Dynamic regulation of the microtubule and actin cytoskeleton in zebrafish epiboly

Gastrulation is a key developmental stage with striking changes in morphology. Coordinated cell movements occur to bring cells to their correct positions in a timely manner. Cell movements and morphological changes are accomplished by precisely controlling dynamic changes in cytoskeletal proteins, microtubules, and actin filaments. Among those cellular movements, epiboly produces the first distinct morphological changes in teleosts. In this review, I describe epiboly and its mechanics, and the dynamic changes in microtubule networks and actin structures, mainly in zebrafish embryos. The factors regulating those cytoskeletal changes will also be discussed.     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses

The immunological synapse is a stable adhesive junction between a polarized immune effector cell and an antigen-bearing cell. Immunological synapses are often observed to have a striking radial symmetry in the plane of contact with a prominent central cluster of antigen receptors surrounded by concentric rings of adhesion molecules and actin-rich projections. There is a striking similarity between the radial zones of the immunological synapse and the dynamic actinomyosin modules employed by migrating cells. Breaking the symmetry of an immunological synapse generates a moving adhesive junction that can be defined as a kinapse, which facilitates signal integration by immune cells while moving over the surface of antigen-presenting cells.     

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.     

Loss of cofilin 1 disturbs actin dynamics, adhesion between enveloping and deep cell layers and cell movements during gastrulation in zebrafish

During gastrulation, cohesive migration drives associated cell layers to the completion of epiboly in zebrafish. The association of different layers relies on E-cadherin based cellular junctions, whose stability can be affected by actin turnover. Here, we examined the effect of malfunctioning actin turnover on the epibolic movement by knocking down an actin depolymerizing factor, cofilin 1, using antisense morpholino oligos (MO). Knockdown of cfl1 interfered with epibolic movement of deep cell layer (DEL) but not in the enveloping layer (EVL) and the defect could be specifically rescued by overexpression of cfl1. It appeared that the uncoordinated movements of DEL and EVL were regulated by the differential expression of cfl1 in the DEL, but not EVL as shown by in situ hybridization. The dissociation of DEL and EVL was further evident by the loss of adhesion between layers by using transmission electronic and confocal microscopy analyses. cfl1 morphants also exhibited abnormal convergent extension, cellular migration and actin filaments, but not involution of hypoblast. The cfl1 MO-induced cell migration defect was found to be cell-autonomous in cell transplantation assays. These results suggest that proper actin turnover mediated by Cfl1 is essential for adhesion between DEL and EVL and cell movements during gastrulation in zebrafish.     

2-O-sulfotransferase regulates Wnt signaling, cell adhesion and cell cycle during zebrafish epiboly

O-sulfotransferases modify heparan sulfate proteoglycans (HSPGs) by catalyzing the transfer of a sulfate to a specific position on heparan sulfate glycosaminoglycan (GAG) chains. Although the roles of specific HSPG modifications have been described in cell culture and invertebrates, little is known about their functions or abilities to modulate specific cell signaling pathways in vertebrate development. Here, we report that 2-O-sulfotransferase (2-OST) is an essential component of canonical Wnt signaling in zebrafish development. 2-OST-deficient embryos have reduced GAG chain sulfation and are refractory to exogenous Wnt8 overexpression. Embryos in which maternally encoded 2-OST is knocked down have normal activation of several zygotic mesoderm, endoderm and ectoderm patterning genes, but have decreased deep cell adhesion and fail to initiate epiboly, which can be rescued by re-expression of 2-OST protein. Reduced cell adhesion and altered cell cycle regulation in 2-OST-deficient embryos are associated with decreased β-catenin and E-cadherin protein levels at cell junctions, and these defects can be rescued by reactivation of the intracellular Wnt pathway, utilizing stabilized β-catenin or dominant-negative Gsk3, but not by overexpression of Wnt8 ligand. Together, these results indicate that 2-OST functions within the Wnt pathway, downstream of Wnt ligand signaling and upstream of Gsk3β and β-catenin intracellular localization and function.     

Cell adhesion and the cytoskeleton

Most cells have macromolecules on their outer surface that are specialized for adhesion. Cells can attach to another cell and/or various extracellular matrix components. When tissue culture cells attach to the substrate, they form a specialized structure called adhesion plaque. At the cytoplasmic side of the adhesion plaque, stress fibers terminate, forming an electron-dense plasma membrane undercoat structure. Integrin is localized to the adhesion plaque and this is a transmembrane protein that connects the cytoskeleton to the extracellular matrix. Endothelial cells in vivo have stress fibers, and we have recently found that the ends of these stress fibers also terminate at a structure similar to the adhesion plaque of cultured cells. It appears, therefore, that endothelial cells in vivo employ similar, if not identical, mechanism for adhesion as the one used by tissue culture cells.     

Zebrafish Dynamin is required for maintenance of enveloping layer integrity and the progression of epiboly

Epiboly, the first morphogenetic cell movement that occurs in the zebrafish embryo, is the process by which the blastoderm thins and spreads to engulf the yolk cell. This process requires the concerted actions of the deep cells, the enveloping layer (EVL) and the extra-embryonic yolk syncytial layer (YSL). The EVL is mechanically coupled to the YSL which acts as an epiboly motor, generating the force necessary to draw the blastoderm towards the vegetal pole though actomyosin flow and contraction of the actomyosin ring. However, it has been proposed that the endocytic removal of yolk cell membrane just ahead of the advancing blastoderm may also play a role. To assess the contribution of yolk cell endocytosis in driving epiboly movements, we used a combination of drug- and dominant-negative-based approaches to inhibit Dynamin, a large GTPase with a well-characterized role in vesicle scission. We show that Dynamin-dependent endocytosis in the yolk cell is dispensable for epiboly of the blastoderm. However, global inhibition of Dynamin function revealed that Dynamin plays a fundamental role within the blastoderm during epiboly, where it maintains epithelial integrity and the transmission of tension across the EVL. The epithelial defects were associated with disrupted tight junctions and a striking reduction of cortically localized phosphorylated ezrin/radixin/moesin (P-ERM), key regulators of epithelial integrity in other systems. Furthermore, we show that Dynamin maintains EVL and promotes epiboly progression by antagonizing Rho A activity.     

Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo

The organization of the actin cytoskeleton was studied in unfertilized porcine oocytes and preimplantation stage embryos from Day 1 through Day 8 of development. Fixed and detergent-extracted oocytes and embryos were analyzed by fluorescence microscopy after staining with either rhodamine-phalloidin to localize filamentous actin or with affinity-purified anti-actin antibodies to localize the total immunodetectable actin. Whereas unfertilized oocytes contain immunoreactive cytoplasmic actin, rhodamine-phalloidin binding is not detected until fertilization when a prominent cortical staining pattern becomes apparent. In early cleavage stage embryos, filamentous actin is concentrated in the cell cortex of blastomeres especially at sites of cell-cell contact. Compacting morulae exhibit a marked accumulation of actin at the margins of blastomeres where numerous interdigitating cell processes are located. The predominantly pericellular distribution of actin becomes a distinguishing feature of trophectodermal cells in the expanding blastocyst at Day 6 of development; these cells form a prominent actin-limited zone circumscribing the inner cell mass. In Day 8 blastocysts, three cell types are present that are readily distinguishable based upon their actin displays among other cytological features. Trophectodermal cells exhibit continuous actin-rich lateral borders and stress fibers along their basal surface. Inner cell mass cells contain a discontinuous actin boundary and prominent foci of actin along their blastocoelic surface. Lining the blastocoel are patches of endodermal cells in which the actin is exclusively cortical. The data are discussed with respect to differences between species and the chronology of actin rearrangements during preimplantation development of the porcine embryo.     

Rearrangement of enveloping layer cells without disruption of the epithelial permeability barrier as a factor in Fundulus epiboly

Silver nitrate staining of blastoderms of Fundulus heteroclitus gastrulae shows that the number of marginal cells of the enveloping layer (EVL) is reduced from 160 to 25 during epiboly. To determine whether this decrease in the number of marginal cells was due to ingression, cell death, or rearrangement of cells, marginal and submarginal regions of the late gastrula were observed directly by time-lapse cinemicrography. Marginal cells rearrange to occupy submarginal positions by first narrowing their boundary with the external yolk syncytial layer (E-YSL), thus becoming tapered in shape. Then, the narrowed marginal boundary retracts from the E-YSL and moves submarginally in the plane of the epithelium. Concurrently, the marginal cells on both sides come into apposition; no gap or break appears in the circum-apical continuity of the epithelial sheet. Marginal cells leave the margin of the EVL during epiboly at a rate of about six per hour. The rate of movement of the EVL cells with respect to one another is about 0.5 to 1.0 micron/min at 21 degrees C. Submarginal cells rearrange in a similar fashion. Although no protrusive activity was seen at the lateral aspects of rearranging cells, the tapering or narrowing associated with rearrangement was accompanied by formation of microfolds on their apical surfaces, and separating or recently separated submarginal cells form "flowers" of microfolds on their apices adjacent to the site of separation. Morphometric analysis shows that about half the narrowing of the margin of the EVL during epiboly is accounted for by cell rearrangement and the other half by the associated tapering and narrowing. These results suggest that epiboly of the EVL may have an active component as well as a passive one.     

Desmosomal cadherins in zebrafish epiboly and gastrulation

Background  

The desmosomal cadherins (DCs), desmocollin (Dsc) and desmoglein (Dsg), are the adhesion molecules of desmosomes, intercellular adhesive junctions of epithelia and cardiac muscle. Both the DCs and desmosomes have demonstrably essential roles in mammalian development. In order to initiate their study in a more tractable developmental system we have characterised zebrafish DCs and examined their roles in early zebrafish development.     

O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development

Background  

The post-translational addition of the monosaccharide O-linked β-N-acetylglucosamine (O-GlcNAc) regulates the activity of a wide variety of nuclear and cytoplasmic proteins. The enzymes O-GlcNAc Transferase (Ogt) and O-GlcNAcase (Oga) catalyze, respectively, the attachment and removal of O-GlcNAc to target proteins. In adult mice, Ogt and Oga attenuate the response to insulin by modifying several components of the signal transduction pathway. Complete loss of ogt function, however, is lethal to mouse embryonic stem cells, suggesting that the enzyme has additional, unstudied roles in development. We have utilized zebrafish as a model to determine role of O-GlcNAc modifications in development. Zebrafish has two ogt genes, encoding six different enzymatic isoforms that are expressed maternally and zygotically.     

Imaging intercellular calcium waves during late epiboly in intact zebrafish embryos

Through the injection of f-aequorin and the use of a photon imaging microscope, we have previously reported that a rhythmic series of intercellular Ca2+ waves circumnavigate zebrafish embryos over a 10 h period during gastrulation and axial segmentation. These waves first appear at about 65% epiboly and continue to arise every 5-10 min up to at least the 16-somite stage. In response to our publication, it was suggested that the waves may be an artefact caused by dechorionation of the embryos and would not be observed during the development of intact embryos (i.e. those with chorions). Here we demonstrate (again initially by aequorin imaging) that the rhythmic intercellular Ca2+ waves that traverse the blastoderm margin can also be observed in embryos that have an intact chorion. In addition, the appearance time, propagation pathway, velocity, duration and Ca2+ rise of the waves, as well as the interwave interval and the timing of wave onset, are approximately the same in both dechorionated embryos and those with an intact chorion. Furthermore, by loading intact embryos with Ca(2+)-green dextran at the single-cell stage and then using scanning confocal microscopy to obtain high-resolution images, we confirm the presence of circumferential Ca2+ waves and show that they pass through a population of deep cells located at the blastoderm margin. The confirmation of these pan-embryonic Ca2+ waves in zebrafish further corroborates our earlier suggestion that such waves might play a fundamental role in normal embryonic patterning during the gastrula period.     

Cell adhesion in embryo morphogenesis

Visualizing and analyzing shape changes at various scales, ranging from single molecules to whole organisms, are essential for understanding complex morphogenetic processes, such as early embryonic development. Embryo morphogenesis relies on the interplay between different tissues, the properties of which are again determined by the interaction between their constituent cells. Cell interactions, on the other hand, are controlled by various molecules, such as signaling and adhesion molecules, which in order to exert their functions need to be spatiotemporally organized within and between the interacting cells. In this review, we will focus on the role of cell adhesion functioning at different scales to organize cell, tissue and embryo morphogenesis. We will specifically ask how the subcellular distribution of adhesion molecules controls the formation of cell-cell contacts, how cell-cell contacts determine tissue shape, and how tissue interactions regulate embryo morphogenesis.     

Regulating the actin cytoskeleton during vesicular transport

Although the actin cytoskeleton is widely believed to play an important role in intracellular protein transport, this role is poorly understood. Recently, progress has been made toward identifying specific actin-binding proteins and signaling molecules involved in regulating actin structures that function in the secretory pathway. Studies on coat protomer I (COPI)-mediated transport at the Golgi apparatus and on clathrin-mediated endocytosis have been particularly informative in identifying such mechanisms. Important similarities between actin regulation at the Golgi and at the plasma membrane have been uncovered. The studies reveal that ADP-ribosylation factor and vesicle coat proteins are able to act through the Rho-family GTP-binding proteins, Cdc42 and Rac, and several specific actin-binding proteins to direct actin assembly through the Arp2/3 complex. Efficient function of the secretory pathway is likely to require precise temporal regulation among transport-vesicle assembly, vesicle scission, and the targeting machinery. It is proposed that numerous actin regulatory mechanisms and the connections between actin signaling and vesicle-coat formation are employed to provide such temporal regulation.     

Endothelial cell cortactin coordinates intercellular adhesion molecule-1 clustering and actin cytoskeleton remodeling during polymorphonuclear leukocyte adhesion and transmigration

Endothelial cell ICAM-1 interacts with leukocyte beta(2) integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.     

Subversion of the actin cytoskeleton during viral infection

Viral infection converts the normal functions of a cell to optimize viral replication and virion production. One striking observation of this conversion is the reconfiguration and reorganization of cellular actin, affecting every stage of the viral life cycle, from entry through assembly to egress. The extent and degree of cytoskeletal reorganization varies among different viral infections, suggesting the evolution of myriad viral strategies. In this Review, we describe how the interaction of viral proteins with the cell modulates the structure and function of the actin cytoskeleton to initiate, sustain and spread infections. The molecular biology of such interactions continues to engage virologists in their quest to understand viral replication and informs cell biologists about the role of the cytoskeleton in the uninfected cell.