In vivo analysis of Drosophila bicoid mRNA localization reveals a novel microtubule-dependent axis specification pathway.

Drosophila bicoid mRNA is synthesized in the nurse cells and transported to the oocyte where microtubules and Exuperantia protein mediate localization to the anterior pole. Fluorescent bicoid mRNA injected into the oocyte displays nonpolar microtubule-dependent transport to the closest cortical surface, and the oocyte microtubule cytoskeleton lacks clear axial asymmetry. Nonetheless, bicoid mRNA injected into the nurse cell cytoplasm, withdrawn, and injected into a second oocyte shows microtubule-dependent transport to the anterior cortex. Nurse cells require microtubules and Exuperantia to support anterior transport of bicoid mRNA, and microtubules are required for bicoid mRNA-Exuperantia particle coassembly. We propose that microtubule-dependent Exuperantia-bicoid mRNA complex formation in the nurse cell cytoplasm allows anterior-specific transport on a grossly nonpolar oocyte microtubule network.     

Miranda couples oskar mRNA/Staufen complexes to the bicoid mRNA localization pathway

The double-stranded RNA binding protein Staufen is required for the microtubule-dependent localization of bicoid and oskar mRNAs to opposite poles of the Drosophila oocyte and also mediates the actin-dependent localization of prospero mRNA during the asymmetric neuroblast divisions. The posterior localization of oskar mRNA requires Staufen RNA binding domain 2, whereas prospero mRNA localization mediated the binding of Miranda to RNA binding domain 5, suggesting that different Staufen domains couple mRNAs to distinct localization pathways. Here, we show that the expression of Miranda during mid-oogenesis targets Staufen/oskar mRNA complexes to the anterior of the oocyte, resulting in bicaudal embryos that develop an abdomen and pole cells instead of the head and thorax. Anterior Miranda localization requires microtubules, rather than actin, and depends on the function of Exuperantia and Swallow, indicating that Miranda links Staufen/oskar mRNA complexes to the bicoid mRNA localization pathway. Since Miranda is expressed in late oocytes and bicoid mRNA localization requires the Miranda-binding domain of Staufen, Miranda may play a redundant role in the final step of bicoid mRNA localization. Our results demonstrate that different Staufen-interacting proteins couple Staufen/mRNA complexes to distinct localization pathways and reveal that Miranda mediates both actin- and microtubule-dependent mRNA localization.     

Swallowing dynein: a missing link in RNA localization?

Localization of bicoid messenger RNA to the anterior cortex of the developing oocyte is essential for correct anterior-posterior patterning of the Drosophila embryo. It now seems that the Swallow protein functions as an adaptor, bridging bicoid mRNA to dynein, a molecular motor that would transport the complex anteriorly along microtubules.     

Changes in bicoid mRNA anchoring highlight conserved mechanisms during the oocyte-to-embryo transition

Intracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a variety of organisms. In Drosophila, bicoid (bcd) mRNA is prelocalized at the oocyte anterior. After fertilization, translation of this RNA produces a Bcd protein gradient that determines anterior cell fates [1] and [2]. Analysis of bcd mRNA during late stages of oogenesis suggested a model for steady-state bcd localization by continual active transport [3]. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4] and [5], or retention of bcd at the anterior in mature oocytes, which can remain dormant for weeks before fertilization [6]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. We show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis. Egg activation triggers bcd release from the anterior cortex for proper deployment in the embryo, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1 mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results thus highlight a conserved mechanism for regulating mRNA anchoring and redeployment during the oocyte-to-embryo transition.     

Redundant RNA recognition events in bicoid mRNA localization.

A cis-acting signal in the 3' UTR of the Drosophila bicoid mRNA directs both the transport of the mRNA from the nurse cells to the oocyte and its anterior localization within the oocyte. Here we demonstrate that the signal mediates redundant RNA recognition events, A and B, that initiate largely overlapping programs of mRNA localization during oogenesis. Recognition event A requires a region encompassing stem-loops IV/V of the predicted secondary structure, and can be eliminated by a single nucleotide mutation. Localization initiated through event B begins slightly later in oogenesis, and requires sequences that have not been narrowly defined. Using forms of the 3' UTR lacking this RNA recognition redundancy, we reexamine the roles of the swallow, staufen, and exuperantia genes, which are all required for normal bicoid mRNA localization. Our results reveal that exuperantia first becomes essential for localization at a time when well-defined microtubule tracks between the nurse cells and oocyte disappear. Thus, exuperantia may specifically facilitate a form of nurse cell-to-oocyte mRNA transport not dependent on the microtubule tracks.     

The molecular motor dynein is involved in targeting swallow and bicoid RNA to the anterior pole of Drosophila oocytes

Localization of bicoid (bcd) messenger RNA to the anterior pole of the Drosophila oocyte requires the exuperantia ( exu), swallow (swa) and staufen (stau) genes. We show here that Swa protein transiently co-localizes with bcd RNA in mid-oogenesis. Swa also localizes to the anterior pole of the oocyte in the absence of bcd RNA. This localization does not require Exu, but depends on intact microtubules. In mutant ovaries with duplicated polarity of microtubules, Swa and bcd RNA are ectopically localized at the posterior pole, as well as being present at the anterior pole. We identify dynein light chain-1 (Ddlc-1), a component of the minus-end-directed microtubule motor cytoplasmic dynein, as a Swa-binding protein. We propose that Swa acts as an adaptor for the dynein complex and thereby enables dynein to transport bcd RNA along microtubules to their minus ends at the anterior pole of the oocyte.     

Microtubules mediate the localization of bicoid RNA during Drosophila oogenesis.

We have examined cytoskeletal requirements for bicoid (bcd) RNA localization during Drosophila oogenesis. bcd is an anterior morphogen whose proper function relies on the localization of its messenger RNA to the anterior cortex of the egg. Drugs that depolymerize microtubules perturb all aspects of bcd RNA localization. During recovery from drug treatment, bcd RNA relocalizes to the oocyte cortex, suggesting that the localization machinery is a component of the cortical cytoskeleton. Taxol, a drug that stabilizes microtubules, also effectively disrupts bcd RNA localization, and the effects of taxol treatments on exuperantia and swallow mutants suggest general roles for these gene products in the multi-step bcd RNA localization process.     

Reorganization of the cytoskeleton during Drosophila oogenesis: implications for axis specification and intercellular transport.

Inhibitor studies have implicated microtubules in at least three important developmental processes during Drosophila oogenesis: oocyte determination and growth during stages 1 through 6, positioning of the anterior determinant bicoid mRNA during stages 9 through 12, and ooplasmic streaming during stages 10b through 12. We have used fluorescence cytochemistry together with laser scanning confocal microscopy to identify distinct microtubule structures at each of the above three periods that are likely to be involved in these processes. During stages 1 through 7, maternal components synthesized in nurse cells are transported through cytoplasmic bridges to the oocyte. At this time, microtubules that appear to originate in the oocyte pass through these cytoplasmic bridges into the adjacent nurse cells; these microtubules are likely to serve as a polarized scaffold on which maternal RNAs and proteins are transported. During stages 7 and 8, microtubules in the oocyte cortex reorganize to form an anterior-to-posterior gradient, suggesting a role for microtubules in the localization of morphogenetic determinants. Finally, when ooplasmic streaming begins during stage 10 b, it is accompanied by the assembly of subsurface microtubule arrays that spiral around the oocyte; these arrays disassemble as the oocyte matures and streaming stops. During ooplasmic streaming, many vesicles are closely associated with the subsurface microtubules, suggesting that streaming is driven by vesicle translocation along microtubules. We believe that actin plays a secondary role in each of these morphogenetic events, based on our parallel studies of actin organization during each of the above stages of oogenesis.     

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.     

A functional link between localized Oskar, dynamic microtubules, and endocytosis

Many cell types including developing oocytes, fibroblasts, epithelia and neurons use mRNA localization as a means to establish polarity. The Drosophila oocyte has served as a useful model in dissecting the mechanism of mRNA localization. The polarity of the oocyte is established by the specific localization of three critical mRNAs-oskar, bicoid and gurken. The localization of these mRNAs requires microtubule integrity, and the activity of microtubule motors. However, the precise organization of the oocyte microtubule cytoskeleton remains an open question. In order to examine the polarity of oocyte microtubules, we visualized the localization of canonical microtubule plus end binding proteins, EB1 and CLIP-190. Both proteins were enriched at the posterior of the oocyte, with additional foci detected within the oocyte cytoplasm and along the cortex. Surprisingly, however, we found that this asymmetric distribution of EB1 and CLIP-190 was not essential for oskar mRNA localization. However, Oskar protein was required for recruiting the plus end binding proteins to the oocyte posterior. Lastly, our results suggest that the enrichment of growing microtubules at the posterior pole functions to promote high levels of endocytosis in this region of the cell. Thus, multiple polarity-determining pathways are functionally linked in the Drosophila oocytes.     

Posterior localization of dynein and dorsal-ventral axis formation depend on kinesin in Drosophila oocytes

To establish the major body axes, late Drosophila oocytes localize determinants to discrete cortical positions: bicoid mRNA to the anterior cortex, oskar mRNA to the posterior cortex, and gurken mRNA to the margin of the anterior cortex adjacent to the oocyte nucleus (the "anterodorsal corner"). These localizations depend on microtubules that are thought to be organized such that plus end-directed motors can move cargoes, like oskar, away from the anterior/lateral surfaces and hence toward the posterior pole. Likewise, minus end-directed motors may move cargoes toward anterior destinations. Contradicting this, cytoplasmic dynein, a minus-end motor, accumulates at the posterior. Here, we report that disruption of the plus-end motor kinesin I causes a shift of dynein from posterior to anterior. This provides an explanation for the dynein paradox, suggesting that dynein is moved as a cargo toward the posterior pole by kinesin-generated forces. However, other results present a new transport polarity puzzle. Disruption of kinesin I causes partial defects in anterior positioning of the nucleus and severe defects in anterodorsal localization of gurken mRNA. Kinesin may generate anterodorsal forces directly, despite the apparent preponderance of minus ends at the anterior cortex. Alternatively, kinesin I may facilitate cytoplasmic dynein-based anterodorsal forces by repositioning dynein toward microtubule plus ends.     

Localization of swallow-Green Fluorescent Protein in Drosophila oogenesis and implications for the role of swallow in RNA localization

The localization of a hybrid protein composed of swallow and Green Fluorescent Protein (GFP) during Drosophila oogenesis is reported. I constructed a hybrid gene with GFP inserted into an internal position of swallow. This gene was integrated into the Drosophila genome and provides full swallow+ function, as assayed by the complete rescue of strong swallow mutants. Swallow-GFP is localized at all points along the oocyte cortex from vitellogenic stages of oogenesis through the end of oogenesis. Higher concentrations of swallow-GFP are present at the anterior oocyte cortex than at the lateral and posterior oocyte cortices at Stages 10 and 11, when bicoid and htsN4 mRNA transport from nurse cells and localization in the oocyte are most active. At Stage 9 and at Stages 12-14 swallow-GFP is equally distributed at the anterior, lateral, and posterior oocyte cortices. The position of swallow-GFP in vitellogenic stages is identical to the position of endogenous swallow protein determined by indirect immunofluorescence using an anti-swallow antibody. At the oocyte cortex, swallow-GFP is present in particulate structures that lie within or just internal to the dense cortical actin meshwork. These particles show little or no movement, suggesting that they are attached to or embedded in the oocyte cortex. These observations are most easily interpreted in the context of mRNA anchoring or microtubule organizing functions for the swallow protein.     

Distinct mechanisms for mRNA localization during embryonic axis specification in the wasp Nasonia

mRNA localization is a powerful mechanism for targeting factors to different regions of the cell and is used in Drosophila to pattern the early embryo. During oogenesis of the wasp Nasonia, mRNA localization is used extensively to replace the function of the Drosophila bicoid gene for the initiation of patterning along the antero-posterior axis. Nasonia localizes both caudal and nanos to the posterior pole, whereas giant mRNA is localized to the anterior pole of the oocyte; orthodenticle1 (otd1) is localized to both the anterior and posterior poles. The abundance of differentially localized mRNAs during Nasonia oogenesis provided a unique opportunity to study the different mechanisms involved in mRNA localization. Through pharmacological disruption of the microtubule network, we found that both anterior otd1 and giant, as well as posterior caudal mRNA localization was microtubule-dependent. Conversely, posterior otd1 and nanos mRNA localized correctly to the posterior upon microtubule disruption. However, actin is important in anchoring these two posteriorly localized mRNAs to the oosome, the structure containing the pole plasm. Moreover, we find that knocking down the functions of the genes tudor and Bicaudal-D mimics disruption of microtubules, suggesting that tudor's function in Nasonia is different from flies, where it is involved in formation of the pole plasm.     

The Drosophila hnRNPA/B homolog, Hrp48, is specifically required for a distinct step in osk mRNA localization

The Staufen-dependent localization of oskar mRNA to the posterior of the Drosophila oocyte induces the formation of the pole plasm, which contains the abdominal and germline determinants. In a germline clone screen for mutations that disrupt the posterior localization of GFP-Staufen, we isolated three missense alleles in the hnRNPA/B homolog, Hrp48. These mutants specifically abolish osk mRNA localization, without affecting its translational control or splicing, or the localization of bicoid and gurken mRNAs and the organization of the microtubule cytoskeleton. Hrp48 colocalizes with osk mRNA throughout oogenesis, and interacts with its 5' and 3' regulatory regions, suggesting that it binds directly to oskar mRNA to mediate its posterior transport. The hrp48 alleles cause a different oskar mRNA localization defect from other mutants, and disrupt the formation of GFP-Staufen particles. This suggests a new step in the localization pathway, which may correspond to the assembly of Staufen/oskar mRNA transport particles.     

Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation

Drosophila Staufen protein is required for the localization of oskar mRNA to the posterior of the oocyte, the anterior anchoring of bicoid mRNA and the basal localization of prospero mRNA in dividing neuroblasts. The only regions of Staufen that have been conserved throughout animal evolution are five double-stranded (ds)RNA-binding domains (dsRBDs) and a short region within an insertion that splits dsRBD2 into two halves. dsRBDs 1, 3 and 4 bind dsRNA in vitro, but dsRBDs 2 and 5 do not, although dsRBD2 does bind dsRNA when the insertion is removed. Full-length Staufen protein lacking this insertion is able to associate with oskar mRNA and activate its translation, but fails to localize the RNA to the posterior. In contrast, Staufen lacking dsRBD5 localizes oskar mRNA normally, but does not activate its translation. Thus, dsRBD2 is required for the microtubule-dependent localization of osk mRNA, and dsRBD5 for the derepression of oskar mRNA translation, once localized. Since dsRBD5 has been shown to direct the actin-dependent localization of prospero mRNA, distinct domains of Staufen mediate microtubule- and actin-based mRNA transport.     

Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes

Localization of bicoid (bcd) mRNA to the anterior and oskar (osk) mRNA to the posterior of the Drosophila oocyte is critical for embryonic patterning. Previous genetic studies implicated exuperantia (exu) in bcd mRNA localization, but its role in this process is not understood. We have biochemically isolated Exu and show that it is part of a large RNase-sensitive complex that contains at least seven other proteins. One of these proteins was identified as the cold shock domain RNA-binding protein Ypsilon Schachtel (Yps), which we show binds directly to Exu and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. Surprisingly, the Exu-Yps complex contains osk mRNA. This biochemical result led us to reexamine the role of Exu in the localization of osk mRNA. We discovered that exu-null mutants are defective in osk mRNA localization in both nurse cells and the oocyte. Furthermore, both Exu/Yps particles and osk mRNA follow a similar temporal pattern of localization in which they transiently accumulate at the oocyte anterior and subsequently localize to the posterior pole. We propose that Exu is a core component of a large protein complex involved in localizing mRNAs both within nurse cells and the developing oocyte.     

In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization

oskar mRNA localization to the posterior of the Drosophila oocyte defines where the abdomen and germ cells form in the embryo. Although this localization requires microtubules and the plus end-directed motor, kinesin, its mechanism is controversial and has been proposed to involve active transport to the posterior, diffusion and trapping, or exclusion from the anterior and lateral cortex. By following oskar mRNA particles in living oocytes, we show that the mRNA is actively transported along microtubules in all directions, with a slight bias toward the posterior. This bias is sufficient to localize the mRNA and is reversed in mago, barentsz, and Tropomyosin II mutants, which mislocalize the mRNA anteriorly. Since almost all transport is mediated by kinesin, oskar mRNA localizes by a biased random walk along a weakly polarized cytoskeleton. We also show that each component of the oskar mRNA complex plays a distinct role in particle formation and transport.     

gurken and the I factor retrotransposon RNAs share common localization signals and machinery

Drosophila gurken mRNA is localized by dynein-mediated transport to a crescent near the oocyte nucleus, thus targeting the TGFalpha signal and forming the primary embryonic axes. Here, we show that gurken and the I factor, a non-LTR retrotransposon, share a small consensus RNA stem loop of defined secondary structure, which forms a conserved signal for dynein-mediated RNA transport to the oocyte nucleus. Furthermore, gurken and the I factor compete in vivo for the same localization machinery. I factor transposition leads to its mRNA accumulating near and within the oocyte nucleus, thus causing perturbations in gurken and bicoid mRNA localization and axis specification. These observations further our understanding of the close association of transposable elements with their host and provide an explanation for how I factor transposition causes female sterility. We propose that the transposition of other elements may exploit the host's RNA transport signals and machinery.