Divergent evolutionary methods pre-empt tissue collision in gastrulation

Experimental animals and embryo assortment

D. melanogaster embryos have been collected on apple juice agar plates with yeast paste at 22 °C or on the temperatures indicated under. Laboratory cultures of M. abdita (Sander pressure) have been maintained as described⁵². M. abdita embryos have been collected on apple juice agar plates with fish meals paste at 25 °C. The laboratory cultures of C. riparius (Bergstrom pressure) have been maintained as described⁵². C. riparius embryos have been collected as freshly deposited egg packages at ambient room temperature (23–26 °C). The laboratory cultures of C. albipunctata have been maintained as described^53,54. Embryos have been obtained after dissecting-out grownup feminine ovarioles, adopted by experimental egg activation by way of a hypo-osmotic shock. The laboratory cultures of H. illucens have been established from current cultures within the Schmidt-Ott lab (University of Chicago). To gather H. illucens embryos, females have been decapitated to set off egg laying on the desired time.

A transient laboratory tradition of C. fuscipes was established from wild-caught adults discovered close to the municipal compost plant of the town of Heidelberg. C. fuscipes adults desire to put eggs in small cavities. To gather embryos, outdated tradition plates of C. albipunctata have been used as they conveniently have such cavities. Before utilizing these for C. fuscipes egg assortment, the plates have been decontaminated by freezing them at −20 °C for no less than 1 week, adopted by thawing in a single day at room temperature.

We couldn’t set up a laboratory tradition for any of the species from the household Empididae, as a result of we couldn’t optimise the tradition circumstances. During our tour in Pula (Croatia) we managed to catch a couple of adults of an Empis species (most likely Empis pennipes, known as Empis sp. all through the textual content). As a consequence, we resorted to repeatedly catching adults throughout that point from the wild, after which decapitated the females to set off egg laying.

Drosophila genetics and transgenic strains

D. melanogaster strains used for stay imaging have been MyoII–eGFP (also referred to as Spaghetti-squash–eGFP, or Sqh–eGFP)⁵⁵, Gap43-mCherry⁵⁶, MyoII-mKate2 (ref. ⁵⁷) and mat-tub-3xmScarlet-CaaX (this examine). The membrane imaging line mat-tub-3xmScarlet-CaaX was made by cloning 3xmScarlet-CaaX into the pBabr vector containing the mat-tub promoter (reward from D. St. Johnston, Gurdon Institute, UK)⁵⁸ and the sqh 3′ untranslated area (UTR), adopted by ΨC31 site-directed integration into the attP2 or attP40 touchdown websites at WellGenetics. D. melanogaster mutant alleles used have been eve^R13 (FlyBase ID: FBal0003885), btd^AX (FlyBase ID: FBal0030657), stg^7M53 (FlyBase ID: FBal0016176) and the quadruple mutant³³ knirps^IID48 (FlyBase ID: FBal0005780) hunchback^7M48 (FlyBase ID: FBal0005395) forkhead^E200 (FlyBase ID: FBal0004007) tailless^L10 (FlyBase ID:FBal0016889). Descriptions of phenotypes related to eve^R13 (http://flybase.org/reports/FBal0003885.htm) and btd^AX (http://flybase.org/reports/FBal0030657.htm) have been obtained from FlyBase (launch FB2025_03)⁵⁹. In stay imaging experiments, the mutant embryos have been recognized on the premise of the absence of a balancer-linked reporter assemble, hb0.7-Venus-NLS, inserted on the FM7h, CyO or TM3 balancer¹².

To generate the eve1^KO line, an eve genomic rescue assemble, eve^CH322-103K22-mNeonGreen, was first created utilizing P[acman]^CH322-103K22 (BACPAC Resources Center), a BAC assemble that encompasses all the eve locus, from which the cease codon of eve was changed with a regular protocol^60,61 with mNeonGreen following a linker (N-ter-GSAGSAAGSGEV-C-ter). To utterly get rid of eve expression within the Eve1 area, the stripe1 (+6.6 to +7.4 kb relative to the transcriptional begin website of eve)²⁵ and late aspect (−6.4 to −4.8 kb)²⁴ enhancers have been deleted from eve^CH322-103K22-mNeonGreen by way of homologous recombination utilizing the next homology arm sequences: stripe1 left, GCAAGTCCGAGACAAATCCACAAATATTGTCAACTCTTTGGCTCTAATCTG; proper, CCAAGGCCGCAAAGTCAACAAGTCGGCAGCAAATTTCCCTTTGTCCGGCGA; and late aspect left, TTGCGTTTGAGCTACGTTACTTACATTTTTCCCACATGAGTCGGGCATACA; proper, TCGATGGGTTGGTCACAATGTGGTGGCCTCTCAACATTGCAAGGCTCTTAC. The resultant BAC assemble, eve^CH322-103K22-mNeonGreenΔst1ΔLE (Extended Data Fig. 4a) was built-in into PBac{y[+]-attP-3B}VK00033 at Rainbow Transgenics, and crossed into the eve^R13 mutant line to generate the eve1^KO line. Identification of eve1^KO embryos in stay imaging experiments was carried out as above, on the premise of the absence of a balancer-linked reporter assemble, hb0.7-Venus-NLS, inserted on the CyO¹².

For Insc overexpression, males of UAS-insc have been crossed to females containing two copies of nos-GAL4-GCN4-bcd3’UTR, which directs focused gene expression within the head area of resultant embryos⁶². These feminine flies additionally contained transgenes for the imaging markers Sqh–eGFP and 3xmScarlet-CaaX. The flies have been incubated at 22 °C for embryo assortment. For Opto-DNRho1 experiments, females of UASp-CIBN-CaaX; UASp-CRY2-Rho1[N19, Y189]²⁷ (a present from B. He, Dartmouth College, USA) have been crossed to males of matαTub-Gal4VP16^67C; matαTub-Gal4VP16¹⁵ double driver line that additionally incorporates the transgene for mat-tub-3xmScarlet-CaaX imaging marker. The resultant F1 flies have been used to arrange egg deposition cages that have been stored at 18 °C for assortment of embryos used within the experiments.

Protein tree

Predicted protein sequences of eve and btd have been used as queries to determine intently associated genes in D. melanogaster and putative orthologues in M. abdita and C. riparius utilizing BLAST. Protein alignments have been carried out in Geneious by MUSCLE alignment with normal parameters. The protein tree was assembled utilizing Jukes–Cantor because the genetic distance mannequin and UPGMA (unweighted pair group technique with arithmetic imply) for tree constructing, with a bootstrap of 1,000 replicates.

Cloning, and messenger RNA and double-stranded RNA synthesis

Cri-btd, Cri-eve, Cri-insc, Cri-sqh, Mab-btd and Mab-eve have been recognized from printed transcriptome sequences and cloned after polymerase chain response amplification from complementary DNA. In vivo labelling of cell outlines and MyoII in C. riparius used Gap43-linker–eGFP and Cri-Sqh-linker–eGFP, which have been expressed within the embryo by the injection of in vitro synthesized messenger RNAs. The Gap43-linker–eGFP fusion assemble for mRNA synthesis was generated by in-frame Gibson meeting of the Gap43 encoding sequence, a brief linker (GSAGSAAGSGEV), and a beforehand printed pSP35T expression vector (pSP-Mab-bsg–eGFP) that contained a 3′-terminal eGFP⁶³. Analogously, the Cri-Sqh-linker–eGFP fusion assemble was generated utilizing a full-length fragment of Cri-sqh amplified by polymerase chain response from cDNA. Nascent mRNAs have been generated utilizing SP6 polymerase, adopted by capping and poly(A)-tailing with devoted capping and poly(A) kits (CELLSCRIPT). Synthesized mRNA was dissolved in H₂O.

For btd RNAi experiments in D. melanogaster, double-stranded RNA was synthesized on templates that comprise the T7 promoter sequence (5′-TAATACGACTCACTATAGGGTACT-3′) at every finish utilizing a MEGAscript T7 equipment (Ambion); templates have been amplified from 0–4 h embryonic cDNA utilizing particular primers (5′-AGCAGATGACGACGACAACA-3; 5′-TACTCGGACTTCATGTGGCA-3). For insc RNAi experiments in C. riparius, dsRNA was synthesized as beforehand described⁶³. The dsRNAs comprised the next gene fragments (place 1 refers to first nucleotide within the open studying body): btd, place 1,487 to 1,817; Cri-insc (GenBank PV919477), place 466 to 1,892.

Injections

For dsRNA injections in D. melanogaster, 0–1 h-old (as much as stage 2) embryos have been collected, dechorionated with bleach and mounted on an agar pad. The mounted embryos have been then picked up utilizing a coverslip painted with glue (ready by immersing bits of Scotch tape in heptane), desiccated for 10–14 min utilizing Drierite (W. A. Hammond Drierite Co.) and lined with a combination of Halocarbon oil 700 and 27 (Sigma-Aldrich) at a ratio of three:1. Needles for injection have been ready from micro-capillaries (Drummond Microcaps, outer diameter 0.97 mm, inside diameter 0.7 mm) pulled with a Sutter P-97/IVF and bevelled with a Narishige pipette beveller (EG-44). Injections have been carried out on a Zeiss Axio Observer D1 inverted microscope utilizing a Narishige manipulator (MO-202U) and microinjector (IM300). A quantity of ~144 pl of resolution with a focus of 1.1–1.6 μg μl⁻¹ or 8–12 μg μl⁻¹ dsRNA was injected into the embryo. Embryos have been stored at 25 °C after injection in a moist chamber till early to mid-cellularization, adopted by stay imaging.

For injections in C. riparius, embryos have been collected, ready and injected basically as described beforehand⁵². Embryos have been injected earlier than the beginning of cellularization (~4 h after egg deposition), after which stored in a moist chamber till the onset of gastrulation. Throughout all procedures, embryos have been stored at 25 °C (±1 °C). Owing to their small measurement, C. riparius embryos (200 µm size) have been at all times injected into the centre of the yolk (50% of anterior–posterior axis). Embryos have been injected with dsRNA sometimes at concentrations of 300 to 700 ng ml⁻¹; mRNA was injected sometimes at concentrations of 1.5–2.5 μg μl⁻¹ (Cri-Gap43–eGFP and Cri-Sqh–eGFP). LifeAct-mCherry was injected as a recombinant protein as beforehand described at ~4.5 mg ml⁻¹ (ref. ⁶³).

Live imaging

Live imaging of D. melanogaster embryos was carried out utilizing two-photon scanning microscopy with a 25× water immersion goal (numerical aperture = 1.05) on an upright Olympus FVMPE-4GDRS system (InSight DeepSee pulsed IR Dual-Line laser, Spectra Physics) or an inverted Olympus FVRS-F2SJ system (Maitai and InSight DeepSee lasers), or a Plan-Apochromat 25× oil immersion goal (numerical aperture = 0.8) on a Zeiss LSM980 inverted microscope (Chameleon laser, Coherent Int). Excitation wavelengths have been 920 nm for eGFP, 950 nm for Venus and 1,040 nm (upright) or 1,100 nm (inverted) for mKate2, mCherry or mScarlet. Three imaging settings have been used with the next parameters (complete z depth, xy dimension of the imaging area of curiosity (ROI), z-step measurement, time interval, imaging angle or view): (1) ~80 µm, 539.5 × 185.5 µm, 2 µm, 90 s, whole-embryo lateral or ventral views; (2) ~60 µm, 253.5 × 152 µm, 1.5 µm, 50 s, head area; (3) ~40 µm, 208.3 × 152 µm, 1 µm, 45 s, cell division in head MDs. Embryos have been collected, dechorionated and mounted on coverslips or glass-bottom dishes, and immersed in 1× phosphate-buffered saline for imaging.

Live imaging of C. riparius embryos was carried out on a Leica SP8 confocal utilizing a 63× glycerol immersion goal (numerical aperture = 1.30). z-stacks of ~25 µm depth have been acquired at a z-step measurement of 1 µm and 90 s time interval. All recordings have been carried out at 25 °C.

Time-lapse imaging to visualise GBE was carried out on Nikon Eclipse-Ti microscope in differential interference distinction mode, utilizing a 20× goal (numerical aperture = 0.8) for D. melanogaster, M. abdita, C. riparius and C. albipunctata, with 1 body each 1 min; on a Leica SP5 DMI6000CS inverted confocal microscope in transmission illumination mode, utilizing a 40× goal (numerical aperture = 1.1) for C. fuscipes, with 1 body each 2 min; and on Zeiss Colibri upright microscope in differential interference distinction mode, utilizing a ten× goal (numerical aperture = 0.45) for H. illucens and a 20× goal (numerical aperture = 0.5) for Empis sp., with 1 body each 3 min. All recordings have been carried out at 25 °C.

Optogenetics

The Opto-DNRho1 system²⁷ was used as beforehand reported. To forestall undesirable photo-activation, Fly crosses and cages have been stored at the hours of darkness and embryos have been processed, staged and mounted in a darkish room with a lightweight supply lined by a lightweight crimson filter (no. 182, Lee Filters). Imaging was carried out on an Olympus FVMPE-RS (InSight DeepSee pulsed IR Dual-Line laser system, Spectra Physics) with a 25× (numerical aperture = 1.05) water immersion goal and excitation wavelength of 1,040 nm for the membrane marker 3xmScarlet-CaaX. The efficacy of MyoII inhibition with the Opto-DNRho1 system was first benchmarked on ventral furrow formation to substantiate that it resulted in a whole blockage of apical constriction²⁷.

Two photo-activation protocols have been used: protocol no. 1 used a 405 nm diode laser at 0.1% energy (5.48 µW) and protocol no. 2 used a 458 nm diode laser at 0.5% energy (27.14 µW), each scanned at 2 µs per pixel. Sham controls have been carried out at 0% laser energy. The photo-activation ROI was illuminated for 3 s in all experiments.

Three experimental designs have been used: (1) lateral imaging with unilateral photo-activation (Fig. 2h,i, Extended Data Fig. 5a and Supplementary Video 4) used protocol no. 1 on a 28.15 × 197.05 µm ROI (50 × 350 pixels) centred on ‘the pre-CF domain’⁶⁴ overlaying all the area of CF initiation alongside the dorso-ventral circumference, starting 16–33 min earlier than gastrulation and repeated each 90 s; (2) ventral imaging with bilateral photo-activation (Fig. 4a,b and Supplementary Video 7) used protocol no. 1 on two 33.78 × 33.78 µm ROIs (60 × 60 pixels) every overlaying one facet of the CF, starting 18–30 min earlier than gastrulation and repeated each 180 s; and (3) ventral imaging with bilateral photo-activation and long-term imaging (Fig. 4d,e and Supplementary Video 8) used protocol no. 2 on two 28.8 × 28.8 µm ROIs (40 × 40 pixels) every overlaying one facet of the CF, starting 15–30 min earlier than gastrulation and repeated each 180 s for 1 h, adopted by time-lapse imaging at 10 or 20 min per body for 18–23 h.

Immunofluorescence and glued imaging

For antibody staining, embryos have been mounted by a warmth–methanol technique⁶⁵ and immunostained with mouse monoclonal anti-Neurotactin (1:20, BP106, Developmental Studies Hybridoma Bank, USA), rabbit polyclonal anti-Eve (1:500, reward from M. Biggin, Lawrence Berkeley National Laboratory, USA), and rat polyclonal anti-Btd (1: 500, reward from E. Wieschaus, Princeton University, USA), adopted by DAPI staining to visualise nuclei. Imaging was carried out on a Leica SP8 system utilizing a 20× (numerical aperture = 0.75) multi-immersion goal with oil immersion (complete z depth: 60–90 µm, z-step measurement: 1.04 µm).

For DNA staining, embryos have been mounted by warmth and devitellinized as described⁶⁶, adopted by staining with DRAQ5 (1:1,000 for 1 h, Thermo Fisher Scientific, catalogue quantity 62251). Imaging was carried out on a Leica SP8 system with a 20× glycerol goal (numerical aperture = 0.75) for D. melanogaster, M. abdita, H. illucens and C. albipunctata, and a 63× glycerol goal (numerical aperture = 1.3) for C. fuscipes and C. riparius, with a z-step measurement of 1 µm in a z-range that covers no less than half of the embryo.

For the hybridization chain response (HCR), embryos have been mounted by warmth and devitellinized as described⁵⁴, probes for Cri-btd and Cri-eve have been generated utilizing beforehand printed software program⁶⁷ (https://github.com/rwnull/insitu_probe_generator) and ordered by way of Sigma-Aldrich. HCR amplifiers (B1-Alexa488 for Cri-eve; B2-Alexa594 for Cri-btd) have been obtained from Molecular Instruments. Devitellinized embryos have been re-hydrated in a collection of 1× phosphate-buffered saline with Tween (PBT) and post-fixed for 40 min with 4% paraformaldehyde in PBT on a shaker. Following PBT washes, we adopted the In situ HCR v.3.0 protocol⁶⁸ for whole-mount fruit fly embryos Revision 9 (13 February 2023) from Molecular Instruments. We then stained the embryos with DRAQ5 in 5× saline-sodium citrate with Tween (1:1,000 for 1 h) and mounted the embryos in 50% glycerol in 5× saline-sodium citrate with Tween. Imaging was carried out as above for DNA staining in Chironomus riparius.

For in situ hybridization, embryos have been mounted by a warmth–formaldehyde technique⁶³. Transcripts have been detected histochemically or fluorescently as described⁶⁹, utilizing RNA probes for Mab-btd (comprising 1,473 nucleotides from +1 to 1,473, with place +1 referring to first nucleotide within the open studying body), Mab-eve (comprising 984 nucleotides, from place 365 to 996 of the putative coding sequence and 351 nucleotides of the three′ UTR), and Cri-insc (comprising 1,427 nucleotides from 466 to 1,892) labelled with both digoxigenin or fluorescein. M. abdita embryos have been additionally stained with DAPI to visualise nuclei.

Image processing and quantification

Images have been processed, assembled into figures and transformed into movies utilizing FIJI, Affinity Designer, Adobe Illustrator and HandBrake. Quantitative knowledge have been analysed and processed utilizing Excel, or custom-made ImageJ or FIJI macros and Python scripts utilizing Numpy, Pandas and SciPy libraries. Plots have been generated in GraphPad Prism or with Python scripts utilizing Matplotlib and Seaborn graphic libraries. Detailed descriptions of picture processing and evaluation procedures are supplied in Supplementary Methods.

Statistical analyses

All of the statistical particulars of experiments, together with the variety of experiments (n), which represents the variety of embryos used until in any other case famous, are given within the determine legends. Python scripts utilizing SciPy library have been carried out to carry out one-way ANOVA adopted by Tukey’s a number of comparability put up hoc take a look at for evaluating means from greater than two teams, and Mann–Whitney U-test was used as a non-parametric impartial take a look at for evaluating two means. GraphPad Prism was used: (1) to carry out statistical analyses to check the blastoderm cell densities throughout species, together with the calculation of medians and the 95% confidence intervals on the median, and one-way ANOVA with Kruskal–Wallis non-parametric take a look at, with out correcting for a number of comparisons (uncorrected Dunn’s take a look at); and (2) to carry out Fisher’s actual take a look at with Bonferroni correction for pie chart distributions. For cell and area space evaluation, Microsoft Excel was used to carry out paired and unpaired t-tests and to plot normal errors.

Reporting abstract

Further info on analysis design is offered within the Nature Portfolio Reporting Summary linked to this text.