摘要:

Summary1. Amphibian eggs are spherical, while the embryos are bilaterally symmetrical. The latter is manifested morphologically when gastrulation begins with the formation of the blastopore at a bilaterally symmetrical (vegetal‐dorsal) location on the surface of the embryo. To account for this change in symmetry two polarities (vectors or axes) are required. These need not go through the centre, but if they do, one will go through two poles, called ‘animal’ and ‘vegetal’ in the amphibian embryo, and the other will pass through two points on opposite sides of the egg, one at the ‘dorsal’ and one at the ‘ventral’ side. Together these two polarities define a plane of bilateral symmetry.2. It may be assumed that one polarity determines that gastrulation begins in the vegetal hemisphere, and the other that it begins at the dorsal side.3. Judging from the distribution of pigment in the cortex of the egg and that of the yolk‐hyaloplasm in the interior, an animal‐vegetal polarity is already present in the unfertilized egg. That cytoplasmic components are actually part of the material substrate of this polarity is evident from the fact that the pattern of gastrulation may be upset if the distribution of yolk‐hyaloplasm is deranged.4. At fertilization the pigment border is raised at the side opposite the fertilizing sperm, giving rise to the ‘grey crescent’. The latter confers the first visible bilateral symmetry on the egg, and in fact it determines the presumptive median plane, for blastopore formation begins in the midline of the grey crescent.The dorso‐ventral polarity imposed by the sperm is not irreversibly determined. By various experimental means, e.g. restriction of the oxygen supply, it may be inverted.5. In order to understand the mechanism of the polarities it is necessary to study the processes on which the effects of the polarities are exerted, viz. the process of invagination associated with the formation of the blastopore. It has been known for a long time that at the bottom of the blastoporal groove are located some large flask‐shaped cells, called ‘Ruffini's cells’. Various arguments can be mobilized to support the notion that these cells actually are engaged in pulling in the embryonic surface.6. These cells are the first representatives of a cell type different from the spherical cells which are typical of the early embryo. It may therefore be presumed that Ruffini's cells are the products of the first cell differentiation occurring during amphibian embryogenesis. And it may further be assumed that the polarities somehow control this process.7. A number of observations suggest that the animal‐vegetal polarity is in direct control of the differentiation, ensuring that Ruffini's cells are formed only in the vegetal hemisphere. This point has been corroborated by isolating in cultures small aggregates from various regions of the blastula. When this is done it is found that the only path of differentiation available to animal cells is the formation of small spherical aggregates composed of a mixture of ciliated and non‐ciliated cells. In contrast, in cultures of vegetal cells an outgrowth of cells occurs, and these cells share a number of properties with Ruffini's cells, and it is suggested that they are representatives of this cell type.8. The formation of these cells is suppressed by inhibitors of RNA synthesis and by anaerobiosis induced by KCN. Since oxidative metabolism is apparently required for the differentiation of Ruffini's cells ‐ gastrulation in the intact embryo is suppressed by anaerobiosis ‐ a number of carbohydrate metabolites were scrutinized for their effect on the formation on Ruffini's cells. It was found that at 10 mm lactate completely suppresses their appearance, and indeed all the other cell differentiations that can otherwise be observed in our cell cultures. Since there is a very steep animal‐vegetal cytoplasmic gradient in carbohydrate, the content being lowest at the vegetal pole, lactate might potentially be the agent of the animal‐vegetal polarity, but there are a number of facts which do not readily support this idea.9. If animal cells are explanted together with a few vegetal cells, some of the aggregates do not become ciliated, but rather exhibit an outgrowth similar to the one observed with vegetal cells. These animal cells have the same general shape as the vegetal Ruffini's cells, but they are smaller and more pigmented, typical ‘animal’ features. When the cultures are preserved, the cells undergo further differentiation, becoming either ‘mesenchyme’ cells, nerve cells, pigment cells and sometimes even muscle cells may be observed. In the normal embryo these differentiation patterns occur in that part of the animal hemisphere which becomes induced through contact with the vegetal material entering the blastocoel during gastrulation. Thus there is reason to assume that the induction occurring in our cultures is a miniature of the normal induction process.10. Just as in the sea‐urchin embryo, the animal cells in amphibia may become ‘vegetalized’ by addition of Li+to the culture medium.11. For various reasons it is likely that Ruffini's cells contain heparan sulphate, and in the belief that this substance might be the inductor proper, its effect was tested on animal cells. It turned out that in a concentration of 0·1 ppm it can alter the differentiation pattern of these cells, and we suggest that heparan sulphate, for the time being, is the most likely candidate for the role of primary inductor in the amphibian embryo.12. The edges of the blastoporal groove, and hence the formation of Ruffini's cells, proceeds gradually around the circumference of the embryo. The effect of the dorso‐ventral polarity therefore appears to be concerned with the time at which the cells undergo differentiation, imposing a spatial and a temporal gradient on this phenomenon. The second overt manifestation of the dorso‐ventral polarity, next to the formation of the grey crescent, concerns the size of the embryonic cells, the dorsal ones being always smaller than the ventral. This fact suggests the possibility that the polarity may exert its effect by interfering with the process of cell division.13. The cell divisions in the early embryo are distinguished by being synchronous; all cells are either undergoing mitosis or they are in interphase. The duration of the latter is typically very short. After a certain number of cell divisions, around 10, when the embryos are in the mid‐blastula stage, the synchrony is gradually lost, while the interphase becomes considerably prolonged. This peculiar behaviour suggests that the cytoplasm of the early embryonic cells contain some factor which ensures the synchrony. The well‐known presence in the early embryo of deoxyriboside‐containing material, in an amount corresponding roughly to the total amount of DNA residing in the cell nuclei after 10 cell divisions hinted that deoxyribosides might indeed be the ‘synchrony factor’.14. This idea was tested first on intact embryos. An excess of deoxyribonucleotides was injected into very early embryos. The result was developmental arrest at a pregastrula stage (no Ruffini's cells formed) in a large percentage of embryos. However, the number of cells was greater than in the controls, and the rate of cell division higher, indicating a delay in the transition to synchrony, thus supporting the proposed mechanism.Furthermore, the deoxynucleotides inhibited cell differentiation and an explanation of this was found in the fact that they also strongly inhibited RNA synthesis.15. The studies were extended to cell cultures. It was found that deoxyribosides inhibit the differentiation of animal as well as vegetal cells; instead, the cells go on dividing at least for another two rounds. The utilization of added deoxyribosides does not demonstrate that the endogenous substances are similarly utilized. That they are, was indicated by the following experiment: In the presence of cytosine arabinoside, an inhibitor of DNA synthesis de novo, the explanted cells go on dividing an unknownferentiation. But in either case these cells are larger (about four times) than the controls. This result suggests that in the experimental cultures the cells go on dividing as long as the cytoplasmic deoxyribosides last and then stop, while the controls synthesize their own DNA for two rounds of division before they undergo differentiation.16. It is now possible to suggest a mechanism for the dorso‐ventral polarity. First it affects the cell size such that the dorsal cells are the smallest. If the cytoplasmic deoxyribosides are evenly distributed at the outset, then small cells must be nearer exhaustion than large ones. A dorso‐ventral gradient in cell sue will therefore automatically imply a dorso‐ventral gradient in the time at which the cells reach the state in which they can undergo differenti

ISSN:1464-7931    

DOI:10.1111/j.1469-185X.1978.tb00991.x

出版商:Blackwell Publishing Ltd    

年代:1978

数据来源: WILEY

摘要:

Summary1.Because the calcium (Ca) ion is intimately associated with so many biochemical and physiological phenomena, it is fundamental to understand how intracellular Ca is maintained and controlled. This review draws attention to the vital role played by mitochondria in controlling intracellular Ca and describes how transport of the ion into and out of mitochondria may itself be controlled.2.The heterogeneous distribution of Ca is a property of most, if not all cells. This arises because the ion binds strongly to a variety of biological compounds, especially those containing oxyanions, which themselves have a heterogeneous distribution in cells, but mostly because of the existence in the cell of specific Ca‐ion transport systems.3.Although the concentration of total Ca in the cell may be quite high, a very large proportion of it is bound and non‐diffusible; a small fraction is diffusible but unionized. The proportion of Ca that is ionized is probably much less than I% of the total4.The mechanisms by which Ca is transported into and out of the mitochondrial matrix are discussed. Inward movement of the ion occurs in response to the membrane potential (negative inside) generated by respiration. The process is carrier‐mediated and exhibits characteristics such as substrate specificity, high affinity for Ca, satur‐ability, cooperativity, stimulation by permeant anions and is specifically inhibited by low concentrations of Ruthenium Red and lanthanum. The properties of the Ca carrier are geared therefore to facilitate rapid inward movement of Ca into the mitochondria. Such a carrier system is found in mitochondria isolated from a wide variety of tissues and species.5.Ionized Ca appears not to be distributed across the inner mitochondrial membrane according to the Nernst equation, so the possibility exists that the ion is transported as Ca/H+antiport or as Ca/anion symport. Alternatively, an efflux system coupled to inward movement of a cation may serve to prevent the [Ca ion]in/[Ca ion]outfrom attaining equilibrium. These components together contribute to a Ca‐translocation cycle that permits Considerable flexibility in the overall control of Ca flux.6.Evidence for Ca cycling in mitochondria is presented and the influence of physiological agents such as Mg, phosphoenolpyruvate, inorganic phosphate and adenine nucleotides, on the influx and efflux components are discussed in some detail. Moreover, various hormones administered in vivo are able to induce changes in mitochondrial Ca cycling. One important feature that emerges from this collection of data is that the ability of mitochondria to retain Ca is associated with their ability to retain also their adenine‐nucleotide complement.7.Various lines of research provide convincing evidence in support of the view that mitochondria play a major role in controlling cell Ca in vivo. Especially significant are the observations that the ‘activity’ of mitochondrial Ca transport can change during development in both insect and mammalian tissue, can depend on the hormonal status of the tissue and undergoes a permanent change in certain tumour cells.8.Finally, consideration is given as to how the mitochondrial Ca transport system is able to modify Ca‐sensitive enzyme activities by regulating the Ca concentration in specific environments. Some biological activities that might be susceptible to such cont

ISSN:1464-7931    

DOI:10.1111/j.1469-185X.1978.tb00992.x

出版商:Blackwell Publishing Ltd    

年代:1978

数据来源: WILEY

摘要:

Summary1. Simple and reliable methods are now available for growing rat and mouse embryos in culture at all stages of organogenesis. Primitive‐streak embryos can be maintained for up to 5 days in culture while they develop to early foetal stages. Older embryos are maintained for progressively shorter periods and the most advanced stage that can be supported is equivalent to the rat foetus of 15 days' gestation.2. The rates of protein synthesis and differentiation of the younger embryos in vitro are similar, and of head‐fold embryos identical, to those in vivo. After the formation of the limb buds growth is slower, with protein synthesis more retarded than differentiation, resulting in embryos or foetuses that are well formed but smaller than in vivo. This slowing of growth of the older embryos in culture is probably caused by the lack of a functional allantoic placenta.3. The embryos of some other species, including the guinea‐pig, hamster, rabbit and opossum have also been maintained in culture during organogenesis but the results are not yet as good as those for rats and mice.4. Maximum growth of rat embryos explanted with the visceral yolk sac intact is obtained in undiluted homologous serum, though adequate growth for many studies can be maintained in mixtures of serum with chemically defined tissue‐culture media. The best results are obtained in serum prepared from blood centrifuged before clotting has occurred (I.C. serum) and heat‐inactivated. The importance of a high concentration of serum in the culture medium may be related to the mechanisms for uptake, transport and digestion of macromolecules by the rodent yolk sac.5. There is no convincing evidence for a changing rate of oxygen consumption during organogenesis but there is strong evidence for changes in energy metabolism. At the beginning of organogenesis, the embryo shows a high rate of anaerobic glycolysis and of pentose‐shunt activity. During the following days these decline while activity of the Krebs' cycle and electron‐transport system increases. Anoxia, or exposure of the embryo to carbon monoxide, increases glycolysis and reduces growth rate.6. The earliest stages of the formation of the heart and blood circulation can be closely observed in culture. The heart rate of the 111/2‐day rat embryo is about 160 beats per minute at 38°C, and falls by about 7% per degree for lower temperatures. Several drugs that are cardioactive in the adult also affect the frequency of the heartbeat in the embryo, and the pattern of response suggests that the adrenergic receptors in the embryo develop before the cholinergic receptors. Experiments in which embryo and yolk sac were cultured separately, as well as together, have indicated that haemopoiesis can occur in the embryo only after a migration of stem cells from the yolk sac.7. Microsurgery has been successfully applied to embryos in culture in studies on morphogenetic movements, heart development, axial rotation, limb‐bud regeneration and placenta formation. Biochemical studies of normal morphogenesis have been few, but one has shown a high rate of hyaluronate synthesis by the embryo which may be related to the maintenance and expansion of extracellular spaces and the formation of the neural folds.8. Embryos are particularly sensitive to teratogenic agents during organogenesis. Teratogens that have been studied on whole embryos in culture include trypan blue, antisera, hyperthermia, anaesthetics, and abnormal concentrations of vitamins, oxygen and glucose. Many of the malformations induced have been similar to those obtained after administration of the same agents in vivo and have demonstrated a direct teratogenic effect on the embryo independent of the maternal metabolism. It is suggested that culture methods may provide a valuable additional screening procedure for new drugs and other potentially e

ISSN:1464-7931    

DOI:10.1111/j.1469-185X.1978.tb00993.x

出版商:Blackwell Publishing Ltd    

年代:1978

数据来源: WILEY

ISSN:1464-7931    

DOI:10.1111/j.1469-185X.1978.tb00994.x

出版商:Blackwell Publishing Ltd    

年代:1978

数据来源: WILEY

ISSN:1464-7931    

DOI:10.1111/j.1469-185X.1978.tb00995.x

出版商:Blackwell Publishing Ltd    

年代:1978

数据来源: WILEY