Relocations of cell convergence sites and formation of pharyngula-like shapes in mechanically relaxed <Emphasis Type="Italic">Xenopus</Emphasis> embryos

Influence of the relaxation of mechanical tensions upon collective cell movements, shape formation, and expression patterns of tissue-specific genes has been studied in Xenopus laevis embryos. We show that the local relaxation of tensile stresses within the suprablastoporal area (SBA) performed at the early-midgastrula stage leads to a complete arrest of normal convergent cell intercalation towards the dorsal midline. As a result, SBA either remains nondeformed or protrudes a strip of cells migrating ventralwards along one of the lateral lips of the opened blastopore. Already, few minutes later, the tissues in the ventral lip vicinity undergo abnormal transversal contraction/longitudinal extension resulting in the abnormal cell convergence toward ventral (rather than dorsal) embryo midline. Within a day, the dorsally relaxed embryos acquire pharyngula-like shapes and often possess tail-like protrusions. Their antero-posterior and dorso-ventral polarity, as well as expression patterns of pan-neural (Sox3), muscular cardiac actin, and forebrain (Otx2) genes substantially deviate from the normal ones. We suggest that normal gastrulation is permanently controlled by mechanical stresses within the blastopore circumference. The role of tissue tensions in regulating collective cell movements and creating pharyngula-like shapes are discussed.     

Local shifts in position and polarized motility drive cell rearrangement during sea urchin gastrulation

This study examines the mechanisms of epithelial cell rearrangement during archenteron elongation in the sea urchin embryo using scanning electron microscopy, differential interference contrast videomicroscopy, cell marking, and fluorescently labeled chimaeric clones. Archenteron elongation involves two major processes: local shifts in position of cells in the archenteron wall and polarized motility of the cells as they rearrange. Fluorescently labeled chimaeric clones introduced into the archenteron of Lytechinus pictus are initially 4-5 cells wide; by the end of gastrulation the clones elongate and narrow, so that they are one cell wide in the narrowest region of the archenteron. The extent of clonal mixing indicates that cells in the archenteron change their relative positions by only 1-2 cell diameters during cell rearrangement. Cells at the blastopore rearrange concomitantly with cells in the archenteron, resulting in a 35% decrease in blastopore diameter. Endoderm cells undergo polarized, stage-specific changes in shape and motility as they rearrange; (1) they flatten markedly along their apical-basal axis throughout archenteron elongation; (2) just prior to the onset of cell rearrangement, basal surfaces of all cells in the archenteron extend long, polarized lamellipodial protrusions along the axis of extension of the archenteron; (3) as cell rearrangement begins, basal surfaces round up and the cells become isodiametric; (4) by the 3/4 gastrula stage the cells become stretched along the animal-vegetal axis, apparently due to filopodial traction, and finally (5) they continue to rearrange, returning to a less elongated shape by the end of gastrulation. Direct observation of gastrulation in the cidaroid Eucidaris tribuloides indicates that in this species cell rearrangement is accomplished by progressive circumferential intercalation of cells without upwardly directed filopodia. This intercalation is accompanied by explosive, apparently stochastic, cortical blebbing activity at the boundaries between cells, suggesting that in addition to whatever cell rearrangement may be generated by filopodial tension, such activity is an important component of the active rearrangement process.     

Experimental analysis of the extension of the dorsal marginal zone in Pleurodeles waltl gastrulae

The capacity for extension of the dorsal marginal zone (DMZ) in Pleurodeles waltl gastrulae was studied by scanning electron microscopy and grafting experiments. At the onset of gastrulation, the cells of the animal pole (AP) undergo important changes in shape and form a single layer. As gastrulation proceeds, the arrangement of cells also changes in the noninvoluted DMZ: radial intercalation leads to a single layer of cells. Grafting experiments involving either AP or DMZ explants were performed using a cell lineage tracer. When rotated 90 degrees or 180 degrees, grafted DMZ explants were able to involute normally and there was extension according to the animal-vegetal axis of the host. In contrast, neither single nor bilayered explants from AP involutes completely, and neither extends when grafted in place of the DMZ. Furthermore, when inside of the host, these AP grafts curl up and inhibit the closure of the blastopore. Once transplanted to the AP region, the DMZ showed no obvious autonomous extension. DMZs cultured in vitro showed little extension and this only from the late gastrula stage onward. Removal of blastocoel roof blocked involution to a varied extent, depending on the developmental stage of the embryos. From these results, it is argued that differences could well exist in the mechanism of gastrulation between anuran and urodele embryos. That migrating mesodermal cells play a major role in urodele gastrulation is discussed.     

Effects of mechanical stretching of embryonic tissues on axial structure formation in <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Effect of mechanical stretch on the differentiation of axial anlages and Chordin gene expression was studied in sandwich explants prepared from embryonic tissues of Xenopus laevis at the early gastrula stage in two variants: with dissected or intact dorso-medial region. In the first case, convergent cell movements were suppressed and properly organized axial organs (notochord and somites) were almost completely absent. However, they developed if the explants of such type were artificially stretched in the ventro-dorsal direction. In this case, axial organs elongated in the line of stretching, that is in the direction vertical to their normal orientation. Segmented mesoderm was always in contact with the chord anlage. In situ hybridization revealed that the area of Chordin gene expression was also extended in the direction of stretching. PCR showed that Chordin gene expression in stretched explants with disrupted dorso-medial region was statistically at the same level as in the explants with intact dorso-medial region. At the same time, the corresponding gene expression in unstretched explants with disrupted dorso-medial region was statistically higher. The obtained data indicate that mechanical stretch and associated cell movements are a necessary and sufficient condition for the formation of proper histological structure of axial organs and regulation of Chordin gene expression.     

Role of cooperative cell movements and mechano-geometric constrains in patterning of axial rudiments in <Emphasis Type="Italic">Xenopus laevis</Emphasis> embryos

The role of cooperative cell movements has been explored in establishment of regular segregation of the marginal zone of Xenopus laevis embryos into the main axial rudiments: notochord, somites and neural tissue. For this purpose, the following operations were performed at the late blastula-early gastrula stages: (1) isolation of marginal zones, (2) addition of the ventral zone fragments to the marginal zones, (3) dissection of isolated marginal zones along either ventral (a) or dorsal (b) midlines, (4) immediate retransplantation of excised fragments of the suprablastoporal area to the same places without rotation or after 90° rotation, (5) Π-shaped separation of the suprablastoporal area either anteriorly or posteriorly. In experiments 1, 4, and 5, lateromedial convergent cell movements and differentiation of the axial rudiments were suppressed. In experiments 4 and 5, cell movements were reoriented ventrally, the entire embryo architecture was extensively reconstructed, and the axial rudiments were relocated to the blastopore lateral lips. In experiment 3, convergent cell movements were restored and oriented either towards the presumptive embryo midline (a), or in the perpendicular direction (b). In both cases, well developed axial rudiments elongated perpendicularly to cell convergences were formed. If the areas of axial rudiment formation were curved, mesodermal somites and neural tissue were always located on the convex (stretched) and concave (compressed) sides, respectively. We conclude that no stable prepatterning of the marginal zone takes place until at least the midgastrula stage. This prepatterning requires cooperative cell movements and associated mechano-geometric constrains.     

Geometry of movement of the outer surface of the embryo during <Emphasis Type="Italic">Xenopus</Emphasis> gastrulation

The surface of Xenopus laevis embryos was marked with carbon particles, after which the location of mark groups was recorded by time-lapse video imaging and subsequent image analysis until their disappearance in the depth of gastric invagination. Measuring the distances between individually identifiable marks whose size is smaller than the size of a single cell makes it possible to quantitatively analyze the geometry of collective cell movement without any external coordinate system. During the dorsal blastopore lip (DBL) formation, the invagination of surface cells fundamentally differs from the preceding and subsequent lateromedial (LM) intercalation, being associated with a decrease in the meridional distance and an increase in the latitudinal distance between the marked surface sites. The sites that began to move towards the DBL later overtake the areas that started movement earlier, which leads to a “plug” in the movement of cells. Pushing the “plug” into the inner layers by changing the DBL shape becomes the rate-limiting stage of gastrulation; then, the directed cell movement is replaced by epiboly based on LM intercalation when the marks remaining on the outer surface of the marginal zone diverge along its meridians without directed migration towards the blastopore. As a result, directional movement of cells and LM intercalation become successive phases of collective cell movement, and the entire morphogenesis of DBL is the direct consequence of epiboly deceleration occurring upon gastric invagination.     

Mediolateral cell intercalation in the dorsal, axial mesoderm of Xenopus laevis

The pattern of mediolateral cell intercalation in mesodermal tissues during gastrulation and neurulation of Xenopus laevis was determined by tracing cells labeled with fluorescein dextran amine (FDA). Patches of the involuting marginal zone (IMZ) of early gastrula stage embryos, labeled by injection of FDA at the one-cell stage, were grafted to the corresponding regions of unlabeled host embryos. The host embryos were fixed at several stages, serially sectioned, and examined with fluorescence microscopy and three-dimensional reconstruction. Patterns of mixing of labeled and unlabeled cells show that mediolateral cell intercalation occurs in the posterior, dorsal mesoderm as this region undergoes convergent extension and differentiates into somites and notochord. In contrast, it does not occur in any dorsoventral sector of the anterior, leading edge of the mesodermal mantle. These results, taken with other evidence, suggest that the mesoderm of Xenopus consists of two subpopulations, each with a characteristic morphogenetic movement, cell behavior, and tissue fate. The migrating mesoderm (1) does not show convergent extension; (2) migrates and spreads on the blastocoel roof; (3) is dependent on this substratum for its morphogenesis; (4) shows little mediolateral intercalation; (5) consists of the anterior, early-involuting region of the mesodermal mantle; and (6) differentiates into head, heart, blood island, and lateral body wall mesoderm. The extending mesoderm (1) shows convergent extension; (2) is independent of the blastocoel roof in its morphogenesis; (3) shows extensive mediolateral intercalation; (4) consists of the posterior, late-involuting parts of the mesodermal mantle; and (5) differentiates into somite and notochord.     

Cytological events in explants of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> during early callogenesis

Leaf explants of diploid (2n = 2x = 10) and autotetraploid (2n = 4x = 20) plants of Arabidopsis thaliana ecotype Columbia were cytologically and cytogenetically analysed to determine the time and the mechanisms of the process of polyploidization. The first polyploid cells were observed after the third day of culture in both genotypes of explants. Polyploid cells were the result of pre-existing mixoploidy in explants of A. thaliana. Other factors such as endoreduplication, endomitosis, abnormal microtubules arrangement and DNA damage may have induced polyploidization during early stages of callogenesis.     

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.     

Monopolar protrusive activity: a new morphogenic cell behavior in the neural plate dependent on vertical interactions with the mesoderm in Xenopus

We compared the type and patterning of morphogenic cell behaviors driving convergent extension of the Xenopus neural plate in the presence and absence of persistent vertical signals from the mesoderm by videorecording explants of deep neural tissue with involuted mesoderm attached and of deep neural tissue alone. In deep neural-over-mesoderm explants, neural plate cells express monopolar medially directed motility and notoplate cells express randomly oriented motility, two new morphogenic cell behaviors. In contrast, in deep neural explants (without notoplate), all cells express bipolar mediolateral cell motility. Deep neural-over-mesoderm and deep neural explants also differ in degree of neighbor exchange during mediolateral cell intercalation. In deep neural-over-mesoderm explants, cells intercalate conservatively, whereas in deep neural explants cells intercalate more promiscuously. Last, in both deep neural-over-mesoderm and deep neural explants, morphogenic cell behaviors differentiate in an anterior-to-posterior and lateral-to-medial progression. However, in deep neural-over-mesoderm explants, morphogenic behaviors first differentiate in intervals along the anteroposterior axis, whereas in deep neural explants, morphogenic behaviors differentiate continuously from the anterior end of the tissue posteriorly. These results describe new morphogenic cell behaviors driving neural convergent extension and also define roles for signals from the mesoderm, up to and beyond late gastrulation, in patterning these cell behaviors.     

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.     

Changes in the shape of epithelial embryonic cells of the spur-toed frog upon deformation of the cell layer

A quantitative study was made of changes in the shape of cells in double explants of the blastocoel roof of the clawed frog gastrula within the first four hours after artificial bending of explants. It was found that, on the concave (contracted) side of explants, epithelial cells stretched out, and in many of them the apical surface contracted, whereas on the convex (stretched) side the cells remained isodiametric. The maximal difference in the apical index between epithelial cells located on the concave and convex sides was observed after 2 h of explant cultivation; by 2 h the artificially produced curvature of the explant further increased. Endocytosis on the concave side was more active than on the convex side. Experiments with inhibitors modulating the behavior of the actomyosin complex showed that unimpeded functioning of myosin II is more important for the apical contraction and elongation of cells than proper structural organization of the actin backbone.     

Time-lapse cinemicrographic analysis of superficial cell behavior during and prior to gastrulation in Xenopus laevis

Time-lapse cinemicrography was used to show what changes in the number, size, shape, arrangement and what movements of apices of superficial cells occur during epiboly, extension, convergence and blastopore formation in the blastula or gastrula of Xenopus laevis. Epiboly of the animal region occurs by apical expansion of superficial cells at a nearly constant rate from the midblastula to the midgastrula stage. Egression of deep cells into the superficial layer does not occur. Extension of the dorsal marginal zone begins in the late blastula stage with the rapid spreading of the apices of cells in this region and this continues until the onset of neurulation when rapid shrinkage begins. Extension and convergence of the dorsal marginal zone occurs by a rearrangement in which individual cells exchange neighbors and by a change in the shape of the cell apices. Regional differences in apical expansion are accompanied by differences in rate of anticlinal division of superficial cells such that cells in all sectors of the animal region and the marginal zone show similar patterns of decrease in apparent apical area. Shrinkage of the apices of bottle cells during blastopore formation is described. From this and other studies, a model of the cellular behavior of epiboly, extension and convergence is constructed and several hypotheses as to how these activities might generate the mechanical forces of the gastrulation movements are presented.     

Regional requirements for Dishevelled signaling during Xenopus gastrulation: separable effects on blastopore closure, mesendoderm internalization and archenteron formation

During amphibian gastrulation, the embryo is transformed by the combined actions of several different tissues. Paradoxically, many of these morphogenetic processes can occur autonomously in tissue explants, yet the tissues in intact embryos must interact and be coordinated with one another in order to accomplish the major goals of gastrulation: closure of the blastopore to bring the endoderm and mesoderm fully inside the ectoderm, and generation of the archenteron. Here, we present high-resolution 3D digital datasets of frog gastrulae, and morphometrics that allow simultaneous assessment of the progress of convergent extension, blastopore closure and archenteron formation in a single embryo. To examine how the diverse morphogenetic engines work together to accomplish gastrulation, we combined these tools with time-lapse analysis of gastrulation, and examined both wild-type embryos and embryos in which gastrulation was disrupted by the manipulation of Dishevelled (Xdsh) signaling. Remarkably, although inhibition of Xdsh signaling disrupted both convergent extension and blastopore closure, mesendoderm internalization proceeded very effectively in these embryos. In addition, much of archenteron elongation was found to be independent of Xdsh signaling, especially during the second half of gastrulation. Finally, even in normal embryos, we found a surprising degree of dissociability between the various morphogenetic processes that occur during gastrulation. Together, these data highlight the central role of PCP signaling in governing distinct events of Xenopus gastrulation, and suggest that the loose relationship between morphogenetic processes may have facilitated the evolution of the wide variety of gastrulation mechanisms seen in different amphibian species.     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

Effects of growth factors on proliferation of basal and luminal cells in human breast epithelial explants in serum-free culture

Summary A method of culturing human breast epithelium is described in which viable explants can be maintained in protein-free medium while retaining the capacity of responding to added hormones and growth factors for at least 7 days. Culture parameters were chosen to provide maximum sensitivity of detection of proliferative responses by autoradiography. Under basal conditions, the mean thymidine labeling index of the explants was 0.08%. After stimulation with insulin, hydrocortisone, and cholera toxin (I,H,CT), a combination known to stimulate proliferation in human breast epithelium in vitro, the mean labeling index was 15.7%. Stimulation of explants with epidermal growth factor (EGF) and transforming growth factor (TGF)-α resulted in mean labeling indices of 6.6 and 10.8%, respectively. Autoradiography at the ultrastructural level demonstrated that in I,H,CT-stimulated explants the majority of the labeled cells were luminal, with only 1.5% being basal cells. In contrast, after EGF and TGF-α basal cells accounted for 11.5 and 18.5% of the labeled population. These results indicate that this system provides an in vitro assay of proliferative activity in the normal human breast that enables comparisons to be made between both the luminal and the basal cells in the explants and their counterparts in monolayer culture prepared from flow sorted cells. Thus, growth responses dependent on cell-to-cell interactions or stromal modulation can be identified.     

Oriented cell division in vertebrate embryogenesis

Tissue morphogenesis depends on the spatial arrangement of cells during development. A number of mechanisms have been described to contribute to the final shape of a tissue or organ, ranging from cell intercalation to the response of cells to chemotactic cues. One such mechanism is oriented cell division. Oriented cell division is determined by the position of the mitotic spindle. Indeed, there is increasing evidence implicating spindle misorientation in tissue and organ misshaping, which underlies disease conditions such as tumorigenesis or polycystic kidneys. Here we review recent studies addressing how the direction of tissue growth is determined by the orientation of cell division and how both extrinsic and intrinsic cues control the position of the mitotic spindle.     

Unravelling the distinct contribution of cell shape changes and cell intercalation to tissue morphogenesis: the case of the Drosophila trachea

Intercalation allows cells to exchange positions in a spatially oriented manner in an array of diverse processes, spanning convergent extension in embryonic gastrulation to the formation of tubular organs. However, given the co-occurrence of cell intercalation and changes in cell shape, it is sometimes difficult to ascertain their respective contribution to morphogenesis. A well-established model to analyse intercalation, particularly in tubular organs, is the Drosophila tracheal system. There, fibroblast growth factor (FGF) signalling at the tip of the dorsal branches generates a ‘pulling’ force believed to promote cell elongation and cell intercalation, which account for the final branch extension. Here, we used a variety of experimental conditions to study the contribution of cell elongation and cell intercalation to morphogenesis and analysed their mutual requirements. We provide evidence that cell intercalation does not require cell elongation and vice versa. We also show that the two cell behaviours are controlled by independent but simultaneous mechanisms, and that cell elongation is sufficient to account for full extension of the dorsal branch, while cell intercalation has a specific role in setting the diameter of this structure. Thus, rather than viewing changes in cell shape and cell intercalation as just redundant events that add robustness to a given morphogenetic process, we find that they can also act by contributing to different features of tissue architecture.     

Dorsoventral Differences in Cell-Cell Interactions Modulate the Motile Behaviour of Cells from the Xenopus Gastrula

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.     

Epithelial Cell Wedging and Neural Trough Formation Are Induced Planarly inXenopus,without Persistent Vertical Interactions with Mesoderm

In this study we investigate the induction of the cell behaviors underlying neurulation in the frog,Xenopus laevis.Although planar signals from the organizer can induce convergent extension movements of the posterior neural tissue in explants, the remaining morphogenic processes of neurulation do not appear to occur in absence of vertical interactions with the organizer (R. Kelleret al.,1992,Dev. Dyn.193, 218–234). These processes include: (1) cell elongation perpendicular to the plane of the epithelium, forming the neural plate; (2) cell wedging, which rolls the neural plate into a trough; (3) intercalation of two layers of neural plate cells to form one layer; and (4) fusion of the neural folds. To allow planar signaling between all the inducing tissues of the involuting marginal zone and the responding prospective ectoderm, we have designed a “giant sandwich” explant. In these explants, cell elongation and wedging are induced in the superficial neural layer by planar signals without persistent vertical interactions with underlying, involuted mesoderm. A neural trough forms, and neural folds form and approach one another. However, the neural folds do not fuse with one another, and the deep cells of these explants do not undergo their normal behaviors of elongation, wedging, and intercalation between the superficial neural cells, even when planar signals are supplemented with vertical signaling until the late midgastrula (stage 11.5). Vertical interactions with mesoderm during and beyond the late gastrula stage were required for expression of these deep cell behaviors and for neural fold fusion. These explants offer a way to regulate deep and superficial cell behaviors and thus make possible the analysis of the relative roles of these behaviors in closing the neural tube.