry-PCP fusion proteins (Fig. 4 and Movie S4).As expected, very little MS2 signal was detected in the vicinity

of stripe 1 during the initial phases of nc 14 due to the removal ofthe stripe 1 enhancer. Only the PP7 allele was expressed in thisterritory, while the MS2 allele was activated in the stripe 1 domainat later stages due to the late eve enhancer (Fig. 4B) (27). How-ever, eve stripe 2 expression from the deficiency allele (green)appeared earlier and extended more anteriorly into the interstriperegion compared with the wild-type stripe 2 pattern (red). Thisbroader pattern persisted for 10–15 min before refining into amore or less normal stripe 2 pattern (Fig. 4 B and C). These re-sults suggest that eve stripe 2 takes over as the dominant stripe onremoval of the stripe 1 enhancer. It will be interesting to seewhether removal of both the stripe 1 and stripe 2 enhancers wouldresult in dominance of stripe 3 expression. Dominance of anteriorstripes is consistent with the anterior-to-posterior coordination inthe stripe shifts seen for both eve and ftz. It is also possible that this

coordination depends on enhancer–enhancer interactions withinthe eve locus.In summary, we have presented evidence for dynamic shifts of ftz

and eve stripes. These shifts are slightly offset, with the ftz stripesshifting and refining faster than the corresponding eve stripes. Thesetemporal dynamics have the potential to augment the positional in-formation encoded by the segmentation regulatory hierarchy.

Materials and MethodsFly Strains. eve-MS2 and eve-PP7 were generated using CRISPR/Cas9-basedinsertion of 24× MS2 RNA stem loop or 24× PP7 RNA stem loop into the 3′UTR of endogenous eve. In brief, approximately 1-kb DNA fragments of 5′and 3′ homology arm sequences were PCR-amplified from the genomic DNAand then inserted into the pBS-MS2-loxP-dsRed-loxP and pBS-PP7-loxP-dsRed-loxP donor plasmids (28). These plasmids were coinjected with thepCFD3 gRNA expression plasmid to nos-Cas9 embryos (29). ftz-MS2 and ftz-PP7 have been described previously (28).

Genome Editing by CRISPR/Cas9. For insertion of MS2 and PP7 stem loops intothe 3′ UTR of eve locus, pCFD3 gRNA expression plasmid and pBS-dsReddonor plasmid were coinjected to nos-Cas9 embryos (29). Microinjectionwas performed as described previously (30). 3xP3-dsRed was used for sub-sequent screening. For deletion of eve stripe 1 enhancer, two pCFD3 gRNAexpression plasmids, pBS-GFP donor plasmid, and pBS-Hsp70-Cas9 plasmid(Addgene; 46294) were coinjected to eve-MS2 embryos. 3xP3-GFP was usedfor subsequent screening.

Single-Color MS2 Live Imaging. MCP-GFP, His2Av-mRFP virgins were matedwith homozygous males carrying MS2 alleles to image ftz-MS2 or eve-MS2embryos (Figs. 1 and 2, SI Appendix, Figs. S2–S5, and Movies S1 and S2). Theresulting embryos were dechorinated, mounted between a semipermeablemembrane (In Vitro Systems & Services) and a coverslip (18 mm × 18 mm), andthen embedded in Halocarbon oil 27 (Sigma-Aldrich). Embryos were imagedwith a Zeiss LSM 880 laser scanning confocal microscope at room temperature,using a Plan-Apochromat 40×/1.3 NA oil immersion objective. At each timepoint, a stack of 26 images separated by 0.5 μm were captured in 16 bits.

Dual-Color MS2/PP7 Live Imaging.Dual-color MS2/PP7 live imagingwas used toimage embryos carrying ftz-pp7/eve-MS2, eve-PP7/eve-Δstripe 1 enhancer-MS2,

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Fig. 4. Dynamics of eve in the absence of the stripe 1enhancer. (A, Top) Schematic of eve locus and enhancers,with eve stripe 1 enhancer deleted via CRISPR/Cas9. Here,24 copies of MS2 stem loops are inserted at the 3′ UTR.(A, Bottom) Schematic of wild-type eve locus and en-hancers with 24 copies of PP7 stem loops inserted at the3′ UTR. (B) Snapshots from an embryo expressing eve-Δstripe 1-MS2 (green) and eve-PP7 (red). Overlap of MS2and PP7 alleles makes false-colored nuclei appear yellow.Nuclei were visualized with His2Av-eBFP2 and are shownin blue. Time represents minutes after the onset of nc 14.Note the precocious MS2 expression anterior to the evestripe 2. (C) Snapshots from an embryo expressing eve-MS2 (green) and eve-PP7 (red). Overlap of MS2 and PP7alleles makes false-colored nuclei appear yellow. Nucleiwere visualized with His2Av-eBFP2 and are shown inblue. Time represents minutes after the onset of nc 14.

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and eve-PP7/eve-MS2 alleles (Figs. 3 and 4 and Movies S3–S5). MCP-GFP,mCherry-PCP, His2Av-eBFP2 virgins were mated with homozygous malescarrying either the PP7 or MS2 allele (28). Resulting trans-heterozygotevirgins were collected and mated with homozygous males carrying theMS2 or PP7 allele. The resulting embryos were prepared as described abovefor single-color live imaging.

In Situ Hybridization. Embryos were dechorionated and fixed in fixationbuffer (0.5× PBS, 25 mM EGTA, 4% formaldehyde, and 50% heptane) for20 min at room temperature. Antisense RNA probes labeled with digoxigenin(DIG RNA Labeling Mix 10× concentration; Roche) and biotin (Biotin RNALabeling Mix 10× concentration; Roche) were used to detect ftz and eveRNAs, respectively. Template DNA for ftz probes was amplified from geno-mic DNA using primers (5′-CGT AAT ACG ACT CAC TAT AGG GTG GGG AAGAGA GTA ACT GAG CAT CGC-3′) and (5′-ATT CGC AAA CTC ACC AGC GT-3′).Template DNA for eve probes was amplified from genomic DNA using pri-mers (5′-CGT AAT ACG ACT CAC TAT AGG GGT GTG TGG ATC GCG GGC TTACGC C-3′) and (5′-ACA CTC GAG CTG TGA CCG CCG C-3′). Hybridization wasperformed at 55 °C overnight in hybridization buffer (50% formamide, 5×SSC, 50 μg/mL heparin, 100 μg/mL salmon sperm DNA, and 0.1% Tween-20).Subsequently, embryos were washed with hybridization buffer at 55 °C andincubated with Western Blocking Buffer (Roche) at room temperature for1 h. Then the embryos were incubated with sheep anti-digoxigenin (Roche)and mouse anti-biotin primary antibodies (Invitrogen) at 4 °C for overnight,followed by incubation with Alexa Fluor 488 donkey anti-sheep (Invitrogen)and Alexa Flour 555 goat anti-mouse (Invitrogen) fluorescent secondaryantibodies at room temperature for 2 h. DNA was stained with Hoechst33342 (Thermo Fisher Scientific), and embryos were mounted in ProLongGold Antifade Mountant (Thermo Fisher Scientific). Imaging was performedwith a Zeiss LSM 880 confocal microscope, and maximum projections of Z-stacks are shown.

Image Analysis. All the processing and analysis were implemented in MATLABR2017b (MathWorks).

Nuclei Segmentation and Tracking. His2Av-mRFP and His2Av-eBFP2 were usedto segment nuclei for single-color and dual-color imaging, respectively.Nuclei-labeled channel images were preprocessed with Gaussian filtering andadaptive histogram equalization to enhance the signal-to-noise contrast.Nuclei were then watershedded to further separate and distinguish neigh-boring nuclei. The number and positions of separate nuclei within a framewere obtained. Nuclei tracking within nc 14 was done by finding the objectwith minimal movement across the frames of interest (from approximately4 min into nc 14 to the onset of gastrulation).

Recording of MS2 and PP7 Signals. Maximum projections of raw images wereused to record fluorescence intensities. Fluorescence intensities within eachsegmented nucleus was extracted. After subtracting the background nuclearsignal, the signals of MS2 and PP7 transcription foci were determined bytaking an average of the top three pixels with the highest fluorescence in-tensity within each nucleus.

Spatial Profile of ftz and eve Transcriptional Activity and mRNA AccumulationAlong the AP Axis. To obtain spatial profiles of ftz and eve transcriptionalactivity along the AP axis, each embryo was divided into bins with equalwidth (10 μm per bin). In addition, to minimize the effect of ftz or evecurvature on the spatial profile, the middle 40-μm region along thedorsoventral axis was defined as the region of interest. Then, for each binwith a size of 10 μm × 40 μm, MS2 or PP7 intensity of all the nuclei withinthe bin was obtained. The average intensity value per bin was plotted foreach frame, with an error bar representing the SEM of all the nuclei withinthe bin. mRNA accumulation over a specified period was obtained bytaking the area of transcriptional trajectory per nucleus over the desig-nated time. Then the spatial plot of mRNA accumulation was plotted inthe same way, by taking the average mRNA accumulation value from allthe nuclei within each bin.

False Coloring. Active nuclei were defined as those with an MS2 or PP7 signalabove a threshold value. The threshold value was determined by taking 15%ofthemaximum fluorescence intensity of each embryo throughout the entirety ofnc 14. For each frame, all the nuclei with an MS2 or PP7 signal above thethreshold value were identified. Using the segmentation mask, these activenuclei were colored either red or green, with a fixed pixel intensity, and layeredover the raw His2Av-mRFP or His2Av-eBFP2 image in a given frame. Mature ftzand eve domains were defined as the nuclei showing active MS2 or PP7 signalsabove the threshold value after 40 min into nc 14 for longer than 2 min. In-dices of these nuclei were obtained, and for each frame these nuclei werecolored in a single color with a fixed intensity. False coloring of mRNA accu-mulation was done by coloring the nuclei with the pixel intensity value cor-responding linearly to its mRNA accumulation.

ACKNOWLEDGMENTS. We thank Evangelos Gatzogiannis for help withconfocal imaging, Anna Hakes and Louis Maddison for help with cloning andimaging, and members of the M.L. laboratory for helpful discussions. T.F.is the recipient of a Human Frontier Science Program Long-Term Fellow-ship. T.H. is supported by funding from National Human Genome Re-search Institute of the National Institutes of Health Grant T32HG003284. Thisstudy was funded by National Institutes of Health Grants U01 EB021239 andGM118147.

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r 10

, 202

0