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.     

用实验方法反转原肠胚外胚层细胞的极性(英文)

过去的工作指出,在紧密连接形成的时候,紧密连接本身也经历了极性化的过程,而且这一过程在时间上和囊胚腔的出现和扩张是相互关连的。为了进一步探讨紧密连接的极性化和外胚层细胞极性之间的关系,进行了原肠胚外胚层细胞内外反转的实验,结合扫描和透射电镜的观察,指出反转后的外胚层细胞表面发生了明显的变化,在原来没有紧密连接的内层细胞的外缘形成了发育完善的紧密连接。内外反转的这一实验操作,使外胚层细胞和内外环境的关系发生了变化。很可能外胚层细胞的极性先发生了反转,然后影响紧密连接在新的向外的一极形成。     

CHANGES IN THE ELECTROPHORETIC MOBILITY OF PRESUMPTIVE ECTODERM CELLS TREATED WITH MESODERMAL INDUCING SUBSTANCES

Using gastrula ectoderm of Cynops pyrrhogaster , the electrophoretic mobility (EPM) of cells was measured. Before EPM measurement, the cells were treated with either LiCl or swimbladder tissue, which are known to possess endo- and mesodermal inducing activity. The EPM of the treated cells increased after 48 hr. A significant difference was observed between the control and experimental series. In addition to the measurements of EPM, histological sections of the explants were examined. Before obvious tissue differentiation occurred in the explants, definitive changes in the EPM values were recorded at 48 and 72 hr after treatment.     

CELL CONTACT REFORMATION OF DISSOCIATED CELLS FROM EMBRYONIC CELLS OF STRONGYLOCENTROTUS PURPURATUS

Disaggregated single cells from the gastrula of Strongylocentrotus purpuratus were studied as they reaggregated and reformed quasi-normal embryos. In this investigation emphasis was placed on the structural events involved during the reformation of cell contacts vis-a-vis cell migration. The early cell contacts are non-junctional cell appositions, which are characterized by non-parallel apposing membranes. Between post-migratory epithelial cells, there is a shift from non-parallel to parallel apposing membranes. These cell appositions are found between the overlapping lamellapodia along the apical margins of the epithelial cells during blastocoel enlargement. Incipient continuous junctions are formed by the deposition of an electron dense material in the intermembrane space. As the junction develops, electron dense plaques form in the cytoplasm immediately subjacent to the junction and septa form between the apposing membranes.     

INDUCING TIME FOR NEURAL TISSUES IN GASTRULA ECTODERM OF CYNOPS PYRRHOGASTER

The inducing times for spinal cord and deuterencephalon in Cynops gastrula were determined by the sandwich method. The extreme posterior of the archenteron roof at the slit-blastopore stage (tail organizer) was used as an inducer. First, the presumptive ectoderm of the earliest gastrula (0-hr stage) was put in contact with the organizer for 6 to 24 hr. Spinal cord and deuterencephalon were induced in almost all explants after 24 and 21 hr of contact, respectively, indicating that 24 hr is enough time for differentiations of both spinal cord and deuterencephalon. Next, presumptive ectoderm of 6- to 21-hr exogastrulae was put in contact with the organizer until the 24-hr stage. Results showed that the net inducing times for spinal cord and deuterencephalon were 18 and 15 hr, respectively, and that neural competence appeared in the presumptive ectoderm at the 6-hr stage (straight-blastopore stage).     

THE INFLUENCE OF ELECTROLYTES UPON THE ELECTROPHORETIC MIGRATION OF BACTERIA AND OF YEAST CELLS

1. It seems first of all clear from our results that the effect of electrolytes upon electrophoretic charge is essentially the same, whether one is dealing with silica dust, bacteria, or yeast cells, although certain quantitative differences appear which will later be discussed. 2. The normal negative charge on the suspended particles appears to be slightly increased by very low concentrations of electrolytes, markedly so in the case of yeast cells. Increase in charge due to minimal concentrations of electrolytes has been recorded by Loeb (1922) for collodion particles. 3. Higher concentrations of electrolytes cause a marked and progressive decrease in negative charge, sometimes leading to an isopotential condition and sometimes to a complete reversal of charge with active migration toward the cathode. This effect is apparently due to the cation alone and increases with the valency of the cation, except that the H ion shows specially marked activity, between that of bivalent and trivalent ions. Since NaOH behaves like an ordinary univalent salt, increased alkalinity of a solution does not further depress the charge already depressed by salts; but, since the H ion is much more active than other univalent or bivalent ions, increased acidity does cause a further progressive depression of charge, even in salt solutions. Certain electrolytes appear to show individual peculiarities due to something else than their valency. Thus KCl for example is distinctly more effective than NaCl. Sodium chloride in general appears to exert less influence upon electrophoretic charge, either in low or high dilution, than do other compounds of univalent ions studied. This depressing effect of moderately high concentrations of electrolytes is much less marked with yeast cells than with Bacterium coli. Silica dust is still less affected by monovalent and bivalent ions than are the yeast cells but appears to be more affected than either yeast or Bacterium coli by AlCl₃. 4. Very high concentrations of AlCl₃ (above 10^–2 M) show a third effect, a decrease of the positive charge produced by concentrations of moderate molar strength. This is analogous to phenomena observed for trivalent salts by Northrop and De Kruif (1921–22) and for acid by Winslow, Falk, and Caulfield (1923–24). 5. Organic substances, such as glucose, glycerol, and saponin produce no effect on electrophoretic velocity until they reach a concentration at which viscosity changes are involved. 6. The first two results observed,—(a) the increase in charge as a result of slight additions of electrolytes, and (b) the marked decrease in charge with further concentration of electrolytes, depending on the valency of the cation, so far as vegetable cells are concerned, are entirely in accord with the theory of the Donnan equilibrium as worked out by Loeb (1922). We might assume in explaining such phenomena that the plant cell contains a certain proportion of unbound protein material and that the first modicum of cation which enters the cell is bound by the protein, leading to an increase in the relative negative charge of the cell as compared with its menstruum, while subsequent increments of cation remain unbound in the cell and thus lower its charge. When we find, however, that the same phenomena are apparent with collodion particles, as shown by Loeb, and with silica dust, it seems difficult to apply such a theory, involving the conceptions of a permeable membrane and unbound organic compounds. Loeb (1923–24) suggests that the primary increase may be due to an aggregation of anions in the part of the electrical double layer adjacent to the suspended particles; but why there should be first an aggregation of anions and later (with increasing concentration) an aggregation of cations, is not easy to conceive. The third result,—the reversion to a more negative charge in the presence of a marked excess of trivalent ions,—is again difficult to explain. Loeb, in this connection, postulates the existence of complex ion-protein compounds, which can scarcely be assumed in the case of the silica particles.     

HORMONE SECRETION BY CELLS DISSOCIATED FROM RAT ANTERIOR PITUITARIES

A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are ~55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [³H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K⁺ and dibutyryl cyclic AMP) is impaired, but after a 6–12-h culture period, the cells apparently recover and discharge 24% and 52%, respectively, of their content of prelabeled growth hormone over a 3-h period in response to these two secretogogues. This represents a stimulation of 109% and 470% over that released by cells incubated in control medium. The results demonstrate that function and morphologic integrity are preserved in this cell system. Therefore it is suitable for the study of various aspects of pituitary secretion and its control.     

INDUCTION OF CELL DIFFERENTIATION IN DISSOCIATED CELLS AND FRAGMENTS OF CALLITHAMNION ROSEUM

Branching in regenerates of Callithamnion roseum is a phenomenon of cellular differentiation. Starting from single cells or filaments, a gradual morphological change in cell types occurs: from parallel cross-walled, branchless stalk cells to oblique cross-walled, branch-bearing main axis cells. The development of a plating technique and a synthetic sea water medium permits quantitative study of cell differentiation (or branching) in populations of regenerating cells or fragments. The distribution of regenerating fragments on a given plate is non-random as seen from the fact that it does not conform to the Poisson series. Clustering and attachment of neighboring fragments increases the likelihood of branch initiation. The change with age in the distribution of plated regenerating fragments is presented. Developmental potentialities of 5 fragment types were examined. Each fragment type could give rise to branching regenerates. Although apical fragments composed of more than 10 cells usually branched first in a plate, single, large, basal cells seem to have a greater regenerative capacity than do small apical fragments.     

EFFECT OF AGING ON NEURAL COMPETENCE OF PRESUMPTIVE ECTODERM, AND EFFECT OF AGED ECTODERM ON DIFFERENTIATION OF THE ORGANIZER IN CYNOPS PYRRHOGASTER II. ROLE OF EXTREME POSTERIOR OF THE ARCHENTERIC ROOF IN THE SLIT-BLASTOPORE STAGE IN NORMAL DEVELOPMENT

The effect of aging on the neural competence of the presumptive ectoderm in gastrulae of Cynops pyrrhogaster and the effect of aged ectoderm on differentiation of the extreme posterior of the archenteric roof in the slit-blastopore stage were examined by a sandwich method in which this organizer was wrapped in the presumptive ectoderm taken from the 0- to 42-hr aged exogastrulae. Vital staining showed that this organizer becomes mainly tail notochord. Therefore it should be called tail or trunk-tail organizer. In 0- to 18-hr explants, typical trunk-tail structures were formed. With further aging of the presumptive ectoderm, a decrease of spinal cord and muscle with a concomitant increase of mesenchyme and mesothelium was observed. In 36- (corresponding to the slit-blastopore-initial neural stage) and 42-hr explants, neural competence had disappeared markedly. The notochord appeared in all explants, indicating this organizer is more firmly determined than the uninvaginated dorsal lip in small yolk-plug stage. Conclusively, this organizer does not play an important role in the induction of the neural plate, but induces the tail in normal development.     

SOME PHYSICOCHEMICAL PROPERTIES OF DISSOCIATED SPONGE CELLS

1. The activity of the hydrogen ion, in a system containing 0.00280 mols of NaAc, 0.520 mols of NaCl per liter, and varied amounts of HCl or NaOH has been investigated. The average value of pK'' for acetic add in this system is about 4.37. 2. The effect of the addition of various amounts of HCl and NaOH to a system containing 0.00280 mols of NaAc, 0.520 mols of NaCl, and a known number of cells of either Microciona prolifera or Cliona celata was then studied. It was found that in weak acid solutions Microciona behaves as a stronger base than Cliona, the former being practically saturated with base at a pH of 7.5. Similar behavior is shown by suspensions of cells to which no acid or base was added: the cells of Cliona are more acidic than the cells of Microciona. 3. The microscopic examinations of the cells subjected to the treatment with acid or base indicate that the cells of Microciona remain alive down to pH 4.50; the cells of Cliona sustain greater acidity,— a,t pH 3.7 they exhibit no signs of cytolysis. Tests for aggregation of these cells showed that this phenomenon is greatly inhibited even by slightly acid solutions. 4. The conclusion is drawn that the concentration of cells being equal, the suspensions of cells of Microciona and Cliona differ from each other in their physicochemical properties, the comparison being made on suspensions of specified composition.     

EFFECT OF PROTEINS ON ELECTROPHORETIC MOBILITY AND SEDIMENTATION VELOCITY OF RED CELLS

The isoelectric point of normal red cells cannot be measured but is certainly lower than that of any plasma protein. Red cells are easily damaged so that they will adsorb proteins from low concentrations. Normal red cells do not adsorb protein even from concentrated solutions, as is evidenced by the finding that the ratio of the mobility of the cells to that of the proteins themselves is at least as high in concentrated casein, albumin, gelatin, or fibrinogen solutions as in dilute. The finding that the observed mobility of red cells is unchanged or only slightly decreased when bulk viscosity is increased by added protein is interpreted as indicating that the red cell surfaces are hydrated. The aggregating effect of certain proteins has been determined and is assumed to be due to their dehydrating effect on the cells. Some types of cells, as beef, are not aggregated, presumably because they are resistant to this dehydrating effect. The difference in the behavior of different types of red cells demonstrates the importance of the nature of the cell as well as of the medium in determining the rate of aggregation and therefore of sedimentation.     

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.     

小鼠胚胎原始外胚层细胞的发育能力(英文)

本文利用胚泡注射技术研究了小鼠胚胎原始外胚层细胞(primitive ectoderm cells)的发育能力。从交配后第五天的129/SV-ter(灰色,GPI-1~a/~a)小鼠的胚胎中分离出原始外胚层细胞并将之注射到交配后第四天的C_(57)BL/6 J(黑色,GPI-1~b/~b)小鼠的胚泡腔内。经过显微操作后的胚泡被移回昆明白假孕鼠内发育,其出生率为83.3%,毛色嵌合体(chimeras)比例为100%。这些嵌合小鼠的磷酸葡萄糖异构酶(GPI-1)分析结果表明,注射的原始外胚层细胞参与了内、中、外三个胚层所衍生的组织和器官(如脑、血液、心脏、肾脏、生殖腺、肌肉、脾、旰等)的胚胎发生。嵌合体与C_(57)BL/6 J小鼠交配后所得的结果表明,原始外胚层细胞在嵌合体内能形成有功能的配子。上述结果说明,原始外胚层细胞与内细胞团(ICM)细胞、体外培养的胚胎干细胞(embryoderivedstem cells)一样,具有发育全能性。导入胚泡后,不仅能参与嵌合体中各种体细胞的分化,并且能经历配子发生产生有功能的雌雄配子。此外,本文还对胚泡注射技术进行了改进,改进后的方法不仅比已报道的各种方法简便,并且使注射嵌合体的比例提高到35.7%。     

A UNIQUE JUNCTIONAL STRUCTURE FOUND IN BLASTULA CELLS OF THE NEWT, TRITURUS PYRRHOGASTER

The outermost cell layer of the animal half of the newt blastula ( Triturus pyrrhogaster ) was examined to investigate intercellular junctions by transmission and scanning electron microscopy. A unique structure is observed at the terminal region of the intercellular junction. The structures are cytoplasmic ridges elevated from the cell surfaces, and their inner part is filled with spaces of various sizes. It is supposed that these ridges result from the encounter of cytoplasmic folds protruding from two neighboring cells.
Below the ridges, there is a short close junctional area which is followed by a long region of intercellular space intermittently bridged by cytoplasmic projections. Microvillus-like cytoplasmic processes on the apical cell surfaces, and microfilaments and microtubules in subsurface regions are observed in this material as in many other embryonic cells of amphibians.