Frizzled-7 signalling controls tissue separation during Xenopus gastrulation.

Cell signalling through Frizzled receptors has evolved to considerable complexity within the metazoans. The Frizzled-dependent signalling cascade comprises several branches, whose differential activation depends on specific Wnt ligands, Frizzled receptor isoforms and the cellular context. In Xenopus laevis embryos, the canonical beta-catenin pathway contributes to the establishment of the dorsal-ventral axis. A different branch, referred to as the planar cell polarity pathway, is essential for cell polarization during elongation of the axial mesoderm by convergent extension. Here we demonstrate that a third branch of the cascade is independent of Dishevelled function and involves signalling through trimeric G proteins and protein kinase C (PKC). During gastrulation, Frizzled-7 (Fz7)-dependent PKC signalling controls cell-sorting behaviour in the mesoderm. Loss of zygotic Fz7 function results in the inability of involuted anterior mesoderm to separate from the ectoderm, which leads to severe gastrulation defects. This result provides a developmentally relevant in vivo function for the Fz/PKC pathway in vertebrates.

Mechanisms of mesendoderm internalization in the Xenopus gastrula: lessons from the ventral side.

Two main processes are involved in driving ventral mesendoderm internalization in the Xenopus gastrula. First, vegetal rotation, an active movement of the vegetal cell mass, initiates gastrulation by rolling the peripheral blastocoel floor against the blastocoel roof. In this way, the leading edge of the internalized mesendoderm is established, that remains separated from the blastocoel roof by Brachet's cleft. Second, in a process of active involution, blastopore lip cells translocate on arc-like trails around the tip of Brachet's cleft. Hereby the lower, Xbra-negative part of the lip moves toward the interior, to contribute mainly to endoderm. In contrast, the upper, Xbra-expressing part moves toward the blastocoel roof-apposed surface of the involuted mesoderm, and eventually becomes inserted into this surface. Vegetal rotation and active mesoderm surface insertion persist over much of gastrulation ventrally. Both processes are also active dorsally. In fact, internalization processes generally spread from dorsal to ventral, though at different rates, which suggests that they are independently controlled. Ventrally and laterally, mesoderm occurs not only in the marginal zone, but also in the adjacent blastocoel roof. Such blastocoel roof mesoderm shares properties with the remaining, ectodermal roof, that are related to its function as substratum for mesendoderm migration. It repels involuted mesoderm, thus contributing to separation of cell layers, and it assembles a fibronectin matrix. These properties change as the blastocoel roof mesoderm moves into the blastopore lip during gastrulation.

Development and control of tissue separation at gastrulation in Xenopus.

During Xenopus gastrulation, the internalizing mesendodermal cell mass is brought into contact with the multilayered blastocoel roof. The two tissues do not fuse, but remain separated by the cleft of Brachet. This maintenance of a stable interface is a precondition for the movement of the two tissues past each other. We show that separation behavior, i.e., the property of internalized cells to remain on the surface of the blastocoel roof substratum, spreads before and during gastrulation from the vegetal endoderm into the anterior and eventually the posterior mesoderm, roughly in parallel to internalization movement. Correspondingly, the blastocoel roof develops differential repulsion behavior, i.e., the ability to specifically repell cells showing separation behavior. From the effects of overexpressing wild-type or dominant negative XB/U or EP/C cadherins we conclude that separation behavior may require modulation of cadherin function. Further, we show that the paired-class homeodomain transcription factors Mix.1 and gsc are involved in the control of separation behavior in the anterior mesoderm. We present evidence that in this function, Mix.1 and gsc may cooperate to repress transcription.

Vegetal rotation, a new gastrulation movement involved in the internalization of the mesoderm and endoderm in Xenopus.

A main achievement of gastrulation is the movement of the endoderm and mesoderm from the surface of the embryo to the interior. Despite its fundamental importance, this internalization process is not well understood in amphibians. We show that in Xenopus, an active distortion of the vegetal cell mass, vegetal rotation, leads to a dramatic expansion of the blastocoel floor and a concomitant turning around of the marginal zone which constitutes the first and major step of mesoderm involution. This vigorous inward surging of the vegetal region into the blastocoel can be analyzed in explanted slices of the gastrula, and is apparently driven by cell rearrangement. Thus, the prospective endoderm, previously thought to be moved passively, provides the main driving force for the internalization of the mesendoderm during the first half of gastrulation. For further involution, and for normal positioning of the involuted mesoderm and its rapid advance toward the animal pole, fibronectin-independent interaction with the blastocoel roof is required.

Establishment of substratum polarity in the blastocoel roof of the Xenopus embryo.

The fibronectin fibril matrix on the blastocoel roof of the Xenopus gastrula contains guidance cues that determine the direction of mesoderm cell migration. The underlying guidance-related polarity of the blastocoel roof is established in the late blastula under the influence of an instructive signal from the vegetal half of the embryo, in particular from the mesoderm. Formation of an oriented substratum depends on functional activin and FGF signaling pathways in the blastocoel roof. Besides being involved in tissue polarization, activin and FGF also affect fibronectin matrix assembly. Activin treatment of the blastocoel roof inhibits fibril formation, whereas FGF modulates the structure of the fibril network. The presence of intact fibronectin fibrils is permissive for directional mesoderm migration on the blastocoel roof extracellular matrix.

Conditions for fibronectin fibril formation in the early Xenopus embryo.

Fibronectin fibril formation on a multilayered cohesive cell sheet is studied in the Xenopus embryo. In the blastula, secreted fibronectin accumulates in the blastocoel, where it associates with mucous material. At the onset of gastrulation, a fibrillar fibronectin matrix develops on the blastocoel roof. Cells engage in this process stochastically within a 2-hr period. Fibril network formation requires more than 60 microg/ml of fibronectin, but the timing of fibrillogenesis is not regulated through the availability of fibronectin. With the exception of a few isolated mesoderm cells, only the cells of the blastocoel roof are able to form fibronectin fibrils. However, this requires that cells are provided with a free surface and, at the same time, with lateral adhesive cell contacts, i.e. fibril assembly occurs only on the surface of cohesive cells aggregates. This explains the observed restriction of fibronectin matrix formation to the inner surface of the blastocoel roof in the embryo. In addition, a minimum blastocoel roof size is required for fibril formation.

Patterns and control of cell motility in the Xenopus gastrula.

By comparing cells with respect to several motility-related properties and the ability to migrate on fibronectin, three cell types can be distinguished in the Xenopus gastrula. These occur in a distinct spatial pattern, thus defining three motility domains which do not correspond to the prospective germ layers. Migratory behavior is confined to a region encompassing the anterior mesoderm and endoderm. When stationary animal cap cells are induced to migrate by treatment with activin, cells become adhesive at low concentrations of fibronectin, show polarized protrusive activity, and form lamellipodia. Adhesion and polarization, but not lamellipodia formation, are mimicked by the immediate early response gene Mix.1. Goosecoid, another immediate early gene, is without effect when expressed alone in animal cap cells, but it acts synergistically with Mix.1 in the control of adhesion, and antagonistically in the polarization of protrusive activity. bFGF also induces migration, lamellipodia formation and polarization in animal cap cells, but has no effect on adhesion. By the various treatments of animal cap cells, new combinations of motile properties can be generated, yielding cell types which are not found in the embryo.

Structure and cytoskeletal organization of migratory mesoderm cells from the Xenopus gastrula.

Prospective mesoderm cells from the Xenopus gastrula exhibit interesting motile behavior, e.g., a transition from a nonmigratory state to an active translocation that can be induced experimentally, and directional substrate-guided locomotion. We examine the cytoskeletal organization of these embryonic cells. We show that the large, globular cells are enclosed in a triple shell consisting of an actomyosin cortex, a peripheral cytokeratin layer, and a peculiar microtubule basket that surrounds the cell body and constrains the distribution of large inclusions such as yolk platelets. Consistent with the migratory phenotype of these cells, no stress fibers or focal contacts are present. Mesoderm cells possess typical lamellipodia with fine protruding filopodia. The leading edge of the lamellar part exposes binding sites for the fodrin SH3 domain. Lamellipodia are connected to the cell body through actin filament bundles of the upper cell cortex. Myosin II is present in the cell body and extends to varying degrees into lamellipodia. We present indirect evidence that myosin II is located in the upper part of lamellipodia and propose a model that involves myosin II in a dynamic linkage between lamellipodium and the cortex of the cell body.

Fibronectin, mesoderm migration, and gastrulation in Xenopus.

The role of fibronectin in mesoderm cell migration and and the importance of mesoderm migration for gastrulation in Xenopus are examined. To allow for migration, a stable interface must exist between migrating mesoderm cells and the cells of the substrate layer, the blastocoel roof. We show that maintenance of this interface does not depend on fibronectin. We further demonstrate that fibronectin contributes to, but is not essential for, mesoderm cell adhesion to the blastocoel roof. However, interaction with fibronectin is necessary for cell spreading and the formation of lamelliform cytoplasmic protrusions. Apparently, the specific role of fibronectin in mesoderm migration is to control cell protrusive activity. Consequently, when fibronectin function is blocked by GRGDSP peptide or antibodies, mesoderm cell migration is inhibited. Nevertheless, gastrulation proceeds nearly normally in inhibitor-treated embryos. It appears that in Xenopus, mesoderm migration is not essential for gastrulation.

Mesoderm migration in the Xenopus gastrula.

During Xenopus gastrulation, the mesoderm involutes at the blastopore lip and moves on the inner surface of the BCR toward the animal pole of the embryo. Active cell migration is involved in this mesoderm translocation. In vitro, mesoderm cells migrate non-persistently and intermittently by extending and retracting multiple lamellipodia, which pull the cell body in their direction. Lamellipodia formation is induced by FN. FN fibrils are present on the BCR as part of the in vivo substrate of mesoderm migration. Mesoderm cells can attach to the BCR independently of FN, but interaction with FN is required for lamellipodia extension and cell migration on the BCR. In contrast to preinvolution mesoderm, involuted migrating mesoderm always stays on the surface of the BCR cell layer: migrating mesoderm cells do not mix with BCR cells, and a stable interface between tissues is maintained. A corresponding change in cell sorting behavior occurs during mesoderm involution. In Xenopus, the mesoderm moves as a multilayered coherent cell mass held together by cadherin-mediated cell adhesion. Aggregate formation changes mesoderm cell behavior, rendering it more continuous, persistent and directional, i.e. more efficient. The mesoderm possesses an intrinsic tissue polarity which biases the direction of its movement. In addition, the fibrillar FN matrix of the BCR contains guidance cues which also direct the mesoderm toward the animal pole. Haptotaxis is most likely not involved in this substrate-dependent guidance of the mesoderm, but intact FN fibrils seem to be required. A polarity of the BCR cell layer which underlies this anisotropy of the BCR matrix develops under the influence of the marginal zone in the late blastula. Although in other amphibian species, gastrulation depends critically on mesoderm cell migration, in Xenopus, convergent extension of the axial mesoderm seems to provide the main driving force for gastrulation.

Fibronectin fibril growth in the extracellular matrix of the Xenopus embryo.

We show that the mechanism of fibronectin fibril formation on the blastocoel roof of the Xenopus embryo is comparable to that in other systems. Fibril assembly is inhibited by RGD peptide, by an amino-terminal fragment of fibronectin, and by cytochalasin B. When added exogenously, intact fibronectin, but not a 110 kDa cell binding fragment of fibronectin, is incorporated into fibrils. Thus, the blastocoel roof of Xenopus represents a valid model system for the study of fibronectin fibril formation in situ. Moreover, we show that fibril formation can be induced experimentally in this system. Examination of fibril elongation by double-labelling experiments reveals that individual, unbranched fibronectin fibrils grow only at one end, i.e. in a unipolar fashion. The rate of elongation is 4.7 microns/min. Most fibrils grow only for a short time, and the increase in total fibril length per cell is driven by the repeated initiation of new fibrils. Assembly of fibronectin into fibrils precedes cross-linking of fibronectin into multimers in this system.

Cell interaction and its role in mesoderm cell migration during Xenopus gastrulation.

In the Xenopus gastrula, the mesoderm moves as a coherent cell aggregate across the blastocoel roof toward the animal pole. We show that the cohesion of the mesoderm is not only mechanically necessary, but that aggregate formation has profound effects on the migratory behavior of mesoderm cells. Whereas isolated mesoderm cells are bi- or multipolar, move stepwise and change their direction of movement frequently, aggregated mesoderm cells migrating on their in vivo substrate appear unipolar and move continuously and persistently. Moreover, only mesoderm cell aggregates, but not single cells, can follow guidance cues present in the extracellular matrix of the blastocoel roof substrate. Thus, the cohesion of the mesodermal cell mass is an essential feature of mesoderm migration during Xenopus gastrulation. We show that the Ca(2+)-dependent cell adhesion molecule U-cadherin is involved in mediating this cohesion.

Motile behavior and protrusive activity of migratory mesoderm cells from the Xenopus gastrula.

During Xenopus gastrulation, the mesoderm migrates across a fibronectin (FN)-containing substrate, the inner surface of the blastocoel roof (BCR). A possible role for FN is to promote the extension of cytoplasmic processes which serve as locomotory organelles for mesoderm cells. To test this idea, the interaction of prospective head mesoderm (HM) cells with FN was examined in vitro. Nonattached HM cells extend filiform processes from an active region of the cell surface. This spontaneous activity is modulated by cell attachment to FN. Additional active regions appear, and cytoplasmic lamellae extend from these sites, leading to cell spreading and translocation. Thus, although FN seems not to induce processes de novo, it modulates a spontaneous protrusive activity to yield the extension of lamellae along the substrate surface. As putative locomotory organelles, HM cell protrusions were characterized functionally. They adhere rapidly and selectively to in situ substrates, preferentially to FN, and retract upon attachment. During translocation, the passive cell body is moved by the activity of the protrusions. Lamellae continuously extend, retract, or split into parts. This leads to an intermittent, nonpersistent mode of translocation. The polarity of HM cells, as expressed in the arrangement of protrusions, bears no constant relationship to the orientation of the cell body, and a cell can change its direction of movement without a corresponding rotation of the cell body. This may be relevant with respect to the mechanism by which mesoderm cells translate guidance cues of the BCR into a polarized, oriented cell structure during directional migration in situ.

Directional mesoderm cell migration in the Xenopus gastrula.

The movement of the dorsal mesoderm across the blastocoel roof of the Xenopus gastrula is examined. We show that different parts of the mesoderm which can be distinguished by their morphogenetic behavior in the embryo are all able to migrate independently on the inner surface of the blastocoel roof. The direction of mesoderm cell migration is determined by guidance cues in the extracellular matrix of the blastocoel roof and by an intrinsic tissue polarity of the mesoderm. The mesodermal polarity shows the same orientation as the external guidance cues and is strongly expressed in the more posterior mesoderm. The guidance cues of the extracellular matrix are recognized by all parts of the dorsal mesoderm and even by nonmesodermal cells from other regions of the embryo. The extracellular matrix consists of a network of fibronectin-containing fibrils. The adhesiveness of this matrix does not vary along the axis of mesoderm movement, excluding haptotaxis as a guidance mechanism in this system. However, an intact fibronectin fibril structure is necessary for directional mesoderm cell migration. When the assembly of fibronectin into fibrils is inhibited, mesoderm explants still migrate on the amorphous extracellular matrix, but no longer directionally. It is proposed that polarized extracellular matrix fibrils may normally guide the migrating mesoderm to its target region.

Mesodermal cell migration during Xenopus gastrulation.

The adhesive glycoprotein fibronectin (FN), which is a component of the network of extracellular matrix fibrils on the inner surface of the blastocoel roof (BCR), has been proposed to play a major role in directing mesodermal cell migration during amphibian gastrulation. In the first part of this paper, the adhesion of Xenopus mesodermal cells to FN in vitro is examined. Cells from several mesoderm regions, which differ in developmental fate and morphogenetic activity, are able to bind specifically to the RGD cell-binding site of FN. Dorsal mesodermal cell adhesion to FN varies along the anterior-posterior (a-p) axis: adhesion is strongest in the anterior head mesoderm, and gradually decreases posteriorly. This a-p gradient of mesodermal adhesiveness to FN does not change during mesodermal involution, and is reflected in the morphology of mesodermal explants on FN. An a-p strip of mesoderm develops a spreading, leading anterior margin and a compact, retracting posterior end, thus moving slowly and directionally over the FN substrate at about 0.8 micron/min. Although dissociated cells from all levels of the dorsal mesodermal axis adhere to FN, only the anterior, leading prospective head mesoderm cells migrate as single cells on a FN substrate in vitro. Locomotion by means of lamelliform protrusions occurs at an average rate of about 1.5 micron/min. Cells of the more posterior axial mesoderm merely shift position at random without substantial net translocation, and preinvolution mesoderm cells are completely stationary. On the BCR, the in vivo substrate for mesodermal cell migration, dissociated prospective head mesoderm cells spread and migrate as on FN in vitro, at 2.2 microns/min. In the presence of an RGD peptide which inhibits cell-FN interaction, cells remain globular and do not spread. They are still motile, but change direction frequently, which leads to less efficient net translocation. Apparently, interaction with the RGD cell-binding site of FN and concomitant spreading of head mesoderm cells is required for the stabilization of cell locomotion. In contrast to the directional migration of the mesoderm cell population toward the animal pole in the embryo, the pathways of dissociated cells on the BCR are randomly oriented. Coherent explants of migratory mesoderm do not move at all on the BCR, although they translocate on FN in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Development of the lateral line system in Xenopus.

The lateral line system of fishes and amphibians consists of numerous epidermal mechano-receptors which are distributed over the whole body surface. As in other amphibians, the lateral line system of Xenopus develops from epidermal placodes situated on the head region of the embryo. The dorsolateralis placodes form a rostro-caudal series of epidermal thickenings centered around the otic placode. In this series, placodes remaining within the epidermis and forming lateral line primordia alternate with lateral line ganglion forming placodes. Each lateral line primordium elongates and migrates within the epidermis along a well-defined pathway, leaving behind a row of small cell groups, the primary lateral line organs. As the ganglion which supplies a given row of organs and the corresponding lateral line primordium originate in spatial contiguity, and as the axons of the lateral line nerve grow out together with the migrating primordium, the lateral line neurones remain in contact with their target cells throughout development. After segregation of a primary organ from a migrating primordium, cell differentiation occurs. Receptor cells establish afferent and efferent synaptic contacts with axons from the lateral line nerve. Apically, a bundle of stereocilia and a single, microtubule-containing kinocilium protrude from the surface of a receptor cell into a jelly-like cupula, which extends into the surrounding fluid. Displacement of the cupula and the concomitant bending of the cilia stimulates the receptor cells. The cilia of a receptor cell are asymmetrically arranged, and this structural polarity is related to the directional sensitivity of the cells. Two types of receptor cells, with opposite orientations, are intermingled within each organ, giving the whole organ a bidirectional sensitivity. The number of lateral line organs is increased by the process of accessory organ formation, where primary organs grow and divide to produce secondary organs. In this way, existing rows of organs are extended. Moreover, single primary organs are transformed into elongate plaques of closely apposed organs. The lateral line system has reached its greatest extent at late larval stages. During metamorphosis, the number of organ plaques is reduced in some lines, and one line even disappears completely. Two large, myelinated afferent fibers innervate a whole organ plaque. They branch repeatedly to supply every organ of the plaque, and each fiber is thought to innervate only receptor cells of the same polarity.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential interaction of Xenopus embryonic cells with fibronectin in vitro.

Two distinct cell types from the amphibian gastrula were compared with regard to their interactions in vitro with fibronectin (FN). Xenopus embryonic endoderm cells attach to FN substrates in a way characteristic of most cell types studied so far; that is, adhesion increases abruptly at a certain threshold concentration of FN, and maximal binding of cells already occurs at low FN concentrations (10 micrograms/ml). In contrast, embryonic ectodermal cells bind maximally to FN substrates only at unusually high concentrations of FN (200 micrograms/ml). This peculiar mode of attachment to FN has been characterized more closely. It is shown that the adhesion of ectodermal cells is modified by their interaction with a heparin-binding domain of the FN molecule. Furthermore, ectodermal cell adhesion increases very slowly with increasing FN concentrations. Despite these characteristic differences, both ectodermal and endodermal cells attach to the normal RGD cell-binding site of FN, as can be shown by competitive inhibition of adhesion by a hexapeptide containing the RGD sequence of amino acids.

Cell proliferation in the ectoderm of the Xenopus embryo: development of substratum requirements for cytokinesis.

The requirements for cell division in ectodermal blastomeres of the early Xenopus embryo were studied. Isolated blastomeres divide autonomously on nonadhesive agar in a simple salt solution up to the midblastula stage. After the midblastula transition, cell-cell contact is required for blastomere division. In isolated blastomeres of that stage, cytokinesis fails, but nuclear division continues normally for some time. Cell-cell contact as a prerequisite for blastomere division can be replaced by culturing blastomeres on an appropriate substratum. Clonal growth of isolated blastomeres is supported by a variety of protein substrata, indicating rather unspecific substratum requirements. Different substrata which do not support blastomere division can affect different steps in cytokinesis.

Development of the lateral line system in Xenopus laevis. III. Development of the supraorbital system in triploid embryos and larvae.

During normal development of the supraorbital lateral line system of Xenopus, an elongated streak of primordial cells becomes subdivided into a linear series of cell groups containing only about eight cells each, thus forming a row of primary lateral line organs (Winklbauer & Hausen, 1983a,b). In triploid Xenopus embryos, cell size is 1.5 X normal. When the formation of lateral line organs occurs in triploid primordia, the nascent organs contain only about five or six cells each, i.e. about two thirds of normal. Thus, the increase in cell size is compensated for by a corresponding reduction in cell number, keeping constant the organ size in terms of total cell mass or volume. This result excludes a cell counting mechanism for determining organ size. In diploids, the primary organs, although being of equal size initially, differ vastly in their final size and exhibit a peculiar frequency distribution of organ sizes. A detailed quantitative model for supraorbital lateral line development has been proposed, which accounts for this characteristic frequency distribution (Winklbauer & Hausen, 1983b). This model makes precise predictions as to the frequency distribution of the final size of triploid lateral line organs, where the initial organ size is reduced to five or six cells. These predictions were verified experimentally.