The maternal gene nanos has a central role in posterior pattern formation of the Drosophila embryo

A group of maternal genes, the posterior group, is required for the development of the abdominal region in the Drosophila embryo. We have used genetic as well as cytoplasmic transfer experiments to order seven of the posterior group genes (nanos, pumilio, oskar, valois, vasa, staufen and tudor) into a functional pathway. An activity present in the posterior pole plasm of wild-type embryos can restore normal abdominal development in posterior group mutants. This activity is synthesized during oogenesis and the gene nanos most likely encodes this activity. The other posterior group genes have distinct accessory functions: pumilio acts downstream of nanos and is required for the distribution or stability of the nanos-dependent activity in the embryo. Staufen, oskar, vasa, valois and tudor act upstream of nanos. Embryos from females mutant for these genes lack the specialized posterior pole plasm and consequently fail to form germ-cell precursors. We suggest that the products of these genes provide the physical structure necessary for the localization of nanos-dependent activity and of germ line determinants.     

Staufen, a gene required to localize maternal RNAs in the Drosophila egg.

The posterior group gene staufen is required both for the localization of maternal determinants to the posterior pole of the Drosophila egg and for bicoid RNA to localize correctly to the anterior pole. We report the cloning and sequencing of staufen and show that staufen protein is one of the first molecules to localize to the posterior pole of the oocyte, perhaps in association with oskar RNA. Once localized, staufen is found in the polar granules and is required to hold other polar granule components at the posterior pole. By the time the egg is laid, staufen protein is also concentrated at the anterior pole, in the same region as bicoid RNA.     

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.     

Maternal-effect mutations altering the anterior-posterior pattern of the Drosophila embryo

Summary Mutations in seven different maternal-effect loci on the second chromosome of Drosophila melanogaster all cause alterations in the anterior-posterior pattern of the embryo. Mutations in torso (tor) and trunk (trk) delete the anterior- and posterior-most structures of the embryo. At the same time they shift cellular fates which are normally found in the subterminal regions of the embryo towards the poles. Mutations in vasa (vas), valois (vls), staufen (stau) and tudor (tud) cause two embryonic defects. For one they result in absence of polar plasm, polar granules and pole cells in all eggs produced by mutant females. Secondly, embryos developing inside such eggs show deletions of abdominal segments. In addition, embryos derived from staufen mothers lack anterior head structures, embryos derived from valois mothers frequently fail to cellularize properly. Mutations in exuperantia (exu) cause deletions of anterior head structures, similar to torso, trunk and staufen. However in exu, these head structures are replaced by an inverted posterior end which comprises posterior midgut, proctodeal region, and often malpighian tubules.The effects of all mutations can be traced back to the beginning stages of gastrulation, indicating that the alterations in cellular fates have probably taken place by that time. Analysis of embryos derived from double mutant mothers suggests that these three phenotypic groups of mutants interfere with three different, independent pathways. All three pathways seem to act additively on the system which specifies anterior-posterior cellular fates within the egg.     

Mitochondrial small ribosomal RNA is present on polar granules in early cleavage embryos of Drosophila melanogaster

In Drosophila, formation of the germline progenitors, the pole cells, is induced by polar plasm localized in the posterior pole region of early embryos. The polar plasm contains polar granules, which act as a repository for the factors required for pole cell formation. It has been postulated that the factors are stored as mRNA and are later translated on polysomes attached to the surface of polar granules. Here, the identification of mitochondrial small ribosomal RNA (mtsrRNA) as a new component of polar granules is described. The mtsrRNA was enriched in the polar plasm of the embryos immediately after oviposition and remained in the polar plasm throughout the cleavage stage until pole cell formation. In situ hybridization at an ultrastructural level revealed that mtsrRNA was enriched on the surface of polar granules in cleavage embryos. Furthermore, the localization of mtsrRNA in the polar plasm depended on the normal function of oskar, vasa and tudor genes, which are all required for pole cell formation. The temporal and spatial distribution of mtsrRNA is essentially identical to that of mitochondrial large ribosomal RNA (mtlrRNA), which has been shown to be required for pole cell formation. Taken together, it is speculated that mtsrRNA and mtlrRNA are part of the translation machinery localized to polar granules, which is essential for pole cell formation.     

Germline autonomy of maternal-effect mutations altering the embryonic body pattern of Drosophila

Nine maternal-effect loci Drosophila melanogaster were tested in germline mosaics to determine whether the wildtype gene activity is required in somatic or germline components of the maternal ovary. Mutations in these loci affect the anterior-posterior or dorso-ventral body pattern. In all nine loci (torso, trunk, exuperantia, vasa, valois, staufen, tudor, dorsal, Toll) a mutant genotype in the germ cells is sufficient to produce all aspects of the mutant embryonic phenotype, even when those germ cells are surrounded by wildtype somatic tissues.     

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.     

Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole

The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end-directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex.     

Oskar organizes the germ plasm and directs localization of the posterior determinant nanos

Oskar is one of seven Drosophila maternal-effect genes that are necessary for germline and abdomen formation. We have cloned oskar and show that oskar RNA is localized to the posterior pole of the oocyte when germ plasm forms. This polar distribution of oskar RNA is established during oogenesis in three phases: accumulation in the oocyte, transport toward the posterior, and finally maintenance at the posterior pole of the oocyte. The colocalization of oskar and nanos in wild-type and bicaudal embryos suggests that oskar directs localization of the posterior determinant nanos. We propose that the pole plasm is assembled stepwise and that continued interaction among its components is required for germ cell determination.     

Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos

In Drosophila, polar plasm contains polar granules, which deposit the factors required for the formation of pole cells, germ line progenitors. Polar granules are tightly associated with mitochondria in early embryos, suggesting that mitochondria could contribute to pole cell formation. We have previously reported that mitochondrial large and small rRNAs (mtrRNAs) are transported from mitochondria to polar granules prior to pole cell formation and the large rRNA is essential for pole cell formation. Here we show that the localization of mtrRNAs is diminished in embryos laid by tudor mutant females, although the polar granules are maintained. We also found that Tud protein was colocalized with mtrRNAs at the boundaries between mitochondria and polar granules when the transport of mtrRNAs takes place. These observations suggest that Tud mediates the transport of mtrRNAs from mitochondria to polar granules.     

tudor, a gene required for assembly of the germ plasm in Drosophila melanogaster

Developmental analysis of a newly isolated maternal effect grandchildless mutant, tudor (tud), in Drosophila melanogaster indicates that tud+ activity is required during oogenesis for the determination and/or formation of primordial germ cells (pole cells) and for normal embryonic abdominal segmentation. Regardless of their genotype, progeny of females homozygous for strong alleles (tud1 and tud3) never form pole cells, apparently lack polar granules in the germ plasm, and approximately 40% of them die during late embryogenesis exhibiting severe abdominal segmentation pattern defects. Females carrying weak allele, tud4, produce progeny with some functional pole cells and form polar granules approximately one-third the size of those observed in wild-type oocytes and embryos. No segmentation abnormalities are observed in the inviable embryos derived from tud4/tud4 females.     

Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation.

Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton.     

Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patterning

Pole cells and posterior segmentation in Drosophila are specified by maternally encoded genes whose products accumulate at the posterior pole of the oocyte. Among these genes is tudor (tud). Progeny of hypomorphic tud mothers lack pole cells and have variable posterior patterning defects. We have isolated a null allele to further investigate tud function. While no pole cells are ever observed in embryos from tud-null mothers, 15% of these embryos have normal posterior patterning. OSKAR (OSK) and VASA (VAS) proteins, and nanos (nos) RNA, all initially localize to the pole plasm of tud-null oocytes and embryos from tud-null mothers, while localization of germ cell-less (gcl) and polar granule component (pgc), is undetectable or severely reduced. In embryos from tud-null mothers, polar granules are greatly reduced in number, size, and electron density. Thus, tud is dispensable for somatic patterning, but essential for pole cell specification and polar granule formation.     

Identification of a component of Drosophila polar granules

Information necessary for the formation of pole cells, precursors of the germ line, is provided maternally and localized to the posterior pole of the Drosophila egg. The maternal origin and posterior localization of polar granules suggest that they may be associated with pole cell determinants. We have generated an antibody (Mab46F11) against polar granules. In oocytes and early embryos, the Mab46F11 antigen is sharply localized to the posterior embryonic pole. In pole cells, it becomes associated with nuclear bodies within, and nuage around, the nucleus. Immunoreactivity remains associated with cells of the germ line throughout the life cycle of both males and females. This antibody recognizes a 72-74 X 10(3) Mr protein and is useful both as a pole lineage marker and in biochemical studies of polar granules.     

Nanos is the localized posterior determinant in Drosophila

Segmental pattern in the Drosophila embryo is established by two maternal factors localized to the anterior and posterior poles of the egg cell. Here we provide molecular evidence that the localized posterior factor is the RNA of the nanos (nos) gene. nos RNA is localized to the posterior pole of early embryos, and nos protein acts at a distance to direct abdomen formation. Synthetic nos RNA has biological activity identical to that of the posterior pole plasm. Injection of nos RNA rescues the segmentation defect of embryos derived from females mutant for all nine known posterior group genes. Injection of nos RNA into the anterior is able to direct formation of ectopic posterior structures. Our results demonstrate that a localized source of nos RNA is sufficient to specify abdominal segmentation and imply that other posterior group genes are required for localization, stabilization, or distribution of the nos gene product.     

The fat facets gene is required for Drosophila eye and embryo development.

In a screen for mutations affecting Drosophila eye development, we have identified a gene called fat facets (faf) which is required for cell interactions that prevent particular cells in the developing eye from becoming photoreceptors. Analysis of eyes mosaic for faf+ and faf- cells shows that faf is required in cells near to, but outside, normal developing photoreceptors and also outside of the ectopic photoreceptors in mutant facets. faf is also essential during oogenesis, and we show that a faf-lacZ hybrid protein is localized via the first 392 amino acids of faf to the posterior pole of oocytes. Posterior localization of faf-lacZ depends on oskar. oskar encodes a key organizer of the pole plasm, a specialized cytoplasm at the posterior pole of embryos. The pole plasm is required for germ cell formation and contains the determinant of posterior polarity, encoded by nanos. Although other pole plasm components are required for localization of nanos RNA or for nanos protein function, faf is not. We have cloned the faf gene, and have shown that it encodes two similar large (approximately 300 x 10(3) M(r)) proteins that are unique with respect to other known proteins.     

poirot,a new regulatory gene of Drosophila oskar acts at the level of the short Oskar protein isoform

Embryonic germ cell formation and abdomen development in Drosophila requires localisation and site specific translation of oskar mRNA in the posterior part of the oocyte. Targeting of oskar function to the posterior pole of the oocyte needs a large set of proteins and RNAs, encoded by posterior group genes. Consequently, mutations in the posterior group genes can result in embryos without abdomens and/or germ cells. During a systematic hobo-mediated mutant isolation screen, we identified poirot, a novel posterior group gene, owing to its germ cell-less phenotype. We show that the lack of poirot activity dramatically decreases OSK protein levels, without affecting the oskar mRNA distribution. In poirot mutant oocytes, delocalised OSK protein is observed, indicating that wild-type poirot has a role in the anchoring process of the OSK protein at the posterior pole. Furthermore, we demonstrate that poirot acts in an isoform-specific manner, only the short OSK isoform is affected, while the long OSK isoform remains at wild-type levels in poirot mutants.     

Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos.

The mago nashi (mago) locus is a newly identified strict maternal effect, grandchildless-like, gene in Drosophila melanogaster. In homozygous mutant mago females reared at 17 degrees C, mago+ function is reduced, the inviable embryos lack abdominal segments and 84-98% of the embryos die. In contrast, at 25 degrees C, some mago alleles produce a novel gene product capable of inducing the formation of symmetrical double abdomen embryos. Reciprocal temperature-shift experiments indicate that the temperature-sensitive period is during oogenetic stages 7-14. Furthermore, embryos collected from mago1 homozygous females contain no apparent functional posterior determinants in the posterior pole. In viable F1 progeny from mago mutant females, regardless of genotype and temperature, polar granules are reduced or absent and germ cells fail to form (the grandchildless-like phenotype). Thus, we propose that the mago+ product is a component of the posterior determinative system, required during oogenesis, both for germ cell determination and delineation of the longitudinal axis of the embryo.