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.     

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.     

Shaping the zebrafish notochord

Promptly after the notochord domain is specified in the vertebrate dorsal mesoderm, it undergoes dramatic morphogenesis. Beginning during gastrulation, convergence and extension movements change a squat cellular array into a narrow, elongated one that defines the primary axis of the embryo. Convergence and extension might be coupled by a highly organized cellular intermixing known as mediolateral intercalation behavior (MIB). To learn whether MIB drives early morphogenesis of the zebrafish notochord, we made 4D recordings and quantitatively analyzed both local cellular interactions and global changes in the shape of the dorsal mesodermal field. We show that MIB appears to mediate convergence and can account for extension throughout the dorsal mesoderm. Comparing the notochord and adjacent somitic mesoderm reveals that extension can be regulated separately from convergence. Moreover, mutational analysis shows that extension does not require convergence. Hence, a cellular machine separate from MIB that can drive dorsal mesodermal extension exists in the zebrafish gastrula. The likely redundant control of morphogenesis may provide for plasticity at this critical stage of early development.     

Cell rearrangement and segmentation in Xenopus: direct observation of cultured explants

We make use of a novel system of explant culture and high resolution video-film recording to analyse for the first time the cell behaviour underlying convergent extension and segmentation in the somitic mesoderm of Xenopus. We find that a sequence of activities sweeps through the somitic mesoderm from anterior to posterior during gastrulation and neurulation, beginning with radial cell intercalation or thinning, continuing with mediolateral intercalation and cell elongation, and culminating in segmentation and somite rotation. Radial intercalation at the posterior tip lengthens the tissue, while mediolateral intercalation farther anterior converges it toward the midline. This extension of the somitic mesoderm helps to elongate the dorsal side of intact neurulae. By separating tissues, we demonstrate that cell rearrangement is independent of the notochord, but radial intercalation - and thus the bulk of extension - requires the presence of an epithelium, either endodermal or ectodermal. Segmentation, on the other hand, can proceed in somitic mesoderm isolated at the end of gastrulation. Finally, we discuss the relationship between cell rearrangement and segmentation.     

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.     

Cell intercalation during notochord development in Xenopus laevis

Morphometric data from scanning electron micrographs (SEM) of cells in intact embryos and high-resolution time-lapse recordings of cell behavior in cultured explants were used to analyze the cellular events underlying the morphogenesis of the notochord during gastrulation and neurulation of Xenopus laevis. The notochord becomes longer, narrower, and thicker as it changes its shape and arrangement and as more cells are added at the posterior end. The events of notochord development fall into three phases. In the first phase, occurring in the late gastrula, the cells of the notochord become distinct from those of the somitic mesoderm on either side. Boundaries form between the two tissues, as motile activity at the boundary is replaced by stabilizing lamelliform protrusions in the plane of the boundary. In the second phase, spanning the late gastrula and early neurula, cell intercalation causes the notochord to narrow, thicken, and lengthen. Its cells elongate and align mediolaterally as they rearrange. Both protrusive activity and its effectiveness are biased: the anterioposterior (AP) margins of the cells advance and retract but produce much less translocation than the more active left and right ends. The cell surfaces composing the lateral boundaries of the notochord remain inactive. In the last phase, lasting from the mid- to late neurula stage, the increasingly flattened cells spread at all their interior margins, transforming the notochord into a cylindrical structure resembling a stack of pizza slices. The notochord is also lengthened by the addition of cells to its posterior end from the circumblastoporal ring of mesoderm. Our results show that directional cell movements underlie cell intercalation and raise specific questions about the cell polarity, contact behavior, and mechanics underlying these movements. They also demonstrate that the notochord is built by several distinct but carefully coordinated processes, each working within a well-defined geometric and mechanical environment.     

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.     

Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis

The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by "relamination" (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.     

Cells remain competent to respond to mesoderm-inducing signals present during gastrulation in Xenopus laevis

During gastrulation, the vertebrate embryo is patterned and shaped by complex signaling pathways and morphogenetic movements. One of the first regions defined during gastrulation is the prospective notochord, which exhibits specific cell behaviors that drive the extension of the embryonic axis. To examine the signals involved in notochord formation in Xenopus laevis and the competence of cells to respond to these signals, we performed cell transplantation experiments during gastrulation. Labeled cells from the prospective notochord, somitic mesoderm, ventrolateral mesoderm, neural ectoderm, and epidermis, between stages 9 (pregastrulation) and 12 (late gastrulation), were grafted into the prospective notochord region of the early gastrula. We show that cells from each region are competent to respond to notochord-inducing signals and differentiate into notochordal tissue. Cells from the prospective neural ectoderm are the most responsive to notochord-inducing signals, whereas cells from the ventrolateral and epidermal regions are the least responsive. We show that at the end of gastrulation, while transplanted cells lose their competence to form notochord, they remain competent to form somites. These results demonstrate that at the end of gastrulation cell fates are not restricted within germ layers. To determine whether notochord-inducing signals are present throughout gastrulation, grafts were made into progressively older host embryos. We found that regardless of the age of the host, grafted cells from each region give rise to notochordal tissue. This indicates that notochord-inducing signals are present throughout gastrulation and that these signals overlap with somite-inducing signals at the end of gastrulation. We conclude that it is the change of competence that restricts cells to specific tissues rather than the regulation of the inducing signals.     

The presumptive floor plate (notoplate) induces behaviors associated with convergent extension in medial but not lateral neural plate cells of Xenopus

In previous work (Elul, T., Keller, R., 2000. Monopolar protrusive activity: a new morphogenic cell behavior in the neural plate dependent on vertical interactions with the mesoderm in Xenopus. Dev. Biol. 224, 3-19; Ezin, A.M., Skoglund, P. Keller, R. 2003. The midline (notochord and notoplate) patterns the cell motility underlying convergence and extension of the Xenopus neural plate. Dev. Biol. 256, 100-114), the midline tissues of notochord and overlying notoplate were found to induce the monopolar, medially directed protrusive activity of deep neural cells. This behavior is thought to drive the mediolateral intercalation and convergent extension of the neural plate in Xenopus. Here we address the issue of whether the notochord, the notoplate, or both is essential for this induction. Our strategy was to remove the notochord, leaving the overlying notoplate intact, and determine whether it alone can induce the monopolar, medially directed cell behavior. We first establish that the notoplate (presumptive floor plate), when separated from the underlying notochord in the early neurula (stages 13-14), will independently mature into a floor plate as assayed three criteria: (1) continued expression of an early marker, sonic hedgehog, and a later, marker, F-spondin; (2) the display of the notoplate/floor plate-specific randomly oriented protrusive activity; (3) the characteristic lack of mixing of cells between the notoplate and lateral neural plate. Under these conditions, in the presence of a mature notoplate/floor plate and in the absence of the notochord, the characteristic monopolar, medially directed behavior occurred, but only locally near the midline. These results show that the notoplate/floor plate capacity to induce the medially directed motility is limited in range, and they suggest that the notochord is necessary for the normally observed longer range induction in lateral neural plate cells. This work helps to further the understanding of molecular and tissue interactions required for convergent extension.     

The midline (notochord and notoplate) patterns the cell motility underlying convergence and extension of the Xenopus neural plate

We investigated the role of the dorsal midline structures, the notochord and notoplate, in patterning the cell motilities that underlie convergent extension of the Xenopus neural plate. In explants of deep neural plate with underlying dorsal mesoderm, lateral neural plate cells show a monopolar, medially directed protrusive activity. In contrast, neural plate explants lacking the underlying dorsal mesoderm show a bipolar, mediolaterally directed protrusive activity. Here, we report that "midlineless" explants consisting of the deep neural plate and underlying somitic mesoderm, but lacking a midline, show bipolar, mediolaterally oriented protrusive activity. Adding an ectopic midline to the lateral edge of these explants restores the monopolar protrusive activity over the entire extent of the midlineless explant. Monopolarized cells near the ectopic midline orient toward it, whereas those located near the original, removed midline orient toward this midline. This behavior can be explained by two signals emanating from the midline. We postulate that one signal polarizes neural plate deep cells and is labile and short-lived and that the second signal orients any polarized cells toward the midline and is persistent.     

Cell motility driving mediolateral intercalation in explants of Xenopus laevis.

In Xenopus, convergence and extension are produced by active intercalation of the deep mesodermal cells between one another along the mediolateral axis (mediolateral cell intercalation), to form a narrower, longer array. The cell motility driving this intercalation is poorly understood. A companion paper shows that the endodermal epithelium organizes the outermost mesodermal cells immediately beneath it to undergo convergence and extension, and other evidence suggests that these deep cells are the most active participants in mediolateral intercalation (Shih, J. and Keller, R. (1992) Development 116, 887-899). In this paper, we shave off the deeper layers of mesodermal cells, which allows us to observe the protrusive activity of the mesodermal cells next to the organizing epithelium with high resolution video microscopy. These mesodermal cells divide in the early gastrula and show rapid, randomly directed protrusive activity. At the early midgastrula stage, they begin to express a characteristic sequence of behaviors, called mediolateral intercalation behavior (MIB): (1) large, stable, filiform and lamelliform protrusions form in the lateral and medial directions, thus making the cells bipolar; (2) these protrusions are applied directly to adjacent cell surfaces and exert traction on them, without contact inhibition; (3) as a result, the cells elongate and align parallel to the mediolateral axis and perpendicular to the axis of extension; (4) the elongate, aligned cells intercalate between one another along the mediolateral axis, thus producing a longer, narrower array. Explants of essentially a single layer of deep mesodermal cells, made at stage 10.5, converge and extend by mediolateral intercalation. Thus by stage 10.5 (early midgastrula), expression of MIB among deep mesodermal cells is physiologically and mechanically independent of the organizing influence of the endodermal epithelium, described previously (Shih, J. and Keller, R. (1992) Development 116 887-899), and is the fundamental cell motility underlying mediolateral intercalation and convergence and extension of the body axis.     

Inductive signals from the somatopleure mediated by bone morphogenetic proteins are essential for the formation of the sternal component of avian ribs

The posterior five pairs of avian ribs are composed of vertebral and sternal components, both derived from the somitic mesoderm. For the patterning of the rib cartilage, inductive signals from neighboring tissues on the somitic mesoderm have been suggested to play critical roles. The notochord and surface ectoderm overlying the somitic mesoderm are essentially required for the development of proximal and distal regions of the ribs, respectively. Involvement of the somatopleure in rib development has already been suggested but is less understood than those of the notochord and surface ectoderm. In this study, we reinvestigated the role of the somatopleure during rib development. We first identified the chicken homologue of the mouse Mesenchymal forkhead-1 (cMfh-1) gene based on sequence similarities. cMfh-1 was observed to be expressed in the nonaxial mesoderm, including the somitic mesoderm, and, subsequently, in cartilage forming the ribs, vertebrae, and appendicular skeletal system. In the interlimb region, corresponding to somites 21-25 (or 26), cMfh-1-positive somitic mesoderm was seen penetrating the somatopleure of E4 embryos, and cMfh-1 was used as a molecular marker demarcating prospective rib cartilage. A series of experiments affecting the penetration of the somitic mesoderm into the somatopleure was performed in the present study, resulting in defects in sternal rib formation. The inductive signals emanating from the somatopleure mediated by BMP family proteins were observed to be essentially involved in the ingrowth of the somitic mesoderm. BMP4 alone, however, could not completely replace inductive signals from the somatopleure, suggesting the involvement of additional signals for rib formation.     

Chongmague reveals an essential role for laminin-mediated boundary formation in chordate convergence and extension movements

Although cell intercalation driven by non-canonical Wnt/planar cell polarity (PCP) pathway-dependent mediolateral cell polarity is important for notochord morphogenesis, it is likely that multiple mechanisms shape the notochord as it converges and extends. Here we show that the recessive short-tailed Ciona savignyi mutation chongmague (chm) has a novel defect in the formation of a morphological boundary around the developing notochord. chm notochord cells initiate intercalation normally, but then fail to maintain their polarized cell morphology and migrate inappropriately to become dispersed in the larval tail. This is unlike aimless (aim), a mutation in the PCP pathway component Prickle, which has a severe defect in early mediolateral intercalation but forms a robust notochord boundary. Positional cloning identifies chm as a mutation in the C. savignyi ortholog of the vertebrate alpha 3/4/5 family of laminins. Cs-lamalpha3/4/5 is highly expressed in the developing notochord, and Cs-lamalpha3/4/5 protein is specifically localized to the outer border of the notochord. Notochord convergence and extension, reduced but not absent in both chm and aim, are essentially abolished in the aim/aim; chm/chm double mutant, indicating that laminin-mediated boundary formation and PCP-dependent mediolateral intercalation are each able to drive a remarkable degree of tail morphogenesis in the absence of the other. These mechanisms therefore initially act in parallel, but we also find that PCP signaling has an important later role in maintaining the perinotochordal/intranotochordal polarity of Cs-lamalpha3/4/5 localization.     

Mediolateral patterning of somites: multiple axial signals, including Sonic hedgehog, regulate Nkx-3.1 expression

The axial structures, the notochord and the neural tube, play an essential role in the dorsoventral patterning of somites and in the differentiation of their many cell lineages. Here, we investigated the role of the axial structures in the mediolateral patterning of the somite by using a newly identified murine homeobox gene, Nkx-3.1, as a medial somitic marker in explant in vitro assays. Nkx-3.1 is dynamically expressed during somitogenesis only in the youngest, most newly-formed somites at the caudal end of the embryo. We found that the expression of Nkx-3.1 in pre-somitic tissue explants is induced by the notochord and maintained in newly-differentiated somites by the notochord and both ventral and dorsal parts of the neural tube. We showed that Sonic hedgehog (Shh) is one of the signaling molecules that can reproduce the effect of the axial structures by exposing explants to either COS cells transfected with a Shh expression construct or to recombinant SHH. Shh could induce and maintain Nkx-3.1 expression in pre-somitic mesoderm and young somites but not in more mature, differentiated ones. The effects of Shh on Nkx-3.1 expression were antagonized by a forskolin-induced increase in the activity of cyclic AMP-dependent protein kinase A. Additionally, we confirmed that the expression of the earliest expressed murine myogenic marker, myf 5, is also regulated by the axial strucutres but that Shh by itself is not capable of inducing or maintaining it. We suggest that the establishment of somitic medial and lateral compartments and the early events in myogenesis are governed by a combination of positive and inhibitory signals derived from the neighboring structures, as has previously been proposed for the dorsoventral patterning of somites.     

beta-Catenin controls cell sorting at the notochord-somite boundary independently of cadherin-mediated adhesion

In Xenopus laevis, patterning of the trunk mesoderm into the dorsal notochord and lateral somites depends on differential regulation of Wnt-beta-catenin signaling. To study the cellular requirements for the physical separation of these tissues, we manipulated beta-catenin activity in individual cells that were scattered within the trunk mesoderm. We found that high activity led to efficient cell sorting from the notochord to the somites, whereas reduced activity led to sorting in the opposite direction. Analysis of individual cells overexpressing beta-catenin revealed that these cells were unable to establish stable contacts with notochord cells but could freely cross the boundary to integrate within the somitic tissue. Interference with cadherin-mediated adhesion disrupted tissue architecture, but it did not affect sorting and boundary formation. Based on these results, we propose that the boundary itself is the result of cell-autonomous changes in contact behavior that do not rely on differences in absolute levels of adhesion.     

Directional migration of leading-edge mesoderm generates physical forces: Implication in Xenopus notochord formation during gastrulation

Gastrulation is a dynamic tissue-remodeling process occurring during early development and fundamental to the later organogenesis. It involves both chemical signals and physical factors. Although much is known about the molecular pathways involved, the roles of physical forces in regulating cellular behavior and tissue remodeling during gastrulation have just begun to be explored. Here, we characterized the force generated by the leading edge mesoderm (LEM) that migrates preceding axial mesoderm (AM), and investigated the contribution of LEM during Xenopus gastrulation. First, we constructed an assay system using micro-needle which could measure physical forces generated by the anterior migration of LEM, and estimated the absolute magnitude of the force to be 20–80 nN. Second, laser ablation experiments showed that LEM could affect the force distribution in the AM (i.e. LEM adds stretch force on axial mesoderm along anterior–posterior axis). Third, migrating LEM was found to be necessary for the proper gastrulation cell movements and the establishment of organized notochord structure; a reduction of LEM migratory activity resulted in the disruption of mediolateral cell orientation and convergence in AM. Finally, we found that LEM migration cooperates with Wnt/PCP to form proper notochord.     

Control of intercalation is cell-autonomous in the notochord of Ciona intestinalis

Dishevelled signaling plays a critical role in the control of cell intercalation during convergent extension in vertebrates. This study presents evidence that Dishevelled serves a similar function in the Ciona notochord. Embryos transgenic for mutant Dishevelled fail to elongate their tails, and notochord cells fail to intercalate, though notochord cell fates are unaffected. Analysis of mosaic transgenics revealed that the effects of mutant Dishevelled on notochord intercalation are cell-autonomous in Ciona, though such defects have nonautonomous effects in Xenopus. Furthermore, our data indicate that notochord cell intercalation in Ciona does not require the progressive signals which coordinate cell intercalation in the Xenopus notochord, highlighting an important difference in how mediolateral cell intercalation is controlled in the two animals. Finally, this study establishes the Ciona embryo as an effective in vivo system for the study of the molecular control of morphogenetic cell movements in chordates.     

Live Imaging-Based Model Selection Reveals Periodic Regulation of the Stochastic G1/S Phase Transition in Vertebrate Axial Development

In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.     

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.