代谢抑制剂对有尾类胚胎表皮细胞兴奋性的影响

It has been reported that the cells in atypical epidermis, which developed from the in vitro cultured ectoderm isolated at early gastrula, showed very low excitability or were even non-excitable at 6 V when examined electro-physiologically. If non-excitable explants were treated with energy supplying substances, such as glucose, the action potential (AP) appeared quickly. It indicates that, the excitability of epidermis cells is related to their energy metabolism. In order to verify the above proposition the effects of metabolic inhibitors on the excitability of the epidermis cells were examined using electrophysiological technique. Two kinds of explants were used: explants which developed from the epidermis underlaid with mesoderm isolated at early neurula (epidermis vesicle) and explant which developed from the ectoderm isolated at early gastrula (atypical epidermis). In all experiments explants were stimulated extracellularly and APs were recorded intracellularly. The specimens were stimulated with electric stimulus at 6 V first, and, if they displayed AP, the strength was lowered to determine the stimulus threshold to evoke AP. The duration of stimulus was fixed at 1 ms. The ratio of the resting potential value during treatment to the original value was taken as index of change level of the resting potential (RP). During treatment of epidermis vesicle with 1 mM NaN3 or 1 mM NaCN or 0.1 mM 2,4-dinitrophenol (DNP), the excitability of the epidermis cells was reduced: the stimulus threshold gradually increased and the cells in most explants lost the excitability. The cells became excitable after the drugs were washed out.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogeneous energy supply and excitability of cells in embryonic atypical epidermis of Cynops cultured in vitro

Cells of in vitro cultured epidermis explants of ectoderm isolated at early gastrula stage,showed only weak excitability or even non-excitable at 6V when examined electrophysiologically.If non-excitable explants were treated with 100 mM glucose,the action potential (AP) appeared and within 1 hr reached its maximum.At the same time,their stimulus threshold became lowered gradually.And,if the glucose was washed out,AP gradually disappeared.If explants were treated with glucose of different concentrations,the percentage of explants which displayed AP increased with the increase of glucose concentration.When explants with approximately the same original stimulus threshold were treated with glucose of different concentrations,the stimulus threshold became lowered more in the more concentrated solution.If explants with different original stimulus thresholds were treated with glucose of the same concentration,the lowering of stimulus threshold was more obvious in those with higher original stimulus threshold.Other energy supplying substances used showed similar effect.     

Mesodermalizing Factors in Fetal Calf Serum

The inducing activities of fetal calf serum (FCS) and fetal calf serum heated for a short time (HFCS) were tested with early gastrula presumptive ectoderm of the newt, Cynops pyrrhogaster as a reactor. Inducing activity was measured by sitting-drop culture of pieces of ectoderm before and after treatment with Ca, Mg-free solution (CMF) in culture medium contained FCS or HFCS (10–50%) as inductor. Results showed that FCS did not induce mesodermalization of ectoderm, before or after treatment with CMF, and that HFCS induced mesodermalization only of ectoderm treated with CMF. The treated ectoderm cells differentiated into mesoderm cells such as muscle and notochord. The induction increased with increase in the duration of CMF treatment and with the concentration of HFCS in the medium. This indicates that FCS changes into mesodermalizing material when heated for a short time and that mesodermal induction by HFCS depends on some effect of CMF on presumptive ectoderm. Since CMF was found to be a neuralizing factor, activation of presumptive ectoderm with a neuralizing factor is probably a prerequisite for mesodermal induction with HFCS.     

Distribution of differentiation potentials and the conditions for their realization in the amphibian neuroectoderm

The X. laevis neuroectoderm (NE) at the mid and late gastrula stages is capable to form mesoderm in vitro after its separation from mesoderm. This capacity is inherent in posterior 2/3 of NE underlied by axial mesoderm in the embryo and forming deuterencephalic and trunk regions of the brain in the normal development. The archencephalic 1/3 of NE of the late gastrula, underlied in the embryo by prechordal plate, is capable of differentiation into archencephalic regions of the brain, rather than into mesoderm. For the typical differentiation of archencephalic NE to be realized, it should be surrounded by the outer ectoderm layer. In the absence of the latter, the whole explant develops into retina and brain only. Inside the closed explants, ectomesenchyme and melanophores arise and the eye material is subdivided into retina and pigmented epithelium. The archencephalic NE, dissociated to individual cells and wrapped into epidermis, forms much more ectomesenchyme and melanophores than the usual NE explants.     

In vitro pancreas formation from Xenopus ectoderm treated with activin and retinoic acid

In the present study, isolated presumptive ectoderm from Xenopus blastula was treated with activin and retinoic acid to induce differentiation into pancreas. The presumptive ectoderm region of the blastula consists of undifferentiated cells and is fated to become epidermis and neural tissue in normal development. When the region is isolated and cultured in vitro, it develops into atypical epidermis. Isolated presumptive ectoderm was treated with activin and retinoic acid. The ectoderm frequently differentiated into pancreas-like structures accompanied by an intestinal epithelium-like structure. Sections of the explants viewed using light and electron microscopy showed some cells clustered and forming an acinus-like structure, including secretory granules. The pancreas-specific molecular markers insulin and XIHbox8 were also expressed in the treated explants. The pancreatic hormones, insulin and glucagon, were detected in the explants using immunohistochemistry. Therefore, sequential treatment with activin and retinoic acid can induce presumptive ectoderm to differentiate into a morphological and functional pancreas in vitro. When ectoderm was immediately treated with retinoic acid after treatment with activin, well-differentiated pronephric tubules were seen in a few of the differentiated pancreases. Treatment with retinoic acid 3-5 h after activin treatment induced frequent pancreatic differentiation. When the time lag was longer than 15h, the explants developed into axial mesoderm and pharynx. The present study provides an effective system for analyzing pancreas differentiation in vertebrate development.     

Excitability changes of presumptive ectoderm following mesodermal induction

Dissociated ectodermal cells of the early newt gastrula which have been treated with CMF (Ca-Mg-free saline) for 5 hr differentiate into muscle cells when cultured in HFCS (heated fetal calf serum) for up to 9-12 days. Similarly dissociated cells placed into FCS (fetal calf serum) culture differentiate into epidermis. Differences in cell-cluster formation have been found between HFCS and FCS in early cell cultures (6 hr), and membrane excitability phenomena associated with the differentiation of these clusters into the muscle cells or epidermal cells have been investigated, respectively. The HFCS cultures consist of cell clusters which have few of microvilli at their surfaces and which form loose contacts by means of lamellipodia. FCS cultures consist of cell clusters which have numerous microvilli at their surfaces and which make tight contacts between cells by means of ridge-structure precursors. The different reaggregation pattern of dissociated ectoderm cells in HFCS reflects changes in the cell membrane surface induced by HFCS. The sequential genesis of action potentials in cells destined to form muscle cells in HFCS is very similar to those produced by somitic muscle cells in vivo and their ionic dependence for generating action potentials is related to epidermal action potentials in vitro (FCS).     

Homoiogenetic regulation through the ectoderm on localized expression of the hatching gland phenotype in the head area of Xenopus embryos

Ectoderm pieces explanted from embryos of Xenopus laevis were cultured and examined for differentiation of hatching gland cells, using immunoreactivity against anti-XHE (Xenopus hatching enzyme) as a marker. The anterio-dorsal ectoderm excised from stage 12-13 (mid-late gastrula) embryos developed hatching gland cells. Meanwhile, the posterio-, but not the anterio-dorsal ectoderm from stage 11 (early gastrula) embryos developed these cells, although it is not fated to do so during normogenesis. This hatching gland cell differentiation from stage 11 posterior ectoderm was not affected by conjugated sandwich culture with the mesoderm but was suppressed when explants contained an anterior portion of the ectoderm. Conjugated cultures of anterior and posterior portions of the ectoderm in various combinations indicated that differentiation of hatching gland cells from stage 11 posterior and stage 12 anterior portions was suppressed specifically by stage 11 anterior ectoderm. Northern blot analyses of cultured explants showed that XHE was expressed in association with XA-1, suggesting its dependence on the anteriorized state. These results indicate that the planar signal(s) emanating from stage 11 anterior ectoderm participates in suppression of the expression of the anteriorized phenotype so that an ordered differentiation along the anteroposterior axis of the surface ectoderm is accomplished.     

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.     

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.     

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.