Tissue-specific changes in nuclear RNA content during early development of Triturus vulgaris

Summary During early development of Triturus vulgaris, as a measure for nuclear activity in neuroectoderm, mesoderm and endoderm, nuclear RNA content was determined by cytochemical methods. In the first stages of gastrulation, that is to say during the early phase of neural induction, the RNA content of the inducing system is considerably higher than in the reacting system. Then, with a phase-shift of about 10 h, the RNA content of the neuroectoderm increases quickly also. In the following stages the nuclear RNA content of both regions is reduced. A second continuous increase in the RNA amount coincides with the formation of the neural tube. In the mesoderm, enhancement of RNA content correlates with cytodifferentiation of the chorda. In all stages the RNA content of the endoderm is higher than in the other tissues and it becomes successively diminished from the early gastrula to the tailbud stage.     

Tissue-specific expression of SORBITOL DEHYDROGENASE in apple fruit during early development

Sorbitol, the primary photosynthate and translocated carbohydrate in apple (Malusxdomestica Borkh.), is converted to fructose by sorbitol dehydrogenase (SDH; EC 1.1.1.14) which is active in apple fruit throughout development. In the apple genome, nine SDH genes have been isolated and their sequences characterized, but their individual expression patterns during apple fruit set and development have not been determined. The objective of this work was to ascertain if SDH genes are differentially expressed and how their patterns of expression may relate to SDH activity in apple seed and cortex during early fruit development. Seed SDH activity was found to be much higher than cortex SDH activity per mg and g fresh weight (FW), and seed SDH activity contributed significantly to whole fruit SDH activity during weeks 2-5 after bloom. Five of the nine SDH genes present in the apple genome were expressed in apple fruit. Two SDH genes, SDH1 and SDH3, were expressed in both seed and cortex tissues. SDH2 expression was limited to cortex, while SDH6 and SDH9 were expressed in seed tissues only. SDH isomeric proteins of different pI values were detected in apple fruit. SDH isomers with pI values of 4.2, 4.8, 5.5, and 6.3 were found in seeds, and SDH isomers with pI values of 5.5, 6.3, 7.3, and 8.3 were found in cortex. The present work is the first to show that SDH is highly active in apple seed and that SDH genes are differentially expressed in seed and cortex during early development.     

Tissue-specific requirements for FGF8 during early inner ear development

Several members of the FGF gene family have been shown to intervene from various tissue sources to direct otic placode induction and otic vesicle formation. In this study we define the roles of FGF8, found in different expression domains during this process, in mice and chickens. By conditional inactivation of Fgf8 in distinct tissue compartments we demonstrate that Fgf8 is required in the mesoderm and endoderm during early inner ear development. In the chicken embryo, overexpression of Fgf8 from various tissue sources during otic specification leads to a loss of otic tissue. In contrast ectopic overexpression of Fgf10, a major player during murine otic induction, does not influence otic vesicle formation in chicken embryos but results in the formation of ectopic structures with a non-otic character. This study underlines the crucial role of a defined Fgf8 expression pattern controlling inner ear formation in vertebrates.     

Relative characteristics of the duration of embryonic development in Triturus vulgaris

The data of Valouch et al. (1971) on tau 0 values in the smooth newt at different temperatures are presented as a curve. Using this curve, the absolute (in hours and days) duration of successive developmental stages provided by Glaesner (1925) is transformed in a relative one, expressed in the number of tau 0 (tau n/tau 0).     

RNA polymerase during early development in mouse embryo

RNA polymerase activity in mouse embryo homogenates has been measured at various stages of pre-implantation development. The amount of enzyme/embryo appears to increase in the period under consideration. On a per cell basis a decline in the level of polymerase was, however, observed from the 2-cell to the early blastocyst stages.     

Messenger RNA synthesis during early ascidian development.

RNA synthesis during early embryogenesis of the ascidian Ciona intestinalis was studied. Embryonic polyribosomes labeled with uridine from 5 to 7 hr after fertilization were isolated and the labeled RNA species were characterized by oligo(dT)-cellulose chromatography and sucrose gradient sedimentation analysis. Since at least 50% of the labeled RNA was polyadenylated and all of it sedimented heterogeneously, it was concluded that mRNA was synthesized during the labeling period. Further, the synthesis of heterogeneously sedimenting, polyadenylated RNA at various stages of development from midcleavage to metamorphosis indicated that gene activity and perhaps mRNA synthesis occurred at earlier and later stages of development as well. Autoradiographic studies showed that the embryonic genome was the site of this activity, since uridine incorporation was localized in embryonic cells and not in accessory cells. Finally, under the labeling conditions employed (2-hr pulses), rRNA synthesis was not detected until larvae hatched.     

Identification of pigment cells during early amphibian development (Triturus alpestris,Ambystoma mexicanum)

Summary The purpose of the present investigation was to provide and apply a methodological manual with which the distribution, patterning and relationship of melanophores and xanthophores can be analyzed during early amphibian development. For demonstration of the methods, which include ultrastructural, histochemical and biochemical approaches, Triturus alpestris and Ambystoma mexicanum (axolotl) embryos are used. These two species differ conspicuously in their larval pigment patterns, showing alternating melanophore bands in horizontal (T. alpestris) and vertical (axolotl) arrangements. With transmission- and scanning electron microscopy melanophores and xanthophores were distinguished by their different pigment organelles and surface structures. The presence of phenol oxidase (tyrosinase) was used to reveal externally invisible or faintly visible melanophores by applying an excess of 3,4 dihydroxy-phenylalanine (dopa). Xanthophores were made visible in fixed and living embryos by demonstrating their pterin fluorescence. In addition, pterins were analyzed by HPLC in embryos before and after pigmentation was visible.Abbreviations DOPA dihydroxy-phenylalanine - FCS fetal calf serum - FIF formaldehyde-induced fluorescence - FITC fluorescein isothiocyanate - HPLC high performance liquid chromatography Dedicated to the memory of Dr. Michael Claviez     

Fluoropyrimidine-mediated changes in small nuclear RNA

Studies were completed to examine the effects of the antineoplastic agent 5-Fluorouridine (FUrd) on the metabolism of the small molecular weight nuclear RNA (snRNA). Cultured Sarcoma-180 murine tumor cells were exposed to FUrd concurrent with [3H]cytidine for 6 h, the drug was removed, and the RNA was isolated at 0, 24, or 48 h following the drug treatment. The results of these studies demonstrated that FUrd produced three dose-dependent changes in snRNA metabolism. The electrophoretic migration of the U4 and U6 snRNA was altered in nondenaturing 10% polyacrylamide slab gels. These results were not observed in denaturing gels or when RNA was extracted at temperatures exceeding 25 degrees C, suggesting that the incorporation of 5-fluorouracil induced secondary structural changes in these RNA. A dose- and time-dependent selective reduction in the turnover of the U1 snRNA synthesized in the presence of FUrd was observed as well, with levels over 100% higher than control cells at 48 h after exposure to 10 microM FUrd. These changes in snRNA metabolism may contribute to the reported alterations in large molecular weight RNA metabolism that also result in fluoropyrimidine-treated cells, or to cytotoxicity.     

Localization of actin messenger RNA during early ascidian development

The spatial distribution of RNA sequences during early development of the ascidian, Styela plicata, was determined by in situ hybridization with poly(U) and cloned DNA probes. Styela eggs and embryos contain three colored cytoplasmic regions of specific morphogenetic fates, the ectoplasm, endoplasm, and myoplasm. These cytoplasmic regions participate in ooplasmic segregation after fertilization and are distributed to different cell lineages during early embryogenesis. n situ hybridization with poly(U) suggests that poly(A)+RNA is unevenly distributed in eggs and embryos, with about 45% in the ectoplasm, 50% in the endoplasm, and only 5% in the myoplasm. In situ hybridization with a histone DNA probe showed that histone RNA sequences were not localized in eggs or embryos and distributed between the three cytoplasmic regions according to their volumes. In situ hybridization with an actin DNA probe showed actin RNA was localized in the myoplasm and ectoplasm of eggs and embryos with about 45% present in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm. These results suggest that a large proportion of the egg actin mRNA is localized in the myoplasm, participates in ooplasmic segregation after fertilization, and is differentially distributed to the mesodermal cell lineages during embryogenesis. Analysis of the translation products of egg mRNA suggests that the localized mRNA codes for a cytoplasmic actin isoform.     

Chromosomal location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

The 5S ribosomal RNA genes have been localized in mitotic and lampbrush chromosomes of Triturus vulgaris meridionalis by in situ hybridization. These genes are clustered in a single locus in an intercalary position of the long arm of chromosome XI. In lampbrush chromosome XI the 5S genes are located near a loop landmark mapped at 66 units.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

The mitotic chromosomes of six specimens from Triturus vulgaris meridionalis have been examined by both in situ hybridization with ³H 18S+28S rRNA and AS-SAT staining method. The results of these two sets of experiments can be summarized as follows: 1) in each specimen the NORs and the additional ribosomal sites, which react positively to in situ hybridization with ³H 18S + 28S rRNA, are also stained by silver; 2) other chromosomal regions, which do not hybridize in situ with ³H 18S+28S rRNA, are on the other hand stained by the AS-SAT method. These latter Ag-positive sites show a species-specific pattern of chromosomal distribution.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with ³H 18S+28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA the DNA sequences coding for 18S+28S rRNA plus the intervening spacer sequences - SSC 0.15 M sodium chloride, 0.015 sodium citrate, pH 7