Characterization of three synuclein genes in Xenopus laevis.

The synuclein family consists of three small intracellular proteins mainly expressed in neural tissues, and has been associated with human neurodegenerative diseases. We have examined the spatial and temporal expression patterns of three synuclein genes during embryogenesis of Xenopus laevis. The Xenopus synucleins were firstly expressed in the developing nervous system at the tail bud stages. At tadpole stages, Xenopus snca was expressed in the brain, branchial arch and somite, and sncbb signals were detected in entire brain and spinal cord. However, sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion. RT-PCR indicated that expression of synucleins was up-regulated at the end of neurulation, and then maintained at later examined stages. Our study provides the spatiotemporal expression patterns of the synuclein family genes in Xenopus embryos, and forms a basis for further functional analysis of synucleins.

Xenopus X-box binding protein 1, a leucine zipper transcription factor, is involved in the BMP signaling pathway.

We describe a novel basic leucine zipper transcription factor, XXBP-1, which interacts with BMP-4 in a positive feedback loop. It is a maternal factor and is zygotically expressed in the dorsal blastopore lip and ventral ectoderm with the exception of the prospective neural plate during gastrulation. Overexpression of XXBP-1 leads to ventralization of early embryos as described for BMP-4, and inhibits neuralization of dissociated ectoderm. Consistent with mediating BMP signaling, we show that the ectopic expression of XXBP-1 recovers the expression of epidermal keratin and reverses the dorsalization imposed by truncated BMP receptor type I, indicating that it may act downstream of the BMP receptor. Its effects can be partially mimicked by a fusion construct containing the VP16 activator domain and the XXBP-1 DNA-binding domain. In contrast, fusing the DNA-binding domain to the even-skipped repressor domain leads to upregulation of the neural markers NCAM and nrp-1 in animal cap assay. Taken together, the results suggest a role for XXBP-1 in the control of neural differentiation, possibly as an activator.

Four decades of teaching developmental biology in Germany.

I have taught developmental biology in Essen for 30 years. Since my department is named Zoophysiologie (Zoophysiology), besides Developmental Biology, I also have to teach General Animal Physiology. This explains why the time for teaching developmental biology is restricted to a lecture course, a laboratory course and several seminar courses. However, I also try to demonstrate in the lecture courses on General Physiology the close relationship between developmental biology, physiology, morphology, anatomy, teratology, carcinogenesis, evolution and ecology (importance of environmental factors on embryogenesis). Students are informed that developmental biology is a core discipline of biology. In the last decade, knowledge about molecular mechanisms in different organisms has exponentially increased. The students are trained to understand the close relationship between conserved gene structure, gene function and signaling pathways, in addition to or as an extension of, classical concepts. Public reports about the human genome project and stem cell research (especially therapeutic and reproductive cloning) have shown that developmental biology, both in traditional view and at the molecular level, is essential for the understanding of these complex topics and for serious and non-emotional debate.

XETOR regulates the size of the proneural domain during primary neurogenesis in Xenopus laevis.

The interaction between early proneural genes and lateral inhibition determines the number of primary neurons. The mechanism for regulating the size of the proneural domain, however, has not been clarified. We show here that inhibition of the function of XETOR in Xenopus, a homolog of human oncoprotein ETO/MTG8, leads to a neurogenic phenotype of expanded proneural domain without alteration in the density of primary neurons. This result suggests that XETOR is a prerequisite for regulating the size of the proneural domain. We further show that such a regulation is accomplished by establishing a negative feedback loop between XETOR and proneural genes except Xngnr-1, as well as by antagonism between XETOR and lateral inhibition.

Determination, induction and pattern formation in early amphibian embryos.

Determination (inducing) factors, the extracellular matrix, signaling pathways, transcription factors and genes interact in pattern formation and neural induction. Genes can either be activated or repressed. The animalvegetal and dorso-ventral polarities are determined in very early developmental stages. Factors of the TGF-ß superfamily in a graded distribution are involved in the determination of endoderm, mesoderm and ectoderm. The differentiation of mesoderm also depends on the animal ectoderm. Neural inducing factors have been partially purified.

Basic fibroblast growth factor can induce exclusively neural tissue in Triturus ectoderm explants.

Ectoderm was isolated from early gastrulae of Triturus alpestris and induced with recombinant basic fibroblast growth factor (b-FGF). Neural tissue differentiated in about 38% of the explants which were induced by 2,5 µg/ml FGF. These explants do not contain other tissues, or contain only small amounts of mesenchyme and melanophores which are probably derived from induced neural crest. It is therefore unlikely that these neural tissues are secondarily induced. The other explants contain predominantly blastema tissue, endothelium/ mesothelium, small amounts of skeletal muscle and, rarely, notochord besides neural tissues. The mitotic rate was enhanced in about 20% of the induced explants. Possible mechanisms for the unexpected neural-inducing activity of b-FGF in Triturus ectoderm are discussed.

Urea is Necessary for the Culture of Embryos of the Marsupial Frog Gastrotheca riobambae, and is Tolerated by Embryos of the Aquatic Frog Xenopus laevis: (ureotelism/urea concentration in frog blood/culture of frog embryos/mesoderm induction).

Urea was found in the capsular fluid that bathes Gastrotheca riobambae embryos during incubation in the maternal pouch. The urea concentration in this fluid is higher than in blood from the mother, indicating that urea is accumulated by the embryo during the period of maternal incubation. Gastrotheca tadpoles tolerate up to 500 mM urea with 86% survival after 24 hours and die in solutions of 0.5 mM ammonia. These findings suggest that urea plays a role in the adaptation of G. riobambae embryos to the conditions of water stress within the maternal pouch. To improve the in vitro culture conditions of early embryos taken from the maternal pouch, a saline solution that contains urea was designed (GRS). GRS plus 30 mM urea was used for the culture of cleavage to the neurula stage embryos of G. riobambae. During organogenesis, the urea concentration was raised to 60 mM. Early embryos of Xenopus laevis tolerate urea, and in addition, no inducing effects of urea have been detected in animal cap explants of Xenopus.

The Dorsalization of Spermann's Organizer Takes Place during Gastrulation in Xenopus laevis Embryos: (Spemann's organizer/dorsal mesoderm/neural induction/suramin/inhibition of notochord formation).

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.

Spatial and temporal localization of FGF receptors in Xenopus laevis.

Mesoderm formation is a result of cell-cell interactions between the vegetal and animal hemisphere and is thought to be mediated by inducing peptide growth factors including members of the FGF and TGFß superfamilies. Our immunochemical study analyses the distribution of FGF receptors coded by the human flg gene during embryogenesis of Xenopus laevis. Immunostaining was detected in the dorsal and ventral ectoderm and also in the marginal zone of early cleavage, blastula and gastrula stages. Signals were very strong in the mid and late blastula (stage 8 and 9) and declined slightly in the early gastrula (stage 10). A dramatic decrease was observed up to the late gastrula (stage 11+). In stage 13 embryos, immunostaining was only found in cells around the blastopore. Isolated ectoderm cultured in vitro showed a similar temporal expression and decrease of the signal as the normal embryos. These results indicate that receptor expression is independent of the interaction of the animal cells with the vegetal part of the embryo. Of interest is the fact that the signal cannot only be found at or near the cell surface but also within the cell. This suggests the presence of an intracellular isoform of the receptor resulting from the endogenous expression of splice variants and the internalization of transmembrane receptor. Taken together our results suggest that the loss of competence (for bFGF around stage 10) is not directly correlated with the presence of receptors. The possible roles of heparan sulphate glucosaminoglycans (low affinity receptors) and control mechanisms in the intracellular signalling pathway downstream of the receptor level should be taken into consideration.

Homoiogenetic Neural Inducing Activity of the Presumptive Neural Plate of Xenopus Laevis: (Xenopus laevis/neural induction/homoiogenetic induction/heteroplastic transplantation/Xenopus borealis).

Heteroplastic combinations were made between Xenopus laevis presumptive neural plate and competent ectoderm of Xenopus borealis. Primarily induced presumptive neural plate cells (Xenopus laevis) can easily be distinguished from Xenopus borealis cells by specific quinacrine fluorescence of the nuclei. It was clearly shown that presumptive neural plate, which has primarily been induced by the underlying chordamesoderm exerts homoiogenetic inducing activity on competent ectoderm. The inducing activity is increased in pieces of presumptive neural plates, when the superficial layer has been removed from the adjacent deep layers. The enhancement can be explained by the fact that the removal of the superficial layer acting as barrier allows the inducing stimulus to be easily propagated from the apical (distal) side of the deep layers of the presumptive neural plate.

A mesoderm-inducing factor from a Xenopus laevis cell line : Chemical properties and relation to the vegetalizing factor from chicken embryos.

We have compared the chemical properties and biological activities of the mesoderm-inducing factor that is secreted by the Xenopus XTC cell line with the vegetalizing factor from chicken embryos. The inducing activity of the factors was tested in different concentrations on totipotent ectoderm either by implantation into early gastrulae of Triturm alpestris or by application of solutions to isolated ectoderm of early gastrulae of Xenopus laevis. Both factors have similar properties. They are not irreversibly inactivated after treatment with 6 M urea or with phenol at 60° C. Reduction with thioglycolic acid inactivates the factors completely. The inducing activity of XTC-conditioned medium decreases only slightly after treatment with 50% formic acid. The apparent molecular mass and the isoelectric point of the factors are similar. The XTC factor was partially purified by size-exclusion and reversed-phase high-pressure liquid chromatography and by isoelectric focusing. The possible relationship of these factors to transforming growth factor ß is discussed.

Crystallins during Xenopus laevis free lens formation.

The ontogeny and localization of crystallins during free lens development (i.e. lens development without the optic vesicle) were investigated in Xenopus laevis using the indirect immunofluorescence staining method with an antiserum raised against homologous total lens soluble proteins. Since the developing free lenses pass through stages similar to those of the lenses regenerated from the inner cell layer of the outer cornea following lentectomy in the same species Freeman's classification was used to identify the stages of free lens development. The first appearance of a positive reaction occurred at early stage IV in a number of cells in an area where future lens fibre cells would develop. With further differentiation of the free lens more and more cells in the fibre area started to show a positive reaction and the first positive reaction in the epithelium was observed late in stage V. Histological examination revealed that a fully differentiated free lens and a normally developed lens are similar but that the free lens is smaller.

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions.

Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.

Electron microscope study of the binding of Con A-gold to superficial and inner ectoderm layers ofXenopus laevis and its relation to the neural-inducing activity of this lectin.

Isolated competent amphibian ectoderm differentiates into neural (archencephalic) structures when treated with the plant lectin concanavalin A (Con A). While the inner ectoderm layer ofXenopus laevis forms brain structures after incubation with Con A, the outer ectoderm layer differentiates into ciliated epidermis only. This difference can be correlated with the pattern of Con A bound to the plasma membrane. With gold-labelled Con A it could be shown by transmission electron microscopy (TEM) that the outer ectoderm binds substantially less lectin than the inner layer. Furthermore we observed characteristic differences at the apical and basal surfaces of the cells of the same layer, i.e. on the apical cell surface of the superficial layer almost no Con A-gold could be found. In contrast, we observed a lot of gold particles on the basal cell side of the superficial layer. However, the number on both surfaces (apical and basal side of the cell) of the inner ectoderm layer was essentially higher, which could explain its biological reaction to the Con A stimulus and the differentiation into neural structures. The data presented in this paper indicate that early and late gastrula ectoderm bind similar amounts of Con A and support the view that the decrease in competence is not correlated with a loss of receptors for inducing factors. Furthermore, we describe the binding and the internalization of Con A via receptor-mediated endocytosis and the further fate of the Con A-gold-receptor complex inside the target cell.

The activation of a neuralizing factor in the neural plate is correlated with its homoiogenetic-inducing activity.

Neural plates which are induced in the dorsal ectoderm of Triturus by the underlying mesoderm acquire, in turn, neural-inducing activity. This process is correlated with the appearance of neural-inducing activity in the microsomal fraction of the neural plate homogenate. The high-speed supernatant also acquires inducing activity after neural induction, but to a lesser extent. The experiments suggest that a masked neuralizing factor, which is already present in the ectoderm, is in part activated and exported from the inducing neural plate cells.

The inducing capacity of the presumptive endoderm of Xenopus laevis studied by transfilter experiments.

The inducing capacity of the vegetal hemisphere of early amphibian blastulae was studied by placing a Nucleopore filter (pore size 0.4 µm) between isolated presumptive endoderm and animal (ectodermal) caps. The inducing effect was shown to traverse the Nucleopore membrane. The reacting ectoderm differentiated into mainly ventral mesodermal derivatives. Expiants consisting of five animal caps also formed dorsal mesodermal and neural structures. Those results together with data published elsewhere suggest that, in addition to a vegetalizing factor, different mesodermal factors must be taken into consideration for the induction of either the ventral or the dorsal mesodermal derivatives. The neural structures are thought to be induced by the primarily induced dorsal mesodermal tissue. Electron microscopic (TEM) examination did not reveal any cell processes in the pores of the filter. The results indicate that transmissible factors rather than signals via cytoplasmic contacts or gap junctions are responsible for the mesodermal induction of ectodermal cells. The data support the view that in normogenesis the mesoderm is determined by the transfer of inducing factors from vegetal blastomeres to cells of the marginal zone (presumptive mesodermal cells).

Change in the differentiation pattern ofXenopus laevis ectoderm by variation of the incubation time and concentration of vegetalizing factor.

Early amphibian gastrula ectoderm (Xenopus laevis) has been treated with vegetalizing factor using the sandwich technique, varying the period of incubation and the inducer concentration.The pattern of induced tissues depends on three factors: the inducer concentration, the size of inducer pellet and the time of exposure of ectodermal target cells to inducer.Short treatment with inducer will result in the formation of blood cells and heart structures. An increase in incubation time or inducer concentration, or both, will cause the formation of increasing amounts of such dorsal mesodermal structures as pronephros, somites and notochord. Neural structures can only be observed in explants with considerable amounts of somites and notochord.Ectoderm treated with high concentrations of vegetalizing factor for the whole period of competence will differentiate into endoderm.Furthermore, the results show thatX. laevis ectoderm does not show any autoneuralizing tendency under our experimental conditions. It therefore seems to be a suitable tool for the study of primary embryonic induction.

A new method for the isolation of plasma membranes from amphibian embryos.

A method for the isolation of plasma membranes is described. N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP), a heterobifunctional reagent, is covalently linked to protein amino groups in plasma membranes of intact cells. After homogenization of the cells the plasma membranes can be separated from other cell components by selective coupling to reduced Thiopropyl-Sepharose 6B and then recovered after treatment with 2-mercaptoethanol.

Biological activity of the vegetalizing factor: Decrease after coupling to polysaccharide matrix and enzymatic recovery of active factor.

The inducing activity of the vegetalizing factor decreases after covalent coupling to CNBr-Sepharose with reduced binding capacity. The residual inducing activity is probably due to the release of a small amount of the factor from Sepharose beads. Covalent coupling to activated CH-Sepharose completely inactivated the vegetalizing factor, whereas the neuralizing factor retained its full activity. The biological activity was also very much reduced when the vegetalizing factor was bound to Sephadex beads, a derivative of dextran. Fully active factor was recovered after enzymatic degradation of the dextran matrix with dextranase. The experiments suggest that the neuralizing factor acts on the cell surface of ectoderm cells, whereas the vegetalizing factor must probably be internalized to become biologically active.