MitoTrack, a user-friendly semi-automatic software for lineage tracking in living embryos

International audienceMotivation: During development, progenitor cells undergo multiple rounds of cellular divisions during which tran-scriptional programs must be faithfully propagated. Investigating the timing of transcriptional activation, which is a highly stochastic phenomenon, requires the analysis of large amounts of data. In order to perform automatic image analysis of transcriptional activation, we developed a software that segments and tracks both small and large objects, leading the user from raw data up to the results in their final form. Results: MitoTrack is a user-friendly open-access integrated software that performs the specific dual task of reporting the precise timing of transcriptional activation while keeping lineage tree history for each nucleus of a living developing embryo. The software works automatically but provides the possibility to easily supervise, correct and validate each step. Availability and implementation: MitoTrack is an open source Python software, embedded within a graphical user interface (download here)

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.Fil: Espínola, Sergio Martín. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Université de Montpellier. Centre de Biologie Structurale; FranciaFil: Götz, Markus. Université de Montpellier. Centre de Biologie Structurale; Francia. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Bellec, Maelle. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Messina, Olivier. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; Francia. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Fiche, Jean Bernard. Université de Montpellier. Centre de Biologie Structurale; Francia. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; FranciaFil: Houbron, Christophe. Université de Montpellier. Centre de Biologie Structurale; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Centre National de la Recherche Scientifique; FranciaFil: Dejean, Matthieu. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; FranciaFil: Reim, Ingolf. Universitat Erlangen Nuremberg; AlemaniaFil: Cardozo Gizzi, Andres Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto Alberto C. Taquini de Investigaciones en Medicina Traslacional - Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas "Prof. Dr. Alberto C. Taquini". Instituto Alberto C. Taquini de Investigaciones en Medicina Traslacional; ArgentinaFil: Lagha, Mounia. Centre National de la Recherche Scientifique; Francia. Université de Montpellier. Institut de Génétique Moléculaire de Montpellier; FranciaFil: Nollmann, Marcelo. Centre National de la Recherche Scientifique; Francia. Institut National de la Santé et de la Recherche Médicale; Francia. Université de Montpellier. Centre de Biologie Structurale; Franci

Transcriptome analyses based on genetic screens for Pax3 myogenic targets in the mouse embryo

<p>Abstract</p> <p>Background</p> <p>Pax3 is a key upstream regulator of the onset of myogenesis, controlling progenitor cell survival and behaviour as well as entry into the myogenic programme. It functions in the dermomyotome of the somite from which skeletal muscle derives and in progenitor cell populations that migrate from the somite such as those of the limbs. Few Pax3 target genes have been identified. Identifying genes that lie genetically downstream of <it>Pax3 </it>is therefore an important endeavour in elucidating the myogenic gene regulatory network.</p> <p>Results</p> <p>We have undertaken a screen in the mouse embryo which employs a <it>Pax3^GFP</it>allele that permits isolation of Pax3 expressing cells by flow cytometry and a <it>Pax3^PAX3-FKHR</it>allele that encodes PAX3-FKHR in which the DNA binding domain of Pax3 is fused to the strong transcriptional activation domain of FKHR. This constitutes a gain of function allele that rescues the <it>Pax3 </it>mutant phenotype. Microarray comparisons were carried out between <it>Pax3^GFP/+</it>and <it>Pax3^{GFP/PAX3-FKHR}</it>preparations from the hypaxial dermomyotome of somites at E9.5 and forelimb buds at E10.5. A further transcriptome comparison between Pax3-GFP positive and negative cells identified sequences specific to myogenic progenitors in the forelimb buds. Potential Pax3 targets, based on changes in transcript levels on the gain of function genetic background, were validated by analysis on loss or partial loss of function <it>Pax3 </it>mutant backgrounds. Sequences that are up- or down-regulated in the presence of PAX3-FKHR are classified as somite only, somite and limb or limb only. The latter should not contain sequences from Pax3 positive neural crest cells which do not invade the limbs. Verification by whole mount <it>in situ </it>hybridisation distinguishes myogenic markers. Presentation of potential Pax3 target genes focuses on signalling pathways and on transcriptional regulation.</p> <p>Conclusions</p> <p>Pax3 orchestrates many of the signalling pathways implicated in the activation or repression of myogenesis by regulating effectors and also, notably, inhibitors of these pathways. Important transcriptional regulators of myogenesis are candidate Pax3 targets. Myogenic determination genes, such as <it>Myf5 </it>are controlled positively, whereas the effect of <it>Pax3 </it>on genes encoding inhibitors of myogenesis provides a potential brake on differentiation. In the progenitor cell population, <it>Pax7 </it>and also <it>Hdac5 </it>which is a potential repressor of <it>Foxc2</it>, are subject to positive control by <it>Pax3</it>.</p

The entrepreneur’s personal networks embeddedness in business incubators: A multi-level approach

International audienc

Regulation of myogenic cell fate in mouse embryos (Pax3 target genes)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

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

A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb

We address the molecular control of myogenesis in progenitor cells derived from the hypaxial somite. Null mutations in Pax3, a key regulator of skeletal muscle formation, lead to cell death in this domain. We have developed a novel allele of Pax3 encoding a Pax3–engrailed fusion protein that acts as a transcriptional repressor. Heterozygote mouse embryos have an attenuated mutant phenotype, with partial conservation of the hypaxial somite and its myogenic derivatives, including some hindlimb muscles. At these sites, expression of Myf5 is compromised, showing that Pax3 acts genetically upstream of this myogenic determination gene. We have characterized a 145-base-pair (bp) regulatory element, at −57.5 kb from Myf5, that directs transgene expression to the mature somite, notably to myogenic cells of the hypaxial domain that form ventral trunk and limb muscles. A Pax3 consensus site in this sequence binds Pax3 in vitro and in vivo. Multimers of the 145-bp sequence direct transgene expression to sites of Pax3 function, and an assay of its activity in the chick embryo shows Pax3 dependence. Mutation of the Pax3 site abolishes all expression controlled by the 145-bp sequence in transgenic mouse embryos. We conclude that Pax3 directly regulates Myf5 in the hypaxial somite and its derivatives

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

Myogenic progenitor cells in the mouse embryo are marked by the expression of Pax3/7 genes that regulate their survival and myogenic potential.

The transcription factors Pax3 and Pax7 are important regulators of myogenic cell fate, as demonstrated by genetic manipulations in the mouse embryo. Pax3 lies genetically upstream of MyoD and has also been shown recently to directly control Myf5 transcription in derivatives of the hypaxial somite, where it also plays an important role in ensuring cell survival. Both Pax3 and Pax7 are expressed in myogenic progenitor cells derived from the central dermomyotome that make a major contribution to skeletal muscle growth. In Pax3/Pax7 double mutants, the myogenic determination genes, Myf5 and MyoD, are not activated in these cells which become incorporated into other tissues or die. This again demonstrates the dual function of Pax factors in regulating the entry of progenitor cells into the myogenic programme and in ensuring their survival. Pax3 expression marks cells in the dermomyotome that either become myogenic or downregulate Pax3 and assume another cell fate. The latter include the smooth muscle cells of the dorsal aorta that share a common clonal origin with the skeletal muscle of the myotome, thus illustrating the initial multipotency of Pax3 expressing cells