BMP antagonism by Spemann's organizer regulates rostral-caudal fate of mesoderm

Recent revisions to the Xenopus fate map challenge the interpretation of previous maps and current models of amphibian axial patterning (Lane, M.C., Smith, W.C., 1999. The origins of primitive blood in Xenopus: implications for axial patterning. Development 126 (3), 423-434.; Lane, M.C., Sheets, M.D., 2000. Designation of the anterior/posterior axis in pregastrula Xenopus laevis. Dev. Biol. 225, 37-58). We determined the rostralmost contributions to both dorsal and ventral mesoderm concomitantly from marginal zone progenitors in stage 6 embryos. Data reveal an unequivocal rostral-to-caudal progression of both dorsal and ventral mesoderm across the pre-gastrula axis historically called the dorsal-ventral axis, and a dorsal-to-ventral progression from animal-to-vegetal in the marginal zone. These findings support the proposed revisions to the fate and axis orientation maps. Most importantly, these results raise questions about the role of the organizer grafts and organizer-derived BMP antagonists in the "induction" of secondary axes. We re-examine both phenomena, and find that organizer grafts and BMP antagonists evoke caudal-to-rostral mesodermal fate transformations, and not ventral-to-dorsal transformations as currently believed. We demonstrate that BMP antagonism evokes a second axis because it stimulates precocious mediolateral intercalation of caudal, dorsal mesoderm. The implications of these findings for models of organizer function in vertebrate axial patterning are discussed.     

The anterior extent of dorsal development of the Xenopus embryonic axis depends on the quantity of organizer in the late blastula

In amphibian gastrulae, the cell population of the organizer region of the marginal zone (MZ) establishes morphogenesis and patterning within itself and within surrounding regions of the MZ, presumptive neurectoderm, and archenteron roof. We have tested the effects on pattern of reducing the amount of organizer region by recombining halves of Xenopus laevis late blastulae cut at different angles from the bilateral plane. When regions within 30 degrees of the dorsal midline are excluded from recombinants, ventralized embryos develop lacking the entire anterior-posterior sequence of dorsal structures, suggesting that the organizer is only 60 degrees wide (centered on the dorsal midline) at the late blastula stage. As more and more dorsal MZ (organizer) is included in the recombinant, progressively more anterior dorsal structures are formed. In all cases, when any dorsal structures are missing they are deleted serially from the anterior end. Thus, we suggest that the amount (lateral width) of the organizer in the MZ determines the anterior extent of dorsal development.     

Chordin is required for the Spemann organizer transplantation phenomenon in Xenopus embryos

We analyzed the Chordin requirement in Xenopus development. Targeting of both chordin Xenopus laevis pseudoalleles with morpholino antisense oligomers (Chd-MO) markedly decreased Chordin production. Embryos developed with moderately reduced dorsoanterior structures and expanded ventroposterior tissues, phenocopying the zebrafish chordino mutant. A strong requirement for Chordin in dorsal development was revealed by experimental manipulations. First, dorsalization by lithium chloride treatment was completely blocked by Chd-MO. Second, Chd-MO inhibited elongation and muscle differentiation in Activin-treated animal caps. Third, Chd-MO completely blocked the induction of the central nervous system (CNS), somites, and notochord by organizer tissue transplanted to the ventral side of host embryos. Unexpectedly, transplantations into the dorsal side revealed a cell-autonomous requirement of Chordin for neural plate differentiation.     

The epithelium of the dorsal marginal zone of Xenopus has organizer properties.

We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.     

Lithium induces dorsal-type migration of mesodermal cells in the entire marginal zone of urodele amphibian embryos

Summary The effect of lithium (Li⁺) on gastrulation movements was investigated during the development of the urodele amphibianPleurodeles waltl. Attention was focused on mesodermal cell migration. Under conditions of Li⁺ treatment providing a maximal enhancement of dorsoanterior structures, it was found that the dorsoventral polarity of gastrulation was abolished. In particular, vital staining and scanning electron microscopy observations on embryo fractures showed that mesodermal cells migrated radially after Li⁺ treatment, which led to the formation of rounded embryos. Epiboly movements thus were accelerated. Nevertheless, contrasting with the precocious disappearance of the early-formed yolk plug, archenteron invagination was constantly retarded and commenced with a delay of several hours as compared to control gastrulae. Cell-lineage analysis of the progenies from ventral or dorsal equatorial blastomeres of 32-cell-stage embryos provided evidence that both dorsal and ventral mesoderm contributed to notochordal tissue after Li⁺ treatment. Dorsalization of the entire marginal zone was confirmed by the ability of the entire mesoderm rudiment to behave as a dorsal organiser after Li⁺ treatment. Comparison of the migratory behaviour of isolated animal hemispheres from Li⁺-treated or control embryos cultured on fibronectin-coated substrate indicated that all marginal cells acquired the autonomous capacity for migration of dorsal marginal cells under the action of lithium.     

Hyperdorsoanterior embryos from Xenopus eggs treated with D2O

Excessively dorsalized embryos of Xenopus laevis develop from eggs treated with 30-70% D2O for a few minutes within the first third of the cell cycle following fertilization. As the concentration of D2O and the duration of exposure are increased, the anatomy of these embryos shifts in the direction of enlarged dorsal and anterior structures and reduced ventral and posterior ones. Twinning of dorsoanterior structures is frequent. Intermediate forms include embryos with large heads but no trunks or tails. The limit form of the series has cylindrical symmetry, with circumferential bands of eye pigment and cement gland, a core of notochord-like tissue, and a centrally located beating heart. D2O treatment seems to increase the egg's sensitivity to the dorsalizing effects of cortical rotation and to stimulate the egg to initiate two or more directions of rotation. Such eggs probably establish thereafter a widened and/or duplicated Nieuwkoop center in the vegetal hemisphere, with the subsequent induction of a widened and/or duplicated Spemann organizer region in the marginal zone, which leads to excessive dorsal development. The existence of these anatomical forms indicates the potential of the egg to undertake dorsal development at all positions of its circumference and suggests that normal patterning depends on the limited and localized activation or disinhibition of this widespread potential.     

Cell rearrangement during gastrulation of Xenopus: direct observation of cultured explants.

We have analyzed cell behavior in the organizer region of the Xenopus laevis gastrula by making high resolution time-lapse recordings of cultured explants. The dorsal marginal zone, comprising among other tissues prospective notochord and somitic mesoderm, was cut from early gastrulae and cultured in a way that permits high resolution microscopy of the deep mesodermal cells, whose organized intercalation produces the dramatic movements of convergent extension. At first, the explants extend without much convergence. This initial expansion results from rapid radial intercalation, or exchange of cells between layers. During the second half of gastrulation, the explants begin to converge strongly toward the midline while continuing to extend vigorously. This second phase of extension is driven by mediolateral cell intercalation, the rearrangement of cells within each layer to lengthen and narrow the array. Toward the end of gastrulation, fissures separate the central notochord from the somitic mesoderm on each side, and cells in both tissues elongate mediolaterally as they intercalate. A detailed analysis of the spatial and temporal pattern of these behaviors shows that both radial and mediolateral intercalation begin first in anterior tissue, demonstrating that the anterior-posterior timing gradient so evident in the mesoderm of the neurula is already forming in the gastrula. Finally, time-lapse recordings of intact embryos reveal that radial intercalation takes places primarily before involution, while mediolateral intercalation begins as the mesoderm goes around the lip. We discuss the significance of these findings to our understanding of both the mechanics of gastrulation and the patterning of the dorsal axis.     

Responses of embryonic Xenopus cells to activin and FGF are separated by multiple dose thresholds and correspond to distinct axes of the mesoderm.

The potent mesoderm-inducing factors activin and FGF are present as maternally synthesized proteins in embryos of X. laevis. We show that activin can act on explanted blastomeres to induce at least five different cell states ranging from posterolateral mesoderm to dorsoanterior organizer mesoderm. Each state is induced in a narrow dose range bounded by sharp thresholds. By contrast, FGF induces only posterolateral markers and does so over relatively broad dose ranges. FGF can modulate the actions of activin, potentiating them and broadening the threshold-bounded dose windows. Our results indicate that orthogonal gradients of activin and FGF would be sufficient to specify the main elements of the body plan.     

The marginal zone of the 32-cell amphibian embryo contains all the information required for chordamesoderm development.

The formation of the amphibian organizer is evidenced by the ability of cells of the dorsal marginal zone (DMZ) to self-differentiate to form notochord and to induce the formation of other axial structures from neighboring regions of the embryo. We have attempted to determine when these abilities are acquired in the urodele, Ambystoma mexicanum (axolotl), and in the anuran, Xenopus laevis, by removing the mesodermalizing influence of the vegetal hemisphere at different stages of development and culturing the animal hemisphere isolate. This was possible, even at the 32 and 64-cell stage, through the use of embryos with rare cleavage patterns. Cultured isolates were analyzed for morphological differentiation of mesodermal and neural structures, and for biochemical differentiation of the tissue-specific enzyme, acetylcholinesterase (AChE). Large amounts of mesodermal and neural structures, and normal expression of AChE were found in isolates made as early as the 32-cell stage in both species. Only a small increase in the percentage of isolates developing mesoderm was detected when isolations were made at later cleavage or blastula stages. The amount of mesoderm formed did not depend on the stage of isolation. Mesoderm differentiation was usually limited to the notocord and muscle. The isolates rarely formed pronephros, mesothelium, or mesenchyme, derivatives of ventral mesoderm, during normal development. The results indicate that the marginal zone of the cleavage-stage embryo contains all of the information needed for the formation of the organizer. The formation of dorsal mesoderm does not require subsequent interaction with the cells of the vegetal hemisphere, although the presence of those cells is likely to play a role in normal pattern formation.     

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center.

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.     

Gastrulation in Xenopus laevis: involution—a current view

In this article, we describe some of the morphogenetic movements reshaping the Xenopus laevis embryo during gastrulation. We have learned a great deal about these movements in recent years through advances made in explant culture techniques. Here, we will focus on involution, the process by which mesoderm is internalized and placed in between ectoderm and endoderm. Our aim is to present our current view of how involution takes place in the dorsal involuting marginal zone of the Xenopus embryos.     

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.     

Separation of an anterior inducing activity from development of dorsal axial mesoderm in large-headed frog embryos

The body of a vertebrate arises through a series of inductive interactions in the embryo. Macrocephaly is a distortion of the body in which a disproportionate amount of tissue is devoted to the head. This syndrome occurs in certain hybrids between frog species and appears to be due to an alteration of inductive relationships. Chimeric blastulae between normal and hybrid embryos developed macrocephaly when the marginal zone was derived from the hybrid. In these cases, a large cement gland, characteristic of the hybrid head, was induced to form from normal ectoderm. When hybrid zygotes were irradiated with ultraviolet (uv) light, all dorsoanterior structures, including notochord, somites, and central nervous system, were eliminated, but the most anterior-induced structure, the cement gland, remained. Embryos without dorsoanterior structures but with cement glands were also produced by injecting germinal vesicle extracts into the blastocoel of uv-irradiated nonhybrid embryos. These results demonstrate that an anterior inducing activity can be uncoupled from development of the neural tube and dorsal axial mesoderm.     

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.     

Developmental basis of pronephric defects in Xenopus body plan phenotypes.

We have used monoclonal antibodies that recognize the pronephric tubules or pronephric duct to explore the induction of the embryonic kidney in developing Xenopus embryos. Morphogenesis of the pronephros was examined in UV-ventralized and lithium-dorsalized embryos. We find that the pronephric tubules are present in all but the strongest UV-induced phenotypes, but absent from relatively moderate lithium phenotypes. Interestingly the pronephric duct, which develops from the ventroposterior portion of the pronephric anlage, is missing from more of the mild UV phenotypes than are pronephric tubules. The loss of the capacity to form pronephroi in UV-ventralized embryos is caused by the loss of tissues capable of inducing the pronephric mesoderm, as marginal zone explants from ventralized embryos are still competent to respond to pronephric-inductive signals. Explant recombination experiments indicate that the tissue responsible for both the loss of pronephroi in UV-ventralized embryos and the induction of pronephroi during normal development is the anterior somites. The absence of pronephroi in relatively mild lithium phenotypes has a developmental basis different from that of the UV phenotype, as explants from lithium-treated embryos are effective inducers of pronephroi in recombinants with competent mesoderm, even though they themselves do not form pronephroi in isolation. Together these data indicate that dorsal tissues, especially the anterior somites, are responsible for the establishment of the intermediate mesoderm and the induction of the embryonic kidneys and that even mild dorsalization destroys the capacity to form cells competent to receive this signal.     

Distribution of dorsal-forming activity in precleavage embryos of the Japanese newt, Cynops pyrrhogaster: effects of deletion of vegetal cytoplasm, UV irradiation, and lithium treatment

Two types of axis-deficient embryos developed after deletion of the vegetal cytoplasm: wasp-shaped embryos and permanent-blastula-type embryos. In situ hybridization revealed that neither type of axis-deficient embryo expressed goosecoid or pax-6. brachyury was expressed in the constricted waist region of the wasp-shaped embryos but was not expressed in the permanent-blastula-type embryos. Further, we examined the effect of UV irradiation on Japanese newt embryos. Surprisingly, UV-irradiated Japanese newt eggs formed hyperdorsalized embryos. These embryos gastrulated in an irregular circular fashion with goosecoid expression in the circular equatorial region. At tailbud stage, these embryos formed a proboscis which is very reminiscent of that formed in hyperdorsalized Xenopus embryos. Transplantation of the marginal region of the UV-irradiated embryos revealed that the entire marginal zone had organizer activity. Thus we conclude that UV hyperdorsalizes Japanese newt embryos. Finally, lithium treatment of normal embryos at the 32-cell stage also resulted in hyperdorsalization. Lithium treatment of vegetally deleted embryos had two distinct results. Lithium treatment of permanent-blastula-type embryos did not result in the formation of dorsal axial structures, while the same treatment reinduced gastrulation and dorsal axis formation in the wasp-shaped embryos. Based on these results, we propose a model for early axis specification in Japanese newt embryos. The model presented here is fundamentally identical to the Xenopus model, with some important modifications. The vegetally located determinants required for dorsal development (dorsal determinants, DDs) are distributed over a wider region at fertilization in Japanese newt embryos than in Xenopus embryos. The marginal region of the Japanese newt embryo at the beginning of development overlaps with the field of the DDs. Gastrulation is very likely to be a dorsal marginal-specific property, while self-constriction is most probably a ventral marginal-specific property in Japanese newt embryos.