RNA sorting in Drosophila oocytes and embryos.

Many RNAs involved in determination of the oocyte, specification of embryonic axes, and establishment of germ cells in Drosophila are localized asymmetrically within the developing egg or syncytial embryo. Here I review the current state of knowledge about the cis-acting sequences involved in RNA targeting, RNA binding proteins; gene activities implicated in localizing specific RNAs, and the role of the tubulin and actin cytoskeletons in RNA sorting within the oocyte. Targeted RNAs are often under complex translational control, and the translational control of two RNAs that localize to the posterior of the oocyte, oskar and nanos, is also discussed. Prospects for filling gaps in our knowledge about the mechanisms of localizing RNAs and the importance of RNA sorting in regulating gene expression are also explored.     

Specific control of messenger translation in Drosophila oocytes and embryos

The distribution of messenger RNA between polysomes and mRNP in oocytes and embryos of Drosophila melanogaster has been studied by in vitro translational analysis. Poly(A)⁺ RNA was purified from polysomes or mRNA from mature oocytes and young embryos. The messenger populations were translated in vitro and the peptides synthesized were separated by two-dimensional electrophoresis. Analysis of the 2D gel patterns enabled the detection of three peptides coded by messengers present predominantly in the mRNA pools of mature oocytes. When DNA-binding peptides were selected from the in vitro translation products, they showed, after separation by two-dimensional electrophoresis, less than 100 spots. The analysis of the 2D gels indicated that three DNA-binding peptides are coded by messengers present only in the mRNP of the oocytes. These messengers are later found in the polysomal fraction of embryos.     

Secretory and inductive properties of Drosophila wingless protein in Xenopus oocytes and embryos.

Like its vertebrate homologues, Xenopus wnt-8 and murine wnt-1, we find that Drosophila wingless (wg) protein causes axis duplication when overexpressed in embryos of Xenopus laevis after mRNA injection. In many cases, the secondary axes contain eyes and cement glands, which reflect the induction of the most dorsoanterior mesodermal type, prechordal mesoderm. We show that the extent of axis duplication is dependent on the embryonic site of expression, with ventral expression leading to a more posterior point of axis bifurcation. The observed duplications are due to de novo generation of new axes as shown by rescue of UV-irradiated embryos. The true dorsal mesoderm-inducing properties of wg protein are indicated by its ability to generate extensive duplications after mRNA injection into D-tier cells of 32-cell embryos. As revealed by lineage mapping, the majority of these D cell progeny populate the endoderm; injections into animal blastomeres at this stage are far less effective in inducing secondary axes. However, when expressed in isolated animal cap explants, wg protein induces only ventral mesoderm, unless basic fibroblast growth factor is added, whereupon induction of muscle and occasionally notochord is seen. We conclude that in intact embryos, wg acts in concert with other factors to cause axis duplication. Immunolocalisation studies in embryos indicate that wg protein remains localised to the blastomeres synthesizing it and has a patchy, often perinuclear distribution within these cells, although some gets to the surface. In oocytes, the pool of wg protein is entirely intracellular and relatively unstable. When the polyanion suramin is added, most of the intracellular material is recovered in the external medium.     

An RNA-binding protein from Xenopus oocytes is associated with specific message sequences

Monoclonal antibodies directed against an RNA-binding protein from Xenopus oocytes were used to immunoselect messenger ribonucleoprotein (mRNP) particles. RNA was extracted from both the immunoselected and nonselected fractions and was used to direct the synthesis of oligo (dT)-primed 32P-cDNA. These two cDNA preparations were then used to probe Xenopus stage-1 oocyte cDNA libraries to identify sequences that had been specifically coimmunoselected by the antibodies. Three cDNA clones were shown to be derived specifically from the antibody-selected mRNPs. During very early oogenesis (stage 1-2), the RNA-binding protein and the three coselected mRNAs sediment in the nontranslating mRNP region of a sucrose gradient. By oocyte stage 6, the binding protein concentration decreases by as much as 22-fold relative to polyadenylated RNA. At this stage of development, the three mRNAs are found predominantly in the polysome region of a sucrose gradient. These data demonstrate that Xenopus oocytes contain an RNA-binding protein which binds specific message sequences and may regulate their expression.     

The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader

When Drosophila cells are heat shocked, the translation of normal cellular mRNAs is repressed, while mRNAs encoding the heat-shock proteins are translated at high rates. We have found that the hsp70 message is not translated at high temperatures when its leader sequence is deleted. This message is translated when the cells are allowed to recover at 25 degrees C, but the translation ceases when the cells are given a second heat shock. A message with an extra 39 bases added onto the 5' end of the leader behaves in the same way. However, if either of two conserved sequence elements in the leader is deleted, the message is still translated during heat shock. Although the specific feature responsible for the preferential translation of heat-shock messages is not yet identified, we conclude that it must reside in the 5' untranslated leader.     

Change in message sequences during erythrodifferentiation.

The change in the poly A(+) mRNA population during erythrodifferentiation was analyzed by cDNA-RNA hybridization. Poly A(+) RNA was isolated from spleen erythroblasts. When mice became anemic, the amount of globin mRNA increased to 50% of the total poly A(+) mRNA. cDNA from anemic spleen erythroblasts that did not contain globin mRNA sequences was cross-hybridized with mRNAs from mouse reticulocytes and cultured Friend leukemia (FL) cells. Only half the spleen cDNA hybridized with reticulocyte mRNA, whereas most of it hybridized with mRNA from FL cells. The results suggest that decrease in the complexity of the message population and increase in the concentration of globin mRNA are important in erythrodifferentiation.     

RNA sorting in Xenopus oocytes and embryos.

Cytoplasmic localization of mRNA molecules has emerged as a powerful mechanism for generating spatially restricted gene expression. This process is an important contributor to cell polarity in both somatic cells and oocytes, and can provide the basis for patterning during embryonic development. In vertebrates, this phenomenon is perhaps best documented in the frog, Xenopus laevis, where polarity along the animal-vegetal axis coincides with the localization of numerous mRNA molecules. Research over the last several years has made exciting progress toward understanding the molecular mechanisms underlying cytoplasmic mRNA localization.     

Microfilament distribution in cold-treated Drosophila embryos.

Cold treatment of Drosophila embryos is observed to result in general alteration of microfilament distribution leading to deformation of the surface caps and to perturbation of the process of cleavage furrow extension. After exposure to low temperature the cortical actin caps underwent several morphological changes, despite the arrested nuclear cycle. These observations are discussed in relation to centrosome behavior during the cell cycle.     

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.     

Ultrastructural studies of oocytes and embryos derived from females flies carrying the grandchildless mutation in Drosophila subobscura

The maternal effect mutant grandchildless in Drosophila subobscura has been analyzed with the electron microscope. The original mutation was linked to a visible genetic marker and established in a balanced stock. Oocytes and early embryos were examined by both transmission and scanning electron microscopy. The earliest defect is seen in mutant eggs and occurs at the end of oogenesis. In the cortex, at both the anterior and the posterior tips, regions appear which are free of ribosomes, mitochondria, and other cytoplasmic organelles. Most of the polar granules are included in these regions at the posterior tip. Following oviposition, this cytoplasmic segregation is no longer observed and most polar granules have disappeared. The few remaining granules are presumed to derive from the peripheral polar plasm which does not become segregated. During embryogenesis there is a retarded movement of nuclei to the anterior and posterior cortices. At the posterior tip nuclei are delayed in reaching the lateral sides and never move directly into the posterior polar plasm. Pole cells never form. After the last syncytial division the lateral nuclei move under the posterior polar plasm to complete the blastoderm. The posterior polar plasm itself protrudes during blastoderm formation as long cytoplasmic extensions which separate from the blastoderm as cytoplasmic blebs. Neither polar granules nor mitochondria are found in these blebs. The grandchildless phenotype is due to the failure of nuclei to migrate directly into the posterior polar plasm. The defect in the polar plasm presumably is related to the process in mature eggs whereby portions of the cortex become segregated at both anterior and posterior tips. This process may change the properties of the posterior polar plasm so that nuclei do not penetrate into it.     

Non-random position of the A-T rich DNA sequences in early embryos of Drosophila virilis

Examination of early embryos of Drosophila virilis by light and electron microscopy has shown that the A-T rich satellite DNA sequences have a non-random distribution within the nuclei. As observed by 33258 Hoechst staining and fluorescent microscopy, these sequences are consistently found to be located on the sides of the nuclei nearest to the vitelline membrane. This arrangement of the A-T rich sequences has been observed from the syncytial balstoderm stage into the gastrula stage where, in each nucleus, the satellite DNA sequences remain at a point nearest the topological outside of the organism.     

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.