Capillarity and active cell movement at mesendoderm translocation in the Xenopus gastrula.

During Xenopus gastrulation, leading edge mesendoderm (LEM) advances animally as a wedge-shaped cell mass over the vegetally moving blastocoel roof (BCR). We show that close contact across the BCR-LEM interface correlates with attenuated net advance of the LEM, which is pulled forward by tip cells while the remaining LEM frequently separates from the BCR. Nevertheless, lamellipodia persist on the detached LEM surface. They attach to adjacent LEM cells and depend on PDGF-A, cell-surface fibronectin and cadherin. We argue that active cell motility on the LEM surface prevents adverse capillary effects in the liquid LEM tissue as it moves by being pulled. It counters tissue surface-tension effects with oriented cell movement and bulges the LEM surface out to keep it close to the curved BCR without attaching to it. Proximity to the BCR is necessary, in turn, for the maintenance and orientation of lamellipodia that permit mass cell movement with minimal substratum contact. Together with a similar process in epithelial invagination, vertical telescoping, the cell movement at the LEM surface defines a novel type of cell rearrangement: vertical shearing.

Cell-cell contact landscapes in Xenopus gastrula tissues.

Molecular and structural facets of cell-cell adhesion have been extensively studied in monolayered epithelia. Here, we perform a comprehensive analysis of cell-cell contacts in a series of multilayered tissues in the Xenopus gastrula model. We show that intercellular contact distances range from 10 to 1,000 nm. The contact width frequencies define tissue-specific contact spectra, and knockdown of adhesion factors modifies these spectra. This allows us to reconstruct the emergence of contact types from complex interactions of the factors. We find that the membrane proteoglycan Syndecan-4 plays a dominant role in all contacts, including narrow C-cadherin-mediated junctions. Glypican-4, hyaluronic acid, paraxial protocadherin, and fibronectin also control contact widths, and unexpectedly, C-cadherin functions in wide contacts. Using lanthanum staining, we identified three morphologically distinct forms of glycocalyx in contacts of the Xenopus gastrula, which are linked to the adhesion factors examined and mediate cell-cell attachment. Our study delineates a systematic approach to examine the varied contributions of adhesion factors individually or in combinations to nondiscrete and seemingly amorphous intercellular contacts.

PDGF-A suppresses contact inhibition during directional collective cell migration.

The leading-edge mesendoderm (LEM) of the Xenopus gastrula moves as an aggregate by collective migration. However, LEM cells on fibronectin in vitro show contact inhibition of locomotion by quickly retracting lamellipodia upon mutual contact. We found that a fibronectin-integrin-syndecan module acts between p21-activated kinase 1 upstream and ephrin B1 downstream to promote the contact-induced collapse of lamellipodia. To function in this module, fibronectin has to be present as puncta on the surface of LEM cells. To overcome contact inhibition in LEM cell aggregates, PDGF-A deposited in the endogenous substratum of LEM migration blocks the fibronectin-integrin-syndecan module at the integrin level. This stabilizes lamellipodia preferentially in the direction of normal LEM movement and supports cell orientation and the directional migration of the coherent LEM cell mass.

Tissue cohesion and the mechanics of cell rearrangement.

Morphogenetic processes often involve the rapid rearrangement of cells held together by mutual adhesion. The dynamic nature of this adhesion endows tissues with liquid-like properties, such that large-scale shape changes appear as tissue flows. Generally, the resistance to flow (tissue viscosity) is expected to depend on the cohesion of a tissue (how strongly its cells adhere to each other), but the exact relationship between these parameters is not known. Here, we analyse the link between cohesion and viscosity to uncover basic mechanical principles of cell rearrangement. We show that for vertebrate and invertebrate tissues, viscosity varies in proportion to cohesion over a 200-fold range of values. We demonstrate that this proportionality is predicted by a cell-based model of tissue viscosity. To do so, we analyse cell adhesion in Xenopus embryonic tissues and determine a number of parameters, including tissue surface tension (as a measure of cohesion), cell contact fluctuation and cortical tension. In the tissues studied, the ratio of surface tension to viscosity, which has the dimension of a velocity, is 1.8 µm/min. This characteristic velocity reflects the rate of cell-cell boundary contraction during rearrangement, and sets a limit to rearrangement rates. Moreover, we propose that, in these tissues, cell movement is maximally efficient. Our approach to cell rearrangement mechanics links adhesion to the resistance of a tissue to plastic deformation, identifies the characteristic velocity of the process, and provides a basis for the comparison of tissues with mechanical properties that may vary by orders of magnitude.

EphA4-dependent Brachyury expression is required for dorsal mesoderm involution in the Xenopus gastrula.

Xenopus provides a well-studied model of vertebrate gastrulation, but a central feature, the movement of the mesoderm to the interior of the embryo, has received little attention. Here, we analyze mesoderm involution at the Xenopus dorsal blastopore lip. We show that a phase of rapid involution - peak involution - is intimately linked to an early stage of convergent extension, which involves differential cell migration in the prechordal mesoderm and a new movement of the chordamesoderm, radial convergence. The latter process depends on Xenopus Brachyury, the expression of which at the time of peak involution is controlled by signaling through the ephrin receptor, EphA4, its ligand ephrinB2 and its downstream effector p21-activated kinase. Our findings support a conserved role for Brachyury in blastopore morphogenesis.

Polarized contact behavior in directionally migrating Xenopus gastrula mesendoderm.

The control of cell-cell adhesion and detachment is essential for collective migration and cell rearrangement. Here, we have used the contact behavior of Xenopus gastrula mesoderm explants migrating directionally on ectoderm conditioned substratum to study the regulation of active cell-cell detachment. When colliding laterally, explants repelled each other, whereas they fused front-to-back when aligned in the direction of migration. For this mesoderm polarization by the substratum, we identified three control modules. First, PDGF-A signaling normally suppresses contact-induced collapse of lamellipodia in a polarized manner. Disruption of PDGF-A function, or of Xwnt6, decreased the polarization of explant contact behavior. Second, the Wnt receptor Xfz7 acted upstream of the kinase Pak1 to control explant fusion independently of PDGF-A-promoted lamellipodia stability. Third, ephrinB1 acted with Dishevelled (Dvl) in front-to-back explant fusion. The second and third modules have been identified previously as regulators of tissue separation at the ectoderm-mesoderm boundary. On non-polarizing, fibronectin-coated substratum, they controlled repulsion between explants in the same way as between tissues during boundary formation. However, explant repulsion/fusion responses were reversed on conditioned substratum by the endogenous guidance cues that also control oriented contact inhibition of lamellipodia. Together, control modules and substratum-bound guidance cues combine preferential front-back adhesion and diminished lateral adhesion of cells to promote collective directional mesoderm migration.

PAPC mediates self/non-self-distinction during Snail1-dependent tissue separation.

Cleft-like boundaries represent a type of cell sorting boundary characterized by the presence of a physical gap between tissues. We studied the cleft-like ectoderm-mesoderm boundary in Xenopus laevis and zebrafish gastrulae. We identified the transcription factor Snail1 as being essential for tissue separation, showed that its expression in the mesoderm depends on noncanonical Wnt signaling, and demonstrated that it enables paraxial protocadherin (PAPC) to promote tissue separation through two novel functions. First, PAPC attenuates planar cell polarity signaling at the ectoderm-mesoderm boundary to lower cell adhesion and facilitate cleft formation. Second, PAPC controls formation of a distinct type of adhesive contact between mesoderm and ectoderm cells that shows properties of a cleft-like boundary at the single-cell level. It consists of short stretches of adherens junction-like contacts inserted between intermediate-sized contacts and large intercellular gaps. These roles of PAPC constitute a self/non-self-recognition mechanism that determines the site of boundary formation at the interface between PAPC-expressing and -nonexpressing cells.

Role of p21-activated kinase in cell polarity and directional mesendoderm migration in the Xenopus gastrula.

The p21 activated kinases (Paks) are prominently involved in the regulation of cell motility. Using a kinase-dead mutant of xPak1, we show that during Xenopus gastrulation, the kinase activity of Pak1 is required upstream of Cdc42 for the establishment of cell polarity in the migrating mesendoderm. Overactivation of Pak1 function by the expression of constitutively active xPak1 compromises the maintenance of cell polarity, by indirectly inhibiting RhoA function. Inhibition of cell polarization does not affect the migration of single mesendoderm cells. However, Pak1 inhibition interferes with the guidance of mesendoderm migration by directional cues residing in the extracellular matrix of the blastocoel roof, and with mesendoderm translocation in the embryo.

Control of gastrula cell motility by the Goosecoid/Mix.1/ Siamois network: basic patterns and paradoxical effects.

In the vegetal half of the Xenopus gastrula, cell populations differ with respect to migration on fibronectin substratum. We show that the paired-class homeodomain transcription factors Goosecoid (Gsc), Mix.1, and Siamois (Sia) are involved in the modulation of migration velocity and cell polarity. Mix.1 is expressed in the whole vegetal half and serves as a competence factor that is necessary, but not sufficient, for rapid cell migration and polarization. In the head mesoderm, Gsc and Sia are coexpressed with Mix.1, promoting rapid cell migration and polarization. Ectopic expression of Gsc and Sia in both vegetal and ventral regions often generates paradoxical effects; if a factor activates a certain motility trait in one region, it inhibits it in the other. Migration velocity and cell polarity are regulated independently. Fast and efficiently migrating multipolar cells and slow-moving polarized cells can be obtained by ectopic expression of these transcription factors in different combinations.

Migrating anterior mesoderm cells and intercalating trunk mesoderm cells have distinct responses to Rho and Rac during Xenopus gastrulation.

Rho GTPases have been shown recently to be important for cell polarity and motility of the trunk mesoderm during gastrulation in Xenopus embryos. This work demonstrated that Rho and Rac have both distinct and overlapping roles in regulating cell shape, and the dynamic properties, polarity, and type of protrusive activity of these cells. Overexpression of activated or inhibitory versions of these GTPases also disrupts development of the head in Xenopus embryos. In this study, we have undertaken a detailed analysis of Rho and Rac function in migrating anterior mesendoderm cells. Scanning electron micrographs of these cells in situ revealed that their normal shingle arrangement is disrupted and both the cells and their lamellipodia are disoriented. Anterior mesendoderm explants plated on their natural blastocoel roof matrix, however, still migrated towards the animal pole, although the tendency to move in this direction is reduced compared to controls. Analysis of a number of parameters in time-lapse recordings of dissociated cells indicated that Rho and Rac also have both distinct and overlapping roles in the motility of the prospective head mesoderm; however, their effects differ to those previously seen in the trunk mesoderm. Both GTPases appear to modulate cell polarization, migration, and protrusive activity. Rho alone, however, regulates the retraction of the lagging edge of the cell. We propose that within the gastrulating Xenopus embryo, two types of mesoderm cells that undergo different motilities have distinct responses to Rho GTPases.

Guidance of mesoderm cell migration in the Xenopus gastrula requires PDGF signaling.

In vertebrates, PDGFA and its receptor, PDGFRalpha, are expressed in the early embryo. Impairing their function causes an array of developmental defects, but the underlying target processes that are directly controlled by these factors are not well known. We show that in the Xenopus gastrula, PDGFA/PDGFRalpha signaling is required for the directional migration of mesodermal cells on the extracellular matrix of the blastocoel roof. Blocking PDGFRalpha function in the mesoderm does not inhibit migration per se, but results in movement that is randomized and no longer directed towards the animal pole. Likewise, compromising PDGFA function in the blastocoel roof substratum abolishes directionality of movement. Overexpression of wild-type PDGFA, or inhibition of PDGFA both lead to randomized migration, disorientation of polarized mesodermal cells, decreased movement towards the animal pole, and reduced head formation and axis elongation. This is consistent with an instructive role for PDGFA in the guidance of mesoderm migration.