Activation of muscle-specific actin genes in Xenopus development by an induction between animal and vegetal cells of a blastula

Muscle gene expression is induced a few hours after vegetal cells of a Xenopus blastula are placed in contact with animal cells that normally develop into epidermis and nerve cells. We have used a muscle-specific actin gene probe to determine the timing of gene activation in animal-vegetal conjugates. Muscle actin RNA is first transcribed in a minority of animal cells at a stage equivalent to late gastrula. The time of muscle gene activation is determined by the developmental stage of the responding (animal) cells, and not by the time when cells are first placed in contact. The minimal cell contact time required for induction is between 1 1/2 and 2 1/2 hr, and the minimal time for gene activation after induction is 5-7 hr.     

Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm

Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.     

Relationship of vegetal cortical dorsal factors in the Xenopus egg with the Wnt/beta-catenin signaling pathway

In Xenopus, the dorsal factor in the vegetal cortical cytoplasm (VCC) of the egg is responsible for axis formation of the embryo. Previous studies have shown that VCC dorsal factor has properties similar to activators of the Wnt/beta-catenin-signaling pathway. In this study, we examined the relationship of the VCC dorsal factor with components of the pathway. First, we tested whether beta-catenin protein, which is known to be localized on the dorsal side of early embryos, accounts for the dorsal axis activity of VCC. Reduction of beta-catenin mRNA and protein in oocytes did not diminish the activity of VCC to induce a secondary axis in recipient embryos. The amount of beta-catenin protein was not enriched in VCC compared to animal cortical cytoplasm, which has no dorsal axis activity. These results indicate that beta-catenin is unlikely to be the VCC dorsal axis factor. Secondly, we examined the effects of four Wnt-pathway-interfering constructs (dominant-negative Xdsh, XGSK3, Axin, and dominant-negative XTcf3) on the ability of VCC to induce expression of the early Wnt target genes, Siamois and Xnr3. The activity of VCC was inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. We also showed that VCC decreased neither the amount nor the activity of exogenous XGSK3, suggesting that the VCC dorsal factor does not act by affecting XGSK3 directly. Finally, we tested six Wnt-pathway activating constructs (Xwnt8, Xdsh, dominant negative XGSK3, dominant negative Axin, XAPC and beta-catenin) for their responses to the four Wnt-pathway-interfering constructs. We found that only XAPC exhibited the same responses as VCC; it was inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. Although the connection between XAPC and the VCC dorsal factor is not yet clear, the fact that APC binds Axin suggests that the VCC dorsal factor could act on Axin rather than XGSK3.     

Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus.

Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid (gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury (Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction.     

The role of the dorsal lip in the induction of heart mesoderm in Xenopus laevis

We have examined the tissue interactions responsible for the expression of heart-forming potency during gastrulation. By comparing the specification of different regions of the marginal zone, we show that heart-forming potency is expressed only in explants containing both the dorsal lip of the blastopore and deep mesoderm between 30 degrees and 45 degrees lateral to the dorsal midline. Embryos from which both of these 30 degrees-45 degrees dorsolateral regions have been removed undergo heart formation in two thirds of cases, as long as the dorsal lip is left intact. If the dorsal lip is removed along with the 30 degrees-45 degrees regions, heart formation does not occur. These results indicate that the dorsolateral deep mesoderm must interact with the dorsal lip in order to express heart-forming potency. Transplantation of the dorsal lip into the ventral marginal zone of host embryos results in the formation of a secondary axis; in over half of cases, this secondary axis includes a heart derived from the host mesoderm. These findings suggest that the establishment of heart mesoderm is initiated by a dorsalizing signal from the dorsal lip of the blastopore.     

Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.     

Hermes is a localized factor regulating cleavage of vegetal blastomeres in Xenopus laevis

We have identified the RNA-binding protein Hermes in a screen for vegetally localized RNAs in Xenopus oocytes. The RNA localizes to the vegetal cortex through both the message transport organizer (METRO) and late pathways. Hermes mRNA and protein are both detected at the vegetal cortex of the oocyte; however, the protein is degraded within a several hour period during oocyte maturation. Injection of antisense morpholino oligonucleotides (HE-MO) against Hermes caused a precocious reduction in Hermes protein present during maturation and resulted in a phenotype characterized by cleavage defects in vegetal blastomeres. The phenotype can be partially rescued by injecting Hermes mRNA. These results demonstrate that the localized RNA-binding protein Hermes functions during oocyte maturation to regulate the cleavage of specific vegetally derived cell lineages. Hermes most likely performs its function by regulating the translation or processing of one or more target RNAs. This is an important mechanism by which the embryo can generate unique cell lineages. The regulation of region-specific cell division is a novel function for a localized mRNA.     

Boundaries and functional domains in the animal/vegetal axis of Xenopus gastrula mesoderm

Patterning of the Xenopus gastrula marginal zone in the axis running equatorially from the Spemann organizer-the so--called "dorsal/ventral axis"--has been extensively studied. It is now evident that patterning in the animal/vegetal axis also needs to be taken into consideration. We have shown that an animal/vegetal pattern is apparent in the marginal zone by midgastrulation in the polarized expression domains of Xenopus brachyury (Xbra) and Xenopus nodal-related factor 2 (Xnr2). In this report, we have followed cells expressing Xbra in the presumptive trunk and tail at the gastrula stage, and find that they fate to presumptive somite, but not to ventrolateral mesoderm of the tailbud embryo. From this, we speculate that the boundary between the Xbra- and Xnr2-expressing cells at gastrula corresponds to a future tissue boundary. In further experiments, we show that the level of mitogen-activated protein kinase (MAPK) activation is polarized along the animal/vegetal axis, with the Xnr2-expressing cells in the vegetal marginal zone having no detectable activated MAPK. We show that inhibition of MAPK activation in Xenopus animal caps results in the conversion of Xnr2 from a dorsal mesoderm inducer to a ventral mesoderm inducer, supporting a role for Xnr2 in induction of ventral mesoderm.     

Minor-class splicing occurs in the nucleus of the Xenopus oocyte

A small fraction of premessenger RNA introns in certain eukaryotes is excised by the minor spliceosome, which contains low-abundance small nuclear ribonucleoproteins (snRNPs). Recently, it was suggested that minor-class snRNPs are localized to and function in the cytoplasm of vertebrate cells. To test whether U12-type splicing occurs in the cytoplasm of Xenopus oocytes, we performed microinjections of the well-characterized P120 minor-class splicing substrate into the nucleus or into the cytoplasm. Our results demonstrate that accurate splicing of this U12-dependent intron occurs exclusively in the nuclear compartment of the oocyte, where U12 and U6atac snRNPs are primarily localized. We further demonstrate that splicing of both a major-class and a minor-class intron is inhibited after nuclear envelope breakdown during meiosis.     

Delivery of germinal granules and localized RNAs via the messenger transport organizer pathway to the vegetal cortex of Xenopus oocytes occurs through directional expansion of the mitochondrial cloud

During Xenopus oogenesis, the message transport organizer (METRO) pathway delivers germinal granules and localized RNAs to the vegetal cortex of the oocyte via the mitochondrial cloud (Balbiani body). According to the traditional model, the mitochondrial cloud is thought to break up at the onset of vitellogenesis and the germinal granules and METRO-localized RNAs are transported within the mitochondrial cloud fragments to the vegetal cortex of the oocyte. We used light and electron microscopy in situ hybridization and three-dimensional reconstruction to show that germinal granules and METRO-localized RNAs are delivered to the oocyte cortex before the onset of mitochondrial cloud fragmentation and that the delivery involves accumulation of localized RNAs and aggregation of germinal granules at the vegetal tip of the mitochondrial cloud and subsequent internal expansion of the mitochondrial cloud between its animal (nuclear) and vegetal tips, which drives the germinal granules and METRO-localized RNAs toward the vegetal cortex. Thus the fragmentation of the cloud that occurs later in oogenesis is irrelevant to the movement of METRO-localized RNAs and germinal granules. On the basis of these findings, we propose here a revised model of germinal granule and localized RNAs delivery to the oocyte vegetal cortex via the METRO pathway.     

Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes

Localization of a specific subset of maternal mRNAs to the vegetal cortex of Xenopus oocytes is important for the regulation of germ layer formation and germ cell development. It is driven by vegetal localization complexes that are formed with the corresponding signal sequences in the untranslated regions of the mRNAs and with a number of different so-called localization proteins. In the context of the present study, we incorporated tagged variants of the known localization protein Vg1RBP into vegetal localization complexes by means of oocyte microinjection. Immunoprecipitation of the corresponding RNPs allowed for the identification of novel Vg1RBP-associated proteins, such as the embryonic poly(A) binding protein, the Y-box RNA-packaging protein 2B and the oocyte-specific version of the elongation factor 1α (42Sp50). Incorporation of 42Sp50 into localization RNPs could be confirmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50. Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNAse-sensitive complexes, as well as to associate specifically with several vegetally localizing mRNAs but not with nonlocalized control RNAs. Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between the nucleus and cytoplasm. Taken together, these observations provide evidence for a novel function of 42Sp50 in the context of vegetal mRNA transport in Xenopus oocytes.     

The specification of heart mesoderm occurs during gastrulation in Xenopus laevis

The establishment of heart mesoderm during Xenopus development has been examined using an assay for heart differentiation in explants and explant combinations in culture. Previous studies using urodele embryos have shown that the heart mesoderm is induced by the prospective pharyngeal endoderm during neurula and postneurula stages. In this study, we find that the specification of heart mesoderm must begin well before the end of gastrulation in Xenopus embryos. Explants of prospective heart mesoderm isolated from mid- or late neurula stages were capable of heart formation in nearly 100% of cases, indicating that the specification of heart mesoderm is complete by midneurula stages. Moreover, inclusion of pharyngeal endoderm had no statistically significant effect upon either the frequency of heart formation or the timing of the initiation of heartbeat in explants of prospective heart mesoderm isolated after the end of gastrulation. When the superficial pharyngeal endoderm was removed at the beginning of gastrulation, experimental embryos formed hearts, as did explants of prospective heart mesoderm from such embryos. These results indicate that the inductive interactions responsible for the establishment of heart mesoderm occur prior to the end of gastrulation and do not require the participation of the superficial pharyngeal endoderm.     

Involvement of cyclophilin D in mitochondrial permeability transition induction in intact cells

The mitochondrial permeability transition (MPT) is involved in both Ca²⁺ signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis-trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca²⁺ concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca²⁺ in intact cells.     

Erythropoietin protects red blood cells from TRAIL1-induced cell death during red blood cell transition in Xenopus laevis

In anuran amphibians, larval red blood cells (RBCs) are replaced by adult-type RBCs during metamorphosis. We previously showed that tumor necrosis factor-related apoptosis-inducing ligand 1 (TRAIL1) induces apoptosis in larval-, but not adult-type RBCs in Xenopus laevis. We also found that protein kinase C (PKC) activation is involved in establishing resistance to TRAIL1-induced apoptosis in adult-type RBCs. Here, we investigated whether erythropoietin (EPO), which induces PKC activation in mammalian erythroblasts, is involved in the RBC transition in X. laevis. RT-PCR analysis revealed that epo mRNA was upregulated in the lung, from the metamorphic climax (stage 60) onward. In an RBC culture system, EPO pretreatment significantly attenuated the TRAIL1-induced death of larval- and adult-type RBCs isolated from tadpoles and adults, probably due partly to PKC activation. In samples from froglets undergoing RBC transition, which included both larval- and adult-type RBCs, EPO exhibited a stronger protective effect on the adult-type than the larval-type RBCs. Newly differentiated RBCs isolated from tadpoles treated with a hemolytic reagent were more resistant to TRAIL1-induced cell death than non-treated controls. These results suggest that EPO functions to protect adult-type RBCs from TRAIL1-induced cell death during RBC transition, and that the protective effect might decrease as RBCs age.     

Ectopic induction of dorsal mesoderm by overexpression of Xwnt-8 elevates the neural competence of Xenopus ectoderm.

The ectoderm of early Xenopus gastrula is competent to become induced to neural tissue, but dorsal ectoderm is more neural competent than ventral ectoderm. It is a tenable, but as yet untested possibility that the higher neural competence of dorsal gastrula ectoderm is dependent on the presence of the dorsal mesoderm. To test this hypothesis we overexpressed Xwnt-8 in order to ectopically induce dorsal mesoderm in the ventral side of the embryo. We found that this elevated the level of neural competence of ventral ectoderm to that of dorsal ectoderm. The effect of Xwnt-8 on neural competence of ventral ectoderm was strictly correlated with its ability to enhance the amount of dorsal structures. The data indicate that the presence of dorsal mesoderm is a prerequisite for establishing the differences in neural competence between gastrula dorsal and ventral ectoderm.     

Regulation of the Dorsal Axial Structures in Cell-Deficient Embryos of Xenopus laevis

To study the regulation of the dorsal axial structures, we removed the right animal dorsal and the right vegetal dorsal cells from an 8-cell embryo of Xenopus laevis .
Most of the right dorsal cell-deficient embryos developed to normally proportioned tailbud embryos. No detectable delay was observed in their development. Examinations of serial sections revealed that they had restored bilateral symmetry. The cell numbers of the somite and the notochord had recovered to more than 90% and 70%, respectively, those of controls. Since the right dorsal cell-deficient embryo retained roughly three-quarters of the prospective region for the somites and half of that for the notochord, respectively, the cell number was more than that expected from the remaining prospective regions. Cell lineage analyses showed that progeny of the right ventral cells had formed almost all of the right dorsal axial structures, which are normally formed by the progeny of the right dorsal cells. However, almost all the notochord cells had been derived from the remaining left dorsal cells.
These results indicate that some quantitative aspects of regulation as expressed in terms of the cell number were different between the two tissues examined.