Search

During Drosophila oogenesis, the posterior determinant, Oskar, is tightly localized at the posterior pole of the oocyte. The exclusive accumulation of Oskar at this site is ensured by localization-dependent translation of oskar mRNA: translation of oskar mRNA is repressed during transport and activated upon localization at the posterior cortex. Previous studies have suggested that oskar translation is poly(A)-independent. We show that a long poly(A) tail is required for efficient oskar translation, both in vivo and in vitro, but is not sufficient to overcome BRE-mediated repression. Moreover, we show that accumulation of Oskar activity requires the Drosophila homolog of Cytoplasmic Polyadenylation Element Binding protein (CPEB), Orb. As posterior localization of oskar mRNA is an essential prerequisite for its translation, it was critical to identify an allele of orb that does localize oskar mRNA to the posterior pole of the oocyte. We show that flies bearing the weak mutation orbmel localize oskar transcripts with a shortened poly(A) that fails to enhance oskar translation, resulting in reduced Oskar levels and posterior patterning defects. We conclude that Orb-mediated cytoplasmic polyadenylation stimulates oskar translation to achieve the high levels of Oskar protein necessary for posterior patterning and germline differentiation.

After its specification, the Drosophila oocyte undergoes a critical polarization event that involves a reorganization of the microtubules (MT) and relocalization of the determinant Orb within the oocyte. This polarization requires Par-1 kinase and the PDZ-containing Par-3 homolog, Bazooka (Baz). Par-1 has been observed on the fusome, which degenerates before the onset of oocyte polarization. How Par-1 acts to polarize the oocyte has been unclear. Here we show that Par-1 becomes restricted to the oocyte in a MT-dependent fashion after disappearance of the fusome. At the time of polarization, the kinase itself and the determinant BicaudalD (BicD) are relocalized from the anterior to the posterior of the oocyte. Par-1 and BicD are interdependent and require MT and the minus end-directed motor Dynein for their relocalization. We show that baz is required for Par-1 relocalization within the oocyte and that the distributions of Baz and Par-1 in the Drosophila oocyte are complementary and strikingly reminiscent of the two PAR proteins in the C. elegans embryo. We propose that, through the combined actions of the fusome, MT, and Baz, Par-1 is selectively enriched and localized within the oocyte, where, in conjunction with BicD, Egalitarian (Egl), and Dynein, it acts on the MT cytoskeleton to effect polarization.

Localization of the maternal determinant Oskar at the posterior pole of Drosophila melanogaster oocyte provides the positional information for pole plasm formation. Spatial control of Oskar expression is achieved through the tight coupling of mRNA localization to translational control, such that only posterior-localized oskar mRNA is translated, producing the two Oskar isoforms Long Osk and Short Osk. We present evidence that this coupling is not sufficient to restrict Oskar to the posterior pole of the oocyte. We show that Long Osk anchors both oskar mRNA and Short Osk, the isoform active in pole plasm assembly, at the posterior pole. In the absence of anchoring by Long Osk, Short Osk disperses into the bulk cytoplasm during late oogenesis, impairing pole cell formation in the embryo. In addition, the pool of untethered Short Osk causes anteroposterior patterning defects, owing to the dispersion of pole plasm and its abdomen-inducing activity throughout the oocyte. We show that the N-terminal extension of Long Osk is necessary but not sufficient for posterior anchoring, arguing for multiple docking elements in Oskar. This study reveals cortical anchoring of the posterior determinant Oskar as a crucial step in pole plasm assembly and restriction, required for proper development of Drosophila melanogaster.

Recent advances shed light on the cellular processes that cooperate during oogenesis to produce a fully patterned egg, containing all the maternal information required for embryonic development. Progress has been made in defining the early steps in oocyte specification and it has been shown that progression of oogenesis is controlled by a meiotic checkpoint and requires active maintenance of the oocyte cell fate. The function of Gurken signalling in patterning the dorsal-ventral axis later in oogenesis is better understood. Anterior-posterior patterning of the embryo requires activities of bicoid and oskar mRNAs, localised within the oocyte. A microtubule motor, Kinesin, is directly implicated in localisation of oskar mRNA to the posterior pole of the oocyte.

A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation

Morphogenesis and cell differentiation in multicellular organisms often require accurate control of cell divisions. We show that a novel cell cycle regulator, tribbles, is critical for this control during Drosophila development. During oogenesis, the level of tribbles affects the number of germ cell divisions as well as oocyte determination. The mesoderm anlage enters mitosis prematurely in tribbles mutant embryos, leading to gastrulation defects. We show that Tribbles acts by specifically inducing degradation of the CDC25 mitotic activators String and Twine via the proteosome pathway. By regulating CDC25, Tribbles serves to coordinate entry into mitosis with morphogenesis and cell fate determination.

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. In this study we demonstrate that, when oskar mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5′ region of the mRNA. We demonstrate that this novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the oskar 3′ UTR is required for activation of oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction betweentrans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. We also show specific interaction of two proteins with the oskar mRNA 5′ region; one of these also recognizes the 3′ repressor element. We discuss the possible involvement of these factors as well as known genes in the process of localization-dependent translation.

Nuclear hormone receptors and homeodomain proteins are two classes of transcription factor that regulate major developmental processes. Both depend on interactions with other proteins for specificity and activity. The Drosophila gene fushi tarazu (ftz)y which encodes a homeodomain protein1 (Ftz), is required zygo-tically for the formation of alternate segments in the developing embryo2. Here we show that the orphan nuclear receptor α Ftz-Fl (ref. 3), which is deposited in the egg during oogenesis4, is an obligatory cofactor for Ftz. The two proteins interact specifically and directly, both in vitro and in vivo, through a conserved domain in the Ftz polypeptide. This interaction suggests that other nuclear receptor/homeodomain protein interactions maybe important and common in developing organisms.

At the posterior pole of the Drosophila oocyte, oskar induces a tightly localized assembly of pole plasm. This spatial restriction of oskar activity has been thought to be achieved by the localization of oskar mRNA, since mislocalization of the RNA to the anterior induces anterior pole plasm. However, ectopic pole plasm does not form in mutant ovaries where oskar mRNA is not localized, suggesting that the unlocalized mRNA is inactive. As a first step towards understanding how oskar activity is restricted to the posterior pole, we analyzed oskar translation in wild type and mutants. We show that the targeting of oskar activity to the posterior pole involves two steps of spatial restriction, cytoskeleton-dependent localization of the mRNA and localization-dependent translation. Furthermore, our experiments demonstrate that two isoforms of Oskar protein are produced by alternative start codon usage. The short isoform, which is translated from the second in-frame AUG of the mRNA, has full oskar activity. Finally, we show that when oskar RNA is localized, accumulation of Oskar protein requires the functions of vasa and tudor, as well as oskar itself, suggesting a positive feedback mechanism in the induction of pole plasm by oskar.

THE localization of oskar (osk) RNA to the posterior pole of the developing fruit fly (Drosophila) oocyte induces the assembly of pole plasm, causing development of the abdomen and germ line1,2. Failure to localize oskar RNA results in embryos that lack abdomen and germ cells. Conversely, mis-targeting of oskar RNA to the anterior of the oocyte causes formation of ectopic abdomen and germ cells at the anterior pole3. Maternal mutants that have reduced pole plasm activity produce sterile adults with normal abdominal development, suggesting that germ cells are more sensitive than abdomen to defects in pole plasm assembly4. Thus mutations in genes that reduce oskar RNA localization or activity can be recovered as viable sterile adults. In a screen for mutants defective in germ cell formation, we isolated nine alleles of the tropomyosin II gene5. Here we show that mutations in tropomyosin II (TmII) virtually abolish oskar RNA localization to the posterior pole, suggesting an involvement of the actin network in oskar RNA localization.

In organisms as diverse as frogs, worms and flies germline precursor cells are set aside from the somatic cells early in development. It has been proposed that specific molecules, referred to as germ cell determinants, are deposited in the egg and direct the germ cell fate, but the molecular nature and function of these determinants is not fully understood. Genetic and molecular analysis in Drosophila melanogaster indicates that germ cell determination involves not only the synthesis of specific germ cell factors but also the proper localization and assembly of a morphologically distinct germ plasm. A pathway for germ plasm assembly has been established in which the oskar gene has a central role. The amount of oskar product in the embryo controls the number of germ cells formed and mislocalization of oskar RNA and protein in the egg cell leads to the formation of ectopic germ cells in the embryo. In addition to its role in anchoring germ cell-specific signals, the germ plasm also serves as the source of abdomen-specific signal. Such a colocalization of morphogenetic signals involved in germ cell formation and in the specification of the body axis is not unique to Drosophila but is also found in Caenorhabditis elegans and Xenopus.

The oskar gene directs germ plasm assembly and controls the number of germ cell precursors formed at the posterior pole of the Drosophila embryo. Mislocalization of oskar RNA to the anterior pole leads to induction of germ cells at the anterior. Of the eight genes necessary for germ cell formation at the posterior, only three, oskar, vasa and tudor, are essential at an ectopic site.

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.

The introns separating the variable and constant regions of active immunoglobulin genes contain tissue-specific transcriptional enhancer elements, DNA segments which act in cis in an orientation- and distance-independent (up to a few kilobases (kb)) manner to enhance transcription initiation at adjacent promoters. The immunoglobulin heavy-chain enhancer is active only in lymphoid cells: in transfection assays it is capable of controlling in cis transcription from the simian virus 40 (SV40) T-antigen, rabbit beta-globin and immunoglobulin gene promoters up to at least 2 kb away. Genetic deletion analysis suggests that a region of as few as 140 base pairs (bp) is sufficient for the enhancement effect. These functional characteristics and DNA sequences are conserved between mouse and man. However, it is not known whether tissue-specific proteins bind to the enhancer. Proteins that interact with DNA at specific sequences can prevent or enhance the reactions of individual guanines or adenines with dimethyl sulphate (DMS), and this property has been used to display the DNA contacts of various regulatory proteins. Here we apply this DMS strategy in experiments involving single-copy genes within intact mammalian nuclei using genomic sequencing.

Intracellular mRNA localization is an effective mechanism for protein targeting leading to functional polarization of the cell. The mechanisms controlling mRNA localization and specifically how the actin and microtubule (MT) cytoskeletons cooperate in this process are not well understood. In Drosophila, Oskar protein accumulation at the posterior pole of the oocyte is required for embryonic development 1, 2, 3 and 4 and is achieved by the transport of oskar mRNA and its exclusive translation at the posterior pole 1, 5, 6 and 7. oskar mRNA localization requires the activity of the MT-based motor Kinesin [8], as well as the formation of a transport-competent ribonucleoprotein (RNP) complex [9]. Here, we show that didum, encoding the Drosophila actin-based motor Myosin-V 10 and 11, is a new posterior group gene that promotes posterior accumulation of Oskar. Myosin-V associates with the oskar mRNA transport complex preferentially at the oocyte cortex, revealing a short-range actomyosin-based mechanism that mediates the local entrapment of oskar at the posterior pole. Our results also show that Myosin-V interacts with Kinesin heavy chain and counterbalances Kinesin function, preventing ectopic accumulation of oskar in the cytoplasm. Our findings reveal that a balance of microtubule- and actin-based motor activities regulates oskar mRNA localization in the Drosophila oocyte.

The Ser/Thr kinases of the PAR-1/MARK/Kin1 family are conserved regulators of polarity in epithelial and non-epithelial cells [1]. Drosophila PAR-1 localizes laterally in the follicular epithelium of the ovary [2, 3], where it has been shown to function at two distinct levels: It stabilizes the cytoskeleton [3, 4] and it regulates apical-basal polarity by directly inhibiting lateral assembly of the apical aPKC/Bazooka/PAR-6 complex [5, 6]. However, it has been unclear how lateral localization of Drosophila PAR-1 is achieved and whether this localization contributes to epithelial polarity in vivo. Here we show that, through its spacer domain, Drosophila PAR-1 accumulates on the lateral plasma membrane (PM) in cells of the follicular epithelium (FE). Rescue experiments indicate that in FE cells PAR-1 kinase activity is essential for all the described functions of PAR-1. In contrast, the spacer domain of PAR-1 is required for apical-basal polarity and growth control but is dispensable for microtubule (MT) stabilization. Our data indicate that the spacer domain of PAR-1 is required for lateral PM localization of PAR-1 kinase and for development of a polarized FE.

SummaryIn Drosophila, localized activity of oskar at the posterior pole of the oocyte induces germline and abdomen formation in the embryo. Oskar has two isoforms, a short isoform encoding the patterning determinant and a long isoform of unknown function. Here, we show by immuno-electron microscopy that the two Oskar isoforms have different subcellular localizations in the oocyte: Short Oskar mainly localizes to polar granules, and Long Oskar is specifically associated with endocytic membranes along the posterior cortex. Our cell biological and genetic analyses reveal that Oskar stimulates endocytosis, and that its two isoforms are required to regulate this process. Furthermore, we describe long F-actin projections at the oocyte posterior pole that are induced by and intermingled with Oskar protein. We propose that Oskar maintains its localization at the posterior pole through dual functions in regulating endocytosis and F-actin dynamics.

The mouse heavy chain immunoglobulin gene contains a tissue-specific enhancer. The enhancer and flanking sequences were studied in vivo by carrying out dimethyl sulfate protection experiments on living cells, in combination with genomic sequencing. Relative to reactions on naked DNA, there are changes (protections and enhancements) in the reactivity of guanine residues to dimethyl sulfate within the enhancer sequence in myeloma, B, and early B cells, whereas virtually no alterations appear in cells of non-B lineage. Most of the affected residues are in four clusters, in sequences homologous to the octamer 5'CAGGTGGC 3' (C, cytosine; A, adenine; G. guanine; T, thymine). The alterations in the pattern of G reactivity are consistent with the tissue-specific binding of molecules to the mouse immunoglobulin heavy chain enhancer.

The coupled regulation of oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of oskar mRNA, a mechanism whereby oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3′untranslated region (UTR) of oskar mRNA. We have established a cell-free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that this ovary extract, containing endogenous Bruno, is able to recapitulate oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, we show that a Drosophila embryo extract lacking Bruno efficiently translates oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in oskar mRNA translation. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.

Par-1 kinase is critical for polarization of the Drosophila melanogaster oocyte and the one-cell Caenorhabditis elegans embryo. Although Par-1 localizes specifically to the posterior pole in both cells, neither its targets nor its function at the posterior pole have been elucidated. Here we show that Drosophila Par-1 phosphorylates the posterior determinant Oskar (Osk) and demonstrate genetically that Par-1 is required for accumulation of Osk protein. We show in cell-free extracts that Osk protein is intrinsically unstable and that it is stabilized after phosphorylation by Par-1. Our data indicate that posteriorly localized Par-1 regulates posterior patterning by stabilizing Osk.

The fushi tarazu pair-rule gene is required for the formation of alternating parasegmental boundaries in the Drosophila embryo. fushi tarazu encodes a homeodomain protein necessary for transcription of the engrailed gene in even-numbered parasegments. Here we report that, within an engrailed enhancer, adjacent and conserved binding sites for the Fushi tarazu protein and a cofactor are each necessary, and together sufficient, for transcriptional activation. Footprinting shows that the cofactor site can be bound specifically by Ftz-F1, a member of the nuclear receptor superfamily. Ftz-F1 and the Fushi tarazu homeodomain bind the sites with 4-to 8-fold cooperativity, suggesting that direct contact between the two proteins may contribute to target recognition. Even parasegmental reporter expression is dependent on Fushi tarazu and maternal Ftz-F1, suggesting that these two proteins are indeed the factors that act upon the two sites in embryos. The two adjacent binding sites are also required for continued activity of the engrailed enhancer after Fushi tarazu protein is no longer detectable, including the period when engrailed, and the enhancer, become dependent upon wingless. We also report the existence of a separate negative regulatory element that apparently responds to odd-skipped.

Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.