Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos.

A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis.This is the final version. It was first published by The Company of Biologists in Development at http://dev.biologists.org/content/141/20/4006.long

Invaginating endoderm pulls on the extending ectoderm

How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using Drosophila germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole Drosophila embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.This work was funded by a Biotechnology and Biological Sciences Research Council grant BB/ J010278/1 to GBB, RJA and BS, a Wellcome Trust Investigator Award 099234/Z/12/Z to BS and a Herchel Smith Postdoctoral Fellowship from the University of Cambridge to C

Mechanical Coupling between Endoderm Invagination and Axis Extension in <i>Drosophila</i>

<div><p>How genetic programs generate cell-intrinsic forces to shape embryos is actively studied, but less so how tissue-scale physical forces impact morphogenesis. Here we address the role of the latter during axis extension, using <i>Drosophila</i> germband extension (GBE) as a model. We found previously that cells elongate in the anteroposterior (AP) axis in the extending germband, suggesting that an extrinsic tensile force contributed to body axis extension. Here we further characterized the AP cell elongation patterns during GBE, by tracking cells and quantifying their apical cell deformation over time. AP cell elongation forms a gradient culminating at the posterior of the embryo, consistent with an AP-oriented tensile force propagating from there. To identify the morphogenetic movements that could be the source of this extrinsic force, we mapped gastrulation movements temporally using light sheet microscopy to image whole <i>Drosophila</i> embryos. We found that both mesoderm and endoderm invaginations are synchronous with the onset of GBE. The AP cell elongation gradient remains when mesoderm invagination is blocked but is abolished in the absence of endoderm invagination. This suggested that endoderm invagination is the source of the tensile force. We next looked for evidence of this force in a simplified system without polarized cell intercalation, in acellular embryos. Using Particle Image Velocimetry, we identify posteriorwards Myosin II flows towards the presumptive posterior endoderm, which still undergoes apical constriction in acellular embryos as in wildtype. We probed this posterior region using laser ablation and showed that tension is increased in the AP orientation, compared to dorsoventral orientation or to either orientations more anteriorly in the embryo. We propose that apical constriction leading to endoderm invagination is the source of the extrinsic force contributing to germband extension. This highlights the importance of physical interactions between tissues during morphogenesis.</p></div

The in vitro dynamics of pseudo-vascular network formation

Pseudo-vascular network formation in vitro is considered a key characteristic of vasculogenic mimicry. While many cancer cell lines form pseudo-vascular networks, little is known about the spatiotemporal dynamics of these formations. Here, we present a framework for monitoring and characterising the dynamic formation and dissolution of pseudo-vascular networks in vitro. The framework combines time-resolved optical microscopy with open-source image analysis for network feature extraction and statistical modelling. The framework is demonstrated by comparing diverse cancer cell lines associated with vasculogenic mimicry, then in detecting response to drug compounds proposed to affect formation of vasculogenic mimics. Dynamic datasets collected were analysed morphometrically and a descriptive statistical analysis model was developed in order to measure stability and dissimilarity characteristics of the pseudo-vascular networks formed. Melanoma cells formed the most stable pseudo-vascular networks and were selected to evaluate the response of their pseudo-vascular networks to treatment with axitinib, brucine and tivantinib. Tivantinib has been found to inhibit the formation of the pseudo-vascular networks more effectively, even in dose an order of magnitude less than the two other agents. Our framework is shown to enable quantitative analysis of both the capacity for network formation, linked vasculogenic mimicry, as well as dynamic responses to treatment

Apical constriction of the posterior endoderm primordium generates a tensile stress in acellular embryos.

<p>(A, B) Examples of movies of the posterior lateral surface of acellular embryos, with the actomyosin cytoskeleton labelled with <i>sqh-GFP</i> (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.s020" target="_blank">S8 Movie</a>, from which example A is taken). The Myosin II signal forms a disorganized meshwork at the apical surface of the embryo, which concentrates in the region close to the PC (see arrows in B). Occasionally, the meshwork becomes more cable-like, orienting towards the presumptive posterior endoderm (identified by the position of the PC). (A’, B’) Particle Imaging Velocimetry (PIV) tracking of the Myosin II signal reveals flows towards the presumptive posterior endoderm. Note that the ventralward flows also seen here move towards the ventral presumptive mesoderm (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.s020" target="_blank">S8 Movie</a>). Arrows represent the displacement from the previous timepoint, scaled by a factor of four. Magnitude is shown using a heat scale, with fastest flows in red. Times shown are from the start of the movies. Scale bars are 20 microns. (C) Cross section of the posterior of an acellular embryo stained for Myosin II (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.s012" target="_blank">S6 Fig</a>), showing the concentration of Myosin II where the apical surface has contracted and the beginning of an invagination. PC are indicated. (D) Schematics showing the position of the laser cuts performed on the lateral surface of the presumptive germband in acellular embryos (approximately to scale). The cuts are along a line 20 microns long, positioned either at the anterior (ant.) or at the posterior (post.) of the embryo, either orthogonal to the posterior flows (magenta, called DV thereafter for simplicity) or parallel to them (blue, called AP thereafter). (E) Dot plot with box plot overlaid showing the normalized relaxation velocities (corrected for displacement) for each category of cuts (<i>n</i> = 13 for ant. AP; n = 11, ant. DV; <i>n</i> = 16, post. AP; <i>n</i> = 12, post. DV). A two sample <i>t</i> test was used for the statistics (comparison ant. AP and ant. DV, ns: <i>p</i> = 0.070; comparison ant. AP and post. AP, ns: <i>p</i> = 0.1225; *: <i>p</i> = 0.0173; **: <i>p</i> = 0.0011). For the box plots, the central red line is the median, the edges of the box are the 25th and 75th percentile, the whiskers extend to the most extreme points not considered outliers, and outliers are plotted individually (Data points considered outliers are those more than 2.7 standard deviations from the mean). Data associated with this Figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.s006" target="_blank">S6 Data</a>.</p

AP cell elongation patterns form an AP gradient.

<p>(A, B) AP cell length change shown for each analyzed cell, for timepoints 7.5, 10, and 12.5 min after GBE onset in anterior (A, wtLB009) and posterior views (B, wtCL051010). The color of the dot at the center of each cell corresponds to the scale bar shown. (A’, B’) Spatial maps summarizing AP cell length change over the 7.5–12.5 min time interval, as a function of the position of cells in the AP (<i>x</i>-axis) and DV (<i>y</i>-axis) embryonic axes, for anterior (A’) and posterior views (B’) (average for five and four embryos per views, respectively). (C, D) Graphs summarizing AP cell length change over the 7.5–12.5 min time interval, as a function of cell position in the AP axis, for anterior (C) and posterior views (D). (E) Graphs summarizing AP cell length change (<i>y</i>-axis), at 2.5, 5, and 7.5 min after the onset of GBE, as a function of cell position in the AP axis (<i>x</i>-axis) for posterior views (average for four wild-type embryos). The ribbon’s width shows the standard error (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#sec009" target="_blank">Materials and Methods</a>). Data associated with this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.s002" target="_blank">S2 Data</a>.</p

Schematic summary.

<p>Our findings indicate that apical constriction and invagination of the endoderm primordium (red region) causes a tensile stress that is propagated to the germband and elongates the cells in AP (pink). Passive AP cell elongation and genetically-programmed polarized cell intercalation (blue) contribute together to <i>Drosophila</i> GBE (green arrow). endo, endoderm.</p

The AP cell elongation gradient is present in <i>twi</i> mutant embryos.

<p>(A) Spatiotemporal map summarizing AP cell length change contributing to GBE, over the first 30 mins of GBE (<i>y</i>-axis), and as a function of cell position in the AP axis (<i>x</i>-axis), for <i>twi</i> mutants in anterior views (average for five embryos). (A’) Graph comparing AP cell length change as a function of time for the first 30 min of GBE, in wild-type (blue) and <i>twi</i> mutants (red) for anterior views (average for five embryos each). In these graphs and thereafter, the ribbon’s width indicates the standard error, and the grey-shaded boxes show where a difference is statistically significant (<i>p</i> < 0.05, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#sec009" target="_blank">Materials and Methods</a>). (B, B’) Equivalent plots as A, A’ for posterior views. (C, D) AP cell length change shown for each analyzed cell, for timepoints 7.5, 10, and 12.5 min after GBE onset in <i>twi</i> mutant embryos, for movie frames of an anterior (C, twiLB012) and a posterior view (D, twiCL140411). The color of the dot at the center of each cell corresponds to the scale bar shown. (C’, D’) Spatial maps summarizing AP cell length change over the 7.5–12.5 mins time interval, as a function of the position of cells in the AP (<i>x</i>-axis) and DV (<i>y</i>-axis) embryonic axes, for anterior and posterior views in <i>twi</i> mutants (average of five and three embryos per view, respectively). (E, F) Graphs comparing AP cell length change over the 7.5–12.5 min time interval, as a function of cell position in the AP axis, for wild-type (blue) and <i>twi</i> mutant (red) embryos, for anterior and posterior views. (G–G”) Graphs summarizing AP length change (<i>y</i>-axis), at 2.5, 5, and 7.5 min after the onset of GBE, as a function of cell position in the AP axis (<i>x</i>-axis) for wild-type (blue) and <i>twi</i> mutant (red) embryos, for posterior views (average for four and three embryos per genotype, respectively). Data associated with this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.s003" target="_blank">S3 Data</a>.</p

Cell shape change analysis during <i>Drosophila</i> axis extension.

<p>(A) Scanning electron microscopy micrographs (from Flybase, [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.ref036" target="_blank">36</a>]) showing lateral views of gastrulating <i>Drosophila</i> embryos at stage six, seven, and eight. Anterior is to the left. The part of the germband undergoing convergent extension is labelled in purple and the main direction of extension indicated with a yellow arrow. The following landmarks or morphogenetic events are indicated: PC, pole cells; CF, cephalic furrow; ATF, anterior transverse furrow; PTF, posterior transverse furrow; PMI, posterior midgut invagination (also called posterior endoderm invagination). The box represents the ventral surface that we optically sectioned by confocal microscopy. (A’) Scanning electron microscopy micrograph showing a ventral view of a gastrulating embryo at stage eight, with the approximate position of the anterior and posterior field of views that we analyzed. Both views are bissected by the ventral furrow (VF) through which the mesoderm invaginates. (B) Schematics representing the small neighborhood of cells considered by the tracking algorithms. Cell shape changes (strain rates) are calculated by comparing two timepoints before and after a given time. A strain rate is the ratio of the change in length to the original length, divided by the time interval, with units of proportion per minute (pp/min). The cell shape change is represented here by two orthogonal vectors, showing elongation in one direction (blue, positive) and shrinkage in the perpendicular direction (red, negative). (C, C’) Example frames of movies of wild-type embryos at t = 10 min after GBE onset labeled with <i>ubi-DE-cad-GFP</i>, showing an anterior (C, wtLB009) and a posterior view (C’ wtCL051010). (C) For anterior views, the cephalic furrow (arrow) is used as an anterior landmark, and the scale shows distance from this landmark. The purple shading shows the region removed from the analysis, where cells stretch behind the cephalic furrow. (C’) For posterior views, the posterior-most edge of the embryo seen in the confocal stack is used as posterior landmark (arrow), with distance from it indicated in the scale. (D, D’) Outcome of tracking for anterior (D) and posterior views (D’), showing the polygons describing the cell outlines and the cell centroids from which are drawn the tracks giving the cell positions for the previous 2.5 min (5 timepoints). The tracks shown are track retained for the analysis, after removing tracks from mesodermal cells, mesectoderm cells, and for anterior views, from cells deformed by the cephalic furrow (corresponding to purple region in C). (E, E’) Spatiotemporal heat maps summarizing AP cell length change contributing to GBE, over the first 30 min of GBE (<i>y</i>-axis) and as a function of cell position in the AP axis (<i>x</i>-axis), for anterior (E) and posterior views (E’), averaged for five and four embryos, respectively. (F, F’) Graphs summarizing AP cell length change as a function of time for the first 30 min of GBE, for anterior (F) and posterior views (F’), averaged for five and four embryos, respectively. The ribbon’s width shows the standard error (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#sec009" target="_blank">Materials and Methods</a>). Data associated with this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002292#pbio.1002292.s001" target="_blank">S1 Data</a>.</p