Comparison of induction during development between Xenopus tropicalis and Xenopus laevis

Several in vitro systems exist for the induction of animal caps using growth factors such as activin. In this paper, we compared the competence of activin-treated animal cap cells dissected from the late blastulae of Xenopus tropicalis and Xenopus laevis. The resultant tissue explants from both species differentiated into mesodermal and endodermal tissues in a dose-dependent manner. In addition, RT-PCR analysis revealed that organizer and mesoderm markers were expressed in a similar temporal and dose-dependent manner in tissues from both organisms. These results indicate that animal cap cells from Xenopus tropicalis have the same competence in response to activin as those from Xenopus laevis.     

The Xenopus receptor tyrosine kinase Xror2 modulates morphogenetic movements of the axial mesoderm and neuroectoderm via Wnt signaling

The Spemann organizer plays a central role in neural induction, patterning of the neuroectoderm and mesoderm, and morphogenetic movements during early embryogenesis. By seeking genes whose expression is activated by the organizer-specific LIM homeobox gene Xlim-1 in Xenopus animal caps, we isolated the receptor tyrosine kinase Xror2. Xror2 is expressed initially in the dorsal marginal zone, then in the notochord and the neuroectoderm posterior to the midbrain-hindbrain boundary. mRNA injection experiments revealed that overexpression of Xror2 inhibits convergent extension of the dorsal mesoderm and neuroectoderm in whole embryos, as well as the elongation of animal caps treated with activin, whereas it does not appear to affect cell differentiation of neural tissue and notochord. Interestingly, mutant constructs in which the kinase domain was point-mutated or deleted (named Xror2-TM) also inhibited convergent extension, and did not counteract the wild-type, suggesting that the ectodomain of Xror2 per se has activities that may be modulated by the intracellular domain. In relation to Wnt signaling for planar cell polarity, we observed: (1) the Frizzled-like domain in the ectodomain is required for the activity of wild-type Xror2 and Xror2-TM; (2) co-expression of Xror2 with Xwnt11, Xfz7, or both, synergistically inhibits convergent extension in embryos; (3) inhibition of elongation by Xror2 in activin-treated animal caps is reversed by co-expression of a dominant negative form of Cdc42 that has been suggested to mediate the planar cell polarity pathway of Wnt; and (4) the ectodomain of Xror2 interacts with Xwnts in co-immunoprecipitation experiments. These results suggest that Xror2 cooperates with Wnts to regulate convergent extension of the axial mesoderm and neuroectoderm by modulating the planar cell polarity pathway of Wnt.     

Ventral ectoderm of Xenopus forms neural tissue, including hindbrain, in response to activin.

The peptide growth factor Activin A has been shown to induce complete axial structures in explanted blastula animal caps. However, it is not understood how much this response to activin depends upon early signals that prepattern the ectoderm. We have therefore asked what tissues can be induced in blastula animal caps by activin in the absence of early dorsal signals. Using whole-mount in situ hybridization, we compare the expression of three neural markers, N-CAM, En-2 and Krox-20 in activin-treated ectoderm from control and ventralized embryos. In response to activin, both normal and ventralized animal caps frequently form neural tissue (and express N-CAM) and express the hindbrain marker Krox-20. However, the more anterior marker, En-2, is expressed in only a small fraction of normal animal caps and rarely in ventralized animal caps; the frequency of expression does not increase with higher doses of activin. In all cases En-2 and Krox-20 are expressed in coherent patches or stripes in the induced caps. Although mesoderm is induced in both control and ventralized animal caps, notochord is found in response to activin at moderate frequency in control caps, but rarely in ventralized animal caps. These results support the idea that in the absence of other signals, activin treatment elicits hindbrain but not notochord or anterior neural tissue; and thus, the anterior and dorsal extent of tissues formed in response to activin depends on a prior prepatterning or previous inductions.     

Endoderm differentiation and inductive effect of activin-treated ectoderm in Xenopus

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk-rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin-treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.     

Induction of cells expressing vascular endothelium markers from undifferentiated Xenopus presumptive ectoderm by co-treatment with activin and angiopoietin-2

Activin is a potent inducer of mesoderm in amphibian embryos. We previously reported that low concentrations of activin could induce the formation of blood cells from Xenopus explants (animal caps). Both hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblasts. In this study, we tried to induce differentiation of vascular endothelial cells in aggregates derived from Xenopus animal caps. Aggregates formed from cells that were co-treated with activin and angiopoietin-2 expressed the vascular endothelial markers, X-msr, Xtie2 and Xegfl7. However, none of these aggregates expressed the hematopoietic marker genes, globin alpha T3, alpha T5, alpha A or GATA-1. We used microarray analysis to compare the gene expression profiles of aggregates treated with activin alone or with activin and angiopoietin. The combination, but not activin alone, induced expression of vascular-related genes such as Xl-fli and VEGF. These results demonstrate that treatment of dissociated animal cap cells with activin and angiopoietin-2 can induce differentiation of endothelial cells, and provides a promising model system for the in vitro study of blood vessel induction in vertebrates.     

In vitro control of organogenesis and body patterning by activin during early amphibian development

In the process of amphibian development, an embryonic body plan is established through cell division, sequential gene expression, morphogenesis and cell differentiation. The mechanism of body patterning is complex and includes multiple induction events. Activin, a TGF-beta family protein, can induce several kinds of mesodermal and endodermal tissues in animal cap explants in a dose-dependent manner. In a recent study of the role of activin in organogenesis, we succeeded in raising a beating heart by treating animal caps with a high concentration of activin. Activin also participates in kidney organogenesis in combination with retinoic acid. An embryonic kidney induced by activin and retinoic acid in vitro can function in vivo when it is transplanted into a larva in which pronephros rudiments have already been removed. Further, the activin-treated animal caps clearly show organizer actions that are closely related to body patterning along the anteroposterior axis. These experiments will help to serve as a model system for understanding organogenesis and body patterning at the cellular and molecular levels.     

Cortical Actin Filaments Potentially Interact with Cortical Microtubules in Regulating Polarity of Cell Expansion in Primary Roots of Maize (Zea mays L.)

Evidence is accumulating implicating cortical microtubules in the directional control of cell expansion. However, the role of actin filaments in this process is still uncertain. To determine the involvement of actin in cell elongation, the organization of actin filaments in primary roots of maize (Zea mays L.) was examined by use of an improved fluorochrome-conjugated phalloidin-labeling method. With this method, a previously undetected state of actin organization was revealed in the elongation and maturation zone of maize roots. Fine transversely oriented cortical actin was observed in all cells of the elongation zone, including the epidermis, cortex, and vascular tissues. The orientation of cortical actin shifted from a predominantly transverse orientation to oblique, longitudinal, and/or random arrangements as the cells matured. The reorientation of cortical actin in maturing root cells mimics the behavior of cortical microtubules reported in other studies. Furthermore, roots treated with the microtubule-stabilizing drug taxol improved the quality of actin preservation as evidenced by the thicker bundles of cortical actin. This suggested that taxol was also capable of stabilizing the cortical actin networks. The elongation of roots exposed to 1 micromole Latrunculin B, an actin-disrupting drug, was inhibited, and after 24 h the roots exhibited moderate swelling particularly along the elongation zone. Latrunculin B also caused microtubules to reorient from transverse to oblique arrays. The results from this study provide evidence that cortical microtubules and actin filaments respond in a coordinated way to environmental signals and may well depend on both elements of the cytoskeleton.     

A role for Xlim-1 in pronephros development in Xenopus laevis

Xlim-1, a LIM class homeobox gene expressed in Xenopus laevis, is one of the earliest known marker genes of pronephros development and is expressed in pronephros rudiment. In this study, we examined the role of Xlim-1 in pronephros development. Temporal expression of Xlim-1 in explants was analyzed in a series of induction assays using RT-PCR analysis. Xlim-1 was expressed 9 to 15 h after activin/retinoic acid treatment, corresponding to pronephros differentiation in explants. We further examined the role of Xlim-1 using a series of microinjection experiments. Presumptive pronephric anlagen of embryos were injected with various Xlim-1 mutants, and effects of these Xlim-1 mutants on pronephrogenesis in embryos and in explants were analyzed by RT-PCR and immunohistochemistry. Dominant-negative Xlim-1 inhibited differentiation of pronephros in activin/retinoic acid-treated animal caps. In embryos injected with a dominant-negative form of Xlim-1, development of pronephric tubules was inhibited at the late tail-bud stage. Our results suggest that Xlim-1 may not initiate differentiation of the pronephros, but that it is necessary for growth and elongation in the development of pronephric tubules.     

Activin-treated ectoderm has complete organizing center activity in Cynops embryos

The differentiation and organizer activity of newt ectoderm treated with activin A was studied in explantation and transplantation experiments. In the explantation experiments, ectoderm dissected from late morulae–early gastrulae stage embryos treated with a high concentration of activin A (100 ng/mL) formed only yolk-rich endodermal cells. Mesodermal tissues, such as notochord and muscle, were seldom found in these explants. When they were transplanted into the blastocoele of other early gastrulae, they formed part of the endoderm of the host embryo and induced a secondary axis with only posterior characters (including axial mesoderm and neural tissues). In contrast, whole secondary axes were induced when activin-treated ectoderm was transplanted into the ventral marginal zone (VMZ) of early blastulae. The transplanted pieces invaginated by themselves and differentiated into foregut structures including pharynx, stomach, and liver. These phenomena were also observed in experiments in which presumptive foregut was used instead of activin-treated ectoderm. These findings show that activin-treated ectoderm can act as the complete organizing center in Cynops .     

Blood cell induction in Xenopus animal cap explants: Effects of fibroblast growth factor, bone morphogenetic proteins, and activin

 Cultures of Xenopus blastula animal caps were used to explore the haematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expanded into egg-shaped structures, and beneath an outer layer of epidermis, a ventral mesodermal lining surrounded a fluid-filled cavity containing ”blood-like cells”. Immunocytochemistry identified some of these cells as early leukocytes, but erythrocytes were rare. BMP-2 or BMP-4 induced primitive erythrocytes as well as leukocytes, and a high concentration was required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induced ventral mesoderm concomitantly. High concentrations of activin A dorsalized explants, which contained infrequent leukocytes, and an optimal combination of activin A and bFGF caused differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increased the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contained a well organized cell mass in which yolk-rich cells mixed with blood cells and pigmented cells did not. BMP-2 plus bFGF also induced numerous leukocytes and fewer erythrocytes, but BMP-4 antagonized the leukopoietic effect of bFGF. The data suggest that the signalling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination. Received: 3 August 1998 / Accepted: 7 October 1998     

Head and trunk-tail organizing effects of the gastrula ectoderm of Cynops pyrrhogaster after treatment with activin A

Differentiation tendency and the inducing ability of the presumptive ectoderm of newt early gastrulae were examined after treatment with activin A at a high concentration (100 ng/ml). The activin-treated ectoderm differentiated preferentially into yolk-rich endodermal cells. Combination explants consisting of three pieces of activin-treated ectoderm formed neural tissues and axial mesoderm along with endodermal cells. However, the neural tissue was poorly organized and never showed any central nervous system characteristics. When the activin-treated ectoderm was sandwiched between two sheets of untreated ectoderm, the sandwich explants differentiated into trunk-tail or head structures depending on the duration of preculture of activin-treated ectoderm in Holtfreter's solution. Short-term (0–5 h) precultured ectoderm induced trunk-tail structures accompanied by axial organs, alimentary canal and beating heart. The arrangement of the explant tissues and organs was similar to that of normal embryos. However, archencephalic structures, such as forebrain and eye, were lacking or deficient. On the other hand, long-term (10–25 h) precultured ectoderm induced archencephalic structures in addition to axial organs. Lineage analysis of the sandwich explants using fluorescent dyes revealed that the activin-treated ectoderm mainly differentiated into endodermal cells and induced axial mesoderm and central nervous system in the untreated ectoderm. These results suggest that activin A is one of the substances involved in triggering endodermal differentiation and that the presumptive ectoderm induced to form endoderm displays trunk-tail organizer or head organizer effects, depending on the duration of preculture.     

Direct activation of phospholipase C-gamma by fibroblast growth factor receptor is not required for mesoderm induction in Xenopus animal caps.

Members of the fibroblast growth factor (FGF) family induce mesoderm formation in explants of Xenopus embryonic ectoderm (animal caps). Recent studies have been directed at determining signaling pathways downstream of the FGF receptor that are important in mesoderm induction. We have recently shown that a point mutation in the FGF receptor changing tyrosine 766 to phenylalanine (Y/F mutation) abolishes phospholipase C-gamma (PLC-gamma) activation in mammalian cells. To explore the role of PLC-gamma activation in FGF-stimulated mesoderm induction, we constructed two chimeric receptors, each consisting of the extracellular portion of the platelet-derived growth factor beta receptor, with one having the transmembrane and intracellular portions of the wild-type FGF receptor 1 (PR-FR wt) and the other having the corresponding region of the Y/F766 mutant FGF receptor 1 (PR-FR Y/F766). When expressed in Xenopus oocytes, only PR-FR wt was able to mediate PLC gamma phosphorylation, inositol-1,4,5-trisphosphate accumulation, and calcium efflux in response to platelet-derived growth factor stimulation. However, both receptors mediated mesoderm induction in Xenopus animal caps as measured by cap elongation, muscle-specific actin mRNA induction, and skeletal muscle formation. These results demonstrate that PLC gamma activation by the FGF receptor is not required for FGF-stimulated mesoderm induction.     

In Vitro Control of the Embryonic Form of Xenopus laevis by Activin A: Time and Dose-Dependent Inducing Properties of Activin-Treated Ectoderm

The inducing properties of activin-treated ectoderm of Xenopus laevis were examined by the preculture and sandwich culture methods. Presumptive ectodermal sheets of the late blastula were treated with 10–100 ng/ml of activin A and precultured for 0–7 hr in Steinberg's solution. They were then sandwiched between two sheets of ectoderm from other late blastulae. Ectoderm precultured for a short term induced trunk-tail structures, whereas that precultured for a long term induced head structures in addition to trunk-tail structures. These time-dependent changes in inducing properties occurred more rapidly when the concentration of activin A was higher. These results suggest that the activin-treated ectoderm functioned as a "head organizer" or "trunk-tail organizer" depending upon the concentration of activin A and the duration of preculture.
To trace the cell lineage of the sandwich explants, activin-treated ectoderm labeled with fluorescein-dextran-amine (FDA) was used in this study. The explants sandwiching the long term-precultured ectoderm formed head structures equipped with non-labeled neural tissues (brain and eye) as well as FDA-labeled mesodermal tissues. These results suggest that the activin-treated ectoderm mainly differentiates into mesodermal tissues and induces neural tissues as the organizer does in normal development.     

Regulation of Xenopus gastrulation by ErbB signaling

During Xenopus gastrulation, mesendodermal cells are internalized and display different movements. Head mesoderm migrates along the blastocoel roof, while trunk mesoderm undergoes convergent extension (C&E). Different signals are implicated in these processes. Our previous studies reveal that signals through ErbB receptor tyrosine kinases modulate Xenopus gastrulation, but the mechanisms employed are not understood. Here we report that ErbB signals control both C&E and head mesoderm migration. Inhibition of ErbB pathway blocks elongation of dorsal marginal zone explants and activin-treated animal caps without removing mesodermal gene expression. Bipolar cell shape and cell mixing in the dorsal region are impaired. Inhibition of ErbB signaling also interferes with migration of prechordal mesoderm on fibronectin. Cell-cell and cell-matrix interaction and cell spreading are reduced when ErbB signaling is blocked. Using antisense morpholino oligonucleotides, we show that ErbB4 is involved in Xenopus gastrulation morphogenesis, and it partially regulates cell movements through modulation of cell adhesion and membrane protrusions. Our results reveal for the first time that vertebrate ErbB signaling modulates gastrulation movements, thus providing a novel pathway, in addition to non-canonical Wnt and FGF signals, that controls gastrulation. We further demonstrate that regulation of cell adhesive properties and cell morphology may underlie the functions of ErbBs in gastrulation.     

A novel gene, BENI is required for the convergent extension during Xenopus laevis gastrulation

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF-beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENI mRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression.