Neurula Cells Research Articles

Towards gene banking amphibian maternal germ lines: short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.

Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40–60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca2+-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both—indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.

Analysis of Histones in Xenopus laevis: I. A DISTINCT INDEX OF ENRICHED VARIANTS AND MODIFICATIONS EXISTS IN EACH CELL TYPE AND IS REMODELED DURING DEVELOPMENTAL TRANSITIONS

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.

Half-Life of Histone H4 mRNA in Xenopus laevis Embryonic Cells at Different Stages: (histone H4 mRNA/Northern blotting/Xenopus embryonic cells/ half-life/actinomycin D).

By Northern blotting methods we have recently found that the relative amount of histone H4 mRNA per Xenopus embryo is constant during cleavage, doubles during the gastrula stage, then decreases at the neurula stage to the level slightly lower than that of the cleavage-embryo. At each developmental stage, the level of mRNA will depend on three parameters: amount of maternal mRNA remaining, rate of the synthesis of the mRNA and rate of the decay of the mRNA. In the present experiment, we compared rates of the decay of H4 mRNA in Xenopus embryonic cells at different stages by pulse-chase experiments with actinomycin D as an inhibitor of transcription. Under the conditions used, the half-life of H4 mRNA was estimated to be 90, 80, and 100 min, for the late blastula, late gastrula and neurula stages, respectively. The decay of H4 mRNA with a half-life of 90 min at the late blastula stage predicts exhaustion of most of maternal H4 mRNA by the early gastruia stage. The slightly longer half-life of H4 mRNA in neurula than in late gastrula cells suggests that H4 mRNA is more stable in neurula cells than in late gastrula cells, and therefore, the large decrease in the level of H4 mRNA observed at the neurula stage does not depend on the increase in the turnover-rate of H4 mRNA. Probably, neurula cells synthesize less H4 mRNA than late gastrula cells but utilize it for longer time.

Kinetic behavior of 30S rRNA intermediate labeled in developing embryonic cells of Xenopus laevis.

In Xenopus neurula cells, "30S" RNA was found to be labeled with 3H-uridine after a relatively short labeling period. Results obtained from cumulative labeling and pulse-labeling and chase experiments with cells from late gastrulae, yolk plug-stage embryos, and neurulae showed that the 30S RNA is an intermediate in rRNA processing and is derived from 40S pre-rRNA and processed to 28S rRNA. The half-life of the 30S rRNA intermediate was about 7.5 min or less at the three stages examined.

Ammonium lon as a possible regulator of the commencement of rRNA synthesis in Xenopus laevis embryogenesis

Recently, we have shown that ammonium salts and amines at an external concentration of 3 m M selectively inhibit rRNA synthesis in Xenopus disaggregated neurula cells. We studied here the change in the amount of ammonia within the embryo and its inhibitory action on the commencement of rRNA synthesis which normally occurs at the blastula stage of development. Ammonia exists at ca. 50 ng/egg (or ca. 3.0 m M at an intra-egg concentration) in the unfertilized egg. This level was maintained during cleavage and then sharply decreased during the blastula stage to the level of ca. 20 ng/embryo (or 1.2 m M) in postblastular stages. Ammonia was extracted from cleavage embryos in a form of ammonium chloride and confirmed to selectively inhibit rRNA synthesis in neurula cells. With authentic ammonium chloride, ammonia was found to be promptly incorporated into cells and to inhibit rRNA synthesis within 1 hr after treatment. In blastula cells, ammonium salts reversibly inhibited the commencement of the synthesis of rRNA, but not hnRNA, 5 S RNA and U1, U2, and U5 snRNAs. The inhibition was at the step of transcription of 40 S pre-rRNA but not the processing or degradation of the processed rRNA. Ammonium salts did not inhibit DNA synthesis, protein synthesis, cell division, and cellular reaggregation. These observations suggest that ammonium ion may be involved in the regulation of the commencement of rRNA synthesis in Xenopus embryogenesis, although it is not yet clear if the ammonium ion exerts its effect directly upon the rDNA transcription system.

Effects of various ammonium salts, amines, polyamines and α-methylornithine on rRNA synthesis in neurula cells of Xenopus laevis and Xenopus borealis

Xenopus neurula cells were cultured in a medium that contained ammonium salts, amines, polyamines or α-methylornithine, and their rRNA synthesis was examined. All the ammonium salts and amines, but not polyamines, were strong and selective inhibitors of rRNA synthesis at 1.25–5.0 mM. α-Methylornithine did not inhibit rRNA synthesis, although it inhibited ornithine decarboxylase, an enzyme claimed to be a direct stimulator of rRNA synthesis. During the treatment ammonium ions and monomethylamines were accumulated within the treated cells. However, monomethylamines did not induce the accumulation of ammonium ions, and vice versa. Ammonium salts and amines also selectively inhibited rRNA synthesis in Xenopus borealis neurula cells.

Demonstration that inhibitor of rRNA synthesis in “charcoal extracts” of Xenopus embryos is artifactually produced ammonium perchlorate

A perchloric acid extract of Xenopus cleavage embryos partially purified with a charcoal column selectively inhibits rRNA synthesis in Xenopus neurula cells. The active substance has long been assumed to be nucleotide-related material, because it appeared to bind to a charcoal column. However, the active substance was proved to be ammonium perchlorate that was produced artifactually when a perchloric acid extract of cleavage embryos was eluted from a charcoal column with ammonia-alcohol. Ammonium ion, but not perchlorate ion, was responsible for the inhibitory activity.

Pattern of protein synthesis in dissociated embryonic cells of Xenopus laevis whose reaggregation was inhibited by Ca 2+ -deprivation and by mechanical interference

Protein synthesis was studied in dissociated Xenopus embryonic cells whose reaggregation was inhibited either chemically (deprivation of Ca 2+) or mechanically (cultivation on sintered glass filter). Under reaggregation-inhibited conditions, synthesis of several proteins was inhibited in neurula but not in blastula and gastrula cells. Under reaggregating conditions, pattern of protein synthesis in blastula cells became ‘neurula-cell-type’ after 17 h. However, under non-reaggregating conditions, activation of many proteins was inhibited. The inhibition was mostly reversible and similar to that observed in neurula cells inhibited by actinomycin D. Thus, synthesis of many proteins directed by new transcripts at the neurula stage appears to depend on the maintenance of cell-to-cell contact.

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos. VII. Inhibition of 40s pre-rRNA synthesis in Xenopus neurula cells.

A fraction containing acid-soluble materials (PCA-extract) from Xenopus early blastulae preferentially inhibits the incorporation of [3H]uridine into 18S and 28S rRNA in Xenopus neurula cells. Pulse- laneling experiments revealed that whereas tubercidin, a known inhibitor of rRNA processing, produced a marked accumulation of the label in 40S pre-rRNA, Xenopus inhibitor suppressed labeling of the pre-rRNA. When tubercidin was added to cells whose activity for rRNA synthesis had been lowered by the Xenopus inhibitor, there was still an accumulation of label in the pre-rRNA. These results indicate that Xenopus inhibitor suppresses the synthesis of rRNA at transcription rather than at processing.

Contribution of maternal mRNA for maintenance of Ca 2+-dependent reaggregating activity in dissociated cells of Xenopus laevis embryos

Dissociated Xenopus laevis blastula cells, where reaggregation was inhibited in Ca 2+-free medium, reaggregated immediately after the addition of Ca 2+. This reaggregation was not inhibited by cordycepin or actinomycin D treatment during culture, although cycloheximide and puromycin were inhibitory. The reaggregation was not inhibited even when fertilized eggs were microinjected with cordycepin and their RNA synthesis was continuously inhibited through cleavage to blastula stages. In neurula cells, cordycepin treatment induced significant reduction in sizes of aggregates formed. These results suggest that the Ca 2+-dependent reaggregating activity of blastula cells is maintained by the translation of maternal, rather than newly synthesized, mRNA.

Locomotion and adhesion of amphibian gastrula and neurula cells cultured on substrata of varied surface charge

This paper reports experiments to determine the effects of electrostatic repulsion on cell adhesion and locomotion, and to demonstrate quantitative differences in adhesiveness among cell populations of the amphibian gastrula, where it may be a factor controlling morphogenesis. Rana pipiens gastrula presumptive germ layer cells and neural crest were cultured on substrata of known surface charge and composition; cell-substratum adhesion assayed, and locomotory rate measured. Because electrokinetic mobilities of R. pipiens gastrula cells are known and that of neural crest has been measured here, various cell-substratum combinations have been ranked in order of the electrostatic force between them. Experimental methods include time-lapse cinematography and single-cell electrophoresis. The results indicate differential cell-substratum adhesion for R. pipiens inner neural ectoderm (INE), chordamesoderm (CHM) and neural crest (NC). In addition, it was found that increase in electrostatic repulsion decreases cell-substratum adhesion, and at first increases rate of locomotion, but then, as repulsion continues to increase, locomotor rate decreases. Increase in the surface potential of presumptive pharyngeal endoderm and chordamesoderm may bring about a decrease in cohesiveness thought to be necessary for the beginning of amphibian gastrulation.

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos : VI. Occurrence in mature oocytes and unfertilized eggs

Occurrence of a factor(s) which can selectively inhibit ribosomal RNA synthesis in isolated neurula cells of Xenopus laevis was examined in oocytes, unfertilized eggs, and embryos of Xenopus laevis. It was found that acid-soluble materials from full-sized oocytes, white-banded mature oocytes, unfertilized eggs, and pregastrular embryos were all active in significantly reducing the relative ratio of the [ 3H]uridine incorporation into 18S and 28S ribosomal RNA to that into 4S RNA from the control value. These results suggest that the inhibitor appears in the terminal step of oogenesis and, hence, may be assumed as a maternal regulator.

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos : IV. The release of inhibitor from cultured cells

Previous reports from our laboratory have shown that a culture medium, conditioned by the growth of isolated cells of Xenopus laevis blastulae, contains a low-molecular-weight substance which selectively inhibits 18 and 28S ribosomal RNA (rRNA) synthesis. Although the occurrence of an inhibitor in an acid-soluble fraction of blastulae has recently been demonstrated, our observation of an inhibitor in a conditioned medium has not been confirmed by other laboratories. To resolve this discrepancy, we have reexamined the effects of conditioned media and acid-soluble extracts on rRNA synthesis by neurula cells. (1) The inhibitory activity for rRNA synthesis can consistently be observed in blastula-conditioned media, provided some of the cells have been broken down during conditioning. If cell rupture is avoided, an inactive conditioned medium is obtained. (2) A homogenate of blastulae inhibits total RNA synthesis and shows no selective inhibition of rRNA synthesis. (3) Charcoal treatment of the conditioned medium and homogenate enhances their specificity for rRNA synthesis. It is then likely that cell breakdown may be involved in the release of the inhibitor into the medium and that some differences in the methods of preparation of conditioned medium may account for the above discrepancy.

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos : V. Inability of inhibitor to repress 5S RNA synthesis

A specific inhibitor of ribosomal RNA (rRNA) synthesis was partially purified from an acid-soluble fraction of Xenopus laevis blastulae. Effects of this inhibitor on 5S rRNA synthesis of isolated neurula cells of the same species were investigated. The results show that the synthesis of both 5S rRNA and 4S RNA proceeds normally when both 18 and 28S rRNA are almost completely inhibited. Failure of the inhibitor to suppress 5S rRNA synthesis suggests that it plays an important role in the regulation of 18 and 28S rRNA synthesis during development and that the synthesis of 5S rRNA is not coordinated to that of 18 and 28S rRNA.

Inhibition of ribosomal RNA synthesis in neurula cells by extracts from blastulae of Xenopus laevis

There have been contradictory reports on the demonstration of an inhibitor of ribosomal RNA synthesis in blastulae of Xenopus laevis. Our attempts to repeat the experiments of Yamana and Shiokawa (1966) and Shiokawa and Yamana (1967b) have failed to demonstrate such an inhibitor. However, direct extraction of blastulae with perchloric acid, as suggested by Yamana, followed by partial purification of the extract by adsorption to charcoal yielded an inhibitor with the properties described by Yamana and Shiokawa. It selectively inhibited the incorporation of uridine and guanosine into cells. The extraction of a factor with these properties from blastulae was highly reproducible. When neurulae were extracted by the same procedure the extract did not selectively inhibit incorporation into ribosomal RNA.

A developmentally significant antigen in the surface of frog embryo cells

Data demonstrating the role of yolk pseudoglobulin in maintaining the integrity of frog neurula cells in hanging drop cultures were recalled. Evidence was presented to demonstrate that the cytolysis of these cultured cells by antineurula sera was preceded by in situ lysis of yolk platelets. It was shown that washed yolk platelets directly exposed to the cytolytic antisera as well as to other antisera were not lysed. This led to the conclusion that the cytolysis of the cultured cells must be attributed to the action of the antisera on cell membrane antigens with pseudoglobulin specificity rather than to the passage of antibody into the cells.