Simulating convergent extension by way of anisotropic differential adhesion

Simulations using the Extended Potts Model suggest that anisotropic differential adhesion can account for convergent extension, as observed during embryonic development of the frog Xenopus laevis for example. During gastrulation in these frogs, convergent extension produces longitudinal tissue growth from latitudinal elongation and migration of aligned constituent cells. The Extended Potts Model employs clustered points on a grid to represent subdivided cells with probabilistic displacement of cell boundaries such that small changes in energy drive gradual tissue development. For modeling convergent extension, simulations include anisotropic differential adhesion: the degree of attachment between adjacent elongated cells depends on their relative orientation. Without considering additional mechanisms, simulations based on anisotropic differential adhesion reproduce the hallmark stages of convergent extension in the correct sequence, with random fluctuations as sufficient impetus for cell reorganization.     

Emergent morphogenesis: Elastic mechanics of a self-deforming tissue

Multicellular organisms are generated by coordinated cell movements during morphogenesis. Convergent extension is a key tissue movement that organizes mesoderm, ectoderm, and endoderm in vertebrate embryos. The goals of researchers studying convergent extension, and morphogenesis in general, include understanding the molecular pathways that control cell identity, establish fields of cell types, and regulate cell behaviors. Cell identity, the size and boundaries of tissues, and the behaviors exhibited by those cells shape the developing embryo; however, there is a fundamental gap between understanding the molecular pathways that control processes within single cells and understanding how cells work together to assemble multicellular structures. Theoretical and experimental biomechanics of embryonic tissues are increasingly being used to bridge that gap. The efforts to map molecular pathways and the mechanical processes underlying morphogenesis are crucial to understanding: (1) the source of birth defects, (2) the formation of tumors and progression of cancer, and (3) basic principles of tissue engineering. In this paper, we first review the process of tissue convergent extension of the vertebrate axis and then review models used to study the self-organizing movements from a mechanical perspective. We conclude by presenting a relatively simple “wedge-model” that exhibits key emergent properties of convergent extension such as the coupling between tissue stiffness, cell intercalation forces, and tissue elongation forces.     

Antagonistic regulation of convergent extension movements in Xenopus by Wnt/beta-catenin and Wnt/Ca2+ signaling

Convergent extension movements are the main driving force of Xenopus gastrulation. A fine-tuned regulation of cadherin-mediated cell-cell adhesion is thought to be required for this process. Members of the Wnt family of extracellular glycoproteins have been shown to modulate cadherin-mediated cell-cell adhesion, convergent extension movements, and cell differentiation. Here we show that endogenous Wnt/beta-catenin signaling activity is essential for convergent extension movements due to its effect on gene expression rather than on cadherins. Our data also suggest that XLEF-1 rather than XTCF-3 is required for convergent extension movements and that XLEF-1 functions in this context in the Wnt/beta-catenin pathway to regulate Xnr-3. In contrast, activation of the Wnt/Ca2+ pathway blocks convergent extension movements, with potential regulation of the Wnt/beta-catenin pathway at two different levels. PKC, activated by the Wnt/Ca2+ pathway, blocks the Wnt/beta-catenin pathway upstream of beta-catenin and phosphorylates Dishevelled. CamKII, also activated by the Wnt/Ca2+ pathway, inhibits the Wnt/beta-catenin signaling cascade downstream of beta-catenin. Thus, an opposing cross-talk of two distinct Wnt signaling cascades regulates convergent extension movements in Xenopus.     

The planar cell polarity gene strabismus regulates convergence and extension and neural fold closure in Xenopus

We cloned Xenopus Strabismus (Xstbm), a homologue of the Drosophila planar cell or tissue polarity gene. Xstbm encodes four transmembrane domains in its N-terminal half and a PDZ-binding motif in its C-terminal region, a structure similar to Drosophila and mouse homologues. Xstbm is expressed strongly in the deep cells of the anterior neural plate and at lower levels in the posterior notochordal and neural regions during convergent extension. Overexpression of Xstbm inhibits convergent extension of mesodermal and neural tissues, as well as neural tube closure, without direct effects on tissue differentiation. Expression of Xstbm(DeltaPDZ-B), which lacks the PDZ-binding region of Xstbm, inhibits convergent extension when expressed alone but rescues the effect of overexpressing Xstbm, suggesting that Xstbm(DeltaPDZ-B) acts as a dominant negative and that both increase and decrease of Xstbm function from an optimum retards convergence and extension. Recordings show that cells expressing Xstbm or Xstbm(DeltaPDZ-B) fail to acquire the polarized protrusive activity underlying normal cell intercalation during convergent extension of both mesodermal and neural and that this effect is population size-dependent. These results further characterize the role of Xstbm in regulating the cell polarity driving convergence and extension in Xenopus.     

Convergent extension by intercalation without mediolaterally fixed cell motion

We construct and implement a stochastic model of convergent extension, using a minimal set of assumptions on cell behavior. In addition to the basic assumptions of volume conservation, random cell motion, and cell-cell and cell-ECM adhesion, and a non-standard assumption that cytoskeletal polymerization generates an internal pressure tending to keep cells convex, we find that we need only two conditions for convergent extension. (1) Each cell type has a particular aspect ratio towards which it regulates its geometry. We do not require that cells align in a specific orientation, e.g. to be oriented mediolaterally. (2) The elongating tissue is composed of cells that prefer to be elongated, and these cells must be accompanied by cells which prefer to be round. The latter effectively provide a boundary to capture. In simulations, our model tissue extends and converges to a stacked arrangement of elongated cells one cell wide, an arrangement which is seen in ascidian notochords, but which has not been observed in other models. This arrangement is achieved without any direct mediolateral bias other than that which is provided by the physical edge of the adjacent tissue.     

Stabilizing the regionalisation of the developing vertebrate central nervous system

During embryonic development, a number of tissues are patterned by their subdivision into domains with distinct regional identity. An important question is how sharp interfaces are established and maintained between adjacent domains despite the potential for scrambling due to cell intermingling during tissue growth. Two mechanisms have been found to underlie the maintenance of sharp interfaces: the specific restriction of cell mixing across boundaries, or the switching of identity of cells that cross between domains. We review the evidence for these mechanisms at distinct boundaries in the developing vertebrate central nervous system, and discuss what is known about their molecular mediators.     

Titin Extensibility In Situ: Entropic Elasticity of Permanently Folded and Permanently Unfolded Molecular Segments

Abstract. Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293–296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (~2.0 μm) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from ~2.0 to 2.7 μm, the “contracted” tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond ~2.7 μm, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112–1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force–SL relation that is not achievable with a single elastic segment.     

Effect of cellular rearrangement time delays on the rheology of vertex models for confluent tissues

Large-scale tissue deformation during biological processes such as morphogenesis requires cellular rearrangements. The simplest rearrangement in confluent cellular monolayers involves neighbor exchanges among four cells, called a T1 transition, in analogy to foams. But unlike foams, cells must execute a sequence of molecular processes, such as endocytosis of adhesion molecules, to complete a T1 transition. Such processes could take a long time compared to other timescales in the tissue. In this work, we incorporate this idea by augmenting vertex models to require a fixed, finite time for T1 transitions, which we call the “T1 delay time”. We study how variations in T1 delay time affect tissue mechanics, by quantifying the relaxation time of tissues in the presence of T1 delays and comparing that to the cell-shape based timescale that characterizes fluidity in the absence of any T1 delays. We show that the molecular-scale T1 delay timescale dominates over the cell shape-scale collective response timescale when the T1 delay time is the larger of the two. We extend this analysis to tissues that become anisotropic under convergent extension, finding similar results. Moreover, we find that increasing the T1 delay time increases the percentage of higher-fold coordinated vertices and rosettes, and decreases the overall number of successful T1s, contributing to a more elastic-like—and less fluid-like—tissue response. Our work suggests that molecular mechanisms that act as a brake on T1 transitions could stiffen global tissue mechanics and enhance rosette formation during morphogenesis.     

Do lamellipodia have the mechanical capacity to drive convergent extension?

Convergent extension (CE), a kinematic motif associated with several important morphogenetic movements in embryos, entails narrowing of a tissue in one in-plane direction and elongation in the other. Although the cell elongation and intercalation which accompany this process have been investigated and relevant genes and biochemical pathways have been studied in multiple organisms, a fundamental question that has not yet been answered is "Do the lamellipodia thought to drive these motions actually have the mechanical capacity to do so?" Here, we address this and a number of related issues using a state-of-the-art computational model which can replicate cell motions, changes in cell shape and tissue deformations. The model is based on the cell-level finite element approach of Chen and Brodland, but has additional features which allow it to model lamellipodium formation and contraction. In studying CE, computational models provide an important complement to molecular approaches because they reveal the "mechanical pathways" through which gene products must ultimately act in order to produce physical movements. The model shows that lamellipodia can drive CE, that they do so through cell intercalations and that the elongated cells characteristic of CE arise only when adjacent tissues resist convergence, a result which we confirm experimentally.     

Bmp activity gradient regulates convergent extension during zebrafish gastrulation

During vertebrate gastrulation, a ventral to dorsal gradient of bone morphogenetic protein (Bmp) activity establishes cell fates. Concomitantly, convergent extension movements narrow germ layers mediolaterally while lengthening them anteroposteriorly. Here, by measuring movements of cell populations in vivo, we reveal the presence of three domains of convergent extension movements in zebrafish gastrula. Ventrally, convergence and extension movements are absent. Lateral cell populations converge and extend at increasing speed until they reach the dorsal domain where convergence speed slows but extension remains strong. Using dorsalized and ventralized mutants, we demonstrate that these domains are specified by the Bmp activity gradient. In vivo cell morphology and behavior analyses indicated that low levels of Bmp activity might promote extension with little convergence by allowing mediolateral cell elongation and dorsally biased intercalation. Further, single cell movement analyses revealed that the high ventral levels of Bmp activity promote epibolic migration of cells into the tailbud, increasing tail formation at the expense of head and trunk. We show that high Bmp activity limits convergence and extension by negatively regulating expression of the wnt11 (silberblick) and wnt5a (pipetail) genes, which are required for convergent extension but not cell fate specification. Therefore, during vertebrate gastrulation, a single gradient of Bmp activity, which specifies cell fates, also regulates the morphogenetic process of convergent extension.     

Mechanisms of convergence and extension by cell intercalation

The cells of many embryonic tissues actively narrow in one dimension (convergence) and lengthen in the perpendicular dimension (extension). Convergence and extension are ubiquitous and important tissue movements in metazoan morphogenesis. In vertebrates, the dorsal axial and paraxial mesodermal tissues, the notochordal and somitic mesoderm, converge and extend. In amphibians as well as a number of other organisms where these movements appear, they occur by mediolateral cell intercalation, the rearrangement of cells along the mediolateral axis to produce an array that is narrower in this axis and longer in the anteroposterior axis. In amphibians, mesodermal cell intercalation is driven by bipolar, mediolaterally directed protrusive activity, which appears to exert traction on adjacent cells and pulls the cells between one another. In addition, the notochordal-somitic boundary functions in convergence and extension by 'capturing' notochordal cells as they contact the boundary, thus elongating the boundary. The prospective neural tissue also actively converges and extends parallel with the mesoderm. In contrast to the mesoderm, cell intercalation in the neural plate normally occurs by monopolar protrusive activity directed medially, towards the midline notoplate-floor-plate region. In contrast, the notoplate-floor-plate region appears to converge and extend by adhering to and being towed by or perhaps migrating on the underlying notochord. Converging and extending mesoderm stiffens by a factor of three or four and exerts up to 0.6 microN force. Therefore, active, force-producing convergent extension, the mechanism of cell intercalation, requires a mechanism to actively pull cells between one another while maintaining a tissue stiffness sufficient to push with a substantial force. Based on the evidence thus far, a cell-cell traction model of intercalation is described. The essential elements of such a morphogenic machine appear to be (i) bipolar, mediolaterally orientated or monopolar, medially directed protrusive activity; (ii) this protrusive activity results in mediolaterally orientated or medially directed traction of cells on one another; (iii) tractive protrusions are confined to the ends of the cells; (iv) a mechanically stable cell cortex over the bulk of the cell body which serves as a movable substratum for the orientated or directed cell traction. The implications of this model for cell adhesion, regulation of cell motility and cell polarity, and cell and tissue biomechanics are discussed.     

Calcium signaling during convergent extension in Xenopus

BACKGROUND: During Xenopus gastrulation, cell intercalation drives convergent extension of dorsal tissues. This process requires the coordination of motility throughout a large population of cells. The signaling mechanisms that regulate these movements in space and time remain poorly understood. RESULTS: To investigate the potential contribution of calcium signaling to the control of morphogenetic movements, we visualized calcium dynamics during convergent extension using a calcium-sensitive fluorescent dye and a novel confocal microscopy system. We found that dramatic intercellular waves of calcium mobilization occurred in cells undergoing convergent extension in explants of gastrulating Xenopus embryos. These waves arose stochastically with respect to timing and position within the dorsal tissues. Waves propagated quickly and were often accompanied by a wave of contraction within the tissue. Calcium waves were not observed in explants of the ventral marginal zone or prospective epidermis. Pharmacological depletion of intracellular calcium stores abolished the calcium dynamics and also inhibited convergent extension without affecting cell fate. These data indicate that calcium signaling plays a direct role in the coordination of convergent extension cell movements. CONCLUSIONS: The data presented here indicate that intercellular calcium signaling plays an important role in vertebrate convergent extension. We suggest that calcium waves may represent a widely used mechanism by which large groups of cells can coordinate complex cell movements.     

The zebrafish glypican knypek controls cell polarity during gastrulation movements of convergent extension.

Mutations in the zebrafish knypek locus impair gastrulation movements of convergent extension that narrow embryonic body and elongate it from head to tail. We demonstrate that knypek regulates cellular movements but not cell fate specification. Convergent extension movement defects in knypek are associated with abnormal cell polarity, as mutant cells fail to elongate and align medio-laterally. Positional cloning reveals that knypek encodes a member of the glypican family of heparan sulfate proteoglycans. Double mutant and overexpression analyses show that Knypek potentiates Wnt11 signaling, mediating convergent extension. These studies provide experimental and genetic evidence that glypican Knypek acts during vertebrate gastrulation as a positive modulator of noncanonical Wnt signaling to establish polarized cell behaviors underlying convergent extension movements.     

The contribution made by cells from a single somite to tissues within a body segment and assessment of their integration with similar cells from adjacent segments

Somites represent the first visual evidence of segmentation in the developing vertebrate embryo and it is becoming clear that this segmental pattern of the somites is used in the initial stages of development of other segmented systems such as the peripheral nervous system. However, it is not known whether the somites continue to contribute to the maintenance of the segmental pattern after the dispersal of the somitic cells. In particular, the extent to which cells from a single somite contribute to all of the tissues of a single body segment and the extent to which they mix with cells from adjacent segments during their migration is not known. In this study, we have replaced single somites in the future cervical region of 2-day-old chick embryos with equivalent, similarly staged quail somites. The chimerae were then allowed to develop for a further 6 days when they were killed. The cervical region was dissected and serially sectioned. The sections were stained with the Feulgen reaction for DNA to differentiate between the chick and quail cells. The results showed that the cells from a single somite remained as a clearly delimited group throughout their migration. Furthermore, the sclerotome, dermatome and myotome portions from the single somites could always be recognised as being separate from similar cells from other somites. The somitic cells formed all of the tissues within a body segment excluding the epidermis, notochord and neural tissue. There was very little mixing of the somitic cells between adjacent segments. The segmental pattern of the somites is therefore maintained during the migration of the somitic cells and this might be fundamental to a mechanism whereby the segmentation of structures, such as the peripheral nervous system, is also maintained during development.     

Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

Serotonin synchronises convergent extension of ectoderm with morphogenetic gastrulation movements in Drosophila.

During Drosophila gastrulation, convergent extension of the ectoderm is required for germband extension. Adhesive heterogeneity within ectodermal cells has been proposed to trigger the intercalation of cells responsible for this movement. Segmentation genes would impose this heterogeneity by establishing a pair-rule pattern of cell adhesion properties. We previously reported that the serotonin receptor (5-ht(2Dro)) is expressed in the presumptive ectoderm with a pair-rule pattern. Here, we show that the peaks of 5-ht(2Dro) expression and serotonin synthesis coincide precisely with the onset of convergent extension of the ectoderm. Gastrulae genetically depleted of serotonin or the 5-ht(2Dro) receptor do not extend their germband properly, and the ectodermal movements becomes asynchronous with the morphogenetic movements in the endoderm and mesoderm. Associated with the beginning of this desynchronisation, is an altered subcellular localisation of adherens junctions within the ectoderm. Combined, these data highlight the role of the ectoderm in Drosophila gastrulation and support the notion that serotonin signalling through the 5-HT(2Dro) receptor triggers changes in cell adhesiveness that are necessary for cell intercalation.     

Morphogenetic pattern formation during ascidian notochord formation is regulative and highly robust.

The ascidian notochord forms through simultaneous invagination and convergent extension of a monolayer epithelial plate. Here we combine micromanipulation with time lapse and confocal microscopy to examine how notochord-intrinsic morphogenetic behaviors and interactions with surrounding tissues, determine these global patterns of movement. We show that notochord rudiments isolated at the 64-cell stage divide and become motile with normal timing; but, in the absence of interactions with non-notochordal tissues, they neither invaginate nor converge and extend. We find that notochord formation is robust in the sense that no particular neighboring tissue is required for notochord formation. Basal contact with either neural plate or anterior endoderm/lateral mesenchyme or posterior mesoderm are each alone sufficient to ensure that the notochord plate forms and extends a cylindrical rod. Surprisingly, the axis of convergent extension depends on the specific tissues that contact the notochord, as do other patterns of cell shape change, movement and tissue deformation that accompany notochord formation. We characterize one case in detail, namely, embryos lacking neural plates, in which a normal notochord forms but by an entirely different trajectory. Our results show ascidian notochord formation to be regulative in a fashion and to a degree never before appreciated. They suggest this regulative behavior depends on a complex interplay between morphogenetic tendencies intrinsic to the notochord plate and instructive and permissive interactions with surrounding tissues. We discuss mechanisms that could account for these data and what they imply about notochord morphogenesis and its evolution within the chordate phylum.