Culturing of avian embryos for time-lapse imaging

Monitoring morphogenetic processes, at high resolution over time, has been a longstanding goal of many developmental cell biologists. It is critical to image cells in their natural environment whenever possible; however imaging many warm-blooded vertebrates, especially mammals, is problematic. At early stages of development, birds are ideal for imaging, since the avian body plan is very similar to that of mammals. We have devised a culturing technique that allows for the acquisition of high-resolution differential interference contrast and epifluorescence images of developing avian embryos in a 4-D (3-D + time) system. The resulting information, from intact embryos, is derived from an area encompassing several millimeters, at micrometer resolution for up to 30 h

A random cell motility gradient downstream of FGF controls elongation of amniote embryos

Vertebrate embryos are characterized by an elongated antero-posterior (AP) body axis, which forms by progressive cell deposition from a posterior growth zone in the embryo. Here, we used tissue ablation in the chicken embryo to demonstrate that the caudal presomitic mesoderm (PSM) has a key role in axis elongation. Using time-lapse microscopy, we analysed the movements of fluorescently labelled cells in the PSM during embryo elongation, which revealed a clear posterior-to-anterior gradient of cell motility and directionality in the PSM. We tracked the movement of the PSM extracellular matrix in parallel with the labelled cells and subtracted the extracellular matrix movement from the global motion of cells. After subtraction, cell motility remained graded but lacked directionality, indicating that the posterior cell movements associated with axis elongation in the PSM are not intrinsic but reflect tissue deformation. The gradient of cell motion along the PSM parallels the fibroblast growth factor (FGF)/mitogen-activated protein kinase (MAPK) gradient1, which has been implicated in the control of cell motility in this tissue2. Both FGF signalling gain- and loss-of-function experiments lead to disruption of the motility gradient and a slowing down of axis elongation. Furthermore, embryos treated with cell movement inhibitors (blebbistatin or RhoK inhibitor), but not cell cycle inhibitors, show a slower axis elongation rate. We propose that the gradient of random cell motility downstream of FGF signalling in the PSM controls posterior elongation in the amniote embryo. Our data indicate that tissue elongation is an emergent property that arises from the collective regulation of graded, random cell motion rather than by the regulation of directionality of individual cellular movements

The role of polysaccharide intercellular adhesin (PIA) in Staphylococcus epidermidis adhesion to host tissues and subsequent antibiotic tolerance

The aim of this study was to determine the role of polysaccharide intercellular adhesin (PIA) in Staphylococcus epidermidis adhesion to host tissues and subsequent antibiotic tolerance. The adherence of S. epidermidis 1457 and the mutant defective in PIA production (1457-M10) to urinary epithelium and endothelium was estimated by colony counting. Minimum bactericidal concentration and mean reduction of cellular activity (XTT) following antibiotic exposure was determined for planktonic and adhered bacteria. S. epidermidis 1457 adhered to a greater extent to both cells than the mutant strain. The adhered strains had a significantly higher antimicrobial tolerance than their planktonic counterparts. The mutant strain was, in general, the most susceptible to the antibiotics assayed. In conclusion, PIA may influence S. epidermidis adherence to host tissues and their antimicrobial susceptibility. Initial adhesion may be the main step for the acquisition of resistance in S. epidermidis

Using a game to engage stakeholders in extreme event attribution science

The impacts of weather and climate-related disasters are increasing, and climate change can exacerbate many disasters. Effectively communicating climate risk and integrating science into policy requires scientists and stakeholders to work together. But dialogue between scientists and policymakers can be challenging given the inherently multidimensional nature of the issues at stake when managing climate risks. Building on the growing use of serious games to create dialogue between stakeholders, we present a new game for policymakers called Climate Attribution Under Loss and Damage: Risking, Observing,co-Negotiating (CAULDRON). CAULDRON aims to communicate understanding of the science attributing extreme events to climate change in a memorable and compelling way, and create space for dialogue around policy decisions addressing changing risks and loss and damage from climate change. We describe the process of developing CAULDRON, and draw on observations of players and their feedback to demonstrate its potential to facilitate the interpretation of probabilistic climate information and the understanding of its relevance to informing policy. Scientists looking to engage with stakeholders can learn valuable lessons in adopting similar innovative approaches. The suitability of games depends on the policy context but, if used appropriately, experiential learning can drive co-produced understanding and meaningful dialogue

Dynamic Analysis of Vascular Morphogenesis Using Transgenic Quail Embryos

Background: One of the least understood and most central questions confronting biologists is how initially simple clusters or sheet-like cell collectives can assemble into highly complex three-dimensional functional tissues and organs. Due to the limits of oxygen diffusion, blood vessels are an essential and ubiquitous presence in all amniote tissues and organs. Vasculogenesis, the de novo self-assembly of endothelial cell (EC) precursors into endothelial tubes, is the first step in blood vessel formation [1]. Static imaging and in vitro models are wholly inadequate to capture many aspects of vascular pattern formation in vivo, because vasculogenesis involves dynamic changes of the endothelial cells and of the forming blood vessels, in an embryo that is changing size and shape. Methodology/Principal Findings: We have generated Tie1 transgenic quail lines Tg(tie1:H2B-eYFP) that express H2B-eYFP in all of their endothelial cells which permit investigations into early embryonic vascular morphogenesis with unprecedented clarity and insight. By combining the power of molecular genetics with the elegance of dynamic imaging, we follow the precise patterning of endothelial cells in space and time. We show that during vasculogenesis within the vascular plexus, ECs move independently to form the rudiments of blood vessels, all while collectively moving with gastrulating tissues that flow toward the embryo midline. The aortae are a composite of somatic derived ECs forming its dorsal regions and the splanchnic derived ECs forming its ventral region. The ECs in the dorsal regions of the forming aortae exhibit variable mediolateral motions as they move rostrally; those in more ventral regions show significant lateral-to-medial movement as they course rostrally. Conclusions/Significance: The present results offer a powerful approach to the major challenge of studying the relative role(s) of the mechanical, molecular, and cellular mechanisms of vascular development. In past studies, the advantages of the molecular genetic tools available in mouse were counterbalanced by the limited experimental accessibility needed for imaging and perturbation studies. Avian embryos provide the needed accessibility, but few genetic resources. The creation of transgenic quail with labeled endothelia builds upon the important roles that avian embryos have played in previous studies of vascular development

Advanced optical imaging in living embryos

Developmental biology investigations have evolved from static studies of embryo anatomy and into dynamic studies of the genetic and cellular mechanisms responsible for shaping the embryo anatomy. With the advancement of fluorescent protein fusions, the ability to visualize and comprehend how thousands to millions of cells interact with one another to form tissues and organs in three dimensions (xyz) over time (t) is just beginning to be realized and exploited. In this review, we explore recent advances utilizing confocal and multi-photon time-lapse microscopy to capture gene expression, cell behavior, and embryo development. From choosing the appropriate fluorophore, to labeling strategy, to experimental set-up, and data pipeline handling, this review covers the various aspects related to acquiring and analyzing multi-dimensional data sets. These innovative techniques in multi-dimensional imaging and analysis can be applied across a number of fields in time and space including protein dynamics to cell biology to morphogenesis

Attribution of 2012 extreme climate events: does air-sea interaction matter?

In 2012, extreme anomalous climate conditions occurred around the globe. Large areas of North America experienced an anomalously hot summer, with large precipitation deficits inducing severe drought. Over Europe, the summer of 2012 was marked by strong precipitation anomalies with the UK experiencing its wettest summer since 1912 while Spain suffered severe drought. What caused these extreme climate conditions in various regions in 2012? This study compares attribution conclusions for 2012 climate anomalies relative to a baseline period (1964–1981) based on two sets of parallel experiments with different model configurations (with coupling to an ocean mixed layer model or with prescribed sea surface temperatures) to assess whether attribution conclusions concerning the climate anomalies in 2012 are sensitive to the representation of air-sea interaction. Modelling results indicate that attribution conclusions for large scale surface air temperature (SAT) changes in both boreal winter and summer are generally robust and not very sensitive to air-sea interaction. This is especially true over southern Europe, Eurasia, North America, South America, and North Africa. Some other responses also appear to be insensitive to air-sea interaction: for example, forced increases in precipitation over northern Europe and Sahel, and reduced precipitation over North America and the Amazon in boreal summer. However, the attribution of circulation and precipitation changes for some other regions exhibits a sensitivity to air-sea interaction. Results from the experiments including coupling to an ocean mixed layer model show a positive NAO-like circulation response in the Atlantic sector in boreal winter and weak changes in the East Asian summer monsoon and precipitation over East Asia. With prescribed sea surface temperatures, some different responses arise over these two regions. Comparison with observed changes indicates that the coupled simulations generally agree better with observations, demonstrating that attribution methods based on atmospheric general circulation models have limitations and may lead to erroneous attribution conclusions for regional anomalies in circulation, precipitation and surface air temperature

Exploratory Analysis in Learning Analytics

This article summarizes the methods, observations, challenges and implications for exploratory analysis drawn from two learning analytics research projects. The cases include an analysis of a games-based virtual performance assessment and an analysis of data from 52,000 students over a 5-year period at a large Australian university. The complex datasets were analyzed and iteratively modeled with a variety of computationally intensive methods to provide the most effective outcomes for learning assessment, performance management and learner tracking. The article presents the research contexts, the tools and methods used in the exploratory phases of analysis, the major findings and the implications for learning analytics research methods

Cytomegalovirus Replicon-Based Regulation of Gene Expression In Vitro and In Vivo

There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed