Visualization of RNA virus infection in a marine protist with a universal biomarker.

Half of the marine virosphere is hypothesized to be RNA viruses (kingdom Orthornavirae) that infect abundant micro-eukaryotic hosts (e.g. protists). To test this, quantitative approaches that broadly track infections in situ are needed. Here, we describe a technique-dsRNA-Immunofluorescence (dsRIF)-that uses a double-stranded RNA (dsRNA) targeting monoclonal antibody to assess host infection status based on the presence of dsRNA, a replicative intermediate of all Orthornavirae infections. We show that the dinoflagellate Heterocapsa circularisquama produces dsRIF signal ~ 1000 times above background autofluorescence when infected by the + ssRNA virus HcRNAV. dsRNA-positive virocells were detected across > 50% of the 48-h infection cycle and accumulated to represent at least 63% of the population. Photosynthetic and chromosomal integrity remained intact during peak replication, indicating HcRNAV infection does not interrupt these processes. This work validates the use of dsRIF on marine RNA viruses and their hosts, setting the stage for quantitative environmental applications that will accelerate understanding of virus-driven ecosystem impacts.

BMP3 inhibits TGFß2-mediated myofibroblast differentiation during wound healing of the embryonic cornea.

Often acute damage to the cornea initiates drastic tissue remodeling, resulting in fibrotic scarring that disrupts light transmission and precedes vision impairment. Very little is known about the factors that can mitigate fibrosis and promote scar-free cornea wound healing. We previously described transient myofibroblast differentiation during non-fibrotic repair in an embryonic cornea injury model. Here, we sought to elucidate the mechanistic regulation of myofibroblast differentiation during embryonic cornea wound healing. We found that alpha-smooth muscle actin (αSMA)-positive myofibroblasts are superficial and their presence inversely correlates with wound closure. Expression of TGFß2 and nuclear localization of pSMAD2 were elevated during myofibroblast induction. BMP3 and BMP7 were localized in the corneal epithelium and corresponded with pSMAD1/5/8 activation and absence of myofibroblasts in the healing stroma. In vitro analyses with corneal fibroblasts revealed that BMP3 inhibits the persistence of TGFß2-induced myofibroblasts by promoting disassembly of focal adhesions and αSMA fibers. This was confirmed by the expression of vinculin and pFAK. Together, these data highlight a mechanism to inhibit myofibroblast persistence during cornea wound repair.

Transmural pressure signals through retinoic acid to regulate lung branching.

During development, the mammalian lung undergoes several rounds of branching, the rate of which is tuned by the relative pressure of the fluid within the lumen of the lung. We carried out bioinformatics analysis of RNA-sequencing of embryonic mouse lungs cultured under physiologic or sub-physiologic transmural pressure and identified transcription factor-binding motifs near genes whose expression changes in response to pressure. Surprisingly, we found retinoic acid (RA) receptor binding sites significantly overrepresented in the promoters and enhancers of pressure-responsive genes. Consistently, increasing transmural pressure activates RA signaling, and pharmacologically inhibiting RA signaling decreases airway epithelial branching and smooth muscle wrapping. We found that pressure activates RA signaling through the mechanosensor Yap. A computational model predicts that mechanical signaling through Yap and RA affects lung branching by altering the balance between epithelial proliferation and smooth muscle wrapping, which we test experimentally. Our results reveal that transmural pressure signals through RA to balance the relative rates of epithelial growth and smooth muscle differentiation in the developing mouse lung and identify RA as a previously unreported component in the mechanotransduction machinery of embryonic tissues.

Mesenchymal proteases and tissue fluidity remodel the extracellular matrix during airway epithelial branching in the embryonic avian lung.

Reciprocal epithelial-mesenchymal signaling is essential for morphogenesis, including branching of the lung. In the mouse, mesenchymal cells differentiate into airway smooth muscle that wraps around epithelial branches, but this contractile tissue is absent from the early avian lung. Here, we have found that branching morphogenesis in the embryonic chicken lung requires extracellular matrix (ECM) remodeling driven by reciprocal interactions between the epithelium and mesenchyme. Before branching, the basement membrane wraps the airway epithelium as a spatially uniform sheath. After branch initiation, however, the basement membrane thins at branch tips; this remodeling requires mesenchymal expression of matrix metalloproteinase 2, which is necessary for branch extension but for not branch initiation. As branches extend, tenascin C (TNC) accumulates in the mesenchyme several cell diameters away from the epithelium. Despite its pattern of accumulation, TNC is expressed exclusively by epithelial cells. Branch extension coincides with deformation of adjacent mesenchymal cells, which correlates with an increase in mesenchymal fluidity at branch tips that may transport TNC away from the epithelium. These data reveal novel epithelial-mesenchymal interactions that direct ECM remodeling during airway branching morphogenesis.

Building branched tissue structures: from single cell guidance to coordinated construction.

Branched networks are ubiquitous throughout nature, particularly found in tissues that require large surface area within a restricted volume. Many tissues with a branched architecture, such as the vasculature, kidney, mammary gland, lung and nervous system, function to exchange fluids, gases and information throughout the body of an organism. The generation of branched tissues requires regulation of branch site specification, initiation and elongation. Branching events often require the coordination of many cells to build a tissue network for material exchange. Recent evidence has emerged suggesting that cell cooperativity scales with the number of cells actively contributing to branching events. Here, we compare mechanisms that regulate branching, focusing on how cell cohorts behave in a coordinated manner to build branched tissues.This article is part of the themed issue 'Systems morphodynamics: understanding the development of tissue hardware'.

Wounded embryonic corneas exhibit nonfibrotic regeneration and complete innervation.

PURPOSE: Wound healing in adult corneas is characterized by activation of keratocytes and extracellular matrix (ECM) synthesis that results in fibrotic scar formation and loss of transparency. Since most fetal wounds heal without scaring, we investigated the regenerative potential of wounded embryonic corneas. METHODS: On embryonic day (E) 7 chick corneas were wounded by making a linear incision traversing the epithelium and anterior stroma. Wounded corneas were collected between E7 and E18, and analyzed for apoptosis, cell proliferation, staining of ECM components, and corneal innervation. RESULTS: Substantial wound retraction was observed within 16-hours postwounding (hpw) and partial re-epithelialized by 5-days postwounding (dpw). Corneal wounds were fully re-epithelialized by 11 dpw with no visible scars. There was no difference in the number of cells undergoing apoptosis between wounded and control corneas. Cell proliferation was reduced in the wounded corneas, albeit mitotic cells in the regenerating epithelium. Staining for alpha-smooth muscle actin (α-SMA), tenascin, and fibronectin was vivid but transient at the wound site. Staining for procollagen I, perlecan, and keratan sulfate proteoglycan was reduced at the wound site. Wounded corneas were fully regenerated by 11 dpw and showed similar patterns of staining for ECM components, albeit an increase in perlecan staining. Corneal innervation was inhibited during wound healing, but regenerated corneas were innervated similar to controls. CONCLUSIONS: These data show that minimal keratocyte activation, rapid ECM reconstruction, and proper innervation occur during nonfibrotic regeneration of the embryonic cornea.