Septate junction proteins are required for cell shape changes, actomyosin reorganization and cell adhesion during dorsal closure in Drosophila

Septate junctions (SJs) serve as occluding barriers in invertebrate epithelia. In Drosophila, at least 30 genes are required for the formation or maintenance of SJs. Interestingly, loss-of-function mutations in core SJ components are embryonic lethal, with defects in developmental events such as head involution and dorsal closure (DC) that occur prior to the formation of a mature SJ, indicating a role for these proteins in mid-embryogenesis independent of their occluding function. To understand this novel function in development, we examined loss-of-function mutations in three core SJ proteins during the process of DC. DC occurs during mid-embryogenesis to seal a dorsal gap in the epidermis following germ band retraction. Closure is driven by contraction of the extraembryonic amnioserosa cells that temporarily cover the dorsal surface and by cell shape changes (elongation) of lateral epidermal cells that bring the contralateral sheets together at the dorsal midline. Using live imaging and examination of fixed tissues, we show that early events in DC occur normally in SJ mutant embryos, but during later closure, coracle, Macroglobulin complement-related and Neurexin-IV mutant embryos exhibit slower rates of closure and display aberrant cells shapes in the dorsolateral epidermis, including dorsoventral length and apical surface area. SJ mutant embryos also show mild defects in actomyosin structures along the leading edge, but laser cutting experiments suggest similar tension and viscoelastic properties in SJ mutant versus wild type epidermis. In a high percentage of SJ mutant embryos, the epidermis tears free from the amnioserosa near the end of DC and live imaging and immunostaining reveal reduced levels of E-cadherin, suggesting that defective adhesion may be responsible for these tears. Supporting this notion, reducing E-cadherin by half significantly enhances the penetrance of DC defects in coracle mutant embryos

MIFA: Metadata, Incentives, Formats, and Accessibility guidelines to improve the reuse of AI datasets for bioimage analysis

Artificial Intelligence methods are powerful tools for biological image analysis and processing. High-quality annotated images are key to training and developing new methods, but access to such data is often hindered by the lack of standards for sharing datasets. We brought together community experts in a workshop to develop guidelines to improve the reuse of bioimages and annotations for AI applications. These include standards on data formats, metadata, data presentation and sharing, and incentives to generate new datasets. We are positive that the MIFA (Metadata, Incentives, Formats, and Accessibility) recommendations will accelerate the development of AI tools for bioimage analysis by facilitating access to high quality training data.Comment: 16 pages, 3 figure

The Role of Cell Mechanics in Embryonic Wound Repair: Staggered Contraction at the Leading Edge

Epithelia are physical barriers against pathogens. Therefore, the ability of multicellular organisms to self-repair epithelial wounds is critical for survival. In embryos, wound repair is mediated by the assembly of a contractile supracellular cable at the wound margin composed of filamentous actin and the molecular motor non-muscle myosin II. It has been proposed that the contraction of the actomyosin cable acts as a "purse-string" to coordinate the movement of cells into the damaged area. Here, I analyze the physical basis of the "purse string" in Drosophila embryos. Using quantitative image analysis I found that, opposing the idea of a uniform "purse string", the distribution of cytoskeletal molecules at the wound margin is heterogeneous with areas of high and low protein density. Furthermore, I showed that mutants for the non-receptor tyrosine kinase Abelson (Abl) display a homogeneous distribution of actin at the wound margin that results in slow wound repair. To investigate the role of actomyosin heterogeneity in wound healing I used biophysical tools to quantify that forces around wounds are also heterogeneous, and patches of the wound edge with heterogeneous actomyosin levels contract faster than homogeneous patches. I developed a mathematical model of wound repair that predicted that actomyosin heterogeneity benefits wound closure if myosin dynamics are directed by tension and strain. To test this idea in vivo, I inhibited stretch-activated ion channels during wound closure, which resulted in disrupted myosin dynamics and impaired tissue repair. Together these results suggest that, instead of a "purse-string", staggered contractility regulates myosin dynamics to coordinate cell movements and to drive fast wound healing.Ph.D

The role of tissue maturity and mechanical state in controlling cell extrusion

Epithelia remove dying or excess cells by extrusion, a process that seamlessly squeezes cells out of the layer without disrupting their barrier function. New studies shed light into the intricate relationship between extrusion, tissue mechanics, and development. They emphasize the importance of whole tissue-mechanics, rather than single cell-mechanics in controlling extrusion. Tissue compaction, stiffness, and cell–cell adhesion can impact the efficiency of cell extrusion and mechanisms that drive it, to adapt to different conditions during development or disease

Physical confinement promotes mesenchymal trans-differentiation of invading transformed cells in vivo

Metastasis is tightly linked with poor cancer prognosis, yet it is not clear how transformed cells become invasive carcinomas. We previously discovered that single KRas(V12)-transformed cells can invade directly from the epithelium by basal cell extrusion. During this process, cells de-differentiate by mechanically pinching off their epithelial determinants, but how they trans-differentiate into a migratory, mesenchymal phenotype is not known. Here, we demonstrate that basally extruded KRas(V12)-expressing cells become significantly deformed as they invade the zebrafish body. Decreasing the confinement that cells experience after they invade reduces the percentage of KRas(V12) cells that trans-differentiate into mesenchymal cell types, while higher confinement increases this percentage. Additionally, increased confinement promotes accumulation of internal masses over time. Altogether, our results suggest that mechanical forces drive not only de-differentiation of KRas(V12)-transformed epithelial cells as they invade but also their re-differentiation into mesenchymal phenotypes that contribute to distant metastases

Tension regulates myosin dynamics during Drosophila embryonic wound repair

Embryos repair epithelial wounds rapidly in a process driven by collective cell movements. Upon wounding, actin and the molecular motor non-muscle myosin II are redistributed in the cells adjacent to the wound, forming a supracellular purse string around the lesion. Purse string contraction coordinates cell movements and drives rapid wound closure. By using fluorescence recovery after photobleaching in Drosophila embryos, we found that myosin turns over as the purse string contracts. Myosin turnover at the purse string was slower than in other actomyosin networks that had a lower level of contractility. Mathematical modelling suggested that myosin assembly and disassembly rates were both reduced by tension at the wound edge. We used laser ablation to show that tension at the purse string increased as wound closure progressed, and that the increase in tension was associated with reduced myosin turnover. Reducing purse string tension by laser-mediated severing resulted in increased turnover and loss of myosin. Finally, myosin motor activity was necessary for its stabilization around the wound and for rapid wound closure. Our results indicate that mechanical forces regulate myosin dynamics during embryonic wound repair.This research was supported by the Ontario Ministry of Economic Development and Innovation (ER14-10-170 to R.F.-G., and Trillium Scholarship to T.Z.-C.), the Natural Sciences and Engineering Research Council of Canada (418438-13), the Canada Foundation for Innovation (30279), the Delta Kappa Gamma Society International (World Fellowship to T.Z.-C.), and University of Toronto, Institute of Biomaterials and Biomedical Engineering Wildcat and International Scholarships (to A.B.K.)

Automated multidimensional image analysis reveals a role for Abl in embryonic wound repair

The embryonic epidermis displays a remarkable ability to repair wounds rapidly. Embryonic wound repair is driven by the evolutionary conserved redistribution of cytoskeletal and junctional proteins around the wound. Drosophila has emerged as a model to screen for factors implicated in wound closure. However, genetic screens have been limited by the use of manual analysis methods. We introduce MEDUSA, a novel image-analysis tool for the automated quantification of multicellular and molecular dynamics from time-lapse confocal microscopy data. We validate MEDUSA by quantifying wound closure in Drosophila embryos, and we show that the results of our automated analysis are comparable to analysis by manual delineation and tracking of the wounds, while significantly reducing the processing time. We demonstrate that MEDUSA can also be applied to the investigation of cellular behaviors in three and four dimensions. Using MEDUSA, we find that the conserved nonreceptor tyrosine kinase Abelson (Abl) contributes to rapid embryonic wound closure. We demonstrate that Abl plays a role in the organization of filamentous actin and the redistribution of the junctional protein β-catenin at the wound margin during embryonic wound repair. Finally, we discuss different models for the role of Abl in the regulation of actin architecture and adhesion dynamics at the wound margin.This work was supported by an Ontario Trillium Scholarship to T.Z.-C., a Connaught Fund New Investigator Award to R.F.-G., and grants from the University of Toronto Faculty of Medicine Dean’s New Staff Fund, the Canada Foundation for Innovation [#30279], the Ontario Research Fund and the Natural Sciences and Engineering Research Council of Canada Discovery Grant program [#418438-13 to R.F.-G.]

Cell–cell and cell–extracellular matrix adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure

Tissue morphogenesis relies on the coordinated action of actin networks, cell–cell adhesions, and cell–extracellular matrix (ECM) adhesions. Such coordination can be achieved through cross-talk between cell–cell and cell–ECM adhesions. Drosophila dorsal closure (DC), a morphogenetic process in which an extraembryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell–cell and cell–ECM adhesions. However, whether the functions of these two types of adhesions are coordinated during DC is not known. Here we analyzed possible interdependence between cell–cell and cell–ECM adhesions during DC and its effect on the actomyosin network. We find that loss of cell–ECM adhesion results in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell–cell adhesion caused up-regulation of cell–ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. Our results show how interdependence between cell–cell and cell–ECM adhesions is important in regulating cell behaviors, force generation, and force transmission critical for tissue morphogenesis.This study was supported by a CIHR Operating Grant to G.T. (MOP-285391) and a grant from the Canada Foundation for Innovation (30279) to R.F.G. T.Z.C. was supported by an Ontario Trillium Scholarship and a Delta Kappa Gamma Society International World Fellowship. We thank Uli Tepass and the Bloomington Drosophila Stock Center for fly lines