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.     

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.     

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     

Xwnt-8 modifies the character of mesoderm induced by bFGF in isolated Xenopus ectoderm.

In Xenopus, growth factors of the TGF-beta, FGF and Wnt oncogene families have been proposed to play a role in generating embryonic pattern. In this paper we examine potential interactions between the bFGF and Xwnt-8 signaling pathways in the induction and dorsal-ventral patterning of mesoderm. Injection of Xwnt-8 mRNA into 2-cell Xenopus embryos does not induce mesoderm formation in animal cap ectoderm isolated from these embryos at the blastula stage, but alters the response of this tissue to mesoderm induction by bFGF. While animal cap explants isolated from non-injected embryos differentiate to form ventral types of mesoderm and muscle in response to bFGF, explants from Xwnt-8 injected embryos form dorsal mesodermal and neural tissues in response to the same concentration of bFGF, even if the ectoderm is isolated from the prospective ventral sides of embryos or from UV-ventralized animals. Our results support a model whereby dorso-ventral mesodermal patterning can be attained by a single mesoderm inducing agent, possibly bFGF, which is uniformly distributed across the prospective dorsal-ventral axis, and which acts in concert with a dorsally localized signal, possibly a Wnt protein, which either alters the response of ectoderm to induction or modifies the character of mesoderm after its induction.     

Cytoplasmic and cortical determinants interact to specify ectoderm and mesoderm in the leech embryo.

In leech embryos, segmental ectoderm and mesoderm are produced by a pair of sister cells located near the animal and vegetal poles, respectively. We have investigated the mechanism that localizes ectodermal and mesodermal fates along the animal-vegetal axis. The results of cleavage arrest and cell ablation experiments suggest that the full range of normal cell interactions are not required for this process. However, when the animal and vegetal hemispheres are separated by re-orientation of the first cleavage plane from meridional to equatorial, the ectodermal fate co-segregates with the animal hemisphere and the mesodermal fate with the vegetal hemisphere. Two pools of yolk-deficient cytoplasm, called teloplasm, are located at the animal and vegetal poles of the zygote, but separation of the animal and vegetal teloplasms is not sufficient for the segregation of ectodermal and mesodermal fates. Rather, complete segregation of fates requires an equatorial cleavage orientation that separates not only the two teloplasms, but also the animal and vegetal cortical regions. This, in conjunction with previous findings, indicates that ectodermal determinants are localized to the cell cortex in the animal hemisphere of the zygote. We propose that these determinants segregate to the ectodermal precursor and interact with factors in teloplasm to transform the fate of this cell from a mesodermal ground state to ectoderm.     

A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos

The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues.     

DVR-4 (bone morphogenetic protein-4) as a posterior-ventralizing factor in Xenopus mesoderm induction.

Establishment of mesodermal tissues in the amphibian body involves a series of inductive interactions probably elicited by a variety of peptide growth factors. Results reported here suggest that mesodermal patterning involves an array of signalling molecules including DVR-4, a TGF-beta-like molecule. We show that ectopic expression of DVR-4 causes embryos to develop with an overall posterior and/or ventral character, and that DVR-4 induces ventral types of mesoderm in animal cap explants. Moreover, DVR-4 overrides the dorsalizing effects of activin. DVR-4 is therefore the first molecule reported both to induce posteroventral mesoderm and to counteract dorsalizing signals such as activin. Possible interactions between these molecules resulting in establishment of the embryonic body plan are discussed.     

Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos

In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.     

Ectodermal control of the avascular zone of the peripheral mesoderm in the chick embryo

Prospective skin ectoderm is underlaid by a relatively thick (100 +/- 20 micrometer) avascular zone of mesoderm in most regions of the early embryo. To determine whether or not the ectoderm exercises a role in the establishment and maintenance of the avascular zone, trypsin-isolated pieces of backskin ectoderm from chick or quail embryos were implanted as a sheet into a slit cut deep into the capillary bed of the wing bud of host chick embryos of stages 19-23. In sham operations, slits were cut at various anteroposterior levels, and the wing was allowed to heal. At intervals of 3-48 hr after these operations, embryos were injected with India ink, fixed, and cleared. Implants formed flattened vesicles, usually in continuity with host ectoderm, but sometimes completely internalized. Periderm cells from each side of the vesicle faced each other, and the cells of the cuboidal layer faced an avascular mesodermal layer at least 100 micrometer thick at all points. The implantation of prospective skin ectoderm resulted in the formation of an avascular zone in normally vascularized mesoderm of the wing bud. In contrast, the vascular bed of the limb bud abutted directly on implants of Millipore filters or of Silastic silicone (Dow Corning). Likewise, the capillary bed came in direct contact with implants of retinal pigment epithelium, an ectodermal derivative normally in close contact with the vascular choroid coat of the eye. These results, taken in conjunction with earlier experiments that show the necessity of the apical ectodermal ridge for the formation of the marginal veins of the limb bud, suggest that epithelial-mesenchymal interactions are involved in important aspects of vasculogenesis in early embryos.     

Abnormal anteroposterior and dorsoventral patterning of the limb bud in the absence of retinoids.

We describe here how the early limb bud of the quail embryo develops in the absence of retinoids, including retinoic acid. Retinoid-deficient embryos develop to about stage 20/21, thus allowing patterns of early gene activity in the limb bud to be readily examined. Genes representing different aspects of limb polarity were analysed. Concerning the anteroposterior axis, Hoxb-8 was up-regulated and its border was shifted anteriorly whereas shh and the mesodermal expression of bmp-2 were down-regulated in the absence of retinoids. Concerning the apical ectodermal genes, fgf-4 was down-regulated whereas fgf-8 and the ectodermal domain of bmp-2 were unaffected. Genes involved in dorsoventral polarity were all disrupted. Wnt-7a, normally confined to the dorsal ectoderm, was ectopically expressed in the ventral ectoderm and the corresponding dorsal mesodermal gene Lmx-1 spread into the ventral mesoderm. En-1 was partially or completely absent from the ventral ectoderm. These dorsoventral patterns of expression resemble those seen in En-1 knockout mouse limb buds. Overall, the patterns of gene expression are also similar to the Japanese limbless mutant. These experiments demonstrate that the retinoid-deficient embryo is a valuable tool for dissecting pathways of gene activity in the limb bud and reveal for the first time a role for retinoic acid in the organisation of the dorsoventral axis.     

Mesoderm induction in the future tail region of Xenopus

Summary We have used interspecific grafts between Xenopus borealis and Xenopus laevis to study the signalling system that produces tail mesoderm. Early gastrula ectoderm grafted into the posterior neural plate region of neurulae responds to a mesodermal inducing signal in this region and forms mainly tail somites; this signal persists until at least the early tail bud stage. Ventral ectoderm grafted into the posterior neural plate loses its competence to respond to this signal after stage 10 1/2. We have established the specification of anterior and posterior neural plate ectoderm. In ectodermal sandwiches or when grafted into unusual positions, anterior regions gave rise to mainly nervous system and posterior regions to large amounts of muscle, together with some nervous system. Thus it was impossible to assess the competence of posterior neural plate ectoderm to form further mesoderm and hence to establish if mesodermal induction continues during neurulation in unmanipulated embryos.     

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.     

Scanning Electron Microscopy (SEM) of the Chick Embryo Primitive Streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2–5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the primitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arranged cells with long, thin processes.
Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.