Developing Frog Embryos Research Articles

Effects of cytochalasin H on developing frog embryos: reversibility of action.

Cytochalasin H (CH) brought about cellular disaggregation resulting into arrest of development in the embryos of the frog, Microhyla ornata. The effects were dependent on the dose of CH, duration of the treatment as well as on developmental stage of the embryo at the beginning of the treatment. The embryos were protected to some extent if the vitelline membranes were left intact. The reversibility of CH-action, after transfer of treated embryos to CH-free medium, was dependent on the extent of cellular disaggregation brought about by CH and on the presence or absence of an intact vitelline membrane.

Lethal effects of diquat and paraquat on developing frog embryos and 15-day-old tadpoles, Rana pipiens.

Lethal effects of diquat and paraquat on developing frog embryos and 15-day-old tadpoles, Rana pipiens.

Effects of cytochalasins on developing frog embryos: protection with L-cysteine and α-D-glucosamine.

Cytochalasins A, B and H (CA, CB and CH) brought about cellular disaggregation and mortality in the developing embryos of the frog, Microhyla ornata. All three cytochalasins exhibited a dose-response relationship. CA was the most potent and its effects were significantly reduced by simultaneous additon of L-cysteine, a sulph-hydryl compound. α-D-Glucosamine, a precursor of complex macromolecules important in cell adhesion, protected against the effects of CH. The effects of CB, however, were influenced neither by L-cysteine nor by α-D-glucosamine. The results revealed differences in the mechanism of action of these three cytochalasins.

Anti-sense RNA injections in fertilized eggs as a test for the function of localized mRNAs

ABSTRACT In general, it is difficult to identify genes that play critical roles in developmental decisions in vertebrates because it is not possible to perform exhaustive screens for mutations that affect developmental processes. While some genes have been identified and analysed by conventional genetic methods, for example the mouse T locus (Bennett, 1975) or the axolotl o mutation (Brothers, 1976), it is likely that many genes that control vertebrate development will have to be identified by other means. In recent years, two methods for identifying such genes have been utilized. First, genes have been selected for study because they are expressed at a particular time or place during embryogenesis. For example, genes that are expressed only during gastrulation (Sargent & Dawid, 1983; Krieg & Melton, 1985) or at the cellular blastoderm stage (Roark, Mahoney, Graham & Lengyel, 1985) have been cloned and there are numerous examples of genes being studied because they are expressed in some, but not all tissues (e.g. Lynn et al. 1983) or are spatially localized within single cells (Jeffery, Tomlinson & Brodeurer, 1983; King & Barklis, 1985; Rebagliati, Weeks, Harvey & Melton, 1985). Second, genes that are potentially important for development have been cloned by virtue of their homology to Drosophila segmentation and homeotic genes. Several homeobox-containing genes have been isolated from frogs (Carrasco, McGinnis, Gehring & DeRobertis, 1984; Harvey, Tabin & Melton, 1986), mice and humans (reviewed by Gehring, 1985) and there is considerable interest in the possibility that these genes have a function in vertebrates homologous to their known functions in Drosophila. In all these cases it is necessary to have some test for the function of the cloned genes. We have explored the possibility of studying gene function by preventing the expression of the gene product in vivo by injecting anti-sense RNA. This report summarizes our recent efforts to prevent mRNA translation in vivo by injecting RNA that is complementary to messenger RNA, so called anti-sense RNA, into developing frog embryos.

Major transitions in histone gene expression do not occur during development in Xenopus laevis

In light of the parallels that exist in the structure of histone genes in sea urchins and in the frog, Xenopus laevis, and in the early development of these animals, it has been thought that Xenopus histone gene expression might be subject to the type of developmental regulation observed in sea urchins. We have examined the patterns of histone mRNA accumulation in Xenopus oocytes and embryos by primer extension and S1 nuclease protection techniques. The data demonstrate that histone genes which are active in Xenopus oocytes, and which contribute to large pools of histone mRNA in the absence of DNA replication, are also transcriptionally active in late embryos and in cultured cells. These results suggest that, rather than activating distinct sets of histone genes at different developmental stages, the developing frog embryo reprograms the expression of histone genes active in nondividing oocytes so that their expression becomes coupled to DNA replication subsequently during embryogenesis.

Factors controlling the size of DNA loops in frog embryos and Friend erythroleukemia cells

Measurements were made of the average size of fluorescent halos of nucleoids of developing frog embryos, mouse fibroblasts (A9 cells) and Friend erythroleukemia cells cultured with various compounds. These halos are thought to represent relaxed lengths of loops of DNA attached to the nuclear matrix. Measurement of transcription of RNA from nuclei in vitro suggested that cells with more loops (smaller halos) had more initiation sites for transcription. DNA loop number decreases during development of the frog embryos, and red blood cells of adults have even fewer and larger loops. One or two days of culture of Friend cells in various compounds that induce differentiation resulted in more DNA loops than for control cells, but after 5 days of culture the differentiated erythrocytes had fewer DNA loops than control cells at stationary phase. Stationary phase cells have almost twice as many DNA loops as log phase cells. Various compounds that stall cells at the G1/S border induced more DNA loops, but subsequently the number of DNA loops decreased as the cells entered S phase. A number of compounds which slow the rate of cell division cause the formation of more DNA loops, perhaps due to longer G1 periods.

Teratogenic and lethal effects of paraquat on developing frog embryos (Rana pipiens).

Paraquat (l,l'dimethyl-4, 4'-bipyridinium dichloride) has been used for a number of years in the United States and other countries as a herbicide in the control of broadleaf weeds and grasses in crop fields, orchards, and berry patches. It is utilized as both a pre-emergence and post-emergence herbicide. Paraquat may also be used in aquatic weed control in some countries but not in the United States. The recommended field application rate is usually 0.25 to 1.0 lb. paraquat cation/acre. The rate of application for aquatic weed control ranges from about 0.I to 2.0 parts per million (ppm), by weight in water (Newman 1970; Calderbank 1972). Little is known of the effects of paraquat on embryonic development in aquatic animals. Murry and Schreiweis (1977) have shown teratogenic malformations in embryos of Oryzias latipes and Paulov [1977) has carried out developmental and body protein studies of the effects of paraquat in Rana temporaria. The present study was conducted to observe lethal and gross teratogenic effects of a range of concentrations of paraquat on Rana pipiens embryos, including concentrations at the recommended aquatic field application rates.

Relative base composition of initiation sites for transcription in developing frog embryos

The use of an inhibitor (0.4 N (NH 4) 2SO 4) and an activator (sodium polydextran sulfate) of initiation of transcription in nuclei of frog embryos in vitro indicated that initiation sites for transcription are relatively A—T-rich. Comparison of incorporation of labeled ATP, UTP, and GTP, when initiation of transcription is prevented, indicates that initiation sites become relatively less A—T-rich as development proceeds. Inhibition of initiation of transcription in isolated nuclei blocks the normal increase in rate of transcription that occurs when cells enter the S phase, showing that an increased level of transcription depends upon a greater number of initiation sites.

Heterogeneous nuclear RNA (HnRNA) size in developing frog embryos

Different stages of developing frog embryos were briefly labeled with [ 3H]uridine and the rapidly labeled RNA was denatured and sized by sucrose density gradient centrifugation. Studies of the accumulation of labeled nuclear and cytoplasmic RNA were employed to learn the extent of turnover of nuclear RNA. Heterogeneous nuclear RNA (HnRNA) size increases during development and this is correlated with an increasing degree of turnover of HnRNA. Size differences of HnRNA exist between axial and belly regions of tailbud embryos. These data suggest that the differences in sizes of nuclear RNA may be important for post-transcriptional processing of HnRNA and control of gene expression in developing frog embryos.

Effects of exposure to N-phenyl-alpha-naphthylamine, octyl-phenyl-alpha-naphthylamine, and dioctyldiphenylamine on the development of frog embryos.

Effects of exposure to N-phenyl-alpha-naphthylamine, octyl-phenyl-alpha-naphthylamine, and dioctyldiphenylamine on the development of frog embryos.

Evidence for the lack of feedback regulation of gene activity and for the absence of subunit exchange between lactate dehydrogenase tetramers in vivo.

Frog and rat lactate dehydrogenase (LDH, L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isozymes, highly purfied by affinity chromatography, were injected into newly fertilized from (Rana pipiens) eggs to study the effect of the injected isozymes on the expression of the LDH genes of the developing frog embryo. From these experiments three conclusions can be drawn: (i) homologous and heterologous LDH, even when present in more than twice the normal amount of LDH, does not play any role in the switching on or off of the frog LDH genes or in regulating the level of gene activity. No evidence was found in support of any feedback regulation of LDH synthesis by the LDH molecule itself. (ii) Injected rat LDH is very stable in the frog embryo, and the contrary to what can be demonstrated in vitro, all the isozymes show the same stability. No evidence was found for selective degradation of any of the injected five isozymes. (iii) The fact that the injected isozymes disappear 12 days after the injection without forming hybrid molecules between themselves or with the stored or newly synthesized frog LDH is evidence in favor of the hypothesis that the tetramer is the degradative unit of LDH, not the monomer as others have postulated. The LDH tetramers do not dissociate the recombine under the physiological conditions present in frog cells.

Messenger activity of nuclear RNA of frog embryos

Equal amounts of neurula or larval nuclear RNA of developing frog embryos were injected into Xenopus oocytes that were incubated in a medium containing a labeled amino acid. Neurula nuclear RNA stimulated protein synthesis more than larval nuclear RNA.

The presence of the Lucké herpesvirus genome in induced tadpole tumors and its oncogenicity: Koch-Henle postulates fulfilled.

Herpesvirus extracted from a naturally occurring frog renal carcinoma (Lucké tumor) induced virus-free Lucké tumors in developing frogs. Herpesvirus recovered from an induced tumor after incubation at low temperature of tumor fragments cultured in vitro was oncogenic when injected into developing frog embryos. With the exception of the "pure culture" requirement, this experiment fulfills Koch-Henle postulates for the identification of the causative agent of the Lucké tumor.

Replicon growth rates during DNA replication in developing frog embryos

The rate of replicon growth during DNA replication was determined for unsynchronized and partially synchronized explants of developing frog embryos ( Rana pipiens). The replicon growth rates for unsynchronized gastrulae, neurulae and tailbuds are 0.06, 0.08 and 0.10 μm/min, respectively. In partially synchronized embryos the replicon growth rates during the early and late S phase are 0.10 and 0.02 μm/min in neurulae and 0.11 and 0.09 μm/min in tailbud embryos, respectively. There were no differences in growth rate between the dorsal axial and belly regions of neurulae and tailbuds. From these results the size and number of replicons was calculated.

Relative transcription from DNA of different degrees of redundancy in developing frog embryos

Frog DNA was separated into highly repetitious, moderately repetitious and least repetitious fractions which were hybridized to labeled RNA of isolated nuclei and whole cells of developing frog embryos and adult frog liver. There was a decrease in the number of kinds of D-RNA transcribed by all three classes of DNA in the nuclei during development, but an increase in the number of kinds of D-RNA in whole embryos during development. The thermal stability of heterologous hybrids of mouse and frog DNA indicate that it is the more repetitious sequences which are more conservative. The speculation is advanced that the general transcriptional pattern during development is based on the redundancy of the genome and the time of acquisition of genes during evolution.

Stimulation of RNA synthesis in developing frog embryos by microinjection of ribosomes.

AbstractOocyte ribosomes stimulate H3–5–uridine incorporation into RNA when microinjected into the vegetal cells of early cleavage embryos. Preparations with a low percentage of monosomes, such as those of swimming larvae and adult frog liver, give little stimulation. E. coli ribosomes fail to affect RNA synthesis. Short term labeling experiments at the gastrula stage in which only nuclear RNA was labeled suggest that added ribosomes may increase nuclear transcription and not cytoplasmic conservation of D‐RNA in polysomes.

Collagen synthesis in Xenopus oocytes after injection of nuclear RNA of frog embryos.

A messenger RNA fraction from polysomes of frog larvae or RNA preparations from isolated nuclei of developing frog embryos were injected into growing Xenopus laevis oocytes that were incubated with labeled proline. Column chromatography of protein hydrolyzates revealed labeled hydroxyproline after injection of the messenger RNA fraction and neurula nuclear RNA, indicating that the injected material had promoted collagen synthesis.

Incorporation of deoxythymidine triphosphate into nuclear DNA of developing frog embryos in vitro

The rate of incorporation of labeled precursor into DNA of isolated nuclei decreased from gastrulation to the tailbud stage in developing frog embryos. The addition of saturating amounts of calf thymus DNA revealed that the activities of the enzyme(s) which incorporates deoxythymidine triphosphate (dTTP) into DNA in vitro decreased over this period. Native DNA was a better template than denatured DNA. The addition of a purified microbial DNA polymerase to gastrula and tailbud nuclei showed that the chromatin DNA of the later stage is a poorer template for the enzyme. The activity of the enzyme(s) which accounts for dTTP incorporation into DNA in vitro was higher during the early S period of neurulae than for late S and higher for late S than early S of tailbuds when nuclei were isolated from partially synchronized cells. For both the randomly dividing and partially synchronized cells, the activity of the enzyme(s) incorporating dTTP into DNA in vitro is correlated with the level of incorporation of labeled precursor into DNA in vivo.

Nuclear DNA-like RNA of regions of developing frog embryos.

A comparison of equal amounts of labeled nuclear RNA of dorsal ectodermmesoderm and endoderm cells of frog neurulae by DNA-RNA hybridizations revealed more kinds of DNA-like RNA in the ectoderm-mesoderm nuclei.

Nuclear DNA-like RNA in developing frog embryos

Abstract The number of kinds of DNA-like RNA transcribed in nuclei of developing Rana pipiens embryos decreases from the early neurula to the larval stage. Approx. 16% of the DNA hybridized with RNA extracted from nuclei of early neurulae while only 2% of the DNA hybridized with RNA from larval nuclei. Competition experiments reveal that neurula nuclei contain copies of all the kinds of DNA-like RNA found in swimming larvae. Exhaustion experiments indicate that neurula nuclei contain a greater number of representatives of the class of D-RNA which is present in only a few copies relative to its homologous DNA sequences, as well as the D-RNA species which are present in numerous copies relative to their homologous DNA sequences, than do nuclei of later stages.