Converting lateral scanning into axial focusing to speed up three-dimensional microscopy.

Funder: MedImmune, and Infinitus (China) Ltd.In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10-100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies

Impacts of both reference population size and inclusion of a residual polygenic effect on the accuracy of genomic prediction

<p>Abstract</p> <p>Background</p> <p>The purpose of this work was to study the impact of both the size of genomic reference populations and the inclusion of a residual polygenic effect on dairy cattle genetic evaluations enhanced with genomic information.</p> <p>Methods</p> <p>Direct genomic values were estimated for German Holstein cattle with a genomic BLUP model including a residual polygenic effect. A total of 17,429 genotyped Holstein bulls were evaluated using the phenotypes of 44 traits. The Interbull genomic validation test was implemented to investigate how the inclusion of a residual polygenic effect impacted genomic estimated breeding values.</p> <p>Results</p> <p>As the number of reference bulls increased, both the variance of the estimates of single nucleotide polymorphism effects and the reliability of the direct genomic values of selection candidates increased. Fitting a residual polygenic effect in the model resulted in less biased genome-enhanced breeding values and decreased the correlation between direct genomic values and estimated breeding values of sires in the reference population.</p> <p>Conclusions</p> <p>Genetic evaluation of dairy cattle enhanced with genomic information is highly effective in increasing reliability, as well as using large genomic reference populations. We found that fitting a residual polygenic effect reduced the bias in genome-enhanced breeding values, decreased the correlation between direct genomic values and sire's estimated breeding values and made genome-enhanced breeding values more consistent in mean and variance as is the case for pedigree-based estimated breeding values.</p

Endothelial Cell Self-fusion during Vascular Pruning

During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell-cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment

KRas-transformed epithelia cells invade and partially dedifferentiate by basal cell extrusion

Metastasis is the main cause of carcinoma-related death, yet we know little about how it initiates due to our inability to visualize stochastic invasion events. Classical models suggest that cells accumulate mutations that first drive formation of a primary mass, and then downregulate epithelia-specific genes to cause invasion and metastasis. Here, using transparent zebrafish epidermis to model simple epithelia, we can directly image invasion. We find that KRas-transformation, implicated in early carcinogenesis steps, directly drives cell invasion by hijacking a process epithelia normally use to promote death—cell extrusion. Cells invading by basal cell extrusion simultaneously pinch off their apical epithelial determinants, endowing new plasticity. Following invasion, cells divide, enter the bloodstream, and differentiate into stromal, neuronal-like, and other cell types. Yet, only invading KRas(V12) cells deficient in p53 survive and form internal masses. Together, we demonstrate that KRas-transformation alone causes cell invasion and partial dedifferentiation, independently of mass formation

Dynamic of lumen collapse in a unicellular tube.

<p>(A) Stills from a time-lapse movie illustrating lumen collapse in a unicellular tube in a transgenic embryo Tg(<i>fliep</i>:GFF)^ubs3,(UAS:mRFP), (<i>5xUAS</i>:<i>cdh5-EGFP</i>)^ubs12. A single lumenized “last link” cell connects two major branches (1). The white/black arrow marks lumen length. The asterisk marks the nucleus; the red arrow points to the narrowest lumen part (next to the nucleus). The lumen splits first next to the nucleus (2, red arrow), forming two distinct luminal compartments within the cell (2, white arrows) that are separated by a nonlumenized part (grey dotted line). The nonlumenized part increases in length as the lower luminal compartment collapses (3). The cell body (nucleus, asterisk) moves towards the upper major branch. The last cell extension (gray line) contacts the lower major branch with a spot-like junction (4, arrow). (B) Stills from a time-lapse movie showing lumen collapse in a unicellular tube in a transgenic embryo TgBAC(<i>kdrl</i>:mKate-CAAX)^ubs16. Black arrows show continuous lumen, gray dotted lines show nonlumenized unicellular regions, the red arrow shows the point of lumen breakage, and asterisks mark the nucleus, where clearly distinguishable. Lumen breaks at the contact site to the lower major branch, next to the nucleus (1–3). The luminal compartment deflates and inflates again (4–5, arrows). After complete lumen collapse, the last connection is resolved (6–8). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s021" target="_blank">S14 Movie</a>. (C) Stills from a time-lapse movie showing lumen collapse in higher time resolution. Inflated luminal compartments (1, arrows) are framed by apical membrane (marked by mKate2-CAAX) and separated by a thin bridge of cell body, most likely the nucleus (1, asterisk). The lumen expands and two apical membranes touch (2, arrow) and fuse (3), but the lumen does not completely inflate (4). The lumen breaks again (5–7) and reconnects in a similar fashion (8–11) within a short time. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s022" target="_blank">S15 Movie</a>. (D) A schematic representing luminal instability, based on still pictures in C. The apical membrane is black, the cell body is dark gray, and the lumen is light gray.</p

Cell rearrangements during pruning of type I and II.

<p>Stills from time-lapse movies illustrating cell rearrangements in type I pruning with lumen collapse before cell rearrangements (A) and type II pruning with cell rearrangements before lumen collapse (B) in transgenic embryos Tg(<i>fliep</i>:GFF)^ubs3,(UAS:mRFP),(<i>5xUAS</i>:<i>cdh5-EGFP</i>)^ubs12. Cell–cell junctions are green (VE-cad-EGFP), and cell cytoplasm is red. Black-and-white pictures show respective channels alone. Key steps of pruning are shown. Green arrows mark multicellular contacts (cell–cell junction length), white arrows mark transcellular lumen, and grey dotted lines mark unicellular fragments without lumen. Asterisks mark nuclei of cells contributing to the branch. (A) Pruning type I. A small, lumenized branch is made of two cells connected by two parallel lines of junctions (1). Lumen collapses when the branch is still multicellular (2–3); after lumen collapse, cells move away from each other, and cell–cell contact surface shrinks, generating a nonlumenized, ajunctional segment (4). Eventually, only the last bridging cell remains (5, grey arrow) prior to final detachment (not shown). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s015" target="_blank">S8</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s016" target="_blank">S9</a> Movies and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s004" target="_blank">S3 Fig</a>. (B) Pruning type II. Cellular architecture of a multicellular branch (1) is simplified to a branch made mainly by two cells connected by parallel lines of junctions (2). Further cell rearrangements lead to the formation of a partially unicellular tube (3). The lumen eventually collapses (4), and the cell body migrates towards the left-side major branch (5) until only a last, narrow cell extension connects two major branches (6). See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s017" target="_blank">S10 Movie</a>. (C) A graph representing percentage of pruning type I (dark blue) and II (light blue) in all pruning events analyzed in transgenic embryos of Tg(<i>fliep</i>:GFF)^ubs3,(UAS:mRFP),(<i>5xUAS</i>:<i>cdh5-EGFP</i>)^ubs12 and TgBAC(<i>kdrl</i>:mKate-CAAX)^ubs16, respectively. Scale bar: 10 μm.</p

Analyses of endothelial cell nuclei during pruning.

<p>Analyses of the nuclei number and behavior in double transgenic embryos Tg(BAC:<i>kdrl</i>:mKate2-CAAX)^UBS16; Tg(<i>kdrl</i>:EGFPnls) ^UBS1. Nuclei were marked and quantified within a region of interest around the pruning segments (nuclei are encircled in dark blue, and green arrows point to a regressing branch). New nuclei derived from cell divisions happening between the time points are marked with light-blue circles and connected to the mother nuclei with a blue line (B–D). Nuclei that leave the field of view in the next time point are marked with asterisks (B–D). The nuclei changed positions in a way corresponding to cell rearrangements. The number of nuclei increased over time because of cell divisions. Nuclei numbers are indicated in the bottom right corner: in dark blue is the number of counted nuclei, in light blue the number of new nuclei (B–D), and in white the nuclei in view. No apoptotic nuclei were observed. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s013" target="_blank">S6 Movie</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s024" target="_blank">S2 Table</a>. Scale bar: 20 μm.</p

SIV development and maturation.

<p><b>(</b>A–D) A model representing four phases of SIV plexus development in the zebrafish embryo between ~36 and 84 hpf. The SIV plexus is blue, and the dorsal aorta (DA), posterior cardinal vein (PCV), and intersegmental vessels are marked in grey. Single endothelial cells sprout ventrally and separate from the PCV (A) to form a primary SIV branch (B). Angiogenic sprouts grow out of the primary SIV and fuse to each other, forming a reticular plexus with multiple cross branches (C, red) and vascular loops (C, green); simultaneously, the plexus grows and moves ventrally. Eventually, the cross branches (and hence the loops) are removed, and the plexus simplifies, forming a set of parallel vertical branches draining into a large ventral SIV branch (D). (A’–E’) Stills from a SPIM time-lapse movie representing five phases of SIV development corresponding to models in A. (A”–E”) Stills from a SPIM time-lapse movie representing SIV development in a <i>silent heart</i> morphant embryo corresponding to models in A. In this case, the SIV keeps its reticular structure because of impaired pruning. (F) A graph comparing the SIV vascular loop formation and remodeling in a wild-type (grey) and silent heart embryo (orange), based on SPIM time-lapse experiments between 36 and 84 hpf. From the left, showing the number of cross branches pruned during remodeling phase, the number of cross branches/loops closed via collateral fusion, the number of cross branches/loops remaining until the end of the movie, and the sum of all loops observed throughout the movie. The values are average numbers per SIV plexus with standard deviation (<i>n</i> = 19 for wild type [WT] and <i>n</i> = 9 for <i>silent heart</i> [SIH]). *** <i>p</i> < 0.001. (G) A graph showing the percentage contribution of pruned (grey), closed by collateral fusion (orange), and remaining (blue) vascular loops to all events observed in WT versus <i>silent heart</i> embryos. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s002" target="_blank">S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s003" target="_blank">S2</a> Figs, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s008" target="_blank">S1</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s011" target="_blank">S4 Movies</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s023" target="_blank">S1 Table</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002126#pbio.1002126.s001" target="_blank">S1 Data</a>.</p