Embryonic induction and cation concentrations in amphibian embryos

Explanted ectoderm from early gastrulae of Triturus alpestris was treated with the Na-K ionophore gramicidin (10(-9) to 10(-5) M) and the Ca-ionophore A 23187 (10(-7) to 10(-5) M). The ectoderm developed almost exclusively to atypical epidermis as in the control explants. When the ectoderm was treated with ouabain (10(-4) M), intracellular Na+ increased about 4.4-fold and K+ was reduced by half. Mesenchyme cells in small number differentiated in about 40% of the ouabain-treated explants. The time course of total Na+ and K+ ion concentrations was measured over a period of 72 h in ectoderm of T. alpestris after induction with vegetalizing factor and in control explants. In the first 15 h after explantation, no significant differences between control and induced explants were found. Thereafter, the steady state concentration of K+ decreased in the induced explants, whereas the steady-state concentration of Na+ slightly increased. The membrane resting potential recorded intracellularly of ectoderm sandwiches from early gastrula stages was found to be -41.3 mV in control and -59.3 mV in induced explants. From the specific conductances and permeabilities of non-induced and induced cells it is concluded that the induction process leads to a differentiation of the cell membrane, which acquires the characteristics of ionic selectivity. Ectoderm from Ambystoma mexicanum forms neural or neuroid tissue, mesenchyme and melanophores after explantation in salt solution in up to 50% of the explants without any additions. Isolated Ambystoma ectoderm is therefore not suitable for test experiments.     

Types of Neural Tissues Induced through the Presumptive Notochord of Newt Embryo

Neural induction through the presumptive notochord was tested by means of the sandwich method. The result disclosed that the notochord was a potent inducer of neural tissues not only in the ectoderm of gastrula but also in the ventral ectoderm of neurula and early tail-bud embryos. Structures formed by the induced neural tissue varied greatly. They can be classified into three types. (1) Tubular: the neural tissues induced in explants containing abundant mesenchymes always formed long tubular structures. The shapes of these neural tubes showed considerable variation; moreover, they were atypical and none formed the regular structure of the spinal cord. This type was most frequent, being found in about 50% of the explants. (2) Inverted: this type was produced when the explant contained mesenchymal component. Consequently, the epithelium of explants was missing. Nevertheless, a considerable mass of neural tissue was always induced. It was noticed that the induced neural tissues were invariably inside out; this type was found in about 30% of the explants. (3) Archencephalic: this was the only type to form the regular structure, i.e., the archencephalon. Formation of the archencephalon was limited solely to those explants containing only a few mesenchymes; this type was found in about 20% of the cases. As described above, it was found that the neural tissues induced by the same inducer of the notochord were not uniform but varied in type. Further, it was shown that the types of neural tissue differed according to different quantities of the surrounding mesenchyme. Based on these facts, it is to be concluded that it is not the inducer of notochord, but the surrounding mesenchyme that is of primary importance for the determination of the types of neural tissue.     

Head and trunk-tail organizing effects of the gastrula ectoderm of Cynops pyrrhogaster after treatment with activin A

Differentiation tendency and the inducing ability of the presumptive ectoderm of newt early gastrulae were examined after treatment with activin A at a high concentration (100 ng/ml). The activin-treated ectoderm differentiated preferentially into yolk-rich endodermal cells. Combination explants consisting of three pieces of activin-treated ectoderm formed neural tissues and axial mesoderm along with endodermal cells. However, the neural tissue was poorly organized and never showed any central nervous system characteristics. When the activin-treated ectoderm was sandwiched between two sheets of untreated ectoderm, the sandwich explants differentiated into trunk-tail or head structures depending on the duration of preculture of activin-treated ectoderm in Holtfreter's solution. Short-term (0–5 h) precultured ectoderm induced trunk-tail structures accompanied by axial organs, alimentary canal and beating heart. The arrangement of the explant tissues and organs was similar to that of normal embryos. However, archencephalic structures, such as forebrain and eye, were lacking or deficient. On the other hand, long-term (10–25 h) precultured ectoderm induced archencephalic structures in addition to axial organs. Lineage analysis of the sandwich explants using fluorescent dyes revealed that the activin-treated ectoderm mainly differentiated into endodermal cells and induced axial mesoderm and central nervous system in the untreated ectoderm. These results suggest that activin A is one of the substances involved in triggering endodermal differentiation and that the presumptive ectoderm induced to form endoderm displays trunk-tail organizer or head organizer effects, depending on the duration of preculture.     

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.     

The Ectomesenchymal-endodermal Interaction System (EEIS) of Triturus alpestris in Tissue Culture

Differentiation of visceral cartilage was studied in the Ectomesenchymal-endodermal Interaction System (EEIS), a hanging-drop culture system containing a piece of head neural fold tissue from behind the prospective ear region of a Triturus alpestris neurula together with a piece of ventrolateral pharynx endoderm from the same embryo. Cartilage cells differentiated only from those neural crest cells which have been in contact with the pharynx endoderm. Cartilage differentiation occurred neither in cultures containing neural fold or pharynx endoderm alone nor in distance cultures in which both explants were separated from each other at a greater distance than usual. However, in some critical cultures of the EEIS containing pharynx endoderm, excised more ventrally from the regular site, accumulations of ectomesenchymal cells were observed near the endoderm which were not transformed into cartilage.
The differentiation of cartilage was classified into three stages: (1) prechondroblasts (day 1 to 5), (2) chondroblasts (about day 6) and (3) chondrocytes (about day 10).
The data obtained suggested that cellular contact between the two inductively correlated tissues was a prerequisite for the transmission mechanism while mitotic cell divisions were mainly responsible for the spread of the inductive message within the responding ectomesenchymal cells.     

Positive and Negative Regulation of the Differentiation of Ventral Mesoderm for Erythrocytes in Xenopus laevis

To elucidate the mechanism of determination and regulation of hemopoiesis in the early Xenopus embryo, explants of dorsal and ventral mesoderm from various stage embryos were cultured alone or combined with various tissues derived from the same stage embryo. Western blot analysis of larvae-specific globin expression using monoclonal antibody L5.41 revealed that extensive erythropoiesis occurred in the explants of ventral mesoderm from st. 22 tailbud embryo, but not in those of dorsal mesoderm. Experiments using combined explants at this stage demonstrated that the in vitro differentiation of erythrocytes in the ventral mesoderm could be completely inhibited by the dorsal tissue, including neural tube, notochord, and somite mesoderm, but not by other mesoderms, gut endoderm, or forebrain. Subsequent explant studies showed that the notochord alone is sufficient for this inhibition. Furthermore, the ventral mesoderm explant from the st. 10⁺ early gastrula embryo was not able to differentiate into erythroid cells. However, small amounts of globin were expressed if ventral mesoderm of this stage was combined with animal pole cells which were mainly differentiated to epidermis. This stimulation was enhanced when both tissues were excised together without separation, while none of the other parts of st. 10⁺ embryo had this stimulatory effect. These observations found in the combined explants suggest that in vivo interactions between the ventral mesoderm and adjacent tissues are important for normal development of erythroid precursor cells.     

Composition of proteoglycans synthesized by rabbit aortic explants in culture and the effect of experimental atherosclerosis

The synthesis of proteoglycans by aorta explants from rabbits with diet-induced atherosclerosis and controls was studied by 35S-incorporation. Proteoglycans were isolated under dissociative conditions from incubation medium and from arterial explants. Additionally, the tissue proteoglycans that were not extracted by 4 M guanidine-HCl were solubilized by digestion of the tissue by elastase in the presence of proteinase inhibitors. The residual tissue was hydrolyzed by papain and glycosaminoglycans were isolated. The atherosclerotic aorta tissue incorporated twice the amount of 35S into proteoglycans than observed for controls; in both groups about 70% of the label incorporated into the tissue was noted in the proteoglycans extracted by guanidine-HC;, while about 30% of the total 35S-labeled proteoglycans synthesized by the explants were found in the media. Atherosclerotic tissue incorporated 35S predominantly into chondroitin sulfate proteoglycans when compared to control tissue. The chondroitinase ABC-digestable proteoglycans that were extracted by guanidine-HCl from atherosclerotic tissues were of larger molecular size than those from control tissue, but the core proteins from these preparations were similar. The heparan sulfate proteoglycan that was obtained by dissociative extraction from atherosclerotic tissue had greater amounts of N-acetyl and lesser amounts of N-sulfate ester groups than the preparation from control tissue. Digestion of the tissue by elastase yielded heparan sulfate proteoglycan as the major constituent in both groups, although atherosclerotic tissue contained relatively small amounts of this proteoglycan. The residual tissue from both groups contained chondroitin sulfate and heparan sulfate as the major glycosaminoglycans with the latter showing a decrease with atherosclerosis. Atherosclerotic tissue secreted into the medium about two-fold more 35S-labeled proteoglycans with larger molecular size than control tissue; proteoglycans of the heparan sulfate and chondroitin sulfate types were the major constituents in the culture medium of both tissues. Thus, proteoglycans undergo both quantitative and qualitative changes in atherosclerosis, reflecting the enhanced smooth muscle cell activity. These changes are potentially important in modulating lipoprotein binding and hemostatic properties, as well as fibrillogenesis of the arterial wall.     

Homoiogenetic Neural Induction in Xenopus Chimeric Explants

We previously raised monoclonal antibodies specific for epidermis (7) and neural tissue (8) of Xenopus for use as markers of tissue differentiation in induction experiments (8). Here we have used these monoclonal antibodies to examine homoiogenetic neural induction, by which cells induced to differentiate to neural tissues can in turn induce competent ectoderm to do the same. Presumptive anterior neural plate excised from late gastrulae of Xenopus laevis was conjugated with competent ectoderm from the initial gastrula of Xenopus borealis , either side by side or with their inner surfaces together. The chimeric explants enabled us to distinguish induced neural tissues from inducing neural tissues. In both types of explant, neural tissues identified by the neural tissue-specific antibody, NEU-1, were induced in the competent ectoderm by the presumptive anterior neural plate. The results suggest that homoiogenetic neural induction does occur in Xenopus embryos.     

Mesoderm and Neural Inductions on Newt Ectoderm by Activin A

Mesoderm-inducing activity of human recombinant activin A was examined on presumptive ectoderm of the Japanese newt, Cynops pyrrhogaster , by using the animal cap assay, Activin A induced neural tissues and mesodermal tissues such as brain, neural tube, notochord, muscle, mesenchyme, coelomic epithelium and blood-like cells after 14 days cultivation. These tissues were induced by activin A at concentrations ranging from 0.5– 100 ng/ml. Dose-dependent inducing activity of activity A on newt ectoderm was slightly different from that on other animals, including Xenopus . Wide range of concentration of activin A (0.5– 100 ng/ml) could induce the neural tube, notochord, mesenchyme and coelomic epithelium on the newt ectoderm. Though the percentage of induced explants (two out of 23 explants, 8.7%) was low, the pulsating heart was induced. This paper showed first that activin could induce the mesodermal and neural tissues in newt presumptive ectoderm. Since activin homologues were present In Xenopus and chick embryos, it is likely that activin may be one of the natural inducers in a wide range of species.     

DIFFERENCE IN INDUCTIVE EFFECT OF LIVER TISSUES WITH AND WITHOUT PERISINUSOIDAL BASEMENT MEMBRANE

Differential inductive capacities among liver tissues of several animals were examined by anticipating the correlation between the capacity and the completness of perisinusoidal basement membrane.
The reacting tissue was competent ectoderm of gastrula of Triturus pyrrhogaster , and the inductive effects of livers on the ectoderm were tested by explantation method. The inductive effect of livers being devoid of the membrane (chick and guinea pig) was neural and the tissues having the dense well-developed membrane (reptiles) produced an assembly of neural and meso-dermal tissues, such as notochord and somite or muscle. The livers with the membrane being of intermediate grade of development ( calf, Triturus and mouse) induce mesodermal tissues, but not frequently, together with neural tissue or alone. The liver tissue was more active in mesodermal induction in proportion to the completeness of the perisinusoidal basement membrane.
On the basis of these data the difference in inductive capacity among liver tissues from different kinds of animals were discussed.     

Sucrose-regulated expression of a chimeric potato tuber gene in leaves of transgenic tobacco plants

Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato tubers. Class-I patatin genes encode 98 to 99% of the patatin mRNA in tubers, but are not normally expressed in other tissues. They are not totally tuber-specific; however, since they can be induced to express at high levels in other tissues under conditions of sink limitation or in explants cultured on medium containing elevated levels of sucrose. To examine the evolution of the mechanisms that regulate patatin gene expression, we introduced a chimeric patatin--glucuronidase (GUS) gene containing 2.5 kb of 5 flanking sequence from the Class-I potato patatin gene PS20 into tobacco plants. The construct was not expressed at significant levels in leaves of juvenile plants or plantlets cultured in vitro, but was expressed at high levels in explants cultured on medium containing 0.3 to 0.4 M sucrose. While there were differences in the expression of the chimeric gene between transgenic tobacco and potato plants, the pattern of sucrose induction was very similar. These results suggest that the mechanism that controls patatin gene expression in potato tubers evolved from a widely distributed mechanism in which gene expression is regulated by the level of available photosynthate.     

Localization and activity of various peptidases in germinating barley

Summary Germinating barley grains contain at least eight different peptidases: three carboxypeptidase (pH optima 4.8, 5.2, and 5.7), three aminopeptidases which act on aminoacyl--naphthylamides (pH opitima in the hydrolysis of di- and tripeptides at pH 5.8–6.5), and two peptidases which hydrolyse Ala-Gly and Leu-Tyr optimally at pH 7.8 and 8.6 respectively. We have determined the activities of these enzymes in the different tissues of non-germinated grains and followed the changes in the activities during a 5-day germination at 16°C.The aleurone layers contain high activities of all three groups of peptidases; there are no changes in the activities of the five aminopeptidases on germination, while the carboxypeptidases exhibit a small increase of activity. The starchy endosperms contain high carboxypeptidase activities, which increase during germination, but are totally devoid of the five aminopeptidases.All the peptidases exhibit high activities in the scutella; the carboxypeptidases and the enzymes acting on Ala-Gly and Leu-Tyr increase in activity during germination, while the naphthylamidase activities remain constant.The three peptidase groups occur in the seedling as well, but compared to the other tissues the carboxypeptidase activities are very small and the naphthylamidase activities are very high. The last-named enzymes seem to be characteristic for growing tissues.The starchy endosperm contains about two thirds of the total reserve proteins of the grain. Its internal pH during germination is 5.0–5.2, a value at which all the carboxypeptidases are highly active. As these enzymes are present in high concentrations in this tissue, it is probable that they have a central role in the mobilization of the reserve proteins during germination. The high peptidase activities of the scutellum, on the other hand, suggest that some of the hydrolysis products are absorbed as peptides and these are further hydrolysed to amino acids in this tissue.Abbreviations used DTT dithiothreitol - GA₃ gibberellic acid - -NA -naphthylamide - TNBS 2,4,6-trinitrobenzene sulphonic acid - Z- N-carbobenzoxy     

Improvement of adventitious shoot formation from carnation leaf explants

Adventitious shoot formation from leaf explants of carnation (Dianthus caryophyllus L.) was investigated. The two leaves from one node of in vitro-grown plants showed different shoot-forming potential, depending on the order in which the leaves were removed from the stem. The leaf removed second formed more shoots and also had a large amount of adhering stem tissue. Explants with equal amounts of adhering stem tissue were obtained by making two incisions through the fused leaf bases, prior to their removal, resulting in an improved shoot formation. The procedure developed for leaf explants from in vitro-grown plants was also applied to leaf explants from greenhousegrown plants. Shoot formation from leaf explants taken from greenhouse-grown plants was further improved by cutting the leaf explant longitudinally into two parts.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid     

Interaction of induced and uninduced cells during primary induction

Summary UsingTriturus pyrrhogaster embryos, the effects of uninduced cells on the differentiation of induced cells were investigated. The inducing stimulus was given to the presumptive ectoderm of early gastrulae by treatment with protein sooution from guinea pig bone-marrow. Mesodermal induction was evoked in the ectodermal explants. After the treatment, some of the ectodermal explants were cut into pieces 1/8 of their original size and combined with untreated presumptive ectoderm. Mesodermal tissues were differentiated in the combined explants too, but the mesodermal tissues evoked in these combined ectodermal explants were different in their regional characters from these in uncombined explants; dorsal structures, such as notochrod and muscle, were observed predominatly in the latter, whereas the dominant structures observed in the former were ventral ones, such as mesothelium and mesenchyme. The shifting of the regional characters in the combined explants was regarded as the result of an unknown effect from the uninduced cells.     

In Vitro Control of the Embryonic Form of Xenopus laevis by Activin A: Time and Dose-Dependent Inducing Properties of Activin-Treated Ectoderm

The inducing properties of activin-treated ectoderm of Xenopus laevis were examined by the preculture and sandwich culture methods. Presumptive ectodermal sheets of the late blastula were treated with 10–100 ng/ml of activin A and precultured for 0–7 hr in Steinberg's solution. They were then sandwiched between two sheets of ectoderm from other late blastulae. Ectoderm precultured for a short term induced trunk-tail structures, whereas that precultured for a long term induced head structures in addition to trunk-tail structures. These time-dependent changes in inducing properties occurred more rapidly when the concentration of activin A was higher. These results suggest that the activin-treated ectoderm functioned as a "head organizer" or "trunk-tail organizer" depending upon the concentration of activin A and the duration of preculture.
To trace the cell lineage of the sandwich explants, activin-treated ectoderm labeled with fluorescein-dextran-amine (FDA) was used in this study. The explants sandwiching the long term-precultured ectoderm formed head structures equipped with non-labeled neural tissues (brain and eye) as well as FDA-labeled mesodermal tissues. These results suggest that the activin-treated ectoderm mainly differentiates into mesodermal tissues and induces neural tissues as the organizer does in normal development.     

DIFFERENTIATION OF PARTIALLY MESODERMALIZED ECTODERM; HOMOIOGENETIC AND HETEROGENETIC INDUCTION BY PRIMARILY INDUCED PART OF THE ECTODERM

Using embryos of the Japanese newt, Cynops pyrrhogaster , homoiogenetic and heterogenetic induction were investigated in the partially mesodermaelzed presumptive ectoderm. Half of the isolated presumptive ectoderm was placed in contact with the swimbladder of the crucian carp, Carasius auratus , for 15 or 60 min, while the other half was stained with Nile blue sulfate at the same time. The distribution of the stained cells in the tissues evoked in the explants was examined after cultivation for 10 days.
Some mesodermal tissues were composed of both stained and unstained cells. This indicates homoiogenetic induction by the primarily induced part of the ectoderm on the other half. The neural and epidermal tissues in the explants were composed of stained cells only, except in one case. We conclude that the neural tissues are derived from cells not placed in contact with the swimbladder and that they are induced by the primarily induced part of the ectoderm.     

Electromotility of outer hair cells from the cochlea of the echolocating bat,Carollia perspicillata

Isolated outer hair cells (OHCs) and explants ot the organ of Corti were obtained from the cochlea of the echolocating bat, Carollia perspicillata, whose hearing range extends up to about 100 kHz. The OHCs were about 10–30 m long and produced resting potentials between-30 to -69 mV. During stimulation with a sinusoidal extracellular voltage field (voltage gradient of 2 mV/m) cyclic length changes were observed in isolated OHCs. The displacements were most prominent at the level of the cell nucleus and the cuticular plate. In the organ of Corti explants, the extracellular electric field induced a radial movement of the cuticular plate which was observed using video subtraction and photodiode techniques. Maximum displacements of about 0.3–0.8 m were elicited by stimulus frequencies below 100 Hz. The displacement amplitude decreased towards the noise level of about 10–30 nm for stimulus frequencies between 100–500 Hz, both in apical and basal explants. This compares well with data from the guinea pig, where OHC motility induced by extracellular electrical stimulation exhibits a low pass characteristic with a corner frequency below 1 kHz. The data indicate that fast OHC movements presumably are quite small at ultrasonic frequencies and it remains to be solved how they participate in amplifying and sharpening cochlear responses in vivo.Abbreviations BM basilar membrane - FFT fast Fourier Transfer - IHC inner hair cell - OHC outer hair cell     

Amino acid transport and protein synthesis in induced ectoderm of amphibian embryos

Summary Isolated gastrula ectoderm ofTriturus alpestris orAmbystoma mexicanum was induced by the vegetalizing factor. Protein synthesis in the induced and uninduced control explants was measured by double labelling with³H-and¹⁴C-amino acids after different periods of cultivation. Slight differences were observed in the pattern of nuclear proteins after 12 h of cultivation and in the pattern of cytoplasmic proteins after 48 h of cultivation.The uptake of leucine started to increase in induced explants after 48 h of cultivation and after 96 h was about 50 times greater than in uninduced control explants. The uptake is reduced under partially anaerobic conditions. Ouabain inhibits the uptake by about 50%.     

Influence of cyclic nucleotides on amphibian ectoderm

Summary Isolated amphibian (Triturus alpestris) gastrula ectoderm was treated with cyclic nucleotides for 24 h and cultured up to 12 days. Explants treated with$cyclic N⁶-Monobutyryl-adenosine-35-monophosphate, cyclic Dibutyryladenosine-35-monophosphate and cyclic Dibutyrylguanosine-35-monophosphate in a concentration of 10^–3 and 10^–5 M did not differentiate into mesoderm- or endoderm-derived tissues. The number of explants with small neural and neuroid structures did not exceed the percentage found in the control series. Inductions could also not be obtained when ectoderm was dissociated prior to the treatment with cyclic nucleotides, or when theophylline (which inhibits phosphodiesterase) was added to the culture medium. The results are discussed with regard to the possible mode of action of the vegetalizing factor.