The nanos is a maternal effect gene required for the development of the fruit fly Drosophila melanogaster. nanos is required during oogenesis for the development and maintenance of the germ line, in the embryo for patterning of the abdomen, and in the primordial germ cells for their migration to the embryonic gonad. nanos messenger RNA is a model system for the study of RNA localization and translation mediated by the 3′-untranslated region (UTR), and the Nanos protein is studied for its ability to repress translation of mRNAs through sequences within their 3′ -untranslated regions (see Translational repressors).
1. Protein and RNA Structure
The nanos gene encodes a 3-kb primary transcript, containing two introns, that is processed to a mature 2.4-kb mRNA (1). The 3 ‘ -untranslated region contains sequences required for localization and translational repression of the transcript (2). nanos mRNA is detected at high levels in ovaries, 0- to 8-hour embryos, and adult female flies (1). Low levels of the transcript are also detected in adult male flies, but there is no genetic evidence of a role for nanos in males. The Nanos protein is a 400-amino-acid-residue polypeptide chain with a predicted molecular weight of 43 kDa. It contains several polyglutamine and polyasparagine repeats, which are common to Drosophila proteins. Analysis of nanos mutants reveals that a small carboxyl-terminal domain is required for nanos function in vivo. Within this region are two Cys-Cys-His-Cys motifs that have been shown to bind metal in vitro and closely resemble the retroviral nucleocapsid class of zinc-finger proteins (3).
2. Oogenesis
A Drosophila melanogaster ovary consists of approximately 16 ovarioles, each representing an independent egg assembly line (for review see Ref. 4). Every ovariole is a linear array of developing egg chambers beginning at the anterior with the germarium, where the progeny of the germ-line and somatic stem cells are organized into egg chambers, and progressing to more mature egg chambers as they move toward the posterior. In the germarium, a germ-line stem cell produces a cystoblast by asymmetric division. The cystoblast undergoes four rounds of division with incomplete cytokinesis, resulting in 16 cells connected by cytoplasmic bridges; one of these cells becomes the oocyte while the other 15 become nurse cells. An egg chamber that has left the germarium consists of the oocyte and nurse cells surrounded by somatically derived follicle cells. As the egg chambers mature, the nurse cells synthesize and deposit factors into the oocyte that are required for the development of the oocyte and early embryo. Late in oogenesis, the nurse cells contract and dump the contents of their cytoplasm into the oocyte. Nanos protein is expressed at high levels in the germ-line cysts of the germarium and at lower levels in the germline stem cells and dividing cystoblasts.
Females trans-heterozygous for strong nanos alleles lay a few eggs, but rapidly become sterile due to a loss of germ-line stem cells from the ovary. Analysis of nanos mutant ovaries demonstrates a number of defects (5). In most instances, ovarioles completely lack germ line, suggesting that zygotic nanos, likepumilio, is required for incorporation of germ-line cells into the developing ovarioles. Less frequently, germ-line cells appear to be incorporated into the ovariole. In some of these ovarioles, germ-line stem cells are absent and germ-line cells may develop directly into egg chambers, while in other cases stem cells seem to be established but the subsequent development of the cystoblast and maintenance of the stem cells are impaired. This suggests that nanos is required for the proliferation and viability of the germ-line stem cells and the developing germ-line cysts (5).
Once developing egg chambers leave the germarium, Nanos protein is no longer detectable until late in oogenesis, when Nanos protein is expressed at high levels in the nurse cells, but not the oocyte (6). The function of nanos at this stage is not clear, because egg chambers that lack this expression develop normally. nanos does not appear to play a role in spermatogenesis.
3. Embryonic Patterning
In Drosophila melanogaster, the anterior-posterior axis is defined by three independent systems: the terminal group of genes that controls the formation of the unsegmented termini at the anterior and posterior ends of the embryo; the anterior group of genes that controls development of the head and thorax; and the posterior group of genes that determines the abdomen (for review see Ref. 7). Each system requires the localized activity of a maternally supplied factor that functions as a spatial signal resulting in a morphogenetic gradient. The terminal and anterior systems require the activities of torso and bicoid, respectively. nanos is the posterior determinant; in the absence of nanos, embryos fail to form abdominal segments. nanos mRNA is localized to the posterior pole, where its translation results in a gradient of Nanos protein from the posterior pole to approximately 50% of the egg length (8). Mislocalization of nanos RNA to an ectopic site leads to ectopic abdomen formation (8). The only required function for nanos in abdomen formation is the repression of maternal hunchback translation at the posterior—resulting in a gradient of Hunchback protein, with its high point at the anterior pole (9-11). Hunchback is a zinc-finger containing transcription factor that represses translation of abdominal gap genes; consequently, repressing hunchback in the posterior allows the ordered expression of the abdominal gap genes and abdomen formation.
3.1. Regulation of nanos mRNA Localization and Translation
Throughout oogenesis, nanos RNA is synthesized in the nurse cells and deposited in the developing oocyte (6). Late in oogenesis, nanos RNA becomes enriched at the posterior pole of the embryo (1). This process requires a localization control element located within the first 400 nucleotides of nanos 3′-untranslated region and the ordered activity of the posterior group genes including oskar, a gene necessary for organization of the pole plasm (12, 13). oskar mutant embryos lack pole plasm and fail to localize nanos mRNA to the posterior pole (14). Ectopic expression of oskar at the anterior pole results in ectopic pole plasm formation and in nanos mRNA being mislocalized to the anterior (14). nanos mRNA that is localized to the posterior pole plasm is translated, whereas unlocalized nanos mRNA is translationally repressed, suggesting that nanos mRNA localization and translation are linked (2). A 90-nucleotide translational control element in the 3′UTR of nanos is both necessary and sufficient for translational repression of nanos. The translational control element can function independently of the localization element, and RNA localization can occur in the absence of the translational control element (12, 13,15). Translational repression can be overcome by removing the 3′ -UTR, localizing the transcript to the anterior pole via heterologous RNA localization sequences from the bicoid 3′UTR, or by specifically mutating the translational control element. A 135-kDa protein, Smaug, has been shown to bind to the translational control element in vitro (15). Mutational analysis of the translational-control-element indicates that failure to bind Smaug in vitro correlates with lack of translational repression in vivo (15). It is not yet understood how the translational control element and Smaug mediate repression, or how this repression is relieved at the posterior pole.
3.2. Function of nanos in Embryonic Patterning
Although the maternal hunchback transcript is distributed throughout the early embryo, it is only translated in the anterior half of the embryo (16). Repression of maternal hunchback translation in the posterior requires sequences within the hunchback 3′ -untranslated region (termed nanos response elements or NREs) and the activity of nanos and pumilio (17). Mutation of the NRE, or loss of nanos or pumilio activity, results in a loss of abdominal segmentation (17). Recently it has been shown that Pumilio is an RNA-binding protein that interacts specifically with the NRE (18-20). Although Nanos can bind RNA with high affinity, it does so with low specificity (3). It has therefore been proposed that Pumilio acts as a sequence-specific adaptor for Nanos that provides the spatial aspects of hunchback translational regulation (19). The mechanisms by which Pumilio and Nanos regulate hunchback translation are unclear. Maternally supplied hunchback RNA is deadenylated in the presence of Nanos and Pumilio (21) which suggests that NRE-directed removal of the poly (A) tail may lead to translational repression. Ectopic expression of nanos in the eye imaginal disc results in disruption of the development of the adult eye (20). This activity requires endogenous pumilio expression. Furthermore, ectopic nanos can repress the expression of reporter genes bearing the NREs, suggesting that the machinery needed for nanos -mediated repression may be present in all cells (20). Finally, ectopic nanos mediates repression of a reporter gene containing internal ribosome entry sites. This suggests that nanos-mediated translational repression may occur in a 5 ‘-cap-independent manner (20).
When Nanos is ectopically localized to the anterior pole of the embryo, in addition to hunchback, translation of the homeodomain transcription factor bicoid is also repressed (8). Repression of bicoid translation results in the loss of head structures, while repression of hunchback translation at the anterior causes a mirror image duplication of the abdomen, resulting in a bicaudal phenotype (8). As with hunchback, translational repression of bicoid RNA is mediated by a NRE in the bicoid 3 ‘ -untranslated region, and the poly (A) tail of bicoid mRNA is shortened in the presence of nanos and pumilio (21).
Cyclin B also contains a sequence in its 3′-untranslated region that is similar to the nanos response element. cyclin B is not translated at the posterior pole, and removal of this element is sufficient to activate translation of cyclin B mRNA at the posterior pole. The role of nanos and pumilio in the regulation of cyclin B translation has not been demonstrated (22).
4. Germ Cell Migration
After fertilization, the Drosophila embryo develops as a syncytium where nuclear rather than cellular divisions follow each other rapidly and in synchrony. The primordial germ cells are the first cells to form. They cellularize at the posterior pole of the embryo, encapsulating the pole plasm in the process (reviewed in Ref. 23). During gastrulation, the germ cells are carried along the dorsal surface of the embryo inside the invaginating posterior midgut pocket. Subsequently, the primordial germ cells migrate through the posterior midgut and move toward the somatic component of the gonad, the gonadal mesoderm. Finally, the germ cells align with the gonadal mesoderm and coalesce to form the embryonic gonad.
Nanos protein and RNA are incorporated into the germ cells as they form at the posterior pole (6). Nanos protein is present in the germ cells until gonad coalescence (6). nanos is not expressed in the soma, and wild-type germ cells transplanted into nanos mutant embryos migrate to and populate the gonad. In nanos mutant embryos, the germ cells form normally and are carried into the midgut pocket. However, while wild-type germ cells migrate from the midgut to the mesoderm, nanos mutant germ cells fail to migrate appropriately and often remain associated with the midgut (5). Analysis of nanos mutant germ cells demonstrates that several germ-cell markers, which are normally expressed within the gonad, are transcribed prematurely during germ-cell migration (24). These data suggest a role for nanos in the specification or maintenance of the germ-cell identity during migration. Although Pumilio protein is also encapsulated in the germ cells when they form, it is not known if pumilio and nanos share a common function in the germ cells as they do in abdomen formation.
5. Homologues
Based on similarity to the carboxyl-terminal domain, nanos homologues have been identified from three other Dipteran species (Drosophila virilis, the housefly Musca domestica, and the midge Chironomous samoensis ), the leech Helobdella robusta, the African clawed frog Xenopus laevis, and the nematode Caenorhabditis elegans (25-27). Analysis of the distribution of the homologues that have been studied so far suggests that nanos is a localized factor important for establishing embryonic polarity and/or development of the germ line. The expression pattern of nanos RNA is virtually indistinguishable among the different Dipteran species analyzed (25). Injection of nanos RNA isolated from the different Dipteran species into D. melanogaster embryos is able to rescue the abdominal defects associated with loss of nanos in D. melanogaster, suggesting that these homologues share a common, conserved function (25).
