Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue-scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.publishedVersio

Supracellular organization confers directionality and mechanical potency to migrating pairs of cardiopharyngeal progenitor cells

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that multicellular arrangements confer biochemical and biomechanical properties contributing to tissue-scale organization. The Ciona cardiopharyngeal progenitors provide the simplest model of collective cell migration, with cohesive bilateral cell pairs polarized along the leader-trailer migration path while moving between the ventral epidermis and trunk endoderm. We use the Cellular Potts Model to computationally probe the distributions of forces consistent with shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we identify cardiopharyngeal progenitors as the simplest cell collective maintaining supracellular polarity with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy to align collective polarity with the direction of migration, as observed with three or more cells in silico and in vivo. Our approach reveals emerging properties of the migrating collective: cell pairs are more persistent, migrating longer distances, and presumably with higher accuracy. Simulations suggest that cell pairs can overcome mechanical resistance of the trunk endoderm more effectively when they are polarized collectively. We propose that polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis

UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph Polarize F-Actin during Embryonic Morphogenesis by Regulating the WAVE/SCAR Actin Nucleation Complex

<div><p>Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the <em>Caenorhabditis elegans</em> embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in <em>C. elegans</em>. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.</p> </div

Guidance pathway proteins regulate F-actin organization and levels in migrating embryonic cells.

<p>Embryos are oriented with anterior to the left. (A) Polymerized actin visualized in 4D using the <i>plin26::vab10ActinBindingDomain::gfp</i> transgene (<i>mcIs51</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Patel1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Gally1" target="_blank">[27]</a>. Embryos imaged at 2-minute intervals for 2 hours, beginning at 240 minutes, at 23°C. Embryos at Early, Middle, and Late stages, as related to F-actin-dependent events (B), are shown. t = minutes after first cleavage. Dots outline unenclosed regions of the embryo. Leading Cells (LCs) are boxed and magnified. White dashed line: leading edge. Embryos were pseudo-colored using GLOWormJ “Fire” setting, from low (blue) to high (yellow) intensity. (B) Time intervals between actin-dependent events. Time “0” corresponds to the first appearance of epidermal pocket cells protrusions, at approximately 250 minutes after first cleavage in wild type and mutants. (C) The intensity of F-actin in the LCs analyzed at the time of actin enrichment at the leading edge. The two LCs (yellow boxed region shown in A) were compared. Additional details about F-actin measurements in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#s4" target="_blank">Materials and Methods</a>. (D). Distribution of actin peaks in the ventral half compared with the dorsal half of LCs. Close-ups of representative embryos at Late stages (∼290 min.) are shown. V = Ventral, bottom. D = Dorsal, top. A ventral to dorsal line was drawn through the cell using the Plot Profile tool in GLOWormJ and fluorescent intensity was measured. Peaks, defined as regions at least 10 fluorescent units higher than the background, were counted and are marked by asterisks. Dashed red lines mark half the cell's length. (E) Ratios of ventral to dorsal actin distribution based on the actin peaks measured as in D during 40 minutes beginning with the enrichment of actin at the leading edge. Error bars show SEM. Asterisks mark statistical significance, * = p<.05, *** = p<0.001 as determined by a One-way Anova test followed by the Tukey test.</p

Distribution of embryonic morphogenesis phenotypes.

<p>Distribution of Full and Partial Gex phenotypes observed in single and double genetic mutants. All strains were cultivated at 20°C unless otherwise indicated. Statistically significant changes in phenotype relative to wild type for single mutants, or relative to the two single mutants for double mutants are indicated, calculated by a One-Way ANOVA test followed by the Tukey test.</p>*<p> = p<0.05.</p>**<p> = p<0.01.</p>***<p> = p<0.001,</p>****<p> = p<0.0001.</p

The dynamic turnover of F-actin protrusions is altered in morphogenesis mutants.

<p>F-actin protrusions produced by the two Leading Cells (LCs) on one side during epidermal cell migration were analyzed using the <i>plin26::vab10 Actin Binding Domain::gfp</i> transgene (<i>mcIs51</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Patel1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Gally1" target="_blank">[27]</a>. Micrographs are close-ups of migrating LCs. Arrows point to protrusions. Asterisks denote site of protrusion retraction. Four consecutive time points are shown with an arbitrary timing of t0 set as 2 minutes before protrusion formation. Bar graph shows the average duration of the F-actin protrusions in minutes. Error bars show SEM. Asterisks mark statistical significance, * = p<.05, *** = p<0.001 as determined by a One-way Anova test followed by the Tukey test.</p

Guidance receptors affect subcellular distribution and levels of WAVE/SCAR.

<p>Embryos are oriented with anterior to the left and dorsal up. (A) Embryos carrying the integrated, rescuing <i>gfp::wve-1</i> (<i>pjIs1</i>) transgene were double-stained with antibodies to GFP (Abcam, ab6556, polyclonal) and AJM-1. Boxed regions are amplified and enhanced equally for contrast. Number of embryos that showed the represented phenotype are indicated. Dotted line outlines the basolateral region of the epidermal cells where the region is discernable. (B) Embryos carrying the rescuing integrated <i>gfp::wve-1</i> transgene were double stained with mAb to GFP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Noegel1" target="_blank">[49]</a> and basolaterally localized UNC-70/beta spectrin <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Moorthy1" target="_blank">[33]</a>. Readings were taken across two cell junctions using the line tool in ImageJ for both the UNC-70 and the GFP signal. 5 readings were taken per cell and averaged and plotted in IPad Prism. SEM is shown. (C) Total levels of endogenous WVE-1 in whole worm lysates, or embryonic lysates (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#s4" target="_blank">Materials and Methods</a>) measured with a polyclonal antibody to WVE-1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Patel1" target="_blank">[12]</a>. Levels of WVE-1 normalized to tubulin and relative to WT are shown below the graph, based on the average of 4 blots from 3 sets of lysates. (D) Subcellular distribution of WVE-1 in fractionated lysates, measured using an antibody to endogenous WVE-1. Lysates were spun at increasing speeds and duration. Pellets were resuspended to match the volume of their partner Supernatant fraction. Equal volumes of each S and P fraction were loaded so that relative amounts of protein in the S vs. P fraction could be compared <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Bernadskaya1" target="_blank">[34]</a> (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#s4" target="_blank">Materials and Methods</a>). 10 µl of each fraction were loaded. Numbers below each band represent the relative percentage of total protein found in each fraction. Numbers represent average of three blots (one set of lysates). S = supernatant, P = pellet. Graph shows average of total protein in S1 and P1 based on 3 blots. SEM is shown. Red rectangles indicate fractions with most significant changes compared to WT.</p

Axonal guidance receptors are required for embryonic epidermal morphogenesis.

<p>(A) Comparison of morphogenesis in embryos with wild type, Partial Gex or Full Gex phenotypes. In wild type the epidermal cells (blue) enclose the embryo by ∼400 minutes after first cleavage at 20°C. By ∼600 minutes the embryos elongate to the three-fold stage. In Partial Gex embryos epidermal cells do not fully enclose the embryo by 400 minutes, and by 600 minutes the internal organs (green = pharynx, red = intestine) partially extrude through epidermal gaps. In Full Gex embryos the epidermal cells completely fail cell migration and by 600 minutes the internal organs are fully extruded to the surface of the embryo. (B) Embryonic morphogenesis defects visualized using DIC optics and the <i>dlg-1::gfp</i> (<i>xnIs16</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Totong1" target="_blank">[25]</a> transgene that marks junctions of epithelial tissues. Embryos are oriented with anterior to the left. Representative images from the ventral (left columns) or lateral (right columns) view and corresponding ventral and lateral images of DLG-1::GFP are shown. <i>DIC images</i>. Arrows: anterior pharynx. Block arrows: anterior intestine. White brackets: extruded internal organs. <i>DLG-1::GFP images</i>. Asterisks: ventral gap between epidermal cells. Open arrows in right panels: leading edge of epidermis. Dots outline the unenclosed regions of embryos. Alleles shown are putative nulls, including deletion mutations in <i>ced-10</i>, <i>unc-6</i>, <i>sax-3</i> and <i>vab-1</i>, with the exception of <i>ced-10(n1993)</i> a hypomorph <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Hedgecock1" target="_blank">[1]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Zallen1" target="_blank">[3]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-George1" target="_blank">[8]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002863#pgen.1002863-Ellis1" target="_blank">[50]</a>. (C) Morphogenesis failure in genetic doubles of axonal guidance mutants. Labeled as in (B). Most <i>unc-40(e1430); vab-1(dx31)</i> and <i>vab-1(e2); sax-3(ky123)</i> doubles die with the Partial Gex phenotype. <i>unc-40(e1430); sax-3(ky200ts)</i> doubles show a synergistic increase in the number of Full Gex embryos. (D) Summary of the proposed contribution of the axonal guidance receptors, the CED-10/Rac1 GTPase, the WAVE/SCAR complex and the Arp2/3 complex to embryonic viability (% lethality) and epidermal morphogenesis (% Full Gex).</p