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.     

影响有尾类离体胚胎表皮细胞兴奋性的实验

It has been reported that in Cynops orientalis the cells in the atypical epidermis resulted from isolation of the ectoderm from the early gastrula and cultured in vitro possesses very low excitability. The great majority of the explants didn't display action potential (AP) with electric stimulus up to 6 V duration 1 ms). If, however, the epidermis was taken from the flank region of a neurula with the underlying mesoderm, in such explant the epidermis cells became excitable at a lower threshold of stimulus. The mesoderm seemed to exert some influence upon the epidermis. In order to study whether tissues other than mesoderm possess similar effect on the excitability of epidermis cells, different combinations of explants were made with the experimental embryological technique and their excitability was examined electrophysiologically. In all experiments the explants were stimulated extracellularly and the action potential was recorded intracellularly. The specimens were stimulated with electric stimulus at 6 V at first, and, if they displayed AP the strength was lowered to determine the threshold of stimulus to evoke AP. The duration of stimulus was fixed at 1 ms. For the control of all series, the ectoderm or epidermis was isolated at the same stage and cultured for the same length of period as the experimental explants but without any treatment. 1). The effect of calf serum. In this group the effect of calf serum was examined and following series were carried out: a) Gastrula ectoderm cultured in Holtfreter solution containing 10% calf serum; b) calf serum + agar as implant: calf serum was mixed with agar (1:1) and cut into small pieces when cooled, and the serum + agar pieces were wrapped with ectoderm; c) heated serum + agar, heated serum was mixed 1:1 with agar and the small pieces were wrapped with ectoderm. In all the experiments the excitability of the epidermis cells was raised more or less but the agar pieces containing fresh serum seemed to be more effective, perhaps due to the higher concentration of the serum and the intimate contact with the epidermis cells (Tab. 1) In the series with serum + agar as implant and in the series with heated serum + agar as implant, it occurred frequently that during cultivation the implants will be extruded and after the extrusion of the implant the remaining epidermis became less sensitive to stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neural differentiation of amphibian gastrula ectoderm exposed to phorbol ester

Summary Ectoderm from early gastrula stages of amphibians was isolated and treated with phorbol 12-myristate 13-acetate. The ectoderm formed neural tissue and in a few cases also mesenchyme and melanophores. The control explants formed atypical epidermis. In explants treated with phorbol 12-myristate 13-acetate the mitotic rate was increased.     

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.     

蝾螈胚胎表皮细胞膜的静息电位,输入电阻与其兴奋性的关系

用微电极细胞内记录技术研究了东方蝾螈胚胎表皮细胞膜的静电位、输入电阻与其兴奋性的关系,在兴奋性形成期间正常胚胎表皮细胞的静息膜电位逐渐增大,膜的输入电阻逐渐减小。与不显示兴奋性的离体非典型胚胎表皮细胞相比,显示兴奋性的膜电位较高,膜电阻较低。用葡萄糖处理非典型表皮,在兴奋性出现同时,细胞膜超极化,膜电阻减小。用哇巴因处理表皮囊泡,在兴奋性消失同时,细胞膜去极化。结果表明,细胞能量供应不足所造成的膜     

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.     

NEURAL COMPETENCE AND CELL LINEAGE OF GASTRULA ECTODERM OF NEWT EMBRYO

The change in the capacity to form neural structures was quantitatively analyzed in both intact and isolated ectoderms of Cynops pyrrhogaster gastrula. The frequency of explants with induced neural structures abruptly decreases between stage 12c and stage 13b in intact ectoderm, and between 12 hr and 18 hr preculture in isolated ectoderm. The quantitative analysis also made clear that the size of the cell population of induced neural structures was gradually reduced with the aging of the ectoderm. The authors simultaneously examined the cell proliferation of early gastrula ectoderm and confirmed that all ectodermal cells divided at least once within 18 hr at 23°C, after which the neural competence of the ectoderm completely disappeared.
The relationships between neural competence and cell lineage (cell generation) of the ectoderm are discussed in the light of these findings.     

INDUCTION OF THE FROG HATCHING GLAND CELL FROM EXPLANTED PRESUMPTIVE ECTODERMAL TISSUE BY LICL

When cells of the superficial layer explanted from the presumptive ectoderm of a Rana japonica early gastrula embryo at stage 10 were cultured in standard salt solution for 4–7 days, they differentiated into cement gland cells (CGCs), cilia cells (CCs) and common epidermal cells (CECs). When, however, these explants were treated with LiCl and transferred to Barth's solution, hatching gland cells (HGCs) and pigment cells were induced.
The optimum condition for inducing differentiation of HGC was treatment with 70 mM LiCl for 6–8 hr at 18°C. The best ability to react to the HGC-inducing stimuli resided in the superficial layer of the dorsal presumptive epidermis of the embryo at stage 10. Upon repeated stimulation, explants from stage 8 embryos underwent differentiation into nerve and pigment cells, whereas those from stage 11 embryos differentiated into CCs and CECs. Under optimum conditions, the total volume of HGCs induced amounted to about 70% of the explanted tissue. The culture media from LiCl-induced HGCs showed an apparent jelly-digesting activity, strongly indicating that the cells were functionally identical with those differentiated in situ .     

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.