A distinct cardiopharyngeal mesoderm genetic hierarchy establishes antero-posterior patterning of esophagus striated muscle

In most vertebrates, the upper digestive tract is composed of muscularized jaws linked to the esophagus that permits food ingestion and swallowing. Masticatory and esophagus striated muscles (ESM) share a common cardiopharyngeal mesoderm (CPM) origin, however ESM are unusual among striated muscles as they are established in the absence of a primary skeletal muscle scaffold. Using mouse chimeras, we show that the transcription factors Tbx1 and Isl1 are required cell-autonomously for myogenic specification of ESM progenitors. Further, genetic loss-of-function and pharmacological studies point to MET/HGF signaling for antero-posterior migration of esophagus muscle progenitors, where Hgf ligand is expressed in adjacent smooth muscle cells. These observations highlight the functional relevance of a smooth and striated muscle progenitor dialogue for ESM patterning. Our findings establish a Tbx1-Isl1-Met genetic hierarchy that uniquely regulates esophagus myogenesis and identify distinct genetic signatures that can be used as framework to interpret pathologies arising within CPM derivatives.Peer reviewe

Direct Reprogramming of Mouse Fibroblasts into Functional Skeletal Muscle Progenitors

Summary Skeletal muscle harbors quiescent stem cells termed satellite cells and proliferative progenitors termed myoblasts, which play pivotal roles during muscle regeneration. However, current technology does not allow permanent capture of these cell populations in vitro. Here, we show that ectopic expression of the myogenic transcription factor MyoD, combined with exposure to small molecules, reprograms mouse fibroblasts into expandable induced myogenic progenitor cells (iMPCs). iMPCs express key skeletal muscle stem and progenitor cell markers including Pax7 and Myf5 and give rise to dystrophin-expressing myofibers upon transplantation in vivo. Notably, a subset of transplanted iMPCs maintain Pax7 expression and sustain serial regenerative responses. Similar to satellite cells, iMPCs originate from Pax7+ cells and require Pax7 itself for maintenance. Finally, we show that myogenic progenitor cell lines can be established from muscle tissue following small-molecule exposure alone. This study thus reports on a robust approach to derive expandable myogenic stem/progenitor-like cells from multiple cell types

The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

Summary The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface has a so far overlooked function for replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity and we propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities

The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

Abstract The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface, beyond its well-acknowledged function in cap-snatching during transcription initiation, has also a pivotal role in replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity. We observe successive conformational changes to switch from a transcriptase to a replicase conformation in the presence of the bound RNPAII C-terminal domain and propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities

Type B and type A influenza polymerases have evolved distinct binding interfaces to recruit the RNA polymerase II CTD

International audienceDuring annual influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs), can become predominant and cause severe morbidity and mortality. Phylogenetic analyses suggest that IAVs (primarily avian viruses) and IBVs (primarily human viruses) have diverged over long time scales. Identifying their common and distinctive features is an effective approach to increase knowledge about the molecular details of influenza infection. The virus-encoded RNA-dependent RNA polymerases (FluPol B and FluPol A ) are PB1-PB2-PA heterotrimers that perform transcription and replication of the viral genome in the nucleus of infected cells. Initiation of viral mRNA synthesis requires a direct association of FluPol with the host RNA polymerase II (RNAP II), in particular the repetitive C-terminal domain (CTD) of the major RNAP II subunit, to enable “cap-snatching” whereby 5’-capped oligomers derived from nascent RNAP II transcripts are pirated to prime viral transcription. Here, we present the first high-resolution co-crystal structure of FluPol B bound to a CTD mimicking peptide at a binding site crossing from PA to PB2. By performing structure-based mutagenesis of FluPol B and FluPol A followed by a systematic investigation of FluPol-CTD binding, FluPol activity and viral phenotype, we demonstrate that IBVs and IAVs have evolved distinct binding interfaces to recruit the RNAP II CTD, despite the CTD sequence being highly conserved across host species. We find that the PB2 627 subdomain, a major determinant of FluPol-host cell interactions and IAV host-range, is involved in CTD-binding for IBVs but not for IAVs, and we show that FluPol B and FluPol A bind to the host RNAP II independently of the CTD. Altogether, our results suggest that the CTD-binding modes of IAV and IBV may represent avian- and human-optimized binding modes, respectively, and that their divergent evolution was shaped by the broader interaction network between the FluPol and the host transcriptional machinery

A polarized cell system amenable to subcellular resolution imaging of influenza virus infection

Abstract The life cycle of influenza A viruses (IAV), and notably intracellular trafficking of the viral genome, depend on multiple interactions with the cellular cytoskeleton and endomembrane system. A limitation of the conventional cellular models used for mechanistic study and subcellular imaging of IAV infection is that they are cultured in two dimensions (2D) under non-polarizing conditions, and therefore they do not recapitulate the intracellular organization of the polarized respiratory epithelial cells naturally targeted by IAVs. To overcome this limitation, we developed an IAV-infection assay in a 3D cell culture system which allows imaging along the baso-lateral axis of polarized cells, with subcellular resolution. Here we describe a protocol to grow polarized monolayers of Caco2-TC7 cells on static Cytodex-3 microcarrier beads, infect them with IAV, and subsequently perform immunostaining and confocal imaging, or electron microscopy, on polarized IAV-infected cells. This method can be extended to other pathogens that infect human polarized epithelial cells

Accumulation of influenza vRNPs at the apical membrane of polarized Caco-2/TC7 cells.

Accumulation of influenza vRNPs at the apical membrane of polarized Caco-2/TC7 cells.</p

Fig 4 -

Influenza virus-induced arrays of endoplasmic reticulum structures parallel to the apical membrane of polarized Caco-2/TC7 cells. A. Caco-2/TC7 cells grown on Cytodex 3 beads were infected at an estimated MOI of 10 PFU/cell with the A/WSN/33 virus or mock-infected. At 15 hpi they were fixed with glutaraldehyde, post-fixed with osmium tetroxide, dehydrated and resin-embedded for transmission electron microscopy. Representative ultrathin sections going through the center of the Cytodex 3 beads are shown. The lower panels represent a higher magnification of the region defined by the dotted box within the upper panels. Endoplasmic and Golgi apparatus membranes, as well as mitochondrion and tight junctions, and, in the case of infected cells, extracellular virions, are indicated by arrows. Scale bar: 1μm. Scale bar for insets: 0.5 μm. B-C. Distribution of the length of endoplasmic reticulum (ER) structures as observed in IAV-infected versus mock-infected cells. Images in the.ser format were opened using the TIA reader plugin of Image J to determine the pixel size. Free-hand lines were drawn on apically located ER-like structures and their length was measured. Two independent experiments were performed. Five cells were used for each condition, 108 and 127 ER-like structures were measured for mock-treated and IAV-infected samples respectively. Significance was tested with an unpaired t test using GraphPad Prism. *** p-value<0.001.</p

Fig 2 -

Influenza A virus infection of polarized Caco-2/TC7 cells. A. 3D-reconstructed z-stack of a Cytodex 3 microcarrier bead obtained by confocal imaging with a 40x objective (NA = 1.25, WD = 335 μm), segmented for DAPI-stained nuclei. The number of nuclei was determined using the Imaris software for cell segmentation. Scale bar: 40μm. B. 3D-reconstructed z-stack of half a Cytodex 3 microcarrier bead obtained by confocal imaging with a 40x objective (NA = 1.25, WD = 335 μm) upon immunostaining for the viral NP. Caco-2/TC7 cells grown on Cytodex 3 beads were infected at a MOI of 10 PFU/cell with the A/WSN/33 virus. At 8 hours post-infection (hpi) they were fixed with 4% paraformaldehyde and stained with a mix of antibodies specific for the viral NP or the cellular PALS1 protein, and DAPI for nuclear staining. Blue, red and green colour: DAPI, NP and PALS1 immunostaining, respectively. Step size: 0.3 μm, pixel size: 0.4 μm, speed: 400 Hz. Scale bar: 20 μm. C. Production of infectious IAV particles. Caco-2/TC7 cells grown on Cytodex 3 beads were infected at an estimated MOI of 0.001 PFU/cell with the A/WSN/33 virus, in the presence or absence of 0.1 μM of the viral inhibitor baloxavir. The supernatants were collected at 24, 48 and 72 hpi and titrated by plaque assay. The data are represented as the mean +/- SD of four independent experiments. The dashed line represents the limit of detection (L.O.D.) of 250 PFU/mL.</p

Fig 1 -

Polarization of Caco-2/TC7 cells grown on Cytodex 3 microcarrier beads. A. Schematic representation of the microtubule cytoskeleton organization in non-polarized versus polarized epithelial cells. In non-polarized cells the microtubules (in blue) grow from the centrosomes (pink cylinders) located near the nucleus and the Golgi apparatus towards the plasma membrane, whereas in polarized cells microtubules grow towards the basal pole. As a consequence, in non-polarized cells the molecular motors that move cargoes towards the plasma membrane are kinesins, the (+) end motors, whereas dynein, the (-) end motor, is moving cargoes towards the apical membrane in polarized cells. B. Cellular morphology as observed with brightfield microscopy. A 10x objective was used (NA = 0.3, WD = 550mm). A bead cross-section is shown in the inset to illustrate the columnar morphology of Caco-2/TC7 cells at 14 days post-seeding on the beads. Scale bars: 100 μm. C. Confocal imaging upon immunostaining for the PALS1 apical membrane marker. A 40x objective (NA = 1.25, WD = 335 μm) was used. Blue and green colour: DAPI and PALS1 immunostaining, respectively. Step size: 0.3 μm, pixel size: 0.4 μm, speed: 400 Hz. Scale bar: 25 μm. D. Confocal imaging upon immunostaining for the ZO-1 marker of tight junctions labelled with ATTO647n. A 93x Leica glycerol objective (NA = 1.3, WD = 300 μm) was used. Blue colour: ZO-1 immunostaining. Step size: 0.18 μm, pixel size: 61 nm, speed: 600 Hz. Scale bar: 10 μm. E. Electron microscopy of an ultrathin section. Caco-2/TC7 cells grown on Cytodex 3 beads were fixed with glutaraldehyde, post-fixed with osmium tetroxide, dehydrated and resin-embedded for transmission electron microscopy. A representative ultrathin section going through the center of the Cytodex 3 beads is shown. Endoplasmic and Golgi apparatus membranes, as well as a mitochondrion and a tight junction, are indicated by arrows. Scale bar: 1 μm.</p