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.

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.

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.

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.

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.

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.

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.

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.

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.

Change of the differentiation pattern of amphibian ectoderm after the increase of the initial cell mass.

Early amphibian gastrula ectoderm (Triturus alpestris) has been treated with vegetalizing factor. While normal sandwiches (animal caps of two eggs) differentiated mainly into endoderm derived tissues, giant-sandwiches (a combination of 8 animal caps) formed mesodermal and neural tissues in addition. The results support the interpretation that ectoderm will differentiate into endoderm derived tissues when all or nearly all cells are induced (presumably depending on certain threshold concentrations of the inducer). This is the case in the normal sandwich after treatment with high concentrations of vegetalizing factor for 24 h. However, in a giantsandwich it must be assumed that only the cells in the vicinity of the inducer will be triggered to differentiate into endoderm derived tissues. Mesodermal structures will be formed by secondary interactions between the induced ectoderm (endoderm) and non induced ectodermal cells. The induction of neural structures could be explained as a further interaction between mesodermalized and non induced ectodermal cells. This chain of events is compared with the steps of determination in normogenesis.

[Experimental studies on competence in early development of amphibian ectoderm]. / Experimentelle Untersuchungen über die Kompetenzverhältnisse früher Entwicklungsstadien des Amphibien-Ektoderms.

The inductive effect of lithium chloride was examined on isolated presumptive ectoderm from different developmental stages (16-32-cell stage up to the early middle gastrula stage) of Triturus vulgaris and Ambystoma mexicanum. The following results were obtained: 1. Nearly the same temporal sequence of differentiation tendencies were found for treated ectoderm of comparable stages of Ambystoma- and Triturus ectoderm. The lithium treatment brought about mesodermal differentiations in the morula stage up to the early gastrula stage (at Triturus also two cases in the 16-32-cell stage). The most frequent and largest mesodermal inductions were obtained in the middle and late blastula. 2. In both species entodermal differentiations were formed in all examined stages. Entodermal inductions decreased in the late blastula to the early middle gastrula. In the morula, early blastula and in the early middle gastrula ectoderm of Ambystoma and Triturus entodermal differentiations appeared independent of mesodermal tissues. 3. There are clear differences in the degree of the differentiation of entodermal structures in relation to the developmental stage. Intestine and peripheral entoderm were more frequent in the older stages than in the earlier ones. 4. Comparing the results of Ambystoma and Triturus there are significant differences with respect to the regionality of the induced tissue complexes. Ambystoma ectoderm forms entodermal and spinocaudal structures, while Triturus ectoderm brings about entodermal, mesodermal and deuterencephalic inductions. The parts and the composition of the analyzed tissue formations correspond with certain regions of the young larva. Thus the treated ectoderm of Ambystoma forms tissues corresponding to the region of the tail, while Triturus ectoderm produces differentiations of the posterior and middle part of the trunk of the young larva.

[Inhibition of aggregation of dissociated embryonic Amphibian cells by inhibitors of protein and RNA synthesis]. / Hemmung der Reaggregation dissoziierter Amphibienzellen durch Inhibitoren der RNS- und Proteinsynthese.

1. Aggregation of embryonic Amphibian cells dissociated by Trypsin or EDTA is supressed reversibly by actidion (cycloheximid 0.5-2 Μg/ml), an inhibitor of protein synthesis. Protein synthesis is inhibited more than 90% at these concentrations. The inhibition by actidion was still reversible after 48 hours, when the cells were transferred to normal medium. Actidion added to cells, cultured in Flickinger solution for 3 h, was no longer able to stop further aggregation. 2. Puromycin reversibly suppresses aggregation at a concentration of 20 (Μg/ml, when protein synthesis is inhibited to 66 %. 3. After EDTA-dissociation the formation of initial small clusters of 5-10 cells (primary aggregation) occurred even in the presence of inhibitors of protein synthesis. The primary aggregation is however suppressed if actidion or puromycin are already added during the dissociation procedure. 4. In the presence of actinomycin-D (1-2 Μg/ml) the cells continue to aggregate like the controls. After 30 h further aggregation is stopped and the aggregates loose single cells. 5. Differences in the aggregation of cells after EDTA and Trypsin treatment are discussed.

[Relationship between competence and protein synthesis in amphibian ectoderm]. / Abhängigkeit der Kompetenz des Amphibien-Ektoderms von der Proteinsynthese.

Isolated Amphibian gastrula ectoderm was pretreated in different ways and induced with a mesodermal/neural (Series I) or a mesodermal/endodermal (Series II) fraction ("sandwich" method).In the controls (K0) the inducer was implanted directly into early gastrula ectoderm. The explants of series K1 were cultured in vitro for 20 (Series I) or 24 hours, respectively (Series II), and then combinated with the inducer. In series Ak the explants were treated with an inhibitor of protein synthesis (2µg/ml Actidion = Cycloheximid) for 20 or 24 hours and then cultured with inducer for 12 days.Ectoderm, cultured for 24 hours in vitro (Series K1) lost its ability to respond to inducing factor. There is no loss of competence when ectoderm is treated with actidion for the same time and then treated with inducer.

The ultrastructure of amphibian ectoderm treated with an inductor or actinomycin D.

Amphibian (Triturus alpestris) ectoderm was isolated and after 2-16 days in culture examined by electron microscopy. It has been shown that the ectoderm, formerly called "undifferentiated ectoderm", forms in part ciliated epithelial cells, which cannot be distiguished from the cells of the epidermis, which has developed in situ (except for the alignment of the cells which depends on the mesenchyme underlying the epidermis). The ultrastructure of the cilia is similar to that of cilia of protozoa or sperms of insects or vertebrates. A zone at the periphery of the epidermal cells, free of yolk platelets, mitochondria and pigment granules (embryonic pigment) is observed after 4 days. This area is rich in vacuolous structures and basal bodies of the cilia.Ectoderm, which was treated with the vegetalizing factor differentiates into mesodermal and endodermal tissues. Cilia, as well as the characteristic peripheral zone, are not formed in the induced ectoderm.In ectoderm treated with actinomycin D (1 µg/ml for 6 h) the differentiation of the peripheral zone and the cilia is delayed.

[Influence of inhibitors of RNA and protein synthesis and inductors upon cell affinity of amphibian tissues]. / Einflu\ von Inhibitoren der RNS- und Protein-Synthese und Induktoren auf die ZellaffinitÄt von Amphibiengewebe.

Isolated Amphibian gastrula ectoderm, pretreated in different ways, was combined with endoderm. 1. Untreated ectoderm separates from endoderm after several days. 2. Ectoderm, treated with the vegetalizing inducing factor, is surrounded by endoderm, which spreads over the treated ectoderm as an uni- or multicellular layer. 3. Treatment of ectoderm with actidion (2 Μg/ml for 5 h or 1 Μg/ml for 20 h), an inhibitor of protein synthesis, prior to induction prevents the spreading of endoderm over ectoderm. 4. Treatment of ectoderm with AcD, an inhibitor of RNA synthesis, prior to induction does not prevent the spreading of endoderm over ectoderm. 5. Treatment of ectoderm with AcD without inductor changes the affinity of ectoderm to endoderm. Ectoderm, treated with 1 Μg/ml AcD, spreads over the endoderm as a thin layer. After treatment with 2 Μg/ml AcD, in some cases endoderm spreads over the ectoderm.

Differentiation of the four animal and the four vegetal blastomeres of the eight-cell-stage ofTriturus alpestris.

The 4 animal and 4 vegetal blastomeres of the eight-cell-stage ofTriturus alpestris were isolated and cultured for up to 12 days. Because of the difficulty of obtaining intact animal and vegetal blastomeres of the same embryo, we either cut off the vegetal blastomeres or sucked off the animal blastomeres. The culture of early embryonic amphibian cells is improved by the use of 50% Leibovitz-medium with added fetal calf serum providing a stable pH and optimal osmotic pressure.Isolated animal blastomeres differentiated to irregularly shaped ciliated epidermis. 30% of the cases showed small amounts of myotomes, notochord and neuroid cells in addition to irregular epidermis. The vegetal blastomeres formed trunk and tail structures but only 6% of all cases formed nearly complete head structures in addition.From the results we conclude that the vegetal blastomeres as well as the animal blastomeres of the eight-cell-stage are already determined as to their future fate. The possibility of partial regulation and the influence of asymmetric or irregular cleavage on the further development of isolated blastomeres 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 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).

Changes of the cell surface charge of amphibian ectoderm after induction.

Isolated ectoderm of early gastrula stages ofTriturus alpestris was treated with vegetalizing factor for 24 h employing the sandwich method (induced ectoderm). Controls were incubated for the same period with γ-globulin which has no inducing activity. Explants of both series were labelled with cationized ferritin, which binds to negatively charged groups at physiological pH. In non-induced ectoderm, ferritin particles can be found as a thin layer all over the plasma membranes. In induced ectoderm the total amount of ferritin bound to the plasma membrane is much lower than in non-induced ectoderm. Ferritin is located in restricted areas only. In contrast to the controls, other membrane areas are free of ferritin particles. The correlation between these results and the change of cell affinity after induction with vegetalizing factor is discussed.