Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity.

Many amniote vertebrate species including humans can form identical twins from a single embryo, but this only occurs rarely. It has been suggested that the primitive-streak-forming embryonic region emits signals that inhibit streak formation elsewhere but the signals involved, how they are transmitted and how they act has not been elucidated. Here we show that short tracks of calcium firing activity propagate through extraembryonic tissue via gap junctions and prevent ectopic primitive streak formation in chick embryos. Cross-regulation of calcium activity and an inhibitor of primitive streak formation (Bone Morphogenetic Protein, BMP) via NF-κB and NFAT establishes a long-range BMP gradient spanning the embryo. This mechanism explains how embryos of widely different sizes can maintain positional information that determines embryo polarity. We provide evidence for similar mechanisms in two different human embryo models and in Drosophila, suggesting an ancient evolutionary origin.

The extra-embryonic area opaca plays a role in positioning the primitive streak of the early chick embryo.

Classical studies have established that the marginal zone, a ring of extra-embryonic epiblast immediately surrounding the embryonic epiblast (area pellucida) of the chick embryo, is important in setting embryonic polarity by positioning the primitive streak, the site of gastrulation. The more external extra-embryonic region (area opaca) was thought to have only nutritive and support functions. Using experimental embryology approaches, this study reveals three separable functions for this outer region. First, juxtaposition of the area opaca directly onto the area pellucida induces a new marginal zone from the latter; this induced domain is entirely posterior in character. Second, ablation and grafting experiments using an isolated anterior half of the blastoderm and pieces of area opaca suggest that the area opaca can influence the polarity of the adjacent marginal zone. Finally, we show that the loss of the ability of such isolated anterior half-embryos to regulate (re-establish polarity spontaneously) at the early primitive streak stage can be rescued by replacing the area opaca by one from a younger stage. These results uncover new roles of chick extra-embryonic tissues in early development.

'Neighbourhood watch' model: embryonic epiblast cells assess positional information in relation to their neighbours.

In many developing and regenerating systems, tissue pattern is established through gradients of informative morphogens, but we know little about how cells interpret these. Using experimental manipulation of early chick embryos, including misexpression of an inducer (VG1 or ACTIVIN) and an inhibitor (BMP4), we test two alternative models for their ability to explain how the site of primitive streak formation is positioned relative to the rest of the embryo. In one model, cells read morphogen concentrations cell-autonomously. In the other, cells sense changes in morphogen status relative to their neighbourhood. We find that only the latter model can account for the experimental results, including some counter-intuitive predictions. This mechanism (which we name the 'neighbourhood watch' model) illuminates the classic 'French Flag Problem' and how positional information is interpreted by a sheet of cells in a large developing system.

Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel that plays a key role in controlling transepithelial fluid movement. Excessive activation results in intestinal fluid loss during secretory diarrheas, whereas CFTR mutations underlie cystic fibrosis (CF). Anion permeability depends both on how well CFTR channels work (permeation/gating) and on how many are present at the membrane. Recently, treatments with two drug classes targeting CFTR-one boosting ion-channel function (potentiators) and the other increasing plasma membrane density (correctors)-have provided significant health benefits to CF patients. Here, we present an image-based fluorescence assay that can rapidly and simultaneously estimate both CFTR ion-channel function and the protein's proximity to the membrane. We monitor F508del-CFTR, the most common CF-causing variant, and confirm rescue by low temperature, CFTR-targeting drugs and second-site revertant mutation R1070W. In addition, we characterize a panel of 62 CF-causing mutations. Our measurements correlate well with published data (electrophysiology and biochemistry), further confirming validity of the assay. Finally, we profile effects of acute treatment with approved potentiator drug VX-770 on the rare-mutation panel. Mapping the potentiation profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-770 might allow an open-channel conformation with an alternative arrangement of domain interfaces. The assay is a valuable tool for investigation of CFTR molecular mechanisms, allowing accurate inferences on gating/permeation. In addition, by providing a two-dimensional characterization of the CFTR protein, it could better inform development of single-drug and precision therapies addressing the root cause of CF disease.