An early role of maternal mRNA in establishing the dorsoventral pattern in pelle mutant Drosophila embryos

Mutations of the maternal effect locus pelle (pll) cause dorsalized Drosophila embryos. In extreme mutants, the embryo develops into a long hollow tube of dorsal cuticular structures with no sign of ventral pattern elements. Injection of wild-type cytoplasm or poly(A)+RNA into mutant pll embryos partially restores the normal pattern. Rescuing activity is present in the wild-type cytoplasm until the late blastoderm stage, but is already absent from the poly(A)+RNA fraction by the time of pole cell formation. At the same time, pll embryos fail to respond to injected biologically active poly(A)+RNA. This indicates that pll+ mRNA is lost early from the pool of maternal RNA and that there is a non-RNA component of rescue. This component, most likely the pll+ protein, appears to be unequally distributed in wild-type embryos.     

Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm

A technique for transplantation of cytoplasm between Drosophila eggs is described. Polar cytoplasm of newly laid eggs was first made ineffective for the determination of germ cells by UV irradiation. The sterility which results from this UV irradiation could be prevented by the injection of polar cytoplasm, but not by the injection of anterior cytoplasm from unirradiated donor eggs. The results provide the first demonstration of transplantation of agents causing determination in an insect and also provide a bioassay for these agents.Microscopical observations of UV irradiated eggs with and without transplanted polar cytoplasm revealed that UV irradiation delays the migration of cleavage nuclei into the posterior periplasm and prevents cytoplasmic protrusions at the posterior pole from becoming isolated from the periplasm to form pole cells. Transplantation of polar cytoplasm repairs these defects.     

Analysis of cytoplasmic activity dependent on the Drosophila terminal pattern gene torso

Cytoplasm from wildtype Drosophila embryos was transplanted into torso (tor) mutant embryos to determine the distribution of terminal rescuing activity at the cleavage stage. Although posterior and lateral wildtype cytoplasm contained rescuing activity that restored posterior terminal (telson) structures Klingler et al. (1988, Nature (London) 335, 275-277) this rescuing activity was not found in anterior cytoplasm. Similarly, transplantation of anterior and lateral wildtype cytoplasm into the anterior of tor embryos rescued anterior terminal (acron) structures, whereas posterior cytoplasm did not. This failure of reciprocal rescue is due to the presence of the products of the anterior and posterior classes of genes, because anterior cytoplasm from bicoid mutant embryos restored the telson in the posterior as well as the acron in the anterior of tor embryos, and because posterior cytoplasm from nanos embryos rescued the acron in the anterior as well as the telson in the posterior of tor embryos. Therefore terminal rescuing activity is evenly distributed throughout the cleavage stage embryo as anticipated from molecular studies.     

Functions of maternal mRNA as a cytoplasmic factor responsible for pole cell formation in Drosophila embryos

Injection of mRNA extracted from Drosophila cleavage embryos or mature oocytes restored pole cell-forming ability to embryos that had been deprived of this ability by uv irradiation. However, mRNA extracted from blastoderms did not show the restoration activity. Pole cells thus formed in uv-irradiated embryos bear similarities to normal pole cells both in their morphology and their ability to migrate to the gonadal rudiments. But this mRNA does not appear to be capable of rescuing uv-induced sterility, or inducing pole cells in the anterior polar region.     

Injection of wild-type cytoplasm and poly(A)+ RNA provokes phenotype rescue in spätzle mutant Drosophila embryos

Summary spätzle (spz), a maternal effect gene of Drosophila, is involved in the establishment of the dorso-ventral axis during embryogenesis. Eggs from females lacking the spz gene product develop into completely dorsalized embryos, i.e. the ventral and lateral pattern elements fail to develop. Upon injection of either cytoplasm or poly(A)⁺ RNA from early wild-type embryos, spz embryos develop lateral pattern elements represented by Filzkörper and in the case of injected cytoplasm additional ventral pattern elements represented by ventral setae. Wild-type cytoplasm retains the rescuing activity longer than the poly(A)⁺ RNA fraction does, and cytoplasm is always more effective in provoking the rescue than poly(A)⁺ RNA. Mosaic females containing spz germ cells surrounded by spz ⁺ tissues were generated by pole cell transplantations; a mutant genotype in the germ cells is sufficient to produce all aspects of the spz mutant phenotype, suggesting that the maternal source of spz gene product is the germ line.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.     

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization. Evidence for a nuclear origin

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)+RNA was therefore investigated. In gel electrophoresis poly(A)+RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3 . 10(5) and 5 . 10(5) Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)+RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)+RNA component of approx. 5 . 10(5) Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)+RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)+RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)+RNA from developing embryos. The hybridization of labelled, nuclear poly(A)+RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)+RNA in dormant cysts is essentially of nuclear origin. The 5 . 10(5)-Da component is largely homologous with the corresponding component of nuclear poly(A)+RNA at later stages.     

Abdominal segmentation, pole cell formation, and embryonic polarity require the localized activity of oskar, a maternal gene in Drosophila

Embryos derived from oskar females lack pole cells and the specialized pole plasm including polar granules. In addition, the abdominal region remains unsegmented and eventually dies. Transplantation of cytoplasm from normal embryos into mutant embryos reveals that osk-dependent activity is strictly localized at the posterior pole and has three distinct functions. In mutant embryos the activity will normalize pole cell formation when transplanted into the posterior pole and abdominal segmentation after transplantation to a more anterior, the prospective abdominal, region. Furthermore, osk activity can provoke the formation of a second "posterior center" at the anterior. The participation of the osk product in the establishment of a source of morphogenetic activity in the posterior pole plasm is discussed.     

Poly(A)-containing RNA in early embryogenesis of sea urchins

The content, biosynthesis and template activity of poly(A)+ RNA in the early stages of sea urchin development have been studied. The amount of poly(A)+ RNA reaches a maximum at the middle blastula stage in polyribosomes and at the 8-blastomere stage in the cytoplasm. Poly(A)+ RNA synthesis becomes noticeable at the 64-blastomere stage and the spectrum of newly synthesized molecules is different from that at the middle blastula stage. The products of translation in vitro of poly(A)+ RNA at all the stages studied show insignificant differences and contain a major group of polypeptides of molecular mass 10-20 kDa.     

Changes in the apparent localization of anterior determinants during early embryogenesis (Smittia spec., Chironomidae,Diptera)

Summary InSmittia embryos, anterior UV irradiation causes the formation of double abdomens in which head and thorax are replaced by a mirror-image duplication of abdominal segments. The transformation is ascribed to the inactivation of cytoplasmic ribonucleoprotein particles acting as anterior determinants. During the first 5 h after egg deposition (h.a.d.), high yields of double abdomens are produced by microbeam irradiation of a small target area (diameter 20 m) behind the anterior pole. This localization coincides with a yolk-free cytoplasmic cone observed behind the anterior pole of newly depositedSmittia eggs (Zissler and Sander 1973). Irradiation of this area becomes less efficient between 5 and 7 h.a.d., while double abdomen yields increase after irradiation of adjacent target areas. A dramatic increase in the double abdomen yield is also observed after irradiation of the entire anterior surface. Under these conditions, the UV fluence required for a given yield decreases by a factor of 3.9 between 5.5. and 7 h.a.d. This can be explained quantitatively by the assumption that the effective targets move an average distance of 8 from the strongly absorbing endoplasm towards the egg surface. During the period of apparent target deshielding, the distance of nuclei from the egg surface actually decreases from 11 to 3 m. However, the timing and direction of the nuclear migration and of the apparent deshielding differ, indicating that the shift of effective targets may be triggered by, but does not exactly follow, the nuclear movement. The data suggest that a major fraction of anterior determinants shifts from a small volume behind the anterior pole to a spread-out distribution over the surface of the anterior half.     

In vitro synthesis of full-length influenza virus complementary RNA.

Influenza virus-specific RNA has been synthesized in vitro, using cytoplasmic or microsomal fractions of influenza virus-infected MDCK cells. The RNA polymerase activity was stimulated 5-30 times by priming with ApG. About 20-30% of the product was polyadenylated. Most of the in vitro product was of positive polarity, as shown by hybridization to strand specific probes and by T1 fingerprinting of the poly(A)+ and poly(A)- RNA segments encoding haemagglutinin and nucleoprotein. The size of poly(A)- RNA segments, determined on sequencing gels, was indistinguishable from that of virion RNA, whereas poly(A)+ RNA segments contain poly(A) tails approximately 50 nucleotides long. The size of in vitro synthesized RNA segments was also determined by gel electrophoresis of S1-treated double-stranded RNAs, obtained by hybridization of poly(A)+ or poly(A)- RNA fractions with excess of unlabelled virion RNA. The results of these experiments indicate that poly(A)- RNA contains full-length complementary RNA. This conclusion is further substantiated by the presence of additional oligonucleotides in the T1 fingerprints of in vitro synthesized poly(A)- haemagglutinin or nucleoprotein RNA, selected by hybridization to cloned DNA probes corresponding to the 3' termini of the genes.     

Localization of nanos RNA controls embryonic polarity.

Anterior-posterior polarity of the Drosophila embryo is initiated during oogenesis through differential maternal RNA localization. The RNA of the anterior morphogen bicoid is localized to the anterior pole of the embryo, where bicoid protein controls head and thorax development. The RNA of the posterior morphogen nanos is localized to the posterior pole, where nanos protein is required for abdomen formation. Here we show that the nanos 3' untranslated region, like that of the bicoid RNA, is sufficient for RNA localization. We have used the bicoid RNA localization signal to mislocalize nanos, producing embryos with two sources of nanos protein. Such embryos form two abdomens with mirror image symmetry. Embryos with nanos RNA localized only to the anterior have greater nanos gene activity than embryos with nanos RNA localized posteriorly. We propose a role for RNA localization in regulating nanos activity.     

Cloning and transcriptional analysis of the segmentation gene fushi tarazu of Drosophila

In the course of studying the Antennapedia (Antp) locus, we found that one of the 3' Antp exons has weak cross-homology to another gene affecting segmentation, fushi tarazu (ftz; meaning "not enough segments"), which is 30 kb to the left of Antp. Homozygous ftz- embryos die before hatching and lack alternate body segments. The reduced number of segments results from the fusion of the anterior portion of one segment with the posterior portion of the next segment. The ftz gene encodes a single 1.9 kb poly(A)+ RNA expressed exclusively from the early blastoderm to gastrula stages of embryonic development. The structure of the ftz gene has been analyzed by S1 nuclease mapping and by restriction mapping of a cDNA clone. The ftz gene consists of two exons, and it is the 3' exon that cross-hybridizes with the 3' exon of Antp. The role of ftz in cell determination is discussed.     

Double abdomen induction by UV in Bradysia tritici (syn. Sciara ocellaris,Sciaridae): sensitive stages and conditions for photoreversal

Summary The pattern anomaly double abdomen was induced in embryos of Bradysia tritici (syn. Sciara ocellaris) by irradiation of the anterior egg pole with far UV (254 or 285 nm) using low UV fluences. The maximum yield of 18% of double abdomens was obtained when 2.5 h embryos were irradiated (late intravitelline cleavage stage); earlier irradiation failed to yield double abdomens, as did irradiations after the early syncytial blastoderm stage. Exposing irradiated embryos to photoreverting light (366 nm) reduced the yield of malformations. Most double abdomens were symmetrical and the number of segments ranged from 3 to 8 in each set, with the mean value at 6.4 segments.     

Messenger RNA during early embryogenesis in Artemia salina: altered translatability and sequence complexity

RNA from developing embryos of Artemia salina (5, 10, and 20 h after re-initiation of development) was translated 3-10 times more efficiently in a rabbit reticulocyte lysate cell-free protein synthesizing system than RNA from dormant gastrulae. The latter did not appear to contain any significant amount of translation inhibitor activity. Ninety percent of the translatable activity in dormant gastrulae was recovered as poly(A)--RNA, whereas 80% of that in post-gastrular developing embryos was present as poly(A)+-RNA. The size of most polypeptides coded for by dormant gastrular RNA was less than 130,000 daltons whereas the size of those coded for by developing embryonic RNA was up to 200,000 daltons, which correlated with a corresponding shift to poly A-containing RNA of higher molecular weight. Two major polypeptides of about 37,000 daltons coded for by dormant gastrular RNA disappeared at 20 h after resumption of development. Hybridization of complementary DNA (cDNA) to a 1000-fold excess of the homologous poly(A)+-RNA revealed the presence of three complexity classes of mRNA. Forty-five percent, 30%, and 25% of RNA in dormant gastrulae were present as high, middle, and low abundance classes comprising about 10, 80, and 9700 species, respectively whereas in the nauplii there were 10, 150, and 7900 species of high, middle, and low abundancy sequences, respectively. Heterologous hybridizations using cDNA complementary to highly abundant messenger population of nauplii (isolated by chromatography on hydroxyapatite) to poly(A)+-RNA from dormant cysts showed considerably divergence in this class of messengers from the two developmental stages. Re-initiation of development of dormant Artemia gastrulae is thus characterized by a "re-programming" seen as a simultaneous and rapid increase in the polyadenylation and translatability of poly(A)+-RNA accompanied by a qualitative change in its sequence complexity.