The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos

Suramin, a polyanionic compound, which is thought to inhibit the binding of growth factors to their receptors, prevents the differentiation of the dorsal blastopore lip of early gastrulae into dorsal mesodermal structures as notochord and somites. Suramin treated blastopore lips form ventral mesodermal structures, mainly heart structures. Several cases showed rythmic contractions ("beating hearts"). Of special interest is the fact that blastopore lips isolated from middle gastrulae followed by suramin treatment differentiate in about 50% of the cases brain structures without the presence of notochord. These data suggest that suramin prevents the differentiation of the dorsal blastopore lip into notochord up to the early middle gastrula stage but no longer the formation of head mesoderm, which is the prequisite for the induction of archencephalic brain structures. Treated chordamesoderm with overlaying ectoderm from late gastrulae will differentiate as untreated controls, namely into dorsal axial structures like notochord, somites and brain structures. The results indicate that primarily a more general or ventral mesodermal signal is transferred from the dorsal vegetal blastomeres (Nieuwkoop center) to the dorsal marginal zone. The dorsalization, which enables the blastopore lip to differentiate into head mesoderm and notochord and in turn to acquire neuralizing activity, takes place during the early steps of gastrulation.     

Bottle cell formation in relation to mesodermal patterning in the Xenopus embryo

The appearance of bottle cells at the dorsal vegetal/marginal boundary of Xenopus embryos marks the onset of blastopore formation. The conditions leading to this epithelial activity were investigated by inducing bottle cells ectopically in the animal region with VegT or different members of the transforming growth factor (TGF)-beta family. Morphological studies on the ectopic bottle cells indicate their close similarity to the endogenous bottle cells at the dorsal blastopore lip. The subepithelial cells of the induced animal region express mesodermal genes in a pattern reminiscent to that observed on the dorsal lip. Relating this expression pattern to the position of the ectopic bottle cells leads to the conclusion that bottle cells form in regions of high TGF-beta signalling. The specific inhibitory effects of cerberus on ectopically induced bottle cells revealed that nodal related growth factors are the intrinsic signals that elicit bottle cell formation in the normal embryo. In addition, fibroblast growth factor signalling is an essential precondition for this epithelial response as it is for mesoderm formation. We conclude that bottle cell formation in the epithelial layer of the gastrula is closely linked to mesodermal patterning in the subepithelial tissues.     

Experimental reversal of the normal dorsal-ventral timing of blastopore formation does not reverse axis polarity in Xenopus laevis embryos

During gastrulation in Xenopus laevis, the dorsal lip of the blastopore normally appears before the ventral lip. Metabolic gradient models propose that the dorsal lip develops from the region of highest metabolic activity and somehow dominates other regions to prevent them from becoming dorsal. To test these ideas, I applied a temperature gradient of 12 degrees C across the embryo. Localized heating of the prospective ventral vegetal region from early in the first cleavage period until gastrulation causes the blastopore lip to form first by 2 hr at the prospective ventral meridian rather than at the prospective dorsal meridian. Despite this reversal of the timing of blastopore formation, gastrulation is completed, and the neural plate forms at its usual position on the prospective dorsal meridian. This demonstrates that the earliest gastrulating regions of the blastopore do not necessarily become dorsal, nor do they inhibit dorsal development by other regions. It is unlikely that axis polarity is based on regional differences in energy metabolism.     

Amphibian embryos as a model system for organ engineering: in vitro induction and rescue of the heart anlage.

Beating hearts can be induced under in vitro conditions when the dorsal blastopore lip (including the zone of Spemann organizer) is treated with Suramin. In contrast, untreated organizer forms dorsal mesodermal derivatives as notochord and somites. When those in vitro produced heart precursor tissues are transplanted ectopically in the posterior trunk area of early larvae, secondary beating heart structures will be formed. Furthermore, the replacement of the heart primordium of the host embryo by heart tissue induced under in vitro conditions will result in the rescue of the heart anlage. This model could be a valuable tool for the study of the multi-step molecular mechanisms of heart structure induction under in vitro conditions and vasculogenesis after transplantation into the host embryo.     

Effects of Inducers on Inner and Outer Gastrula Ectoderm Layers of Xenopus laevis

Abstract. Gastrula ectoderm, isolated from Xenopus laevis , was cultured in Holtfreter solution or modified Leibovitz medium (L-15) by the sandwich-method with or without inducer. The ectoderm (SD cell layers) consists of two cell sheets, representing a superficial (S) and a deep (D) layer. In the L-15 medium rather than in Holtfreter solution, the two cell layers separate out into distinct cell masses. This difference in cell affinity under certain experimental conditions could indicate that the deep layer contains endodermal cells. However, an endodermal character of the deep layer can be ruled out by induction experiments with vegetalizing factor or dorsal blastopore lip as inducers. Under the influence of vegetalizing factor the outer as well as the inner ectoderm layer differentiated into mesodermal derivatives such as notochord and somites. The results of the experiments with dorsal blastopore lip as inducer indicate that both inner and outer ectoderm layers are responsive to the neural stimulus. The lower neural competence of the outer ectoderm layer observed by several authors in normogenesis is discussed with regard to the hypothesis about short distance diffusion of the neuralizing factor and/or close cell-to-cell contact between inducing tissue and ectodermal target cells.     

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.     

Ca2+ as a messenger of dorsal--ventral polarity formation in frog (Rana temporaria) eggs.

Three methods of Ca2+ administration were used to influence the location of the grey crescent and the dorsal lip of blastopore in R temporaria eggs, ie a Ca2+ microinjection into the subcortical cytoplasm, egg pricking in high Ca2+ solutions and Ca2+ ionophore A23187 microinjection and application. The treatments all induced grey crescent and dorsal lip of blastopore formation near the Ca2+ administration site. Inositol trisphosphate injections gave similar results. Colchicine injections into the eggs inhibited the appearance of both natural and Ca(2+)-induced grey crescents.     

Microfolds in the suprablastoporal zone of the frog gastrula and their relationship to the geometry and mechanics of gastrulation

Spatial distribution and orientation of microfolds arising during invagination of the outer layer of suprablastoporal zone into the blastopore dorsal lip and changes of the lip shape were studied in Rana ridibunda embryos using statistical analysis of a normal individual variability. Active invagination of the cells into the lip correlated with deviation of the orientation of microfolds from the normal in the points of their intersection with the zone of dorsal lip inflection and their orientation is normalized upon transition of the cells across the inflection zone. Frequency distribution of the angle of microfold deviation from the normal is close to the exponential and, therefore, the angle of deviation is an analog of the potential energy of cells-components of the microfold: the bigger the deviation angle, the higher the potential energy. The minimum potential energy is observed at the normal orientation of microfolds, i.e., when it coincides with the radius of the dorsal lip curvature at the point of intersection with the microfold. The following mechanism of dorsal lip formation has been proposed: equatorial contraction of cells upon their invagination into the dorsal lip causes deviation of cell flux orientation from the normal orientation and the normal orientation is restored through an increase in the local curvature of dorsal lip. When the orientation of cell fluxes is normalized, invagination of cells in the dorsal lip ceases. The wave of normalization overtakes the wave of cell invagination into the dorsal lip at the lip angle length 120 degrees. At this moment, the archenteron roof is mechanically detached from the superficial cells of the suprablastoporal zone and lateral blastopore lips and this determines separation of the presumptive notochord.     

Furrowing surface contraction wave coincident with primary neural induction in amphibian embryos

We predicted, and have now observed, a surface contraction wave in axolotl (Ambystoma mexicanum) embryos that appears to coincide temporally and spatially with primary neural induction and homoiogenetic induction, and with involution of the chordomesoderm. The wave starts from a focus anterior to the dorsal lip of the blastopore and spreads as an ellipse, until part of it encounters the rim of the blastopore and vanishes there. The remaining are then continues over the dorsal hemisphere until it reforms an ellipse that decreases in size. About 9 to 12 hours after it begins, the wave vanishes at a focus diametrically opposite its point of origin. The wave involves both local contraction and furrowing in the monolayer ectoderm. To a good approximation, the hemispherical portion of the ectoderm traversed by the wave becomes neuroepithelium, while the ectoderm not transversed by the wave becomes epidermis. The wave might provide a mechanism to determine the time and location at which neuroepithelial differentiation occurs. © 1994 Wiley-Liss, Inc.     

Signals from the dorsal blastopore lip region during gastrulation bias the ectoderm toward a nonepidermal pathway of differentiation in Xenopus laevis

Epi 1, a monoclonal antibody, was generated against an epidermal specific epithelial antigen; it does not stain neural epithelium. We have used Epi 1 as a marker to determine when the spatial patterns delineating neural from nonneural epithelium become established. We used ventral ectoderm in a sandwich assay to show that signals from the central blastopore lip region, passing through the plane of the ectoderm sheet, define the pattern and boundary characteristics of Epi 1 expression. The dorsal blastopore lip at stages 10 and 12 are the strongest in inhibiting Epi 1 expression. The involuted chordamesoderm has only a limited inhibitory effect on Epi 1 expression in ventral ectoderm recombinates and does not appear to establish pattern boundaries. We suggest that the blastopore lip region establishes a preneural bias in the adjacent ectoderm prior to the interaction of the latter with chordamesoderm.     

Spemann's organizer--it's origin and derivatives (cellular-tissue and molecular-genetic aspects)

In 1924 H. Spemann and H. Mangold discovered that a piece of the dorsal lip of a blastopore from Triturus cristatus, after transplantation to the ventral side of another embryo, was able to cause the neighbouring tissues to change their fate and participate in the formation of a new embryo. The dorsal lip was termed "the organizer". Since then, for as long as 75 years, attempts have been made to establish the intimate mechanisms of the organizer activity. However, no real advance was achieved in their understanding. Within the last 15 years, genetic and molecular techniques have been vastly improved, to help in tracing the fate of many cell lineages, and in compiling more exactly the fate maps for different parts of the embryo. Using these data, I have attempted to trace the fate of Spemann's organizer after the early gastrula stage. Analysis of data on inductive abilities of the organizer cells, on the use of markers, and on the observation of expression of specific genes allowed to conclude that Spemann's organizer in amphibia and its homologues in other vertebrates too are heterogeneous: they are composed of distinct cell populations able to induce primarity the development of either the head or trunk parts of the embryo. These population, determined to become the head of the trunk organizers still at the blastula stage, may be located either in the single continuous cell layer (as in amphibia and birds) or separated among different tissue germs (as in mammals). When the dorsal-ventral orientation of the embryo is established and the organizer is switched on the very early invaginating cells of the dorsal blastopore lip (in the case of amphibia) move in advance of the entire invaginating mesoderm and by the end of gastrulation occupy the place just in front of the notochord. It is supposed that the early dorsal lip and the prechordal mesoderm (PCM) are one and the same cell population, i.e. during gastrulation Spemann's organizer transfers from the lip of blastopore to the prechordal zone. The PCM seems to play an exclusive role in the formation of a head in vertebrate, because some mutations in genes expressed in the PCM result in the entire head deletion. It is supposed that spreading of differentiating signals from the PCM occurs along the main body axis in both caudal and rostral directions. After the main body plan formation the PCM is replaced by adenohypophysis. This conclusion is drawn not only from the same topology of both these structures, but also from the similarities of a set of specific genetical markers expressed in these, that makes it possible to suppose the existence of deep connections and succession between them. The adenohypophysis seems to arise directly from the PCM, or cells of the ectoderm influenced by the PCM may be subsequently transformed into humoral cells of adenohypophysis. In this interpretation, adenohypophysis and the much earlier established PCM may be considered as derivatives of Spemann's organizer. This inference is supported by the fact that all the three above structures first originate in vertebrates only.     

Developmental biology of amphibians after Hans Spemann in Germany

After the Hans Spemann and Hilde Mangold discovery of the importance of the dorsal blastopore lip for axis formation in the early embryo (Nobelprize for Spemann, 1935), the scientific community tried in a goldrush-like manner to find the inducing factors responsible for the programming of early embyronic determination and differentiation. The slow progress towards a solution of this problem caused a fading of interest on behalf of most laboratories. This article describes the activities of a few laboratories in Finland, Japan and Germany, which continued their studies despite tremendous experimental difficulties. Finally only Heinz Tiedemann's group in Berlin was the first which could isolate a mesoderm/endoderm inducing factor in highly purified form, the so-called vegetalizing factor, now known as activin. Furthermore this article describes the identification of neuralizing factors like Chordin, Cerberus and Dickkopf in the zone of the Spemann-Mangold organizer. The finding that BMP-4 acts as an antagonist to these factors located on the dorsal side led to a new understanding of the mechanisms of action of inducing (neuralizing) factors and early embryonic pattern formation. Moreover, the observations that closely related genes and their products were also found in Drosophila, Zebrafish, Mice and Human were the basis for new concepts of evolutionary mechanisms (dorsal/ventral and anterior/posterior polarity or conserved processes in eye-development of all 7 animal phyla).     

Blastopore formation and dorsal mesoderm induction are independent events in early Cynops embryogenesis

The independent roles of blastopore formation and dorsal mesoderm induction in dorsal axis formation of the Cynops pyrrhogaster embryo were attempted to be clarified. The blastopore-forming (bottle) cells originated mainly from the progeny of the mid-dorsal C and/or D blastomeres of the 32-cell embryo, but were not defined to a fixed blastomere. It was confirmed that the isolated dorsal C and D blastomeres autonomously formed a blastopore. Ultraviolet-irradiated eggs formed an abnormal blastopore and then did not form a dorsal axis, although the lower dorsal marginal zone (LDMZ) still had dorsal mesoderm-inducing activity. Involution of the dorsal marginal zone was disturbed by the abnormal blastopore. These embryos were rescued by artificially facilitating involution of the dorsal marginal zone. Suramin-injected and nocodazole-treated blastulae did not have involution of the dorsal marginal zone, although the blastopore was formed. Neither embryos formed the dorsal axis. The dorsal mesoderm-inducing activity of the LDMZ in the nocodazole-treated gastrulae was still active. In contrast, the LDMZ of the suramin-injected embryos lost its dorsal mesoderm-inducing activity. bra expression was activated in the nocodazole-treated embryos but not in the suramin-injected embryos. The present study suggested that (i) the dorsal determinants consist of blastopore-forming and dorsal mesoderm-inducing factors, which are not always mutually dependent; (ii) both factors are activated during the late blastula stage; (iii) the dorsal marginal zone cannot specify to an organized notochord and muscle without the involution that blastopore formation leads to; and (iv) the localization of both factors in the same place is prerequisite for dorsal axis formation.     

In vitro organogenesis of pancreas in Xenopus laevis dorsal lips treated with retinoic acid

Dorsal lips of Xenopus laevis may differentiate into pancreas after treatment with retinoic acid in vitro. The dorsal lip region is fated to be dorsal mesoderm and anterior endoderm. Dorsal lip cells isolated from stage 10 early gastrula differentiate into tissues such as notochord, muscle and pharynx. However, in the present study, dorsal lips treated with 10(-4) M retinoic acid for 3 h differentiated into pancreas-like structures accompanied by notochord and thick endodermal epithelium. Sections of the explants showed that some cells gathered and formed an acinus-like structure as observed under microscopes. In addition to the morphological changes, expressions of the pancreas-specific molecular markers, XIHbox8 and insulin, were induced in retinoic acid-treated dorsal lip explants. Therefore, it is suggested that retinoic acid may induce the dorsal lip cells to differentiate into a functional pancreas. However, continuous treatment with retinoic acid did not induce pancreas differentiation at any concentration. Dorsal lips treated with retinoic acid within 5 h after isolation differentiated into pancreas-like cells, while those treated after 15 h or more did not. The present study provided a suitable test system for analyzing pancreas differentiation in early vertebrate development.     

The first cleavage plane and the embryonic axis are determined by separate mechanisms in Xenopus laevis. I. Independence in undisturbed embryos

We examined the spatial relationships between the meridian of sperm entry the plane of first cleavage, and the embryonic axis (defined by the neural groove) in eggs of Xenopus laevis. Direct measurement of the angular separations between these embryonic structures in gelatin-embedded eggs confirmed the classical conclusion that the sperm entry point and neural groove tend to form on opposite sides of the egg, and also revealed that the first cleavage plane has a nearly random orientation with respect to the neural groove. We next examined the distortion of the first cleavage plane that results from the normal processes of convergence and extension during gastrulation and neurulation. We permanently marked the first cleavage plane by injecting one blastomere of the two-cell embryo with a fluorescent lineage marker. At the start of gastrulation, the interface between the labeled and unlabeled regions was almost randomly oriented relative to the dorsal blastopore lip, confirming our first set of observations. In embryos with the interface less than 60 degrees to the plane passing through the midline of the dorsal lip, convergent movements of cells produced a confrontation of labeled and unlabeled cells along much of the dorsal midline. Thus, although the first cleavage plane and the bilateral plane were frequently not congruent, the morphogenetic movements of gastrulation and neurulation brought about an apparent congruence in many half-labeled embryos.     

Modification of Dorsal-Ventral Polarity in Xenopus laevis Embryos Following Withdrawal of Egg Contents before First Cleavage

When fertilized Xenopus laevis eggs were pricked just beneath the marginal zone with a thick glass needle prior to the first cleavage, a small amount of cytoplasm escaped into the exudate. Those eggs were placed in a poly L-lysine-coated plastic dish filled with 10% Ficoll solution. The location of the sperm entrance site (SES) of each egg was marked by scratching the surface of the plastic dish. The pricked embryos were anchored to the dish through poly L-lysine, and developed, therefore, without changing their original position. Consequently, development of the dorsalventral polarity was conveniently monitored with respect to the location of the SES. Embryos which developed from eggs pricked on the side opposite the SES showed modification of the dorsal-ventral polarity: Semi-quantitative studies showed that an exudation approximately 1.5–12.5% of the whole egg contents from the presumptive dorsal side caused a reversal of the dorsal-ventral polarity. That is, the dorsal lip of the blastopore formed on the same side of the SES, whereas the dorsal lip formed on the side opposite the SES in the normal control and sham-operated embryos. Half of the embryos which had larger cytoplasmic exudates more than 12.5% of the whole egg contents failed to form the dorsal lip by the time all controls and the embryos with smaller exudates showed normal dorsal lip formation. When eggs were pricked on the SES side, the normal topographic relationship between the SES and future dorsal lip side was reinforced.     

Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes

The upper lip and primary palate form an essential separation between the brain, nasal structures and the oral cavity. Surprisingly little is known about the development of these structures, despite the fact that abnormalities can result in various forms of orofacial clefts. We have uncovered that retinoic acid is a critical regulator of upper lip and primary palate development in Xenopus laevis. Retinoic acid synthesis enzyme, RALDH2, and retinoic acid receptor gamma (RARγ) are expressed in complementary and partially overlapping regions of the orofacial prominences that fate mapping revealed contribute to the upper lip and primary palate. Decreased RALDH2 and RARγ result in a median cleft in the upper lip and primary palate. To further understand how retinoic acid regulates upper lip and palate morphogenesis we searched for genes downregulated in response to RARγ inhibition in orofacial tissue, and uncovered homeobox genes lhx8 and msx2. These genes are both expressed in overlapping domains with RARγ, and together their loss of function also results in a median cleft in the upper lip and primary palate. Inhibition of RARγ and decreased Lhx8/Msx2 function result in decreased cell proliferation and failure of dorsal anterior cartilages to form. These results suggest a model whereby retinoic acid signaling regulates Lhx8 and Msx2, which together direct the tissue growth and differentiation necessary for the upper lip and primary palate morphogenesis. This work has the potential to better understand the complex nature of the upper lip and primary palate development which will lead to important insights into the etiology of human orofacial clefts.