Modeling the roles of cohesotaxis, cell-intercalation, and tissue geometry in collective cell migration of Xenopus mesendoderm.

Collectively migrating Xenopus mesendoderm cells are arranged into leader and follower rows with distinct adhesive properties and protrusive behaviors. In vivo, leading row mesendoderm cells extend polarized protrusions and migrate along a fibronectin matrix assembled by blastocoel roof cells. Traction stresses generated at the leading row result in the pulling forward of attached follower row cells. Mesendoderm explants removed from embryos provide an experimentally tractable system for characterizing collective cell movements and behaviors, yet the cellular mechanisms responsible for this mode of migration remain elusive. We introduce an agent-based computational model of migrating mesendoderm in the Cellular-Potts computational framework to investigate the relative contributions of multiple parameters specific to the behaviors of leader and follower row cells. Sensitivity analyses identify cohesotaxis, tissue geometry, and cell intercalation as key parameters affecting the migration velocity of collectively migrating cells. The model predicts that cohesotaxis and tissue geometry in combination promote cooperative migration of leader cells resulting in increased migration velocity of the collective. Radial intercalation of cells towards the substrate is an additional mechanism to increase migratory speed of the tissue. Summary Statement: We present a novel Cellular-Potts model of collective cell migration to investigate the relative roles of cohesotaxis, tissue geometry, and cell intercalation on migration velocity of Xenopus mesendoderm.

Renin Cell Baroreceptor, a Nuclear Mechanotransducer Central for Homeostasis.

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Extracellular Matrix (ECM) and the Sculpting of Embryonic Tissues.

Extracellular matrices (ECMs) are structurally and compositionally diverse networks of collagenous and noncollagenous glycoproteins, glycosaminoglycans, proteoglycans, and associated molecules that together comprise the metazoan matrisome. Proper deposition and assembly of ECM is of profound importance to cell proliferation, survival, and differentiation, and the morphogenesis of tissues and organ systems that define sequential steps in the development of all animals. Importantly, it is now clear that the instructive influence of a particular ECM at various points in development reflects more than a simple summing of component parts; cellular responses also reflect the dynamic assembly and changing topology of embryonic ECM, which in turn affect its biomechanical properties. This review highlights recent advances in understanding how biophysical features attributed to ECM, such as stiffness and viscoelasticity, play important roles in the sculpting of embryonic tissues and the regulation of cell fates. Forces generated within cells and tissues are transmitted both through integrin-based adhesions to ECM, and through cadherin-dependent cell-cell adhesions; the resulting short- and long-range deformations of embryonic tissues drive morphogenesis. This coordinate regulation of cell-ECM and cell-cell adhesive machinery has emerged as a common theme in a variety of developmental processes. In this review we consider select examples in the embryo where ECM is implicated in setting up tissue barriers and boundaries, in resisting pushing or pulling forces, or in constraining or promoting cell and tissue movement. We reflect on how each of these processes contribute to morphogenesis.

Mechanical and signaling roles for keratin intermediate filaments in the assembly and morphogenesis of Xenopus mesendoderm tissue at gastrulation.

The coordination of individual cell behaviors is a crucial step in the assembly and morphogenesis of tissues. Xenopus mesendoderm cells migrate collectively along a fibronectin (FN) substrate at gastrulation, but how the adhesive and mechanical forces required for these movements are generated and transmitted is unclear. Traction force microscopy (TFM) was used to establish that traction stresses are limited primarily to leading edge cells in mesendoderm explants, and that these forces are balanced by intercellular stresses in follower rows. This is further reflected in the morphology of these cells, with broad lamellipodial protrusions, mature focal adhesions and a gradient of activated Rac1 evident at the leading edge, while small protrusions, rapid turnover of immature focal adhesions and lack of a Rac1 activity gradient characterize cells in following rows. Depletion of keratin (krt8) with antisense morpholinos results in high traction stresses in follower row cells, misdirected protrusions and the formation of actin stress fibers anchored in streak-like focal adhesions. We propose that maintenance of mechanical integrity in the mesendoderm by keratin intermediate filaments is required to balance stresses within the tissue to regulate collective cell movements.

FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm.

Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity.

Cadherin adhesion, tissue tension, and noncanonical Wnt signaling regulate fibronectin matrix organization.

In this study we demonstrate that planar cell polarity signaling regulates morphogenesis in Xenopus embryos in part through the assembly of the fibronectin (FN) matrix. We outline a regulatory pathway that includes cadherin adhesion and signaling through Rac and Pak, culminating in actin reorganization, myosin contractility, and tissue tension, which, in turn, directs the correct spatiotemporal localization of FN into a fibrillar matrix. Increased mechanical tension promotes FN fibril assembly in the blastocoel roof (BCR), while reduced BCR tension inhibits matrix assembly. These data support a model for matrix assembly in tissues where cell-cell adhesions play an analogous role to the focal adhesions of cultured cells by transferring to integrins the tension required to direct FN fibril formation at cell surfaces.

The physical state of fibronectin matrix differentially regulates morphogenetic movements in vivo.

This study demonstrates that proper spatiotemporal expression and the physical assembly state of fibronectin (FN) matrix play key roles in the regulation of morphogenetic cell movements in vivo. We examine the progressive assembly and 3D fibrillar organization of FN and its role in regulating cell and tissue movements in Xenopus embryos. Expression of the 70 kD N-terminal fragment of FN blocks FN fibril assembly at gastrulation but not initial FN binding to integrins at the cell surface. We find that fibrillar FN is necessary to maintain cell polarity through oriented cell division and to promote epiboly, possibly through maintenance of tissue-surface tension. In contrast, FN fibrils are dispensable for convergence and extension movements required for axis elongation. Closure of the migratory mesendodermal mantle was accelerated in the absence of a fibrillar matrix. Thus, the macromolecular assembly of FN matrices may constitute a general regulatory mechanism for coordination of distinct morphogenetic movements.

Live imaging of cell protrusive activity, and extracellular matrix assembly and remodeling during morphogenesis in the frog, Xenopus laevis.

Cell motility and matrix assembly have traditionally been studied in isolation because of a lack of suitable model systems in which both can be observed simultaneously. With embryonic tissues from the gastrulating frog Xenopus laevis we observe stages of fibronectin fibrillogenesis coincident with protrusive activity in the overlying cells. Using live confocal time-lapse images collected from Cy3-tagged fibronectin and plasma membrane tethered green fluorescent protein, we describe the movement and the elaboration of a complex fibrillar network undergoing topological rearrangements of fibrils on the surface of an embryonic tissue. Discrete processes of annealing, polymerization, stretching, breaking, and recoiling are recorded. Elaboration and maintenance of the complex topology of the extracellular matrix appears to require filamentous actin. These findings support a mechanical-model in which cell tractive forces elaborate the complex topological fibrillar network and are part of a homeostatic mechanism for the regulation of the extracellular matrix.

Multiscale computational analysis of Xenopus laevis morphogenesis reveals key insights of systems-level behavior.

BACKGROUND: Tissue morphogenesis is a complex process whereby tissue structures self-assemble by the aggregate behaviors of independently acting cells responding to both intracellular and extracellular cues in their environment. During embryonic development, morphogenesis is particularly important for organizing cells into tissues, and although key regulatory events of this process are well studied in isolation, a number of important systems-level questions remain unanswered. This is due, in part, to a lack of integrative tools that enable the coupling of biological phenomena across spatial and temporal scales. Here, we present a new computational framework that integrates intracellular signaling information with multi-cell behaviors in the context of a spatially heterogeneous tissue environment. RESULTS: We have developed a computational simulation of mesendoderm migration in the Xenopus laevis explant model, which is a well studied biological model of tissue morphogenesis that recapitulates many features of this process during development in humans. The simulation couples, via a JAVA interface, an ordinary differential equation-based mass action kinetics model to compute intracellular Wnt/beta-catenin signaling with an agent-based model of mesendoderm migration across a fibronectin extracellular matrix substrate. The emergent cell behaviors in the simulation suggest the following properties of the system: maintaining the integrity of cell-to-cell contact signals is necessary for preventing fractionation of cells as they move, contact with the Fn substrate and the existence of a Fn gradient provides an extracellular feedback loop that governs migration speed, the incorporation of polarity signals is required for cells to migrate in the same direction, and a delicate balance of integrin and cadherin interactions is needed to reproduce experimentally observed migratory behaviors. CONCLUSION: Our computational framework couples two different spatial scales in biology: intracellular with multicellular. In our simulation, events at one scale have quantitative and dynamic impact on events at the other scale. This integration enables the testing and identification of key systems-level hypotheses regarding how signaling proteins affect overall tissue-level behavior during morphogenesis in an experimentally verifiable system. Applications of this approach extend to the study of tissue patterning processes that occur during adulthood and disease, such as tumorgenesis and atherogenesis.

Using Xenopus embryos to investigate integrin function.

Xenopus embryos are a useful and important system for cell biological studies of integrin adhesion and signaling. Explants prepared from gastrulating embryos undergo normal morphogenetic movements when cultured in simple salt solutions. These preparations are accessible to a variety of experimental perturbations and time-lapse imaging at high resolution, making it possible to elucidate mechanisms of integrin function in intact tissues and whole embryos. Methods used for the visualization of integrins, cadherins, extracellular matrix, and cytoskeletal linkages in both fixed and live tissues are described. We also discuss the use of a novel explant preparation suitable for following the normal deposition and assembly of fibronectin fibrils by ectoderm and mesoderm at gastrulation.

Multicellular computer simulation of morphogenesis: blastocoel roof thinning and matrix assembly in Xenopus laevis.

In the blastocoel roof (BCR) of the Xenopus laevis embryo, epibolic movements are driven by the radial intercalation of deep cell layers and the coordinate spreading of the overlying superficial cell layer. Thinning of the lateral margins of the BCR by radial intercalation requires fibronectin (FN), which is produced and assembled into fibrils by the inner deep cell layer of the BCR. A cellular automata (CA) computer model was developed to analyze the spatial and temporal movements of BCR cells during epiboly. Simulation parameters were defined based on published data and independent results detailing initial tissue geometry, cell numbers, cell intercalation rates, and migration rates. Hypotheses regarding differential cell adhesion and FN assembly were also considered in setting system parameters. A 2-dimensional model simulation was developed that predicts BCR thinning time of 4.8 h, which closely approximates the time required for the completion of gastrulation in vivo. Additionally, the model predicts a temporal increase in FN matrix assembly that parallels fibrillogenesis in the embryo. The model is capable of independent predictions of cell rearrangements during epiboly, and here was used to predict successfully the lateral dispersion of a patch of cells implanted in the BCR, and increased assembly of FN matrix following inhibition of radial intercalation by N-cadherin over-expression.

Fibrillins can co-assemble in fibrils, but fibrillin fibril composition displays cell-specific differences.

Fibrillins are microfibril-forming extracellular matrix macromolecules that modulate skeletal development. In humans, mutations in fibrillins result in long bone overgrowth as well as other distinct phenotypes. Whether fibrillins form independent microfibrillar networks or can co-polymerize, forming a single microfibril, is not known. However, this knowledge is required to determine whether phenotypes arise because of loss of singular or composite functions of fibrillins. Immunolocalization experiments using tissues and de novo matrices elaborated by cultured cells demonstrated that both fibrillins can be present in the same individual microfibril in certain tissues and that both fibrillins can co-polymerize in fibroblast cultures. These studies suggest that the molecular information directing fibrillin fibril formation may be similar in both fibrillins. Furthermore, these studies provide a molecular basis for compensation of one fibrillin by the other during fetal life. In postnatal tissues, fibrillin-2 antibodies demonstrated exuberant staining in only one location: peripheral nerves. This surprising finding implicates distinct functions for fibrillin-2 in peripheral nerves, because a unique feature in humans and in mice mutant for fibrillin-2 is joint contractures that resolve over time.