Cell surface changes of the presumptive ectoderm following neural-inducing treatment by concanavalin A

Summary Scanning electron microscopic studies revealed that Concanavalin A (ConA) induces characteristic changes of the cell surface and the cell architecture of the presumptive ectoderm associated with differentiation into neural tissues. In Con A-treated cells, the filopodia with which cells were connected to each other disappeared from the interior (blastocoelic) surface and the cellular adhesivity decreased significantly. Thereafter, the cells underwent from those of the control explants. After cultivation for 60 h, a certain pattern of cell arrangement, which resembled the architecture of neural tissues, was observed among randomly arranged cells in the explants treated with Con A. The morphological changes specifically observed in Con A-treated explants were different from those found in explants treated with succinyl Con A (S-Con A) orDolichos biflorus agglutinin (DBA), which is unable to induce formation of the neural tissues. The molecular organization of the plasma membrane appears to be important in the mechanism of neural induction.     

Removal of N-linked oligosaccharides of presumptive ectoderm impairs neural induction in Pleurodeles waltl.

Studies were carried out on the embryo of the amphibian Pleurodeles waltl to investigate the potential role of the N-linked oligosaccharides of the ectodermal cell membrane in the neural induction process. Glycopeptidase F (GPase F) was used to cleave N-linked oligosaccharides on presumptive ectoderm. Removal of oligosaccharide moieties from ectoderm membrane glycoconjugates completely inhibited natural neural induction in vitro. On the other hand, Swainsonine (Sw) and 1-deoxynojirimycin (dNM), specific inhibitors of enzymes involved in glycosylation, provoked strong and persistent changes in the structure of the N-linked oligosaccharides of presumptive ectoderm but did not prevent neuralisation of treated ectoderm. We conclude that N-linked carbohydrates are implicated in the phenomenon of neural induction. However, the structural integrity of N-linked carbohydrates of target tissue is not itself critical in this process. The existence of specific carbohydrates on presumptive ectoderm was still questioned as receptors of neural signal.     

Excitability changes in the crustacean motor axon following activity

It has been shown experimentally that the crustacean motor axon is supernormally excitable following a train of action potentials (Zucker 1974). Such a phenomenon can lead to recruitment of terminals which are unexcited at low rates of stimulation. Although currents underlying the crustacean motor axon have been characterized (Connor et al. 1977), it is not known whether this membrane model accounts for a supernormal period, what might cause superexcitablity in this model, or how excitability might change during repetitive stimulation. In present study, it is demonstrated that the crustacean motor axon model does predict a supernormal period, that the supernormal period results from slow recovery from inactivation of the transient potassium, or A, current, and that supernormal excitability is enhanced by repetitive stimulation.     

From presumptive ectoderm to neural cells in an amphibian

As an immediate consequence of neural induction during gastrulation, some neuroectodermal cells acquire the ability to develop a number of specific neuronal and astroglial features, without requiring subsequent chordamesodermal cues. Thus, cholinergic, dopaminergic, noradrenergic, gabaergic, somatostatinergic, enkephalinergic, etc. traits are expressed in cultures of neural plate and neural fold isolated from amphibian late gastrulae immediately after induction and cultured in a defined medium. These results strongly suggest that at the late gastrula stage, the neural precursor population does not yet constitute a homogeneous set of cells. It was of interest to know the origin of this heterogeneity. Is it a direct result of the process of neural induction itself, stochastic phenomena being involved or not at the cellular level, or does it reflect a pre-existing heterogeneity in the presumptive ectoderm? At the early gastrula state, presumptive ectoderm can be neuralized consecutively to its dissociation into single cells. Using this experimental model, we have demonstrated by means of immunological probes that neuralized presumptive ectodermal cells, without any intervention of the chordamesoderm (natural inducing tissue), can develop autonomously into glial and neuronal lineages. These data suggest the existence of diverse predispositions of presumptive ectodermal cells. Competent ectoderm seems to be a heterogeneous structure with cells presenting distinct neural predispositions that can emerge as a consequence of a permissive inductive signal without real specificity (such as a target tissue dissociation). Moreover, such a differentiated neuronal population includes neurons of the GABAergic and enkephalinergic phenotypes but not of the cholinergic, catecholaminergic, somatostatinergic, etc. phenotypes. These data show that the developmental program of ectodermal cells induced without interaction with the chordamesoderm appears restricted compared to the naturally induced ectoderm. Experiments are now under way to analyze such sequential neural events.     

Long-term culture of Xenopus presumptive ectoderm in a nutrient-supplemented culture medium

Animal cap assay is a useful experimental model for investigating the activity of inducers in amphibian development. This assay has revealed that activin A is a potent mesoderm-inducing factor. However, it has been very difficult to induce highly differentiated tissues such as cartilage in a 3-4 day culture period. It was recently reported that jaw cartilage was induced in vitro in an animal cap that had been cultured for 14 days in Steinberg's solution using the sandwich culture method and activin A. Under these conditions, necrosis was occasionally observed in the explants. In this study, we have achieved long-term animal cap cultures in a nutrient-supplemented culture medium designated RDX. This medium was made by modifying the saline concentration of the RD medium previously developed as a basal medium for the serum-free culture of various kinds of mammalian cells. The explants cultured in RDX grew more vigorously compared with those in Steinberg's solution. RDX medium promoted a wider variety of tissue induction and gene expression in the animal caps than Steinberg's solution, and also increased the frequency of cartilage induction. Therefore, the supplemental nutrients may support and promote the differentiation of cartilage. This long-term culture method using RDX medium is useful for studying the differentiation of tissues or organs such as cartilage in vitro.     

Glycoproteins responsive to the neural-inducing effect of Concanavalin A in Cynops presumptive ectoderm

To examine the possible occurrence of receptors in the ectodermal cell surface which apparently mediates the neural-inducing stimulus, a further experiment by using Con A was done in combination with the enzyme treatments. The presumptive ectoderm explants of Cynops gastrula were first treated with neuraminidase to remove sialic acid. Prior to the Con A treatment, the explants were treated with almond glycopeptidase, which cleaves the asparagine linkage between protein and oligosaccharide in glycoprotein and releases the oligosaccharide moiety intact containing mannose residue from the substrate. No neural induction occurred. When the explants were not treated with almond glycopeptidase, the neural induction frequency was found to be the same as that of the explants treated with only Con A. Biochemical analyses showed that when the fixed ectoderm explants were treated with almond glycopeptidase, several oligosaccharides were released and then fractionated by means of Bio-Gel P-4 filtration. Based on the strict specificity of almond glycopeptidase, these oligosaccharides are unmistakably asparagine-linked oligasaccharides with mannose residues. We discuss the hypothesis of involvement of glycoproteins in the first step of molecular events in the neural induction mechanism.     

Differentiation of presumptive lens ectoderm of the chick in vitro studied with immunofluorescence techniques

The differentiation capacity of presumptive lens ectoderm was studied in the chick by an in vitro technique using the appearance of central nervous system or lens-specific antigens as indicators of differentiation. Handling the explants resulted in 'autodifferentiation' of both antigens, but co-culture with alcohol-killed primitive node or optic cup material could induce much stronger differentiation. Little specificity exists in the reaction and a hypothesis is presented whereby selection between the two differentiation pathways is thought to be due mainly to maturation within the ectoderm and the inducing tissue plays a minor qualitative role.     

Correlation between immunodifferentiation and histodifferentiation in presumptive lens ectoderm of the chick in vitro

The ability of stage-4-9 chick presumptive lens ectoderm to undergo nervous tissue or lens differentiation was studied in vitro. The tissue was cultured alone or co-cultured with alcohol-killed primitive node or optic cup as inducer. Immunofluorescence was studied on paraffin-wax preparations, which were then studied histologically. An attempt was made to correlate immunological and histological differentiation. The presumptive lens ectoderm differentiated both nervous tissue and lens structures in all stages, regardless of the presence or absence of an inducer. The outcome, however, was improved when an inducer was included. The inducers were not qualitatively specific. The stage-4 ectoderm proved to be more apt than older stages to differentiate nervous tissue and form neural tube-like structures. In the former stage, lens differentiation occurred with less readiness. Older stages differentiated lens structures readily and also showed immunological signs of nervous tissue differentiation. No indication of histological differentiation, however, was apparent and no neural tube-like structures formed.     

Vegetalising factor extracted from the fish swimbladder and tested on presumptive ectoderm ofTriturus embryos

Summary Vegetalising factor was isolated from swimbladder of crusian carp (Carassius auratus) by solubilishing with 8 M urea the precipitate obtained after digesting the swimbladder with collagenase. The urea-soluble fraction vegetalised isolated presumptive ectoderm ofTriturus gastrula and produced both undifferentiated mesodermal and endodermal cells. Brief heating of the fraction changed its capacity to produce organised mesodermal tissues, such as notochord and somite, and the frequency of induction of undifferentiated cells was reduced. By inserting the urea-soluble fraction into the blastocoel of an early gastrula, embryos without epidermis were obtained. Some of the embryos consisted of undifferentiated mesodermal and endodermal cells, but in the remaining ones small fragments of notochord, small numbers of somites and pronephros developed, enclosed by endodermal cells.     

Skeletogenic potential of induced secondary mesenchyme cells derived from the presumptive ectoderm in echinoid embryos

 During the normal development of echinoids, an animal cap consisting of 8 mesomeres in a 16-cell stage embryo differentiates exclusively into ectoderm. Micromeres in an embryo at the same stage differentiate into primary mesenchyme cells (PMC) and coelomic pouch constituents. An animal cap and a quartet of micromeres were isolated from a 16-cell stage embryo and recombined to make a chimeric embryo devoid of presumptive endoderm and secondary mesenchyme cells (SMC). The PMC in the chimeric embryo were completely removed at the mesenchyme blastula stage. The PMC-depleted chimeric embryos formed an archenteron derived from the mesomeres. Some secondary mesenchyme-like cells (induced SMC) were released from the archenteron tip. A considerable fraction of the induced SMC formed the typical mesenchyme pattern after migrating into the vegetal region, synthesized skeletogenic mesenchyme cell-surface protein (msp130) and produced the larval skeleton. These findings indicate that induced SMC derived from the presumptive ectoderm have the same nature as natural SMC in both the timing of their release and their skeletogenic potential expressed in the absence of PMC. Received: 14 November 1996 / Accepted: 30 December 1996     

Activin-like signaling activates Notch signaling during mesodermal induction

Both activin-like signaling and Notch signaling play fundamental roles during early development. Activin-like signaling is involved in mesodermal induction and can induce a broad range of mesodermal genes and tissues from prospective ectodermal cells (animal caps). On the other hand, Notch signaling plays important roles when multipotent precursor cells achieve a specific cell fate. However, the relationship between these two signal pathways is not well understood. Here, we show that activin A induces Delta-1, Delta-2 and Notch expression and then activates Notch signaling in animal caps. Also, in vivo, ectopic activin-like signaling induced the ectopic expression of Delta-1 and Delta-2, whereas inhibition of activin-like signaling abolished the expression of Delta-1 and Delta-2. Furthermore, we show that MyoD, which is myogenic gene induced by activin A, can induce Delta-1 expression. However, MyoD had no effect on Notch expression, and inhibited Delta-2 expression. These results indicated that activin A induces Delta-1, Delta-2 and Notch by different cascades. We conclude that Notch signaling is activated when activin-like signaling induces various tissues from homogenous undifferentiated cells.     

The pattern of protein and glycoprotein synthesis in presumptive lens and non-lens ectoderm of the chicken embryo

Summary Lens induction is a classic example of the tissue interactions that lead to cell specialization during early vertebrate development. Previous studies have shown that a large region of head ectoderm, but not trunk ectoderm, of 36 h (stage 10) chicken embryos retains the potential to form lenses and synthesize the protein δ-crystallin under some conditions. We have used polyacrylamide gel electrophoresis and fluorography to examine protein and glycoprotein synthesis in presumptive lens ectoderm and presumptive dorsal (trunk) epidermis to look for differentiation markers for these two regions prior to the appearance of δ-crystallin at 50 h. Although nearly all of the proteins incorporating³H-leucine were shared by presumptive lens ectoderm and trunk ectoderm, these two regions showed more dramatic differences in the incorporation of³H-sugars into glycoproteins. when non-lens head ectoderm that has a capacity for lens formation in vitro was labeled, a hybrid pattern of glycoprotein synthesis was discovered: glycoproteins found in either presumptive lens ectoderm or trunk ectoderm were oftentimes also found in other head ectoderm. Therefore, molecular markers have been identified for three regions of ectoderm committed to different fates (lens and skin), well before features of terminal differentiation begin to appear in the lens.     

The effect of aging on the neural competence of the presumptive ectoderm and the effect of aged ectoderm on the differentiation of the trunk organizer inCynops pyrrhogaster

Summary The effect of aging on the neural competence of the presumptive ectoderm of the early gastrula, and the effect of aged ectoderm on the differentiation of the still uninvaginated dorsal blastoporal lip at the small yolk-plug stage — representing the trunk organizer — were examined by the sandwich method inCynops pyrrhogaster.The presumptive ectoderm to be used as reaction system was taken from 0 to 36 h exogastrulae obtained by operation at the early gastrula stage and combined with trunk organizer. In the 0 to 12 h explants typical trunktail structures were formed. With further aging of the presumptive ectoderm a decrease in frequency of spinal cord, notochord, and muscle and a simultaneous increase in frequency of mesenchyme and mesothelium were observed. In the 30 and 36 h explants neural competence had largely disappeared, the frequency of notochord and muscle become very low and their differentiation very poor, whereas the frequency of mesenchyme and mesothelium reached very high levels.We infer a reciprocal relationship between the induced spinal cord and the differentiation of notochord and muscle, as well as a transformation of notochordal material into mesenchyme and mesothelium under the influence of the aged ectoderm. The mode of action of the trunk organizer in normal development is discussed.     

Changes of the cell surface charge of amphibian ectoderm after induction

Summary 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 -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.     

Unexpected activities of Smad7 in Xenopus mesodermal and neural induction

Neural induction is widely believed to be a direct consequence of inhibition of BMP pathways. Because of conflicting results and interpretations, we have re-examined this issue in Xenopus and chick embryos using the powerful and general TGFβ inhibitor, Smad7, which inhibits both Smad1- (BMP) and Smad2- (Nodal/Activin) mediated pathways. We confirm that Smad7 efficiently inhibits phosphorylation of Smad1 and Smad2. Surprisingly, however, over-expression of Smad7 in Xenopus ventral epidermis induces expression of the dorsal mesodermal markers Chordin and Brachyury. Neural markers are induced, but in a non-cell-autonomous manner and only when Chordin and Brachyury are also induced. Simultaneous inhibition of Smad1 and Smad2 by different approaches does not account for all Smad7 effects, indicating that Smad7 has activities other than inhibition of the TGFβ pathway. We provide evidence that these effects are independent of Wnt, FGF, Hedgehog and retinoid signalling. We also show that these effects are due to elements outside of the MH2 domain of Smad7. Together, these results indicate that BMP inhibition is not sufficient for neural induction even when Nodal/Activin is also blocked, and that Smad7 activity is considerably more complex than had previously been assumed. We suggest that experiments relying on Smad7 as an inhibitor of TGFβ-pathways should be interpreted with considerable caution.