Tension-dependent collective cell movements in the early gastrula ectoderm of Xenopus laevis embryos

Ventral ectodermal explants taken from early gastrula embryos of Xenopus laevis were artificially stretched either by two opposite concentrated forces or by a distributed force applied to the internal explant’s layer. These modes of stretching reflect different mechanical situations taking place in the normal development. Two main types of kinematic response to the applied tensions were detected. First, by 15 min after the onset of concentrated stretching a substantial proportion of the explant’s cells exhibited a concerted movement towards the closest point of the applied stretching force. We define this movement as tensotaxis. Later, under both concentrated and distributed stretching, most of the cell’s trajectories became reoriented perpendicular to the stretching force, and the cells started to intercalate between each other, both horizontally and vertically. This was accompanied by extensive elongation of the outer ectodermal cells and reconstruction of cell-cell contacts. The intercalation movements led first to a considerable reduction in the stretch-induced tensions and then to the formation of peculiar bipolar ”embryoid” shapes. The type and intensity of the morphomechanical responses did not depend upon the orientation of a stretching force in relation to the embryonic axes. We discuss the interactions of the passive and active components in tension-dependent cell movements and their relations to normal morphogenetic events. Received: 26 April 1999 / Accepted: 30 August 1999     

Calcium signalling during neural induction in Xenopus laevis embryos

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.     

Differential induction of regulatory genes during mesoderm formation in Xenopus laevis embryos

Mesoderm development in Xenopus laevis depends on inductive cell interactions mediated by diffusible molecules. The mesoderm inducer activin is capable of redirecting the development of animal explants both morphologically and biochemically. We have studied the induction of four regulatory genes, Mix. 1, goosecoid (gsc), Xlim-1 and Xbra in such explants by activin, and the influence of other factors on this induction. Activin induction of gsc is strongly enhanced by dorsalization of the embryo by LiCl, while expression of the other genes is only slightly enhanced. The protein synthesis inhibitor cycloheximide (CHX) inhibits the activin-dependent induction of Xbra partially, while induction of Mix. 1 and Xlim- 1 is essentially unaffected. In contrast, gsc shows strong superinduction in the presence of activin and CHX, and can be induced in animal explants by CHX alone. Induction and superinduction by CHX have previously been observed for immediate early genes in a variety of systems, notably for the activation of c-fos expression by serum stimulation, but have not been reported in early amphibian embryos. © 1993Wiley-Liss, Inc.     

Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis

Ectodermal placodes, from which many cranial sense organs and ganglia develop, arise from a common placodal primordium defined by Six1 expression. Here, we analyse placodal Six1 induction in Xenopus using microinjections and tissue grafts. We show that placodal Six1 induction occurs during neural plate and neural fold stages. Grafts of anterior neural plate but not grafts of cranial dorsolateral endomesoderm induce Six1 ectopically in belly ectoderm, suggesting that only the neural plate is sufficient for inducing Six1 in ectoderm. However, extirpation of either anterior neural plate or of cranial dorsolateral endomesoderm abolishes placodal Six1 expression indicating that both tissues are required for its induction. Elevating BMP-levels blocks placodal Six1 induction, whereas ectopic sources of BMP inhibitors expand placodal Six1 expression without inducing Six1 ectopically. This suggests that BMP inhibition is necessary but needs to cooperate with additional factors for Six1 induction. We show that FGF8, which is expressed in the anterior neural plate, can strongly induce ectopic Six1 in ventral ectoderm when combined with BMP inhibitors. In contrast, FGF8 knockdown abolishes placodal Six1 expression. This suggests that FGF8 is necessary and together with BMP inhibitors sufficient to induce placodal Six1 expression in cranial ectoderm, implicating FGF8 as a central component in generic placode induction.     

Neural inducing factors from Xenopus laevis eggs and embryos

Large foreheads can be induced by ribonucleoprotein particles from Xenopus laevis eggs and embryos. The host embryos develop only a rudimentary primary axis. A neural inducing factor from the cytosol of gastrula-neurula stages has been partially purified. The factors are associated with other proteins in larger complexes.     

The induction of pyruvate kinase synthesis by heat shock in Xenopus laevis embryos

Heat-shocked Xenopus embryos have an unusually complex heat shock response. The dominant heat shock protein (Hsp) has a relative molecular mass (M_r) of 62,000 D (Hsp62). Affinity-purified IgGs against the glycolytic enzyme pyruvate kinase (PK; EC 2.7.1.40) specifically immunoprecipitated Hsp62 from extracts of embryos that had been heat-shocked at 37°C for 30 min. Thus, Hsp62 and pyruvate kinase are immunologically cross-reacting. Electrophoretic separation of PK isoforms suggests that heat-shocked Xenopus embryos increase synthesis of an isoform of PK. Thermal denaturation studies suggest that this isoform has enhanced thermal stability. The identification of PK as an Hsp is discussed within the context of a physiological requirement for elevated levels of anaerobic glycolysis in heatstressed cells as a vital component of the acquisition of thermotolerance. © 1993Wiley-Liss, Inc.     

Effects of retinoic acid on the endoderm in Xenopus embryos

 When Xenopus embryos from mid-tailbud to early tadpole stages were exposed to retinoic acid (RA), the gut developed with an uncoiled, straight intestinal tube, morphogenesis of the liver and stomach was affected and intestinal epithelial cells developed without a brush border and alkaline phosphatase activity. However, the temporal and spatial expression pattern of XlHbox 8, the only homeobox gene expressed in the endoderm was unaffected. In lateral plate mesodermal cells the expression of α-smooth muscle (SM) actin was delayed. A similar syndrome has been reported in a study of embryos lacking functional FGF receptors in which it was proposed that the uncoiled intestinal tube and the delayed differentiation of the intestinal muscle cells are causally related. Our results support this proposition and further suggest that mesenchymal-epithelial interactions concerned with regional specification of the endoderm may be impaired resulting in other defects in the gut. Received: 3 October 1997 / Accepted: 3 February 1998     

Dorsal induction from dorsal vegetal cells in Xenopus occurs after mid-blastula transition

In a screening for activin-responsive genes, we isolated a Xenopus lefty/antivin-related gene, called Xantivin (Xatv). In the animal cap assay, the expression of Xatv was induced by activin signaling, and in the embryo, by nodal-related genes. Overexpression of Xatv in the marginal zone caused suppression of mesoderm formation and gastrulation defects, and inhibited the secondary axis formation induced by Xnr1 and Xactivin, suggesting that Xatv acted as a feedback inhibitor of activin signaling. However, in the animal cap, Xatv failed to antagonize Xnr1 and Xactivin. This result suggested that Xatv has different responses in the marginal zone and in the animal region, and antagonizes to a higher degree activin signaling in the marginal zone.     

Pyruvate kinase isozymes in oocytes and embryos from the frog Xenopus laevis

1. The kinetic characteristics of pyruvate kinase isozymes from oocytes, embryos, liver and skeletal muscle from the clawed frog Xenopus laevis were measured in cell extracts. 2. The muscle and liver isozymes display Michaelis-Menten kinetics with Kms for phosphoenolpyruvate (PEP) of 0.02 and 0.05 mM, respectively. 3. Pyruvate kinase from oocytes and embryos displays cooperative kinetics for PEP with a Km of about 0.15 mM; the kinetics become hyperbolic and the Km for PEP is reduced to 0.05 mM in the presence of microM concentrations of fructose-1,6-bisphosphate. 4. These data serve to characterize pyruvate kinase activity in oocytes and embryos and the kinetics are compared to mammalian pyruvate kinase isozymes.     

Vegetal pole cells and commitment to form endoderm in Xenopus laevis

In order to compare their states of commitment with their normal developmental fate, single vegetal pole cells from early Xenopus embryos were labeled and transplanted into the blastocoels of host embryos. In a previous study we showed, using this single cell transplantation assay, that vegetal pole cells become committed to endoderm by the early gastrula stage. In this paper we examine some properties of the commitment process. First, we show that it is gradual. When vegetal blastomeres are taken from progressively older embryos an increasing number of them enter only the endoderm, until by the early gastrula stage they all do. Second, we show that commitment can continue in vitro when an appropriate tissue mass is present. We suggest that commitment to form endoderm may be, in the right conditions, a cell autonomous process.     

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.     

Neural-inducing activity of nuclei and nuclear fractions from Xenopus laevis embryos

Summary From embryos (Xenopus laevis) of different developmental stages nuclei were isolated which exert neural inducing activity in the biological test. The active material could partly be extracted from the nuclei. Experiments for the isolation of nuclear ribonucleoprotein (RNP) particles have shown that the activity is localized at least in part in these particles. On the other hand, some neural inducer is not detached from chromatin and the nuclear matrix even with ionic detergents. Inducing activity was found in germinal vesicles and to a higher degree in the cytoplasm of oocytes, but in a masked, biologically inactive state.     

Tubulin:tyrosine ligase in oocytes and embryos of Xenopus laevis

Tubulin:tyrosine ligase (TTL), which catalyzes the post-translational addition of tyrosine to the α chain of tubulin, exists in a wide variety of embryonic and adult vertebrate tissues. In the present study, we report that TTL exists in amphibian oocytes at a time when tubulin is a poor substrate for tyrosination, and when, in immature oocytes, tubulin is not polymerizable. Ligase activity detected at several stages of oogenesis and embryogenesis in Xenopus is compatible with mammalian brain tubulin in the tyrosination reaction. Within 3–5 hr after fertilization, [³H] tyrosine incorporated/μg endogenous tubulin increases approximately 3.5-fold over that in extracts prepared from the largest oocytes obtained. This increase cannot be accounted for by increasing levels of TTL. Ligase activity remains fairly constant throughout oogenesis and early embryogensis and rises significantly (2-fold) only 35–50 hr after fertilization. The late rise in embryonic ligase activity is not accompanied by a change in apparent k_m for tubulin.     

Translational control of activin in Xenopus laevis embryos

Activin is a potent mesoderm inducing factor present in embryos of Xenopus laevis. Recent evidence has implicated activin in the inhibition of neural development in addition to the well-established induction of mesoderm in ectodermal explants. These diverse effects are critically dependent on the concentration of activin yet little is known about the mechanisms regulating the level of activin in the embryo. We report that the 3′ untranslated region (3′ UTR) of activin βB mRNA inhibits the translation of activin in embryos. Microinjection of activin mRNA from which the 3′ UTR has been deleted is 8–10-fold more potent in inducing mesoderm than mRNA containing the 3′ UTR. Truncation of the 3′ UTR also leads to a marked enhancement of activin protein levels in embryos but has no effect when the truncated mRNA is translated in vitro. The 3′ UTR also confers translational inhibition on a heterologous mRNA. These data show that a maternal factor(s) present in X. laevis regulates the translation of injected activin βB mRNA. This factor(s) could be responsible for regulating the levels of endogenous activin βB protein during mesoderm induction and the specification of ectodermal derivatives such as neural and epidermal tissues. © 1995 Wiley-Liss, Inc.