Boosting the toolbox for live imaging of translation.

Live imaging of translation based on tag recognition by a single chain antibody is a powerful technique to assess translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.

Direct observation of translational activation by a ribonucleoprotein granule.

Biomolecular condensates organize biochemical processes at the subcellular level and can provide spatiotemporal regulation within a cell. Among these, ribonucleoprotein (RNP) granules are storage hubs for translationally repressed mRNA. Whether RNP granules can also activate translation and how this could be achieved remains unclear. Here, using single-molecule imaging, we demonstrate that the germ cell-determining RNP granules in Drosophila embryos are sites for active translation of nanos mRNA. Nanos translation occurs preferentially at the germ granule surface with the 3' UTR buried within the granule. Smaug, a cytosolic RNA-binding protein, represses nanos translation, which is relieved when Smaug is sequestered to the germ granule by the scaffold protein Oskar. Together, our findings uncover a molecular process by which RNP granules achieve localized protein synthesis through the compartmentalized loss of translational repression.

A hub of activity.

Imaging experiments reveal the complex and dynamic nature of the transcriptional hubs associated with Notch signaling.

BurstDECONV: a signal deconvolution method to uncover mechanisms of transcriptional bursting in live cells.

Monitoring transcription in living cells gives access to the dynamics of this complex fundamental process. It reveals that transcription is discontinuous, whereby active periods (bursts) are separated by one or several types of inactive periods of distinct lifetimes. However, decoding temporal fluctuations arising from live imaging and inferring the distinct transcriptional steps eliciting them is a challenge. We present BurstDECONV, a novel statistical inference method that deconvolves signal traces into individual transcription initiation events. We use the distribution of waiting times between successive polymerase initiation events to identify mechanistic features of transcription such as the number of rate-limiting steps and their kinetics. Comparison of our method to alternative methods emphasizes its advantages in terms of precision and flexibility. Unique features such as the direct determination of the number of promoter states and the simultaneous analysis of several potential transcription models make BurstDECONV an ideal analytic framework for live cell transcription imaging experiments. Using simulated realistic data, we found that our method is robust with regards to noise or suboptimal experimental designs. To show its generality, we applied it to different biological contexts such as Drosophila embryos or human cells.

The control of transcriptional memory by stable mitotic bookmarking.

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is mediated, in part, by transcription factors (TF) mitotic bookmarking. However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that GAGA pioneer factor (GAF) acts as a stable mitotic bookmarker during zygotic genome activation. We show that, during mitosis, GAF remains associated to a large fraction of its interphase targets, including at cis-regulatory sequences of key developmental genes with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we report that GAF binding establishes competence for rapid activation upon mitotic exit.

Quantitative imaging of transcription in living Drosophila embryos reveals the impact of core promoter motifs on promoter state dynamics.

Genes are expressed in stochastic transcriptional bursts linked to alternating active and inactive promoter states. A major challenge in transcription is understanding how promoter composition dictates bursting, particularly in multicellular organisms. We investigate two key Drosophila developmental promoter motifs, the TATA box (TATA) and the Initiator (INR). Using live imaging in Drosophila embryos and new computational methods, we demonstrate that bursting occurs on multiple timescales ranging from seconds to minutes. TATA-containing promoters and INR-containing promoters exhibit distinct dynamics, with one or two separate rate-limiting steps respectively. A TATA box is associated with long active states, high rates of polymerase initiation, and short-lived, infrequent inactive states. In contrast, the INR motif leads to two inactive states, one of which relates to promoter-proximal polymerase pausing. Surprisingly, the model suggests pausing is not obligatory, but occurs stochastically for a subset of polymerases. Overall, our results provide a rationale for promoter switching during zygotic genome activation.

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during early Drosophila development.

Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (CRMs), for example, enhancers and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly within multicellular developing organisms. In the present study, we employ Hi-M, a single-cell spatial genomics approach, to detect CRM-promoter looping interactions within topologically associating domains (TADs) during early Drosophila development. By comparing cis-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses reveal that multiple CRMs spatially coalesce to form hubs. Loops and CRM hubs are established early during development, before the emergence of TADs. Moreover, CRM hubs are formed, in part, via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach provides insight into the role of CRM-promoter interactions in defining transcriptional states, as well as distinct cell types.

Tyramine induces dynamic RNP granule remodeling and translation activation in the Drosophila brain.

Ribonucleoprotein (RNP) granules are dynamic condensates enriched in regulatory RNA binding proteins (RBPs) and RNAs under tight spatiotemporal control. Extensive recent work has investigated the molecular principles underlying RNP granule assembly, unraveling that they form through the self-association of RNP components into dynamic networks of interactions. How endogenous RNP granules respond to external stimuli to regulate RNA fate is still largely unknown. Here, we demonstrate through high-resolution imaging of intact Drosophila brains that Tyramine induces a reversible remodeling of somatic RNP granules characterized by the decondensation of granule-enriched RBPs (e.g. Imp/ZBP1/IGF2BP) and helicases (e.g. Me31B/DDX-6/Rck). Furthermore, our functional analysis reveals that Tyramine signals both through its receptor TyrR and through the calcium-activated kinase CamkII to trigger RNP component decondensation. Finally, we uncover that RNP granule remodeling is accompanied by the rapid and specific translational activation of associated mRNAs. Thus, this work sheds new light on the mechanisms controlling cue-induced rearrangement of physiological RNP condensates.

Imaging translation dynamics in live embryos reveals spatial heterogeneities.

Much is known about the factors involved in the translation of messenger RNA (mRNA) into protein; however, this multistep process has not been imaged in living multicellular organisms. Here, we deploy the SunTag method to visualize and quantify the timing, location, and kinetics of the translation of single mRNAs in living Drosophila embryos. By focusing on the translation of the conserved major epithelial-mesenchymal transition-inducing transcription factor Twist, we identify spatial heterogeneity in mRNA translation efficiency and reveal the existence of translation factories, where clustered mRNAs are cotranslated preferentially at basal perinuclear regions. Observing the location and dynamics of mRNA translation in a living multicellular organism opens avenues for understanding gene regulation during development.

Lighting Up Gene Activation in Living Drosophila Embryos.

With its rapid development, ease of collection, and the presence of a unique layer of nuclei located close to the surface, the Drosophila syncytial embryo is ideally suited to study the establishment of gene expression patterns during development. Recent improvements in RNA labeling technologies and confocal microscopy allow for visualizing gene activation and quantifying transcriptional dynamics in living Drosophila embryos. Here we review the available tools for mRNA fluorescent labeling and detection in live embryos and precisely describe the overall procedure, from design to mounting and confocal imaging.

Microscopy-Based Chromosome Conformation Capture Enables Simultaneous Visualization of Genome Organization and Transcription in Intact Organisms.

Eukaryotic chromosomes are organized in multiple scales, from nucleosomes to chromosome territories. Recently, genome-wide methods identified an intermediate level of chromosome organization, topologically associating domains (TADs), that play key roles in transcriptional regulation. However, these methods cannot directly examine the interplay between transcriptional activation and chromosome architecture while maintaining spatial information. Here we present a multiplexed, sequential imaging approach (Hi-M) that permits simultaneous detection of chromosome organization and transcription in single nuclei. This allowed us to unveil the changes in 3D chromatin organization occurring upon transcriptional activation and homologous chromosome unpairing during awakening of the zygotic genome in intact Drosophila embryos. Excitingly, the ability of Hi-M to explore the multi-scale chromosome architecture with spatial resolution at different stages of development or during the cell cycle will be key to understanding the mechanisms and consequences of the 4D organization of the genome.

Publisher Correction: Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs.

The original version of this Article contained an error in Fig. 4a, in which the "=" sign of the equation was inadvertently replaced with a "-" sign. This has been corrected in the PDF and HTML versions of the Article.

Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs.

Pioneer transcription factors can engage nucleosomal DNA, which leads to local chromatin remodeling and to the establishment of transcriptional competence. However, the impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains unclear. Here we employ quantitative live imaging methods and mathematical modeling to test the effect of the pioneer factor Zelda on transcriptional dynamics and memory in Drosophila embryos. We demonstrate that increasing the number of Zelda binding sites accelerates the kinetics of nuclei transcriptional activation regardless of their transcriptional past. Despite its known pioneering activities, we show that Zelda does not remain detectably associated with mitotic chromosomes and is neither necessary nor sufficient to foster memory. We further reveal that Zelda forms sub-nuclear dynamic hubs where Zelda binding events are transient. We propose that Zelda facilitates transcriptional activation by accumulating in microenvironments where it could accelerate the duration of multiple pre-initiation steps.

Remembering the past: Mitotic bookmarking in a developing embryo.

During development, transcriptional properties of progenitor cells are stably propagated across multiple cellular divisions. Yet, at each division, chromatin faces structural constraints imposed by the important nuclear re-organization operating during mitosis. It is now clear that not all transcriptional regulators are ejected during mitosis, but rather that a subset of transcription factors, chromatin regulators and epigenetic histone marks are able to 'bookmark' specific loci, thereby providing a mitotic memory. Here we review mechanisms of mitotic bookmarking and discuss their impact on transcriptional dynamics in the context of multicellular developing embryos. We document recent discoveries and technological advances, and present current mathematical models of short-term transcriptional memory.

A Growing Toolbox to Image Gene Expression in Single Cells: Sensitive Approaches for Demanding Challenges.

The spatiotemporal regulation of gene expression is key to many biological processes. Recent imaging approaches opened exciting perspectives for understanding the intricate mechanisms regulating RNA metabolism, from synthesis to decay. Imaging techniques allow their observation at high spatial and temporal resolution, while keeping cellular morphology and micro-environment intact. Here, we focus on approaches for imaging single RNA molecules in cells, tissues, and embryos. In fixed cells, the rapid development of smFISH multiplexing opens the way to large-scale single-molecule studies, while in live cells, gene expression can be observed in real time in its native context. We highlight the strengths and limitations of these methods, as well as future challenges. We present how they advanced our understanding of gene expression heterogeneity and bursting, as well as the spatiotemporal aspects of splicing, translation, and RNA decay. These insights yield a dynamic and stochastic view of gene expression in single cells.

Transcriptional Memory in the Drosophila Embryo.

Transmission of active transcriptional states from mother to daughter cells has the potential to foster precision in the gene expression programs underlying development. Such transcriptional memory has been specifically proposed to promote rapid reactivation of complex gene expression profiles after successive mitoses in Drosophila development [1]. By monitoring transcription in living Drosophila embryos, we provide the first evidence for transcriptional memory in animal development. We specifically monitored the activities of stochastically expressed transgenes in order to distinguish active and inactive mother cells and the behaviors of their daughter nuclei after mitosis. Quantitative analyses reveal that there is a 4-fold higher probability for rapid reactivation after mitosis when the mother experienced transcription. Moreover, memory nuclei activate transcription twice as fast as neighboring inactive mothers, thus leading to augmented levels of gene expression. We propose that transcriptional memory is a mechanism of precision, which helps coordinate gene activity during embryogenesis.

Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium.

Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial-cadherin(+) hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis.

Notch regulation of myogenic versus endothelial fates of cells that migrate from the somite to the limb.

Multipotent Pax3-positive (Pax3(+)) cells in the somites give rise to skeletal muscle and to cells of the vasculature. We had previously proposed that this cell-fate choice depends on the equilibrium between Pax3 and Foxc2 expression. In this study, we report that the Notch pathway promotes vascular versus skeletal muscle cell fates. Overactivating the Notch pathway specifically in Pax3(+) progenitors, via a conditional Pax3(NICD) allele, results in an increase of the number of smooth muscle and endothelial cells contributing to the aorta. At limb level, Pax3(+) cells in the somite give rise to skeletal muscles and to a subpopulation of endothelial cells in blood vessels of the limb. We now demonstrate that in addition to the inhibitory role of Notch signaling on skeletal muscle cell differentiation, the Notch pathway affects the Pax3:Foxc2 balance and promotes the endothelial versus myogenic cell fate, before migration to the limb, in multipotent Pax3(+) cells in the somite of the mouse embryo.