Isolation and characterization of a Xenopus gene (XMLP) encoding a MARCKS-like protein.

We have identified a cDNA coding for a Xenopus MARCKS-like protein (XMLP) from a cDNA library prepared from activin-treated ectoderm. Using whole-mount in situ hybridization and RT-PCR, we found XMLP maternal transcripts during the cleavage stages. After MBT, the signals were restricted to the neural plate. Subsequently XMLP was expressed predominantly in the brain, somites and pronephros. Ectopic expression of XMLP resulted in eye and axis defects and in a change of the expression pattern of Krox 20, a neural marker for rhombomeres 3 and 5. Injected XMLP caused apoptosis. It was characterized by loss of intercellular adhesion contacts, transient plasma membrane ruffling at gastrula, and epithelial disruption attailbud stage. Overexpression of mutant XMLPs showed that this phenotype was correlated with its putative PSD domain and glycine at position 2. The embryos injected with a morpholino oligo complementaryto XMLPmRNA showed malformations of the anterior axis and eye defects. Extirpation experiments indicated that the phenotypes might be correlated with disturbed morphorgenetic movements rather than an inhibition of induction process. Overexpression of XCYP26 resulted in a shift of the expression pattern of XMLP. In the early tailbud stage (stage 20) the signal stripe in the XCYP26 injected half of the embryo got diffuse or even disappeared. This observation suggests that retinoic acid plays an important role in the regulation of XMLP. Our results suggest that XMLP might participate in pattern formation of the embryonic axis and the central nervous system.

Pluripotent cells (stem cells) and their determination and differentiation in early vertebrate embryogenesis.

Mammalian embryonic stem cells can be obtained from the inner cell mass of blastocysts or from primordial germ cells. These stem cells are pluripotent and can develop into all three germ cell layers of the embryo. Somatic mammalian stem cells, derived from adult or fetal tissues, are more restricted in their developmental potency. Amphibian ectodermal and endodermal cells lose their pluripotency at the early gastrula stage. The dorsal mesoderm of the marginal zone is determined before the mid-blastula transition by factors located after cortical rotation in the marginal zone, without induction by the endoderm. Secreted maternal factors (BMP, FGF and activins), maternal receptors and maternal nuclear factors (beta-catenin, Smad and Fast proteins), which form multiprotein transcriptional complexes, act together to initiate pattern formation. Following mid-blastula transition in Xenopus laevis (Daudin) embryos, secreted nodal-related (Xnr) factors become important for endoderm and mesoderm differentiation to maintain and enhance mesoderm induction. Endoderm can be induced by high concentrations of activin (vegetalizing factor) or nodal-related factors, especially Xnr5 and Xnr6, which depend on Wnt/beta-catenin signaling and on VegT, a vegetal maternal transcription factor. Together, these and other factors regulate the equilibrium between endoderm and mesoderm development. Many genes are activated and/or repressed by more than one signaling pathway and by regulatory loops to refine the tuning of gene expression. The nodal related factors, BMP, activins and Vg1 belong to the TGF-beta superfamily. The homeogenetic neural induction by the neural plate probably reinforces neural induction and differentiation. Medical and ethical problems of future stem cell therapy are briefly discussed.

XCL-2 is a novel m-type calpain and disrupts morphogenetic movements during embryogenesis in Xenopus laevis.

We identified a novel cDNA, XCL-2, encoding an m-type calpain, a calcium-dependent intracellular protease. This protein has all characteristic structures and active sites of canonical calpains. Zygotic transcription of the gene was first detected at stage 10. It is expressed exclusively in the ventral circumblastoporal collar and the mesoderm-free zone at the most anterior tip of neural fold in late gastrulae and neurulae. In later stages, expression is only found in cement gland and proctodeum. It is also expressed in a tissue-specific manner. In adult tissues, various levels of expression were detected in brain, eye, heart, intestine, kidney, lung, stomach and testis, but not in liver, muscle, nerve, ovary, skin and spleen. Overexpression of wild-type XCL-2 suggests that this gene is involved in gastrulation movement and convergent extension during gastrulation and neurulation. Overexpression of a dominant-negative mutant caused a phenotype morphologically similar to, but histologically different from, that caused by overexpression of wild-type XCL-2. The mutant phenotype can be rescued by injection of wild-type XCL-2. These data suggest that XCL-2 plays an important role in convergent extension movements during embryogenesis in Xenopus laevis.

Expression of Xenopus L-arginine:glycine amidinotransferase (XAT) during early embryonic development.

We have isolated a full-length cDNA encoding Xenopus L-arginine:glycine amidinotransferase (XAT), which shares a highly conserved sequence with human, chick and rat amidinotransferase. Although there are some studies about its structure and function in energy metabolism of adult tissues in some other species, little is known about its roles during early embryonic development. Characterization of embryonic expression indicates that XAT is differentially expressed around the yolk plug including the dorsal blastopore area at early gastrula stages and is extensively expressed in the midline of the neural plate of early neurula stages. Sections reveal that its transcripts are located in the notochord. In the tailbud stage signals are found both in the notochord and trunk area, whereas only faint signals can be found in the cephalic part.

Tissue-specific expression of an Ornithine decarboxylase paralogue, XODC2, in Xenopus laevis.

Ornithine decarboxylase (ODC) is involved in the biosynthesis of polyamines and hence has been found in almost all types of cells studied. Therefore it is frequently used as internal standard. We isolated a cDNA, XODC2, which is a paralogue to ubiquitous ODC and expressed in a spatial and temporal manner during the early embryogenesis of Xenopus laevis. Expression of XODC2was first detected at the animal pole at stage 9. During neurula stages the signals were found both in the extreme anterior and posterior part of the dorsal body axis. In tailbud stages the expression is further shifted to both the tail and head areas and gradually restricted to distinct tissues: forebrain, inner layer of epidermis of the head area, stomodeal-hypophyseal anlage, frontal gland, ear vesicle, branchial arches, the front tip of neural tube and proctodeum. In addition, signals were also found in the inner layer of epidermis underneath the cement gland during early tailbud stages while in later tailbud stages signals were detected at the apical zone of the cement gland. Comparative studies indeed could confirm that XODC1 in contrast to XODC2 is expressed ubiquitously throughout the whole embryos during early development of Xenopus laevis.

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).

Planar signalling is not sufficient to generate a specific anterior/posterior neural pattern in pseudoexogastrula explants from Xenopus and Triturus.

Early observations on the morphology of total exogastrulae from urodeles (Axolotl) had provided evidence for essential vertical signalling mechanisms in the process of neural induction. Conversely, more recent studies with anurans (Xenopus laevis) making use of molecular markers for neural-specific gene expression appear to support the idea of planar signalling as providing sufficient information for neural differentiation along the anterior-posterior axis. In an attempt to resolve this apparent contradiction, we report on the comparative analysis of morphology and gene expression characteristics with explants prepared from both urodeles (Triturus alpestris) and anurans (Xenopus laevis). For this purpose, we have made use of a refined experimental protocol for the preparation of exogastrulae that is intended to combine the advantages of the Holtfreter type exogastrula and the Keller sandwich techniques, and which we refer to as pseudoexogastrula explants. Analysis of histology and expression of several neural and ectodermal marker genes in such explants suggests that neural differentiation is induced in both species, but only within the intermediate zone between ectoderm and endomesoderm. Therefore, experiments with Xenopus and Triturus explants described in this communication argue against planar signalling events as being sufficient to generate a specific anterior/posterior neural pattern.

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.

Characterization of the Ets-type protein ER81 in Xenopus embryos.

A function for FGF-type peptide growth factors has been implied for early mesodermal patterning events in Xenopus laevis. FGF signalling operates via the MAP kinase cascade that can directly activate the transcription of organizer-expressed genes, such as Xbra and Xegr-1. We have recently provided evidence for a critical role of Ets-type transcription factors in FGF mediated Xegr-1 transcription activation. Here, we report on the identification of the Xenopus Ets-type protein ER81 that is expressed in a pattern overlapping with the ones of Xegr-1 and Xbra during gastrulation. Microinjection in XER81 encoding mRNA into ventral blastomeres of Xenopus embryos results in the induction of ectopic, tail-like protrusions, whereas dorsal overexpression results in disturbed eye development. In the animal cap assay, ectopic expression of XER81 is found to interfere with activin mediated induction of Xegr-1 and gsc, but not with the Xbra response to activin.

The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals.

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk-tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.

Regionalized metabolic activity establishes boundaries of retinoic acid signalling.

The competence of a cell to respond to the signalling molecule retinoic acid (RA) is thought to depend largely on its repertoire of cognate zinc finger nuclear receptors. XCYP26 is an RA hydroxylase that is expressed differentially during early Xenopus development. In Xenopus embryos, XCYP26 can rescue developmental defects induced by application of exogenous RA, suggesting that the enzymatic modifications introduced inhibit RA signalling activities in vivo. Alterations in the expression pattern of a number of different molecular markers for neural development induced upon ectopic expression of XCYP26 reflect a primary function of RA signalling in hindbrain development. Progressive inactivation of RA signalling results in a stepwise anteriorization of the molecular identity of individual rhombomeres. The expression pattern of XCYP26 during gastrulation appears to define areas within the prospective neural plate that develop in response to different concentrations of RA. Taken together, these observations appear to reflect an important regulatory function of XCYP26 for RA signalling; XCYP26-mediated modification of RA modulates its signalling activity and helps to establish boundaries of differentially responsive and non-responsive territories.

Neural induction in embryos.

Neural differentiation of the ectoderm is inhibited by bone morphogenetic protein 4 (BMP-4) in amphibia as well as mammalia. This inhibition is released by neural inducing factor(s), which are secreted from the dorsal mesoderm. Masked neuralizing factor(s) are already present in the ectoderm before induction. In homogenates from Xenopus oocytes and embryos neural inducing factors were found in the supernatant (centrifuged at 105000 g), in small vesicles and a ribonucleoprotein fraction. A neuralizing factor, which is a protein of small size, has been partially purified from Xenopus gastrulae. Genes that are expressed in the dorsal mesoderm and involved in the de novo synthesis of neuralizing factor(s) have been cloned. The differentiation of cells with a neuronal fate starts in the neural plate immediately after neural induction. Genes homologous to the Notch and Delta genes of lateral inhibition in insects are involved in this process.

Anterior specification of embryonic ectoderm: the role of the Xenopus cement gland-specific gene XAG-2.

In a search for novel developmental genes expressed in a spatially restricted pattern in dorsal ectoderm of Xenopus we have identified XAG-2, a cement gland-specific gene with a putative role in ectodermal patterning. XAG-2 encodes a secreted protein, which is expressed in the anterior region of dorsal ectoderm from late gastrula stages onwards. Activation of XAG-2 transcription is observed in response to organizer-secreted molecules including the noggin, chordin, follistatin and cerberus gene products. Overexpression of XAG-2 but not of the related cement gland marker XAG-1 induces both cement gland differentiation and expression of anterior neural marker genes in the absence of mesoderm formation. Further, we show that XAG-2 signaling depends on an intact fibroblast growth factor (FGF) signal transduction pathway and that XAG-2-induced anterior neural fate of ectodermal cells can be transformed to a more posterior character by retinoic acid. Based on these findings we propose a role for XAG-2 in the specification of dorsoanterior ectodermal fate, i.e. in the formation of cement gland and induction of forebrain fate of Xenopus.

The final determination of Xenopus ectoderm depends on intrinsic and external positional information.

Traditionally the whole animal cap (ventral plus dorsal ectoderm) of amphibian blastula and gastrula stages was considered as a homogeneous cell mass, because both the isolated dorsal and ventral ectoderm without induction differentiated into ciliated (atypical) epidermis. Recent results suggest a predisposition of the dorsal and ventral ectoderm. We used a special experimental approach, i.e. injection of activin as inducer into the blastocoel of intact Xenopus blastulae before the isolation of animal caps and fluorescein-dextran-amine (FDA) as a lineage tracer. In recombinants of FDA-labeled and unlabeled ectoderm we showed that the cells of the dorsal ectoderm mainly differentiate into neural tissue and notochord when they remain at their original dorsal position. In contrast, when small pieces of dorsal ectoderm are transplanted to the ventral part of animal caps, most of the descendants form epidermis. However, when small pieces of the ventral ectoderm are transplanted to the dorsal side, they significantly contribute to neural tissue and notochord. These results suggest that the prepattern in Xenopus animal caps of the late blastula and early gastrula stages is labile and reversible. Still more important is the fact that the fate of individual cells depends on the site of their localization within the animal cap. This means that cells in the dorsal most or ventral most part of the animal cap, respectively, will not randomly differentiate into all cell types, but predominantly into dorsal or ventral derivatives, respectively.

Characterization and early embryonic expression of a neural specific transcription factor xSOX3 in Xenopus laevis.

Using the powerful RDA-PCR-technique we could identify a novel Xenopus specific Sox-gene (xSox3) a transcription factor closely related to the sox sub-group B, which contains a HMG box. In normogenesis the xSox3 gene is expressed in the presumptive central nervous system. Furthermore a maternal component is also found in oocytes and in early cleavage stages in the animal hemisphere only. By whole-mount in situ hybridization the first zygotic transcription activities can be detected in the late blastula in the dorsal ectoderm and the dorsal and lateral part of the marginal zone. The expression reaches the highest level atthe late gastrula till the late neurula and fades after stage 30. The expression is restricted from gastrulation onwards to the presumptive brain area and the lens epithelium. Furthermore we could show that the gene is expressed in isolated Spemann organizer with adjacent neuroectoderm. The signal can be suppressed by suramin treatment, which inhibits neural development and causes a shift of dorsal to ventral mesoderm. The treatment of whole embryos with LiCl and UV results in an overexpression or an inhibition of the expression, respectively. In exogastrulae (pseudo-exogastrulae) the gene is expressed in the close vicinity to the endomesoderm only, but not in the distal most part of the ectoderm. This result indicates that it is unlikely that the gene can be activated by planar signals. The gene can also be activated in dissociated gastrula ectoderm without mesodermal or neural inducers. That means that the gene can be expressed in ectodermal cells in a cell autonomous manner.

Factors responsible for the establishment of the body plan in the amphibian embryo.

A central topic of embryology is the establishment of the body plan during embryogenesis. Starting with maternal factors distributed in the early cleavage stages in distinct patterns and gradients cell-to-cell interactions including early embryonic induction result in the formation of mesoderm and the organizer area. While many facts are known about the role of growth factors like activin (closely related to the vegetalizing factor), processed Vg1, BMPs and FGF for mesoderm formation, the establishment of the central nervous system is not yet well understood. However, there is growing evidence that neural induction is a multistep process at the level of the dorsal mesoderm (organizer) and the reacting neuroectoderm. Therefore the existence of only one neuralizing factor is unlikely. We report about data that follistatin protein is not a direct neural inducer. Furthermore our comparative studies of Xenopus and Triturus exogastrulae indicate that planar signals are unlikely in the Triturus embryo (urodeles) during the early steps of neural induction. Vertical signals emanating from the chordamesoderm are essential for the terminal neuralization and regionalization of the central nervous system during gastrulation for both Xenopus and Triturus. The putative role of neuralizing factors and BMP/activin-like molecules for the stabilization or shift of neuroectoderm into different pathways of differentiation (epidermis or neural default state) is discussed.

The role of vertical and planar signals during the early steps of neural induction.

The classical Einsteck-test (Spemann and Mangold, Roux Arch. Dev. Biol. 100: 599-638, 1924) and data from total exogastrulae (Holtfreter, 1933) suggest that vertical signals are transmitted between the chordamesoderm (organizer) and reacting ectoderm in the early phase of neural induction. In contrast to these results with Axoloti (urodeles), several authors observed the expression of neural specific genes in Xenopus exogastrulae, isolated dorsal blastopore lip with adjacent ectoderm (open-face explants) and Keller-sandwiches. Our data with Xenopus (anurans) also show that the expression of neural specific genes takes place in exogastrulae. However, when we prepared open face explants and exogastrula-like structures by microdissection at very early gastrula stage, the signal of a class II beta-tubulin, characteristic of terminal neural differentiation, is not found in the ectoderm. These results suggest that planar signals transmitted from the chordamesoderm into the ectodermal part can fairly be excluded under these experimental conditions. In similar experiments with Triturus alpestris we could not observe either the differentiation of neural structures in the ectodermal part of exogastrulae. These results confirm earlier experiments of Holtfreter performed with Ambystoma mexicanum (Axoltl) embryos. On the basis of the published data of different authors and our results, we cannot exclude the existence of planar signals for early and/or transient expressed genes before the onset of gastrulation in Xenopus, which make the neuroectoderm susceptible for the response to vertical signals during gastrulation. On the other hand our experiments with Triturus alpestris suggest that planar neural signals are unlikely in this species. These differences between Triturus and Xenopus embryos are discussed in the context of the peculiarities in morphological structure, competence and speed of development of the two species.