Biology subjects

Bellec, M.

Publications and source records attributed to Bellec, M..

5 recordsLinked to original sources

bioRxiv2026

Real-time single-molecule imaging in zebrafish embryos uncovers non-canonical translation

Precise spatiotemporal control of protein synthesis is essential during embryogenesis, yet directly measuring translation kinetics in vivo remains challenging in vertebrates. In particular, it remains unclear how translation efficiency is determined for key developmental regulators, and which kinetic steps limit their production. Here, we used ALFA array-based nascent chain labeling combined with lattice light-sheet microscopy to visualize bmp2b translation in real time and at single-molecule resolution in early zebrafish embryos. When combined with MS2/MCP labeling to visualize all bmp2b mRNAs, we found that only some of them are being actively translated, suggesting that limited mRNA translation competence contributes to overall translation efficiency. We found that bmp2b translation operates below a maximal initiation regime, as replacement of its UTRs with viral UTRs increases ribosome loading. Furthermore, the bmp2b, but not actb2, 5'UTR supports cap-independent translation with ribosome loading comparable to cap-dependent initiation. Altogether, this approach provides a quantitative in vivo framework to dissect translation kinetics during early vertebrate development.

developmental biology↗

bioRxiv2023

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.

developmental biology↗

bioRxiv2021

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 thought to be mediated, in part, by mitotic bookmarking by transcription factors (TF). 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 the pioneer-like transcription factor GAF acts as stable mitotic bookmarker during zygotic genome activation. We report that 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 provide evidence that GAF binding establishes competence for rapid activation upon mitotic exit.

developmental biology↗

bioRxiv2020

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

During development, naïve cells gradually acquire distinct cell fates, through sophisticated mechanisms of precise spatio-temporal gene regulation. Acquisition of cell fate is thought to rely on the specific interaction of remote cis-regulatory modules (e.g. enhancers, silencers) (CRM) and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly in single cells within multicellular developing organisms. Here we employ Hi-M, a single-cell spatial genomics approach, to systematically detect CRM-promoter looping interactions within topological associating domains (TADs) during 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 revealed the existence of local interactions between multiple remote CRMs to form hubs. We found that loops and CRM hubs are established early during development, prior to the emergence of TADs. Moreover, CRM hubs are formed via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach offers a new perspective on the role of CRM-promoter interactions in defining transcriptional activation and repression states, as well as distinct cell types.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2020

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 co-translated 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.

developmental biology↗