Jeremy Dufourt
email: jeremy.dufourt@igmm.cnrs.fr

Maelle Bellec

Antonio Trullo

Matthieu Dejean

Sylvain De Rossi

Mounia Lagha
email: mounia.lagha@igmm.cnrs.fr

Affiliation

Imaging translation dynamics in live embryos reveals spatial heterogeneities

The translation of individual mRNA molecules is a key biological process, yet this multi-step process has never been imaged in living multicellular organisms. Here we deploy the recently developed Suntag method to visualize and quantify translation dynamics of single mRNAs in living Drosophila embryos. By focusing on the translation of the conserved major epithelial-mesenchymal transition (EMT)-inducing transcription factor Twist, we identified spatial heterogeneity in mRNA translation efficiency and reveal the existence of translation factories, where clustered mRNAs are cotranslated preferentially at basal perinuclear regions. Simultaneous visualization of transcription and translation dynamics in a living multicellular organism opens exciting new avenues for understanding of gene regulation during development.

Introduction

During the development of multicellular organisms, gene expression must be precisely regulated in time and space. Decades of genetic manipulations in Drosophila have dissected the gene regulatory networks responsible for the establishment of precise patterns of gene expression [START_REF] Wieschaus | Positional Information and Cell Fate Determination in the Early Drosophila Embryo[END_REF]. The establishment of these patterns has been primarily studied at the transcriptional/mRNA level in fixed embryos using fluorescent in situ hybridization (FISH), or in living embryos via the recent adoption of mRNA labeling technologies such as the MS2/MCP system [START_REF] Garcia | Quantitative imaging of transcription in living Drosophila embryos links polymerase activity to patterning[END_REF][START_REF] Lucas | Live imaging of bicoiddependent transcription in Drosophila embryos[END_REF][START_REF] Pichon | A Growing Toolbox to Image Gene Expression in Single Cells: Sensitive Approaches for Demanding Challenges[END_REF]. In the context of a developing embryo, precision in mRNA production is functionally relevant only if it leads to precision in protein abundance.

While this central dogma has been established

over the last 60 years, the non-linearity between the levels of a given mRNA and the amount of protein it encodes has only recently started to emerge [START_REF] Gabut | Ribosome and Translational Control in Stem Cells[END_REF][START_REF] Becker | Quantifying posttranscriptional regulation in the development of Drosophila melanogaster[END_REF][START_REF] Liu | On the Dependency of Cellular Protein Levels on mRNA Abundance[END_REF][START_REF] Casas-Vila | The developmental proteome of Drosophila melanogaster[END_REF]. One explanation for this poor correlation resides in the spatial control of translation that could lead to differential protein output. It is indeed well established that mRNAs can be localized to particular subcellular compartments [START_REF] Lecuyer | Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function[END_REF]. This subcellular targeting is evolutionary conserved and has been described in a variety of organisms from bacteria to mammals [START_REF] Chin | RNA localization: Making its way to the center stage[END_REF].

However, the consequences of such localization in terms of translational output are much less thoroughly understood. In some cases, localization of mRNAs favors their translation, as exemplified by oskar at the posterior pole of Drosophila oocytes [START_REF] Halstead | Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals[END_REF] or β-actin at neuronal branching points [START_REF] Wong | RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo[END_REF][START_REF] Wu | Translation dynamics of single mRNAs in live cells and neurons[END_REF]. But in other cases, mRNAs can assemble in multimolecular complexes to prevent their translation, such as with mRNAs located in stress granules [START_REF] Moon | Multicolour singlemolecule tracking of mRNA interactions with RNP granules[END_REF].

The non-linearity between mRNA and protein distributions has primarily been studied with genomic approaches on populations of cells where spatial constraints are eroded. Indeed, while live imaging of mRNA has been possible since 1998 [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF], a similar method to image many cycles of translation (Suntag) was only deployed in 2016 in cultured cells [START_REF] Wu | Translation dynamics of single mRNAs in live cells and neurons[END_REF][START_REF] Yan | Dynamics of Translation of Single mRNA Molecules In Vivo[END_REF][START_REF] Morisaki | Real-time quantification of single RNA translation dynamics in living cells[END_REF][START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF][START_REF] Wang | Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells[END_REF],

and has yet to be introduced in an intact developing organism. Here we have implemented the Suntag labeling method in Drosophila and quantified the translational dynamics of endogenous twist (twi) mRNA molecules in living embryos. We uncovered a broad range of translation efficiencies of the twi mRNA population. In particular, cytoplasm located below nuclei fosters enhanced translation, in part via directed-mRNA clustering in translation factories. The revealed translation factories of twi mRNAs are primarily located in the basal perinuclear space and exhibit reduced mobilities.

Moreover we identified that single mRNA molecules located in this subcellular compartment are more efficiently translated than identical mRNA located apically.

Localized and enhanced translation of transcription factor encoding mRNAs at the basal perinuclear cytoplasm may facilitate their nuclear import and limit their diffusion, two essential features allowing precise (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted April 30, 2020. . https://doi.org/10.1101/2020.04. [START_REF] Metzler | Anomalous diffusion models and their properties: non-stationarity, nonergodicity, and ageing at the centenary of single particle tracking[END_REF].058974 doi: bioRxiv preprint formation of primordial embryonic germ layers.

Results

Implementation of Suntag method to visualize translation in a developing organism

In the Suntag system, the multimerization of a GCN4-based epitope (named suntag), with high affinity for a single chain antibody scFv coupled to a fluorescent protein (sfGFP) [START_REF] Tanenbaum | A protein-tagging system for signal amplification in gene expression and fluorescence imaging[END_REF] allows multiple scFv-GFP to bind (Figure 1a). This signal amplification creates a bright fluorescent spot above the background signal.

We focused our analysis on the twi gene, which encodes a key conserved transcriptional activator of the mesodermal gene network and the EMT program in metazoans that is frequently deregulated in a large number of metastatic cancers [START_REF] Yang | Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis[END_REF].

Initially, to test if the Suntag labeling system could be functional in a living organism we created a twi_suntag transgene whereby 32 suntag repeats [START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF] were inserted immediately after the start codon (Figure 1b). In addition, we created various scFv-GFP lines for maternal deposition, in embryos, of the scFv detector protein (Figure S1a-c plane within the mesoderm of a scFv-GFP-NLS/+ > twi_suntag/+ embryo stained with endogenous scFv-GFP (green) and probes against suntags (red). scFv-GFP foci colocalizing with suntag probes reveal mRNAs in translation (yellow, arrow), while spots that are only stained with suntag probes correspond to non-translating mRNAs (red, arrowhead), scale bars 1μm. d, Snapshots of dorsal and ventral views of a maximum intensity projected Z-stack from a scFv-GFP-NLS/+ > twi_suntag/+ live embryo imaged with a MultiView Selective Plane Illumination Microscope, MuViSPIM, (related movie 2). Associated zoomed images are shown on the right. All embryos are ventral views, oriented with anterior to the left. e, Frames taken from a fast-confocal movie (≈2 frames/sec) of a scFv-GFP-NLS/MCP-TagRFPT-NLS > twi_suntag_MS2/+ embryo (related to movie 4), showing an mRNA molecule (red) colocalizing and moving with a bright scFv-GFP signal (green).

While the suntag signal was restricted specifically to the mesoderm, twi_suntag expression appeared stochastically in cells within this domain, likely due to the presence of only the twi proximal enhancer in the transgene. Thus, our twi transgene did not fully recapitulate twi canonical expression (Figure S1f).

Imaging translation from an endogenous locus

To monitor twi translational dynamics from the endogenous locus, we created a twi_suntag_CRISPR allele (Figure 2a) and a twi_suntag_MS2_CRISPR allele (Figure S2a).

With its complete set of regulatory regions, twi_suntag_CRISPR and twi_suntag_MS2_CRISPR were activated throughout the mesoderm (Figure 2b, Figure S2b). The scFv-GFP protein labeling completely overlapped that of twi mRNAs labelled with smFISH probes directed against the suntag (Figure 2b) or MS2 sequences (Figure S2b), and bright scFv-GFP spots are observed in nc14. At single molecule resolution, bright foci of translating individual twi mRNA are detected in ≈64% of total pool of single mRNA molecules in nc14 (n=12022 from four smFISH), along with untranslated single mRNAs and single molecules of Twi protein (Figure 2c, Figure S2c). Using live imaging of these twi_CRISPR alleles, we examined the timing of twi translation. To image the entire embryo without compromising on the temporal resolution, we employed light sheet microscopy (MuViSPIM) [START_REF] Schmied | Sample Preparation and Mounting of Drosophila Embryos for Multiview Light Sheet Microscopy[END_REF][START_REF] Krzic | Multiview lightsheet microscope for rapid in toto imaging[END_REF]. This revealed that twi translation was strongly induced during nc14

(Figure 2d, 2e, Figure S2d, movie 5 and S2).

This result was confirmed by smFISH and confocal microscopy (Figure S2e-f). Bright but rare scFv-GFP foci appeared as early as nc12 (Figure S2e-f) and we observe a persistent translation during mitoses (Figure 2f). Following the initial syncytial development stage, the plasma membrane progressively invaginates between adjacent nuclei during nc14 with an apico-basal directionality, a process named cellularization. Interestingly, the large wave of twi translation during nc14 occurs prior to the completion of cellularization.

Thus, twi mRNA are efficiently translated during a developmental window where short range diffusion between neighboring 'pseudocells' is possible [START_REF] Gregor | Diffusion and scaling during early embryonic pattern formation[END_REF]. The timing of twi translation is consistent with dynamics of twi mRNA production. twist is a zygotic gene which is among the first genes to be transcribed (~nc11) [START_REF] Sandler | Quantitative Single-Embryo Profile of Drosophila Genome Activation and the Dorsal-Ventral Patterning Network[END_REF]. The number of cytoplasmic twi transcripts peaks early in nc14( 26) and seems to precede the stage when twi translation is at maximum (Figure 2e).Moreover, the timing of twi translation is consistent with the timing of the appearance of nuclear Twi protein (Figure 2g). Given the pivotal role of Twi protein acting as a major transcriptional activator of the mesodermal fate [START_REF] Sandmann | A core transcriptional network for early mesoderm development in Drosophila melanogaster[END_REF], this relatively late translation was not expected.

By combining suntag and MS2 labeling, it is possible to image transcription and translation from an endogenous gene within a single embryo (Figure S2g and movie S3). Thus, double labeling with suntag/MS2 repeats provides a method to quantify the precise chronology of events and possible flow of information from transcription to translation.

Spatial heterogeneity of twist translation

Having determined the precise timing of twi translation, we then investigated its subcellular twi_suntag_CRISPR/+ embryos expressing scFv-GFP labelled with suntag smFISH probes. d, Zoomed z-projected confocal images taken from the apical and basal perinuclear compartment of scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ Drosophila embryos expressing scFv-GFP (green) and-labelled with suntag smFISH probes (red). Top panels show two z-projected images of the apical compartments. Bottom panels are equivalent z-projected images located basally on which it is possible to distinguish single mRNA molecules not in translation (white stars), single mRNA molecule engaged in translation (white arrows), and large GFP foci colocalizing with cluster of mRNAs (arrowhead). e, Violin plot of the distribution of the number of mRNA molecules per cluster from images taken apically (n=523; blue) and basally (n=1384; green) from four nc14 embryos scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ labelled with suntag smFISH probes. Centered black bar represents the median, dashed black lines represent quartiles. * * * * p < 0.0001 with a two-tailed Welch's t-test. f, Percentage of mRNA in translation located in the apical or basal cytoplasm(blue or green respectively) or not translating (light blue or light green respectively) in each class of mRNA pool (see main text and methods), from four scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ nc14 embryos labelled with suntag smFISH probes.g, Violin plot of the distribution of scFv-GFP intensities colocalizing with single mRNA molecules located apically (n=2380; blue) and basally (n=4202; green) from four nc14 scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ embryos labelled with suntag smFISH probes (see methods). Centered black bar represents the median, dashed black lines represent quartiles. * * * * p < 0.0001 with a two-tailed Welch's t-test.

Remarkably, similar clustering of mRNA was also detected with the twi_suntag transgene (Figure S3e-f), where the mRNA distribution is different from that of the twi_CRISPR alleles, owing to an initial erratic activation pattern (Figure S1f). Thus, mRNA clustering and cotranslation do not seem to depend on mRNA concentration. To quantify these clusters and their ability to be translated, we divided the total pool of mRNAs into 4 distinct classes of mRNA densities with 1, 2, 3 or ≥4 mRNAs (Figure 3f) and scored their ability to undergo translation.

Remarkably, molecules located in a large mRNA cluster were systematically engaged in translation. This is particularly true for mRNA clusters located at the basal perinuclear space (Figure 3f).

Given the enhanced clustering in this compartment, we questioned the properties of this basal compartment on translational efficiency. To estimate the efficiency of translation, we extracted the intensity of the scFv-GFP signal overlapping each individual mRNA molecule (not contained within a cluster) (see methods). We found that in the basal perinuclear space, a single molecule of mRNA is on average 50% more intense than a single molecule located apically, suggesting an enhanced efficiency of translation (Figure 3g).

Collectively our results suggest that the basal perinuclear cytoplasm fosters an enhanced twi translation via the formation of co-translated mRNA clusters and via an enhanced efficiency of single mRNA molecules translation.

Tracking of translating mRNPs reveals distinct mobilities

To quantitatively characterize the diffusive properties of twi_suntag_CRISPR mRNPs, we imaged, the scFv-GFP signal in the apical and basal perinuclear region in the mesoderm at high speed and employed single particle tracking (Figure 4a and movie S5, S6). The trajectories show clear differences in particle mobility between the apical and basal compartments (Figure 4b-c and Figure S4). The mRNPs located above nuclei tend to displace faster than those located below as quantified by 1D displacement without preferential drift (Figure S4b). This difference in behavior could be due to a differential confinement or alternatively and nonexclusively to a lower diffusion capacity of the mRNPs located at the basal perinuclear space.

To discriminate between these two scenarios, we quantified the mean square displacement (MSD). MSD curves reveal clear distinct displacement properties among these two subpopulations (Figure 4d). In both populations, the sublinear growth of the MSD curves suggests a sub-diffusive behavior (also called anomalous diffusion) [START_REF] Woringer | Protein motion in the nucleus: from anomalous diffusion to weak interactions[END_REF]. Indeed, a linear fitting of the MSD curves was unsatisfactory (R 2 <0.8) (Figure S4 c). We therefore used a non-linear model (anomalous fitting) [START_REF] Woringer | Protein motion in the nucleus: from anomalous diffusion to weak interactions[END_REF][START_REF] Metzler | Anomalous diffusion models and their properties: non-stationarity, nonergodicity, and ageing at the centenary of single particle tracking[END_REF] to fit to our data and estimated two parameters: the anomalous diffusion exponent (alpha parameter) and the diffusion coefficient. The anomalous alpha parameter was estimated to be <1 (Figure 4e), confirming the non-linearity and the confinement of the diffusion process. However, this parameter appears comparable in both compartments.

Thus, the differential mobility of mRNPs between the apical and basal cytoplasmic compartments cannot be solely attributed to a distinctive confinement.

We therefore examined the diffusion coefficient. Sites of translation located apically show an average diffusion coefficient of 0.051µm 2 s -1 , a value comparable to what has been reported in cell culture experiments using Suntag [START_REF] Wu | Translation dynamics of single mRNAs in live cells and neurons[END_REF][START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF] or by spt-PALM on ribosomes [START_REF] Katz | Mapping translation 'hotspots' in live cells by tracking single molecules of mRNA and ribosomes[END_REF]. Remarkably, the diffusion coefficient of mRNPs located basally is three-times lower, showing a significantly lower mobility (Figure 4f). In conclusion, these kinetic measurements of live translation spots (Figure 4) are consistent with the conclusions obtained from fixed tissues (Figure 3) and strongly suggest that in the basal perinuclear cytoplasm, translation sites primarily consist of slow diffusing clusters of mRNAs engaged in translation, referred to as translation factories. Low mobility of translation factories can be attributed to their larger size. However, an anchoring to particular organelles, specific to the basal perinuclear cytoplasm, could also slow down the diffusion of mRNPs and remains to be demonstrated.

Conclusion

The Suntag method provides a versatile approach to measure translation kinetics of single mRNAs in a developing embryo. This [START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF] or in dividing yeast cells [START_REF] Pizzinga | Translation factor mRNA granules direct protein synthetic capacity to regions of polarized growth[END_REF]. Given the widespread subcellular localization of mRNA during development [START_REF] Lecuyer | Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function[END_REF], we expect that our newly identified twi translation factories represent one example among many more to come. Co-translating multiple mRNAs within a local microenvironment may allow a higher efficiency e.g via ribosome recycling [START_REF] Gerovac | Control of mRNA Translation by Versatile ATP-Driven Machines[END_REF][START_REF] Marshall | Ribosome recycling induces optimal translation rate at low ribosomal availability[END_REF].

Secondly, Drosophila twi translation factories are not randomly distributed but rather concentrate in the basal perinuclear space. We discovered that in this compartment, the efficiency of translation of single mRNA molecules was significantly higher than that of identical mRNAs located elsewhere. This spatial bias might be supported by the already reported higher availability of mitochondria in this perinuclear compartment [START_REF] Chowdhary | Analysis of mitochondrial organization and function in the Drosophila blastoderm embryo[END_REF], in line with the fact that protein synthesis is one of the most energy-consuming processes in the cell(35-

37).

In the context of a syncytial embryo, compartmentalization of the cytoplasm could be important for the confinement of protein

Methods

Drosophila stocks and genetics

The yw stock was used as a control.

Cloning and Transgenesis

The twi-suntag transgene was synthesized Plan-Apochromat (0.8NA) air objective lens.

GFP were excited using a 488nm laser, Cy3 and Quasar570 were excited using a 561nm laser, Quasar670 was excited using a 633nm laser.

Image analysis

Analysis of smFISH data related to figure 3 The first parameter extracted is the percentage of mRNA in translation: given by the ratio between the number of mRNA spots overlapping a translation spot, divided by the total number of mRNA spots.

We also extracted the efficiency of translation. We then classified all the spots in terms of intensity, assigning a value of 1 to a spot if its intensity is < µ + 3σ, value 2 if its intensity I, is such that µ + 3σ < I < 2 * (µ + 3σ) and so on. The ortho slicer tool was used to show cross sections of embryos with ≈5µm extended section thickness for movie 6 and S4 and ≈10µm extended section thickness for movie S3.

Live Imaging

Embryos were dechorionated with tape and mounted between a hydrophobic membrane and a coverslip as described previously 14 .

Movies for scFv-GFP-noNLS (related to movie S1) and scFv-GFP-NLS (related to movie 1)

were acquired using a Zeiss LSM780 confocal microscope with a Plan-Apochromat 40x/1. Each movie was max intensity Z-projected using Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF]. Single particle trajectories were generated with MatLab 2015b (Mathworks Inc., USA) using SLIMfast, which implements the Multiple-Target-Tracing algorithm [START_REF] Liu | 3D imaging of Sox2 enhancer clusters in embryonic stem cells[END_REF][START_REF] Serge | Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes[END_REF].

Individual spots were localized using a blob diameter of 9 pixels. They were then tracked using maximal frame gap of 0 frame and a maximal expected diffusion coefficient set to (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Dmax = 0,5 µm 2 .s -1 . Tracked movies were then evaluated using the script evalSPT [START_REF] Normanno | Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher[END_REF]. Each tracked spots was visually checked and poorly tracked spots were manually removed using evalSPT. In total we obtained 237 traces from

  and movie 1and S1). To ensure minimal levels of free scFv in the cytoplasm, we added a nuclear localization signal (NLS; Figure S1c and movie 1), referred to as scFv-GFP. We also generated a graded scFv-GFP line with high expression at the anterior pole that diminishes towards the posterior region (scFv-GFP-bcd 3'UTR) (Figure S1d). A diffuse, soluble GFP signal is detected in all scFv lines, showing that scFv can be genetically encoded in Drosophila without forming aggregates (Figure S1 b, c and movie 1 and S1).In the presence of the twi_suntag transgene and scFv-GFP detector protein, clear fluorescent spots are detected in fixed embryos at nuclear cycle (nc) 14 specifically in the mesoderm, where twist is transcribed (FigureS1e).

  Figure 1: Imaging translation with suntag labeling in Drosophila embryos a, Principle of the Suntag system: labeled antibody probes, here single-chain variable fragments coupled to sfGFP (scFv-GFP), bind to repeated suntag epitopes, allowing visualization of nascent translation. Tandem array of suntag peptides are shown in orange, scFv-GFP molecules in green and ribosomes in purple. b, Schematic representation of twist_Suntag transgene. c, Zoomed confocal single

  By performing single molecule mRNA labeling using single molecule fluorescent in situ hybridization (smFISH) with the simultaneous detection of native scFv-GFP, we could detect two populations of cytoplasmic single mRNA molecules: those co-localizing with a bright GFP signal, ≈56% of total mRNA in nc14 (n=6231 from three smFISH), corresponding to mRNAs in translation, and those devoid of a GFP signal and corresponding to untranslated mRNAs (Figure 1c). By live imaging, distinct spots were detected clearly above background within the presumptive mesoderm of twi_suntag transgenic embryos (Figure 1d and movie 2). These bright spots correspond to nascent sites of translation, as they disappear upon puromycin injection into live embryos (Figure S1g and movie 3). In order to visualize both mRNA and their translation in living embryos, we created another transgene where, in addition to the suntag sequences, MS2 repeats were inserted in the 3'UTR region (Figure S1h). To amplify the fluorescent signal produced by single molecules of mRNA, we employed a 128-loop array of a new generation of optimized MS2 loops(22). Similar to the twi_suntag transgene, twi_suntag_MS2 transcripts were expressed in the presumptive mesoderm (Figure S1i) and we could detect two populations of cytoplasmic mRNAs, the first corresponding to mRNAs in translation (arrow) and the second to mRNAs not in translation (arrowhead) (Figure S1j). Using two-color live imaging of the twi_suntag_MS2 transgene (with scFv-GFP and MCP-TagRFPT detector transgenes), we occasionally detected mRNA spots colocalizing and traveling with suntag signal in the cytoplasm of living embryos (Figure 1e and movie 4). However, fast diffusion of mRNA molecules and rapid photobleaching of the fluorescent tag precluded their tracking. In conclusion, we have developed and validated the genetic tools to image translation in intact live Drosophila embryos. We can thus image translation in live embryos with scFv-GFP and use smFISH to detect and quantify single molecules of cytoplasmic mRNAs.

  spatial localization. Using a reconstructed transverse view of a developing embryo by MuViSPIM, the sites of translation in nc14 appeared much more prominent in the basal perinuclear region (i.e. towards the inside of the embryo), although translation was also observed in the apical perinuclear region (Figure 3a and Figure S3a and movie 6 and S4). To confirm the observation that translation is heterogeneously distributed, we quantified the scFv-GFP signal in these two compartments in living embryos from nc11 to nc14 by confocal microscopy (Figure S3b and S3c). Contrary to early developmental stages, where translation seems equivalent in the apical and basal cytoplasmic space, there is a striking difference in the behavior in nc14. In nc14, the largest and brightest spots of twi translation appeared mainly in the cytoplasm located below the nuclei (basal) (Figure 3a, S3a-c). To better characterize these large scFv-GFP foci with their associated mRNA corresponding to messenger ribonucleoproteins (mRNPs), we systematically quantified the number of translated and non-translated mRNAs in fixed samples in 3D (Figure 3b and S3d). While mRNA molecules were present along the entire depth of a cell volume (Figure 3c), their intensity was clearly enhanced at the level of the basal perinuclear space (Figure 3c-d). A similar spatial heterogeneity is observed for the scFv-GFP translation signal, which peaks in the basal perinuclear space (Figure 3c-d). We then determined the intensity of a single mRNA molecule from smFISH experiments and identified clusters of multiple molecules of mRNAs (see methods). These mRNA clusters were of varying sizes ranging from 2 to 6mRNAs and were significantly more frequent in the basal perinuclear cytoplasm (Figure3e).

  Figure3:Revealing a spatial heterogeneity of endogenous twist mRNAs translation a, Cross section of a transverse plane from reconstructed images by MuViSPIM of a His2Av-mRFP/+; scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ embryo (related movie 6). Ventral mesodermal nuclei are located at the bottom. Nuclei are observed with the His2Av-mRFP (magenta) and sites of translation with the scFv-GFP (green dots), scale bars 30μm. b, Maximum intensity projection of three z-planes (≈1 μm) of confocal images of nc13 and nc14 scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ embryos expressing scFv-GFP (green) labelled with suntag smFISH probes (red) and DAPI (blue), scale bars 5μm. c, Intensity quantification of detected mRNA spots (red) and scFv-GFP spots (greenin zplanes (0.5 μm apart) of four nc14 scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ embryos expressing scFv-GFP labelled with suntag smFISH probes. d, Zoomed z-projected confocal images taken from the apical and basal perinuclear compartment of scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ Drosophila embryos expressing scFv-GFP (green) and-labelled with suntag smFISH probes (red). Top panels show two z-projected images of the apical compartments. Bottom panels are equivalent z-projected images located basally on which it is possible to distinguish single mRNA molecules not in

Figure4: Quantifying the mobilities of twist mRNPs translation foci a ,

 a Figure4: Quantifying the mobilities of twist mRNPs translation foci a, Typical confocal images of translation foci located in theapical and basal perinuclear cytoplasm at nc14 used for single particle tracking. Images extracted from a fast movie at a rate of 2 frames s -1 of a living scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ embryo (related movie S5-6). Scale bars 5μm. b, c, Examples of color-coded trajectories of translation foci, taken from fast movies as shown in a. d, Graph representing the mean Mean Square Displacement (MSD) as a function of time for particles located apically (n=237 traces, three movies; blue) or basally (n=263 traces, three movies; green) nuclei. Error bars represent SEM. e, Violin plots of the alpha parameter for the particles located apically (blue) and basally (green) using anomalous fitting (centered black bar represent median, dashed lines represent quartiles). f, Violin plots of the estimated diffusion coefficient distribution of the particles located apically and basally of living scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ nc14 embryos (centered black bar represents median, dashed lines represent quartiles). g,

  labeling technology can be combined with mRNA labeling methods for the simultaneous quantitative imaging of transcription and translation dynamics. By focusing on twi mRNAs as a paradigm for transcription factor encoding transcripts, we have uncovered fundamental features of translation in a living organism. First, we have shown the existence of translation factories in an embryo, whereby clusters of several mRNAs were co-translated in a microenvironment, echoing what has recently been shown in cultured human cells

  synthesis and subsequent delivery to final functional destinations. In the case of twi mRNAs, which encodes a critical transcription factor, a combination of slow mRNPs mobility and enhanced efficiency of translation in the basal perinuclear space could limit the protein's diffusion outside of the presumptive mesoderm and favor rapid import of neo-synthesized Twi protein to the nucleus. Rapid nuclear availability of this key transcription factor might be essential for rapid activation of the hundreds of direct target genes instructing the mesodermal fate(27). Enhancement of translation efficiency in dedicated cytoplasmic microenvironments could be employed by other developmental genes to ensure gene expression precision. Thus, precision in the establishment of developmental patterns(38), such as the presumptive mesoderm, cannot only be attributed to a precision in transcriptional activation. Future work will elucidate if other developmentally regulated mRNAs are translated via translation factories. We anticipate that the Suntag labeling method, combined with recently developed optogenetic manipulations(39, 40), will pave the way towards hitherto inaccessible translation modalities in intact developing organisms, much like the revolution triggered by the deployment of mRNA detection in living organisms.

  -His2Av-mRFP comes from[START_REF] Dufourt | Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs[END_REF]. For most experiments, female virgins expressing scFv-sfGFP-NLS were crossed to yw to obtain scFv-sfGFP-NLS/+ flies. scFv-sfGFP-NLS/+ virgins were crossed with males containing suntag repeats and/or MS2 constructs.

  was performed by custom-made algorithms developed in Python TM . Briefly, a blob detection was performed on the scFv-GFP and mRNA channels (green and red respectively) separately. Raw data were filtered frame by frame with a two-dimensional Difference of Gaussian Filter whose kernels size are determined as in(38) and the filtered images were thresholded with a user defined threshold value; the choice of the threshold was driven by visual inspection thanks to an interactive graphical tool. All the 3D connected components of the resulting binary images are considered as spots, which are then filtered in size with a volume threshold of 10 pixels. All spots with centroids in the same frame where the nuclei were detected were removed so as to only analyze mRNAs located at the apical and basal perinuclear cytoplasmic part. Within the detected mRNA pool, three populations could be distinguished by volume or intensity. As expected, smFISH allowed us to detect single molecules of mRNA, mRNAs in clusters and mRNAs undergoing decay. By looking at the mRNA spots not engaged in translation (red spots not overlapping green spots), a separable population can be characterized by a very low spot volume. We speculate that these small spots are mRNA in decay that are unable to be translated. To remove these small mRNA spots (possibly in decay), we refined the volume threshold of a single molecule mRNA by looking at the histogram of the volume of the mRNA spots in translation (red spots overlapping with at least one pixel of a green spot). This led to the detection of two major populations on the histogram: single molecules of mRNA, representing the larger population shown in the histogram, and mRNA clusters, characterized by a smaller population and a bigger volume. We performed a double Gaussian fitting on the histogram to differentiate the single molecule and cluster populations, then we defined a threshold value for the lower bound of the single molecule volume as µ -3σ, where µ and σ are, respectively, the mean and the standard deviation of the Gaussian function fitting single molecule mRNA spots. Having obtained the correct threshold value for the volume, we reanalyzed the mRNA channel to connect the spots detection results from both channels. We obtained two clear populations of mRNAs that we divided into two subpopulations based on position above or below the nucleus.

For

  this, we estimated mRNA single molecule intensity separately for above and below the nucleus by performing a Gaussian fitting on the histogram of intensities of all detected mRNA spots. Single molecules are by far the widest population, so a single Gaussian fitting gives an estimate of the single molecule intensity, not influenced by the population of mRNA clusters, also present in the histogram. We used µ + 3σ for the single molecule upper intensity bound where µ and σ are the mean and the standard deviation of the fitting respectively. For translation spots we used the GFP intensity divided by background, where the background is calculated for each spot as the average intensity value of the pixels surrounding the spot itself. This allowed us to rescale by the background, because in this case we have a bath of free diffusing GFP (provided by the nos-scFv-GFP-NLS transgene). To score differential clustering of mRNAs, above and below nuclei, all mRNA spots intensities above µ + 3σ (mRNA clusters) were rescaled by the mean intensity of a single mRNA molecule (extracted from the Gaussian fitting), which allowed us to express the signal of each mRNA entity (cluster of varying sizes) as a function of a single molecule. For translation efficiency and clustering of mRNA, outliers were removed using the ROUT method(43), embedded in GraphPad Prism 8 software, with Q set to 0.1%.

For

  each spot category, we checked the percentage of mRNA spots overlapping a translational spot, meaning that they are engaged in translation. For smFISH of the CRISPR alleles, analysis above the nucleus contained between 5 and 8 confocal Z-planes spaced 0.5μm apart, and analysis of the region below the nucleus contained 17 to 28 Z-planes spaced 0.5μm apart. For smFISH analysis with twi_suntag transgene, only one filtering with a volume threshold of 10 pixels was performed as only two populations appeared (single molecule mRNA and mRNA clusters). Analysis for above the nucleus contained 6 to 12 confocal Z-planes spaced 0.5μm apart and analysis below the nucleus contain 36 to 41 confocal Z-planes spaced 0.5μm apart. Light-Sheet Microscopy For light-sheet imaging (related to movie 2,5,6 and S3-4), we employed the MuViSPIM (Luxendo, a Brüker company). This setup provides two-sided illumination with two Nikon 10x/0.3 water objectives and two-sided detection with two Olympus 20x/1.0 W objectives. The light sheet is generated by a scanning of a gaussian beam. We used the line illumination tool for improved background suppression. Images are acquired by two ORCA Flash 4.0 (C91440) from Hamamatsu and processed by LuxControl v1.10.2. A 50ms exposure time was used for the green and red channel with a 488nm and 561nm laser excitation respectively. Maximum intensity projection were processed with Fiji(44). Fusion is processed by a software solution from Luxendo (Image Processor v2.9.0) Deconvolution was performed after the fusion process and executed with Huygens Professional v 19.10 (Scientific Volume Imaging B.V). We used the gaussian multi-view light sheet parameters for processing of 3D+t images. 3D reconstruction was done with Imaris v9.5.0 (Bitplane, an Oxford company).

  3 oil objective lens, with GFP excitation using a 488nm laser. A GaAsP detector was used to detect GFP fluorescence with the following settings: 1024x 1024 pixels, each Z-stacks comprised 6 planes, 16-bit and zoom 2.0. Movies for scFv-GFP-Bcd3'UTR-NLS were acquired using a Zeiss LSM780 confocal microscope with a Plan-Apochromat 40x/1.3 Oil objective and the following settings: 512x 512 pixels, each Z-stacks comprised 12 planes, 16-bit. Movies of scFv-GFP-NLS/+> twi_suntag_CRISPR/+ or twi_suntag_MS2_CRISPR/+ were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm laser with the following settings: 640x 640 pixel images, each Z-stacks comprised 50-60 planes spaced 0.5μm apart and zoom 2.5x. Movies were then processed to remove frame outside of the embryos or containing the membrane signal to correct drifting, and processed stacks were max intensity projected using a custom made software, developed in Python TM . Movies of scFv-GFP-NLS/MCP-TagRFPT-NLS >twi_suntag_MS2/+ transgene (related movie 4) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP and RFP were excited using a 488nm and 561nm laser respectively with the following settings: 276x 316 pixel images, each Z-stacks comprised 2 planes spaced 0.5μm apart and zoom 14x. Puromycin Injection Embryos were dechorionated with tape, lined up on a hydrophobic membrane covered in heptane glue with desiccation at room temperature for 10 minutes prior to being covered in Voltalef Oil 10S (VWR), and injected with 10mg/mL puromycin (InvivoGen) using a FemtoJet ***5427*** (Eppendorf) microinjector and associated Femtotips® I (Eppendorf) needles. Embryos were injected in the lateral region immediately prior to coverslip positioning. Time lapse images (related to Movie 3) were acquired using a confocal LSM780 (Zeiss) microscope. A GaAsP detector was used to detect the GFP fluorescence excited using a 488nm laser. Detection and tracking of single particles For single particle tracking (related movie S5-6), movies were acquired with a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens, GFP was excited using a 488nm laser and the following settings: 132 x 132 pixels, each Z-stacks comprised 10 planes 0.5μm apart, Zoom 8x. Under these conditions a zstack was acquired every ≈500ms.

  three movies above nuclei (Apical) and 263 traces from three movies below nuclei (Basal).1D displacement in x and y were obtained from evalSPT tool for each movie. Mean square displacements (MSD) were calculated for tracks present for at least 10 consecutive frames using the MSDanalyzer MatLab script(48). Coefficients diffusion were first obtained by fitting 0 to 20 seconds of the MSD of individual trajectories with a linear model as follows: y = a*x+b To estimate MSD, according to Einstein's theory, the MSD of Brownian motion is described as MSD(t) = 2dDt with d representing dimensionality, in our case, d = 2. D represents the diffusion coefficient. Then, MSD of individual trajectories were fitted with a non-linear model, as follows: If x = log(D) and y = where 'D' are the delays at which the MSD is calculated, then this method fits y = f(x) by a straight line y = α * x + γ so that we approximate the MSD curves by MSD = γ * D α . According to a previous modeling study(49), an α value of 1 indicates free diffusive movement, a value of α close to 2 indicates movement by active transport, and a value of α <1 indicates a motion constrained in space.

Figure

  Figure S2: Capturing the timing of translation of endogenous twist mRNAs a, Schematic of the twi_suntag_MS2_CRISPR targeting strategy. b, c, Maximum intensity projection of confocal images of nc14 twi_suntag_MS2_CRISPR Drosophila embryos expressing scFv-GFP (green) labelled with suntag probes (red), scale bars 100μm. c, Zoomed view within the mesoderm shows two distinct mRNA (red) pools: those engaged in translation (co-localizing with scFv-GFP signal, arrows) and untranslated single molecules (red, arrowheads). Scale bars 1μm. d, Maximum intensity projection snapshots of His2Av-mRFP/+; scFv-GFP-NLS/+ > twi_suntag_MS2_CRISPR/+ embryo imaged with a MuVi-SPIM, (related movie S2). Upper panels show the dorsal part of the embryo while the lower panels show the ventral mesoderm part of the embryo, where twi is expressed. Nuclei are shown in magenta and sites of translation in green. e, Maximum intensity projection of confocal images from nc12, nc13 and nc14 twi_suntag_MS2_CRISPR embryos expressing scFv-GFP (green) and labelled with suntag probes (red) and DAPI (blue). Scale bar, 10m. f, Maximum intensity projection of confocal images of a scFv-GFP-NLS/+ > twi_suntag_MS2_CRISPR/+ live embryo imaged from nc12 to nc14 showing twi activation. Scale bars 10μm. g, Sagittal view of a confocal time lapse movie of a scFv-GFP-NLS/MCP-TagRFPT >Twi_suntag_MS2_CRISPR/+ embryo (related movieS3). Sites of nascent transcription appear as bright red spots (arrows) and sites of translation as green spots (arrowheads). Left panel: schematic of a sagittal section throughout an embryo, with the dorso-ventral axis (D, V) and the mesoderm (blue cells) indicated.

Figure

  Figure S3: Revealing a spatial heterogeneity in translation sites in the mesoderm a, Representative cross section of a transverse plane from reconstructed MuViSPIM images of a His2Av-mRFP/+; scFv-GFP-NLS/+ > twi_suntag_MS2_CRISPR/+ embryo (movie S4). Ventral mesodermal nuclei are located at the bottom. Nuclei are observed with a His2Av-mRFP transgene (magenta) and sites of translation with the scFV-GFP (green dots). Scale bars 30μm. b, Confocal images of a scFv-GFP-NLS/+ > live embryo imaged from nc11 to nc14. Upper panels show a maximum intensity projection of four z-stacks located apically (≈2 μm). Lower panels show a maximum intensity projection of 21 z-stacks located basally (≈10 μm). c, Quantification of total intensities of GFP foci apically (light green) and basally (dark green) shown in panel b. A schematic representation of the two compartments analyzed is provided on the left. d, Maximum intensity projection of three z-stacks (≈1 μm) of confocal images of nc13 and nc14 scFv-GFP-NLS/+ > twi_suntag_MS2_CRISPR/+ Drosophila embryos expressing scFv-GFP (green) labelled with suntag probes (red) and DAPI (blue), scale bars 5μm. e, Maximum intensity projection of three z-stacks (≈1 μm) of confocal images of nc14 scFv-GFP-NLS/+ > twi_suntag/+ transgenic Drosophila embryos expressing scFv-GFP (green) labelled with suntag probes (red) and DAPI (blue), scale bars 5μm. f, Violin plot of the distribution of the number of mRNA molecules per cluster quantified from images taken apically (n=207; blue) and basally (n=688; green) from three nc14 embryos scFv-GFP-NLS/+ > twi_suntag/+ transgene labelled with suntag smFISH probes. Centered black bar represent median, dashed black lines represent quartiles.* * * * p < 0.0001 with a two-tailed Welch's t-test.

Figure S4: Quantifying the mobilities of twi mRNAs translation sites a , 1 : 2 : 3 : 4 : 5 : 6 :

 a123456 Figure S4: Quantifying the mobilities of twi mRNAs translation sites a, 1D displacement distribution in x and y merged measured between two consecutive frames of traces located in the apical (blue) and basal (green) perinuclear cytoplasm of scFv-GFP-NLS/+ > twi_suntag_CRISPR/+ embryos. Gaussian fitting is shown in black. b, 1D displacement distribution x and in y separately measured between two consecutive frames of traces located in the apical perinuclear cytoplasm (top histogram, x (light blue) and y (dark blue)) and basal perinuclear cytoplasm (bottom histogram, x (light green) and y (dark green)). Displacement are equally distributed in x and y showing no drift due to cellular movements. c, Distribution of R 2 a goodness of the linear fit (light grey) and non-linear fit (dark grey) of apical traces (top histogram) and basal traces (bottom histogram). A good fit is considered when R 2 >0.8. d, One representative trajectory of a translation particle located apically (left panel) as well as linear (middle panel) and non-linear (right panel) fitting of its MSD over time. e, One representative trajectory of a translation particle located basally (left panel) as well as linear (middle panel) and non-linear (left panel) fitting of its MSD over time.

Figure2: Capturing the timing of translation of endogenous twist mRNAs

	projection snapshots of His2Av-mRFP/+; scFv-GFP-NLS/+ > twi_suntag_CRISPR/+, of a live embryo imaged with a
	MuViSPIM from nc13 to mid nc14 (Movie 5). Nuclei are shown in magenta and sites of translation in green. e,
	quantification of the scFv-GFP signal in the mesoderm from nc11 to 30 minutes into nc14, from which the 'free' scFv-
	GFP (unbound to suntag) measured in the dorsal side where twi is not expressed, was subtracted (n=3 embryos). f,
	Maximum intensity projection of three z-planes (≈1 μm) of confocal images of mitosis from nc13 to nc14 scFv-GFP-
	NLS/+ > twi_suntag_CRISPR/+ embryos expressing scFv-GFP, labelled with suntag smFISH probes and DAPI, (left panel)
	large view of DAPI staining (blue). Right panels are zoomed views exhibiting two groups of mRNAs (red) molecules:
	those co-localizing with scFv-GFP signal (arrows, yellow in merge) and those not co-localizing with a GFP signal
	(arrowheads, red only), scale bars 5μm. g, wild type fixed embryos at the indicated developmental stage, stained with
	a Twi antibody (red) and DAPI (blue). All embryos are oriented with anterior to the left and ventral on the bottom.
	a,	CRISPR	editing	of
	endogenous twist gene by
	suntag repeats. b, c, Confocal
	images		of	nc14
		twi_suntag_CRISPR
	Drosophila		embryos
	expressing scFv-GFP (green)
	stained with a suntag smFISH
	probe (red). In b, Maximum
	intensity projection of a
	ventral view shows the
	twi_suntag is expressed
	within the entire twi pattern,
	scale bars 100μm. In c,
	zoomed view within the
	mesoderm exhibiting two
	groups of mRNAs (red)
	molecules: those co-localizing
	with scFv-GFP signal that are
	engaged	in	translation
	(arrows, yellow in merge) and
	those not co-localizing with a
	GFP	signal	that	are
	untranslated (arrowheads,
	red only). Single molecule
	proteins are labeled only by
	scFv-GFP (stars). Scale bars
	1μm. d, Dorsal and ventral
	maximum		intensity

Acknowledgements

We are grateful to E. Bertrand and X.Pichon for sharing the 32x suntag plasmid and MS2-Cy3 smFISH probes and helpful discussions. We thank I.Izzedin for providing evalSPT and MSD analyser Matlab scripts. We thank R.Zinzen for sharing the twi antibody. We thank C.Desplan, R.Bordonne, T.Saunders, V.Pimmett, and all members of the Lagha lab for their critical reading of the manuscript and constructive discussions. We acknowledge the MRI imaging facility, a member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). M.B is a recipient of an FRM fellowship. This work was supported by the ERC SyncDev starting grant to M.L and a HFSP-CDA grant to M.L. M.L, J.D and S.D.R are sponsored by the CNRS.

Supplementary figure

scFv-GFP-NLS/+ >twi_suntag/+ transgenic embryo, stained with probes against twist (green) and suntag repeats (red). Scale bars 100μm. g, Maximum intensity projection of frames taken from live imaging (related movie 3) of a scFv-GFP-NLS/+ > twi_suntag/+ embryo injected with puromycin. Red arrowheads indicate the site of drug injection. T0 correspond to ≈25-30min after puromycin injection. Zoomed images from the color-coded indicated regions are provided in the lower panels. h, Schematic of the twi_suntag_MS2 transgene. i, Maximum intensity projection of confocal images from a nc14 scFv-GFP-NLS/+ >twi_suntag_MS2/+ transgenic embryo, stained with endogenous scFv-GFP (green) and smFISH probes against suntag repeats (red). Scale bars 100μm. j, Zoomed confocal plane of a scFv-GFP-NLS/+ > twi_suntag_MS2/+ transgenic embryo stained with endogenous scFv-GFP (green) and probes against suntag (blue) and MS2 (red) repeats. scFv-GFP foci colocalizing with suntag and MS2 probes reveal mRNAs in translation (arrow), while spots that are only stained with suntag and MS2 probes represent mRNAs not in translation (arrowhead), scale bars 1μm.