Developing Frog Embryos Research Articles

Collagen synthesis in frog embryo endoderm cells

A low level of collagen synthesis has been detected in endoderm cells of developing frog embryos. There is little difference between the relative rates of collagen synthesis between the ectoderm-mesoderm and endoderm cells at the gastrula stage. By the tailbud stage most of the collagen being made by the embryo is synthesized by the ectoderm-mesoderm.

Zonal Centrifuge Applied to the Purification of Herpesvirus in the Lucké Frog Kidney Tumor

A series of "winter" and "summer" Lucké kidney tumors of the frog (Rana pipiens) were homogenized and fractionated by differential centrifugation into nuclear, mitochondrial, and mitochondrial supernatant fractions. Winter tumors often contained high concentrations of herpesvirus, whereas no virus was observed in any of the summer tumors. The crude tumor fractions were further purified by rate-zonal sucrose gradient centrifugation in a B-XV zonal rotor. Gradient fractions rich in an enveloped, nucleated form of the herpesvirus from certain winter tumors have induced renal tumors when injected into developing frog embryos. Zonal centrifugation was followed by isopycnic banding of the virus zones for further purification of the different morphological forms of the virus.

Zonal centrifuge applied to the purification of Herpesvirus in the Lucké frog kidney tumor.

A series of "winter" and "summer" Lucké kidney tumors of the frog (Rana pipiens) were homogenized and fractionated by differential centrifugation into nuclear, mitochondrial, and mitochondrial supernatant fractions. Winter tumors often contained high concentrations of herpesvirus, whereas no virus was observed in any of the summer tumors. The crude tumor fractions were further purified by rate-zonal sucrose gradient centrifugation in a B-XV zonal rotor. Gradient fractions rich in an enveloped, nucleated form of the herpesvirus from certain winter tumors have induced renal tumors when injected into developing frog embryos. Zonal centrifugation was followed by isopycnic banding of the virus zones for further purification of the different morphological forms of the virus.

Effect of Rate of Cell Division on RNA Synthesis in Developing Frog Embryos

ENDODERM cells of developing Xenopus laevis1 and Rana pipiens embryos2,3 synthesize more RNA per cell, including DNA-like RNA, than do the more rapidly dividing ectoderm–mesoderm cells. Slowing the rate of cell division by culturing frog embryo explants in LiCl increases the amount of RNA synthesized per cell, including DNA-like RNA2, while increasing the rate of cell division by exposure to NaHCO3 has the opposite effect3. These data suggest that a reduction in the rate of cell division allows higher levels of RNA (including DNA-like RNA) to be synthesized per cell.

Effect of actinomycin D on RNA and protein synthesis in regions of developing frog embryos.

Aux stades neurula et bourgeon caudal du developpement embryonnaire deRana pipiens, traitee a l'actinomycine D, la reduction de la synthese de proteine est proportionellement moindre que celle du RNA. Aux niveaux semblables de l'inhibition de la synthese du RNA, la synthese de proteine a diminue plus fortement dans la region de l'endoderme que dans les cellules de l'ectoderme-mesoderme dorsaux, aux stades embryonnaires mentionnes plus haut. Les donnees suggerent que le D-RNA se conserve mieux dans le cytoplasme des cellules de l'ectoderme-mesoderme dorseaux que dans celui des cellules de l'endoderme.

An inverse relation between the rate of cell division and RNA synthesis per cell in developing frog embryos

ABSTRACT Previous work has shown that endoderm cells synthesize more RNA per cell, including nuclear d-RNA, than do dorsal ectoderm-mesoderm cells (Flickinger, Miyagi, Moser & Rollins, 1967; Woodland & Gurdon, 1968). Both groups found lower levels of DNA synthesis in endoderm cells compared to dorsal ectoderm-mesoderm cells. It has also been found that in developing frog embryos both LiCl and cytosine arabinoside inhibit DNA synthesis and also inhibit RNA synthesis. Since RNA synthesis is inhibited less than DNA synthesis, the levels of RNA synthesis relative to total DNA increase (Flickinger, Miyagi, Moser & Rollins, 1967). A number of investigators have demonstrated that LiCl can cause isolated amphibian gastrula ectoderm to form endoderm and mesoderm (Barth & Barth, 1962; Gebhardt & Nieuwkoop, 1964; Masui, 1966; Ogi, 1961). Such compounds as NaHCO3 (Barth & Barth, 1963) and NaCl (Barth, 1966) can induce isolated frog gastrula ectoderm to form neural tissue.

Changes i chromosomal DNA replication tterns in developing frog embryos.

AbstractContinuous exposure of embryonic explants to thymidine‐H3 was used to determine patterns of late‐replicating DNA synthesis, i.e. DNA synthesized during the last quarter of the S period, in chromosomes from different germ layers of developing frog embryos (Rana pipiens). Autoradiographic localization of silver grains over chromosomes 1, 7 and 8, as well as unkaryotyped chromosomes, revealed that patterns of late‐replicating DNA are similar in determined cells that will undergo different pathways of differentiation. In undetermined early gastrula cells and in differentiated tailbud dorsal axial cells (nerve tube, somites and notochord) more sites of DNA synthesis were late‐replicating in chromosomes 1, 7 and 8 and in unkaryotyped chromosomes than in cells of the neural plate‐dorsal mesoderm of neurulae where differentiation is occurring and in neurula endoderm where determination is taking place.

The composition of extracted nuclei of developing frog embryos used as template material for RNA synthesis in vitro.

Les quantités de protéines basiques et résiduelles, de phosphoprotéine et ded-RNA se trouvant dans les noyaux extraits d'embryons deRana pipiens diminuent en passant du stade de blastula à celui de la larve. Cette évidence suggère que l'accumulation progressive des protéines ou dud-RNA, en masquant le DNA n'offre pas le contrôle principal de la synthèse de l'RNA in vivo. Une réduction de la quantité de l'endogène RNA polymerase fonctionelle incluse dans la protéine résiduelle explique mieux la réduction de la synthèse dud-RNA dans les stades plus avancés du développement embryonnaire.

Protein synthesis in frog embryos and frog liver preparations, and its inhibition by embryo components.

SummaryThe capacity to carry out activities related to protein synthesis has been studied in developing frog embryos and post hatching larvae. Rapid incorporation of labeled amino acids from the external medium into protein could be demonstrated with intact larvae about 5 days before a net increase of total protein was found. Only minor incorporation of labeled amino acid into protein was obtained with cell-free preparations from frog embryos and larvae, even at developmental stages when the total protein content was increasing; however, these preparations did possess a high capacity for amino acid activation. In contrast, analogous cell-free fractions from adult frog liver contained an active system for incorporation of phenylalanine or of an amino acid mixture into protein; the characteristics of the frog liver system were similar to those of cell-free systems for amino acid incorporation described in other species. The low incorporating activity of cell-free preparations from frog embryos or larvae was...

Generation times and DNA replication patterns of cells of developing frog embryos

The method of continuous labeling with thymidine- 3H and autoradiography of tissue squashes was used to examine patterns of DNA synthesis reflected in the chromosomes of developing embryos of Rana pipiens. At the neurula and tailbud stages there are regions of late-replicating DNA at one or both terminal segments of most chromosomes. This pattern was not present at the blastula or gastrula stages. At any stage the duration of S and G 2 are similar for cells in different parts of the body, but the length of G 1 and generation time varies.

Studies on HeLa cell nuclear DNA-like RNA by RNA-DNA hybridization.

A class of rapidly labeled nuclear RNA distinct from ribosomal precursor RNA has recently been characterized in various types of animal cells.1-' This RNA has a base composition resembling DNA (substituting uracil for thynline) and is therefore clearly distinguishable from nucleolar ribosomal RNA precursor molecules. On the basis of sedimentation behavior after various treatments3' B and contour lengths in electron micrographs,2 it has been suggested that this RNA represents molecules of various lengths, including some polyribonucleotide chains as large as 107 molecular weight. We have used the abbreviation HS-nRNA (heterogeneously sedimenting nuclear RNA) to refer to this class of RNA obtained from HeLa cells.5' 6 The cytoplasmic mRNA of HeLa cells comprises molecules which sediment much more slowly than the HS-rnRNA,7' 8 although the base composition of both is DNA-like. It is therefore possible that cytoplasmic mRNA is derived from HS-nRNA by a specific cleavage mechanism(s). Such a mechanism, by which long polynucleotides are converted to specific shorter molecules, has been described in HeLa cells: 28S and 16S ribosomal RNA derive from a 45S ribosomal precursor molecule in the nucleolus of the cell.6' 9, 10 Work on nucleated duck erythroblasts (cells which primarily synthesize hemoglobin) suggests that the majority of HS-nRNA molecules in that cell never leave the nucleus but are apparently constantly synthesized and degraded.2' On the other hand, consideration of the kinetics of pulse-chase experiments have led Brown and Gurdon to suggest that in developing frog embryos at least some of the shorter DNA-like RNA may arise from cleavage of longer molecules. In Hela, cells, the rate of incorporation of H3-uridine into HS-nRNA was compared to what would be expected if the HS-nRNA served as a precursor to cytoplasmic mRNA.5 Those kinetic experiments gave no clear indication of whether any of the HS-nRNA might be converted to cytoplasmic mRNA. To explore further the relationship of these two classes of RNA molecules, we turned to the technique of RNA-DNA hybridization.1 It was anticipated that experiments utilizing competition hybridization would be especially useful. For example, if HS-nRNA or RNA derived from it never entered the cytoplasm, hybrid formation by radioactive HS-nRNA should not be affected by cytoplasmic mRNA. It was found, however, that while unlabeled HeLa cell cytoplasmic RNA would interfere with hybridization of labeled RNA molecules, no true competition for DNA sites by unlabeled RNA molecules could be demonstrated. Therefore, the extent to which nucleotide sequences in these two classes of DINTAlike RNA might be related could niot be determined from such experiments. Materials and Methods.-Labeling procedures and preparation of nucleic acids: Hela cells were grown, labeled, and fractionated into cytoplasmic and nuclear fractions as previously described.5 7t 10 The total nuclear RNA was extracted by a hot phenol-sodium dodecyl sulfate (phenol-SDS) procedure and subjected to zonal sedimentation.A 12 The RNA sedimenting faster than the 45S ribosomal precursor peak (approximately 50-IOO(S) was collected by ethanol pre-

The relation of DNA synthesis to RNA synthesis in developing frog embryos

Frog embryos (gastrulae, neurulae, and tailbuds) were cut into dorsal ectoderm-mesoderm and endoderm regions and the isolated parts were incubated with uridine- 14C. In relation to the size of the acid-soluble pool, the endoderm regions synthesize less total RNA than do dorsal ectoderm and mesoderm cells, while cytosine arabinoside and LiCl inhibit both total RNA and DNA synthesis. The endoderm cells synthesize more RNA in relation to the level of DNA synthesis than do the dorsal ectoderm-mesoderm cells. Unlabeled RNA:DNA ratios were also higher for nuclei and chromatin of the belly cells than for the dorsal axial cells. Embryos in which DNA synthesis was retarded with lithium chloride or cytosine arabinoside synthesized more RNA in relation to the amount of DNA synthesis than did the control embryos. This suggests that there is less inhibition of RNA synthesis, compared to the level of inhibition of DNA synthesis, by cytosine arabinoside and LiCl. LiCl and NaCl (0.25% concentrations) caused an increase in the level of labeled compounds in the acid-soluble pools. The results are discussed in relation to the speculation that the frequency of mitosis can affect the transcription of the DNA of the genome, leading to the synthesis of qualitatively different mRNA molecules which affect the direction of cellular differentiation.

The equivalence of DNA from developing frog embryos

DNA was isolated from gastrulae, neurulae, tailbuds and larvae of developing frog embryos by a method which reduced the level of protein to less than 1 per cent. In the presence of Micrococcus lysodeikticus RNA polymerase and the four ribonucleoside triphosphates, equal amounts of these DNA preparations primed for similar levels of RNA synthesis. The priming activity for RNA synthesis of adult frog liver DNA was greatest when the protein level was reduced to less than 1 per cent. This liver DNA primed for significantly more RNA synthesis than did liver chromatin or nucleoprotein preparations which contained the same amount of DNA. RNase treatment of low-protein DNA did not affect priming for RNA synthesis. DNA preparations from four developmental stages of the frog embryo and adult frog liver showed similar sedimentation constants in the analytical ultracentrifuge and similar melting profiles, indicating similarity of these preparations.

Stimulation of protein synthesis of frog embryo cells by larval and adult frog liver ribonucleic acid preparations.

RECENT work from our laboratory has characterized certain quantitative properties of DNA and chromatin from developing frog embryos in terms of RNA-polymerase-mediated RNA synthesis1,2. Apart from knowing how much RNA can be synthesized using chromatin or DNA as a primer, it would be of interest to know some of the qualitative properties of the RNA synthesized. The present investigation examines two questions: Can added RNA stimulate protein synthesis of intact frog embryo cells? If it can, is this stimulation due to species of tissue-specific RNA molecules directing the synthesis of the appropriate proteins? Liver RNA preparations can stimulate haemin synthesis of minced frog embryos3 or mouse ascites tumour cells4, and Niu et al.5 have claimed that liver RNA induces the synthesis of liver specific proteins in ascites tumour cells.

Hemin synthesis in the developing frog embryo and its stimulation by frog liver RNA

Hemin was synthesized from δ-aminolevulinic acid-4-C 14 by intact and minced frog embryos; the isotopic activity of the hemin samples is a measure of the activity of the enzymes of this pathway of porphyrin biosynthesis. Administration of the isotopic hemin precursor to intact embryos revealed low levels of synthesis until stage 25, but this is attributed to the poor penetration of δ-aminolevulinic acid into the embryos due to their relatively impermeable surface coat. Cutting each embryo into 4 or 5 pieces (mincing) allowed penetration of the hemin precursor into the embryo. It appears there is a synthesis of the enzymes of this porphyrin biosynthetic pathway in the minced embryos, since puromycin inhibited the incorporation of labeled amino acids into protein, and also the incorporation of δ-aminolevulinic acid-4-C 14 into hemin of minced stage 20 embryos. The minced embryos showed a slight increase in hemin synthesis from gastrulation to neurulation and a larger increase to the tailbud stage. The level of hemin formation in minced stage 25 larvae was lower than that of intact stage 25 larvae; at the feeding larval stage the labeled compound can enter the organism and is converted to hemin more efficiently than by minced stage 25 larvae. The real course of hemin synthesis in developing frog embryos, as revealed from the results obtained with both minced and intact embryos, is that of a gradual increase from gastrulation to neurulation, followed by an increased rate of hemin synthesis at tailbud and larval stages. The addition of frog liver RNA (1 mg/ml) to minced stage 20 embryos consistently stimulated the level of hemin synthesis above that of untreated minced embryos. Frog liver DNA and RNA of frog spleens, muscle, and red blood cells, as well as mouse ascites tumor cell RNA (1 mg/ml), did not stimulate hemin synthesis. Stimulation is also evoked by a mixture of the four ribonucleosides. Puromycin inhibits both protein and hemin synthesis of minced embryos while liver RNA stimulates the incorporation of labeled amino acids into the protein fraction. It appears that hemin synthesis is linked to the synthesis of the enzymes of porphyrin biosynthesis in these experiments. Phenol extraction of RNA from centrifugal fractions of liver homogenates revealed that there is active RNA in the ultracentrifugal supernatant fraction and that a transfer RNA fraction isolated from the ultracentrifugal supernatant of frog liver retains the ability to stimulate hemin synthesis of minced stage 20 embryos. Hot phenol extracts of the interphase of cold phenol extracts of frog liver also stimulated hemin synthesis of minced gastrulae cultured for 24 hours, but the stimulation by the four ribonucleosides and the RNA of the ultracentrifugal supernatant argue against an effect of messenger-RNA molecules. It is likely that supplying precursors of RNA synthesis, or transfer RNA, to cut frog embryos can stimulate the synthesis of enzymes synthesizing hemin, as well as the synthesis of other proteins.

Sequential appearance of monoiodotyrosine, diiodotyrosine, and thyroxine in the developing frog embryo

The incorporation of NaI 131 into intact frog embryos, parts taken from the intact embryos, cultured regions of the embryos, and cell-free systems derived from parts of the embryos, has been followed by chromatography in a collidine-H 2O-NH 3 system and counting with a windowless autoscanner. The synthesis of monoiodotyrosine is first detected at the tail-bud stage in both the head and lower jaw regions of the embryos. Monoiodotyrosine and diiodotyrosine are found in the thyroids of feeding larvae of 11 mm length; these two compounds and thyroxine are present in the thyroids of 16-mm larvae. Several explanations for the sequential appearance of these compounds are discussed.

Enzymatic patterns in the developing frog embryo

Methods are presented for the determination of twelve enzymes as found in homogenates of the R. pipiens embryo. The determinations are primarily based on the spectrophotometric or fluorometric estimation of pyridine nucleotides and can be easily performed, if necessary on very small amounts of material. The activities of these twelve enzymes as found in homogenates of the developing embryo are presented. In most cases these activities remain relatively constant until hatching, at which time they begin to increase (aldolase, lactic dehydrogenase, glutamic-aspartic transaminase, malic dehydrogenase, glutamic-alanine transaminase TPNH-cytochrome c reductase, pyruvate kinase). Four enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase, α-glycerophosphate dehydrogenase, isocitric dehydrogenase) begin to increase in activity around neurulation, however, and a fifth enzyme (DPNH-cytochrome c reductase) does not begin to increase in activity until the closing of the larval opercular folds. The significance of the developmental curves obtained is discussed in terms of those factors that would contribute to enzymatic activities as found in crude homogenates of a developing system. The preferential synthesis of an enzyme in a specific area, the existence of multiple enzyme forms, and morphological changes within cells are all seen as factors that would complicate any definite interpretation. These phenomena are highly relevant to differentiating systems, however and need to be extensively investigated after general patterns have been established. At the same time, information is brought to bear on the regulation of metabolic pathways in the early frog embryo. Specifically, it is suggested that isocitric dehydrogenase may be the rate-limiting enzyme of the citric acid cycle and that glucose-6-phosphate dehydrogenase may have a role in the regulation of glycolysis during early development. These enzymes are not considered “regulatory” as such, however, but rather as expressions of a dynamic state.

Localization of lens antigens in developing frog embryos

1. 1. By the use of the precipitin method, utilizing brain absorbed anti-frog and anti-cattle lens sera, antigens with lens-determinate groups were detected in immature frog oocytes, neurulae, the tails of late-stage feeding larvae, the pigmented retina-iris and aqueous humor of adult frog eyes. 2. 2. There was no difference noted in the intensity of the precipitin reaction, with brain absorbed anti-frog lens, comparing embryos raised to the neurula stage at 10 °C or at 25 °C. The heads of both of these temperature groups showed a stronger reaction with the anti-lens serum than did the trunk fractions. At stage 16 the three regions of the head (optic cup, optic vesicle and oral sucker area) all react equally well with anti-frog lens serum. 3. 3. Agar diffusion plates indicated the presence of eight frog lens antigens. One of these was similar to an antigen present in early stage feeding larvae, tails of late stage feeding larvae and the aqueous humor. Two other lens antigens showed reactions of identity with two brain antigens and one of these common lens-brain antigens was also found in the immature oocytes. Yet another lens antigen was similar to a second small oocyte supernate antigen. The antigens in the immature oocytes which react with the antifrog lens serum are different from the antigens in this preparation which react with an anti-testis serum and an antiserum to small oocyte supernate itself.

Calcium in aging and fertilized frog eggs

Fat-soluble, water-soluble and insoluble fractions of unfertilized eggs and developing frog embryos were analyzed for calcium at various times up to 93 hours. Sections of the eggs and embryos were tested for calcium by the microincineration method. No significant amounts of calcium were found in fat-soluble fractions. Changes in water-soluble and insoluble fractions suggested an interchange between the fractions, and showed many similarities between unfertilized eggs and developing embryos. By 93 hours the insoluble calcium per unfertilized egg was about 9 times that in the embryo. The possible significance of calcium in cellular aging is discussed.

Effect of Estrogens on Early Development of Frog Embryos.

SummaryAbnormalities were produced in the developing eggs of Rana pipiens and Rana clamitans by exposure to diethylstilbestro and to estrone in varying concentrations and for varying lengths of time. Higher concentrations of both substances produced more abnormalities, and estrone was found to be more effective than diethylstilbestrol.